Research ArticleEffects of Canonical NF-120581B Signaling Pathway on theProliferation and OdontoOsteogenic Differentiation of HumanStem Cells from Apical Papilla

Junjun Li1 Ming Yan2 Zilu Wang1 Shuanglin Jing2 Yao Li1 Genxia Liu1

Jinhua Yu12 and Zhipeng Fan3

1 Institute of Stomatology Nanjing Medical University 140 Hanzhong Road Nanjing Jiangsu 210029 China2 Endodontic Department School of Stomatology Nanjing Medical University 136 Hanzhong Road Nanjing Jiangsu 210029 China3 Laboratory of Molecular Signaling and Stem Cells Therapy and Molecular Laboratory for Gene Therapy and Tooth RegenerationBeijing Key Laboratory of Tooth Regeneration and Function Reconstruction School of Stomatology Capital Medical University4 Tian Tan Xi Li Beijing 100050 China

Correspondence should be addressed to Jinhua Yu yuziyi yjhhotmailcom andZhipeng Fan fanzhipengwang54384hotmailcom

Received 26 January 2014 Accepted 4 March 2014 Published 23 April 2014

Academic Editor Ruoning Wang

Copyright copy 2014 Junjun Li et alThis is an open access article distributed under the Creative CommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background InformationNF-120581B signaling pathway plays a complicated role in the biological functions of mesenchymal stem cellsHowever the effects of NF-120581B pathway on the odontoosteogenic differentiation of stem cells from apical papilla (SCAPs) remainunclear The present study was designed to evaluate the effects of canonical NF-120581B pathway on the osteoodontogenic capacity ofSCAPs in vitro ResultsWestern blot results demonstrated that NF-120581B pathway in SCAPs was successfully activated by TNF-120572 orblocked by BMS-345541 NF-120581B pathway-activated SCAPs presented a higher proliferation activity compared with control groupsas indicated by dimethyl-thiazol-diphenyl tetrazoliumbromide assay (MTT) and flow cytometry assay (FCM)Wound scratch assayrevealed that NF-120581B pathway-activated SCAPs presented an improved migration capacity enhanced alkaline phosphatase (ALP)activity and upregulated mineralization capacity of SCAPs as compared with control groups Meanwhile the odontoosteogenicmarkers (ALPALP RUNX2RUNX2 OSXOSX OCNOCN OPNOPN BSPBSP DSPPDSP and DMP-1DMP-1) in NF-120581Bpathway-activated SCAPs were also significantly upregulated as compared with control groups at both protein and mRNA levelsHowever NF-120581B pathway-inhibited SCAPs exhibited a lower proliferationmigration capacity and decreased odontoosteogenicability in comparison with control groups Conclusion Our findings suggest that classical NF-120581B pathway plays a paramount rolein the proliferation and committed differentiation of SCAPs

1 Introduction

NF-120581B pathway regulates the expression of a multitude ofgenes involved in the immune system growth inflammationand cancer development [1ndash3] It is commonly believed thatNF-120581B pathway plays an important role during the toothorganogenesis and eruption process [4] Furthermore NF-120581B pathway interacts with other signaling pathways such asNotch signaling and PI3KAkt pathway during the toothdevelopment and inflammation [5 6] The abolition of NF-120581B pathway may result in a developmental arrest of teeth [7]

However the influence of NF-120581B pathway on tooth develop-ment as well as root maturation has not been fully clarified

Tooth root development is an independent process afterthe formation of tooth crown during which stem cells fromapical papilla (SCAPs) are believed to play a crucial role [8]SCAPs were a unique population of stem cells gathered inthe immature root apex with the capacity of self-renewaland multiple differentiation [9] Compared with dental pulpstem cells (DPSCs) SCAPs take on a higher proliferativeability and stronger multipotent differentiation potentialFurthermore the strong expression of CD24 (pluripotency

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 319651 12 pageshttpdxdoiorg1011552014319651

2 BioMed Research International

marker) and DSPP supports the concept that SCAPs are thebest candidate for tooth regeneration and rehabilitation ofdamage [9ndash11] Nevertheless there is no convincing scientificevidence about the effects of NF-120581B pathway on the prolifer-ation and differentiation of SCAPs

NF-120581B pathway can be triggered in dental pulp stemcells under various stimulating factors that is traumainflammatory factors MTA and estrogen [12ndash15] in whichtumor necrosis factor-120572 (TNF-120572) is the putative classicalpathway activator [4 16 17] Meanwhile this pathwaycan be effectively inhibited by 4(21015840-aminoethyl) amino-18-dimethylimidazo(12-a) quinoxaline (BMS-345541 a selectiveinhibitor of IKK) [18 19] In this study we hypothesizethat activation or inhibition of NF-120581B pathway can affectthe pace of proliferation and differentiation in SCAPs Totest this hypothesis SCAPs were isolated from the devel-oping apical papillae and 10 ngmL TNF-120572 or 1 120583molLBMS-345541 was respectively used to activate or inhibitNF-120581B pathway in SCAPs [15 20] The present findingsrevealed that the proliferative ability migration potential andodontoosteogenic differentiation potential of SCAPs can besignificantly affected by the activation or inhibition of NF-120581B pathwayThese results provide novel insights into the roleof classical NF-120581B pathway during the modification of mes-enchymal stem cells and stem cell-based tooth regeneration

2 Materials and Methods

21 Cell Isolation andCulture Theprocedure for cell isolationand culture was performed as described previously [21]Briefly healthy human impacted third molars (119899 = 20)were gathered from sixteen young patients under the ageof 20 in Oral Surgery Department of Jiangsu ProvincialStomatological Hospital Root apical papilla was carefullyisolated from the immature root apex Primary apical papillacells were enzymatically separated according to previousstudy [9] and cultured in alpha minimum essential medium(120572-MEM Gibco Life Technologies Grand Island NY) sup-plementedwith 10 fetal bovine serum (FBS Hyclone USA)100 120583gmL streptomycin and 100 UmL penicillin at 37∘C ina humidified atmosphere of 5 CO

2 Then anti-rabbit IgG

Dynabeads (Dynal Biotech Oslo Norway) and rabbit anti-STRO-1 antibody (Santa Cruz Delaware CA) were used topurify these isolated cells according to the standard operatingprocedures formagnetic activated cell sorting (MACS) TNF-120572 (Peprotech USA) was dissolved in 120572-MEM at the con-centration of 100mgmL and stored at minus20∘C BMS-345541(Sigma-Aldrich MO) was dissolved in DMSO to producea 50120583molL stock solution In consideration of the cellularcytotoxicity of chemical inhibitor at high concentration asrevealed in previous study [15] we selected 1 120583molL BMS-345541 for the subsequent investigation

22 Cell Identification To determine the nature of cul-tured cells isolated cells were immunostained with theantibody against STRO-1 (1 200 Novus Biologicals USA)and cytokeratin (1 100 Bioworld USA) Phosphate bufferedsaline (PBS) was simultaneously used as a control Flow

cytometric analysis of specific surface antigens was alsoused to characterize the cultured cells Cells were harvestedand incubated with various combinations of the follow-ing fluorochrome-conjugated rabbit anti-human antibodiesCD34-FITC CD45-PerCP CD90-PE CD105-APC CD146-APC and CD73-PE (all fromMiltenyi Germany) for 20minat room temperature in the dark The corresponding mouseIgG isotype control antibodies conjugated to FITC PEAPC or PerCP were employed as negative controls in eachexperiment Stained cells were washed twice with 001molLPBS and analyzed using BD FACSCalibur (BD BiosciencesUSA)

23 Proliferation and Migration Assay The proliferation ofSCAPs treated in NF-120581B-activated culture media containing10 ngmL TNF-120572 or NF-120581B-inhibited culture mediacontaining 1 120583molL BMS-345541 was examined by 3-(45-dimethylthiazol-2-yl)-25-diphenyl-25-tetrazoliumbromide(MTT) assay and flow cytometry (FCM) SCAPs were seededinto 96-well plates (Nunc Thermo Fisher Scientific Inc)at an initial density of 2 times 103 cellswell for 24 hours At60 confluence cells were serum starved for 24 hours andtreated with 10 ngmL TNF-120572 or 1 120583molL NF-120581B inhibitorBMS-345541 After 0 1 3 5 7 and 9 days of cocultureMTT assay was performed according to the previousreport [22] The optical absorbance was obtained in anenzyme-linked immunosorbent assay plate reader (TitertekHelsinki Finland) at 490 nm according to the manufacturerrsquosinstruction The data were presented as the means plusmn SD(119899 = 6) and this experiment was repeated in triplicate

SCAPs (1 times 106) in control NF-120581B-activated and NF-120581B-inhibited groups were respectively collected washed twicewith cold 001molL PBS and fixed in 70 ice-cold ethanolovernight at 4∘C in the dark DNA content analysis of thesecells was carried out using FACScan flow cytometer (BDBiosciences San Jose CA)Then cell cycle distributions (G

1

S and G2M phases) were described and compared This

experiment was repeated three timesFor the wound healing assay SCAPs were cultured to

90 confluence in 100mm culture dishes and then woundedby using a pipette tip to scratch the monolayers The initialscratched areas were uniform across the different samplesand permanently marked Floating cells and debris wereremoved and cells were cultured in 120572-MEM supplementedwith 10 ngmL TNF-120572 or 1 120583molL BMS-345541 The markedareas in each group were photographed at 0 6 12 and 24hours after the scratch with an inverted microscope

24 Alkaline Phosphatase (ALP) Activity Assay and AlizarinRed Staining For the evaluation of osteogenic differentia-tion ALP activity was detected by using an ALP kit (NanjingJiancheng Technological Inc Nanjing China) Cells wereplated at a density of 3 times 103 cells per well into 96-well platesThen the media were changed and cells were cultured inthe complete media or the mineralization-inducing media(MM) containing 120572-MEM 10 FBS 100 120583gmL strepto-mycin 100 UmL penicillin 50mgL ascorbic acid 2mmolL

BioMed Research International 3

L-glutamine 10 nmolL dexamethasone and 10mmolL 120573-glycerophosphate (Sigma) In addition activator (TNF-120572) orinhibitor (BMS-345541) was added into experiment groupsALP activity was measured after 3 5 and 7 days of cultureThe relative ALP activity was normalized to the total proteincontent per sample

After 2weeks ofmineralization induction cells were fixedin ice-cold 70 ethanol for 30 minutes and stained withalizarin red (40mM pH = 42 Sigma-Aldrich) for 5min atroom temperature Images were obtained using a scannerCalcium contents were quantitatively analyzed according toour previous method [13] The results were described as themeans plusmn SD and each experiment was performed for threetimes

25 Real-Time Reverse Transcriptase-Polymerase Chain Reac-tion (Real-Time RT-PCR) SCAPs were cultured in completemedia ormineralizationmedia (MM) supplementing activa-tor or inhibitor to experiment groups After 3 days or 7 days ofincubation total RNA was extracted from cells in each groupusing TRIzol reagent (Invitrogen Carlsbad CA) accordingto the manufacturerrsquos instructions Total RNA was subjectedto reverse transcription with a PrimeScript RT Master Mixkit (TaKaRa Dalian China) The mRNA expressions ofseveral osteoblasticdentinogenic markers including ALPosteocalcin (OCN) bone sialoprotein (BSP) osterix (OSX)runt-related transcription factor 2 (RUNX2) osteopontin(OPN) dentin sialophosphoprotein (DSPP) and dentinmatrix protein-1 (DMP-1) were quantified by real-time RT-PCR using SYBR Premix Ex Taq kit (TaKaRa Bio Japan) andABI 7300 real-time PCR system Relative gene expressionvalues were calculated by the 2minusΔΔCt method as previouslydescribed [23] GAPDH (glyceraldehyde-3-phosphate dehy-drogenase) was employed as reference housekeeping gene fornormalizingmRNA levels All PCR reactionswere performedin triplicate and data were expressed as means plusmn SD Primersused for real-time RT-PCR were as follows GAPDH 51015840-GAAGGTGAAGGTCGGAGTC-31015840 and 51015840-GAGATGGTG-ATGGGATTTC-31015840 ALP 51015840-GACCTCCTCGGAAGACAC-TC-31015840 and 51015840-TGAAGGGCTTCTTGTCTGTG-31015840 OCN 51015840-AGCAAAGGTGCAGCCTTTGT-31015840 and 51015840-GCGCCTGGG-TCTCTTCACT-31015840 BSP 51015840-CTATGGAGAGGACGCCAC-GCCTGG-31015840 and 51015840-CATAGCCATCGTAGCCTTGTCCT-31015840 RUNX2 51015840-TCTTAGAACAAATTCTGCCCTTT-31015840 and51015840-TGCTTTGGTCTTGAAATCACA-31015840OSX 51015840-CCTCCT-CAGCTCACCTTCTC-31015840 and 51015840-GTTGGGAGCCCAAAT-AGAAA-31015840 DSPP 51015840-ATATTGAGGGCTGGAATGGGG-A-31015840 and 51015840-TTTGTGGCTCCAGCATTGTCA-31015840 OPN 51015840-CCAAGTAAGTCCAACGAAAG-31015840 and 51015840-GGTGATGTC-CTCGTCTGTA-31015840 DMP-1 51015840-CCCTTGGAGAGCAGT-GAGTC-31015840 and 51015840-CTCCTTTTCCTGTGCTCCTG-31015840

26 Western Blot Analysis Cultured cells in complete mediaormineralizationmedia (MM) with the presence of activatoror inhibitor in experimental groups were dissolved on ice for20min in RIPA lysis buffer (Beyotime China) supplementedwith 1mM phenylmethylsulfonyl fluoride (PMSF Beyotime)

20120583g of protein from each sample was used for western blotanalysis following the protocols in our previous study [14]

As for the detection of signaling pathway SCAPs werecultured in serum-free media for 24 hours followed bythe treatment of 10 ngmL TNF-120572 or 1 120583molL BMS-345541At the indicated time points the cytoplasm protein wasextracted with a Keygen Kit (Keygen Biotech China) andwestern blot was subsequently performed

The primary antibodies in this experiment were as fol-lows OCN (1 1000 Millipore) BSP (1 1000 Abcam) OSX(1 1000 Abcam) RUNX2 (1 1000 Abcam) DSP (1 500Santa Cruz) OPN (1 1000 Abcam) DMP-1 (1 1000 Novus)phosphor-P65 (1 1000 Cell Signaling) P65 (1 1000 CellSignaling) phosphor-I120581B120572 (1 1000 Cell Signaling) I120581B120572(1 1000 Cell Signaling) and 120573-ACTIN (1 1000 Bioworld)Target protein expression was then quantified according tothe band intensity and standardized by the structure protein120573-ACTIN with Image-Proplus 50 software In brief theintegral optical density of the protein bands was calculated byusing Image-Proplus 50 software Then densitometry ratiosbetween the target protein and 120573-ACTIN were obtained ina semiquantitative level and the histogram was then plottedThe experiment was performed for three times

27 Statistical Analysis Statistical analysis was performed bypaired t-test and one-way analysis of variance (ANOVA)Follow-up comparisons between experiment groups andcontrol groupwere then carried out using theDunnet posttestanalysis Values of 119875 lt 005 were regarded as statisticallysignificant

3 Results

31 Identification of SCAPs Immunocytochemistry anal-ysis showed that SCAPs were stained positively for themesenchymal stem cell (MSC) surface molecule STRO-1(Figure 1(a)) but negatively for epithelial cell marker cytok-eratin (Figure 1(b)) Similarly there was a high expression ofMSCmarkers (eg CD29 CD73 CD90 CD105 and CD146)while the hematopoieticmarkers (eg CD34 andCD45)werelow expressed in SCAPs as demonstrated by flow cytometry(Figure 1(d)) These data revealed the stromal origin of theseisolated cells with stem cell characteristics and the absence ofhematopoietic precursor contamination

32 Activation and Inhibition of Canonical NF-120581B SignalingPathway in SCAPs It has been extensively proven that TNF-120572 is a potent activator of canonical NF-120581B pathway whileBMS-345541 is the highly selective inhibitor of NF-120581B Todetermine whether SCAPs treated with TNF-120572 or BMS-345541 can result in the NF-120581B activation or inhibitionrespectively cytoplasm protein was extracted and subjectedto electrophoresis In TNF-120572-treated SCAPs phospho-I120581B120572was obviously elevated in a time-dependent manner andphosphorylated P65 rapidly reached a maximal increasewithin 15 minutes after TNF-120572 stimulation (Figures 2(a) and2(b)) Suppressed NF-120581B activity was detected in SCAPs afterincubation with BMS-345541 as indicated by the decreased

4 BioMed Research International

STRO-1 100120583m

(a)

CK 100120583m

(b)

PBS 100120583m

(c)

100 101 102 103 104

1000

0

037

SSC-

H

CD34 FITC100 101 102 103 104

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084

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184

CD73 PE100 101 102 103 104

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9999

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132

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CD105 APC

9999

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049

CD45 PerCP100 101 102 103 104

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297

100 101 102 103 104

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SSC-

H

112

CD90 PE100 101 102 103 104

CD90 PE

9994

104100 101 102 103

104

CD146 APC100 101 102 103 104

CD146 APC

9184

Isotype control StainingIsotype control Staining

(d)

Figure 1 Characterization of SCAPs (a) isolated SCAPswere positive for STRO-1 by immunocytochemistry (b) isolated SCAPswere negativefor CK by immunocytochemistry (c) PBS served as a negative control (d) flow cytometric analysis revealed that cultured SCAPs are positivefor CD73 (9999) CD105 (9999) CD90 (9994) and CD146 (9184) but negative for CD34 (084) and CD45 (297) Mouse IgGisotype control antibodies conjugated to FITC PE APC or PerCP were used as negative controls Scale bars 100 120583m

phosphorylated I120581B120572 and P65 (Figures 2(e) and 2(f)) Ratiosof phosphorylated to unphosphorylated forms of proteinsfurther confirmed the activation of NF-120581B by TNF-120572 andinhibition of NF-120581B by BMS-345541 (Figures 2(c) 2(d) 2(g)and 2(h) 119875 lt 005)

33 Effects of Canonical NF-120581B Pathway on the Proliferation ofSCAPs As shown in Figure 3(a) SCAPs in activator group

exhibited higher proliferation while the SCAPs-inhibitorgroup showed less proliferation capacity as compared withthe corresponding control groups respectively (119875 lt 005)except for the time points at baseline (day 0) and thefirst day Flow cytometry assay revealed that the activator-treated SCAPs exhibited a higher percentage of cells in Sand G

2M phases (2652) and a lower percentage of cells

in G0G1phase (7348) in comparison with untreated cells

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30min 60min0min 15min

P-P65

P65

P-I120581B120572

I120581B120572

ACTIN

65kDa

65kDa

39kDa

40kDa

43kDa

30min 60min0min 15min

P-P65

P65

P-I120581B120572

I120581B120572

ACTIN

65kDa

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14

12

1

08

06

04

02

0

lowastlowast lowast

lowast

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P-P65P65P-I120581B120572I120581B120572

Prot

eins

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lowastlowastlowast

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INI120581B120572 P-I120581B120572 P65 P-P65

0min15min

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lowastlowast

lowastlowast

lowastlowastlowastlowast

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B120572

lowastlowastlowast

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0min15min

30min60min

353

252

151

1

050

P-P6

5P65

0min15min

30min60min

lowastlowast

lowastlowast

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1614121

1

080604020

lowast

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B120572I120581

B120572

lowastlowastlowastlowastlowastlowast

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lowast4353

252

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050

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30min60min

lowastlowastlowast

(a)

(b) (f)

(e)

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P-P6

5P65

Figure 2 Activation and inhibition of canonical NF-120581B signaling pathway in SCAPs (a)The expression of NF-120581B pathway proteins in TNF-120572-treated SCAPs at different time points (b) Semiquantitative analysis confirmed the upregulation of P-I120581B120572 and P-P65 after the activation ofNF-120581B pathway (c)The ratio of phosphorylated to unphosphorylated form of P65 (d)The ratio of phosphorylated I120581B120572 to unphosphorylatedI120581B120572 (e) The protein levels of I120581B120572 P-I120581B120572 P65 and P-P65 in the cytoplasm of BMS-345541-treated SCAPs at indicated time points (f)Semiquantitative analysis confirmed the declined expression of P-I120581B120572 and P-P65 after the inhibition of NF-120581B pathway (g) The ratio ofphosphorylated to unphosphorylated form of P65 (h) The ratio of P-I120581B120572 to I120581B120572 Values are the means plusmn SD 119899 = 3 lowastlowast119875 lt 001 lowastlowastlowast119875 lt0001

6 BioMed Research International

lowast

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MTT25

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ue (4

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G2M

G0G1 = 8506

G2M = 986

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G0G1 = 7348

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S = 1766S = 508

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Channels (FL2-A)30 60 900 120 150

G0G1 = 9109

G2M = 536

S = 354

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(e)

0h 6h 12h 24h

Control

Inhibitor

(f)

Figure 3 Effects of canonical NF-120581B signaling pathway on the proliferation of SCAPs (a) Cell proliferation in NF-120581B pathway-activatedand control groups was detected by MTT assay (b) Flow cytometry analysis for SCAPs in NF-120581B pathway-activated and untreated groupsThe proliferation index (PI = S + G

2M) in NF-120581B pathway-activated group (2652) was significantly higher than that in control group

(1494) (c) Images of scratch wounds in NF-120581B pathway-activated and untreated groups at indicated time points (d) Growth curves of NF-120581B pathway-inhibited and untreated SCAPs (e) Representative cell cycle distributions of NF-120581B pathway-inhibited and untreated SCAPTheproliferation index (PI = S + G

2M) in NF-120581B pathway-activated group (890) was obviously lower than that in control group (1783)

(f) Cell motility of SCAPs in NF-120581B pathway-inhibited and untreated group assessed by a scratch assay Values were the means plusmn SD 119899 = 6lowast

119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 Scale bars = 100 120583m

(119875 lt 005 Figure 3(b))Therewas a lower percentage (890)of proliferating cells in SG

2Mphases in the inhibitor-treated

SCAPs as compared with the control group (1783) at day 3(Figure 3(e)) which is consistent with the findings in MTTassay (Figure 3(d)) These results indicate that canonical

NF-120581B pathway facilitated the proliferation of SCAPs thatis the cell proliferation rate increasing in pace with theactivation of canonical NF-120581B and is restrained along withthe blockage of NF-120581B Furthermore an improved migrationability was observed in activator-treated SCAPs (Figure 3(c))

BioMed Research International 7

while scratch closure was markedly slower in SCAPs treatedwith inhibitor (Figure 3(f))

34 Effects of Canonical NF-120581B Pathway on the OdontoOsteo-genic Differentiation of SCAPs ALP activities in activator-treated SCAPs in basic medium andmineralization-inducingmedium were elevated at day 5 and day 7 (119875 lt 005Figure 4(a)) as compared with untreated groupsThe densityof calcification nodules was significantly higher in activator-stimulated groups than in the other groups after 14 days ofcoculture (Figure 4(h)) Moreover quantitative calciummea-surement illustrated more calcifications in activator-treatedSCAPs in comparison with untreated groups (Figure 4(i))

Differentially expression levels of related osteoodonto-genic genes were also investigated by real-time RT-PCRassays In activator-treated group expression of specificosteoodontogenic genes (eg ALP OCN BSP OSXRUNX2 DSP OPN and DMP-1) was significantly upregu-lated at days 3 and 7 (Figures 4(b) and 4(c)) At day 3 theexpression of some osteoodontogenic genes in activator-treated groups did not change obviously in complete mediabut was distinctly increased in the presence ofmineralization-inducing media At day 7 the odontoosteo-genic markers were significantly upregulated following theactivator treatment regardless of the presence or absence ofmineralization-inducing media This phenomenon was notobserved in NF-120581B-inhibition group These findings wereconfirmed by western blot assay in which activated classicalNF-120581B signaling pathway induced a significant increase ofrelated protein expression (Figures 4(d)ndash4(g))

As shown in Figure 5(a) the blockage of NF-120581B signalingpathway obviously downregulated the ALP activity at days 35 and 7 (119875 lt 005) At day 14 less calcified nodules weregenerated in inhibitor-treated groups (Figure 5(h)) Calciumquantification also revealed the less calcium deposition ininhibitor and inhibitor + MM groups as compared withcontrol and MM groups respectively (Figure 5(i) 119875 lt 005)There was a remarkable decrease of osteoodontogenic genesat different time points (Figures 5(b) and 5(c)) Western blotanalysis further verified these findings (Figures 5(d)ndash5(g))

4 Discussion

SCAPs are known as a kind of ideal candidates for dentaltissue engineering and have the characteristics of self-renewaland multilineage differentiation potential [9 10] Diversestudies have proved that SCAPs are able to differentiate intoosteoodontoblasts in vitro under appropriate conditions andformbonedentin-like tissues in vivo [24 25] Certainlymanysignaling pathways may be involved in the process of cellproliferation and differentiation including NF-120581B pathway

NF-120581B exists in the cytoplasm in a latent form bindingto inhibitory proteins termed inhibitory 120581B proteins (I120581Bs)[3 26] I120581B complex is the major regulator of NF-120581B pathwayand is composed of catalytic subunits IKK120572 and IKK120573 anda regulatory subunit IKK120574 TNF-120572 is generally known toactivate classical NF-120581B pathway [17] Exposure of cells tosuitable concentrations of TNF-120572 brings about the rapid

phosphorylation ubiquitination and proteolytic degradationof I120581B which allows NF-120581B to translocate to the nucleuswhere it subsequently regulates the gene transcription [27ndash29] BMS-345541 a selective inhibitor of the catalytic subunitsof IKK targets NF-120581B signaling pathway and downregulatesthe activity of NF-120581B [18 19] In this study the expressionof cytoplastic P-P65P-I120581B120572 was noticeably upregulated afterthe treatment of TNF-120572 and downregulated by the inhibitorBMS-345541 indicating the successful establishment of acellular model for the activation or suppression of canonicalNF-120581B pathway

Cell proliferation and migration were indispensable fortissue development and wound healing In the present studyNF-120581B-activated SCAPs exhibited an increased growth ratehigher proliferation index and more effective migrationin wound healing assay Moreover the migration distancesbetween thewound lines in inhibitor-treated cells were longerthan control cells after 24 hours of culture Together withthe weakened proliferation of inhibitor-treated cells it ispredictable that the canonical NF-120581B pathway has a positiveinfluence on the cell multiplication activity and motility

Previous studies have proved that various stimuli canexert an active role on the committed differentiation ofdental stem cells via NF-120581B pathway [28] In the presentstudy the odontoosteogenic capacity remarkably changedalong with the activation or blockage of classical NF-120581Bpathway Activator-treated SCAPs exhibited an enhancedALP activity increased calcium deposition and upregulatedexpression of odontoosteogenic genes and proteins whileinhibitor-treated cells presented the lower ALP activitydecreased mineralization and downregulated expression ofodontoosteoblast markers

It is widely recognized that ALP is a functionalmarker forosteoblast activity and bone formation [30] As an importantextracellular matrix of bone BSP is mainly secreted byosteoblasts whose expression is a crucial symbol in matrixdeposition and mineralization [31 32] OPN is anotherextracellular matrix protein expressed in numerous cell typesincluding odontoblasts in which OPN plays multifacetedroles in a variety of biological and pathological processessuch as osteogenic differentiation tooth mineralization anddental biofilm formation [33 34] OCN is the primarynoncollagenous protein in the bone cells secreted in the latestage of osteogenesis and known as a specific indicator ofosteogenic differentiation RUNX2 is the first transcriptionfactor required for determination of the osteoblast lineageand OSX acts as a downstream gene of RUNX2 that is highlyexpressed in the functional odontoosteoblasts [35] DSPPand DSP are well-known markers of odontoblasts highlyexpressed in dentin or predentin structures and essentialfor dentinogenesis [36] DMP-1 is an acidic extracellularmatrix protein that is primarily found in dentin and boneand has been implicated in dentin mineralization and signaltransduction in the process of odontogenesis [37]

In this study the expression of RUNX2RUNX2 reducedafter 3 days of incubation with the inhibitor BMS-345541and then gradually increased at day 7 while the expressionof DSPPDSP did not compromise after 3 days of incuba-tion but noticeably decreased at day 7 indicating that the

8 BioMed Research International

ALP

3 5 7

(day)

35

3

25

2

15

1

05

0

ControlActivator

MMActivator + MM

lowast

lowastlowastlowastlowastlowastlowast

(sig

ma u

nitm

inm

g pr

otei

n)

(a)

ControlActivator

MMActivator + MM

lowastlowast

lowastlowast

876543210

ALP

OCN BS

P

RUN

X2

OSX

DSP

P

OPN

DM

P-1

Gen

esG

APD

H

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastPCR 3d

(b)

lowast

lowast lowast

lowast

lowast lowastlowast

lowast

25

20

15

10

5

0

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

Gen

esG

APD

H

ControlActivator

MMActivator + MM

ALP

OCN BS

P

RUN

X2

OSX

DSP

P

OPN

DM

P-1

PCR 7d

(c)

MMControl Activator

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

Activator + MM

Western blot 3d

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

(d)

ControlActivator

MMActivator + MM

09

08

07

06

05

04

03

02

01

0

Prot

eins

ACT

IN

OCN BSP RUNX2 OSX DSP OPN DMP-1

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

lowast

lowast

lowast

(e)

MMControl Activator

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

Activator + MM

Western blot 7d

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

(f)

Figure 4 Continued

BioMed Research International 9

14

12

1

08

06

04

02

0

Prot

eins

ACT

IN

OCN BSP RUNX2 OSX DSP OPN DMP-1

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

ControlActivator

MMActivator + MM

(g)

Control Activator

MM Activator + MM

(h)

1

CPC06

05

04

03

02

01

0

lowast

lowastlowastlowast

ControlActivator

MMActivator + MM

(ng

calc

ium

mg

prot

ein)

(i)

Figure 4 Odontoosteogenic differentiation in canonical NF-120581B-activated SCAPs (a) ALP activity of SCAPs in control activator MM andactivator + MM groups at days 3 5 and 7 Values are the means plusmn SD 119899 = 6 lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 (b) Real-time RT-PCRanalysis for odontoosteogenic genes (BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in each group at day 7 (c) Real-time RT-PCRanalysis for odontoosteogenic genes (ALP BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in different groups at day 7 GAPDHserved as a housekeeping gene lowastlowast2minusΔΔCt ge 2 119875 lt 001 lowast1 lt 2minusΔΔCt lt 2 119875 lt 001 119899 = 3 (d) Western blot analyses for the odontoosteogenicproteins (BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in different groups at day 3 120573-ACTIN served as an internal control (e)Semiquantitative analysis demonstrated that the expression of BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1 was stronger in NF-120581Bpathway-activated SCAPs than those in control group at day 3 especially in the presence of mineralization-inducing media (f) Western blotanalyses for the odontoosteogenic proteins (BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in different groups at day 7 120573-ACTINwas used as an internal control (g) Semiquantitative analysis confirmed that the expression of BSP OCN RUNX2 OSX DSP OPN DSPand DMP-1 was upregulated in NF-120581B pathway-activated SCAPs at day 7 regardless of the presence or absence of mineralization-inducingmedia (h) Alizarin red staining of SCAPs after 14 days of induction in different groups (i) Quantitative calcium analysis showed that thecalcium content in activator and activator + MM groups was significantly higher than the control and MM groups respectively Values weredescribed as the means plusmn SD lowast119875 lt 005 lowastlowastlowast119875 lt 0001

expression of RUNX2RUNX2 and DSPPDSP tends to beinversely correlated [38] Moreover the dramatic differenceof these odontoosteogenic markers at both mRNA andprotein levels in response to the regulation of NF-120581B pathwaysuggests that activated canonical NF-120581B pathway favors theodontoosteoblastic differentiation of SCAPs particularly inthe presence of mineralization-inducing media

In summary the findings accumulated here confirmedthe potential involvement of canonical NF-120581B pathway inthe proliferationmigration and committed differentiation ofSCAPs in vitro Both activation and inhibition of the classicalNF-120581B pathway can bring about the permanent changes in

human stem cells Moreover it is commonly believed thatNF-120581B signaling plays a pivotal role not only in the progressof normal physiological process but also in the pathologicalprocess and dysfunction ofNF-120581B is linked to various humandiseases Thus proper balance of intracellular NF-120581B shouldbemaintained in the physiological conditions while intricateinteractivity between NF-120581B and other signaling pathwaysneeds to be extensively investigated

Conflict of Interests

The authors declare no conflict of interests

10 BioMed Research International

ControlInhibitor

MM

ALP3

25

2

15

1

05

03 5 7

(day)

Inhibitor + MM

lowastlowast lowastlowast

lowastlowast

lowast

lowast

(sig

ma u

nitm

inm

g pr

otei

n)

(a)

ControlInhibitor

35

3

25

2

15

1

05

0

Gen

esG

APD

H

ALP OCN BSP RUNX2 OSX DSPP OPN DMP-1

MMInhibitor + MM

lowastlowast

lowast

lowast

lowast lowastlowast

lowast

lowastlowast

lowastlowast

PCR 3d

(b)

ControlInhibitor

MM

1614121086420

Gen

esG

APD

H

ALP OCN BSP RUNX2 OSX DSPP OPN DMP-1

Inhibitor + MM

lowastlowast lowastlowast

lowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowastlowastlowastPCR 7d

(c)

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

MMInhibitorControl Inhibitor + MM

Western blot 3d

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

(d)

Prot

eins

ACT

IN

ControlInhibitor

MM

12

1

08

06

04

02

0OCN BSP RUNX2 OSX DSP OPN DMP-1

Inhibitor + MM

lowast

lowast lowast

lowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

(e)

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

MMInhibitorControl Inhibitor + MM

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

Western blot 7d

(f)

Figure 5 Continued

BioMed Research International 11

Prot

eins

ACT

IN

OCN BSP RUNX2 OSX DSP OPN DMP-1

ControlInhibitor MM

Inhibitor + MM

0504504

035030250201501005

0

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(g)

MM

InhibitorControl

Inhibitor + MM

(h)

06

05

04

03

02

01

0

lowast

lowastlowastCPC

1

MMInhibitorControl

Inhibitor + MM

(ng

calc

ium

mg

prot

ein)

(i)

Figure 5 Odontoosteogenic differentiation in canonical NF-120581B-inhibited SCAPs (a) ALP activity at different time points in different groups(ie control inhibitor MM and inhibitor +MM) (b) Real-time RT-PCR for the detection ofALP BSP OCN RUNX2 OSX DSP OPN DSPandDMP-1 at day 3ThemRNA levels were normalized toGAPDH (c) Real-time RT-PCR for the detection ofALP BSP OCN RUNX2 OSXDSP OPN DSP andDMP-1 of SCAPs respectively in control inhibitor MM and inhibitor +MMgroups at day 7 (d)The odontoosteogenicprotein levels of SCAPs in control inhibitor MM and inhibitor +MM groups were characterized by western blot analysis at day 3 120573-ACTINwas used as a loading control (e) Semiquantitative analysis demonstrated that the expression of BSP OCN RUNX2 OSX DSP OPN DSPand DMP-1 was more downregulated in NF-120581B pathway-inhibited SCAPs than those in control group at day 3 (f) The odontoosteogenicprotein levels of SCAPs in control inhibitor MM and inhibitor + MM groups were characterized by western blot analysis at day 7 (g)Semiquantitative analysis demonstrated that the expression of odontoosteogenic markers (BSP OCN RUNX2 OSX DSP OPN DSP andDMP-1) was significantly more downregulated in NF-120581B pathway-inhibited SCAPs than those in control group at day 7 (h) Alizarin redstaining of SCAPs after 14 days of osteogenic induction (i) Calcium quantification demonstrated the weaker calcium deposition in inhibitorand inhibitor + MM groups as compared with control and MM groups respectively

Acknowledgments

This work was supported by National Natural Science Foun-dation of China (no 81371144) Natural Science Foundationof Jiangsu Province (no BK20131392) and the Priority Aca-demic Program Development of Jiangsu Higher EducationInstitutions (PAPD no 2011-137)

References

[1] AKumar Y TakadaAMBoriek andB BAggarwal ldquoNuclearfactor-120581B its role in health and diseaserdquo Journal of MolecularMedicine vol 82 no 7 pp 434ndash448 2004

[2] F Chen V Castranova X Shi and LM Demers ldquoNew insightsinto the role of nuclear factor-120581B a ubiquitous transcription

factor in the initiation of diseasesrdquo Clinical Chemistry vol 45no 1 pp 7ndash17 1999

[3] C Gasparini and M Feldmann ldquoNF-120581B as a target for modu-lating inflammatory responsesrdquoCurrent Pharmaceutical Designvol 18 no 35 pp 5735ndash5745 2012

[4] A Ohazama and P T Sharpe ldquoTNF signalling in toothdevelopmentrdquo Current Opinion in Genetics amp Development vol14 no 5 pp 513ndash519 2004

[5] Y-H Kuan F-M Huang Y-C Li and Y-C Chang ldquoProin-flammatory activation ofmacrophages by bisphenol A-glycidyl-methacrylate involved NF120581B activation via PI3KAkt pathwayrdquoFood and Chemical Toxicology vol 50 no 11 pp 4003ndash40092012

[6] X Cai P Gong YHuang andY Lin ldquoNotch signalling pathwayin tooth development and adult dental cellsrdquo Cell Proliferationvol 44 no 6 pp 495ndash507 2011

12 BioMed Research International

[7] J-M Courtney J Blackburn and P T Sharpe ldquoThe Ectodys-plasin and NF120581B signalling pathways in odontogenesisrdquoArchives of Oral Biology vol 50 no 2 pp 159ndash163 2005

[8] W Sonoyama Y Liu T Yamaza et al ldquoCharacterization of theapical papilla and its residing stem cells from human immaturepermanent teeth a pilot studyrdquo Journal of Endodontics vol 34no 2 pp 166ndash171 2008

[9] W Sonoyama Y Liu D Fang et al ldquoMesenchymal stem cell-mediated functional tooth regeneration in swinerdquo PLoS ONEvol 1 no 1 article e79 2006

[10] H Chopra M K Hans and S Shetty ldquoStem cells-the hiddentreasure a strategic reviewrdquoDental Research Journal vol 10 no4 pp 421ndash427 2013

[11] R Dong R Yao J Du S Wang and Z Fan ldquoDepletion ofhistone demethylase KDM2A enhanced the adipogenic andchondrogenic differentiation potentials of stem cells from apicalpapillardquo Experimental Cell Research vol 319 no 18 pp 2874ndash2882 2013

[12] Y Wang M Yan Z Wang et al ldquoDental pulp stem cells fromtraumatically exposed pulps exhibited an enhanced osteogenicpotential and weakened odontogenic capacityrdquo Archives of OralBiology vol 58 no 11 pp 1709ndash1717 2013

[13] Y Wang M Yan Z Fan L Ma Y Yu and J Yu ldquoMineraltrioxide aggregate enhances the odontoosteogenic capacity ofstem cells from inflammatory dental pulps via NF-120581B pathwayrdquoOral Diseases 2013

[14] Y Wang M Yan Y Yu J Wu J Yu and Z Fan ldquoEstrogendeficiency inhibits the odontoosteogenic differentiation ofdental pulp stem cells via activation of the NF-120581B pathwayrdquo Celland Tissue Research vol 352 no 3 pp 551ndash559 2013

[15] Y Wang Y Zheng Z Wang et al ldquo10minus7M 17120573-oestradiolenhances odontoosteogenic potency of human dental pulpstem cells by activation of the NF-120581B pathwayrdquo Cell Prolifera-tion vol 46 no 6 pp 677ndash684 2013

[16] Z-G Lu H Liu T Yamaguchi Y Miki and K YoshidaldquoProtein kinase C120575 activates RelAp65 and nuclear factor-120581B signaling in response to tumor necrosis factor-120572rdquo CancerResearch vol 69 no 14 pp 5927ndash5935 2009

[17] G Bonizzi and M Karin ldquoThe two NF-120581B activation pathwaysand their role in innate and adaptive immunityrdquo Trends inImmunology vol 25 no 6 pp 280ndash288 2004

[18] K W McIntyre D J Shuster K M Gillooly et al ldquoA highlyselective inhibitor of I120581B kinase BMS-345541 blocks both jointinflammation and destruction in collagen-induced arthritis inmicerdquo Arthritis amp Rheumatism vol 48 no 9 pp 2652ndash26592003

[19] J R Burke M A Pattoli K R Gregor et al ldquoBMS-345541 is ahighly selective inhibitor of I120581B kinase that binds at an allostericsite of the enzyme and blocks NF-120581B-dependent transcriptionin micerdquoThe Journal of Biological Chemistry vol 278 no 3 pp1450ndash1456 2003

[20] F W Paula-Silva A Ghosh L A Silva and Y L Kapila ldquoTNF-120572 promotes an odontoblastic phenotype in dental pulp cellsrdquoJournal of Dental Research vol 88 no 4 pp 339ndash344 2009

[21] S Wang J Mu Z Fan et al ldquoInsulin-like growth factor 1 canpromote the osteogenic differentiation and osteogenesis of stemcells from apical papillardquo Stem Cell Research vol 8 no 3 pp346ndash356 2012

[22] Y Yu J Mu Z Fan et al ldquoInsulin-like growth factor 1 enhancesthe proliferation and osteogenic differentiation of human peri-odontal ligament stem cells via ERK and JNKMAPKpathwaysrdquoHistochemistry andCell Biology vol 137 no 4 pp 513ndash525 2012

[23] H Aonuma N Ogura K Takahashi et al ldquoCharacteristics andosteogenic differentiation of stemprogenitor cells in the humandental follicle analyzed by gene expression profilingrdquo Cell andTissue Research vol 350 no 2 pp 317ndash331 2012

[24] L Wang M Yan Y Wang et al ldquoProliferation andosteoodontoblastic differentiation of stem cells from dentalapical papilla in mineralization-inducing medium containingadditional KH

2PO4rdquo Cell Proliferation vol 46 no 2 pp

214ndash222 2013[25] G T-J Huang T Yamaza L D Shea et al ldquoStemProgenitor

cell-mediated de novo regeneration of dental pulp with newlydeposited continuous layer of dentin in an in vivomodelrdquoTissueEngineering A vol 16 no 2 pp 605ndash615 2010

[26] J-H Kwak J-K Jung and H Lee ldquoNuclear factor-120581 Binhibitors a patent review (2006ndash2010)rdquo Expert Opinion onTherapeutic Patents vol 21 no 12 pp 1897ndash1910 2011

[27] K Hess A Ushmorov J Fiedler R E Brenner and T WirthldquoTNF120572 promotes osteogenic differentiation of human mes-enchymal stem cells by triggering theNF-120581B signaling pathwayrdquoBone vol 45 no 2 pp 367ndash376 2009

[28] X Feng G Feng J Xing et al ldquoTNF-120572 triggers osteogenicdifferentiation of human dental pulp stem cells via the NF-120581Bsignalling pathwayrdquoCell Biology International vol 37 no 12 pp1267ndash1275 2013

[29] J Chang F Liu M Lee et al ldquoNF-120581B inhibits osteogenicdifferentiation of mesenchymal stem cells by promoting 120573-catenin degradationrdquo Proceedings of the National Academy ofSciences of the United States of America vol 110 no 23 pp9469ndash9474 2013

[30] C Shui and A Scutt ldquoMild heat shock induces proliferationalkaline phosphatase activity and mineralization in humanbone marrow stromal cells and Mg-63 cells in vitrordquo Journal ofBone and Mineral Research vol 16 no 4 pp 731ndash741 2001

[31] X Wei J Ling L Wu L Liu and Y Xiao ldquoExpressionof mineralization markers in dental pulp cellsrdquo Journal ofEndodontics vol 33 no 6 pp 703ndash708 2007

[32] S Z Khan E Kokubu K Matsuzaka and T Inoue ldquoBehaviourof rat-cultured dental pulp cells in three-dimensional collagentype-1 gel in vitro and in vivordquo Australian Endodontic Journalvol 39 no 3 pp 137ndash145 2013

[33] D-X Cao Z-J Li X-O Jiang et al ldquoOsteopontin as potentialbiomarker and therapeutic target in gastric and liver cancersrdquoWorld Journal of Gastroenterology vol 18 no 30 pp 3923ndash39302012

[34] S SchlaferMK Raarup P LWejse et al ldquoOsteopontin reducesbiofilm formation in a multi-species model of dental biofilmrdquoPloS ONE vol 7 no 8 Article ID e41534 2012

[35] S Chen J Gluhak-Heinrich Y HWang et al ldquoRunx2Osx andDspp in tooth developmentrdquo Journal of Dental Research vol 88no 10 pp 904ndash909 2009

[36] S Suzuki N Haruyama F Nishimura and A B KulkarnildquoDentin sialophosphoprotein and dentin matrix protein-1 twohighly phosphorylated proteins inmineralized tissuesrdquoArchivesof Oral Biology vol 57 no 9 pp 1165ndash1175 2012

[37] R S Prescott R Alsanea M I Fayad et al ldquoIn vivo fenerationof dental pulp-like tissue by using dental pulp stem cells a col-lagen scaffold and dentin matrix protein 1 after subcutaneoustransplantation in micerdquo Journal of Endodontics vol 34 no 4pp 421ndash426 2008

[38] T Komori ldquoRegulation of bone development and extracellularmatrix protein genes by RUNX2rdquo Cell and Tissue Research vol339 no 1 pp 189ndash195 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

2 BioMed Research International

marker) and DSPP supports the concept that SCAPs are thebest candidate for tooth regeneration and rehabilitation ofdamage [9ndash11] Nevertheless there is no convincing scientificevidence about the effects of NF-120581B pathway on the prolifer-ation and differentiation of SCAPs

NF-120581B pathway can be triggered in dental pulp stemcells under various stimulating factors that is traumainflammatory factors MTA and estrogen [12ndash15] in whichtumor necrosis factor-120572 (TNF-120572) is the putative classicalpathway activator [4 16 17] Meanwhile this pathwaycan be effectively inhibited by 4(21015840-aminoethyl) amino-18-dimethylimidazo(12-a) quinoxaline (BMS-345541 a selectiveinhibitor of IKK) [18 19] In this study we hypothesizethat activation or inhibition of NF-120581B pathway can affectthe pace of proliferation and differentiation in SCAPs Totest this hypothesis SCAPs were isolated from the devel-oping apical papillae and 10 ngmL TNF-120572 or 1 120583molLBMS-345541 was respectively used to activate or inhibitNF-120581B pathway in SCAPs [15 20] The present findingsrevealed that the proliferative ability migration potential andodontoosteogenic differentiation potential of SCAPs can besignificantly affected by the activation or inhibition of NF-120581B pathwayThese results provide novel insights into the roleof classical NF-120581B pathway during the modification of mes-enchymal stem cells and stem cell-based tooth regeneration

2 Materials and Methods

21 Cell Isolation andCulture Theprocedure for cell isolationand culture was performed as described previously [21]Briefly healthy human impacted third molars (119899 = 20)were gathered from sixteen young patients under the ageof 20 in Oral Surgery Department of Jiangsu ProvincialStomatological Hospital Root apical papilla was carefullyisolated from the immature root apex Primary apical papillacells were enzymatically separated according to previousstudy [9] and cultured in alpha minimum essential medium(120572-MEM Gibco Life Technologies Grand Island NY) sup-plementedwith 10 fetal bovine serum (FBS Hyclone USA)100 120583gmL streptomycin and 100 UmL penicillin at 37∘C ina humidified atmosphere of 5 CO

2 Then anti-rabbit IgG

Dynabeads (Dynal Biotech Oslo Norway) and rabbit anti-STRO-1 antibody (Santa Cruz Delaware CA) were used topurify these isolated cells according to the standard operatingprocedures formagnetic activated cell sorting (MACS) TNF-120572 (Peprotech USA) was dissolved in 120572-MEM at the con-centration of 100mgmL and stored at minus20∘C BMS-345541(Sigma-Aldrich MO) was dissolved in DMSO to producea 50120583molL stock solution In consideration of the cellularcytotoxicity of chemical inhibitor at high concentration asrevealed in previous study [15] we selected 1 120583molL BMS-345541 for the subsequent investigation

22 Cell Identification To determine the nature of cul-tured cells isolated cells were immunostained with theantibody against STRO-1 (1 200 Novus Biologicals USA)and cytokeratin (1 100 Bioworld USA) Phosphate bufferedsaline (PBS) was simultaneously used as a control Flow

cytometric analysis of specific surface antigens was alsoused to characterize the cultured cells Cells were harvestedand incubated with various combinations of the follow-ing fluorochrome-conjugated rabbit anti-human antibodiesCD34-FITC CD45-PerCP CD90-PE CD105-APC CD146-APC and CD73-PE (all fromMiltenyi Germany) for 20minat room temperature in the dark The corresponding mouseIgG isotype control antibodies conjugated to FITC PEAPC or PerCP were employed as negative controls in eachexperiment Stained cells were washed twice with 001molLPBS and analyzed using BD FACSCalibur (BD BiosciencesUSA)

23 Proliferation and Migration Assay The proliferation ofSCAPs treated in NF-120581B-activated culture media containing10 ngmL TNF-120572 or NF-120581B-inhibited culture mediacontaining 1 120583molL BMS-345541 was examined by 3-(45-dimethylthiazol-2-yl)-25-diphenyl-25-tetrazoliumbromide(MTT) assay and flow cytometry (FCM) SCAPs were seededinto 96-well plates (Nunc Thermo Fisher Scientific Inc)at an initial density of 2 times 103 cellswell for 24 hours At60 confluence cells were serum starved for 24 hours andtreated with 10 ngmL TNF-120572 or 1 120583molL NF-120581B inhibitorBMS-345541 After 0 1 3 5 7 and 9 days of cocultureMTT assay was performed according to the previousreport [22] The optical absorbance was obtained in anenzyme-linked immunosorbent assay plate reader (TitertekHelsinki Finland) at 490 nm according to the manufacturerrsquosinstruction The data were presented as the means plusmn SD(119899 = 6) and this experiment was repeated in triplicate

SCAPs (1 times 106) in control NF-120581B-activated and NF-120581B-inhibited groups were respectively collected washed twicewith cold 001molL PBS and fixed in 70 ice-cold ethanolovernight at 4∘C in the dark DNA content analysis of thesecells was carried out using FACScan flow cytometer (BDBiosciences San Jose CA)Then cell cycle distributions (G

1

S and G2M phases) were described and compared This

experiment was repeated three timesFor the wound healing assay SCAPs were cultured to

90 confluence in 100mm culture dishes and then woundedby using a pipette tip to scratch the monolayers The initialscratched areas were uniform across the different samplesand permanently marked Floating cells and debris wereremoved and cells were cultured in 120572-MEM supplementedwith 10 ngmL TNF-120572 or 1 120583molL BMS-345541 The markedareas in each group were photographed at 0 6 12 and 24hours after the scratch with an inverted microscope

24 Alkaline Phosphatase (ALP) Activity Assay and AlizarinRed Staining For the evaluation of osteogenic differentia-tion ALP activity was detected by using an ALP kit (NanjingJiancheng Technological Inc Nanjing China) Cells wereplated at a density of 3 times 103 cells per well into 96-well platesThen the media were changed and cells were cultured inthe complete media or the mineralization-inducing media(MM) containing 120572-MEM 10 FBS 100 120583gmL strepto-mycin 100 UmL penicillin 50mgL ascorbic acid 2mmolL

BioMed Research International 3

L-glutamine 10 nmolL dexamethasone and 10mmolL 120573-glycerophosphate (Sigma) In addition activator (TNF-120572) orinhibitor (BMS-345541) was added into experiment groupsALP activity was measured after 3 5 and 7 days of cultureThe relative ALP activity was normalized to the total proteincontent per sample

After 2weeks ofmineralization induction cells were fixedin ice-cold 70 ethanol for 30 minutes and stained withalizarin red (40mM pH = 42 Sigma-Aldrich) for 5min atroom temperature Images were obtained using a scannerCalcium contents were quantitatively analyzed according toour previous method [13] The results were described as themeans plusmn SD and each experiment was performed for threetimes

25 Real-Time Reverse Transcriptase-Polymerase Chain Reac-tion (Real-Time RT-PCR) SCAPs were cultured in completemedia ormineralizationmedia (MM) supplementing activa-tor or inhibitor to experiment groups After 3 days or 7 days ofincubation total RNA was extracted from cells in each groupusing TRIzol reagent (Invitrogen Carlsbad CA) accordingto the manufacturerrsquos instructions Total RNA was subjectedto reverse transcription with a PrimeScript RT Master Mixkit (TaKaRa Dalian China) The mRNA expressions ofseveral osteoblasticdentinogenic markers including ALPosteocalcin (OCN) bone sialoprotein (BSP) osterix (OSX)runt-related transcription factor 2 (RUNX2) osteopontin(OPN) dentin sialophosphoprotein (DSPP) and dentinmatrix protein-1 (DMP-1) were quantified by real-time RT-PCR using SYBR Premix Ex Taq kit (TaKaRa Bio Japan) andABI 7300 real-time PCR system Relative gene expressionvalues were calculated by the 2minusΔΔCt method as previouslydescribed [23] GAPDH (glyceraldehyde-3-phosphate dehy-drogenase) was employed as reference housekeeping gene fornormalizingmRNA levels All PCR reactionswere performedin triplicate and data were expressed as means plusmn SD Primersused for real-time RT-PCR were as follows GAPDH 51015840-GAAGGTGAAGGTCGGAGTC-31015840 and 51015840-GAGATGGTG-ATGGGATTTC-31015840 ALP 51015840-GACCTCCTCGGAAGACAC-TC-31015840 and 51015840-TGAAGGGCTTCTTGTCTGTG-31015840 OCN 51015840-AGCAAAGGTGCAGCCTTTGT-31015840 and 51015840-GCGCCTGGG-TCTCTTCACT-31015840 BSP 51015840-CTATGGAGAGGACGCCAC-GCCTGG-31015840 and 51015840-CATAGCCATCGTAGCCTTGTCCT-31015840 RUNX2 51015840-TCTTAGAACAAATTCTGCCCTTT-31015840 and51015840-TGCTTTGGTCTTGAAATCACA-31015840OSX 51015840-CCTCCT-CAGCTCACCTTCTC-31015840 and 51015840-GTTGGGAGCCCAAAT-AGAAA-31015840 DSPP 51015840-ATATTGAGGGCTGGAATGGGG-A-31015840 and 51015840-TTTGTGGCTCCAGCATTGTCA-31015840 OPN 51015840-CCAAGTAAGTCCAACGAAAG-31015840 and 51015840-GGTGATGTC-CTCGTCTGTA-31015840 DMP-1 51015840-CCCTTGGAGAGCAGT-GAGTC-31015840 and 51015840-CTCCTTTTCCTGTGCTCCTG-31015840

26 Western Blot Analysis Cultured cells in complete mediaormineralizationmedia (MM) with the presence of activatoror inhibitor in experimental groups were dissolved on ice for20min in RIPA lysis buffer (Beyotime China) supplementedwith 1mM phenylmethylsulfonyl fluoride (PMSF Beyotime)

20120583g of protein from each sample was used for western blotanalysis following the protocols in our previous study [14]

As for the detection of signaling pathway SCAPs werecultured in serum-free media for 24 hours followed bythe treatment of 10 ngmL TNF-120572 or 1 120583molL BMS-345541At the indicated time points the cytoplasm protein wasextracted with a Keygen Kit (Keygen Biotech China) andwestern blot was subsequently performed

The primary antibodies in this experiment were as fol-lows OCN (1 1000 Millipore) BSP (1 1000 Abcam) OSX(1 1000 Abcam) RUNX2 (1 1000 Abcam) DSP (1 500Santa Cruz) OPN (1 1000 Abcam) DMP-1 (1 1000 Novus)phosphor-P65 (1 1000 Cell Signaling) P65 (1 1000 CellSignaling) phosphor-I120581B120572 (1 1000 Cell Signaling) I120581B120572(1 1000 Cell Signaling) and 120573-ACTIN (1 1000 Bioworld)Target protein expression was then quantified according tothe band intensity and standardized by the structure protein120573-ACTIN with Image-Proplus 50 software In brief theintegral optical density of the protein bands was calculated byusing Image-Proplus 50 software Then densitometry ratiosbetween the target protein and 120573-ACTIN were obtained ina semiquantitative level and the histogram was then plottedThe experiment was performed for three times

27 Statistical Analysis Statistical analysis was performed bypaired t-test and one-way analysis of variance (ANOVA)Follow-up comparisons between experiment groups andcontrol groupwere then carried out using theDunnet posttestanalysis Values of 119875 lt 005 were regarded as statisticallysignificant

3 Results

31 Identification of SCAPs Immunocytochemistry anal-ysis showed that SCAPs were stained positively for themesenchymal stem cell (MSC) surface molecule STRO-1(Figure 1(a)) but negatively for epithelial cell marker cytok-eratin (Figure 1(b)) Similarly there was a high expression ofMSCmarkers (eg CD29 CD73 CD90 CD105 and CD146)while the hematopoieticmarkers (eg CD34 andCD45)werelow expressed in SCAPs as demonstrated by flow cytometry(Figure 1(d)) These data revealed the stromal origin of theseisolated cells with stem cell characteristics and the absence ofhematopoietic precursor contamination

32 Activation and Inhibition of Canonical NF-120581B SignalingPathway in SCAPs It has been extensively proven that TNF-120572 is a potent activator of canonical NF-120581B pathway whileBMS-345541 is the highly selective inhibitor of NF-120581B Todetermine whether SCAPs treated with TNF-120572 or BMS-345541 can result in the NF-120581B activation or inhibitionrespectively cytoplasm protein was extracted and subjectedto electrophoresis In TNF-120572-treated SCAPs phospho-I120581B120572was obviously elevated in a time-dependent manner andphosphorylated P65 rapidly reached a maximal increasewithin 15 minutes after TNF-120572 stimulation (Figures 2(a) and2(b)) Suppressed NF-120581B activity was detected in SCAPs afterincubation with BMS-345541 as indicated by the decreased

4 BioMed Research International

STRO-1 100120583m

(a)

CK 100120583m

(b)

PBS 100120583m

(c)

100 101 102 103 104

1000

0

037

SSC-

H

CD34 FITC100 101 102 103 104

CD34 FITC

084

100 101 102 103 104

184

CD73 PE100 101 102 103 104

CD73 PE

9999

100 101 102 103 104

1000

0

SSC-

H

CD105 APC

132

100 101 102 103 104

CD105 APC

9999

100 101 102 103 104

049

CD45 PerCP100 101 102 103 104

CD45 PerCP

297

100 101 102 103 104

1000

0

SSC-

H

112

CD90 PE100 101 102 103 104

CD90 PE

9994

104100 101 102 103

104

CD146 APC100 101 102 103 104

CD146 APC

9184

Isotype control StainingIsotype control Staining

(d)

Figure 1 Characterization of SCAPs (a) isolated SCAPswere positive for STRO-1 by immunocytochemistry (b) isolated SCAPswere negativefor CK by immunocytochemistry (c) PBS served as a negative control (d) flow cytometric analysis revealed that cultured SCAPs are positivefor CD73 (9999) CD105 (9999) CD90 (9994) and CD146 (9184) but negative for CD34 (084) and CD45 (297) Mouse IgGisotype control antibodies conjugated to FITC PE APC or PerCP were used as negative controls Scale bars 100 120583m

phosphorylated I120581B120572 and P65 (Figures 2(e) and 2(f)) Ratiosof phosphorylated to unphosphorylated forms of proteinsfurther confirmed the activation of NF-120581B by TNF-120572 andinhibition of NF-120581B by BMS-345541 (Figures 2(c) 2(d) 2(g)and 2(h) 119875 lt 005)

33 Effects of Canonical NF-120581B Pathway on the Proliferation ofSCAPs As shown in Figure 3(a) SCAPs in activator group

exhibited higher proliferation while the SCAPs-inhibitorgroup showed less proliferation capacity as compared withthe corresponding control groups respectively (119875 lt 005)except for the time points at baseline (day 0) and thefirst day Flow cytometry assay revealed that the activator-treated SCAPs exhibited a higher percentage of cells in Sand G

2M phases (2652) and a lower percentage of cells

in G0G1phase (7348) in comparison with untreated cells

BioMed Research International 5

30min 60min0min 15min

P-P65

P65

P-I120581B120572

I120581B120572

ACTIN

65kDa

65kDa

39kDa

40kDa

43kDa

30min 60min0min 15min

P-P65

P65

P-I120581B120572

I120581B120572

ACTIN

65kDa

65kDa

39kDa

40kDa

43kDa

14

12

1

08

06

04

02

0

lowastlowast lowast

lowast

lowastlowast

P-P65P65P-I120581B120572I120581B120572

Prot

eins

ACT

IN

0min15min

30min60min

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

06

05

04

03

02

01

0

lowastlowast

Prot

eins

ACT

INI120581B120572 P-I120581B120572 P65 P-P65

0min15min

30min60min

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

25

2

15

1

1

05

0

P-I120581

B120572I120581

B120572

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0min15min

30min60min

353

252

151

1

050

P-P6

5P65

0min15min

30min60min

lowastlowast

lowastlowast

lowastlowast

1614121

1

080604020

lowast

P-I120581

B120572I120581

B120572

lowastlowastlowastlowastlowastlowast

0min15min

30min60min

lowast4353

252

151

1

050

0min15min

30min60min

lowastlowastlowast

(a)

(b) (f)

(e)

(c) (g)

(d) (h)

P-P6

5P65

Figure 2 Activation and inhibition of canonical NF-120581B signaling pathway in SCAPs (a)The expression of NF-120581B pathway proteins in TNF-120572-treated SCAPs at different time points (b) Semiquantitative analysis confirmed the upregulation of P-I120581B120572 and P-P65 after the activation ofNF-120581B pathway (c)The ratio of phosphorylated to unphosphorylated form of P65 (d)The ratio of phosphorylated I120581B120572 to unphosphorylatedI120581B120572 (e) The protein levels of I120581B120572 P-I120581B120572 P65 and P-P65 in the cytoplasm of BMS-345541-treated SCAPs at indicated time points (f)Semiquantitative analysis confirmed the declined expression of P-I120581B120572 and P-P65 after the inhibition of NF-120581B pathway (g) The ratio ofphosphorylated to unphosphorylated form of P65 (h) The ratio of P-I120581B120572 to I120581B120572 Values are the means plusmn SD 119899 = 3 lowastlowast119875 lt 001 lowastlowastlowast119875 lt0001

6 BioMed Research International

lowast

lowast

lowast

lowast

lowast lowast

ControlActivator

MTT25

2

15

1

05

0

OD

val

ue (4

90

nm)

0 1 3 5 7 9 11

(day)

(a)

S30 60 90 120

SSSSSSSSSSSSSSSSSSSSSS0

300

900

1200

600

Num

ber

SCAPsChannels (FL2-A)

G2M

G0G1 = 8506

G2M = 986

G0G1

S

30 60 90 120

S

0

Num

ber

Channels (FL2-A)

G2M

G0G1 = 7348

G2M = 886

G0G1

1000

800

600

400

200

00

SCAPs + activator

S = 1766S = 508

(b)

Control

Activator

0h 6h 12h 24h

(c)

ControlInhibitor

25

2

15

1

05

0

OD

val

ue (4

90

nm) lowast

lowast

MTT

0 1 3 5 7 9 11

(day)

lowastlowast

lowastlowastlowast

lowastlowastlowast

(d)

SSSSSSSSSSSSSSSSSSSSSSS

SCAPsChannels (FL2-A)

G0G1

30 60 90 1200

G2M

1600

1200

800

400

0

Num

ber

G0G1 = 8217

G2M = 600

S = 1183

1600

1200

800

400

0

Num

ber

S

G0G1

G2M

Channels (FL2-A)30 60 900 120 150

G0G1 = 9109

G2M = 536

S = 354

SCAPs + inhibitor

(e)

0h 6h 12h 24h

Control

Inhibitor

(f)

Figure 3 Effects of canonical NF-120581B signaling pathway on the proliferation of SCAPs (a) Cell proliferation in NF-120581B pathway-activatedand control groups was detected by MTT assay (b) Flow cytometry analysis for SCAPs in NF-120581B pathway-activated and untreated groupsThe proliferation index (PI = S + G

2M) in NF-120581B pathway-activated group (2652) was significantly higher than that in control group

(1494) (c) Images of scratch wounds in NF-120581B pathway-activated and untreated groups at indicated time points (d) Growth curves of NF-120581B pathway-inhibited and untreated SCAPs (e) Representative cell cycle distributions of NF-120581B pathway-inhibited and untreated SCAPTheproliferation index (PI = S + G

2M) in NF-120581B pathway-activated group (890) was obviously lower than that in control group (1783)

(f) Cell motility of SCAPs in NF-120581B pathway-inhibited and untreated group assessed by a scratch assay Values were the means plusmn SD 119899 = 6lowast

119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 Scale bars = 100 120583m

(119875 lt 005 Figure 3(b))Therewas a lower percentage (890)of proliferating cells in SG

2Mphases in the inhibitor-treated

SCAPs as compared with the control group (1783) at day 3(Figure 3(e)) which is consistent with the findings in MTTassay (Figure 3(d)) These results indicate that canonical

NF-120581B pathway facilitated the proliferation of SCAPs thatis the cell proliferation rate increasing in pace with theactivation of canonical NF-120581B and is restrained along withthe blockage of NF-120581B Furthermore an improved migrationability was observed in activator-treated SCAPs (Figure 3(c))

BioMed Research International 7

while scratch closure was markedly slower in SCAPs treatedwith inhibitor (Figure 3(f))

34 Effects of Canonical NF-120581B Pathway on the OdontoOsteo-genic Differentiation of SCAPs ALP activities in activator-treated SCAPs in basic medium andmineralization-inducingmedium were elevated at day 5 and day 7 (119875 lt 005Figure 4(a)) as compared with untreated groupsThe densityof calcification nodules was significantly higher in activator-stimulated groups than in the other groups after 14 days ofcoculture (Figure 4(h)) Moreover quantitative calciummea-surement illustrated more calcifications in activator-treatedSCAPs in comparison with untreated groups (Figure 4(i))

Differentially expression levels of related osteoodonto-genic genes were also investigated by real-time RT-PCRassays In activator-treated group expression of specificosteoodontogenic genes (eg ALP OCN BSP OSXRUNX2 DSP OPN and DMP-1) was significantly upregu-lated at days 3 and 7 (Figures 4(b) and 4(c)) At day 3 theexpression of some osteoodontogenic genes in activator-treated groups did not change obviously in complete mediabut was distinctly increased in the presence ofmineralization-inducing media At day 7 the odontoosteo-genic markers were significantly upregulated following theactivator treatment regardless of the presence or absence ofmineralization-inducing media This phenomenon was notobserved in NF-120581B-inhibition group These findings wereconfirmed by western blot assay in which activated classicalNF-120581B signaling pathway induced a significant increase ofrelated protein expression (Figures 4(d)ndash4(g))

As shown in Figure 5(a) the blockage of NF-120581B signalingpathway obviously downregulated the ALP activity at days 35 and 7 (119875 lt 005) At day 14 less calcified nodules weregenerated in inhibitor-treated groups (Figure 5(h)) Calciumquantification also revealed the less calcium deposition ininhibitor and inhibitor + MM groups as compared withcontrol and MM groups respectively (Figure 5(i) 119875 lt 005)There was a remarkable decrease of osteoodontogenic genesat different time points (Figures 5(b) and 5(c)) Western blotanalysis further verified these findings (Figures 5(d)ndash5(g))

4 Discussion

SCAPs are known as a kind of ideal candidates for dentaltissue engineering and have the characteristics of self-renewaland multilineage differentiation potential [9 10] Diversestudies have proved that SCAPs are able to differentiate intoosteoodontoblasts in vitro under appropriate conditions andformbonedentin-like tissues in vivo [24 25] Certainlymanysignaling pathways may be involved in the process of cellproliferation and differentiation including NF-120581B pathway

NF-120581B exists in the cytoplasm in a latent form bindingto inhibitory proteins termed inhibitory 120581B proteins (I120581Bs)[3 26] I120581B complex is the major regulator of NF-120581B pathwayand is composed of catalytic subunits IKK120572 and IKK120573 anda regulatory subunit IKK120574 TNF-120572 is generally known toactivate classical NF-120581B pathway [17] Exposure of cells tosuitable concentrations of TNF-120572 brings about the rapid

phosphorylation ubiquitination and proteolytic degradationof I120581B which allows NF-120581B to translocate to the nucleuswhere it subsequently regulates the gene transcription [27ndash29] BMS-345541 a selective inhibitor of the catalytic subunitsof IKK targets NF-120581B signaling pathway and downregulatesthe activity of NF-120581B [18 19] In this study the expressionof cytoplastic P-P65P-I120581B120572 was noticeably upregulated afterthe treatment of TNF-120572 and downregulated by the inhibitorBMS-345541 indicating the successful establishment of acellular model for the activation or suppression of canonicalNF-120581B pathway

Cell proliferation and migration were indispensable fortissue development and wound healing In the present studyNF-120581B-activated SCAPs exhibited an increased growth ratehigher proliferation index and more effective migrationin wound healing assay Moreover the migration distancesbetween thewound lines in inhibitor-treated cells were longerthan control cells after 24 hours of culture Together withthe weakened proliferation of inhibitor-treated cells it ispredictable that the canonical NF-120581B pathway has a positiveinfluence on the cell multiplication activity and motility

Previous studies have proved that various stimuli canexert an active role on the committed differentiation ofdental stem cells via NF-120581B pathway [28] In the presentstudy the odontoosteogenic capacity remarkably changedalong with the activation or blockage of classical NF-120581Bpathway Activator-treated SCAPs exhibited an enhancedALP activity increased calcium deposition and upregulatedexpression of odontoosteogenic genes and proteins whileinhibitor-treated cells presented the lower ALP activitydecreased mineralization and downregulated expression ofodontoosteoblast markers

It is widely recognized that ALP is a functionalmarker forosteoblast activity and bone formation [30] As an importantextracellular matrix of bone BSP is mainly secreted byosteoblasts whose expression is a crucial symbol in matrixdeposition and mineralization [31 32] OPN is anotherextracellular matrix protein expressed in numerous cell typesincluding odontoblasts in which OPN plays multifacetedroles in a variety of biological and pathological processessuch as osteogenic differentiation tooth mineralization anddental biofilm formation [33 34] OCN is the primarynoncollagenous protein in the bone cells secreted in the latestage of osteogenesis and known as a specific indicator ofosteogenic differentiation RUNX2 is the first transcriptionfactor required for determination of the osteoblast lineageand OSX acts as a downstream gene of RUNX2 that is highlyexpressed in the functional odontoosteoblasts [35] DSPPand DSP are well-known markers of odontoblasts highlyexpressed in dentin or predentin structures and essentialfor dentinogenesis [36] DMP-1 is an acidic extracellularmatrix protein that is primarily found in dentin and boneand has been implicated in dentin mineralization and signaltransduction in the process of odontogenesis [37]

In this study the expression of RUNX2RUNX2 reducedafter 3 days of incubation with the inhibitor BMS-345541and then gradually increased at day 7 while the expressionof DSPPDSP did not compromise after 3 days of incuba-tion but noticeably decreased at day 7 indicating that the

8 BioMed Research International

ALP

3 5 7

(day)

35

3

25

2

15

1

05

0

ControlActivator

MMActivator + MM

lowast

lowastlowastlowastlowastlowastlowast

(sig

ma u

nitm

inm

g pr

otei

n)

(a)

ControlActivator

MMActivator + MM

lowastlowast

lowastlowast

876543210

ALP

OCN BS

P

RUN

X2

OSX

DSP

P

OPN

DM

P-1

Gen

esG

APD

H

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastPCR 3d

(b)

lowast

lowast lowast

lowast

lowast lowastlowast

lowast

25

20

15

10

5

0

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

Gen

esG

APD

H

ControlActivator

MMActivator + MM

ALP

OCN BS

P

RUN

X2

OSX

DSP

P

OPN

DM

P-1

PCR 7d

(c)

MMControl Activator

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

Activator + MM

Western blot 3d

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

(d)

ControlActivator

MMActivator + MM

09

08

07

06

05

04

03

02

01

0

Prot

eins

ACT

IN

OCN BSP RUNX2 OSX DSP OPN DMP-1

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

lowast

lowast

lowast

(e)

MMControl Activator

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

Activator + MM

Western blot 7d

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

(f)

Figure 4 Continued

BioMed Research International 9

14

12

1

08

06

04

02

0

Prot

eins

ACT

IN

OCN BSP RUNX2 OSX DSP OPN DMP-1

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

ControlActivator

MMActivator + MM

(g)

Control Activator

MM Activator + MM

(h)

1

CPC06

05

04

03

02

01

0

lowast

lowastlowastlowast

ControlActivator

MMActivator + MM

(ng

calc

ium

mg

prot

ein)

(i)

Figure 4 Odontoosteogenic differentiation in canonical NF-120581B-activated SCAPs (a) ALP activity of SCAPs in control activator MM andactivator + MM groups at days 3 5 and 7 Values are the means plusmn SD 119899 = 6 lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 (b) Real-time RT-PCRanalysis for odontoosteogenic genes (BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in each group at day 7 (c) Real-time RT-PCRanalysis for odontoosteogenic genes (ALP BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in different groups at day 7 GAPDHserved as a housekeeping gene lowastlowast2minusΔΔCt ge 2 119875 lt 001 lowast1 lt 2minusΔΔCt lt 2 119875 lt 001 119899 = 3 (d) Western blot analyses for the odontoosteogenicproteins (BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in different groups at day 3 120573-ACTIN served as an internal control (e)Semiquantitative analysis demonstrated that the expression of BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1 was stronger in NF-120581Bpathway-activated SCAPs than those in control group at day 3 especially in the presence of mineralization-inducing media (f) Western blotanalyses for the odontoosteogenic proteins (BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in different groups at day 7 120573-ACTINwas used as an internal control (g) Semiquantitative analysis confirmed that the expression of BSP OCN RUNX2 OSX DSP OPN DSPand DMP-1 was upregulated in NF-120581B pathway-activated SCAPs at day 7 regardless of the presence or absence of mineralization-inducingmedia (h) Alizarin red staining of SCAPs after 14 days of induction in different groups (i) Quantitative calcium analysis showed that thecalcium content in activator and activator + MM groups was significantly higher than the control and MM groups respectively Values weredescribed as the means plusmn SD lowast119875 lt 005 lowastlowastlowast119875 lt 0001

expression of RUNX2RUNX2 and DSPPDSP tends to beinversely correlated [38] Moreover the dramatic differenceof these odontoosteogenic markers at both mRNA andprotein levels in response to the regulation of NF-120581B pathwaysuggests that activated canonical NF-120581B pathway favors theodontoosteoblastic differentiation of SCAPs particularly inthe presence of mineralization-inducing media

In summary the findings accumulated here confirmedthe potential involvement of canonical NF-120581B pathway inthe proliferationmigration and committed differentiation ofSCAPs in vitro Both activation and inhibition of the classicalNF-120581B pathway can bring about the permanent changes in

human stem cells Moreover it is commonly believed thatNF-120581B signaling plays a pivotal role not only in the progressof normal physiological process but also in the pathologicalprocess and dysfunction ofNF-120581B is linked to various humandiseases Thus proper balance of intracellular NF-120581B shouldbemaintained in the physiological conditions while intricateinteractivity between NF-120581B and other signaling pathwaysneeds to be extensively investigated

Conflict of Interests

The authors declare no conflict of interests

10 BioMed Research International

ControlInhibitor

MM

ALP3

25

2

15

1

05

03 5 7

(day)

Inhibitor + MM

lowastlowast lowastlowast

lowastlowast

lowast

lowast

(sig

ma u

nitm

inm

g pr

otei

n)

(a)

ControlInhibitor

35

3

25

2

15

1

05

0

Gen

esG

APD

H

ALP OCN BSP RUNX2 OSX DSPP OPN DMP-1

MMInhibitor + MM

lowastlowast

lowast

lowast

lowast lowastlowast

lowast

lowastlowast

lowastlowast

PCR 3d

(b)

ControlInhibitor

MM

1614121086420

Gen

esG

APD

H

ALP OCN BSP RUNX2 OSX DSPP OPN DMP-1

Inhibitor + MM

lowastlowast lowastlowast

lowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowastlowastlowastPCR 7d

(c)

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

MMInhibitorControl Inhibitor + MM

Western blot 3d

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

(d)

Prot

eins

ACT

IN

ControlInhibitor

MM

12

1

08

06

04

02

0OCN BSP RUNX2 OSX DSP OPN DMP-1

Inhibitor + MM

lowast

lowast lowast

lowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

(e)

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

MMInhibitorControl Inhibitor + MM

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

Western blot 7d

(f)

Figure 5 Continued

BioMed Research International 11

Prot

eins

ACT

IN

OCN BSP RUNX2 OSX DSP OPN DMP-1

ControlInhibitor MM

Inhibitor + MM

0504504

035030250201501005

0

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(g)

MM

InhibitorControl

Inhibitor + MM

(h)

06

05

04

03

02

01

0

lowast

lowastlowastCPC

1

MMInhibitorControl

Inhibitor + MM

(ng

calc

ium

mg

prot

ein)

(i)

Figure 5 Odontoosteogenic differentiation in canonical NF-120581B-inhibited SCAPs (a) ALP activity at different time points in different groups(ie control inhibitor MM and inhibitor +MM) (b) Real-time RT-PCR for the detection ofALP BSP OCN RUNX2 OSX DSP OPN DSPandDMP-1 at day 3ThemRNA levels were normalized toGAPDH (c) Real-time RT-PCR for the detection ofALP BSP OCN RUNX2 OSXDSP OPN DSP andDMP-1 of SCAPs respectively in control inhibitor MM and inhibitor +MMgroups at day 7 (d)The odontoosteogenicprotein levels of SCAPs in control inhibitor MM and inhibitor +MM groups were characterized by western blot analysis at day 3 120573-ACTINwas used as a loading control (e) Semiquantitative analysis demonstrated that the expression of BSP OCN RUNX2 OSX DSP OPN DSPand DMP-1 was more downregulated in NF-120581B pathway-inhibited SCAPs than those in control group at day 3 (f) The odontoosteogenicprotein levels of SCAPs in control inhibitor MM and inhibitor + MM groups were characterized by western blot analysis at day 7 (g)Semiquantitative analysis demonstrated that the expression of odontoosteogenic markers (BSP OCN RUNX2 OSX DSP OPN DSP andDMP-1) was significantly more downregulated in NF-120581B pathway-inhibited SCAPs than those in control group at day 7 (h) Alizarin redstaining of SCAPs after 14 days of osteogenic induction (i) Calcium quantification demonstrated the weaker calcium deposition in inhibitorand inhibitor + MM groups as compared with control and MM groups respectively

Acknowledgments

This work was supported by National Natural Science Foun-dation of China (no 81371144) Natural Science Foundationof Jiangsu Province (no BK20131392) and the Priority Aca-demic Program Development of Jiangsu Higher EducationInstitutions (PAPD no 2011-137)

References

[1] AKumar Y TakadaAMBoriek andB BAggarwal ldquoNuclearfactor-120581B its role in health and diseaserdquo Journal of MolecularMedicine vol 82 no 7 pp 434ndash448 2004

[2] F Chen V Castranova X Shi and LM Demers ldquoNew insightsinto the role of nuclear factor-120581B a ubiquitous transcription

factor in the initiation of diseasesrdquo Clinical Chemistry vol 45no 1 pp 7ndash17 1999

[3] C Gasparini and M Feldmann ldquoNF-120581B as a target for modu-lating inflammatory responsesrdquoCurrent Pharmaceutical Designvol 18 no 35 pp 5735ndash5745 2012

[4] A Ohazama and P T Sharpe ldquoTNF signalling in toothdevelopmentrdquo Current Opinion in Genetics amp Development vol14 no 5 pp 513ndash519 2004

[5] Y-H Kuan F-M Huang Y-C Li and Y-C Chang ldquoProin-flammatory activation ofmacrophages by bisphenol A-glycidyl-methacrylate involved NF120581B activation via PI3KAkt pathwayrdquoFood and Chemical Toxicology vol 50 no 11 pp 4003ndash40092012

[6] X Cai P Gong YHuang andY Lin ldquoNotch signalling pathwayin tooth development and adult dental cellsrdquo Cell Proliferationvol 44 no 6 pp 495ndash507 2011

12 BioMed Research International

[7] J-M Courtney J Blackburn and P T Sharpe ldquoThe Ectodys-plasin and NF120581B signalling pathways in odontogenesisrdquoArchives of Oral Biology vol 50 no 2 pp 159ndash163 2005

[8] W Sonoyama Y Liu T Yamaza et al ldquoCharacterization of theapical papilla and its residing stem cells from human immaturepermanent teeth a pilot studyrdquo Journal of Endodontics vol 34no 2 pp 166ndash171 2008

[9] W Sonoyama Y Liu D Fang et al ldquoMesenchymal stem cell-mediated functional tooth regeneration in swinerdquo PLoS ONEvol 1 no 1 article e79 2006

[10] H Chopra M K Hans and S Shetty ldquoStem cells-the hiddentreasure a strategic reviewrdquoDental Research Journal vol 10 no4 pp 421ndash427 2013

[11] R Dong R Yao J Du S Wang and Z Fan ldquoDepletion ofhistone demethylase KDM2A enhanced the adipogenic andchondrogenic differentiation potentials of stem cells from apicalpapillardquo Experimental Cell Research vol 319 no 18 pp 2874ndash2882 2013

[12] Y Wang M Yan Z Wang et al ldquoDental pulp stem cells fromtraumatically exposed pulps exhibited an enhanced osteogenicpotential and weakened odontogenic capacityrdquo Archives of OralBiology vol 58 no 11 pp 1709ndash1717 2013

[13] Y Wang M Yan Z Fan L Ma Y Yu and J Yu ldquoMineraltrioxide aggregate enhances the odontoosteogenic capacity ofstem cells from inflammatory dental pulps via NF-120581B pathwayrdquoOral Diseases 2013

[14] Y Wang M Yan Y Yu J Wu J Yu and Z Fan ldquoEstrogendeficiency inhibits the odontoosteogenic differentiation ofdental pulp stem cells via activation of the NF-120581B pathwayrdquo Celland Tissue Research vol 352 no 3 pp 551ndash559 2013

[15] Y Wang Y Zheng Z Wang et al ldquo10minus7M 17120573-oestradiolenhances odontoosteogenic potency of human dental pulpstem cells by activation of the NF-120581B pathwayrdquo Cell Prolifera-tion vol 46 no 6 pp 677ndash684 2013

[16] Z-G Lu H Liu T Yamaguchi Y Miki and K YoshidaldquoProtein kinase C120575 activates RelAp65 and nuclear factor-120581B signaling in response to tumor necrosis factor-120572rdquo CancerResearch vol 69 no 14 pp 5927ndash5935 2009

[17] G Bonizzi and M Karin ldquoThe two NF-120581B activation pathwaysand their role in innate and adaptive immunityrdquo Trends inImmunology vol 25 no 6 pp 280ndash288 2004

[18] K W McIntyre D J Shuster K M Gillooly et al ldquoA highlyselective inhibitor of I120581B kinase BMS-345541 blocks both jointinflammation and destruction in collagen-induced arthritis inmicerdquo Arthritis amp Rheumatism vol 48 no 9 pp 2652ndash26592003

[19] J R Burke M A Pattoli K R Gregor et al ldquoBMS-345541 is ahighly selective inhibitor of I120581B kinase that binds at an allostericsite of the enzyme and blocks NF-120581B-dependent transcriptionin micerdquoThe Journal of Biological Chemistry vol 278 no 3 pp1450ndash1456 2003

[20] F W Paula-Silva A Ghosh L A Silva and Y L Kapila ldquoTNF-120572 promotes an odontoblastic phenotype in dental pulp cellsrdquoJournal of Dental Research vol 88 no 4 pp 339ndash344 2009

[21] S Wang J Mu Z Fan et al ldquoInsulin-like growth factor 1 canpromote the osteogenic differentiation and osteogenesis of stemcells from apical papillardquo Stem Cell Research vol 8 no 3 pp346ndash356 2012

[22] Y Yu J Mu Z Fan et al ldquoInsulin-like growth factor 1 enhancesthe proliferation and osteogenic differentiation of human peri-odontal ligament stem cells via ERK and JNKMAPKpathwaysrdquoHistochemistry andCell Biology vol 137 no 4 pp 513ndash525 2012

[23] H Aonuma N Ogura K Takahashi et al ldquoCharacteristics andosteogenic differentiation of stemprogenitor cells in the humandental follicle analyzed by gene expression profilingrdquo Cell andTissue Research vol 350 no 2 pp 317ndash331 2012

[24] L Wang M Yan Y Wang et al ldquoProliferation andosteoodontoblastic differentiation of stem cells from dentalapical papilla in mineralization-inducing medium containingadditional KH

2PO4rdquo Cell Proliferation vol 46 no 2 pp

214ndash222 2013[25] G T-J Huang T Yamaza L D Shea et al ldquoStemProgenitor

cell-mediated de novo regeneration of dental pulp with newlydeposited continuous layer of dentin in an in vivomodelrdquoTissueEngineering A vol 16 no 2 pp 605ndash615 2010

[26] J-H Kwak J-K Jung and H Lee ldquoNuclear factor-120581 Binhibitors a patent review (2006ndash2010)rdquo Expert Opinion onTherapeutic Patents vol 21 no 12 pp 1897ndash1910 2011

[27] K Hess A Ushmorov J Fiedler R E Brenner and T WirthldquoTNF120572 promotes osteogenic differentiation of human mes-enchymal stem cells by triggering theNF-120581B signaling pathwayrdquoBone vol 45 no 2 pp 367ndash376 2009

[28] X Feng G Feng J Xing et al ldquoTNF-120572 triggers osteogenicdifferentiation of human dental pulp stem cells via the NF-120581Bsignalling pathwayrdquoCell Biology International vol 37 no 12 pp1267ndash1275 2013

[29] J Chang F Liu M Lee et al ldquoNF-120581B inhibits osteogenicdifferentiation of mesenchymal stem cells by promoting 120573-catenin degradationrdquo Proceedings of the National Academy ofSciences of the United States of America vol 110 no 23 pp9469ndash9474 2013

[30] C Shui and A Scutt ldquoMild heat shock induces proliferationalkaline phosphatase activity and mineralization in humanbone marrow stromal cells and Mg-63 cells in vitrordquo Journal ofBone and Mineral Research vol 16 no 4 pp 731ndash741 2001

[31] X Wei J Ling L Wu L Liu and Y Xiao ldquoExpressionof mineralization markers in dental pulp cellsrdquo Journal ofEndodontics vol 33 no 6 pp 703ndash708 2007

[32] S Z Khan E Kokubu K Matsuzaka and T Inoue ldquoBehaviourof rat-cultured dental pulp cells in three-dimensional collagentype-1 gel in vitro and in vivordquo Australian Endodontic Journalvol 39 no 3 pp 137ndash145 2013

[33] D-X Cao Z-J Li X-O Jiang et al ldquoOsteopontin as potentialbiomarker and therapeutic target in gastric and liver cancersrdquoWorld Journal of Gastroenterology vol 18 no 30 pp 3923ndash39302012

[34] S SchlaferMK Raarup P LWejse et al ldquoOsteopontin reducesbiofilm formation in a multi-species model of dental biofilmrdquoPloS ONE vol 7 no 8 Article ID e41534 2012

[35] S Chen J Gluhak-Heinrich Y HWang et al ldquoRunx2Osx andDspp in tooth developmentrdquo Journal of Dental Research vol 88no 10 pp 904ndash909 2009

[36] S Suzuki N Haruyama F Nishimura and A B KulkarnildquoDentin sialophosphoprotein and dentin matrix protein-1 twohighly phosphorylated proteins inmineralized tissuesrdquoArchivesof Oral Biology vol 57 no 9 pp 1165ndash1175 2012

[37] R S Prescott R Alsanea M I Fayad et al ldquoIn vivo fenerationof dental pulp-like tissue by using dental pulp stem cells a col-lagen scaffold and dentin matrix protein 1 after subcutaneoustransplantation in micerdquo Journal of Endodontics vol 34 no 4pp 421ndash426 2008

[38] T Komori ldquoRegulation of bone development and extracellularmatrix protein genes by RUNX2rdquo Cell and Tissue Research vol339 no 1 pp 189ndash195 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 3

L-glutamine 10 nmolL dexamethasone and 10mmolL 120573-glycerophosphate (Sigma) In addition activator (TNF-120572) orinhibitor (BMS-345541) was added into experiment groupsALP activity was measured after 3 5 and 7 days of cultureThe relative ALP activity was normalized to the total proteincontent per sample

After 2weeks ofmineralization induction cells were fixedin ice-cold 70 ethanol for 30 minutes and stained withalizarin red (40mM pH = 42 Sigma-Aldrich) for 5min atroom temperature Images were obtained using a scannerCalcium contents were quantitatively analyzed according toour previous method [13] The results were described as themeans plusmn SD and each experiment was performed for threetimes

25 Real-Time Reverse Transcriptase-Polymerase Chain Reac-tion (Real-Time RT-PCR) SCAPs were cultured in completemedia ormineralizationmedia (MM) supplementing activa-tor or inhibitor to experiment groups After 3 days or 7 days ofincubation total RNA was extracted from cells in each groupusing TRIzol reagent (Invitrogen Carlsbad CA) accordingto the manufacturerrsquos instructions Total RNA was subjectedto reverse transcription with a PrimeScript RT Master Mixkit (TaKaRa Dalian China) The mRNA expressions ofseveral osteoblasticdentinogenic markers including ALPosteocalcin (OCN) bone sialoprotein (BSP) osterix (OSX)runt-related transcription factor 2 (RUNX2) osteopontin(OPN) dentin sialophosphoprotein (DSPP) and dentinmatrix protein-1 (DMP-1) were quantified by real-time RT-PCR using SYBR Premix Ex Taq kit (TaKaRa Bio Japan) andABI 7300 real-time PCR system Relative gene expressionvalues were calculated by the 2minusΔΔCt method as previouslydescribed [23] GAPDH (glyceraldehyde-3-phosphate dehy-drogenase) was employed as reference housekeeping gene fornormalizingmRNA levels All PCR reactionswere performedin triplicate and data were expressed as means plusmn SD Primersused for real-time RT-PCR were as follows GAPDH 51015840-GAAGGTGAAGGTCGGAGTC-31015840 and 51015840-GAGATGGTG-ATGGGATTTC-31015840 ALP 51015840-GACCTCCTCGGAAGACAC-TC-31015840 and 51015840-TGAAGGGCTTCTTGTCTGTG-31015840 OCN 51015840-AGCAAAGGTGCAGCCTTTGT-31015840 and 51015840-GCGCCTGGG-TCTCTTCACT-31015840 BSP 51015840-CTATGGAGAGGACGCCAC-GCCTGG-31015840 and 51015840-CATAGCCATCGTAGCCTTGTCCT-31015840 RUNX2 51015840-TCTTAGAACAAATTCTGCCCTTT-31015840 and51015840-TGCTTTGGTCTTGAAATCACA-31015840OSX 51015840-CCTCCT-CAGCTCACCTTCTC-31015840 and 51015840-GTTGGGAGCCCAAAT-AGAAA-31015840 DSPP 51015840-ATATTGAGGGCTGGAATGGGG-A-31015840 and 51015840-TTTGTGGCTCCAGCATTGTCA-31015840 OPN 51015840-CCAAGTAAGTCCAACGAAAG-31015840 and 51015840-GGTGATGTC-CTCGTCTGTA-31015840 DMP-1 51015840-CCCTTGGAGAGCAGT-GAGTC-31015840 and 51015840-CTCCTTTTCCTGTGCTCCTG-31015840

26 Western Blot Analysis Cultured cells in complete mediaormineralizationmedia (MM) with the presence of activatoror inhibitor in experimental groups were dissolved on ice for20min in RIPA lysis buffer (Beyotime China) supplementedwith 1mM phenylmethylsulfonyl fluoride (PMSF Beyotime)

20120583g of protein from each sample was used for western blotanalysis following the protocols in our previous study [14]

As for the detection of signaling pathway SCAPs werecultured in serum-free media for 24 hours followed bythe treatment of 10 ngmL TNF-120572 or 1 120583molL BMS-345541At the indicated time points the cytoplasm protein wasextracted with a Keygen Kit (Keygen Biotech China) andwestern blot was subsequently performed

The primary antibodies in this experiment were as fol-lows OCN (1 1000 Millipore) BSP (1 1000 Abcam) OSX(1 1000 Abcam) RUNX2 (1 1000 Abcam) DSP (1 500Santa Cruz) OPN (1 1000 Abcam) DMP-1 (1 1000 Novus)phosphor-P65 (1 1000 Cell Signaling) P65 (1 1000 CellSignaling) phosphor-I120581B120572 (1 1000 Cell Signaling) I120581B120572(1 1000 Cell Signaling) and 120573-ACTIN (1 1000 Bioworld)Target protein expression was then quantified according tothe band intensity and standardized by the structure protein120573-ACTIN with Image-Proplus 50 software In brief theintegral optical density of the protein bands was calculated byusing Image-Proplus 50 software Then densitometry ratiosbetween the target protein and 120573-ACTIN were obtained ina semiquantitative level and the histogram was then plottedThe experiment was performed for three times

27 Statistical Analysis Statistical analysis was performed bypaired t-test and one-way analysis of variance (ANOVA)Follow-up comparisons between experiment groups andcontrol groupwere then carried out using theDunnet posttestanalysis Values of 119875 lt 005 were regarded as statisticallysignificant

3 Results

31 Identification of SCAPs Immunocytochemistry anal-ysis showed that SCAPs were stained positively for themesenchymal stem cell (MSC) surface molecule STRO-1(Figure 1(a)) but negatively for epithelial cell marker cytok-eratin (Figure 1(b)) Similarly there was a high expression ofMSCmarkers (eg CD29 CD73 CD90 CD105 and CD146)while the hematopoieticmarkers (eg CD34 andCD45)werelow expressed in SCAPs as demonstrated by flow cytometry(Figure 1(d)) These data revealed the stromal origin of theseisolated cells with stem cell characteristics and the absence ofhematopoietic precursor contamination

32 Activation and Inhibition of Canonical NF-120581B SignalingPathway in SCAPs It has been extensively proven that TNF-120572 is a potent activator of canonical NF-120581B pathway whileBMS-345541 is the highly selective inhibitor of NF-120581B Todetermine whether SCAPs treated with TNF-120572 or BMS-345541 can result in the NF-120581B activation or inhibitionrespectively cytoplasm protein was extracted and subjectedto electrophoresis In TNF-120572-treated SCAPs phospho-I120581B120572was obviously elevated in a time-dependent manner andphosphorylated P65 rapidly reached a maximal increasewithin 15 minutes after TNF-120572 stimulation (Figures 2(a) and2(b)) Suppressed NF-120581B activity was detected in SCAPs afterincubation with BMS-345541 as indicated by the decreased

4 BioMed Research International

STRO-1 100120583m

(a)

CK 100120583m

(b)

PBS 100120583m

(c)

100 101 102 103 104

1000

0

037

SSC-

H

CD34 FITC100 101 102 103 104

CD34 FITC

084

100 101 102 103 104

184

CD73 PE100 101 102 103 104

CD73 PE

9999

100 101 102 103 104

1000

0

SSC-

H

CD105 APC

132

100 101 102 103 104

CD105 APC

9999

100 101 102 103 104

049

CD45 PerCP100 101 102 103 104

CD45 PerCP

297

100 101 102 103 104

1000

0

SSC-

H

112

CD90 PE100 101 102 103 104

CD90 PE

9994

104100 101 102 103

104

CD146 APC100 101 102 103 104

CD146 APC

9184

Isotype control StainingIsotype control Staining

(d)

Figure 1 Characterization of SCAPs (a) isolated SCAPswere positive for STRO-1 by immunocytochemistry (b) isolated SCAPswere negativefor CK by immunocytochemistry (c) PBS served as a negative control (d) flow cytometric analysis revealed that cultured SCAPs are positivefor CD73 (9999) CD105 (9999) CD90 (9994) and CD146 (9184) but negative for CD34 (084) and CD45 (297) Mouse IgGisotype control antibodies conjugated to FITC PE APC or PerCP were used as negative controls Scale bars 100 120583m

phosphorylated I120581B120572 and P65 (Figures 2(e) and 2(f)) Ratiosof phosphorylated to unphosphorylated forms of proteinsfurther confirmed the activation of NF-120581B by TNF-120572 andinhibition of NF-120581B by BMS-345541 (Figures 2(c) 2(d) 2(g)and 2(h) 119875 lt 005)

33 Effects of Canonical NF-120581B Pathway on the Proliferation ofSCAPs As shown in Figure 3(a) SCAPs in activator group

exhibited higher proliferation while the SCAPs-inhibitorgroup showed less proliferation capacity as compared withthe corresponding control groups respectively (119875 lt 005)except for the time points at baseline (day 0) and thefirst day Flow cytometry assay revealed that the activator-treated SCAPs exhibited a higher percentage of cells in Sand G

2M phases (2652) and a lower percentage of cells

in G0G1phase (7348) in comparison with untreated cells

BioMed Research International 5

30min 60min0min 15min

P-P65

P65

P-I120581B120572

I120581B120572

ACTIN

65kDa

65kDa

39kDa

40kDa

43kDa

30min 60min0min 15min

P-P65

P65

P-I120581B120572

I120581B120572

ACTIN

65kDa

65kDa

39kDa

40kDa

43kDa

14

12

1

08

06

04

02

0

lowastlowast lowast

lowast

lowastlowast

P-P65P65P-I120581B120572I120581B120572

Prot

eins

ACT

IN

0min15min

30min60min

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

06

05

04

03

02

01

0

lowastlowast

Prot

eins

ACT

INI120581B120572 P-I120581B120572 P65 P-P65

0min15min

30min60min

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

25

2

15

1

1

05

0

P-I120581

B120572I120581

B120572

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0min15min

30min60min

353

252

151

1

050

P-P6

5P65

0min15min

30min60min

lowastlowast

lowastlowast

lowastlowast

1614121

1

080604020

lowast

P-I120581

B120572I120581

B120572

lowastlowastlowastlowastlowastlowast

0min15min

30min60min

lowast4353

252

151

1

050

0min15min

30min60min

lowastlowastlowast

(a)

(b) (f)

(e)

(c) (g)

(d) (h)

P-P6

5P65

Figure 2 Activation and inhibition of canonical NF-120581B signaling pathway in SCAPs (a)The expression of NF-120581B pathway proteins in TNF-120572-treated SCAPs at different time points (b) Semiquantitative analysis confirmed the upregulation of P-I120581B120572 and P-P65 after the activation ofNF-120581B pathway (c)The ratio of phosphorylated to unphosphorylated form of P65 (d)The ratio of phosphorylated I120581B120572 to unphosphorylatedI120581B120572 (e) The protein levels of I120581B120572 P-I120581B120572 P65 and P-P65 in the cytoplasm of BMS-345541-treated SCAPs at indicated time points (f)Semiquantitative analysis confirmed the declined expression of P-I120581B120572 and P-P65 after the inhibition of NF-120581B pathway (g) The ratio ofphosphorylated to unphosphorylated form of P65 (h) The ratio of P-I120581B120572 to I120581B120572 Values are the means plusmn SD 119899 = 3 lowastlowast119875 lt 001 lowastlowastlowast119875 lt0001

6 BioMed Research International

lowast

lowast

lowast

lowast

lowast lowast

ControlActivator

MTT25

2

15

1

05

0

OD

val

ue (4

90

nm)

0 1 3 5 7 9 11

(day)

(a)

S30 60 90 120

SSSSSSSSSSSSSSSSSSSSSS0

300

900

1200

600

Num

ber

SCAPsChannels (FL2-A)

G2M

G0G1 = 8506

G2M = 986

G0G1

S

30 60 90 120

S

0

Num

ber

Channels (FL2-A)

G2M

G0G1 = 7348

G2M = 886

G0G1

1000

800

600

400

200

00

SCAPs + activator

S = 1766S = 508

(b)

Control

Activator

0h 6h 12h 24h

(c)

ControlInhibitor

25

2

15

1

05

0

OD

val

ue (4

90

nm) lowast

lowast

MTT

0 1 3 5 7 9 11

(day)

lowastlowast

lowastlowastlowast

lowastlowastlowast

(d)

SSSSSSSSSSSSSSSSSSSSSSS

SCAPsChannels (FL2-A)

G0G1

30 60 90 1200

G2M

1600

1200

800

400

0

Num

ber

G0G1 = 8217

G2M = 600

S = 1183

1600

1200

800

400

0

Num

ber

S

G0G1

G2M

Channels (FL2-A)30 60 900 120 150

G0G1 = 9109

G2M = 536

S = 354

SCAPs + inhibitor

(e)

0h 6h 12h 24h

Control

Inhibitor

(f)

Figure 3 Effects of canonical NF-120581B signaling pathway on the proliferation of SCAPs (a) Cell proliferation in NF-120581B pathway-activatedand control groups was detected by MTT assay (b) Flow cytometry analysis for SCAPs in NF-120581B pathway-activated and untreated groupsThe proliferation index (PI = S + G

2M) in NF-120581B pathway-activated group (2652) was significantly higher than that in control group

(1494) (c) Images of scratch wounds in NF-120581B pathway-activated and untreated groups at indicated time points (d) Growth curves of NF-120581B pathway-inhibited and untreated SCAPs (e) Representative cell cycle distributions of NF-120581B pathway-inhibited and untreated SCAPTheproliferation index (PI = S + G

2M) in NF-120581B pathway-activated group (890) was obviously lower than that in control group (1783)

(f) Cell motility of SCAPs in NF-120581B pathway-inhibited and untreated group assessed by a scratch assay Values were the means plusmn SD 119899 = 6lowast

119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 Scale bars = 100 120583m

(119875 lt 005 Figure 3(b))Therewas a lower percentage (890)of proliferating cells in SG

2Mphases in the inhibitor-treated

SCAPs as compared with the control group (1783) at day 3(Figure 3(e)) which is consistent with the findings in MTTassay (Figure 3(d)) These results indicate that canonical

NF-120581B pathway facilitated the proliferation of SCAPs thatis the cell proliferation rate increasing in pace with theactivation of canonical NF-120581B and is restrained along withthe blockage of NF-120581B Furthermore an improved migrationability was observed in activator-treated SCAPs (Figure 3(c))

BioMed Research International 7

while scratch closure was markedly slower in SCAPs treatedwith inhibitor (Figure 3(f))

34 Effects of Canonical NF-120581B Pathway on the OdontoOsteo-genic Differentiation of SCAPs ALP activities in activator-treated SCAPs in basic medium andmineralization-inducingmedium were elevated at day 5 and day 7 (119875 lt 005Figure 4(a)) as compared with untreated groupsThe densityof calcification nodules was significantly higher in activator-stimulated groups than in the other groups after 14 days ofcoculture (Figure 4(h)) Moreover quantitative calciummea-surement illustrated more calcifications in activator-treatedSCAPs in comparison with untreated groups (Figure 4(i))

Differentially expression levels of related osteoodonto-genic genes were also investigated by real-time RT-PCRassays In activator-treated group expression of specificosteoodontogenic genes (eg ALP OCN BSP OSXRUNX2 DSP OPN and DMP-1) was significantly upregu-lated at days 3 and 7 (Figures 4(b) and 4(c)) At day 3 theexpression of some osteoodontogenic genes in activator-treated groups did not change obviously in complete mediabut was distinctly increased in the presence ofmineralization-inducing media At day 7 the odontoosteo-genic markers were significantly upregulated following theactivator treatment regardless of the presence or absence ofmineralization-inducing media This phenomenon was notobserved in NF-120581B-inhibition group These findings wereconfirmed by western blot assay in which activated classicalNF-120581B signaling pathway induced a significant increase ofrelated protein expression (Figures 4(d)ndash4(g))

As shown in Figure 5(a) the blockage of NF-120581B signalingpathway obviously downregulated the ALP activity at days 35 and 7 (119875 lt 005) At day 14 less calcified nodules weregenerated in inhibitor-treated groups (Figure 5(h)) Calciumquantification also revealed the less calcium deposition ininhibitor and inhibitor + MM groups as compared withcontrol and MM groups respectively (Figure 5(i) 119875 lt 005)There was a remarkable decrease of osteoodontogenic genesat different time points (Figures 5(b) and 5(c)) Western blotanalysis further verified these findings (Figures 5(d)ndash5(g))

4 Discussion

SCAPs are known as a kind of ideal candidates for dentaltissue engineering and have the characteristics of self-renewaland multilineage differentiation potential [9 10] Diversestudies have proved that SCAPs are able to differentiate intoosteoodontoblasts in vitro under appropriate conditions andformbonedentin-like tissues in vivo [24 25] Certainlymanysignaling pathways may be involved in the process of cellproliferation and differentiation including NF-120581B pathway

NF-120581B exists in the cytoplasm in a latent form bindingto inhibitory proteins termed inhibitory 120581B proteins (I120581Bs)[3 26] I120581B complex is the major regulator of NF-120581B pathwayand is composed of catalytic subunits IKK120572 and IKK120573 anda regulatory subunit IKK120574 TNF-120572 is generally known toactivate classical NF-120581B pathway [17] Exposure of cells tosuitable concentrations of TNF-120572 brings about the rapid

phosphorylation ubiquitination and proteolytic degradationof I120581B which allows NF-120581B to translocate to the nucleuswhere it subsequently regulates the gene transcription [27ndash29] BMS-345541 a selective inhibitor of the catalytic subunitsof IKK targets NF-120581B signaling pathway and downregulatesthe activity of NF-120581B [18 19] In this study the expressionof cytoplastic P-P65P-I120581B120572 was noticeably upregulated afterthe treatment of TNF-120572 and downregulated by the inhibitorBMS-345541 indicating the successful establishment of acellular model for the activation or suppression of canonicalNF-120581B pathway

Cell proliferation and migration were indispensable fortissue development and wound healing In the present studyNF-120581B-activated SCAPs exhibited an increased growth ratehigher proliferation index and more effective migrationin wound healing assay Moreover the migration distancesbetween thewound lines in inhibitor-treated cells were longerthan control cells after 24 hours of culture Together withthe weakened proliferation of inhibitor-treated cells it ispredictable that the canonical NF-120581B pathway has a positiveinfluence on the cell multiplication activity and motility

Previous studies have proved that various stimuli canexert an active role on the committed differentiation ofdental stem cells via NF-120581B pathway [28] In the presentstudy the odontoosteogenic capacity remarkably changedalong with the activation or blockage of classical NF-120581Bpathway Activator-treated SCAPs exhibited an enhancedALP activity increased calcium deposition and upregulatedexpression of odontoosteogenic genes and proteins whileinhibitor-treated cells presented the lower ALP activitydecreased mineralization and downregulated expression ofodontoosteoblast markers

It is widely recognized that ALP is a functionalmarker forosteoblast activity and bone formation [30] As an importantextracellular matrix of bone BSP is mainly secreted byosteoblasts whose expression is a crucial symbol in matrixdeposition and mineralization [31 32] OPN is anotherextracellular matrix protein expressed in numerous cell typesincluding odontoblasts in which OPN plays multifacetedroles in a variety of biological and pathological processessuch as osteogenic differentiation tooth mineralization anddental biofilm formation [33 34] OCN is the primarynoncollagenous protein in the bone cells secreted in the latestage of osteogenesis and known as a specific indicator ofosteogenic differentiation RUNX2 is the first transcriptionfactor required for determination of the osteoblast lineageand OSX acts as a downstream gene of RUNX2 that is highlyexpressed in the functional odontoosteoblasts [35] DSPPand DSP are well-known markers of odontoblasts highlyexpressed in dentin or predentin structures and essentialfor dentinogenesis [36] DMP-1 is an acidic extracellularmatrix protein that is primarily found in dentin and boneand has been implicated in dentin mineralization and signaltransduction in the process of odontogenesis [37]

In this study the expression of RUNX2RUNX2 reducedafter 3 days of incubation with the inhibitor BMS-345541and then gradually increased at day 7 while the expressionof DSPPDSP did not compromise after 3 days of incuba-tion but noticeably decreased at day 7 indicating that the

8 BioMed Research International

ALP

3 5 7

(day)

35

3

25

2

15

1

05

0

ControlActivator

MMActivator + MM

lowast

lowastlowastlowastlowastlowastlowast

(sig

ma u

nitm

inm

g pr

otei

n)

(a)

ControlActivator

MMActivator + MM

lowastlowast

lowastlowast

876543210

ALP

OCN BS

P

RUN

X2

OSX

DSP

P

OPN

DM

P-1

Gen

esG

APD

H

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastPCR 3d

(b)

lowast

lowast lowast

lowast

lowast lowastlowast

lowast

25

20

15

10

5

0

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

Gen

esG

APD

H

ControlActivator

MMActivator + MM

ALP

OCN BS

P

RUN

X2

OSX

DSP

P

OPN

DM

P-1

PCR 7d

(c)

MMControl Activator

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

Activator + MM

Western blot 3d

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

(d)

ControlActivator

MMActivator + MM

09

08

07

06

05

04

03

02

01

0

Prot

eins

ACT

IN

OCN BSP RUNX2 OSX DSP OPN DMP-1

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

lowast

lowast

lowast

(e)

MMControl Activator

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

Activator + MM

Western blot 7d

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

(f)

Figure 4 Continued

BioMed Research International 9

14

12

1

08

06

04

02

0

Prot

eins

ACT

IN

OCN BSP RUNX2 OSX DSP OPN DMP-1

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

ControlActivator

MMActivator + MM

(g)

Control Activator

MM Activator + MM

(h)

1

CPC06

05

04

03

02

01

0

lowast

lowastlowastlowast

ControlActivator

MMActivator + MM

(ng

calc

ium

mg

prot

ein)

(i)

Figure 4 Odontoosteogenic differentiation in canonical NF-120581B-activated SCAPs (a) ALP activity of SCAPs in control activator MM andactivator + MM groups at days 3 5 and 7 Values are the means plusmn SD 119899 = 6 lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 (b) Real-time RT-PCRanalysis for odontoosteogenic genes (BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in each group at day 7 (c) Real-time RT-PCRanalysis for odontoosteogenic genes (ALP BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in different groups at day 7 GAPDHserved as a housekeeping gene lowastlowast2minusΔΔCt ge 2 119875 lt 001 lowast1 lt 2minusΔΔCt lt 2 119875 lt 001 119899 = 3 (d) Western blot analyses for the odontoosteogenicproteins (BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in different groups at day 3 120573-ACTIN served as an internal control (e)Semiquantitative analysis demonstrated that the expression of BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1 was stronger in NF-120581Bpathway-activated SCAPs than those in control group at day 3 especially in the presence of mineralization-inducing media (f) Western blotanalyses for the odontoosteogenic proteins (BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in different groups at day 7 120573-ACTINwas used as an internal control (g) Semiquantitative analysis confirmed that the expression of BSP OCN RUNX2 OSX DSP OPN DSPand DMP-1 was upregulated in NF-120581B pathway-activated SCAPs at day 7 regardless of the presence or absence of mineralization-inducingmedia (h) Alizarin red staining of SCAPs after 14 days of induction in different groups (i) Quantitative calcium analysis showed that thecalcium content in activator and activator + MM groups was significantly higher than the control and MM groups respectively Values weredescribed as the means plusmn SD lowast119875 lt 005 lowastlowastlowast119875 lt 0001

expression of RUNX2RUNX2 and DSPPDSP tends to beinversely correlated [38] Moreover the dramatic differenceof these odontoosteogenic markers at both mRNA andprotein levels in response to the regulation of NF-120581B pathwaysuggests that activated canonical NF-120581B pathway favors theodontoosteoblastic differentiation of SCAPs particularly inthe presence of mineralization-inducing media

In summary the findings accumulated here confirmedthe potential involvement of canonical NF-120581B pathway inthe proliferationmigration and committed differentiation ofSCAPs in vitro Both activation and inhibition of the classicalNF-120581B pathway can bring about the permanent changes in

human stem cells Moreover it is commonly believed thatNF-120581B signaling plays a pivotal role not only in the progressof normal physiological process but also in the pathologicalprocess and dysfunction ofNF-120581B is linked to various humandiseases Thus proper balance of intracellular NF-120581B shouldbemaintained in the physiological conditions while intricateinteractivity between NF-120581B and other signaling pathwaysneeds to be extensively investigated

Conflict of Interests

The authors declare no conflict of interests

10 BioMed Research International

ControlInhibitor

MM

ALP3

25

2

15

1

05

03 5 7

(day)

Inhibitor + MM

lowastlowast lowastlowast

lowastlowast

lowast

lowast

(sig

ma u

nitm

inm

g pr

otei

n)

(a)

ControlInhibitor

35

3

25

2

15

1

05

0

Gen

esG

APD

H

ALP OCN BSP RUNX2 OSX DSPP OPN DMP-1

MMInhibitor + MM

lowastlowast

lowast

lowast

lowast lowastlowast

lowast

lowastlowast

lowastlowast

PCR 3d

(b)

ControlInhibitor

MM

1614121086420

Gen

esG

APD

H

ALP OCN BSP RUNX2 OSX DSPP OPN DMP-1

Inhibitor + MM

lowastlowast lowastlowast

lowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowastlowastlowastPCR 7d

(c)

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

MMInhibitorControl Inhibitor + MM

Western blot 3d

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

(d)

Prot

eins

ACT

IN

ControlInhibitor

MM

12

1

08

06

04

02

0OCN BSP RUNX2 OSX DSP OPN DMP-1

Inhibitor + MM

lowast

lowast lowast

lowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

(e)

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

MMInhibitorControl Inhibitor + MM

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

Western blot 7d

(f)

Figure 5 Continued

BioMed Research International 11

Prot

eins

ACT

IN

OCN BSP RUNX2 OSX DSP OPN DMP-1

ControlInhibitor MM

Inhibitor + MM

0504504

035030250201501005

0

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(g)

MM

InhibitorControl

Inhibitor + MM

(h)

06

05

04

03

02

01

0

lowast

lowastlowastCPC

1

MMInhibitorControl

Inhibitor + MM

(ng

calc

ium

mg

prot

ein)

(i)

Figure 5 Odontoosteogenic differentiation in canonical NF-120581B-inhibited SCAPs (a) ALP activity at different time points in different groups(ie control inhibitor MM and inhibitor +MM) (b) Real-time RT-PCR for the detection ofALP BSP OCN RUNX2 OSX DSP OPN DSPandDMP-1 at day 3ThemRNA levels were normalized toGAPDH (c) Real-time RT-PCR for the detection ofALP BSP OCN RUNX2 OSXDSP OPN DSP andDMP-1 of SCAPs respectively in control inhibitor MM and inhibitor +MMgroups at day 7 (d)The odontoosteogenicprotein levels of SCAPs in control inhibitor MM and inhibitor +MM groups were characterized by western blot analysis at day 3 120573-ACTINwas used as a loading control (e) Semiquantitative analysis demonstrated that the expression of BSP OCN RUNX2 OSX DSP OPN DSPand DMP-1 was more downregulated in NF-120581B pathway-inhibited SCAPs than those in control group at day 3 (f) The odontoosteogenicprotein levels of SCAPs in control inhibitor MM and inhibitor + MM groups were characterized by western blot analysis at day 7 (g)Semiquantitative analysis demonstrated that the expression of odontoosteogenic markers (BSP OCN RUNX2 OSX DSP OPN DSP andDMP-1) was significantly more downregulated in NF-120581B pathway-inhibited SCAPs than those in control group at day 7 (h) Alizarin redstaining of SCAPs after 14 days of osteogenic induction (i) Calcium quantification demonstrated the weaker calcium deposition in inhibitorand inhibitor + MM groups as compared with control and MM groups respectively

Acknowledgments

This work was supported by National Natural Science Foun-dation of China (no 81371144) Natural Science Foundationof Jiangsu Province (no BK20131392) and the Priority Aca-demic Program Development of Jiangsu Higher EducationInstitutions (PAPD no 2011-137)

References

[1] AKumar Y TakadaAMBoriek andB BAggarwal ldquoNuclearfactor-120581B its role in health and diseaserdquo Journal of MolecularMedicine vol 82 no 7 pp 434ndash448 2004

[2] F Chen V Castranova X Shi and LM Demers ldquoNew insightsinto the role of nuclear factor-120581B a ubiquitous transcription

factor in the initiation of diseasesrdquo Clinical Chemistry vol 45no 1 pp 7ndash17 1999

[3] C Gasparini and M Feldmann ldquoNF-120581B as a target for modu-lating inflammatory responsesrdquoCurrent Pharmaceutical Designvol 18 no 35 pp 5735ndash5745 2012

[4] A Ohazama and P T Sharpe ldquoTNF signalling in toothdevelopmentrdquo Current Opinion in Genetics amp Development vol14 no 5 pp 513ndash519 2004

[5] Y-H Kuan F-M Huang Y-C Li and Y-C Chang ldquoProin-flammatory activation ofmacrophages by bisphenol A-glycidyl-methacrylate involved NF120581B activation via PI3KAkt pathwayrdquoFood and Chemical Toxicology vol 50 no 11 pp 4003ndash40092012

[6] X Cai P Gong YHuang andY Lin ldquoNotch signalling pathwayin tooth development and adult dental cellsrdquo Cell Proliferationvol 44 no 6 pp 495ndash507 2011

12 BioMed Research International

[7] J-M Courtney J Blackburn and P T Sharpe ldquoThe Ectodys-plasin and NF120581B signalling pathways in odontogenesisrdquoArchives of Oral Biology vol 50 no 2 pp 159ndash163 2005

[8] W Sonoyama Y Liu T Yamaza et al ldquoCharacterization of theapical papilla and its residing stem cells from human immaturepermanent teeth a pilot studyrdquo Journal of Endodontics vol 34no 2 pp 166ndash171 2008

[9] W Sonoyama Y Liu D Fang et al ldquoMesenchymal stem cell-mediated functional tooth regeneration in swinerdquo PLoS ONEvol 1 no 1 article e79 2006

[10] H Chopra M K Hans and S Shetty ldquoStem cells-the hiddentreasure a strategic reviewrdquoDental Research Journal vol 10 no4 pp 421ndash427 2013

[11] R Dong R Yao J Du S Wang and Z Fan ldquoDepletion ofhistone demethylase KDM2A enhanced the adipogenic andchondrogenic differentiation potentials of stem cells from apicalpapillardquo Experimental Cell Research vol 319 no 18 pp 2874ndash2882 2013

[12] Y Wang M Yan Z Wang et al ldquoDental pulp stem cells fromtraumatically exposed pulps exhibited an enhanced osteogenicpotential and weakened odontogenic capacityrdquo Archives of OralBiology vol 58 no 11 pp 1709ndash1717 2013

[13] Y Wang M Yan Z Fan L Ma Y Yu and J Yu ldquoMineraltrioxide aggregate enhances the odontoosteogenic capacity ofstem cells from inflammatory dental pulps via NF-120581B pathwayrdquoOral Diseases 2013

[14] Y Wang M Yan Y Yu J Wu J Yu and Z Fan ldquoEstrogendeficiency inhibits the odontoosteogenic differentiation ofdental pulp stem cells via activation of the NF-120581B pathwayrdquo Celland Tissue Research vol 352 no 3 pp 551ndash559 2013

[15] Y Wang Y Zheng Z Wang et al ldquo10minus7M 17120573-oestradiolenhances odontoosteogenic potency of human dental pulpstem cells by activation of the NF-120581B pathwayrdquo Cell Prolifera-tion vol 46 no 6 pp 677ndash684 2013

[16] Z-G Lu H Liu T Yamaguchi Y Miki and K YoshidaldquoProtein kinase C120575 activates RelAp65 and nuclear factor-120581B signaling in response to tumor necrosis factor-120572rdquo CancerResearch vol 69 no 14 pp 5927ndash5935 2009

[17] G Bonizzi and M Karin ldquoThe two NF-120581B activation pathwaysand their role in innate and adaptive immunityrdquo Trends inImmunology vol 25 no 6 pp 280ndash288 2004

[18] K W McIntyre D J Shuster K M Gillooly et al ldquoA highlyselective inhibitor of I120581B kinase BMS-345541 blocks both jointinflammation and destruction in collagen-induced arthritis inmicerdquo Arthritis amp Rheumatism vol 48 no 9 pp 2652ndash26592003

[19] J R Burke M A Pattoli K R Gregor et al ldquoBMS-345541 is ahighly selective inhibitor of I120581B kinase that binds at an allostericsite of the enzyme and blocks NF-120581B-dependent transcriptionin micerdquoThe Journal of Biological Chemistry vol 278 no 3 pp1450ndash1456 2003

[20] F W Paula-Silva A Ghosh L A Silva and Y L Kapila ldquoTNF-120572 promotes an odontoblastic phenotype in dental pulp cellsrdquoJournal of Dental Research vol 88 no 4 pp 339ndash344 2009

[21] S Wang J Mu Z Fan et al ldquoInsulin-like growth factor 1 canpromote the osteogenic differentiation and osteogenesis of stemcells from apical papillardquo Stem Cell Research vol 8 no 3 pp346ndash356 2012

[22] Y Yu J Mu Z Fan et al ldquoInsulin-like growth factor 1 enhancesthe proliferation and osteogenic differentiation of human peri-odontal ligament stem cells via ERK and JNKMAPKpathwaysrdquoHistochemistry andCell Biology vol 137 no 4 pp 513ndash525 2012

[23] H Aonuma N Ogura K Takahashi et al ldquoCharacteristics andosteogenic differentiation of stemprogenitor cells in the humandental follicle analyzed by gene expression profilingrdquo Cell andTissue Research vol 350 no 2 pp 317ndash331 2012

[24] L Wang M Yan Y Wang et al ldquoProliferation andosteoodontoblastic differentiation of stem cells from dentalapical papilla in mineralization-inducing medium containingadditional KH

2PO4rdquo Cell Proliferation vol 46 no 2 pp

214ndash222 2013[25] G T-J Huang T Yamaza L D Shea et al ldquoStemProgenitor

cell-mediated de novo regeneration of dental pulp with newlydeposited continuous layer of dentin in an in vivomodelrdquoTissueEngineering A vol 16 no 2 pp 605ndash615 2010

[26] J-H Kwak J-K Jung and H Lee ldquoNuclear factor-120581 Binhibitors a patent review (2006ndash2010)rdquo Expert Opinion onTherapeutic Patents vol 21 no 12 pp 1897ndash1910 2011

[27] K Hess A Ushmorov J Fiedler R E Brenner and T WirthldquoTNF120572 promotes osteogenic differentiation of human mes-enchymal stem cells by triggering theNF-120581B signaling pathwayrdquoBone vol 45 no 2 pp 367ndash376 2009

[28] X Feng G Feng J Xing et al ldquoTNF-120572 triggers osteogenicdifferentiation of human dental pulp stem cells via the NF-120581Bsignalling pathwayrdquoCell Biology International vol 37 no 12 pp1267ndash1275 2013

[29] J Chang F Liu M Lee et al ldquoNF-120581B inhibits osteogenicdifferentiation of mesenchymal stem cells by promoting 120573-catenin degradationrdquo Proceedings of the National Academy ofSciences of the United States of America vol 110 no 23 pp9469ndash9474 2013

[30] C Shui and A Scutt ldquoMild heat shock induces proliferationalkaline phosphatase activity and mineralization in humanbone marrow stromal cells and Mg-63 cells in vitrordquo Journal ofBone and Mineral Research vol 16 no 4 pp 731ndash741 2001

[31] X Wei J Ling L Wu L Liu and Y Xiao ldquoExpressionof mineralization markers in dental pulp cellsrdquo Journal ofEndodontics vol 33 no 6 pp 703ndash708 2007

[32] S Z Khan E Kokubu K Matsuzaka and T Inoue ldquoBehaviourof rat-cultured dental pulp cells in three-dimensional collagentype-1 gel in vitro and in vivordquo Australian Endodontic Journalvol 39 no 3 pp 137ndash145 2013

[33] D-X Cao Z-J Li X-O Jiang et al ldquoOsteopontin as potentialbiomarker and therapeutic target in gastric and liver cancersrdquoWorld Journal of Gastroenterology vol 18 no 30 pp 3923ndash39302012

[34] S SchlaferMK Raarup P LWejse et al ldquoOsteopontin reducesbiofilm formation in a multi-species model of dental biofilmrdquoPloS ONE vol 7 no 8 Article ID e41534 2012

[35] S Chen J Gluhak-Heinrich Y HWang et al ldquoRunx2Osx andDspp in tooth developmentrdquo Journal of Dental Research vol 88no 10 pp 904ndash909 2009

[36] S Suzuki N Haruyama F Nishimura and A B KulkarnildquoDentin sialophosphoprotein and dentin matrix protein-1 twohighly phosphorylated proteins inmineralized tissuesrdquoArchivesof Oral Biology vol 57 no 9 pp 1165ndash1175 2012

[37] R S Prescott R Alsanea M I Fayad et al ldquoIn vivo fenerationof dental pulp-like tissue by using dental pulp stem cells a col-lagen scaffold and dentin matrix protein 1 after subcutaneoustransplantation in micerdquo Journal of Endodontics vol 34 no 4pp 421ndash426 2008

[38] T Komori ldquoRegulation of bone development and extracellularmatrix protein genes by RUNX2rdquo Cell and Tissue Research vol339 no 1 pp 189ndash195 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 BioMed Research International

STRO-1 100120583m

(a)

CK 100120583m

(b)

PBS 100120583m

(c)

100 101 102 103 104

1000

0

037

SSC-

H

CD34 FITC100 101 102 103 104

CD34 FITC

084

100 101 102 103 104

184

CD73 PE100 101 102 103 104

CD73 PE

9999

100 101 102 103 104

1000

0

SSC-

H

CD105 APC

132

100 101 102 103 104

CD105 APC

9999

100 101 102 103 104

049

CD45 PerCP100 101 102 103 104

CD45 PerCP

297

100 101 102 103 104

1000

0

SSC-

H

112

CD90 PE100 101 102 103 104

CD90 PE

9994

104100 101 102 103

104

CD146 APC100 101 102 103 104

CD146 APC

9184

Isotype control StainingIsotype control Staining

(d)

Figure 1 Characterization of SCAPs (a) isolated SCAPswere positive for STRO-1 by immunocytochemistry (b) isolated SCAPswere negativefor CK by immunocytochemistry (c) PBS served as a negative control (d) flow cytometric analysis revealed that cultured SCAPs are positivefor CD73 (9999) CD105 (9999) CD90 (9994) and CD146 (9184) but negative for CD34 (084) and CD45 (297) Mouse IgGisotype control antibodies conjugated to FITC PE APC or PerCP were used as negative controls Scale bars 100 120583m

phosphorylated I120581B120572 and P65 (Figures 2(e) and 2(f)) Ratiosof phosphorylated to unphosphorylated forms of proteinsfurther confirmed the activation of NF-120581B by TNF-120572 andinhibition of NF-120581B by BMS-345541 (Figures 2(c) 2(d) 2(g)and 2(h) 119875 lt 005)

33 Effects of Canonical NF-120581B Pathway on the Proliferation ofSCAPs As shown in Figure 3(a) SCAPs in activator group

exhibited higher proliferation while the SCAPs-inhibitorgroup showed less proliferation capacity as compared withthe corresponding control groups respectively (119875 lt 005)except for the time points at baseline (day 0) and thefirst day Flow cytometry assay revealed that the activator-treated SCAPs exhibited a higher percentage of cells in Sand G

2M phases (2652) and a lower percentage of cells

in G0G1phase (7348) in comparison with untreated cells

BioMed Research International 5

30min 60min0min 15min

P-P65

P65

P-I120581B120572

I120581B120572

ACTIN

65kDa

65kDa

39kDa

40kDa

43kDa

30min 60min0min 15min

P-P65

P65

P-I120581B120572

I120581B120572

ACTIN

65kDa

65kDa

39kDa

40kDa

43kDa

14

12

1

08

06

04

02

0

lowastlowast lowast

lowast

lowastlowast

P-P65P65P-I120581B120572I120581B120572

Prot

eins

ACT

IN

0min15min

30min60min

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

06

05

04

03

02

01

0

lowastlowast

Prot

eins

ACT

INI120581B120572 P-I120581B120572 P65 P-P65

0min15min

30min60min

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

25

2

15

1

1

05

0

P-I120581

B120572I120581

B120572

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0min15min

30min60min

353

252

151

1

050

P-P6

5P65

0min15min

30min60min

lowastlowast

lowastlowast

lowastlowast

1614121

1

080604020

lowast

P-I120581

B120572I120581

B120572

lowastlowastlowastlowastlowastlowast

0min15min

30min60min

lowast4353

252

151

1

050

0min15min

30min60min

lowastlowastlowast

(a)

(b) (f)

(e)

(c) (g)

(d) (h)

P-P6

5P65

Figure 2 Activation and inhibition of canonical NF-120581B signaling pathway in SCAPs (a)The expression of NF-120581B pathway proteins in TNF-120572-treated SCAPs at different time points (b) Semiquantitative analysis confirmed the upregulation of P-I120581B120572 and P-P65 after the activation ofNF-120581B pathway (c)The ratio of phosphorylated to unphosphorylated form of P65 (d)The ratio of phosphorylated I120581B120572 to unphosphorylatedI120581B120572 (e) The protein levels of I120581B120572 P-I120581B120572 P65 and P-P65 in the cytoplasm of BMS-345541-treated SCAPs at indicated time points (f)Semiquantitative analysis confirmed the declined expression of P-I120581B120572 and P-P65 after the inhibition of NF-120581B pathway (g) The ratio ofphosphorylated to unphosphorylated form of P65 (h) The ratio of P-I120581B120572 to I120581B120572 Values are the means plusmn SD 119899 = 3 lowastlowast119875 lt 001 lowastlowastlowast119875 lt0001

6 BioMed Research International

lowast

lowast

lowast

lowast

lowast lowast

ControlActivator

MTT25

2

15

1

05

0

OD

val

ue (4

90

nm)

0 1 3 5 7 9 11

(day)

(a)

S30 60 90 120

SSSSSSSSSSSSSSSSSSSSSS0

300

900

1200

600

Num

ber

SCAPsChannels (FL2-A)

G2M

G0G1 = 8506

G2M = 986

G0G1

S

30 60 90 120

S

0

Num

ber

Channels (FL2-A)

G2M

G0G1 = 7348

G2M = 886

G0G1

1000

800

600

400

200

00

SCAPs + activator

S = 1766S = 508

(b)

Control

Activator

0h 6h 12h 24h

(c)

ControlInhibitor

25

2

15

1

05

0

OD

val

ue (4

90

nm) lowast

lowast

MTT

0 1 3 5 7 9 11

(day)

lowastlowast

lowastlowastlowast

lowastlowastlowast

(d)

SSSSSSSSSSSSSSSSSSSSSSS

SCAPsChannels (FL2-A)

G0G1

30 60 90 1200

G2M

1600

1200

800

400

0

Num

ber

G0G1 = 8217

G2M = 600

S = 1183

1600

1200

800

400

0

Num

ber

S

G0G1

G2M

Channels (FL2-A)30 60 900 120 150

G0G1 = 9109

G2M = 536

S = 354

SCAPs + inhibitor

(e)

0h 6h 12h 24h

Control

Inhibitor

(f)

Figure 3 Effects of canonical NF-120581B signaling pathway on the proliferation of SCAPs (a) Cell proliferation in NF-120581B pathway-activatedand control groups was detected by MTT assay (b) Flow cytometry analysis for SCAPs in NF-120581B pathway-activated and untreated groupsThe proliferation index (PI = S + G

2M) in NF-120581B pathway-activated group (2652) was significantly higher than that in control group

(1494) (c) Images of scratch wounds in NF-120581B pathway-activated and untreated groups at indicated time points (d) Growth curves of NF-120581B pathway-inhibited and untreated SCAPs (e) Representative cell cycle distributions of NF-120581B pathway-inhibited and untreated SCAPTheproliferation index (PI = S + G

2M) in NF-120581B pathway-activated group (890) was obviously lower than that in control group (1783)

(f) Cell motility of SCAPs in NF-120581B pathway-inhibited and untreated group assessed by a scratch assay Values were the means plusmn SD 119899 = 6lowast

119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 Scale bars = 100 120583m

(119875 lt 005 Figure 3(b))Therewas a lower percentage (890)of proliferating cells in SG

2Mphases in the inhibitor-treated

SCAPs as compared with the control group (1783) at day 3(Figure 3(e)) which is consistent with the findings in MTTassay (Figure 3(d)) These results indicate that canonical

NF-120581B pathway facilitated the proliferation of SCAPs thatis the cell proliferation rate increasing in pace with theactivation of canonical NF-120581B and is restrained along withthe blockage of NF-120581B Furthermore an improved migrationability was observed in activator-treated SCAPs (Figure 3(c))

BioMed Research International 7

while scratch closure was markedly slower in SCAPs treatedwith inhibitor (Figure 3(f))

34 Effects of Canonical NF-120581B Pathway on the OdontoOsteo-genic Differentiation of SCAPs ALP activities in activator-treated SCAPs in basic medium andmineralization-inducingmedium were elevated at day 5 and day 7 (119875 lt 005Figure 4(a)) as compared with untreated groupsThe densityof calcification nodules was significantly higher in activator-stimulated groups than in the other groups after 14 days ofcoculture (Figure 4(h)) Moreover quantitative calciummea-surement illustrated more calcifications in activator-treatedSCAPs in comparison with untreated groups (Figure 4(i))

Differentially expression levels of related osteoodonto-genic genes were also investigated by real-time RT-PCRassays In activator-treated group expression of specificosteoodontogenic genes (eg ALP OCN BSP OSXRUNX2 DSP OPN and DMP-1) was significantly upregu-lated at days 3 and 7 (Figures 4(b) and 4(c)) At day 3 theexpression of some osteoodontogenic genes in activator-treated groups did not change obviously in complete mediabut was distinctly increased in the presence ofmineralization-inducing media At day 7 the odontoosteo-genic markers were significantly upregulated following theactivator treatment regardless of the presence or absence ofmineralization-inducing media This phenomenon was notobserved in NF-120581B-inhibition group These findings wereconfirmed by western blot assay in which activated classicalNF-120581B signaling pathway induced a significant increase ofrelated protein expression (Figures 4(d)ndash4(g))

As shown in Figure 5(a) the blockage of NF-120581B signalingpathway obviously downregulated the ALP activity at days 35 and 7 (119875 lt 005) At day 14 less calcified nodules weregenerated in inhibitor-treated groups (Figure 5(h)) Calciumquantification also revealed the less calcium deposition ininhibitor and inhibitor + MM groups as compared withcontrol and MM groups respectively (Figure 5(i) 119875 lt 005)There was a remarkable decrease of osteoodontogenic genesat different time points (Figures 5(b) and 5(c)) Western blotanalysis further verified these findings (Figures 5(d)ndash5(g))

4 Discussion

SCAPs are known as a kind of ideal candidates for dentaltissue engineering and have the characteristics of self-renewaland multilineage differentiation potential [9 10] Diversestudies have proved that SCAPs are able to differentiate intoosteoodontoblasts in vitro under appropriate conditions andformbonedentin-like tissues in vivo [24 25] Certainlymanysignaling pathways may be involved in the process of cellproliferation and differentiation including NF-120581B pathway

NF-120581B exists in the cytoplasm in a latent form bindingto inhibitory proteins termed inhibitory 120581B proteins (I120581Bs)[3 26] I120581B complex is the major regulator of NF-120581B pathwayand is composed of catalytic subunits IKK120572 and IKK120573 anda regulatory subunit IKK120574 TNF-120572 is generally known toactivate classical NF-120581B pathway [17] Exposure of cells tosuitable concentrations of TNF-120572 brings about the rapid

phosphorylation ubiquitination and proteolytic degradationof I120581B which allows NF-120581B to translocate to the nucleuswhere it subsequently regulates the gene transcription [27ndash29] BMS-345541 a selective inhibitor of the catalytic subunitsof IKK targets NF-120581B signaling pathway and downregulatesthe activity of NF-120581B [18 19] In this study the expressionof cytoplastic P-P65P-I120581B120572 was noticeably upregulated afterthe treatment of TNF-120572 and downregulated by the inhibitorBMS-345541 indicating the successful establishment of acellular model for the activation or suppression of canonicalNF-120581B pathway

Cell proliferation and migration were indispensable fortissue development and wound healing In the present studyNF-120581B-activated SCAPs exhibited an increased growth ratehigher proliferation index and more effective migrationin wound healing assay Moreover the migration distancesbetween thewound lines in inhibitor-treated cells were longerthan control cells after 24 hours of culture Together withthe weakened proliferation of inhibitor-treated cells it ispredictable that the canonical NF-120581B pathway has a positiveinfluence on the cell multiplication activity and motility

Previous studies have proved that various stimuli canexert an active role on the committed differentiation ofdental stem cells via NF-120581B pathway [28] In the presentstudy the odontoosteogenic capacity remarkably changedalong with the activation or blockage of classical NF-120581Bpathway Activator-treated SCAPs exhibited an enhancedALP activity increased calcium deposition and upregulatedexpression of odontoosteogenic genes and proteins whileinhibitor-treated cells presented the lower ALP activitydecreased mineralization and downregulated expression ofodontoosteoblast markers

It is widely recognized that ALP is a functionalmarker forosteoblast activity and bone formation [30] As an importantextracellular matrix of bone BSP is mainly secreted byosteoblasts whose expression is a crucial symbol in matrixdeposition and mineralization [31 32] OPN is anotherextracellular matrix protein expressed in numerous cell typesincluding odontoblasts in which OPN plays multifacetedroles in a variety of biological and pathological processessuch as osteogenic differentiation tooth mineralization anddental biofilm formation [33 34] OCN is the primarynoncollagenous protein in the bone cells secreted in the latestage of osteogenesis and known as a specific indicator ofosteogenic differentiation RUNX2 is the first transcriptionfactor required for determination of the osteoblast lineageand OSX acts as a downstream gene of RUNX2 that is highlyexpressed in the functional odontoosteoblasts [35] DSPPand DSP are well-known markers of odontoblasts highlyexpressed in dentin or predentin structures and essentialfor dentinogenesis [36] DMP-1 is an acidic extracellularmatrix protein that is primarily found in dentin and boneand has been implicated in dentin mineralization and signaltransduction in the process of odontogenesis [37]

In this study the expression of RUNX2RUNX2 reducedafter 3 days of incubation with the inhibitor BMS-345541and then gradually increased at day 7 while the expressionof DSPPDSP did not compromise after 3 days of incuba-tion but noticeably decreased at day 7 indicating that the

8 BioMed Research International

ALP

3 5 7

(day)

35

3

25

2

15

1

05

0

ControlActivator

MMActivator + MM

lowast

lowastlowastlowastlowastlowastlowast

(sig

ma u

nitm

inm

g pr

otei

n)

(a)

ControlActivator

MMActivator + MM

lowastlowast

lowastlowast

876543210

ALP

OCN BS

P

RUN

X2

OSX

DSP

P

OPN

DM

P-1

Gen

esG

APD

H

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastPCR 3d

(b)

lowast

lowast lowast

lowast

lowast lowastlowast

lowast

25

20

15

10

5

0

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

Gen

esG

APD

H

ControlActivator

MMActivator + MM

ALP

OCN BS

P

RUN

X2

OSX

DSP

P

OPN

DM

P-1

PCR 7d

(c)

MMControl Activator

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

Activator + MM

Western blot 3d

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

(d)

ControlActivator

MMActivator + MM

09

08

07

06

05

04

03

02

01

0

Prot

eins

ACT

IN

OCN BSP RUNX2 OSX DSP OPN DMP-1

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

lowast

lowast

lowast

(e)

MMControl Activator

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

Activator + MM

Western blot 7d

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

(f)

Figure 4 Continued

BioMed Research International 9

14

12

1

08

06

04

02

0

Prot

eins

ACT

IN

OCN BSP RUNX2 OSX DSP OPN DMP-1

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

ControlActivator

MMActivator + MM

(g)

Control Activator

MM Activator + MM

(h)

1

CPC06

05

04

03

02

01

0

lowast

lowastlowastlowast

ControlActivator

MMActivator + MM

(ng

calc

ium

mg

prot

ein)

(i)

Figure 4 Odontoosteogenic differentiation in canonical NF-120581B-activated SCAPs (a) ALP activity of SCAPs in control activator MM andactivator + MM groups at days 3 5 and 7 Values are the means plusmn SD 119899 = 6 lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 (b) Real-time RT-PCRanalysis for odontoosteogenic genes (BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in each group at day 7 (c) Real-time RT-PCRanalysis for odontoosteogenic genes (ALP BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in different groups at day 7 GAPDHserved as a housekeeping gene lowastlowast2minusΔΔCt ge 2 119875 lt 001 lowast1 lt 2minusΔΔCt lt 2 119875 lt 001 119899 = 3 (d) Western blot analyses for the odontoosteogenicproteins (BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in different groups at day 3 120573-ACTIN served as an internal control (e)Semiquantitative analysis demonstrated that the expression of BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1 was stronger in NF-120581Bpathway-activated SCAPs than those in control group at day 3 especially in the presence of mineralization-inducing media (f) Western blotanalyses for the odontoosteogenic proteins (BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in different groups at day 7 120573-ACTINwas used as an internal control (g) Semiquantitative analysis confirmed that the expression of BSP OCN RUNX2 OSX DSP OPN DSPand DMP-1 was upregulated in NF-120581B pathway-activated SCAPs at day 7 regardless of the presence or absence of mineralization-inducingmedia (h) Alizarin red staining of SCAPs after 14 days of induction in different groups (i) Quantitative calcium analysis showed that thecalcium content in activator and activator + MM groups was significantly higher than the control and MM groups respectively Values weredescribed as the means plusmn SD lowast119875 lt 005 lowastlowastlowast119875 lt 0001

expression of RUNX2RUNX2 and DSPPDSP tends to beinversely correlated [38] Moreover the dramatic differenceof these odontoosteogenic markers at both mRNA andprotein levels in response to the regulation of NF-120581B pathwaysuggests that activated canonical NF-120581B pathway favors theodontoosteoblastic differentiation of SCAPs particularly inthe presence of mineralization-inducing media

In summary the findings accumulated here confirmedthe potential involvement of canonical NF-120581B pathway inthe proliferationmigration and committed differentiation ofSCAPs in vitro Both activation and inhibition of the classicalNF-120581B pathway can bring about the permanent changes in

human stem cells Moreover it is commonly believed thatNF-120581B signaling plays a pivotal role not only in the progressof normal physiological process but also in the pathologicalprocess and dysfunction ofNF-120581B is linked to various humandiseases Thus proper balance of intracellular NF-120581B shouldbemaintained in the physiological conditions while intricateinteractivity between NF-120581B and other signaling pathwaysneeds to be extensively investigated

Conflict of Interests

The authors declare no conflict of interests

10 BioMed Research International

ControlInhibitor

MM

ALP3

25

2

15

1

05

03 5 7

(day)

Inhibitor + MM

lowastlowast lowastlowast

lowastlowast

lowast

lowast

(sig

ma u

nitm

inm

g pr

otei

n)

(a)

ControlInhibitor

35

3

25

2

15

1

05

0

Gen

esG

APD

H

ALP OCN BSP RUNX2 OSX DSPP OPN DMP-1

MMInhibitor + MM

lowastlowast

lowast

lowast

lowast lowastlowast

lowast

lowastlowast

lowastlowast

PCR 3d

(b)

ControlInhibitor

MM

1614121086420

Gen

esG

APD

H

ALP OCN BSP RUNX2 OSX DSPP OPN DMP-1

Inhibitor + MM

lowastlowast lowastlowast

lowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowastlowastlowastPCR 7d

(c)

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

MMInhibitorControl Inhibitor + MM

Western blot 3d

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

(d)

Prot

eins

ACT

IN

ControlInhibitor

MM

12

1

08

06

04

02

0OCN BSP RUNX2 OSX DSP OPN DMP-1

Inhibitor + MM

lowast

lowast lowast

lowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

(e)

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

MMInhibitorControl Inhibitor + MM

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

Western blot 7d

(f)

Figure 5 Continued

BioMed Research International 11

Prot

eins

ACT

IN

OCN BSP RUNX2 OSX DSP OPN DMP-1

ControlInhibitor MM

Inhibitor + MM

0504504

035030250201501005

0

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(g)

MM

InhibitorControl

Inhibitor + MM

(h)

06

05

04

03

02

01

0

lowast

lowastlowastCPC

1

MMInhibitorControl

Inhibitor + MM

(ng

calc

ium

mg

prot

ein)

(i)

Figure 5 Odontoosteogenic differentiation in canonical NF-120581B-inhibited SCAPs (a) ALP activity at different time points in different groups(ie control inhibitor MM and inhibitor +MM) (b) Real-time RT-PCR for the detection ofALP BSP OCN RUNX2 OSX DSP OPN DSPandDMP-1 at day 3ThemRNA levels were normalized toGAPDH (c) Real-time RT-PCR for the detection ofALP BSP OCN RUNX2 OSXDSP OPN DSP andDMP-1 of SCAPs respectively in control inhibitor MM and inhibitor +MMgroups at day 7 (d)The odontoosteogenicprotein levels of SCAPs in control inhibitor MM and inhibitor +MM groups were characterized by western blot analysis at day 3 120573-ACTINwas used as a loading control (e) Semiquantitative analysis demonstrated that the expression of BSP OCN RUNX2 OSX DSP OPN DSPand DMP-1 was more downregulated in NF-120581B pathway-inhibited SCAPs than those in control group at day 3 (f) The odontoosteogenicprotein levels of SCAPs in control inhibitor MM and inhibitor + MM groups were characterized by western blot analysis at day 7 (g)Semiquantitative analysis demonstrated that the expression of odontoosteogenic markers (BSP OCN RUNX2 OSX DSP OPN DSP andDMP-1) was significantly more downregulated in NF-120581B pathway-inhibited SCAPs than those in control group at day 7 (h) Alizarin redstaining of SCAPs after 14 days of osteogenic induction (i) Calcium quantification demonstrated the weaker calcium deposition in inhibitorand inhibitor + MM groups as compared with control and MM groups respectively

Acknowledgments

This work was supported by National Natural Science Foun-dation of China (no 81371144) Natural Science Foundationof Jiangsu Province (no BK20131392) and the Priority Aca-demic Program Development of Jiangsu Higher EducationInstitutions (PAPD no 2011-137)

References

[1] AKumar Y TakadaAMBoriek andB BAggarwal ldquoNuclearfactor-120581B its role in health and diseaserdquo Journal of MolecularMedicine vol 82 no 7 pp 434ndash448 2004

[2] F Chen V Castranova X Shi and LM Demers ldquoNew insightsinto the role of nuclear factor-120581B a ubiquitous transcription

factor in the initiation of diseasesrdquo Clinical Chemistry vol 45no 1 pp 7ndash17 1999

[3] C Gasparini and M Feldmann ldquoNF-120581B as a target for modu-lating inflammatory responsesrdquoCurrent Pharmaceutical Designvol 18 no 35 pp 5735ndash5745 2012

[4] A Ohazama and P T Sharpe ldquoTNF signalling in toothdevelopmentrdquo Current Opinion in Genetics amp Development vol14 no 5 pp 513ndash519 2004

[5] Y-H Kuan F-M Huang Y-C Li and Y-C Chang ldquoProin-flammatory activation ofmacrophages by bisphenol A-glycidyl-methacrylate involved NF120581B activation via PI3KAkt pathwayrdquoFood and Chemical Toxicology vol 50 no 11 pp 4003ndash40092012

[6] X Cai P Gong YHuang andY Lin ldquoNotch signalling pathwayin tooth development and adult dental cellsrdquo Cell Proliferationvol 44 no 6 pp 495ndash507 2011

12 BioMed Research International

[7] J-M Courtney J Blackburn and P T Sharpe ldquoThe Ectodys-plasin and NF120581B signalling pathways in odontogenesisrdquoArchives of Oral Biology vol 50 no 2 pp 159ndash163 2005

[8] W Sonoyama Y Liu T Yamaza et al ldquoCharacterization of theapical papilla and its residing stem cells from human immaturepermanent teeth a pilot studyrdquo Journal of Endodontics vol 34no 2 pp 166ndash171 2008

[9] W Sonoyama Y Liu D Fang et al ldquoMesenchymal stem cell-mediated functional tooth regeneration in swinerdquo PLoS ONEvol 1 no 1 article e79 2006

[10] H Chopra M K Hans and S Shetty ldquoStem cells-the hiddentreasure a strategic reviewrdquoDental Research Journal vol 10 no4 pp 421ndash427 2013

[11] R Dong R Yao J Du S Wang and Z Fan ldquoDepletion ofhistone demethylase KDM2A enhanced the adipogenic andchondrogenic differentiation potentials of stem cells from apicalpapillardquo Experimental Cell Research vol 319 no 18 pp 2874ndash2882 2013

[12] Y Wang M Yan Z Wang et al ldquoDental pulp stem cells fromtraumatically exposed pulps exhibited an enhanced osteogenicpotential and weakened odontogenic capacityrdquo Archives of OralBiology vol 58 no 11 pp 1709ndash1717 2013

[13] Y Wang M Yan Z Fan L Ma Y Yu and J Yu ldquoMineraltrioxide aggregate enhances the odontoosteogenic capacity ofstem cells from inflammatory dental pulps via NF-120581B pathwayrdquoOral Diseases 2013

[14] Y Wang M Yan Y Yu J Wu J Yu and Z Fan ldquoEstrogendeficiency inhibits the odontoosteogenic differentiation ofdental pulp stem cells via activation of the NF-120581B pathwayrdquo Celland Tissue Research vol 352 no 3 pp 551ndash559 2013

[15] Y Wang Y Zheng Z Wang et al ldquo10minus7M 17120573-oestradiolenhances odontoosteogenic potency of human dental pulpstem cells by activation of the NF-120581B pathwayrdquo Cell Prolifera-tion vol 46 no 6 pp 677ndash684 2013

[16] Z-G Lu H Liu T Yamaguchi Y Miki and K YoshidaldquoProtein kinase C120575 activates RelAp65 and nuclear factor-120581B signaling in response to tumor necrosis factor-120572rdquo CancerResearch vol 69 no 14 pp 5927ndash5935 2009

[17] G Bonizzi and M Karin ldquoThe two NF-120581B activation pathwaysand their role in innate and adaptive immunityrdquo Trends inImmunology vol 25 no 6 pp 280ndash288 2004

[18] K W McIntyre D J Shuster K M Gillooly et al ldquoA highlyselective inhibitor of I120581B kinase BMS-345541 blocks both jointinflammation and destruction in collagen-induced arthritis inmicerdquo Arthritis amp Rheumatism vol 48 no 9 pp 2652ndash26592003

[19] J R Burke M A Pattoli K R Gregor et al ldquoBMS-345541 is ahighly selective inhibitor of I120581B kinase that binds at an allostericsite of the enzyme and blocks NF-120581B-dependent transcriptionin micerdquoThe Journal of Biological Chemistry vol 278 no 3 pp1450ndash1456 2003

[20] F W Paula-Silva A Ghosh L A Silva and Y L Kapila ldquoTNF-120572 promotes an odontoblastic phenotype in dental pulp cellsrdquoJournal of Dental Research vol 88 no 4 pp 339ndash344 2009

[21] S Wang J Mu Z Fan et al ldquoInsulin-like growth factor 1 canpromote the osteogenic differentiation and osteogenesis of stemcells from apical papillardquo Stem Cell Research vol 8 no 3 pp346ndash356 2012

[22] Y Yu J Mu Z Fan et al ldquoInsulin-like growth factor 1 enhancesthe proliferation and osteogenic differentiation of human peri-odontal ligament stem cells via ERK and JNKMAPKpathwaysrdquoHistochemistry andCell Biology vol 137 no 4 pp 513ndash525 2012

[23] H Aonuma N Ogura K Takahashi et al ldquoCharacteristics andosteogenic differentiation of stemprogenitor cells in the humandental follicle analyzed by gene expression profilingrdquo Cell andTissue Research vol 350 no 2 pp 317ndash331 2012

[24] L Wang M Yan Y Wang et al ldquoProliferation andosteoodontoblastic differentiation of stem cells from dentalapical papilla in mineralization-inducing medium containingadditional KH

2PO4rdquo Cell Proliferation vol 46 no 2 pp

214ndash222 2013[25] G T-J Huang T Yamaza L D Shea et al ldquoStemProgenitor

cell-mediated de novo regeneration of dental pulp with newlydeposited continuous layer of dentin in an in vivomodelrdquoTissueEngineering A vol 16 no 2 pp 605ndash615 2010

[26] J-H Kwak J-K Jung and H Lee ldquoNuclear factor-120581 Binhibitors a patent review (2006ndash2010)rdquo Expert Opinion onTherapeutic Patents vol 21 no 12 pp 1897ndash1910 2011

[27] K Hess A Ushmorov J Fiedler R E Brenner and T WirthldquoTNF120572 promotes osteogenic differentiation of human mes-enchymal stem cells by triggering theNF-120581B signaling pathwayrdquoBone vol 45 no 2 pp 367ndash376 2009

[28] X Feng G Feng J Xing et al ldquoTNF-120572 triggers osteogenicdifferentiation of human dental pulp stem cells via the NF-120581Bsignalling pathwayrdquoCell Biology International vol 37 no 12 pp1267ndash1275 2013

[29] J Chang F Liu M Lee et al ldquoNF-120581B inhibits osteogenicdifferentiation of mesenchymal stem cells by promoting 120573-catenin degradationrdquo Proceedings of the National Academy ofSciences of the United States of America vol 110 no 23 pp9469ndash9474 2013

[30] C Shui and A Scutt ldquoMild heat shock induces proliferationalkaline phosphatase activity and mineralization in humanbone marrow stromal cells and Mg-63 cells in vitrordquo Journal ofBone and Mineral Research vol 16 no 4 pp 731ndash741 2001

[31] X Wei J Ling L Wu L Liu and Y Xiao ldquoExpressionof mineralization markers in dental pulp cellsrdquo Journal ofEndodontics vol 33 no 6 pp 703ndash708 2007

[32] S Z Khan E Kokubu K Matsuzaka and T Inoue ldquoBehaviourof rat-cultured dental pulp cells in three-dimensional collagentype-1 gel in vitro and in vivordquo Australian Endodontic Journalvol 39 no 3 pp 137ndash145 2013

[33] D-X Cao Z-J Li X-O Jiang et al ldquoOsteopontin as potentialbiomarker and therapeutic target in gastric and liver cancersrdquoWorld Journal of Gastroenterology vol 18 no 30 pp 3923ndash39302012

[34] S SchlaferMK Raarup P LWejse et al ldquoOsteopontin reducesbiofilm formation in a multi-species model of dental biofilmrdquoPloS ONE vol 7 no 8 Article ID e41534 2012

[35] S Chen J Gluhak-Heinrich Y HWang et al ldquoRunx2Osx andDspp in tooth developmentrdquo Journal of Dental Research vol 88no 10 pp 904ndash909 2009

[36] S Suzuki N Haruyama F Nishimura and A B KulkarnildquoDentin sialophosphoprotein and dentin matrix protein-1 twohighly phosphorylated proteins inmineralized tissuesrdquoArchivesof Oral Biology vol 57 no 9 pp 1165ndash1175 2012

[37] R S Prescott R Alsanea M I Fayad et al ldquoIn vivo fenerationof dental pulp-like tissue by using dental pulp stem cells a col-lagen scaffold and dentin matrix protein 1 after subcutaneoustransplantation in micerdquo Journal of Endodontics vol 34 no 4pp 421ndash426 2008

[38] T Komori ldquoRegulation of bone development and extracellularmatrix protein genes by RUNX2rdquo Cell and Tissue Research vol339 no 1 pp 189ndash195 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Signal TransductionJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

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Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

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Enzyme Research

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International Journal of

Microbiology

BioMed Research International 5

30min 60min0min 15min

P-P65

P65

P-I120581B120572

I120581B120572

ACTIN

65kDa

65kDa

39kDa

40kDa

43kDa

30min 60min0min 15min

P-P65

P65

P-I120581B120572

I120581B120572

ACTIN

65kDa

65kDa

39kDa

40kDa

43kDa

14

12

1

08

06

04

02

0

lowastlowast lowast

lowast

lowastlowast

P-P65P65P-I120581B120572I120581B120572

Prot

eins

ACT

IN

0min15min

30min60min

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

06

05

04

03

02

01

0

lowastlowast

Prot

eins

ACT

INI120581B120572 P-I120581B120572 P65 P-P65

0min15min

30min60min

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

25

2

15

1

1

05

0

P-I120581

B120572I120581

B120572

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0min15min

30min60min

353

252

151

1

050

P-P6

5P65

0min15min

30min60min

lowastlowast

lowastlowast

lowastlowast

1614121

1

080604020

lowast

P-I120581

B120572I120581

B120572

lowastlowastlowastlowastlowastlowast

0min15min

30min60min

lowast4353

252

151

1

050

0min15min

30min60min

lowastlowastlowast

(a)

(b) (f)

(e)

(c) (g)

(d) (h)

P-P6

5P65

Figure 2 Activation and inhibition of canonical NF-120581B signaling pathway in SCAPs (a)The expression of NF-120581B pathway proteins in TNF-120572-treated SCAPs at different time points (b) Semiquantitative analysis confirmed the upregulation of P-I120581B120572 and P-P65 after the activation ofNF-120581B pathway (c)The ratio of phosphorylated to unphosphorylated form of P65 (d)The ratio of phosphorylated I120581B120572 to unphosphorylatedI120581B120572 (e) The protein levels of I120581B120572 P-I120581B120572 P65 and P-P65 in the cytoplasm of BMS-345541-treated SCAPs at indicated time points (f)Semiquantitative analysis confirmed the declined expression of P-I120581B120572 and P-P65 after the inhibition of NF-120581B pathway (g) The ratio ofphosphorylated to unphosphorylated form of P65 (h) The ratio of P-I120581B120572 to I120581B120572 Values are the means plusmn SD 119899 = 3 lowastlowast119875 lt 001 lowastlowastlowast119875 lt0001

6 BioMed Research International

lowast

lowast

lowast

lowast

lowast lowast

ControlActivator

MTT25

2

15

1

05

0

OD

val

ue (4

90

nm)

0 1 3 5 7 9 11

(day)

(a)

S30 60 90 120

SSSSSSSSSSSSSSSSSSSSSS0

300

900

1200

600

Num

ber

SCAPsChannels (FL2-A)

G2M

G0G1 = 8506

G2M = 986

G0G1

S

30 60 90 120

S

0

Num

ber

Channels (FL2-A)

G2M

G0G1 = 7348

G2M = 886

G0G1

1000

800

600

400

200

00

SCAPs + activator

S = 1766S = 508

(b)

Control

Activator

0h 6h 12h 24h

(c)

ControlInhibitor

25

2

15

1

05

0

OD

val

ue (4

90

nm) lowast

lowast

MTT

0 1 3 5 7 9 11

(day)

lowastlowast

lowastlowastlowast

lowastlowastlowast

(d)

SSSSSSSSSSSSSSSSSSSSSSS

SCAPsChannels (FL2-A)

G0G1

30 60 90 1200

G2M

1600

1200

800

400

0

Num

ber

G0G1 = 8217

G2M = 600

S = 1183

1600

1200

800

400

0

Num

ber

S

G0G1

G2M

Channels (FL2-A)30 60 900 120 150

G0G1 = 9109

G2M = 536

S = 354

SCAPs + inhibitor

(e)

0h 6h 12h 24h

Control

Inhibitor

(f)

Figure 3 Effects of canonical NF-120581B signaling pathway on the proliferation of SCAPs (a) Cell proliferation in NF-120581B pathway-activatedand control groups was detected by MTT assay (b) Flow cytometry analysis for SCAPs in NF-120581B pathway-activated and untreated groupsThe proliferation index (PI = S + G

2M) in NF-120581B pathway-activated group (2652) was significantly higher than that in control group

(1494) (c) Images of scratch wounds in NF-120581B pathway-activated and untreated groups at indicated time points (d) Growth curves of NF-120581B pathway-inhibited and untreated SCAPs (e) Representative cell cycle distributions of NF-120581B pathway-inhibited and untreated SCAPTheproliferation index (PI = S + G

2M) in NF-120581B pathway-activated group (890) was obviously lower than that in control group (1783)

(f) Cell motility of SCAPs in NF-120581B pathway-inhibited and untreated group assessed by a scratch assay Values were the means plusmn SD 119899 = 6lowast

119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 Scale bars = 100 120583m

(119875 lt 005 Figure 3(b))Therewas a lower percentage (890)of proliferating cells in SG

2Mphases in the inhibitor-treated

SCAPs as compared with the control group (1783) at day 3(Figure 3(e)) which is consistent with the findings in MTTassay (Figure 3(d)) These results indicate that canonical

NF-120581B pathway facilitated the proliferation of SCAPs thatis the cell proliferation rate increasing in pace with theactivation of canonical NF-120581B and is restrained along withthe blockage of NF-120581B Furthermore an improved migrationability was observed in activator-treated SCAPs (Figure 3(c))

BioMed Research International 7

while scratch closure was markedly slower in SCAPs treatedwith inhibitor (Figure 3(f))

34 Effects of Canonical NF-120581B Pathway on the OdontoOsteo-genic Differentiation of SCAPs ALP activities in activator-treated SCAPs in basic medium andmineralization-inducingmedium were elevated at day 5 and day 7 (119875 lt 005Figure 4(a)) as compared with untreated groupsThe densityof calcification nodules was significantly higher in activator-stimulated groups than in the other groups after 14 days ofcoculture (Figure 4(h)) Moreover quantitative calciummea-surement illustrated more calcifications in activator-treatedSCAPs in comparison with untreated groups (Figure 4(i))

Differentially expression levels of related osteoodonto-genic genes were also investigated by real-time RT-PCRassays In activator-treated group expression of specificosteoodontogenic genes (eg ALP OCN BSP OSXRUNX2 DSP OPN and DMP-1) was significantly upregu-lated at days 3 and 7 (Figures 4(b) and 4(c)) At day 3 theexpression of some osteoodontogenic genes in activator-treated groups did not change obviously in complete mediabut was distinctly increased in the presence ofmineralization-inducing media At day 7 the odontoosteo-genic markers were significantly upregulated following theactivator treatment regardless of the presence or absence ofmineralization-inducing media This phenomenon was notobserved in NF-120581B-inhibition group These findings wereconfirmed by western blot assay in which activated classicalNF-120581B signaling pathway induced a significant increase ofrelated protein expression (Figures 4(d)ndash4(g))

As shown in Figure 5(a) the blockage of NF-120581B signalingpathway obviously downregulated the ALP activity at days 35 and 7 (119875 lt 005) At day 14 less calcified nodules weregenerated in inhibitor-treated groups (Figure 5(h)) Calciumquantification also revealed the less calcium deposition ininhibitor and inhibitor + MM groups as compared withcontrol and MM groups respectively (Figure 5(i) 119875 lt 005)There was a remarkable decrease of osteoodontogenic genesat different time points (Figures 5(b) and 5(c)) Western blotanalysis further verified these findings (Figures 5(d)ndash5(g))

4 Discussion

SCAPs are known as a kind of ideal candidates for dentaltissue engineering and have the characteristics of self-renewaland multilineage differentiation potential [9 10] Diversestudies have proved that SCAPs are able to differentiate intoosteoodontoblasts in vitro under appropriate conditions andformbonedentin-like tissues in vivo [24 25] Certainlymanysignaling pathways may be involved in the process of cellproliferation and differentiation including NF-120581B pathway

NF-120581B exists in the cytoplasm in a latent form bindingto inhibitory proteins termed inhibitory 120581B proteins (I120581Bs)[3 26] I120581B complex is the major regulator of NF-120581B pathwayand is composed of catalytic subunits IKK120572 and IKK120573 anda regulatory subunit IKK120574 TNF-120572 is generally known toactivate classical NF-120581B pathway [17] Exposure of cells tosuitable concentrations of TNF-120572 brings about the rapid

phosphorylation ubiquitination and proteolytic degradationof I120581B which allows NF-120581B to translocate to the nucleuswhere it subsequently regulates the gene transcription [27ndash29] BMS-345541 a selective inhibitor of the catalytic subunitsof IKK targets NF-120581B signaling pathway and downregulatesthe activity of NF-120581B [18 19] In this study the expressionof cytoplastic P-P65P-I120581B120572 was noticeably upregulated afterthe treatment of TNF-120572 and downregulated by the inhibitorBMS-345541 indicating the successful establishment of acellular model for the activation or suppression of canonicalNF-120581B pathway

Cell proliferation and migration were indispensable fortissue development and wound healing In the present studyNF-120581B-activated SCAPs exhibited an increased growth ratehigher proliferation index and more effective migrationin wound healing assay Moreover the migration distancesbetween thewound lines in inhibitor-treated cells were longerthan control cells after 24 hours of culture Together withthe weakened proliferation of inhibitor-treated cells it ispredictable that the canonical NF-120581B pathway has a positiveinfluence on the cell multiplication activity and motility

Previous studies have proved that various stimuli canexert an active role on the committed differentiation ofdental stem cells via NF-120581B pathway [28] In the presentstudy the odontoosteogenic capacity remarkably changedalong with the activation or blockage of classical NF-120581Bpathway Activator-treated SCAPs exhibited an enhancedALP activity increased calcium deposition and upregulatedexpression of odontoosteogenic genes and proteins whileinhibitor-treated cells presented the lower ALP activitydecreased mineralization and downregulated expression ofodontoosteoblast markers

It is widely recognized that ALP is a functionalmarker forosteoblast activity and bone formation [30] As an importantextracellular matrix of bone BSP is mainly secreted byosteoblasts whose expression is a crucial symbol in matrixdeposition and mineralization [31 32] OPN is anotherextracellular matrix protein expressed in numerous cell typesincluding odontoblasts in which OPN plays multifacetedroles in a variety of biological and pathological processessuch as osteogenic differentiation tooth mineralization anddental biofilm formation [33 34] OCN is the primarynoncollagenous protein in the bone cells secreted in the latestage of osteogenesis and known as a specific indicator ofosteogenic differentiation RUNX2 is the first transcriptionfactor required for determination of the osteoblast lineageand OSX acts as a downstream gene of RUNX2 that is highlyexpressed in the functional odontoosteoblasts [35] DSPPand DSP are well-known markers of odontoblasts highlyexpressed in dentin or predentin structures and essentialfor dentinogenesis [36] DMP-1 is an acidic extracellularmatrix protein that is primarily found in dentin and boneand has been implicated in dentin mineralization and signaltransduction in the process of odontogenesis [37]

In this study the expression of RUNX2RUNX2 reducedafter 3 days of incubation with the inhibitor BMS-345541and then gradually increased at day 7 while the expressionof DSPPDSP did not compromise after 3 days of incuba-tion but noticeably decreased at day 7 indicating that the

8 BioMed Research International

ALP

3 5 7

(day)

35

3

25

2

15

1

05

0

ControlActivator

MMActivator + MM

lowast

lowastlowastlowastlowastlowastlowast

(sig

ma u

nitm

inm

g pr

otei

n)

(a)

ControlActivator

MMActivator + MM

lowastlowast

lowastlowast

876543210

ALP

OCN BS

P

RUN

X2

OSX

DSP

P

OPN

DM

P-1

Gen

esG

APD

H

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastPCR 3d

(b)

lowast

lowast lowast

lowast

lowast lowastlowast

lowast

25

20

15

10

5

0

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

Gen

esG

APD

H

ControlActivator

MMActivator + MM

ALP

OCN BS

P

RUN

X2

OSX

DSP

P

OPN

DM

P-1

PCR 7d

(c)

MMControl Activator

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

Activator + MM

Western blot 3d

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

(d)

ControlActivator

MMActivator + MM

09

08

07

06

05

04

03

02

01

0

Prot

eins

ACT

IN

OCN BSP RUNX2 OSX DSP OPN DMP-1

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

lowast

lowast

lowast

(e)

MMControl Activator

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

Activator + MM

Western blot 7d

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

(f)

Figure 4 Continued

BioMed Research International 9

14

12

1

08

06

04

02

0

Prot

eins

ACT

IN

OCN BSP RUNX2 OSX DSP OPN DMP-1

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

ControlActivator

MMActivator + MM

(g)

Control Activator

MM Activator + MM

(h)

1

CPC06

05

04

03

02

01

0

lowast

lowastlowastlowast

ControlActivator

MMActivator + MM

(ng

calc

ium

mg

prot

ein)

(i)

Figure 4 Odontoosteogenic differentiation in canonical NF-120581B-activated SCAPs (a) ALP activity of SCAPs in control activator MM andactivator + MM groups at days 3 5 and 7 Values are the means plusmn SD 119899 = 6 lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 (b) Real-time RT-PCRanalysis for odontoosteogenic genes (BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in each group at day 7 (c) Real-time RT-PCRanalysis for odontoosteogenic genes (ALP BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in different groups at day 7 GAPDHserved as a housekeeping gene lowastlowast2minusΔΔCt ge 2 119875 lt 001 lowast1 lt 2minusΔΔCt lt 2 119875 lt 001 119899 = 3 (d) Western blot analyses for the odontoosteogenicproteins (BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in different groups at day 3 120573-ACTIN served as an internal control (e)Semiquantitative analysis demonstrated that the expression of BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1 was stronger in NF-120581Bpathway-activated SCAPs than those in control group at day 3 especially in the presence of mineralization-inducing media (f) Western blotanalyses for the odontoosteogenic proteins (BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in different groups at day 7 120573-ACTINwas used as an internal control (g) Semiquantitative analysis confirmed that the expression of BSP OCN RUNX2 OSX DSP OPN DSPand DMP-1 was upregulated in NF-120581B pathway-activated SCAPs at day 7 regardless of the presence or absence of mineralization-inducingmedia (h) Alizarin red staining of SCAPs after 14 days of induction in different groups (i) Quantitative calcium analysis showed that thecalcium content in activator and activator + MM groups was significantly higher than the control and MM groups respectively Values weredescribed as the means plusmn SD lowast119875 lt 005 lowastlowastlowast119875 lt 0001

expression of RUNX2RUNX2 and DSPPDSP tends to beinversely correlated [38] Moreover the dramatic differenceof these odontoosteogenic markers at both mRNA andprotein levels in response to the regulation of NF-120581B pathwaysuggests that activated canonical NF-120581B pathway favors theodontoosteoblastic differentiation of SCAPs particularly inthe presence of mineralization-inducing media

In summary the findings accumulated here confirmedthe potential involvement of canonical NF-120581B pathway inthe proliferationmigration and committed differentiation ofSCAPs in vitro Both activation and inhibition of the classicalNF-120581B pathway can bring about the permanent changes in

human stem cells Moreover it is commonly believed thatNF-120581B signaling plays a pivotal role not only in the progressof normal physiological process but also in the pathologicalprocess and dysfunction ofNF-120581B is linked to various humandiseases Thus proper balance of intracellular NF-120581B shouldbemaintained in the physiological conditions while intricateinteractivity between NF-120581B and other signaling pathwaysneeds to be extensively investigated

Conflict of Interests

The authors declare no conflict of interests

10 BioMed Research International

ControlInhibitor

MM

ALP3

25

2

15

1

05

03 5 7

(day)

Inhibitor + MM

lowastlowast lowastlowast

lowastlowast

lowast

lowast

(sig

ma u

nitm

inm

g pr

otei

n)

(a)

ControlInhibitor

35

3

25

2

15

1

05

0

Gen

esG

APD

H

ALP OCN BSP RUNX2 OSX DSPP OPN DMP-1

MMInhibitor + MM

lowastlowast

lowast

lowast

lowast lowastlowast

lowast

lowastlowast

lowastlowast

PCR 3d

(b)

ControlInhibitor

MM

1614121086420

Gen

esG

APD

H

ALP OCN BSP RUNX2 OSX DSPP OPN DMP-1

Inhibitor + MM

lowastlowast lowastlowast

lowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowastlowastlowastPCR 7d

(c)

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

MMInhibitorControl Inhibitor + MM

Western blot 3d

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

(d)

Prot

eins

ACT

IN

ControlInhibitor

MM

12

1

08

06

04

02

0OCN BSP RUNX2 OSX DSP OPN DMP-1

Inhibitor + MM

lowast

lowast lowast

lowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

(e)

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

MMInhibitorControl Inhibitor + MM

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

Western blot 7d

(f)

Figure 5 Continued

BioMed Research International 11

Prot

eins

ACT

IN

OCN BSP RUNX2 OSX DSP OPN DMP-1

ControlInhibitor MM

Inhibitor + MM

0504504

035030250201501005

0

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(g)

MM

InhibitorControl

Inhibitor + MM

(h)

06

05

04

03

02

01

0

lowast

lowastlowastCPC

1

MMInhibitorControl

Inhibitor + MM

(ng

calc

ium

mg

prot

ein)

(i)

Figure 5 Odontoosteogenic differentiation in canonical NF-120581B-inhibited SCAPs (a) ALP activity at different time points in different groups(ie control inhibitor MM and inhibitor +MM) (b) Real-time RT-PCR for the detection ofALP BSP OCN RUNX2 OSX DSP OPN DSPandDMP-1 at day 3ThemRNA levels were normalized toGAPDH (c) Real-time RT-PCR for the detection ofALP BSP OCN RUNX2 OSXDSP OPN DSP andDMP-1 of SCAPs respectively in control inhibitor MM and inhibitor +MMgroups at day 7 (d)The odontoosteogenicprotein levels of SCAPs in control inhibitor MM and inhibitor +MM groups were characterized by western blot analysis at day 3 120573-ACTINwas used as a loading control (e) Semiquantitative analysis demonstrated that the expression of BSP OCN RUNX2 OSX DSP OPN DSPand DMP-1 was more downregulated in NF-120581B pathway-inhibited SCAPs than those in control group at day 3 (f) The odontoosteogenicprotein levels of SCAPs in control inhibitor MM and inhibitor + MM groups were characterized by western blot analysis at day 7 (g)Semiquantitative analysis demonstrated that the expression of odontoosteogenic markers (BSP OCN RUNX2 OSX DSP OPN DSP andDMP-1) was significantly more downregulated in NF-120581B pathway-inhibited SCAPs than those in control group at day 7 (h) Alizarin redstaining of SCAPs after 14 days of osteogenic induction (i) Calcium quantification demonstrated the weaker calcium deposition in inhibitorand inhibitor + MM groups as compared with control and MM groups respectively

Acknowledgments

This work was supported by National Natural Science Foun-dation of China (no 81371144) Natural Science Foundationof Jiangsu Province (no BK20131392) and the Priority Aca-demic Program Development of Jiangsu Higher EducationInstitutions (PAPD no 2011-137)

References

[1] AKumar Y TakadaAMBoriek andB BAggarwal ldquoNuclearfactor-120581B its role in health and diseaserdquo Journal of MolecularMedicine vol 82 no 7 pp 434ndash448 2004

[2] F Chen V Castranova X Shi and LM Demers ldquoNew insightsinto the role of nuclear factor-120581B a ubiquitous transcription

factor in the initiation of diseasesrdquo Clinical Chemistry vol 45no 1 pp 7ndash17 1999

[3] C Gasparini and M Feldmann ldquoNF-120581B as a target for modu-lating inflammatory responsesrdquoCurrent Pharmaceutical Designvol 18 no 35 pp 5735ndash5745 2012

[4] A Ohazama and P T Sharpe ldquoTNF signalling in toothdevelopmentrdquo Current Opinion in Genetics amp Development vol14 no 5 pp 513ndash519 2004

[5] Y-H Kuan F-M Huang Y-C Li and Y-C Chang ldquoProin-flammatory activation ofmacrophages by bisphenol A-glycidyl-methacrylate involved NF120581B activation via PI3KAkt pathwayrdquoFood and Chemical Toxicology vol 50 no 11 pp 4003ndash40092012

[6] X Cai P Gong YHuang andY Lin ldquoNotch signalling pathwayin tooth development and adult dental cellsrdquo Cell Proliferationvol 44 no 6 pp 495ndash507 2011

12 BioMed Research International

[7] J-M Courtney J Blackburn and P T Sharpe ldquoThe Ectodys-plasin and NF120581B signalling pathways in odontogenesisrdquoArchives of Oral Biology vol 50 no 2 pp 159ndash163 2005

[8] W Sonoyama Y Liu T Yamaza et al ldquoCharacterization of theapical papilla and its residing stem cells from human immaturepermanent teeth a pilot studyrdquo Journal of Endodontics vol 34no 2 pp 166ndash171 2008

[9] W Sonoyama Y Liu D Fang et al ldquoMesenchymal stem cell-mediated functional tooth regeneration in swinerdquo PLoS ONEvol 1 no 1 article e79 2006

[10] H Chopra M K Hans and S Shetty ldquoStem cells-the hiddentreasure a strategic reviewrdquoDental Research Journal vol 10 no4 pp 421ndash427 2013

[11] R Dong R Yao J Du S Wang and Z Fan ldquoDepletion ofhistone demethylase KDM2A enhanced the adipogenic andchondrogenic differentiation potentials of stem cells from apicalpapillardquo Experimental Cell Research vol 319 no 18 pp 2874ndash2882 2013

[12] Y Wang M Yan Z Wang et al ldquoDental pulp stem cells fromtraumatically exposed pulps exhibited an enhanced osteogenicpotential and weakened odontogenic capacityrdquo Archives of OralBiology vol 58 no 11 pp 1709ndash1717 2013

[13] Y Wang M Yan Z Fan L Ma Y Yu and J Yu ldquoMineraltrioxide aggregate enhances the odontoosteogenic capacity ofstem cells from inflammatory dental pulps via NF-120581B pathwayrdquoOral Diseases 2013

[14] Y Wang M Yan Y Yu J Wu J Yu and Z Fan ldquoEstrogendeficiency inhibits the odontoosteogenic differentiation ofdental pulp stem cells via activation of the NF-120581B pathwayrdquo Celland Tissue Research vol 352 no 3 pp 551ndash559 2013

[15] Y Wang Y Zheng Z Wang et al ldquo10minus7M 17120573-oestradiolenhances odontoosteogenic potency of human dental pulpstem cells by activation of the NF-120581B pathwayrdquo Cell Prolifera-tion vol 46 no 6 pp 677ndash684 2013

[16] Z-G Lu H Liu T Yamaguchi Y Miki and K YoshidaldquoProtein kinase C120575 activates RelAp65 and nuclear factor-120581B signaling in response to tumor necrosis factor-120572rdquo CancerResearch vol 69 no 14 pp 5927ndash5935 2009

[17] G Bonizzi and M Karin ldquoThe two NF-120581B activation pathwaysand their role in innate and adaptive immunityrdquo Trends inImmunology vol 25 no 6 pp 280ndash288 2004

[18] K W McIntyre D J Shuster K M Gillooly et al ldquoA highlyselective inhibitor of I120581B kinase BMS-345541 blocks both jointinflammation and destruction in collagen-induced arthritis inmicerdquo Arthritis amp Rheumatism vol 48 no 9 pp 2652ndash26592003

[19] J R Burke M A Pattoli K R Gregor et al ldquoBMS-345541 is ahighly selective inhibitor of I120581B kinase that binds at an allostericsite of the enzyme and blocks NF-120581B-dependent transcriptionin micerdquoThe Journal of Biological Chemistry vol 278 no 3 pp1450ndash1456 2003

[20] F W Paula-Silva A Ghosh L A Silva and Y L Kapila ldquoTNF-120572 promotes an odontoblastic phenotype in dental pulp cellsrdquoJournal of Dental Research vol 88 no 4 pp 339ndash344 2009

[21] S Wang J Mu Z Fan et al ldquoInsulin-like growth factor 1 canpromote the osteogenic differentiation and osteogenesis of stemcells from apical papillardquo Stem Cell Research vol 8 no 3 pp346ndash356 2012

[22] Y Yu J Mu Z Fan et al ldquoInsulin-like growth factor 1 enhancesthe proliferation and osteogenic differentiation of human peri-odontal ligament stem cells via ERK and JNKMAPKpathwaysrdquoHistochemistry andCell Biology vol 137 no 4 pp 513ndash525 2012

[23] H Aonuma N Ogura K Takahashi et al ldquoCharacteristics andosteogenic differentiation of stemprogenitor cells in the humandental follicle analyzed by gene expression profilingrdquo Cell andTissue Research vol 350 no 2 pp 317ndash331 2012

[24] L Wang M Yan Y Wang et al ldquoProliferation andosteoodontoblastic differentiation of stem cells from dentalapical papilla in mineralization-inducing medium containingadditional KH

2PO4rdquo Cell Proliferation vol 46 no 2 pp

214ndash222 2013[25] G T-J Huang T Yamaza L D Shea et al ldquoStemProgenitor

cell-mediated de novo regeneration of dental pulp with newlydeposited continuous layer of dentin in an in vivomodelrdquoTissueEngineering A vol 16 no 2 pp 605ndash615 2010

[26] J-H Kwak J-K Jung and H Lee ldquoNuclear factor-120581 Binhibitors a patent review (2006ndash2010)rdquo Expert Opinion onTherapeutic Patents vol 21 no 12 pp 1897ndash1910 2011

[27] K Hess A Ushmorov J Fiedler R E Brenner and T WirthldquoTNF120572 promotes osteogenic differentiation of human mes-enchymal stem cells by triggering theNF-120581B signaling pathwayrdquoBone vol 45 no 2 pp 367ndash376 2009

[28] X Feng G Feng J Xing et al ldquoTNF-120572 triggers osteogenicdifferentiation of human dental pulp stem cells via the NF-120581Bsignalling pathwayrdquoCell Biology International vol 37 no 12 pp1267ndash1275 2013

[29] J Chang F Liu M Lee et al ldquoNF-120581B inhibits osteogenicdifferentiation of mesenchymal stem cells by promoting 120573-catenin degradationrdquo Proceedings of the National Academy ofSciences of the United States of America vol 110 no 23 pp9469ndash9474 2013

[30] C Shui and A Scutt ldquoMild heat shock induces proliferationalkaline phosphatase activity and mineralization in humanbone marrow stromal cells and Mg-63 cells in vitrordquo Journal ofBone and Mineral Research vol 16 no 4 pp 731ndash741 2001

[31] X Wei J Ling L Wu L Liu and Y Xiao ldquoExpressionof mineralization markers in dental pulp cellsrdquo Journal ofEndodontics vol 33 no 6 pp 703ndash708 2007

[32] S Z Khan E Kokubu K Matsuzaka and T Inoue ldquoBehaviourof rat-cultured dental pulp cells in three-dimensional collagentype-1 gel in vitro and in vivordquo Australian Endodontic Journalvol 39 no 3 pp 137ndash145 2013

[33] D-X Cao Z-J Li X-O Jiang et al ldquoOsteopontin as potentialbiomarker and therapeutic target in gastric and liver cancersrdquoWorld Journal of Gastroenterology vol 18 no 30 pp 3923ndash39302012

[34] S SchlaferMK Raarup P LWejse et al ldquoOsteopontin reducesbiofilm formation in a multi-species model of dental biofilmrdquoPloS ONE vol 7 no 8 Article ID e41534 2012

[35] S Chen J Gluhak-Heinrich Y HWang et al ldquoRunx2Osx andDspp in tooth developmentrdquo Journal of Dental Research vol 88no 10 pp 904ndash909 2009

[36] S Suzuki N Haruyama F Nishimura and A B KulkarnildquoDentin sialophosphoprotein and dentin matrix protein-1 twohighly phosphorylated proteins inmineralized tissuesrdquoArchivesof Oral Biology vol 57 no 9 pp 1165ndash1175 2012

[37] R S Prescott R Alsanea M I Fayad et al ldquoIn vivo fenerationof dental pulp-like tissue by using dental pulp stem cells a col-lagen scaffold and dentin matrix protein 1 after subcutaneoustransplantation in micerdquo Journal of Endodontics vol 34 no 4pp 421ndash426 2008

[38] T Komori ldquoRegulation of bone development and extracellularmatrix protein genes by RUNX2rdquo Cell and Tissue Research vol339 no 1 pp 189ndash195 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 BioMed Research International

lowast

lowast

lowast

lowast

lowast lowast

ControlActivator

MTT25

2

15

1

05

0

OD

val

ue (4

90

nm)

0 1 3 5 7 9 11

(day)

(a)

S30 60 90 120

SSSSSSSSSSSSSSSSSSSSSS0

300

900

1200

600

Num

ber

SCAPsChannels (FL2-A)

G2M

G0G1 = 8506

G2M = 986

G0G1

S

30 60 90 120

S

0

Num

ber

Channels (FL2-A)

G2M

G0G1 = 7348

G2M = 886

G0G1

1000

800

600

400

200

00

SCAPs + activator

S = 1766S = 508

(b)

Control

Activator

0h 6h 12h 24h

(c)

ControlInhibitor

25

2

15

1

05

0

OD

val

ue (4

90

nm) lowast

lowast

MTT

0 1 3 5 7 9 11

(day)

lowastlowast

lowastlowastlowast

lowastlowastlowast

(d)

SSSSSSSSSSSSSSSSSSSSSSS

SCAPsChannels (FL2-A)

G0G1

30 60 90 1200

G2M

1600

1200

800

400

0

Num

ber

G0G1 = 8217

G2M = 600

S = 1183

1600

1200

800

400

0

Num

ber

S

G0G1

G2M

Channels (FL2-A)30 60 900 120 150

G0G1 = 9109

G2M = 536

S = 354

SCAPs + inhibitor

(e)

0h 6h 12h 24h

Control

Inhibitor

(f)

Figure 3 Effects of canonical NF-120581B signaling pathway on the proliferation of SCAPs (a) Cell proliferation in NF-120581B pathway-activatedand control groups was detected by MTT assay (b) Flow cytometry analysis for SCAPs in NF-120581B pathway-activated and untreated groupsThe proliferation index (PI = S + G

2M) in NF-120581B pathway-activated group (2652) was significantly higher than that in control group

(1494) (c) Images of scratch wounds in NF-120581B pathway-activated and untreated groups at indicated time points (d) Growth curves of NF-120581B pathway-inhibited and untreated SCAPs (e) Representative cell cycle distributions of NF-120581B pathway-inhibited and untreated SCAPTheproliferation index (PI = S + G

2M) in NF-120581B pathway-activated group (890) was obviously lower than that in control group (1783)

(f) Cell motility of SCAPs in NF-120581B pathway-inhibited and untreated group assessed by a scratch assay Values were the means plusmn SD 119899 = 6lowast

119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 Scale bars = 100 120583m

(119875 lt 005 Figure 3(b))Therewas a lower percentage (890)of proliferating cells in SG

2Mphases in the inhibitor-treated

SCAPs as compared with the control group (1783) at day 3(Figure 3(e)) which is consistent with the findings in MTTassay (Figure 3(d)) These results indicate that canonical

NF-120581B pathway facilitated the proliferation of SCAPs thatis the cell proliferation rate increasing in pace with theactivation of canonical NF-120581B and is restrained along withthe blockage of NF-120581B Furthermore an improved migrationability was observed in activator-treated SCAPs (Figure 3(c))

BioMed Research International 7

while scratch closure was markedly slower in SCAPs treatedwith inhibitor (Figure 3(f))

34 Effects of Canonical NF-120581B Pathway on the OdontoOsteo-genic Differentiation of SCAPs ALP activities in activator-treated SCAPs in basic medium andmineralization-inducingmedium were elevated at day 5 and day 7 (119875 lt 005Figure 4(a)) as compared with untreated groupsThe densityof calcification nodules was significantly higher in activator-stimulated groups than in the other groups after 14 days ofcoculture (Figure 4(h)) Moreover quantitative calciummea-surement illustrated more calcifications in activator-treatedSCAPs in comparison with untreated groups (Figure 4(i))

Differentially expression levels of related osteoodonto-genic genes were also investigated by real-time RT-PCRassays In activator-treated group expression of specificosteoodontogenic genes (eg ALP OCN BSP OSXRUNX2 DSP OPN and DMP-1) was significantly upregu-lated at days 3 and 7 (Figures 4(b) and 4(c)) At day 3 theexpression of some osteoodontogenic genes in activator-treated groups did not change obviously in complete mediabut was distinctly increased in the presence ofmineralization-inducing media At day 7 the odontoosteo-genic markers were significantly upregulated following theactivator treatment regardless of the presence or absence ofmineralization-inducing media This phenomenon was notobserved in NF-120581B-inhibition group These findings wereconfirmed by western blot assay in which activated classicalNF-120581B signaling pathway induced a significant increase ofrelated protein expression (Figures 4(d)ndash4(g))

As shown in Figure 5(a) the blockage of NF-120581B signalingpathway obviously downregulated the ALP activity at days 35 and 7 (119875 lt 005) At day 14 less calcified nodules weregenerated in inhibitor-treated groups (Figure 5(h)) Calciumquantification also revealed the less calcium deposition ininhibitor and inhibitor + MM groups as compared withcontrol and MM groups respectively (Figure 5(i) 119875 lt 005)There was a remarkable decrease of osteoodontogenic genesat different time points (Figures 5(b) and 5(c)) Western blotanalysis further verified these findings (Figures 5(d)ndash5(g))

4 Discussion

SCAPs are known as a kind of ideal candidates for dentaltissue engineering and have the characteristics of self-renewaland multilineage differentiation potential [9 10] Diversestudies have proved that SCAPs are able to differentiate intoosteoodontoblasts in vitro under appropriate conditions andformbonedentin-like tissues in vivo [24 25] Certainlymanysignaling pathways may be involved in the process of cellproliferation and differentiation including NF-120581B pathway

NF-120581B exists in the cytoplasm in a latent form bindingto inhibitory proteins termed inhibitory 120581B proteins (I120581Bs)[3 26] I120581B complex is the major regulator of NF-120581B pathwayand is composed of catalytic subunits IKK120572 and IKK120573 anda regulatory subunit IKK120574 TNF-120572 is generally known toactivate classical NF-120581B pathway [17] Exposure of cells tosuitable concentrations of TNF-120572 brings about the rapid

phosphorylation ubiquitination and proteolytic degradationof I120581B which allows NF-120581B to translocate to the nucleuswhere it subsequently regulates the gene transcription [27ndash29] BMS-345541 a selective inhibitor of the catalytic subunitsof IKK targets NF-120581B signaling pathway and downregulatesthe activity of NF-120581B [18 19] In this study the expressionof cytoplastic P-P65P-I120581B120572 was noticeably upregulated afterthe treatment of TNF-120572 and downregulated by the inhibitorBMS-345541 indicating the successful establishment of acellular model for the activation or suppression of canonicalNF-120581B pathway

Cell proliferation and migration were indispensable fortissue development and wound healing In the present studyNF-120581B-activated SCAPs exhibited an increased growth ratehigher proliferation index and more effective migrationin wound healing assay Moreover the migration distancesbetween thewound lines in inhibitor-treated cells were longerthan control cells after 24 hours of culture Together withthe weakened proliferation of inhibitor-treated cells it ispredictable that the canonical NF-120581B pathway has a positiveinfluence on the cell multiplication activity and motility

Previous studies have proved that various stimuli canexert an active role on the committed differentiation ofdental stem cells via NF-120581B pathway [28] In the presentstudy the odontoosteogenic capacity remarkably changedalong with the activation or blockage of classical NF-120581Bpathway Activator-treated SCAPs exhibited an enhancedALP activity increased calcium deposition and upregulatedexpression of odontoosteogenic genes and proteins whileinhibitor-treated cells presented the lower ALP activitydecreased mineralization and downregulated expression ofodontoosteoblast markers

It is widely recognized that ALP is a functionalmarker forosteoblast activity and bone formation [30] As an importantextracellular matrix of bone BSP is mainly secreted byosteoblasts whose expression is a crucial symbol in matrixdeposition and mineralization [31 32] OPN is anotherextracellular matrix protein expressed in numerous cell typesincluding odontoblasts in which OPN plays multifacetedroles in a variety of biological and pathological processessuch as osteogenic differentiation tooth mineralization anddental biofilm formation [33 34] OCN is the primarynoncollagenous protein in the bone cells secreted in the latestage of osteogenesis and known as a specific indicator ofosteogenic differentiation RUNX2 is the first transcriptionfactor required for determination of the osteoblast lineageand OSX acts as a downstream gene of RUNX2 that is highlyexpressed in the functional odontoosteoblasts [35] DSPPand DSP are well-known markers of odontoblasts highlyexpressed in dentin or predentin structures and essentialfor dentinogenesis [36] DMP-1 is an acidic extracellularmatrix protein that is primarily found in dentin and boneand has been implicated in dentin mineralization and signaltransduction in the process of odontogenesis [37]

In this study the expression of RUNX2RUNX2 reducedafter 3 days of incubation with the inhibitor BMS-345541and then gradually increased at day 7 while the expressionof DSPPDSP did not compromise after 3 days of incuba-tion but noticeably decreased at day 7 indicating that the

8 BioMed Research International

ALP

3 5 7

(day)

35

3

25

2

15

1

05

0

ControlActivator

MMActivator + MM

lowast

lowastlowastlowastlowastlowastlowast

(sig

ma u

nitm

inm

g pr

otei

n)

(a)

ControlActivator

MMActivator + MM

lowastlowast

lowastlowast

876543210

ALP

OCN BS

P

RUN

X2

OSX

DSP

P

OPN

DM

P-1

Gen

esG

APD

H

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastPCR 3d

(b)

lowast

lowast lowast

lowast

lowast lowastlowast

lowast

25

20

15

10

5

0

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

Gen

esG

APD

H

ControlActivator

MMActivator + MM

ALP

OCN BS

P

RUN

X2

OSX

DSP

P

OPN

DM

P-1

PCR 7d

(c)

MMControl Activator

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

Activator + MM

Western blot 3d

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

(d)

ControlActivator

MMActivator + MM

09

08

07

06

05

04

03

02

01

0

Prot

eins

ACT

IN

OCN BSP RUNX2 OSX DSP OPN DMP-1

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

lowast

lowast

lowast

(e)

MMControl Activator

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

Activator + MM

Western blot 7d

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

(f)

Figure 4 Continued

BioMed Research International 9

14

12

1

08

06

04

02

0

Prot

eins

ACT

IN

OCN BSP RUNX2 OSX DSP OPN DMP-1

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

ControlActivator

MMActivator + MM

(g)

Control Activator

MM Activator + MM

(h)

1

CPC06

05

04

03

02

01

0

lowast

lowastlowastlowast

ControlActivator

MMActivator + MM

(ng

calc

ium

mg

prot

ein)

(i)

Figure 4 Odontoosteogenic differentiation in canonical NF-120581B-activated SCAPs (a) ALP activity of SCAPs in control activator MM andactivator + MM groups at days 3 5 and 7 Values are the means plusmn SD 119899 = 6 lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 (b) Real-time RT-PCRanalysis for odontoosteogenic genes (BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in each group at day 7 (c) Real-time RT-PCRanalysis for odontoosteogenic genes (ALP BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in different groups at day 7 GAPDHserved as a housekeeping gene lowastlowast2minusΔΔCt ge 2 119875 lt 001 lowast1 lt 2minusΔΔCt lt 2 119875 lt 001 119899 = 3 (d) Western blot analyses for the odontoosteogenicproteins (BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in different groups at day 3 120573-ACTIN served as an internal control (e)Semiquantitative analysis demonstrated that the expression of BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1 was stronger in NF-120581Bpathway-activated SCAPs than those in control group at day 3 especially in the presence of mineralization-inducing media (f) Western blotanalyses for the odontoosteogenic proteins (BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in different groups at day 7 120573-ACTINwas used as an internal control (g) Semiquantitative analysis confirmed that the expression of BSP OCN RUNX2 OSX DSP OPN DSPand DMP-1 was upregulated in NF-120581B pathway-activated SCAPs at day 7 regardless of the presence or absence of mineralization-inducingmedia (h) Alizarin red staining of SCAPs after 14 days of induction in different groups (i) Quantitative calcium analysis showed that thecalcium content in activator and activator + MM groups was significantly higher than the control and MM groups respectively Values weredescribed as the means plusmn SD lowast119875 lt 005 lowastlowastlowast119875 lt 0001

expression of RUNX2RUNX2 and DSPPDSP tends to beinversely correlated [38] Moreover the dramatic differenceof these odontoosteogenic markers at both mRNA andprotein levels in response to the regulation of NF-120581B pathwaysuggests that activated canonical NF-120581B pathway favors theodontoosteoblastic differentiation of SCAPs particularly inthe presence of mineralization-inducing media

In summary the findings accumulated here confirmedthe potential involvement of canonical NF-120581B pathway inthe proliferationmigration and committed differentiation ofSCAPs in vitro Both activation and inhibition of the classicalNF-120581B pathway can bring about the permanent changes in

human stem cells Moreover it is commonly believed thatNF-120581B signaling plays a pivotal role not only in the progressof normal physiological process but also in the pathologicalprocess and dysfunction ofNF-120581B is linked to various humandiseases Thus proper balance of intracellular NF-120581B shouldbemaintained in the physiological conditions while intricateinteractivity between NF-120581B and other signaling pathwaysneeds to be extensively investigated

Conflict of Interests

The authors declare no conflict of interests

10 BioMed Research International

ControlInhibitor

MM

ALP3

25

2

15

1

05

03 5 7

(day)

Inhibitor + MM

lowastlowast lowastlowast

lowastlowast

lowast

lowast

(sig

ma u

nitm

inm

g pr

otei

n)

(a)

ControlInhibitor

35

3

25

2

15

1

05

0

Gen

esG

APD

H

ALP OCN BSP RUNX2 OSX DSPP OPN DMP-1

MMInhibitor + MM

lowastlowast

lowast

lowast

lowast lowastlowast

lowast

lowastlowast

lowastlowast

PCR 3d

(b)

ControlInhibitor

MM

1614121086420

Gen

esG

APD

H

ALP OCN BSP RUNX2 OSX DSPP OPN DMP-1

Inhibitor + MM

lowastlowast lowastlowast

lowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowastlowastlowastPCR 7d

(c)

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

MMInhibitorControl Inhibitor + MM

Western blot 3d

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

(d)

Prot

eins

ACT

IN

ControlInhibitor

MM

12

1

08

06

04

02

0OCN BSP RUNX2 OSX DSP OPN DMP-1

Inhibitor + MM

lowast

lowast lowast

lowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

(e)

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

MMInhibitorControl Inhibitor + MM

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

Western blot 7d

(f)

Figure 5 Continued

BioMed Research International 11

Prot

eins

ACT

IN

OCN BSP RUNX2 OSX DSP OPN DMP-1

ControlInhibitor MM

Inhibitor + MM

0504504

035030250201501005

0

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(g)

MM

InhibitorControl

Inhibitor + MM

(h)

06

05

04

03

02

01

0

lowast

lowastlowastCPC

1

MMInhibitorControl

Inhibitor + MM

(ng

calc

ium

mg

prot

ein)

(i)

Figure 5 Odontoosteogenic differentiation in canonical NF-120581B-inhibited SCAPs (a) ALP activity at different time points in different groups(ie control inhibitor MM and inhibitor +MM) (b) Real-time RT-PCR for the detection ofALP BSP OCN RUNX2 OSX DSP OPN DSPandDMP-1 at day 3ThemRNA levels were normalized toGAPDH (c) Real-time RT-PCR for the detection ofALP BSP OCN RUNX2 OSXDSP OPN DSP andDMP-1 of SCAPs respectively in control inhibitor MM and inhibitor +MMgroups at day 7 (d)The odontoosteogenicprotein levels of SCAPs in control inhibitor MM and inhibitor +MM groups were characterized by western blot analysis at day 3 120573-ACTINwas used as a loading control (e) Semiquantitative analysis demonstrated that the expression of BSP OCN RUNX2 OSX DSP OPN DSPand DMP-1 was more downregulated in NF-120581B pathway-inhibited SCAPs than those in control group at day 3 (f) The odontoosteogenicprotein levels of SCAPs in control inhibitor MM and inhibitor + MM groups were characterized by western blot analysis at day 7 (g)Semiquantitative analysis demonstrated that the expression of odontoosteogenic markers (BSP OCN RUNX2 OSX DSP OPN DSP andDMP-1) was significantly more downregulated in NF-120581B pathway-inhibited SCAPs than those in control group at day 7 (h) Alizarin redstaining of SCAPs after 14 days of osteogenic induction (i) Calcium quantification demonstrated the weaker calcium deposition in inhibitorand inhibitor + MM groups as compared with control and MM groups respectively

Acknowledgments

This work was supported by National Natural Science Foun-dation of China (no 81371144) Natural Science Foundationof Jiangsu Province (no BK20131392) and the Priority Aca-demic Program Development of Jiangsu Higher EducationInstitutions (PAPD no 2011-137)

References

[1] AKumar Y TakadaAMBoriek andB BAggarwal ldquoNuclearfactor-120581B its role in health and diseaserdquo Journal of MolecularMedicine vol 82 no 7 pp 434ndash448 2004

[2] F Chen V Castranova X Shi and LM Demers ldquoNew insightsinto the role of nuclear factor-120581B a ubiquitous transcription

factor in the initiation of diseasesrdquo Clinical Chemistry vol 45no 1 pp 7ndash17 1999

[3] C Gasparini and M Feldmann ldquoNF-120581B as a target for modu-lating inflammatory responsesrdquoCurrent Pharmaceutical Designvol 18 no 35 pp 5735ndash5745 2012

[4] A Ohazama and P T Sharpe ldquoTNF signalling in toothdevelopmentrdquo Current Opinion in Genetics amp Development vol14 no 5 pp 513ndash519 2004

[5] Y-H Kuan F-M Huang Y-C Li and Y-C Chang ldquoProin-flammatory activation ofmacrophages by bisphenol A-glycidyl-methacrylate involved NF120581B activation via PI3KAkt pathwayrdquoFood and Chemical Toxicology vol 50 no 11 pp 4003ndash40092012

[6] X Cai P Gong YHuang andY Lin ldquoNotch signalling pathwayin tooth development and adult dental cellsrdquo Cell Proliferationvol 44 no 6 pp 495ndash507 2011

12 BioMed Research International

[7] J-M Courtney J Blackburn and P T Sharpe ldquoThe Ectodys-plasin and NF120581B signalling pathways in odontogenesisrdquoArchives of Oral Biology vol 50 no 2 pp 159ndash163 2005

[8] W Sonoyama Y Liu T Yamaza et al ldquoCharacterization of theapical papilla and its residing stem cells from human immaturepermanent teeth a pilot studyrdquo Journal of Endodontics vol 34no 2 pp 166ndash171 2008

[9] W Sonoyama Y Liu D Fang et al ldquoMesenchymal stem cell-mediated functional tooth regeneration in swinerdquo PLoS ONEvol 1 no 1 article e79 2006

[10] H Chopra M K Hans and S Shetty ldquoStem cells-the hiddentreasure a strategic reviewrdquoDental Research Journal vol 10 no4 pp 421ndash427 2013

[11] R Dong R Yao J Du S Wang and Z Fan ldquoDepletion ofhistone demethylase KDM2A enhanced the adipogenic andchondrogenic differentiation potentials of stem cells from apicalpapillardquo Experimental Cell Research vol 319 no 18 pp 2874ndash2882 2013

[12] Y Wang M Yan Z Wang et al ldquoDental pulp stem cells fromtraumatically exposed pulps exhibited an enhanced osteogenicpotential and weakened odontogenic capacityrdquo Archives of OralBiology vol 58 no 11 pp 1709ndash1717 2013

[13] Y Wang M Yan Z Fan L Ma Y Yu and J Yu ldquoMineraltrioxide aggregate enhances the odontoosteogenic capacity ofstem cells from inflammatory dental pulps via NF-120581B pathwayrdquoOral Diseases 2013

[14] Y Wang M Yan Y Yu J Wu J Yu and Z Fan ldquoEstrogendeficiency inhibits the odontoosteogenic differentiation ofdental pulp stem cells via activation of the NF-120581B pathwayrdquo Celland Tissue Research vol 352 no 3 pp 551ndash559 2013

[15] Y Wang Y Zheng Z Wang et al ldquo10minus7M 17120573-oestradiolenhances odontoosteogenic potency of human dental pulpstem cells by activation of the NF-120581B pathwayrdquo Cell Prolifera-tion vol 46 no 6 pp 677ndash684 2013

[16] Z-G Lu H Liu T Yamaguchi Y Miki and K YoshidaldquoProtein kinase C120575 activates RelAp65 and nuclear factor-120581B signaling in response to tumor necrosis factor-120572rdquo CancerResearch vol 69 no 14 pp 5927ndash5935 2009

[17] G Bonizzi and M Karin ldquoThe two NF-120581B activation pathwaysand their role in innate and adaptive immunityrdquo Trends inImmunology vol 25 no 6 pp 280ndash288 2004

[18] K W McIntyre D J Shuster K M Gillooly et al ldquoA highlyselective inhibitor of I120581B kinase BMS-345541 blocks both jointinflammation and destruction in collagen-induced arthritis inmicerdquo Arthritis amp Rheumatism vol 48 no 9 pp 2652ndash26592003

[19] J R Burke M A Pattoli K R Gregor et al ldquoBMS-345541 is ahighly selective inhibitor of I120581B kinase that binds at an allostericsite of the enzyme and blocks NF-120581B-dependent transcriptionin micerdquoThe Journal of Biological Chemistry vol 278 no 3 pp1450ndash1456 2003

[20] F W Paula-Silva A Ghosh L A Silva and Y L Kapila ldquoTNF-120572 promotes an odontoblastic phenotype in dental pulp cellsrdquoJournal of Dental Research vol 88 no 4 pp 339ndash344 2009

[21] S Wang J Mu Z Fan et al ldquoInsulin-like growth factor 1 canpromote the osteogenic differentiation and osteogenesis of stemcells from apical papillardquo Stem Cell Research vol 8 no 3 pp346ndash356 2012

[22] Y Yu J Mu Z Fan et al ldquoInsulin-like growth factor 1 enhancesthe proliferation and osteogenic differentiation of human peri-odontal ligament stem cells via ERK and JNKMAPKpathwaysrdquoHistochemistry andCell Biology vol 137 no 4 pp 513ndash525 2012

[23] H Aonuma N Ogura K Takahashi et al ldquoCharacteristics andosteogenic differentiation of stemprogenitor cells in the humandental follicle analyzed by gene expression profilingrdquo Cell andTissue Research vol 350 no 2 pp 317ndash331 2012

[24] L Wang M Yan Y Wang et al ldquoProliferation andosteoodontoblastic differentiation of stem cells from dentalapical papilla in mineralization-inducing medium containingadditional KH

2PO4rdquo Cell Proliferation vol 46 no 2 pp

214ndash222 2013[25] G T-J Huang T Yamaza L D Shea et al ldquoStemProgenitor

cell-mediated de novo regeneration of dental pulp with newlydeposited continuous layer of dentin in an in vivomodelrdquoTissueEngineering A vol 16 no 2 pp 605ndash615 2010

[26] J-H Kwak J-K Jung and H Lee ldquoNuclear factor-120581 Binhibitors a patent review (2006ndash2010)rdquo Expert Opinion onTherapeutic Patents vol 21 no 12 pp 1897ndash1910 2011

[27] K Hess A Ushmorov J Fiedler R E Brenner and T WirthldquoTNF120572 promotes osteogenic differentiation of human mes-enchymal stem cells by triggering theNF-120581B signaling pathwayrdquoBone vol 45 no 2 pp 367ndash376 2009

[28] X Feng G Feng J Xing et al ldquoTNF-120572 triggers osteogenicdifferentiation of human dental pulp stem cells via the NF-120581Bsignalling pathwayrdquoCell Biology International vol 37 no 12 pp1267ndash1275 2013

[29] J Chang F Liu M Lee et al ldquoNF-120581B inhibits osteogenicdifferentiation of mesenchymal stem cells by promoting 120573-catenin degradationrdquo Proceedings of the National Academy ofSciences of the United States of America vol 110 no 23 pp9469ndash9474 2013

[30] C Shui and A Scutt ldquoMild heat shock induces proliferationalkaline phosphatase activity and mineralization in humanbone marrow stromal cells and Mg-63 cells in vitrordquo Journal ofBone and Mineral Research vol 16 no 4 pp 731ndash741 2001

[31] X Wei J Ling L Wu L Liu and Y Xiao ldquoExpressionof mineralization markers in dental pulp cellsrdquo Journal ofEndodontics vol 33 no 6 pp 703ndash708 2007

[32] S Z Khan E Kokubu K Matsuzaka and T Inoue ldquoBehaviourof rat-cultured dental pulp cells in three-dimensional collagentype-1 gel in vitro and in vivordquo Australian Endodontic Journalvol 39 no 3 pp 137ndash145 2013

[33] D-X Cao Z-J Li X-O Jiang et al ldquoOsteopontin as potentialbiomarker and therapeutic target in gastric and liver cancersrdquoWorld Journal of Gastroenterology vol 18 no 30 pp 3923ndash39302012

[34] S SchlaferMK Raarup P LWejse et al ldquoOsteopontin reducesbiofilm formation in a multi-species model of dental biofilmrdquoPloS ONE vol 7 no 8 Article ID e41534 2012

[35] S Chen J Gluhak-Heinrich Y HWang et al ldquoRunx2Osx andDspp in tooth developmentrdquo Journal of Dental Research vol 88no 10 pp 904ndash909 2009

[36] S Suzuki N Haruyama F Nishimura and A B KulkarnildquoDentin sialophosphoprotein and dentin matrix protein-1 twohighly phosphorylated proteins inmineralized tissuesrdquoArchivesof Oral Biology vol 57 no 9 pp 1165ndash1175 2012

[37] R S Prescott R Alsanea M I Fayad et al ldquoIn vivo fenerationof dental pulp-like tissue by using dental pulp stem cells a col-lagen scaffold and dentin matrix protein 1 after subcutaneoustransplantation in micerdquo Journal of Endodontics vol 34 no 4pp 421ndash426 2008

[38] T Komori ldquoRegulation of bone development and extracellularmatrix protein genes by RUNX2rdquo Cell and Tissue Research vol339 no 1 pp 189ndash195 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 7

while scratch closure was markedly slower in SCAPs treatedwith inhibitor (Figure 3(f))

34 Effects of Canonical NF-120581B Pathway on the OdontoOsteo-genic Differentiation of SCAPs ALP activities in activator-treated SCAPs in basic medium andmineralization-inducingmedium were elevated at day 5 and day 7 (119875 lt 005Figure 4(a)) as compared with untreated groupsThe densityof calcification nodules was significantly higher in activator-stimulated groups than in the other groups after 14 days ofcoculture (Figure 4(h)) Moreover quantitative calciummea-surement illustrated more calcifications in activator-treatedSCAPs in comparison with untreated groups (Figure 4(i))

Differentially expression levels of related osteoodonto-genic genes were also investigated by real-time RT-PCRassays In activator-treated group expression of specificosteoodontogenic genes (eg ALP OCN BSP OSXRUNX2 DSP OPN and DMP-1) was significantly upregu-lated at days 3 and 7 (Figures 4(b) and 4(c)) At day 3 theexpression of some osteoodontogenic genes in activator-treated groups did not change obviously in complete mediabut was distinctly increased in the presence ofmineralization-inducing media At day 7 the odontoosteo-genic markers were significantly upregulated following theactivator treatment regardless of the presence or absence ofmineralization-inducing media This phenomenon was notobserved in NF-120581B-inhibition group These findings wereconfirmed by western blot assay in which activated classicalNF-120581B signaling pathway induced a significant increase ofrelated protein expression (Figures 4(d)ndash4(g))

As shown in Figure 5(a) the blockage of NF-120581B signalingpathway obviously downregulated the ALP activity at days 35 and 7 (119875 lt 005) At day 14 less calcified nodules weregenerated in inhibitor-treated groups (Figure 5(h)) Calciumquantification also revealed the less calcium deposition ininhibitor and inhibitor + MM groups as compared withcontrol and MM groups respectively (Figure 5(i) 119875 lt 005)There was a remarkable decrease of osteoodontogenic genesat different time points (Figures 5(b) and 5(c)) Western blotanalysis further verified these findings (Figures 5(d)ndash5(g))

4 Discussion

SCAPs are known as a kind of ideal candidates for dentaltissue engineering and have the characteristics of self-renewaland multilineage differentiation potential [9 10] Diversestudies have proved that SCAPs are able to differentiate intoosteoodontoblasts in vitro under appropriate conditions andformbonedentin-like tissues in vivo [24 25] Certainlymanysignaling pathways may be involved in the process of cellproliferation and differentiation including NF-120581B pathway

NF-120581B exists in the cytoplasm in a latent form bindingto inhibitory proteins termed inhibitory 120581B proteins (I120581Bs)[3 26] I120581B complex is the major regulator of NF-120581B pathwayand is composed of catalytic subunits IKK120572 and IKK120573 anda regulatory subunit IKK120574 TNF-120572 is generally known toactivate classical NF-120581B pathway [17] Exposure of cells tosuitable concentrations of TNF-120572 brings about the rapid

phosphorylation ubiquitination and proteolytic degradationof I120581B which allows NF-120581B to translocate to the nucleuswhere it subsequently regulates the gene transcription [27ndash29] BMS-345541 a selective inhibitor of the catalytic subunitsof IKK targets NF-120581B signaling pathway and downregulatesthe activity of NF-120581B [18 19] In this study the expressionof cytoplastic P-P65P-I120581B120572 was noticeably upregulated afterthe treatment of TNF-120572 and downregulated by the inhibitorBMS-345541 indicating the successful establishment of acellular model for the activation or suppression of canonicalNF-120581B pathway

Cell proliferation and migration were indispensable fortissue development and wound healing In the present studyNF-120581B-activated SCAPs exhibited an increased growth ratehigher proliferation index and more effective migrationin wound healing assay Moreover the migration distancesbetween thewound lines in inhibitor-treated cells were longerthan control cells after 24 hours of culture Together withthe weakened proliferation of inhibitor-treated cells it ispredictable that the canonical NF-120581B pathway has a positiveinfluence on the cell multiplication activity and motility

Previous studies have proved that various stimuli canexert an active role on the committed differentiation ofdental stem cells via NF-120581B pathway [28] In the presentstudy the odontoosteogenic capacity remarkably changedalong with the activation or blockage of classical NF-120581Bpathway Activator-treated SCAPs exhibited an enhancedALP activity increased calcium deposition and upregulatedexpression of odontoosteogenic genes and proteins whileinhibitor-treated cells presented the lower ALP activitydecreased mineralization and downregulated expression ofodontoosteoblast markers

It is widely recognized that ALP is a functionalmarker forosteoblast activity and bone formation [30] As an importantextracellular matrix of bone BSP is mainly secreted byosteoblasts whose expression is a crucial symbol in matrixdeposition and mineralization [31 32] OPN is anotherextracellular matrix protein expressed in numerous cell typesincluding odontoblasts in which OPN plays multifacetedroles in a variety of biological and pathological processessuch as osteogenic differentiation tooth mineralization anddental biofilm formation [33 34] OCN is the primarynoncollagenous protein in the bone cells secreted in the latestage of osteogenesis and known as a specific indicator ofosteogenic differentiation RUNX2 is the first transcriptionfactor required for determination of the osteoblast lineageand OSX acts as a downstream gene of RUNX2 that is highlyexpressed in the functional odontoosteoblasts [35] DSPPand DSP are well-known markers of odontoblasts highlyexpressed in dentin or predentin structures and essentialfor dentinogenesis [36] DMP-1 is an acidic extracellularmatrix protein that is primarily found in dentin and boneand has been implicated in dentin mineralization and signaltransduction in the process of odontogenesis [37]

In this study the expression of RUNX2RUNX2 reducedafter 3 days of incubation with the inhibitor BMS-345541and then gradually increased at day 7 while the expressionof DSPPDSP did not compromise after 3 days of incuba-tion but noticeably decreased at day 7 indicating that the

8 BioMed Research International

ALP

3 5 7

(day)

35

3

25

2

15

1

05

0

ControlActivator

MMActivator + MM

lowast

lowastlowastlowastlowastlowastlowast

(sig

ma u

nitm

inm

g pr

otei

n)

(a)

ControlActivator

MMActivator + MM

lowastlowast

lowastlowast

876543210

ALP

OCN BS

P

RUN

X2

OSX

DSP

P

OPN

DM

P-1

Gen

esG

APD

H

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastPCR 3d

(b)

lowast

lowast lowast

lowast

lowast lowastlowast

lowast

25

20

15

10

5

0

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

Gen

esG

APD

H

ControlActivator

MMActivator + MM

ALP

OCN BS

P

RUN

X2

OSX

DSP

P

OPN

DM

P-1

PCR 7d

(c)

MMControl Activator

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

Activator + MM

Western blot 3d

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

(d)

ControlActivator

MMActivator + MM

09

08

07

06

05

04

03

02

01

0

Prot

eins

ACT

IN

OCN BSP RUNX2 OSX DSP OPN DMP-1

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

lowast

lowast

lowast

(e)

MMControl Activator

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

Activator + MM

Western blot 7d

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

(f)

Figure 4 Continued

BioMed Research International 9

14

12

1

08

06

04

02

0

Prot

eins

ACT

IN

OCN BSP RUNX2 OSX DSP OPN DMP-1

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

ControlActivator

MMActivator + MM

(g)

Control Activator

MM Activator + MM

(h)

1

CPC06

05

04

03

02

01

0

lowast

lowastlowastlowast

ControlActivator

MMActivator + MM

(ng

calc

ium

mg

prot

ein)

(i)

Figure 4 Odontoosteogenic differentiation in canonical NF-120581B-activated SCAPs (a) ALP activity of SCAPs in control activator MM andactivator + MM groups at days 3 5 and 7 Values are the means plusmn SD 119899 = 6 lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 (b) Real-time RT-PCRanalysis for odontoosteogenic genes (BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in each group at day 7 (c) Real-time RT-PCRanalysis for odontoosteogenic genes (ALP BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in different groups at day 7 GAPDHserved as a housekeeping gene lowastlowast2minusΔΔCt ge 2 119875 lt 001 lowast1 lt 2minusΔΔCt lt 2 119875 lt 001 119899 = 3 (d) Western blot analyses for the odontoosteogenicproteins (BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in different groups at day 3 120573-ACTIN served as an internal control (e)Semiquantitative analysis demonstrated that the expression of BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1 was stronger in NF-120581Bpathway-activated SCAPs than those in control group at day 3 especially in the presence of mineralization-inducing media (f) Western blotanalyses for the odontoosteogenic proteins (BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in different groups at day 7 120573-ACTINwas used as an internal control (g) Semiquantitative analysis confirmed that the expression of BSP OCN RUNX2 OSX DSP OPN DSPand DMP-1 was upregulated in NF-120581B pathway-activated SCAPs at day 7 regardless of the presence or absence of mineralization-inducingmedia (h) Alizarin red staining of SCAPs after 14 days of induction in different groups (i) Quantitative calcium analysis showed that thecalcium content in activator and activator + MM groups was significantly higher than the control and MM groups respectively Values weredescribed as the means plusmn SD lowast119875 lt 005 lowastlowastlowast119875 lt 0001

expression of RUNX2RUNX2 and DSPPDSP tends to beinversely correlated [38] Moreover the dramatic differenceof these odontoosteogenic markers at both mRNA andprotein levels in response to the regulation of NF-120581B pathwaysuggests that activated canonical NF-120581B pathway favors theodontoosteoblastic differentiation of SCAPs particularly inthe presence of mineralization-inducing media

In summary the findings accumulated here confirmedthe potential involvement of canonical NF-120581B pathway inthe proliferationmigration and committed differentiation ofSCAPs in vitro Both activation and inhibition of the classicalNF-120581B pathway can bring about the permanent changes in

human stem cells Moreover it is commonly believed thatNF-120581B signaling plays a pivotal role not only in the progressof normal physiological process but also in the pathologicalprocess and dysfunction ofNF-120581B is linked to various humandiseases Thus proper balance of intracellular NF-120581B shouldbemaintained in the physiological conditions while intricateinteractivity between NF-120581B and other signaling pathwaysneeds to be extensively investigated

Conflict of Interests

The authors declare no conflict of interests

10 BioMed Research International

ControlInhibitor

MM

ALP3

25

2

15

1

05

03 5 7

(day)

Inhibitor + MM

lowastlowast lowastlowast

lowastlowast

lowast

lowast

(sig

ma u

nitm

inm

g pr

otei

n)

(a)

ControlInhibitor

35

3

25

2

15

1

05

0

Gen

esG

APD

H

ALP OCN BSP RUNX2 OSX DSPP OPN DMP-1

MMInhibitor + MM

lowastlowast

lowast

lowast

lowast lowastlowast

lowast

lowastlowast

lowastlowast

PCR 3d

(b)

ControlInhibitor

MM

1614121086420

Gen

esG

APD

H

ALP OCN BSP RUNX2 OSX DSPP OPN DMP-1

Inhibitor + MM

lowastlowast lowastlowast

lowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowastlowastlowastPCR 7d

(c)

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

MMInhibitorControl Inhibitor + MM

Western blot 3d

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

(d)

Prot

eins

ACT

IN

ControlInhibitor

MM

12

1

08

06

04

02

0OCN BSP RUNX2 OSX DSP OPN DMP-1

Inhibitor + MM

lowast

lowast lowast

lowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

(e)

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

MMInhibitorControl Inhibitor + MM

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

Western blot 7d

(f)

Figure 5 Continued

BioMed Research International 11

Prot

eins

ACT

IN

OCN BSP RUNX2 OSX DSP OPN DMP-1

ControlInhibitor MM

Inhibitor + MM

0504504

035030250201501005

0

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(g)

MM

InhibitorControl

Inhibitor + MM

(h)

06

05

04

03

02

01

0

lowast

lowastlowastCPC

1

MMInhibitorControl

Inhibitor + MM

(ng

calc

ium

mg

prot

ein)

(i)

Figure 5 Odontoosteogenic differentiation in canonical NF-120581B-inhibited SCAPs (a) ALP activity at different time points in different groups(ie control inhibitor MM and inhibitor +MM) (b) Real-time RT-PCR for the detection ofALP BSP OCN RUNX2 OSX DSP OPN DSPandDMP-1 at day 3ThemRNA levels were normalized toGAPDH (c) Real-time RT-PCR for the detection ofALP BSP OCN RUNX2 OSXDSP OPN DSP andDMP-1 of SCAPs respectively in control inhibitor MM and inhibitor +MMgroups at day 7 (d)The odontoosteogenicprotein levels of SCAPs in control inhibitor MM and inhibitor +MM groups were characterized by western blot analysis at day 3 120573-ACTINwas used as a loading control (e) Semiquantitative analysis demonstrated that the expression of BSP OCN RUNX2 OSX DSP OPN DSPand DMP-1 was more downregulated in NF-120581B pathway-inhibited SCAPs than those in control group at day 3 (f) The odontoosteogenicprotein levels of SCAPs in control inhibitor MM and inhibitor + MM groups were characterized by western blot analysis at day 7 (g)Semiquantitative analysis demonstrated that the expression of odontoosteogenic markers (BSP OCN RUNX2 OSX DSP OPN DSP andDMP-1) was significantly more downregulated in NF-120581B pathway-inhibited SCAPs than those in control group at day 7 (h) Alizarin redstaining of SCAPs after 14 days of osteogenic induction (i) Calcium quantification demonstrated the weaker calcium deposition in inhibitorand inhibitor + MM groups as compared with control and MM groups respectively

Acknowledgments

This work was supported by National Natural Science Foun-dation of China (no 81371144) Natural Science Foundationof Jiangsu Province (no BK20131392) and the Priority Aca-demic Program Development of Jiangsu Higher EducationInstitutions (PAPD no 2011-137)

References

[1] AKumar Y TakadaAMBoriek andB BAggarwal ldquoNuclearfactor-120581B its role in health and diseaserdquo Journal of MolecularMedicine vol 82 no 7 pp 434ndash448 2004

[2] F Chen V Castranova X Shi and LM Demers ldquoNew insightsinto the role of nuclear factor-120581B a ubiquitous transcription

factor in the initiation of diseasesrdquo Clinical Chemistry vol 45no 1 pp 7ndash17 1999

[3] C Gasparini and M Feldmann ldquoNF-120581B as a target for modu-lating inflammatory responsesrdquoCurrent Pharmaceutical Designvol 18 no 35 pp 5735ndash5745 2012

[4] A Ohazama and P T Sharpe ldquoTNF signalling in toothdevelopmentrdquo Current Opinion in Genetics amp Development vol14 no 5 pp 513ndash519 2004

[5] Y-H Kuan F-M Huang Y-C Li and Y-C Chang ldquoProin-flammatory activation ofmacrophages by bisphenol A-glycidyl-methacrylate involved NF120581B activation via PI3KAkt pathwayrdquoFood and Chemical Toxicology vol 50 no 11 pp 4003ndash40092012

[6] X Cai P Gong YHuang andY Lin ldquoNotch signalling pathwayin tooth development and adult dental cellsrdquo Cell Proliferationvol 44 no 6 pp 495ndash507 2011

12 BioMed Research International

[7] J-M Courtney J Blackburn and P T Sharpe ldquoThe Ectodys-plasin and NF120581B signalling pathways in odontogenesisrdquoArchives of Oral Biology vol 50 no 2 pp 159ndash163 2005

[8] W Sonoyama Y Liu T Yamaza et al ldquoCharacterization of theapical papilla and its residing stem cells from human immaturepermanent teeth a pilot studyrdquo Journal of Endodontics vol 34no 2 pp 166ndash171 2008

[9] W Sonoyama Y Liu D Fang et al ldquoMesenchymal stem cell-mediated functional tooth regeneration in swinerdquo PLoS ONEvol 1 no 1 article e79 2006

[10] H Chopra M K Hans and S Shetty ldquoStem cells-the hiddentreasure a strategic reviewrdquoDental Research Journal vol 10 no4 pp 421ndash427 2013

[11] R Dong R Yao J Du S Wang and Z Fan ldquoDepletion ofhistone demethylase KDM2A enhanced the adipogenic andchondrogenic differentiation potentials of stem cells from apicalpapillardquo Experimental Cell Research vol 319 no 18 pp 2874ndash2882 2013

[12] Y Wang M Yan Z Wang et al ldquoDental pulp stem cells fromtraumatically exposed pulps exhibited an enhanced osteogenicpotential and weakened odontogenic capacityrdquo Archives of OralBiology vol 58 no 11 pp 1709ndash1717 2013

[13] Y Wang M Yan Z Fan L Ma Y Yu and J Yu ldquoMineraltrioxide aggregate enhances the odontoosteogenic capacity ofstem cells from inflammatory dental pulps via NF-120581B pathwayrdquoOral Diseases 2013

[14] Y Wang M Yan Y Yu J Wu J Yu and Z Fan ldquoEstrogendeficiency inhibits the odontoosteogenic differentiation ofdental pulp stem cells via activation of the NF-120581B pathwayrdquo Celland Tissue Research vol 352 no 3 pp 551ndash559 2013

[15] Y Wang Y Zheng Z Wang et al ldquo10minus7M 17120573-oestradiolenhances odontoosteogenic potency of human dental pulpstem cells by activation of the NF-120581B pathwayrdquo Cell Prolifera-tion vol 46 no 6 pp 677ndash684 2013

[16] Z-G Lu H Liu T Yamaguchi Y Miki and K YoshidaldquoProtein kinase C120575 activates RelAp65 and nuclear factor-120581B signaling in response to tumor necrosis factor-120572rdquo CancerResearch vol 69 no 14 pp 5927ndash5935 2009

[17] G Bonizzi and M Karin ldquoThe two NF-120581B activation pathwaysand their role in innate and adaptive immunityrdquo Trends inImmunology vol 25 no 6 pp 280ndash288 2004

[18] K W McIntyre D J Shuster K M Gillooly et al ldquoA highlyselective inhibitor of I120581B kinase BMS-345541 blocks both jointinflammation and destruction in collagen-induced arthritis inmicerdquo Arthritis amp Rheumatism vol 48 no 9 pp 2652ndash26592003

[19] J R Burke M A Pattoli K R Gregor et al ldquoBMS-345541 is ahighly selective inhibitor of I120581B kinase that binds at an allostericsite of the enzyme and blocks NF-120581B-dependent transcriptionin micerdquoThe Journal of Biological Chemistry vol 278 no 3 pp1450ndash1456 2003

[20] F W Paula-Silva A Ghosh L A Silva and Y L Kapila ldquoTNF-120572 promotes an odontoblastic phenotype in dental pulp cellsrdquoJournal of Dental Research vol 88 no 4 pp 339ndash344 2009

[21] S Wang J Mu Z Fan et al ldquoInsulin-like growth factor 1 canpromote the osteogenic differentiation and osteogenesis of stemcells from apical papillardquo Stem Cell Research vol 8 no 3 pp346ndash356 2012

[22] Y Yu J Mu Z Fan et al ldquoInsulin-like growth factor 1 enhancesthe proliferation and osteogenic differentiation of human peri-odontal ligament stem cells via ERK and JNKMAPKpathwaysrdquoHistochemistry andCell Biology vol 137 no 4 pp 513ndash525 2012

[23] H Aonuma N Ogura K Takahashi et al ldquoCharacteristics andosteogenic differentiation of stemprogenitor cells in the humandental follicle analyzed by gene expression profilingrdquo Cell andTissue Research vol 350 no 2 pp 317ndash331 2012

[24] L Wang M Yan Y Wang et al ldquoProliferation andosteoodontoblastic differentiation of stem cells from dentalapical papilla in mineralization-inducing medium containingadditional KH

2PO4rdquo Cell Proliferation vol 46 no 2 pp

214ndash222 2013[25] G T-J Huang T Yamaza L D Shea et al ldquoStemProgenitor

cell-mediated de novo regeneration of dental pulp with newlydeposited continuous layer of dentin in an in vivomodelrdquoTissueEngineering A vol 16 no 2 pp 605ndash615 2010

[26] J-H Kwak J-K Jung and H Lee ldquoNuclear factor-120581 Binhibitors a patent review (2006ndash2010)rdquo Expert Opinion onTherapeutic Patents vol 21 no 12 pp 1897ndash1910 2011

[27] K Hess A Ushmorov J Fiedler R E Brenner and T WirthldquoTNF120572 promotes osteogenic differentiation of human mes-enchymal stem cells by triggering theNF-120581B signaling pathwayrdquoBone vol 45 no 2 pp 367ndash376 2009

[28] X Feng G Feng J Xing et al ldquoTNF-120572 triggers osteogenicdifferentiation of human dental pulp stem cells via the NF-120581Bsignalling pathwayrdquoCell Biology International vol 37 no 12 pp1267ndash1275 2013

[29] J Chang F Liu M Lee et al ldquoNF-120581B inhibits osteogenicdifferentiation of mesenchymal stem cells by promoting 120573-catenin degradationrdquo Proceedings of the National Academy ofSciences of the United States of America vol 110 no 23 pp9469ndash9474 2013

[30] C Shui and A Scutt ldquoMild heat shock induces proliferationalkaline phosphatase activity and mineralization in humanbone marrow stromal cells and Mg-63 cells in vitrordquo Journal ofBone and Mineral Research vol 16 no 4 pp 731ndash741 2001

[31] X Wei J Ling L Wu L Liu and Y Xiao ldquoExpressionof mineralization markers in dental pulp cellsrdquo Journal ofEndodontics vol 33 no 6 pp 703ndash708 2007

[32] S Z Khan E Kokubu K Matsuzaka and T Inoue ldquoBehaviourof rat-cultured dental pulp cells in three-dimensional collagentype-1 gel in vitro and in vivordquo Australian Endodontic Journalvol 39 no 3 pp 137ndash145 2013

[33] D-X Cao Z-J Li X-O Jiang et al ldquoOsteopontin as potentialbiomarker and therapeutic target in gastric and liver cancersrdquoWorld Journal of Gastroenterology vol 18 no 30 pp 3923ndash39302012

[34] S SchlaferMK Raarup P LWejse et al ldquoOsteopontin reducesbiofilm formation in a multi-species model of dental biofilmrdquoPloS ONE vol 7 no 8 Article ID e41534 2012

[35] S Chen J Gluhak-Heinrich Y HWang et al ldquoRunx2Osx andDspp in tooth developmentrdquo Journal of Dental Research vol 88no 10 pp 904ndash909 2009

[36] S Suzuki N Haruyama F Nishimura and A B KulkarnildquoDentin sialophosphoprotein and dentin matrix protein-1 twohighly phosphorylated proteins inmineralized tissuesrdquoArchivesof Oral Biology vol 57 no 9 pp 1165ndash1175 2012

[37] R S Prescott R Alsanea M I Fayad et al ldquoIn vivo fenerationof dental pulp-like tissue by using dental pulp stem cells a col-lagen scaffold and dentin matrix protein 1 after subcutaneoustransplantation in micerdquo Journal of Endodontics vol 34 no 4pp 421ndash426 2008

[38] T Komori ldquoRegulation of bone development and extracellularmatrix protein genes by RUNX2rdquo Cell and Tissue Research vol339 no 1 pp 189ndash195 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

8 BioMed Research International

ALP

3 5 7

(day)

35

3

25

2

15

1

05

0

ControlActivator

MMActivator + MM

lowast

lowastlowastlowastlowastlowastlowast

(sig

ma u

nitm

inm

g pr

otei

n)

(a)

ControlActivator

MMActivator + MM

lowastlowast

lowastlowast

876543210

ALP

OCN BS

P

RUN

X2

OSX

DSP

P

OPN

DM

P-1

Gen

esG

APD

H

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastPCR 3d

(b)

lowast

lowast lowast

lowast

lowast lowastlowast

lowast

25

20

15

10

5

0

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

Gen

esG

APD

H

ControlActivator

MMActivator + MM

ALP

OCN BS

P

RUN

X2

OSX

DSP

P

OPN

DM

P-1

PCR 7d

(c)

MMControl Activator

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

Activator + MM

Western blot 3d

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

(d)

ControlActivator

MMActivator + MM

09

08

07

06

05

04

03

02

01

0

Prot

eins

ACT

IN

OCN BSP RUNX2 OSX DSP OPN DMP-1

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

lowast

lowast

lowast

(e)

MMControl Activator

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

Activator + MM

Western blot 7d

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

(f)

Figure 4 Continued

BioMed Research International 9

14

12

1

08

06

04

02

0

Prot

eins

ACT

IN

OCN BSP RUNX2 OSX DSP OPN DMP-1

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

ControlActivator

MMActivator + MM

(g)

Control Activator

MM Activator + MM

(h)

1

CPC06

05

04

03

02

01

0

lowast

lowastlowastlowast

ControlActivator

MMActivator + MM

(ng

calc

ium

mg

prot

ein)

(i)

Figure 4 Odontoosteogenic differentiation in canonical NF-120581B-activated SCAPs (a) ALP activity of SCAPs in control activator MM andactivator + MM groups at days 3 5 and 7 Values are the means plusmn SD 119899 = 6 lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 (b) Real-time RT-PCRanalysis for odontoosteogenic genes (BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in each group at day 7 (c) Real-time RT-PCRanalysis for odontoosteogenic genes (ALP BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in different groups at day 7 GAPDHserved as a housekeeping gene lowastlowast2minusΔΔCt ge 2 119875 lt 001 lowast1 lt 2minusΔΔCt lt 2 119875 lt 001 119899 = 3 (d) Western blot analyses for the odontoosteogenicproteins (BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in different groups at day 3 120573-ACTIN served as an internal control (e)Semiquantitative analysis demonstrated that the expression of BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1 was stronger in NF-120581Bpathway-activated SCAPs than those in control group at day 3 especially in the presence of mineralization-inducing media (f) Western blotanalyses for the odontoosteogenic proteins (BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in different groups at day 7 120573-ACTINwas used as an internal control (g) Semiquantitative analysis confirmed that the expression of BSP OCN RUNX2 OSX DSP OPN DSPand DMP-1 was upregulated in NF-120581B pathway-activated SCAPs at day 7 regardless of the presence or absence of mineralization-inducingmedia (h) Alizarin red staining of SCAPs after 14 days of induction in different groups (i) Quantitative calcium analysis showed that thecalcium content in activator and activator + MM groups was significantly higher than the control and MM groups respectively Values weredescribed as the means plusmn SD lowast119875 lt 005 lowastlowastlowast119875 lt 0001

expression of RUNX2RUNX2 and DSPPDSP tends to beinversely correlated [38] Moreover the dramatic differenceof these odontoosteogenic markers at both mRNA andprotein levels in response to the regulation of NF-120581B pathwaysuggests that activated canonical NF-120581B pathway favors theodontoosteoblastic differentiation of SCAPs particularly inthe presence of mineralization-inducing media

In summary the findings accumulated here confirmedthe potential involvement of canonical NF-120581B pathway inthe proliferationmigration and committed differentiation ofSCAPs in vitro Both activation and inhibition of the classicalNF-120581B pathway can bring about the permanent changes in

human stem cells Moreover it is commonly believed thatNF-120581B signaling plays a pivotal role not only in the progressof normal physiological process but also in the pathologicalprocess and dysfunction ofNF-120581B is linked to various humandiseases Thus proper balance of intracellular NF-120581B shouldbemaintained in the physiological conditions while intricateinteractivity between NF-120581B and other signaling pathwaysneeds to be extensively investigated

Conflict of Interests

The authors declare no conflict of interests

10 BioMed Research International

ControlInhibitor

MM

ALP3

25

2

15

1

05

03 5 7

(day)

Inhibitor + MM

lowastlowast lowastlowast

lowastlowast

lowast

lowast

(sig

ma u

nitm

inm

g pr

otei

n)

(a)

ControlInhibitor

35

3

25

2

15

1

05

0

Gen

esG

APD

H

ALP OCN BSP RUNX2 OSX DSPP OPN DMP-1

MMInhibitor + MM

lowastlowast

lowast

lowast

lowast lowastlowast

lowast

lowastlowast

lowastlowast

PCR 3d

(b)

ControlInhibitor

MM

1614121086420

Gen

esG

APD

H

ALP OCN BSP RUNX2 OSX DSPP OPN DMP-1

Inhibitor + MM

lowastlowast lowastlowast

lowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowastlowastlowastPCR 7d

(c)

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

MMInhibitorControl Inhibitor + MM

Western blot 3d

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

(d)

Prot

eins

ACT

IN

ControlInhibitor

MM

12

1

08

06

04

02

0OCN BSP RUNX2 OSX DSP OPN DMP-1

Inhibitor + MM

lowast

lowast lowast

lowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

(e)

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

MMInhibitorControl Inhibitor + MM

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

Western blot 7d

(f)

Figure 5 Continued

BioMed Research International 11

Prot

eins

ACT

IN

OCN BSP RUNX2 OSX DSP OPN DMP-1

ControlInhibitor MM

Inhibitor + MM

0504504

035030250201501005

0

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(g)

MM

InhibitorControl

Inhibitor + MM

(h)

06

05

04

03

02

01

0

lowast

lowastlowastCPC

1

MMInhibitorControl

Inhibitor + MM

(ng

calc

ium

mg

prot

ein)

(i)

Figure 5 Odontoosteogenic differentiation in canonical NF-120581B-inhibited SCAPs (a) ALP activity at different time points in different groups(ie control inhibitor MM and inhibitor +MM) (b) Real-time RT-PCR for the detection ofALP BSP OCN RUNX2 OSX DSP OPN DSPandDMP-1 at day 3ThemRNA levels were normalized toGAPDH (c) Real-time RT-PCR for the detection ofALP BSP OCN RUNX2 OSXDSP OPN DSP andDMP-1 of SCAPs respectively in control inhibitor MM and inhibitor +MMgroups at day 7 (d)The odontoosteogenicprotein levels of SCAPs in control inhibitor MM and inhibitor +MM groups were characterized by western blot analysis at day 3 120573-ACTINwas used as a loading control (e) Semiquantitative analysis demonstrated that the expression of BSP OCN RUNX2 OSX DSP OPN DSPand DMP-1 was more downregulated in NF-120581B pathway-inhibited SCAPs than those in control group at day 3 (f) The odontoosteogenicprotein levels of SCAPs in control inhibitor MM and inhibitor + MM groups were characterized by western blot analysis at day 7 (g)Semiquantitative analysis demonstrated that the expression of odontoosteogenic markers (BSP OCN RUNX2 OSX DSP OPN DSP andDMP-1) was significantly more downregulated in NF-120581B pathway-inhibited SCAPs than those in control group at day 7 (h) Alizarin redstaining of SCAPs after 14 days of osteogenic induction (i) Calcium quantification demonstrated the weaker calcium deposition in inhibitorand inhibitor + MM groups as compared with control and MM groups respectively

Acknowledgments

This work was supported by National Natural Science Foun-dation of China (no 81371144) Natural Science Foundationof Jiangsu Province (no BK20131392) and the Priority Aca-demic Program Development of Jiangsu Higher EducationInstitutions (PAPD no 2011-137)

References

[1] AKumar Y TakadaAMBoriek andB BAggarwal ldquoNuclearfactor-120581B its role in health and diseaserdquo Journal of MolecularMedicine vol 82 no 7 pp 434ndash448 2004

[2] F Chen V Castranova X Shi and LM Demers ldquoNew insightsinto the role of nuclear factor-120581B a ubiquitous transcription

factor in the initiation of diseasesrdquo Clinical Chemistry vol 45no 1 pp 7ndash17 1999

[3] C Gasparini and M Feldmann ldquoNF-120581B as a target for modu-lating inflammatory responsesrdquoCurrent Pharmaceutical Designvol 18 no 35 pp 5735ndash5745 2012

[4] A Ohazama and P T Sharpe ldquoTNF signalling in toothdevelopmentrdquo Current Opinion in Genetics amp Development vol14 no 5 pp 513ndash519 2004

[5] Y-H Kuan F-M Huang Y-C Li and Y-C Chang ldquoProin-flammatory activation ofmacrophages by bisphenol A-glycidyl-methacrylate involved NF120581B activation via PI3KAkt pathwayrdquoFood and Chemical Toxicology vol 50 no 11 pp 4003ndash40092012

[6] X Cai P Gong YHuang andY Lin ldquoNotch signalling pathwayin tooth development and adult dental cellsrdquo Cell Proliferationvol 44 no 6 pp 495ndash507 2011

12 BioMed Research International

[7] J-M Courtney J Blackburn and P T Sharpe ldquoThe Ectodys-plasin and NF120581B signalling pathways in odontogenesisrdquoArchives of Oral Biology vol 50 no 2 pp 159ndash163 2005

[8] W Sonoyama Y Liu T Yamaza et al ldquoCharacterization of theapical papilla and its residing stem cells from human immaturepermanent teeth a pilot studyrdquo Journal of Endodontics vol 34no 2 pp 166ndash171 2008

[9] W Sonoyama Y Liu D Fang et al ldquoMesenchymal stem cell-mediated functional tooth regeneration in swinerdquo PLoS ONEvol 1 no 1 article e79 2006

[10] H Chopra M K Hans and S Shetty ldquoStem cells-the hiddentreasure a strategic reviewrdquoDental Research Journal vol 10 no4 pp 421ndash427 2013

[11] R Dong R Yao J Du S Wang and Z Fan ldquoDepletion ofhistone demethylase KDM2A enhanced the adipogenic andchondrogenic differentiation potentials of stem cells from apicalpapillardquo Experimental Cell Research vol 319 no 18 pp 2874ndash2882 2013

[12] Y Wang M Yan Z Wang et al ldquoDental pulp stem cells fromtraumatically exposed pulps exhibited an enhanced osteogenicpotential and weakened odontogenic capacityrdquo Archives of OralBiology vol 58 no 11 pp 1709ndash1717 2013

[13] Y Wang M Yan Z Fan L Ma Y Yu and J Yu ldquoMineraltrioxide aggregate enhances the odontoosteogenic capacity ofstem cells from inflammatory dental pulps via NF-120581B pathwayrdquoOral Diseases 2013

[14] Y Wang M Yan Y Yu J Wu J Yu and Z Fan ldquoEstrogendeficiency inhibits the odontoosteogenic differentiation ofdental pulp stem cells via activation of the NF-120581B pathwayrdquo Celland Tissue Research vol 352 no 3 pp 551ndash559 2013

[15] Y Wang Y Zheng Z Wang et al ldquo10minus7M 17120573-oestradiolenhances odontoosteogenic potency of human dental pulpstem cells by activation of the NF-120581B pathwayrdquo Cell Prolifera-tion vol 46 no 6 pp 677ndash684 2013

[16] Z-G Lu H Liu T Yamaguchi Y Miki and K YoshidaldquoProtein kinase C120575 activates RelAp65 and nuclear factor-120581B signaling in response to tumor necrosis factor-120572rdquo CancerResearch vol 69 no 14 pp 5927ndash5935 2009

[17] G Bonizzi and M Karin ldquoThe two NF-120581B activation pathwaysand their role in innate and adaptive immunityrdquo Trends inImmunology vol 25 no 6 pp 280ndash288 2004

[18] K W McIntyre D J Shuster K M Gillooly et al ldquoA highlyselective inhibitor of I120581B kinase BMS-345541 blocks both jointinflammation and destruction in collagen-induced arthritis inmicerdquo Arthritis amp Rheumatism vol 48 no 9 pp 2652ndash26592003

[19] J R Burke M A Pattoli K R Gregor et al ldquoBMS-345541 is ahighly selective inhibitor of I120581B kinase that binds at an allostericsite of the enzyme and blocks NF-120581B-dependent transcriptionin micerdquoThe Journal of Biological Chemistry vol 278 no 3 pp1450ndash1456 2003

[20] F W Paula-Silva A Ghosh L A Silva and Y L Kapila ldquoTNF-120572 promotes an odontoblastic phenotype in dental pulp cellsrdquoJournal of Dental Research vol 88 no 4 pp 339ndash344 2009

[21] S Wang J Mu Z Fan et al ldquoInsulin-like growth factor 1 canpromote the osteogenic differentiation and osteogenesis of stemcells from apical papillardquo Stem Cell Research vol 8 no 3 pp346ndash356 2012

[22] Y Yu J Mu Z Fan et al ldquoInsulin-like growth factor 1 enhancesthe proliferation and osteogenic differentiation of human peri-odontal ligament stem cells via ERK and JNKMAPKpathwaysrdquoHistochemistry andCell Biology vol 137 no 4 pp 513ndash525 2012

[23] H Aonuma N Ogura K Takahashi et al ldquoCharacteristics andosteogenic differentiation of stemprogenitor cells in the humandental follicle analyzed by gene expression profilingrdquo Cell andTissue Research vol 350 no 2 pp 317ndash331 2012

[24] L Wang M Yan Y Wang et al ldquoProliferation andosteoodontoblastic differentiation of stem cells from dentalapical papilla in mineralization-inducing medium containingadditional KH

2PO4rdquo Cell Proliferation vol 46 no 2 pp

214ndash222 2013[25] G T-J Huang T Yamaza L D Shea et al ldquoStemProgenitor

cell-mediated de novo regeneration of dental pulp with newlydeposited continuous layer of dentin in an in vivomodelrdquoTissueEngineering A vol 16 no 2 pp 605ndash615 2010

[26] J-H Kwak J-K Jung and H Lee ldquoNuclear factor-120581 Binhibitors a patent review (2006ndash2010)rdquo Expert Opinion onTherapeutic Patents vol 21 no 12 pp 1897ndash1910 2011

[27] K Hess A Ushmorov J Fiedler R E Brenner and T WirthldquoTNF120572 promotes osteogenic differentiation of human mes-enchymal stem cells by triggering theNF-120581B signaling pathwayrdquoBone vol 45 no 2 pp 367ndash376 2009

[28] X Feng G Feng J Xing et al ldquoTNF-120572 triggers osteogenicdifferentiation of human dental pulp stem cells via the NF-120581Bsignalling pathwayrdquoCell Biology International vol 37 no 12 pp1267ndash1275 2013

[29] J Chang F Liu M Lee et al ldquoNF-120581B inhibits osteogenicdifferentiation of mesenchymal stem cells by promoting 120573-catenin degradationrdquo Proceedings of the National Academy ofSciences of the United States of America vol 110 no 23 pp9469ndash9474 2013

[30] C Shui and A Scutt ldquoMild heat shock induces proliferationalkaline phosphatase activity and mineralization in humanbone marrow stromal cells and Mg-63 cells in vitrordquo Journal ofBone and Mineral Research vol 16 no 4 pp 731ndash741 2001

[31] X Wei J Ling L Wu L Liu and Y Xiao ldquoExpressionof mineralization markers in dental pulp cellsrdquo Journal ofEndodontics vol 33 no 6 pp 703ndash708 2007

[32] S Z Khan E Kokubu K Matsuzaka and T Inoue ldquoBehaviourof rat-cultured dental pulp cells in three-dimensional collagentype-1 gel in vitro and in vivordquo Australian Endodontic Journalvol 39 no 3 pp 137ndash145 2013

[33] D-X Cao Z-J Li X-O Jiang et al ldquoOsteopontin as potentialbiomarker and therapeutic target in gastric and liver cancersrdquoWorld Journal of Gastroenterology vol 18 no 30 pp 3923ndash39302012

[34] S SchlaferMK Raarup P LWejse et al ldquoOsteopontin reducesbiofilm formation in a multi-species model of dental biofilmrdquoPloS ONE vol 7 no 8 Article ID e41534 2012

[35] S Chen J Gluhak-Heinrich Y HWang et al ldquoRunx2Osx andDspp in tooth developmentrdquo Journal of Dental Research vol 88no 10 pp 904ndash909 2009

[36] S Suzuki N Haruyama F Nishimura and A B KulkarnildquoDentin sialophosphoprotein and dentin matrix protein-1 twohighly phosphorylated proteins inmineralized tissuesrdquoArchivesof Oral Biology vol 57 no 9 pp 1165ndash1175 2012

[37] R S Prescott R Alsanea M I Fayad et al ldquoIn vivo fenerationof dental pulp-like tissue by using dental pulp stem cells a col-lagen scaffold and dentin matrix protein 1 after subcutaneoustransplantation in micerdquo Journal of Endodontics vol 34 no 4pp 421ndash426 2008

[38] T Komori ldquoRegulation of bone development and extracellularmatrix protein genes by RUNX2rdquo Cell and Tissue Research vol339 no 1 pp 189ndash195 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 9

14

12

1

08

06

04

02

0

Prot

eins

ACT

IN

OCN BSP RUNX2 OSX DSP OPN DMP-1

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

ControlActivator

MMActivator + MM

(g)

Control Activator

MM Activator + MM

(h)

1

CPC06

05

04

03

02

01

0

lowast

lowastlowastlowast

ControlActivator

MMActivator + MM

(ng

calc

ium

mg

prot

ein)

(i)

Figure 4 Odontoosteogenic differentiation in canonical NF-120581B-activated SCAPs (a) ALP activity of SCAPs in control activator MM andactivator + MM groups at days 3 5 and 7 Values are the means plusmn SD 119899 = 6 lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 (b) Real-time RT-PCRanalysis for odontoosteogenic genes (BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in each group at day 7 (c) Real-time RT-PCRanalysis for odontoosteogenic genes (ALP BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in different groups at day 7 GAPDHserved as a housekeeping gene lowastlowast2minusΔΔCt ge 2 119875 lt 001 lowast1 lt 2minusΔΔCt lt 2 119875 lt 001 119899 = 3 (d) Western blot analyses for the odontoosteogenicproteins (BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in different groups at day 3 120573-ACTIN served as an internal control (e)Semiquantitative analysis demonstrated that the expression of BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1 was stronger in NF-120581Bpathway-activated SCAPs than those in control group at day 3 especially in the presence of mineralization-inducing media (f) Western blotanalyses for the odontoosteogenic proteins (BSP OCN RUNX2 OSX DSP OPN DSP and DMP-1) in different groups at day 7 120573-ACTINwas used as an internal control (g) Semiquantitative analysis confirmed that the expression of BSP OCN RUNX2 OSX DSP OPN DSPand DMP-1 was upregulated in NF-120581B pathway-activated SCAPs at day 7 regardless of the presence or absence of mineralization-inducingmedia (h) Alizarin red staining of SCAPs after 14 days of induction in different groups (i) Quantitative calcium analysis showed that thecalcium content in activator and activator + MM groups was significantly higher than the control and MM groups respectively Values weredescribed as the means plusmn SD lowast119875 lt 005 lowastlowastlowast119875 lt 0001

expression of RUNX2RUNX2 and DSPPDSP tends to beinversely correlated [38] Moreover the dramatic differenceof these odontoosteogenic markers at both mRNA andprotein levels in response to the regulation of NF-120581B pathwaysuggests that activated canonical NF-120581B pathway favors theodontoosteoblastic differentiation of SCAPs particularly inthe presence of mineralization-inducing media

In summary the findings accumulated here confirmedthe potential involvement of canonical NF-120581B pathway inthe proliferationmigration and committed differentiation ofSCAPs in vitro Both activation and inhibition of the classicalNF-120581B pathway can bring about the permanent changes in

human stem cells Moreover it is commonly believed thatNF-120581B signaling plays a pivotal role not only in the progressof normal physiological process but also in the pathologicalprocess and dysfunction ofNF-120581B is linked to various humandiseases Thus proper balance of intracellular NF-120581B shouldbemaintained in the physiological conditions while intricateinteractivity between NF-120581B and other signaling pathwaysneeds to be extensively investigated

Conflict of Interests

The authors declare no conflict of interests

10 BioMed Research International

ControlInhibitor

MM

ALP3

25

2

15

1

05

03 5 7

(day)

Inhibitor + MM

lowastlowast lowastlowast

lowastlowast

lowast

lowast

(sig

ma u

nitm

inm

g pr

otei

n)

(a)

ControlInhibitor

35

3

25

2

15

1

05

0

Gen

esG

APD

H

ALP OCN BSP RUNX2 OSX DSPP OPN DMP-1

MMInhibitor + MM

lowastlowast

lowast

lowast

lowast lowastlowast

lowast

lowastlowast

lowastlowast

PCR 3d

(b)

ControlInhibitor

MM

1614121086420

Gen

esG

APD

H

ALP OCN BSP RUNX2 OSX DSPP OPN DMP-1

Inhibitor + MM

lowastlowast lowastlowast

lowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowastlowastlowastPCR 7d

(c)

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

MMInhibitorControl Inhibitor + MM

Western blot 3d

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

(d)

Prot

eins

ACT

IN

ControlInhibitor

MM

12

1

08

06

04

02

0OCN BSP RUNX2 OSX DSP OPN DMP-1

Inhibitor + MM

lowast

lowast lowast

lowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

(e)

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

MMInhibitorControl Inhibitor + MM

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

Western blot 7d

(f)

Figure 5 Continued

BioMed Research International 11

Prot

eins

ACT

IN

OCN BSP RUNX2 OSX DSP OPN DMP-1

ControlInhibitor MM

Inhibitor + MM

0504504

035030250201501005

0

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(g)

MM

InhibitorControl

Inhibitor + MM

(h)

06

05

04

03

02

01

0

lowast

lowastlowastCPC

1

MMInhibitorControl

Inhibitor + MM

(ng

calc

ium

mg

prot

ein)

(i)

Figure 5 Odontoosteogenic differentiation in canonical NF-120581B-inhibited SCAPs (a) ALP activity at different time points in different groups(ie control inhibitor MM and inhibitor +MM) (b) Real-time RT-PCR for the detection ofALP BSP OCN RUNX2 OSX DSP OPN DSPandDMP-1 at day 3ThemRNA levels were normalized toGAPDH (c) Real-time RT-PCR for the detection ofALP BSP OCN RUNX2 OSXDSP OPN DSP andDMP-1 of SCAPs respectively in control inhibitor MM and inhibitor +MMgroups at day 7 (d)The odontoosteogenicprotein levels of SCAPs in control inhibitor MM and inhibitor +MM groups were characterized by western blot analysis at day 3 120573-ACTINwas used as a loading control (e) Semiquantitative analysis demonstrated that the expression of BSP OCN RUNX2 OSX DSP OPN DSPand DMP-1 was more downregulated in NF-120581B pathway-inhibited SCAPs than those in control group at day 3 (f) The odontoosteogenicprotein levels of SCAPs in control inhibitor MM and inhibitor + MM groups were characterized by western blot analysis at day 7 (g)Semiquantitative analysis demonstrated that the expression of odontoosteogenic markers (BSP OCN RUNX2 OSX DSP OPN DSP andDMP-1) was significantly more downregulated in NF-120581B pathway-inhibited SCAPs than those in control group at day 7 (h) Alizarin redstaining of SCAPs after 14 days of osteogenic induction (i) Calcium quantification demonstrated the weaker calcium deposition in inhibitorand inhibitor + MM groups as compared with control and MM groups respectively

Acknowledgments

This work was supported by National Natural Science Foun-dation of China (no 81371144) Natural Science Foundationof Jiangsu Province (no BK20131392) and the Priority Aca-demic Program Development of Jiangsu Higher EducationInstitutions (PAPD no 2011-137)

References

[1] AKumar Y TakadaAMBoriek andB BAggarwal ldquoNuclearfactor-120581B its role in health and diseaserdquo Journal of MolecularMedicine vol 82 no 7 pp 434ndash448 2004

[2] F Chen V Castranova X Shi and LM Demers ldquoNew insightsinto the role of nuclear factor-120581B a ubiquitous transcription

factor in the initiation of diseasesrdquo Clinical Chemistry vol 45no 1 pp 7ndash17 1999

[3] C Gasparini and M Feldmann ldquoNF-120581B as a target for modu-lating inflammatory responsesrdquoCurrent Pharmaceutical Designvol 18 no 35 pp 5735ndash5745 2012

[4] A Ohazama and P T Sharpe ldquoTNF signalling in toothdevelopmentrdquo Current Opinion in Genetics amp Development vol14 no 5 pp 513ndash519 2004

[5] Y-H Kuan F-M Huang Y-C Li and Y-C Chang ldquoProin-flammatory activation ofmacrophages by bisphenol A-glycidyl-methacrylate involved NF120581B activation via PI3KAkt pathwayrdquoFood and Chemical Toxicology vol 50 no 11 pp 4003ndash40092012

[6] X Cai P Gong YHuang andY Lin ldquoNotch signalling pathwayin tooth development and adult dental cellsrdquo Cell Proliferationvol 44 no 6 pp 495ndash507 2011

12 BioMed Research International

[7] J-M Courtney J Blackburn and P T Sharpe ldquoThe Ectodys-plasin and NF120581B signalling pathways in odontogenesisrdquoArchives of Oral Biology vol 50 no 2 pp 159ndash163 2005

[8] W Sonoyama Y Liu T Yamaza et al ldquoCharacterization of theapical papilla and its residing stem cells from human immaturepermanent teeth a pilot studyrdquo Journal of Endodontics vol 34no 2 pp 166ndash171 2008

[9] W Sonoyama Y Liu D Fang et al ldquoMesenchymal stem cell-mediated functional tooth regeneration in swinerdquo PLoS ONEvol 1 no 1 article e79 2006

[10] H Chopra M K Hans and S Shetty ldquoStem cells-the hiddentreasure a strategic reviewrdquoDental Research Journal vol 10 no4 pp 421ndash427 2013

[11] R Dong R Yao J Du S Wang and Z Fan ldquoDepletion ofhistone demethylase KDM2A enhanced the adipogenic andchondrogenic differentiation potentials of stem cells from apicalpapillardquo Experimental Cell Research vol 319 no 18 pp 2874ndash2882 2013

[12] Y Wang M Yan Z Wang et al ldquoDental pulp stem cells fromtraumatically exposed pulps exhibited an enhanced osteogenicpotential and weakened odontogenic capacityrdquo Archives of OralBiology vol 58 no 11 pp 1709ndash1717 2013

[13] Y Wang M Yan Z Fan L Ma Y Yu and J Yu ldquoMineraltrioxide aggregate enhances the odontoosteogenic capacity ofstem cells from inflammatory dental pulps via NF-120581B pathwayrdquoOral Diseases 2013

[14] Y Wang M Yan Y Yu J Wu J Yu and Z Fan ldquoEstrogendeficiency inhibits the odontoosteogenic differentiation ofdental pulp stem cells via activation of the NF-120581B pathwayrdquo Celland Tissue Research vol 352 no 3 pp 551ndash559 2013

[15] Y Wang Y Zheng Z Wang et al ldquo10minus7M 17120573-oestradiolenhances odontoosteogenic potency of human dental pulpstem cells by activation of the NF-120581B pathwayrdquo Cell Prolifera-tion vol 46 no 6 pp 677ndash684 2013

[16] Z-G Lu H Liu T Yamaguchi Y Miki and K YoshidaldquoProtein kinase C120575 activates RelAp65 and nuclear factor-120581B signaling in response to tumor necrosis factor-120572rdquo CancerResearch vol 69 no 14 pp 5927ndash5935 2009

[17] G Bonizzi and M Karin ldquoThe two NF-120581B activation pathwaysand their role in innate and adaptive immunityrdquo Trends inImmunology vol 25 no 6 pp 280ndash288 2004

[18] K W McIntyre D J Shuster K M Gillooly et al ldquoA highlyselective inhibitor of I120581B kinase BMS-345541 blocks both jointinflammation and destruction in collagen-induced arthritis inmicerdquo Arthritis amp Rheumatism vol 48 no 9 pp 2652ndash26592003

[19] J R Burke M A Pattoli K R Gregor et al ldquoBMS-345541 is ahighly selective inhibitor of I120581B kinase that binds at an allostericsite of the enzyme and blocks NF-120581B-dependent transcriptionin micerdquoThe Journal of Biological Chemistry vol 278 no 3 pp1450ndash1456 2003

[20] F W Paula-Silva A Ghosh L A Silva and Y L Kapila ldquoTNF-120572 promotes an odontoblastic phenotype in dental pulp cellsrdquoJournal of Dental Research vol 88 no 4 pp 339ndash344 2009

[21] S Wang J Mu Z Fan et al ldquoInsulin-like growth factor 1 canpromote the osteogenic differentiation and osteogenesis of stemcells from apical papillardquo Stem Cell Research vol 8 no 3 pp346ndash356 2012

[22] Y Yu J Mu Z Fan et al ldquoInsulin-like growth factor 1 enhancesthe proliferation and osteogenic differentiation of human peri-odontal ligament stem cells via ERK and JNKMAPKpathwaysrdquoHistochemistry andCell Biology vol 137 no 4 pp 513ndash525 2012

[23] H Aonuma N Ogura K Takahashi et al ldquoCharacteristics andosteogenic differentiation of stemprogenitor cells in the humandental follicle analyzed by gene expression profilingrdquo Cell andTissue Research vol 350 no 2 pp 317ndash331 2012

[24] L Wang M Yan Y Wang et al ldquoProliferation andosteoodontoblastic differentiation of stem cells from dentalapical papilla in mineralization-inducing medium containingadditional KH

2PO4rdquo Cell Proliferation vol 46 no 2 pp

214ndash222 2013[25] G T-J Huang T Yamaza L D Shea et al ldquoStemProgenitor

cell-mediated de novo regeneration of dental pulp with newlydeposited continuous layer of dentin in an in vivomodelrdquoTissueEngineering A vol 16 no 2 pp 605ndash615 2010

[26] J-H Kwak J-K Jung and H Lee ldquoNuclear factor-120581 Binhibitors a patent review (2006ndash2010)rdquo Expert Opinion onTherapeutic Patents vol 21 no 12 pp 1897ndash1910 2011

[27] K Hess A Ushmorov J Fiedler R E Brenner and T WirthldquoTNF120572 promotes osteogenic differentiation of human mes-enchymal stem cells by triggering theNF-120581B signaling pathwayrdquoBone vol 45 no 2 pp 367ndash376 2009

[28] X Feng G Feng J Xing et al ldquoTNF-120572 triggers osteogenicdifferentiation of human dental pulp stem cells via the NF-120581Bsignalling pathwayrdquoCell Biology International vol 37 no 12 pp1267ndash1275 2013

[29] J Chang F Liu M Lee et al ldquoNF-120581B inhibits osteogenicdifferentiation of mesenchymal stem cells by promoting 120573-catenin degradationrdquo Proceedings of the National Academy ofSciences of the United States of America vol 110 no 23 pp9469ndash9474 2013

[30] C Shui and A Scutt ldquoMild heat shock induces proliferationalkaline phosphatase activity and mineralization in humanbone marrow stromal cells and Mg-63 cells in vitrordquo Journal ofBone and Mineral Research vol 16 no 4 pp 731ndash741 2001

[31] X Wei J Ling L Wu L Liu and Y Xiao ldquoExpressionof mineralization markers in dental pulp cellsrdquo Journal ofEndodontics vol 33 no 6 pp 703ndash708 2007

[32] S Z Khan E Kokubu K Matsuzaka and T Inoue ldquoBehaviourof rat-cultured dental pulp cells in three-dimensional collagentype-1 gel in vitro and in vivordquo Australian Endodontic Journalvol 39 no 3 pp 137ndash145 2013

[33] D-X Cao Z-J Li X-O Jiang et al ldquoOsteopontin as potentialbiomarker and therapeutic target in gastric and liver cancersrdquoWorld Journal of Gastroenterology vol 18 no 30 pp 3923ndash39302012

[34] S SchlaferMK Raarup P LWejse et al ldquoOsteopontin reducesbiofilm formation in a multi-species model of dental biofilmrdquoPloS ONE vol 7 no 8 Article ID e41534 2012

[35] S Chen J Gluhak-Heinrich Y HWang et al ldquoRunx2Osx andDspp in tooth developmentrdquo Journal of Dental Research vol 88no 10 pp 904ndash909 2009

[36] S Suzuki N Haruyama F Nishimura and A B KulkarnildquoDentin sialophosphoprotein and dentin matrix protein-1 twohighly phosphorylated proteins inmineralized tissuesrdquoArchivesof Oral Biology vol 57 no 9 pp 1165ndash1175 2012

[37] R S Prescott R Alsanea M I Fayad et al ldquoIn vivo fenerationof dental pulp-like tissue by using dental pulp stem cells a col-lagen scaffold and dentin matrix protein 1 after subcutaneoustransplantation in micerdquo Journal of Endodontics vol 34 no 4pp 421ndash426 2008

[38] T Komori ldquoRegulation of bone development and extracellularmatrix protein genes by RUNX2rdquo Cell and Tissue Research vol339 no 1 pp 189ndash195 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

10 BioMed Research International

ControlInhibitor

MM

ALP3

25

2

15

1

05

03 5 7

(day)

Inhibitor + MM

lowastlowast lowastlowast

lowastlowast

lowast

lowast

(sig

ma u

nitm

inm

g pr

otei

n)

(a)

ControlInhibitor

35

3

25

2

15

1

05

0

Gen

esG

APD

H

ALP OCN BSP RUNX2 OSX DSPP OPN DMP-1

MMInhibitor + MM

lowastlowast

lowast

lowast

lowast lowastlowast

lowast

lowastlowast

lowastlowast

PCR 3d

(b)

ControlInhibitor

MM

1614121086420

Gen

esG

APD

H

ALP OCN BSP RUNX2 OSX DSPP OPN DMP-1

Inhibitor + MM

lowastlowast lowastlowast

lowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowastlowastlowastPCR 7d

(c)

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

MMInhibitorControl Inhibitor + MM

Western blot 3d

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

(d)

Prot

eins

ACT

IN

ControlInhibitor

MM

12

1

08

06

04

02

0OCN BSP RUNX2 OSX DSP OPN DMP-1

Inhibitor + MM

lowast

lowast lowast

lowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

(e)

130kDa

66kDa

53kDa

46kDa

37kDa

35kDa

22kDa

43kDa

MMInhibitorControl Inhibitor + MM

DMP-1

OPN

DSP

OSX

RUNX2

BSP

OCN

ACTIN

Western blot 7d

(f)

Figure 5 Continued

BioMed Research International 11

Prot

eins

ACT

IN

OCN BSP RUNX2 OSX DSP OPN DMP-1

ControlInhibitor MM

Inhibitor + MM

0504504

035030250201501005

0

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(g)

MM

InhibitorControl

Inhibitor + MM

(h)

06

05

04

03

02

01

0

lowast

lowastlowastCPC

1

MMInhibitorControl

Inhibitor + MM

(ng

calc

ium

mg

prot

ein)

(i)

Figure 5 Odontoosteogenic differentiation in canonical NF-120581B-inhibited SCAPs (a) ALP activity at different time points in different groups(ie control inhibitor MM and inhibitor +MM) (b) Real-time RT-PCR for the detection ofALP BSP OCN RUNX2 OSX DSP OPN DSPandDMP-1 at day 3ThemRNA levels were normalized toGAPDH (c) Real-time RT-PCR for the detection ofALP BSP OCN RUNX2 OSXDSP OPN DSP andDMP-1 of SCAPs respectively in control inhibitor MM and inhibitor +MMgroups at day 7 (d)The odontoosteogenicprotein levels of SCAPs in control inhibitor MM and inhibitor +MM groups were characterized by western blot analysis at day 3 120573-ACTINwas used as a loading control (e) Semiquantitative analysis demonstrated that the expression of BSP OCN RUNX2 OSX DSP OPN DSPand DMP-1 was more downregulated in NF-120581B pathway-inhibited SCAPs than those in control group at day 3 (f) The odontoosteogenicprotein levels of SCAPs in control inhibitor MM and inhibitor + MM groups were characterized by western blot analysis at day 7 (g)Semiquantitative analysis demonstrated that the expression of odontoosteogenic markers (BSP OCN RUNX2 OSX DSP OPN DSP andDMP-1) was significantly more downregulated in NF-120581B pathway-inhibited SCAPs than those in control group at day 7 (h) Alizarin redstaining of SCAPs after 14 days of osteogenic induction (i) Calcium quantification demonstrated the weaker calcium deposition in inhibitorand inhibitor + MM groups as compared with control and MM groups respectively

Acknowledgments

This work was supported by National Natural Science Foun-dation of China (no 81371144) Natural Science Foundationof Jiangsu Province (no BK20131392) and the Priority Aca-demic Program Development of Jiangsu Higher EducationInstitutions (PAPD no 2011-137)

References

[1] AKumar Y TakadaAMBoriek andB BAggarwal ldquoNuclearfactor-120581B its role in health and diseaserdquo Journal of MolecularMedicine vol 82 no 7 pp 434ndash448 2004

[2] F Chen V Castranova X Shi and LM Demers ldquoNew insightsinto the role of nuclear factor-120581B a ubiquitous transcription

factor in the initiation of diseasesrdquo Clinical Chemistry vol 45no 1 pp 7ndash17 1999

[3] C Gasparini and M Feldmann ldquoNF-120581B as a target for modu-lating inflammatory responsesrdquoCurrent Pharmaceutical Designvol 18 no 35 pp 5735ndash5745 2012

[4] A Ohazama and P T Sharpe ldquoTNF signalling in toothdevelopmentrdquo Current Opinion in Genetics amp Development vol14 no 5 pp 513ndash519 2004

[5] Y-H Kuan F-M Huang Y-C Li and Y-C Chang ldquoProin-flammatory activation ofmacrophages by bisphenol A-glycidyl-methacrylate involved NF120581B activation via PI3KAkt pathwayrdquoFood and Chemical Toxicology vol 50 no 11 pp 4003ndash40092012

[6] X Cai P Gong YHuang andY Lin ldquoNotch signalling pathwayin tooth development and adult dental cellsrdquo Cell Proliferationvol 44 no 6 pp 495ndash507 2011

12 BioMed Research International

[7] J-M Courtney J Blackburn and P T Sharpe ldquoThe Ectodys-plasin and NF120581B signalling pathways in odontogenesisrdquoArchives of Oral Biology vol 50 no 2 pp 159ndash163 2005

[8] W Sonoyama Y Liu T Yamaza et al ldquoCharacterization of theapical papilla and its residing stem cells from human immaturepermanent teeth a pilot studyrdquo Journal of Endodontics vol 34no 2 pp 166ndash171 2008

[9] W Sonoyama Y Liu D Fang et al ldquoMesenchymal stem cell-mediated functional tooth regeneration in swinerdquo PLoS ONEvol 1 no 1 article e79 2006

[10] H Chopra M K Hans and S Shetty ldquoStem cells-the hiddentreasure a strategic reviewrdquoDental Research Journal vol 10 no4 pp 421ndash427 2013

[11] R Dong R Yao J Du S Wang and Z Fan ldquoDepletion ofhistone demethylase KDM2A enhanced the adipogenic andchondrogenic differentiation potentials of stem cells from apicalpapillardquo Experimental Cell Research vol 319 no 18 pp 2874ndash2882 2013

[12] Y Wang M Yan Z Wang et al ldquoDental pulp stem cells fromtraumatically exposed pulps exhibited an enhanced osteogenicpotential and weakened odontogenic capacityrdquo Archives of OralBiology vol 58 no 11 pp 1709ndash1717 2013

[13] Y Wang M Yan Z Fan L Ma Y Yu and J Yu ldquoMineraltrioxide aggregate enhances the odontoosteogenic capacity ofstem cells from inflammatory dental pulps via NF-120581B pathwayrdquoOral Diseases 2013

[14] Y Wang M Yan Y Yu J Wu J Yu and Z Fan ldquoEstrogendeficiency inhibits the odontoosteogenic differentiation ofdental pulp stem cells via activation of the NF-120581B pathwayrdquo Celland Tissue Research vol 352 no 3 pp 551ndash559 2013

[15] Y Wang Y Zheng Z Wang et al ldquo10minus7M 17120573-oestradiolenhances odontoosteogenic potency of human dental pulpstem cells by activation of the NF-120581B pathwayrdquo Cell Prolifera-tion vol 46 no 6 pp 677ndash684 2013

[16] Z-G Lu H Liu T Yamaguchi Y Miki and K YoshidaldquoProtein kinase C120575 activates RelAp65 and nuclear factor-120581B signaling in response to tumor necrosis factor-120572rdquo CancerResearch vol 69 no 14 pp 5927ndash5935 2009

[17] G Bonizzi and M Karin ldquoThe two NF-120581B activation pathwaysand their role in innate and adaptive immunityrdquo Trends inImmunology vol 25 no 6 pp 280ndash288 2004

[18] K W McIntyre D J Shuster K M Gillooly et al ldquoA highlyselective inhibitor of I120581B kinase BMS-345541 blocks both jointinflammation and destruction in collagen-induced arthritis inmicerdquo Arthritis amp Rheumatism vol 48 no 9 pp 2652ndash26592003

[19] J R Burke M A Pattoli K R Gregor et al ldquoBMS-345541 is ahighly selective inhibitor of I120581B kinase that binds at an allostericsite of the enzyme and blocks NF-120581B-dependent transcriptionin micerdquoThe Journal of Biological Chemistry vol 278 no 3 pp1450ndash1456 2003

[20] F W Paula-Silva A Ghosh L A Silva and Y L Kapila ldquoTNF-120572 promotes an odontoblastic phenotype in dental pulp cellsrdquoJournal of Dental Research vol 88 no 4 pp 339ndash344 2009

[21] S Wang J Mu Z Fan et al ldquoInsulin-like growth factor 1 canpromote the osteogenic differentiation and osteogenesis of stemcells from apical papillardquo Stem Cell Research vol 8 no 3 pp346ndash356 2012

[22] Y Yu J Mu Z Fan et al ldquoInsulin-like growth factor 1 enhancesthe proliferation and osteogenic differentiation of human peri-odontal ligament stem cells via ERK and JNKMAPKpathwaysrdquoHistochemistry andCell Biology vol 137 no 4 pp 513ndash525 2012

[23] H Aonuma N Ogura K Takahashi et al ldquoCharacteristics andosteogenic differentiation of stemprogenitor cells in the humandental follicle analyzed by gene expression profilingrdquo Cell andTissue Research vol 350 no 2 pp 317ndash331 2012

[24] L Wang M Yan Y Wang et al ldquoProliferation andosteoodontoblastic differentiation of stem cells from dentalapical papilla in mineralization-inducing medium containingadditional KH

2PO4rdquo Cell Proliferation vol 46 no 2 pp

214ndash222 2013[25] G T-J Huang T Yamaza L D Shea et al ldquoStemProgenitor

cell-mediated de novo regeneration of dental pulp with newlydeposited continuous layer of dentin in an in vivomodelrdquoTissueEngineering A vol 16 no 2 pp 605ndash615 2010

[26] J-H Kwak J-K Jung and H Lee ldquoNuclear factor-120581 Binhibitors a patent review (2006ndash2010)rdquo Expert Opinion onTherapeutic Patents vol 21 no 12 pp 1897ndash1910 2011

[27] K Hess A Ushmorov J Fiedler R E Brenner and T WirthldquoTNF120572 promotes osteogenic differentiation of human mes-enchymal stem cells by triggering theNF-120581B signaling pathwayrdquoBone vol 45 no 2 pp 367ndash376 2009

[28] X Feng G Feng J Xing et al ldquoTNF-120572 triggers osteogenicdifferentiation of human dental pulp stem cells via the NF-120581Bsignalling pathwayrdquoCell Biology International vol 37 no 12 pp1267ndash1275 2013

[29] J Chang F Liu M Lee et al ldquoNF-120581B inhibits osteogenicdifferentiation of mesenchymal stem cells by promoting 120573-catenin degradationrdquo Proceedings of the National Academy ofSciences of the United States of America vol 110 no 23 pp9469ndash9474 2013

[30] C Shui and A Scutt ldquoMild heat shock induces proliferationalkaline phosphatase activity and mineralization in humanbone marrow stromal cells and Mg-63 cells in vitrordquo Journal ofBone and Mineral Research vol 16 no 4 pp 731ndash741 2001

[31] X Wei J Ling L Wu L Liu and Y Xiao ldquoExpressionof mineralization markers in dental pulp cellsrdquo Journal ofEndodontics vol 33 no 6 pp 703ndash708 2007

[32] S Z Khan E Kokubu K Matsuzaka and T Inoue ldquoBehaviourof rat-cultured dental pulp cells in three-dimensional collagentype-1 gel in vitro and in vivordquo Australian Endodontic Journalvol 39 no 3 pp 137ndash145 2013

[33] D-X Cao Z-J Li X-O Jiang et al ldquoOsteopontin as potentialbiomarker and therapeutic target in gastric and liver cancersrdquoWorld Journal of Gastroenterology vol 18 no 30 pp 3923ndash39302012

[34] S SchlaferMK Raarup P LWejse et al ldquoOsteopontin reducesbiofilm formation in a multi-species model of dental biofilmrdquoPloS ONE vol 7 no 8 Article ID e41534 2012

[35] S Chen J Gluhak-Heinrich Y HWang et al ldquoRunx2Osx andDspp in tooth developmentrdquo Journal of Dental Research vol 88no 10 pp 904ndash909 2009

[36] S Suzuki N Haruyama F Nishimura and A B KulkarnildquoDentin sialophosphoprotein and dentin matrix protein-1 twohighly phosphorylated proteins inmineralized tissuesrdquoArchivesof Oral Biology vol 57 no 9 pp 1165ndash1175 2012

[37] R S Prescott R Alsanea M I Fayad et al ldquoIn vivo fenerationof dental pulp-like tissue by using dental pulp stem cells a col-lagen scaffold and dentin matrix protein 1 after subcutaneoustransplantation in micerdquo Journal of Endodontics vol 34 no 4pp 421ndash426 2008

[38] T Komori ldquoRegulation of bone development and extracellularmatrix protein genes by RUNX2rdquo Cell and Tissue Research vol339 no 1 pp 189ndash195 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 11

Prot

eins

ACT

IN

OCN BSP RUNX2 OSX DSP OPN DMP-1

ControlInhibitor MM

Inhibitor + MM

0504504

035030250201501005

0

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(g)

MM

InhibitorControl

Inhibitor + MM

(h)

06

05

04

03

02

01

0

lowast

lowastlowastCPC

1

MMInhibitorControl

Inhibitor + MM

(ng

calc

ium

mg

prot

ein)

(i)

Figure 5 Odontoosteogenic differentiation in canonical NF-120581B-inhibited SCAPs (a) ALP activity at different time points in different groups(ie control inhibitor MM and inhibitor +MM) (b) Real-time RT-PCR for the detection ofALP BSP OCN RUNX2 OSX DSP OPN DSPandDMP-1 at day 3ThemRNA levels were normalized toGAPDH (c) Real-time RT-PCR for the detection ofALP BSP OCN RUNX2 OSXDSP OPN DSP andDMP-1 of SCAPs respectively in control inhibitor MM and inhibitor +MMgroups at day 7 (d)The odontoosteogenicprotein levels of SCAPs in control inhibitor MM and inhibitor +MM groups were characterized by western blot analysis at day 3 120573-ACTINwas used as a loading control (e) Semiquantitative analysis demonstrated that the expression of BSP OCN RUNX2 OSX DSP OPN DSPand DMP-1 was more downregulated in NF-120581B pathway-inhibited SCAPs than those in control group at day 3 (f) The odontoosteogenicprotein levels of SCAPs in control inhibitor MM and inhibitor + MM groups were characterized by western blot analysis at day 7 (g)Semiquantitative analysis demonstrated that the expression of odontoosteogenic markers (BSP OCN RUNX2 OSX DSP OPN DSP andDMP-1) was significantly more downregulated in NF-120581B pathway-inhibited SCAPs than those in control group at day 7 (h) Alizarin redstaining of SCAPs after 14 days of osteogenic induction (i) Calcium quantification demonstrated the weaker calcium deposition in inhibitorand inhibitor + MM groups as compared with control and MM groups respectively

Acknowledgments

This work was supported by National Natural Science Foun-dation of China (no 81371144) Natural Science Foundationof Jiangsu Province (no BK20131392) and the Priority Aca-demic Program Development of Jiangsu Higher EducationInstitutions (PAPD no 2011-137)

References

[1] AKumar Y TakadaAMBoriek andB BAggarwal ldquoNuclearfactor-120581B its role in health and diseaserdquo Journal of MolecularMedicine vol 82 no 7 pp 434ndash448 2004

[2] F Chen V Castranova X Shi and LM Demers ldquoNew insightsinto the role of nuclear factor-120581B a ubiquitous transcription

factor in the initiation of diseasesrdquo Clinical Chemistry vol 45no 1 pp 7ndash17 1999

[3] C Gasparini and M Feldmann ldquoNF-120581B as a target for modu-lating inflammatory responsesrdquoCurrent Pharmaceutical Designvol 18 no 35 pp 5735ndash5745 2012

[4] A Ohazama and P T Sharpe ldquoTNF signalling in toothdevelopmentrdquo Current Opinion in Genetics amp Development vol14 no 5 pp 513ndash519 2004

[5] Y-H Kuan F-M Huang Y-C Li and Y-C Chang ldquoProin-flammatory activation ofmacrophages by bisphenol A-glycidyl-methacrylate involved NF120581B activation via PI3KAkt pathwayrdquoFood and Chemical Toxicology vol 50 no 11 pp 4003ndash40092012

[6] X Cai P Gong YHuang andY Lin ldquoNotch signalling pathwayin tooth development and adult dental cellsrdquo Cell Proliferationvol 44 no 6 pp 495ndash507 2011

12 BioMed Research International

[7] J-M Courtney J Blackburn and P T Sharpe ldquoThe Ectodys-plasin and NF120581B signalling pathways in odontogenesisrdquoArchives of Oral Biology vol 50 no 2 pp 159ndash163 2005

[8] W Sonoyama Y Liu T Yamaza et al ldquoCharacterization of theapical papilla and its residing stem cells from human immaturepermanent teeth a pilot studyrdquo Journal of Endodontics vol 34no 2 pp 166ndash171 2008

[9] W Sonoyama Y Liu D Fang et al ldquoMesenchymal stem cell-mediated functional tooth regeneration in swinerdquo PLoS ONEvol 1 no 1 article e79 2006

[10] H Chopra M K Hans and S Shetty ldquoStem cells-the hiddentreasure a strategic reviewrdquoDental Research Journal vol 10 no4 pp 421ndash427 2013

[11] R Dong R Yao J Du S Wang and Z Fan ldquoDepletion ofhistone demethylase KDM2A enhanced the adipogenic andchondrogenic differentiation potentials of stem cells from apicalpapillardquo Experimental Cell Research vol 319 no 18 pp 2874ndash2882 2013

[12] Y Wang M Yan Z Wang et al ldquoDental pulp stem cells fromtraumatically exposed pulps exhibited an enhanced osteogenicpotential and weakened odontogenic capacityrdquo Archives of OralBiology vol 58 no 11 pp 1709ndash1717 2013

[13] Y Wang M Yan Z Fan L Ma Y Yu and J Yu ldquoMineraltrioxide aggregate enhances the odontoosteogenic capacity ofstem cells from inflammatory dental pulps via NF-120581B pathwayrdquoOral Diseases 2013

[14] Y Wang M Yan Y Yu J Wu J Yu and Z Fan ldquoEstrogendeficiency inhibits the odontoosteogenic differentiation ofdental pulp stem cells via activation of the NF-120581B pathwayrdquo Celland Tissue Research vol 352 no 3 pp 551ndash559 2013

[15] Y Wang Y Zheng Z Wang et al ldquo10minus7M 17120573-oestradiolenhances odontoosteogenic potency of human dental pulpstem cells by activation of the NF-120581B pathwayrdquo Cell Prolifera-tion vol 46 no 6 pp 677ndash684 2013

[16] Z-G Lu H Liu T Yamaguchi Y Miki and K YoshidaldquoProtein kinase C120575 activates RelAp65 and nuclear factor-120581B signaling in response to tumor necrosis factor-120572rdquo CancerResearch vol 69 no 14 pp 5927ndash5935 2009

[17] G Bonizzi and M Karin ldquoThe two NF-120581B activation pathwaysand their role in innate and adaptive immunityrdquo Trends inImmunology vol 25 no 6 pp 280ndash288 2004

[18] K W McIntyre D J Shuster K M Gillooly et al ldquoA highlyselective inhibitor of I120581B kinase BMS-345541 blocks both jointinflammation and destruction in collagen-induced arthritis inmicerdquo Arthritis amp Rheumatism vol 48 no 9 pp 2652ndash26592003

[19] J R Burke M A Pattoli K R Gregor et al ldquoBMS-345541 is ahighly selective inhibitor of I120581B kinase that binds at an allostericsite of the enzyme and blocks NF-120581B-dependent transcriptionin micerdquoThe Journal of Biological Chemistry vol 278 no 3 pp1450ndash1456 2003

[20] F W Paula-Silva A Ghosh L A Silva and Y L Kapila ldquoTNF-120572 promotes an odontoblastic phenotype in dental pulp cellsrdquoJournal of Dental Research vol 88 no 4 pp 339ndash344 2009

[21] S Wang J Mu Z Fan et al ldquoInsulin-like growth factor 1 canpromote the osteogenic differentiation and osteogenesis of stemcells from apical papillardquo Stem Cell Research vol 8 no 3 pp346ndash356 2012

[22] Y Yu J Mu Z Fan et al ldquoInsulin-like growth factor 1 enhancesthe proliferation and osteogenic differentiation of human peri-odontal ligament stem cells via ERK and JNKMAPKpathwaysrdquoHistochemistry andCell Biology vol 137 no 4 pp 513ndash525 2012

[23] H Aonuma N Ogura K Takahashi et al ldquoCharacteristics andosteogenic differentiation of stemprogenitor cells in the humandental follicle analyzed by gene expression profilingrdquo Cell andTissue Research vol 350 no 2 pp 317ndash331 2012

[24] L Wang M Yan Y Wang et al ldquoProliferation andosteoodontoblastic differentiation of stem cells from dentalapical papilla in mineralization-inducing medium containingadditional KH

2PO4rdquo Cell Proliferation vol 46 no 2 pp

214ndash222 2013[25] G T-J Huang T Yamaza L D Shea et al ldquoStemProgenitor

cell-mediated de novo regeneration of dental pulp with newlydeposited continuous layer of dentin in an in vivomodelrdquoTissueEngineering A vol 16 no 2 pp 605ndash615 2010

[26] J-H Kwak J-K Jung and H Lee ldquoNuclear factor-120581 Binhibitors a patent review (2006ndash2010)rdquo Expert Opinion onTherapeutic Patents vol 21 no 12 pp 1897ndash1910 2011

[27] K Hess A Ushmorov J Fiedler R E Brenner and T WirthldquoTNF120572 promotes osteogenic differentiation of human mes-enchymal stem cells by triggering theNF-120581B signaling pathwayrdquoBone vol 45 no 2 pp 367ndash376 2009

[28] X Feng G Feng J Xing et al ldquoTNF-120572 triggers osteogenicdifferentiation of human dental pulp stem cells via the NF-120581Bsignalling pathwayrdquoCell Biology International vol 37 no 12 pp1267ndash1275 2013

[29] J Chang F Liu M Lee et al ldquoNF-120581B inhibits osteogenicdifferentiation of mesenchymal stem cells by promoting 120573-catenin degradationrdquo Proceedings of the National Academy ofSciences of the United States of America vol 110 no 23 pp9469ndash9474 2013

[30] C Shui and A Scutt ldquoMild heat shock induces proliferationalkaline phosphatase activity and mineralization in humanbone marrow stromal cells and Mg-63 cells in vitrordquo Journal ofBone and Mineral Research vol 16 no 4 pp 731ndash741 2001

[31] X Wei J Ling L Wu L Liu and Y Xiao ldquoExpressionof mineralization markers in dental pulp cellsrdquo Journal ofEndodontics vol 33 no 6 pp 703ndash708 2007

[32] S Z Khan E Kokubu K Matsuzaka and T Inoue ldquoBehaviourof rat-cultured dental pulp cells in three-dimensional collagentype-1 gel in vitro and in vivordquo Australian Endodontic Journalvol 39 no 3 pp 137ndash145 2013

[33] D-X Cao Z-J Li X-O Jiang et al ldquoOsteopontin as potentialbiomarker and therapeutic target in gastric and liver cancersrdquoWorld Journal of Gastroenterology vol 18 no 30 pp 3923ndash39302012

[34] S SchlaferMK Raarup P LWejse et al ldquoOsteopontin reducesbiofilm formation in a multi-species model of dental biofilmrdquoPloS ONE vol 7 no 8 Article ID e41534 2012

[35] S Chen J Gluhak-Heinrich Y HWang et al ldquoRunx2Osx andDspp in tooth developmentrdquo Journal of Dental Research vol 88no 10 pp 904ndash909 2009

[36] S Suzuki N Haruyama F Nishimura and A B KulkarnildquoDentin sialophosphoprotein and dentin matrix protein-1 twohighly phosphorylated proteins inmineralized tissuesrdquoArchivesof Oral Biology vol 57 no 9 pp 1165ndash1175 2012

[37] R S Prescott R Alsanea M I Fayad et al ldquoIn vivo fenerationof dental pulp-like tissue by using dental pulp stem cells a col-lagen scaffold and dentin matrix protein 1 after subcutaneoustransplantation in micerdquo Journal of Endodontics vol 34 no 4pp 421ndash426 2008

[38] T Komori ldquoRegulation of bone development and extracellularmatrix protein genes by RUNX2rdquo Cell and Tissue Research vol339 no 1 pp 189ndash195 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

12 BioMed Research International

[7] J-M Courtney J Blackburn and P T Sharpe ldquoThe Ectodys-plasin and NF120581B signalling pathways in odontogenesisrdquoArchives of Oral Biology vol 50 no 2 pp 159ndash163 2005

[8] W Sonoyama Y Liu T Yamaza et al ldquoCharacterization of theapical papilla and its residing stem cells from human immaturepermanent teeth a pilot studyrdquo Journal of Endodontics vol 34no 2 pp 166ndash171 2008

[9] W Sonoyama Y Liu D Fang et al ldquoMesenchymal stem cell-mediated functional tooth regeneration in swinerdquo PLoS ONEvol 1 no 1 article e79 2006

[10] H Chopra M K Hans and S Shetty ldquoStem cells-the hiddentreasure a strategic reviewrdquoDental Research Journal vol 10 no4 pp 421ndash427 2013

[11] R Dong R Yao J Du S Wang and Z Fan ldquoDepletion ofhistone demethylase KDM2A enhanced the adipogenic andchondrogenic differentiation potentials of stem cells from apicalpapillardquo Experimental Cell Research vol 319 no 18 pp 2874ndash2882 2013

[12] Y Wang M Yan Z Wang et al ldquoDental pulp stem cells fromtraumatically exposed pulps exhibited an enhanced osteogenicpotential and weakened odontogenic capacityrdquo Archives of OralBiology vol 58 no 11 pp 1709ndash1717 2013

[13] Y Wang M Yan Z Fan L Ma Y Yu and J Yu ldquoMineraltrioxide aggregate enhances the odontoosteogenic capacity ofstem cells from inflammatory dental pulps via NF-120581B pathwayrdquoOral Diseases 2013

[14] Y Wang M Yan Y Yu J Wu J Yu and Z Fan ldquoEstrogendeficiency inhibits the odontoosteogenic differentiation ofdental pulp stem cells via activation of the NF-120581B pathwayrdquo Celland Tissue Research vol 352 no 3 pp 551ndash559 2013

[15] Y Wang Y Zheng Z Wang et al ldquo10minus7M 17120573-oestradiolenhances odontoosteogenic potency of human dental pulpstem cells by activation of the NF-120581B pathwayrdquo Cell Prolifera-tion vol 46 no 6 pp 677ndash684 2013

[16] Z-G Lu H Liu T Yamaguchi Y Miki and K YoshidaldquoProtein kinase C120575 activates RelAp65 and nuclear factor-120581B signaling in response to tumor necrosis factor-120572rdquo CancerResearch vol 69 no 14 pp 5927ndash5935 2009

[17] G Bonizzi and M Karin ldquoThe two NF-120581B activation pathwaysand their role in innate and adaptive immunityrdquo Trends inImmunology vol 25 no 6 pp 280ndash288 2004

[18] K W McIntyre D J Shuster K M Gillooly et al ldquoA highlyselective inhibitor of I120581B kinase BMS-345541 blocks both jointinflammation and destruction in collagen-induced arthritis inmicerdquo Arthritis amp Rheumatism vol 48 no 9 pp 2652ndash26592003

[19] J R Burke M A Pattoli K R Gregor et al ldquoBMS-345541 is ahighly selective inhibitor of I120581B kinase that binds at an allostericsite of the enzyme and blocks NF-120581B-dependent transcriptionin micerdquoThe Journal of Biological Chemistry vol 278 no 3 pp1450ndash1456 2003

[20] F W Paula-Silva A Ghosh L A Silva and Y L Kapila ldquoTNF-120572 promotes an odontoblastic phenotype in dental pulp cellsrdquoJournal of Dental Research vol 88 no 4 pp 339ndash344 2009

[21] S Wang J Mu Z Fan et al ldquoInsulin-like growth factor 1 canpromote the osteogenic differentiation and osteogenesis of stemcells from apical papillardquo Stem Cell Research vol 8 no 3 pp346ndash356 2012

[22] Y Yu J Mu Z Fan et al ldquoInsulin-like growth factor 1 enhancesthe proliferation and osteogenic differentiation of human peri-odontal ligament stem cells via ERK and JNKMAPKpathwaysrdquoHistochemistry andCell Biology vol 137 no 4 pp 513ndash525 2012

[23] H Aonuma N Ogura K Takahashi et al ldquoCharacteristics andosteogenic differentiation of stemprogenitor cells in the humandental follicle analyzed by gene expression profilingrdquo Cell andTissue Research vol 350 no 2 pp 317ndash331 2012

[24] L Wang M Yan Y Wang et al ldquoProliferation andosteoodontoblastic differentiation of stem cells from dentalapical papilla in mineralization-inducing medium containingadditional KH

2PO4rdquo Cell Proliferation vol 46 no 2 pp

214ndash222 2013[25] G T-J Huang T Yamaza L D Shea et al ldquoStemProgenitor

cell-mediated de novo regeneration of dental pulp with newlydeposited continuous layer of dentin in an in vivomodelrdquoTissueEngineering A vol 16 no 2 pp 605ndash615 2010

[26] J-H Kwak J-K Jung and H Lee ldquoNuclear factor-120581 Binhibitors a patent review (2006ndash2010)rdquo Expert Opinion onTherapeutic Patents vol 21 no 12 pp 1897ndash1910 2011

[27] K Hess A Ushmorov J Fiedler R E Brenner and T WirthldquoTNF120572 promotes osteogenic differentiation of human mes-enchymal stem cells by triggering theNF-120581B signaling pathwayrdquoBone vol 45 no 2 pp 367ndash376 2009

[28] X Feng G Feng J Xing et al ldquoTNF-120572 triggers osteogenicdifferentiation of human dental pulp stem cells via the NF-120581Bsignalling pathwayrdquoCell Biology International vol 37 no 12 pp1267ndash1275 2013

[29] J Chang F Liu M Lee et al ldquoNF-120581B inhibits osteogenicdifferentiation of mesenchymal stem cells by promoting 120573-catenin degradationrdquo Proceedings of the National Academy ofSciences of the United States of America vol 110 no 23 pp9469ndash9474 2013

[30] C Shui and A Scutt ldquoMild heat shock induces proliferationalkaline phosphatase activity and mineralization in humanbone marrow stromal cells and Mg-63 cells in vitrordquo Journal ofBone and Mineral Research vol 16 no 4 pp 731ndash741 2001

[31] X Wei J Ling L Wu L Liu and Y Xiao ldquoExpressionof mineralization markers in dental pulp cellsrdquo Journal ofEndodontics vol 33 no 6 pp 703ndash708 2007

[32] S Z Khan E Kokubu K Matsuzaka and T Inoue ldquoBehaviourof rat-cultured dental pulp cells in three-dimensional collagentype-1 gel in vitro and in vivordquo Australian Endodontic Journalvol 39 no 3 pp 137ndash145 2013

[33] D-X Cao Z-J Li X-O Jiang et al ldquoOsteopontin as potentialbiomarker and therapeutic target in gastric and liver cancersrdquoWorld Journal of Gastroenterology vol 18 no 30 pp 3923ndash39302012

[34] S SchlaferMK Raarup P LWejse et al ldquoOsteopontin reducesbiofilm formation in a multi-species model of dental biofilmrdquoPloS ONE vol 7 no 8 Article ID e41534 2012

[35] S Chen J Gluhak-Heinrich Y HWang et al ldquoRunx2Osx andDspp in tooth developmentrdquo Journal of Dental Research vol 88no 10 pp 904ndash909 2009

[36] S Suzuki N Haruyama F Nishimura and A B KulkarnildquoDentin sialophosphoprotein and dentin matrix protein-1 twohighly phosphorylated proteins inmineralized tissuesrdquoArchivesof Oral Biology vol 57 no 9 pp 1165ndash1175 2012

[37] R S Prescott R Alsanea M I Fayad et al ldquoIn vivo fenerationof dental pulp-like tissue by using dental pulp stem cells a col-lagen scaffold and dentin matrix protein 1 after subcutaneoustransplantation in micerdquo Journal of Endodontics vol 34 no 4pp 421ndash426 2008

[38] T Komori ldquoRegulation of bone development and extracellularmatrix protein genes by RUNX2rdquo Cell and Tissue Research vol339 no 1 pp 189ndash195 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology