research article members of the cyr61/ctgf/nov protein...

Research ArticleMembers of the Cyr61CTGFNOV Protein FamilyEmerging Players in Hepatic Progenitor Cell Activation andIntrahepatic Cholangiocarcinoma

Qunfeng Wu1 Marda Jorgensen2 Joanna Song2 Junmei Zhou2 Chen Liu1 and Liya Pi2

1Department of Pathology and Laboratory Medicine Rutgers The State University of New Jersey Newark NJ 07103 USA2Department of Pediatrics University of Florida Gainesville FL 32610 USA

Correspondence should be addressed to Liya Pi lpipedsufledu

Received 21 July 2016 Revised 24 September 2016 Accepted 26 September 2016

Academic Editor Andrea C Gardini

Copyright copy 2016 Qunfeng Wu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Hepatic stemprogenitor cells (HPC) reside quiescently in normal biliary trees and are activated in the form of ductular reactionsduring severe liver damage when the replicative ability of hepatocytes is inhibited HPC niches are full of profibrotic stimulifavoring scarring and hepatocarcinogenesis The Cyr61CTGFNOV (CCN) protein family consists of six members CCN1CYR61CCN2CTGF CCN3NOV CCN4WISP1 CCN5WISP2 and CCN6WISP3 which function as extracellular signalingmodulatorsto mediate cell-matrix interaction during angiogenesis wound healing fibrosis and tumorigenesis This study investigatedexpression patterns of CCN proteins in HPC and cholangiocarcinoma (CCA) Mouse HPC were induced by the biliary toxin 35-diethoxycarbonyl-14-dihydrocollidine (DDC) Differential expression patterns of CCN proteins were found in HPC from DDCdamaged mice and in human CCA tumors In addition we utilized reporter mice that carried Ccn2Ctgf promoter driven GFPand detected strong Ccn2Ctgf expression in epithelial cell adhesion molecule (EpCAM)+ HPC under normal conditions and inDDC-induced liver damage Abundant CCN2CTGF protein was also found in cytokeratin 19 (CK19)+ human HPC that weresurrounded by 120572-smooth muscle actin (120572-SMA)+ myofibroblast cells in intrahepatic CCA tumors These results suggest that CCNproteins particularly CCN2CTGF function in HPC activation and CCA development

1 Introduction

The normal liver fosters a small subset of adult stem cells thatreside quiescently in the Canals of Hering which is a transi-tional zone located between the bile canaliculi and interlob-ular ductal systems [1] In response to massive parenchymaldamage or severe impairment if hepatocyte proliferationis arrested the stem cells become activated in the form ofductular reactions and produce atypical ductular cells alsotermedhepatic progenitor cells (HPC) in humans or oval cellsin rodents [2 3] These cells are capable of aiding in bothhepatocytic regeneration and biliary regeneration HPC areheterogeneous populations that expand extensively aroundperiportal areas and express certain biliarymarkers includingepithelial cell adhesion molecule (EpCAM) and cytokeratin19 (CK19) [4 5]The expansion of the HPC population repre-sents an attempt to participate in the regeneration of damaged

liver during chronic liver injury However persistent injuryand the chronic inflammatory milieu activate profibrogenicsignaling resulting in myofibroblast activation and collagendepositionHPC activation is strongly correlatedwith fibrosisprogression in many human chronic liver diseases includingalcoholic and nonalcoholic steatohepatitis chronic hepatitisC cholestatic hepatitis and hereditary haemochromatosis[6ndash11] Moreover HPC within the biliary tree have beengaining relevance in relation to liver cancer particularlyintrahepatic cholangiocarcinoma (CCA) and hepatocellularcarcinoma (HCC) During chronic hepatitis B or C viralinfection with accompanying liver cirrhosis HPC prolifer-ation is directly related to disease severity suggesting thatactivation of the HPC compartment is associated with anincreased risk of liver cancer development [7] Clinical andexperimental animal studies have demonstrated that progen-itor cells can play a critical role in hepatic carcinogenesis and

Hindawi Publishing CorporationGastroenterology Research and PracticeVolume 2016 Article ID 2313850 9 pageshttpdxdoiorg10115520162313850

2 Gastroenterology Research and Practice

recruitment of progenitor cells contributing to liver cancerleading to HCC and intrahepatic CCA [12ndash15] In additionclinical-pathological correlation and risk factors for HCCand intrahepatic CCA support the role of HPC in differentsubtypes of liver cancers Recent evidences indicate that HPCmay be the cell of origin in certain subtypes of CCA suchas cholangiolocarcinoma and the so-called mixed-type CCAor HCC tumors such as CK19+ HCC [13 16ndash18] Lastlycombined hepatocellular cholangiocarcinoma (CHC) a rareform of primary liver cancer that accounts for 04ndash142 ofliver carcinomas [19] exhibits pathological features of HCCand CCA It has been proposed that CHC directly derivesfrom HPC activation and differentiation [20]

Matricellular proteins of the CCN (CYR61CTGFNOV)family are important signal modifiers in stem cell nichesand can modulate multiple signaling pathways mediatedby Notch Wnt and transforming growth factor-120573 (TGF-120573) [3 21 22] The CCN family is composed of six struc-turally conserved secreted proteins CCN1cysteine-rich 61(CYR61) CCN2connective tissue growth factor (CTGF)CCN3nephroblastoma overexpressed (NOV) CCN4Wnt1induced secreted protein-1 (WISP-1) CCN5WISP-2 andCCN6WISP-3 All CCN proteins share a conserved four-modular structure with the exception of CCN5WISP-2which lacks a cysteine knot domain module CCN proteinsexert activity on various types of cells through their broadbinding capabilities to ECM proteins growth factors andcell surface proteins [21] CCN proteins modulate cell-matrixinteraction and mediate cell adhesion migration differenti-ation apoptosis and survival Altered levels of CCN expres-sion or activities contribute to the pathogenesis of many dis-eases that occur when inflammation or tissue injury becomeschronic such as liver fibrosis and liver cancer [21 23ndash25]In order to understand CCN function in HPC activationand CCA development we examined the expression of CCNproteins in mouse HPC induced by 35-diethoxycarbonyl-14-dihydrocollidine (DDC) a biliary toxin that inducesductular reaction and biliary fibrosis [26] The levels ofCCNmRNAswere also quantitativelymeasured fromhumanintrahepatic CAAWithin the family CCN2CTGF exhibitedpredominant expression in both mouse HPC and humanCCA tumors

2 Materials and Methods

21 Human Tissues Human liver tissues were obtainedthrough Shands Hospital according to an approved protocolapproved by the institutional review board at the Universityof FloridaWritten informed consents were obtained from allsubjects Tumor or adjacent nontumor tissues from intrahep-aticCCAcontaining liverswere separated after dissection andsnap-frozen for molecular analyses

22 The DDCMouse Model Transgenic mice carrying Ccn2Ctgf promoter driven GFP (Ctgfp-GFP) were previouslydescribed [27] Wild-type or Ctgfp-GFP mice at 8ndash10 weeksof age were fed with a diet supplemented with 01 DDC(Bio-Serv Frenchtown NJ) to induce cholangitis ductular

reactions and biliary fibrosis All protocols and procedureswere approved by the University of Florida IACUC and werein accordance with National Institutes of Health guidelines

23 Immunohistochemistry Allmouse liver tissueswere fixedovernight in 4 paraformaldehyde to preserve the GFPfluorescence signal Tissues were infiltrated with 20 sucrosebefore being embedded in OCT 6120583m sections were usedin this study GFP staining in combination with EpCAMantibody (eBioscience San Diego CA) or hepatocyte nuclearfactor 4120572 (HNF4120572) antibody (Santa Cruz BiotechnologiesDallas Texas) required antigen retrieval at 37∘C for 10minutes in trypsin (Digest-All 2 Invitrogen Carlsbad CA)Primary antibodies employed were 1 100 rat anti-EpCAM1 100 rabbit anti-HNF4120572 and 1 750 chicken anti-GFP(Aves Labs Tigard OR) Alexa Fluor 488 conjugated goatanti-chicken and Alexa Fluor 594 conjugated donkey anti-rabbit secondary antibodies (Invitrogen Carlsbad CA) wereused at 1 500 for detection Human intrahepatic CCA andHCC tissue arrays were purchased from US Biomax Inc(Rockville MD) Gomorirsquos Trichrome staining for collagenfibers was carried out using aThermo Scientific Chromaviewcollagen blue kit according to themanufacturerrsquos instructions(Richard-Allan Scientific Kalamazoo MI) To evaluate fibro-sis in DDC treated mice liver sections were stained withPicrosirius Red solution (American MasterTech Lodi CA)In addition staining for CK19 CCN2CTGF and 120572-SMArequires antigen retrieval in Trilogy pretreatment solution(Cell Marque Rocklin CA) at 95∘C for 25 minutes Thesections were blocked in 3 horse serum (Vector Laborato-ries) for one hour prior to overnight incubation with anti-CK19 antibody (Abcam) anti-CCN2CTGF antibody (SantaCruz Biotechnologies) and anti-120572SMA antibody (SigmaSt Louis MO) Additional IHC for CCN2CTGF in liversections of human intrahepatic CCA was carried out usinga rabbit antibody (1 200 Abcam) and detected using aVECTASTAINABC-AP kit and Vector Red substrate (VectorLaboratories Burlingame CA)

24 RNA Isolation and Reverse Transcriptase-PolymeraseChain Reaction (RT-PCR) Analysis Total RNAs were ex-tracted using RNeasy Mini kit (Qiagen Valencia CA) fol-lowed by incubation with RQ1 RNase-free DNase (PromegaMadison WI) to remove any genomic DNA contamination2 120583g of total RNA and 50 pmol random hexamer were usedfor Superscript III First-Strand cDNA Synthesis (Invit-rogen) RT-PCR was carried out using 05 120583L cDNAs astemplates 02120583M of each set of primers and REDExtract-N-Amp tissue PCR mixtures (Sigma) Amplification con-ditions consisted of thirty cycles of denaturation at 94∘Cfor 30 sec annealing at 55∘C for 30 sec and extension at72∘C for 30 sec Primer pairs for mouse genes includethe following 51015840-TTTACAGTTGGGCTGGAAGC-31015840 and51015840-CACCGCTCTGAAAGGGATCT-31015840 for Ccn1Cyr61 51015840-GTCTTCACACTGGTGCAGCC-31015840 and 51015840-ACTGGAAGA-CACATTTGGCC-31015840 for Ccn2Ctgf 51015840-CTTGGTGCGGAG-ACACTTTT-31015840 and 51015840-CGCCAGTGTGAGATGGTAAA-31015840 for Ccn3Nov 51015840-TGAGTGCTTGAGCATCCAGA-31015840

Gastroenterology Research and Practice 3

and 51015840-AATGAGAAAGGAGAGAGGCTGT-31015840 for Ccn4Wisp1v 51015840-GTGTCCAAGGACAGGCACAG-31015840 and 51015840-GC-AACCCACTGATCCATCTT-31015840 for Ccn5Wisp2 51015840-AAT-GAGAAAGGAGAGGAGGCTGT-31015840 and 51015840-TGAGTG-CTTGAGCATCCAGA-31015840 for Ccn6Wisp3 51015840-CCACCA-CGCTCTTCTGTCTAC-31015840 and 51015840-AGGGTCTGGGCCAT-AGAACT-31015840 for tumor necrosis factor (TNF120572) 51015840-CCA-CCGCAAATGCTTCTAAGT-31015840 and 51015840-GGCAGGAATG-ATTTGGAAAGG-31015840 for 120572-SMA 51015840-TGTGTTCCCTA-CTCAGCCGTCT-31015840 and 51015840-CATCGGTCATGCTCTCTC-CAA-31015840 for procollagen type 1205721(I) and 51015840-TTGACGGAAG-GGCACCACCAG-31015840 and 51015840-GCACCACCACCCACG-GAATCG-31015840 for 18S ribosomal RNA Wisp1v gene lackingsequence corresponding to Von Willbred Factor type C(VWC) domain is a variant of Wisp1 gene It was chosen inthis study because of its association with cholangiocarcinoma[28]

In real-time RT-PCR analysis cDNAs from CCA sam-ples were analyzed in ABI Prism 7900 HT Fast Real-Time (Applied Biosystems Carlsbad California) Primerpairs for human genes used are as follows 51015840-TCA-CCCTTCTCCACTTGACC-31015840 and 51015840-AGTCCTCGTTGA-GCTGCTTG-31015840 for CCN1CYR61 51015840-TGGAGATTTTGG-GAGTACGG-31015840 and 51015840-CAGGCTAGAGAAGCAGAG-CC-31015840 for CCN2CTGF 51015840-AAGAGCTGTGGTATGGGG-TTC-31015840 and 51015840-GGTGGATGGCTTTGAGTGAC-31015840 forCCN3NOV 51015840-GTAAGATGTGCGCTCAGCAG-31015840 and 51015840-ACTGGGCGTTAACATTGGAG-31015840 for CCN4WISP1v 51015840-AGCCCAAGGACCCCAGTT-31015840 and 51015840-TCTCCAGTC-GGCAGAAGC-31015840 for CCN5WISP2 51015840-CTGGCCTGG-CACAGTTCT-31015840 and 51015840-TCTCTCACCAGGCTCACTCC-31015840 for CCN6WISP3 and 51015840-GATGAGATTGGCATGGCT-TT-31015840 and 51015840-GAGAAGTGGGGTGGCTT-31015840 for 120573-ACTINThe relative amount of target mRNA was calculated usingthe delta CT method and normalized against 120573-ACTIN asreference gene in each sample

25 Statistical Analysis Microsoft Excel software (MicrosoftCorp Redmond WA) was used for statistical analysisData were represented as mean plusmn SD Statistical significance(119875 lt 005) was evaluated using Studentrsquos t-test and one-way analysis of variance (ANOVA) All experiments wereperformed a minimum of three times

3 Results

31 Dynamic Expression of CCN Proteins during HPCActivation and Biliary Fibrosis Induced by DDC Damage inMice DDC is a porphyrinogenic agent widely used to studymechanisms of HPCoval cell activation and proliferation[29] DDC feeding is associated with increased biliaryporphyrin secretion and segmental bile duct obstructionleading to pronounced pericholangitis ductular reactionactivation of periductal myofibroblasts and extensive colla-gen deposition in periportal areas within treated livers [26]To monitor these changes we fed wild-type C56BL6J micewith DDC-containing diets for 0ndash20 days Total RNA wasextracted from liver homogenates As shown in Figure 1(a)

DDC-induced liver damage was verified in RT-PCR analysisbased on upregulation of the proinflammatory gene TNF120572 at5 10 15 and 20 days after treatment Accordingly ductularreactions occurred as early as day 5 indicated by induction ofthe HPC marker EpCAM In addition fibrosis related genesincluding 120572-SMA and procollagen 1205721(I) were upregulated inthe DDC damaged livers suggesting a concomitant fibrosisin response to DDC toxicity The development of ductularreactions and liver fibrosis was confirmed in DDC-fedmice using both HampE staining and Sirius Red staining asshown in Figure 1(b) We also detected sustained inductionof Ccn1Cyr61 Ccn2Ctgf and Ccn4Wisp1v mRNAs from5 to 20 days after DDC feeding In addition Ccn5Wisp2transcript was gradually increased in a temporal and spatialpattern similar to that of TNF120572 and reached a peak atday 20 By contrast Ccn3Nov and Ccn6Wisp3 did notshow significant induction These differential expressionpatterns suggested the involvement ofCcn1Cyr61Ccn2CtgfCcn4Wisp1v and Ccn5Wisp2 in DDC-induced liver injury

32 Altered Expression of CCN Proteins in Intrahepatic CCATumors CCN proteins are important regulators in stemcells and tumorigenesis Expression of CCN family membershas been shown to correlate with the clinical features ofHCC [25] To further determine whether CCN proteins wereinvolved in liver cancer development we extracted total RNAfrom intrahepatic CCA tumors as well as their adjacentnormal counterparts Expression patterns of CCN proteinsin these tissues were compared with normal human livertissues by RT-PCR analysis Consistent with previous reportsdetailing overexpression of CCN2CTGF and CCN4WISP1vin CCA [27 30] we detected significant overexpressionof these two transcripts in all tested tumor tissues fromour tumor samples (Figure 1(c)) In addition inductionof CCN1Cyr61 and CCN5WISP2 was also found in theCCA tumors whereas CCN3NOV and CCN6WISP3 didnot have obvious changes in both the nontumor and tumorsamples from the CCA tissues (Figure 1(c)) These resultsindicate that CCN1Cyr61 CCN2CTGF CCN4WISP1v andCCN5WISP2 are involved in CCA tumorigenesis

33 Specific Promoter Activity of the Ccn2Ctgf Gene inMouse HPC under Both Normal and DDC-Induced LiverDamage Conditions The CCN2CTGF gene had a very highlevel of induction in both DDC damaged mouse livers andCCA tumors as shown in Figures 1(a) and 1(c) To verifythe expression of this gene in HPC we took advantageof reporter mice carrying the Ctgfp-GFP transgene andvisualized Ccn2Ctgf expression in vivo based on GFP flu-orescence Mouse HPC were labeled using an antibody forthe HPC marker EpCAM As shown in Figure 2 confocalmicroscopy analysis detected specific Ccn2Ctgf promoteractivity in EpCAM+ HPC from subsets of small ducts inuntreated mice In response to DDC-induced liver damageEpCAM+ HPC expanded in large numbers within the peri-portal zones The majority of the EpCAM+ HPC were GFPpositive indicating predominant expression of the Ccn2Ctgfgene in activated mouse HPC By contrast only vascular


0 5 10 15 20Days on DDC

18S

EpCAM

Ccn2Ctgf

Ccn1Cyr61

Ccn3Nov

Ccn5Wisp2

Ccn6Wisp3

Ccn4Wisp1v

TNF

-SMA

Collagen 1(I)

(a)

Hamp

ESi

rius R

edDay 0 Day 10 Day 20

(b)CCN2CTGF

NL

NT T NL

NT T

NL

NT TNL

NT T

NL

NT T

CCN3NOV

CCN4WISP1v

CCN5WISP2

CCN6WISP3

00

15

CCN1CRY61

NL

NT T

lowast

lowast

lowast

lowast

0

2

4

6

8

Fold

incr

ease

Fold

incr

ease

0

1

2

3

4

5

Fold

incr

ease

0

1

2

3

4

5

Fold

incr

ease

0

1

2

3

Fold

incr

ease

00

15

Fold

incr

ease

(c)

Figure 1 Dynamic expression of CCN proteins in HPC and human CCA tumors (a) Transcriptional levels of the proinflammatory geneTNF120572 the HPC marker EpCAM and fibrosis related genes 120572-SMA and collagen 1205721(I) were measured by RT-PCR analysis to assess thedevelopment of HPC activation and biliary fibrosis in mice that were fed DDC for 0ndash20 days CCN transcripts were determined in theDDC damaged mouse livers (a) or in human CCA tissues by quantitative RT-PCR analysis (c) (b) HampE staining and Sirius Red stainingshow the development of ductular reaction and liver fibrosis in DDC-fed mice Scale bar 250 120583m (c) Data were calculated from CCA andcorresponding nontumor samples from five human livers as compared to normal livers from three healthy donors NL normal liver fromhealthy donors NT adjacent nontumor tissues from CCA patients T tumor tissues from CCA patients lowast119875 lt 005


EpCAM Ctgfp-GFP Merge

Nor

mal

DD

C

(a)

DAPI MergeCtgfp-GFP HNF4

(b)

Figure 2 Immunofluorescent staining shows strong induction of Ccn2Ctgf promoter activity in DDC-induced mouse HPC (a) Dualstaining for EpCAM and GFP proteins was carried out on liver sections from mice that carried Ccn2Ctgf promoter driven GFP (Ctgfp-GFP) transgene under normal conditions or after 20-day DDC feeding (b) Double staining for HNF4120572 and GFP showed little Ccn2Ctgfpromoter activity in mature hepatocytes of damaged livers from mice that were fed DDC for 20 days Scale bar 35120583m

CCN2CTGFTrichrome

(a)

CCN2CTGFTrichrome

(b)

Figure 3 Immunofluorescent staining detects abundant CCN2CTGF protein in fibrotic human CCA tumors Trichrome or CCN2CTGFstaining was carried out on human intrahepatic CCA tissue arrays Representative images for human CCA tumors (a) or non-CCA controltumors (pheochromocytoma) from adrenal glands (b) were shown Inserts are macroscopic views of the stained tissues Scale bar 100120583mArrowheads indicate the same location in each set of images in Figure 3

endothelial cells (EC) lining central veins exhibited detectableCcn2Ctgf promoter activity inDDCdamaged livers (Supple-mental Figure 1 in Supplementary Material available onlineat httpdxdoiorg10115520162313850) Little Ccn2Ctgfpromoter activity was found in HNF4120572+ hepatocytes locatedat periportal and pericentral areas during DDC-inducedliver injury as shown in Figure 2(b) and SupplementalFigure 1 These observations were further supported byimmunofluorescent staining for glutamine synthetase anenzyme specifically expressed in hepatocytes around zone3 of the liver acinus in the livers [31] Lastly absence ofCcn2Ctgf promoter activity was found inmesenchymal cellsthat expressed vimentin (Supplemental Figure 3) implying

that CCN2CTGF was not directly involved in epithelial-mesenchymal transition during DDC-induced liver injury inmice

34 Overexpression of CCN2CTGF Protein in CK19+ HPCSurrounded by120572-SMA+Myofibroblast Cells in Fibrotic HumanIntrahepatic CCA Tumors The tumor microenvironment isa very important factor in the regulation of angiogene-sis invasion and metastasis We observed thick collagenfibrils in CCA stromal tissues that surrounded the tumorepithelial cells using Trichrome staining in Figure 3(a)Immunohistochemistry on consecutive sections showedabundant CCN2CTGF localization in disorganized hepatic


CCN2CTGF Merge-SMA

(a)

CCN2CTGFCK19 Merge

(b)

IH-CCA

(c)

CK19CCN2CTGF Merge

(d)

HCC

(e)

Figure 4 Localization of CCN2CTGF protein in CK19+ human HPC that are surrounded by intratumoral 120572-SMA+ myofibroblast cellsImmunofluorescent staining for CCN2CTGF in combination with 120572-SMA (a) or CK19 (b) was carried out on human CCA sections (d)Theimmunofluorescent staining revealed CCN2CTGF localization in CK19+ human HCC tumor cells (c and e) IH-CCA and HCC tumors in(b) and (d) were histologically analyzed by HampE staining Scale bar 30120583m

parenchyma characterization by extensive ductular reactionand mixed HPC populations in the IH-CCA (Figure 3(b))This was in contrast to weak staining in pheochromocy-toma from adrenal glands as a negative control Additionalimmunofluorescent staining in Figure 4(a) suggested that120572-SMA+ myofibroblast cells were not the main cellularsource of CCN2CTGF protein CK19 is a marker thatlabels HPC liver cancer stem cells and ductular reactionsin humans Consistent with our recent finding [27] dualstaining detected overexpression of CCN2CTGF protein inCK19+ HPC characterized by atypical ductular cells withoutclearly defined lumens in IH-CCA tumors (Figures 4(b) and4(c)) High level of CCN2CTGF protein was also found inCK19+ HCC tumors (Figures 4(d) and 4(e)) Because CK19positiveHCChas been described as a cancer originating fromHPC [14 17] these results support a role for CCN2CTGF inHPC activation during IH-CCA and HCC tumorigenesis

4 Discussion

CCN family members are small secreted cysteine-rich pro-teins modulating key pathways involved in initiation andresolution of normal wound healing and fibrosis after tissuedamage For example CCN2CTGF is overexpressed inmanykinds of fibrotic disorders [32] During liver damage this pro-tein is produced by a variety of different cell types includinghepatic stellate cells biliary epithelial cells hepatocytes andKupffer cells [32] It promotes fibrogenesis and survival inactivated hepatic stellate cells through direct interaction withvarious subtypes of integrins leading to enhanced adhesivesignaling [33] In vivo overexpression of CCN2CTGF inhepatocytes does not result in hepatic injury or fibrosis perse but renders the livers more susceptible to the injuriousactions of other fibrotic stimuli in mice [34] SimilarlyCCN4WISP1 is profibrotic since blockade of this moleculedecreases experimental liver fibrosis in vivo [35 36] In

contrast CCN1CYR61 and CCN3NOV are inhibitors ofthe fibrotic response [37 38] CCN1CYR61 induces senes-cence in hepatic myofibroblasts leading to attenuated TGF-120573 signaling while its overexpression results in ER stress-related apoptosis in hepatic stellate cells [39] Inhibition ofCCN3NOV using small interfering RNA enhances fibroticgene expression in hepatic stellate cells [40] Given the factthat liver fibrosis occurs as a final outcome of an abnormalwound healing response and is closely associated with HPCactivation andductular reactions during chronic liver diseasewe speculate that the CCN proteins also regulate HPCactivation In supporting this notion a recent report identifiesCCN1CYR61 as a critical regulator in biliary injury repairthrough the integrin 120572v1205735NF-120581BJAG1 signaling axis [41]CCN1CYR61 stimulates Jag1 expression in hepatic stellatecells and promotes HPC differentiation cholangiocyte prolif-eration and ductular reactions [41] CCN2CTGF is anothermember involved in HPC activation and ductular reactionsWe have identified this molecule as one of several differen-tially expressed genes in oval cells from regenerating rat liversfollowing 2-acetylaminofluorene and partial hepatectomy[42] CCN2CTGF binds to fibronectin and promotes rat ovalcell adhesion and migration [43] Moreover CCN2CTGFfunctions together with integrin 120572v1205736 and enhances TGF-120573 activation Conditional deletion of exon 4 of this genereduces ductular reactions and biliary fibrosis [27] In thisstudy we observed that Ccn2Ctgf had a relatively higherlevel of induction than other CCN proteins in DDC damagedmouse livers in Figure 1(a) Specific promoter activity of theCcn2Ctgf gene was found in HPC from normal and DDCdamaged mouse livers (Figure 3) suggesting an importantrole for Ccn2Ctgf in HPC activation and ductular reactionsOn the other hand we observed upregulation ofCcn4Wisp1vand Ccn5Wisp2 in DDC damaged mouse livers as shown inFigure 1(a)Thus Ccn4Wisp1v and Ccn5Wisp2 are potentialmediators for HPC activation and biliary fibrosis although


their cellular sources and functions need to be defined infuture studies

CCN proteins are emerging as a unique group ofextracellular signalingmodulators involved in establishmentgrowth and metastases in liver cancer via interaction ofcancer cells with the intratumor stroma [24] The majorityof CCN family members are induced by growth factorscytokines and cellular stress such as inflammation Alteredexpressions of CCN1CYR61 CCN2CTGF CCN3NOV andCCN4WISP1 were found to correlate with clinical featuresincluding venous invasion cellular differentiation TNMstaging for malignant tumors disease-free survival andoverall survival in HCC [25] Both positive and negativeassociations of CCN1CYR61 with HCC have been reported[25 44 45] CCN2CTGF and CCN3NOV have been shownto promote HCC development [46 47] CCN4WISP1 isnegatively linked with HCC whereas CCN5WISP2 effectsno significant change in HCC development [25] In theCCA tumor setting only CCN2CTGF and CCN4WISP1vhave been studied CCN2CTGF has been identified as anindependent prognostic indicator of both tumor recurrenceand overall survival for intrahepatic CCA patients regardlessof tumor location tumor grade or vascular and perineuralinvasion [30] CCN4WISP1v can stimulate the invasivephenotype of CCA cells with activation of both p38 andp42p44 mitogen-activated protein kinases [28] Overex-pression of CCN4WISP1v is associated with lymphatic andperineural invasion of CCA tumor cells and a poor clinicalprognosis [28] This study showed significant upregulationof CCN2CTGF and CCN4WISP1v mRNAs in our CCAtumors (Figure 1(c)) Immunohistochemistry demonstratedlocalization of CCN2CTGF in HPC and cholangiocyteswithin the CCA tissues (Figures 3 and 4) In addition weobserved the induction of CCN1CYR61 and CCN5WISP2but no significant change of CCN3NOV and CCN6WISP3in CCA (Figure 1(c)) Based on these dynamic expressionpatterns it is consistent to postulate that like CCN2CTGFand CCN4WISP1v CCN1CYR61 and CCN5WISP2 arecritical regulators in CCA development Characterization ofcell types expressing CCN1CYR61 and CCN5WISP2 andinvestigation of exact action mediated by the two moleculeswould be interesting directions to address the molecularmechanism of CCA tumorigenesis in the future

In summary cell-cell and cell-matrix interactions arecrucial for stem cellprogenitor function and liver cancerprogression In the HPC niche myofibroblast cells immunecells cytokines matricellular proteins and inflammatoryproteins are orchestrated along with specialized ECM to pro-mote progenitor cell differentiation and CCA developmentThe differential spatiotemporal expression profiles amongCCN proteins indicate that they might be required at precisetime slots during these pathological processes CCN proteinsmight act alone or in concert to exert overlapped or antago-nistic functions Genetic approaches are a powerful tool indetermining protein function Since germline conditionaland tissue specific knockout mice for several CCN membersare available [27 41 48ndash52] it is anticipated that utilizationof these genetic materials will help address the detailedmechanisms involved in CCN-mediated regulation in the

future And these studies will provide further insight intoHPC activation ductular reactions liver fibrosis and CCAprogression during chronic liver disease

5 Conclusion

We report differential expression patterns of CCN proteinsin HPC from DDC damaged mouse livers and human CCAtumors Strong induction and specific localization indicatethe importance of the profibrotic CCN2CTGF member inHPC activation and CCA development

Abbreviations

CCN Cyr61CTGFNOVCyr61 Cysteine-rich angiogenic inducer 61

proteinCTGF Connective tissue growth factorNOV Nephroblastoma overexpressed proteinWISP1v WNT1 inducible signaling pathway

protein 1 variantWISP2 WNT1 inducible signaling pathway

protein 2WISP3 WNT1 inducible signaling pathway

protein 3CCA CholangiocarcinomaHCC Hepatocellular carcinomaHPC Hepatic progenitor cellCHC Combined hepatocellular

cholangiocarcinomaEpCAM Epithelial cell adhesion moleculeCK19 Cytokeratin 19

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this manuscript

Authorsrsquo Contributions

Conception and design were done by Liya Pi Chen Liuand Qunfeng Wu Human liver samples were collectedby Qunfeng Wu and Chen Liu The DDC mouse modelcharacterization and data analysis were performed byMardaJorgensen Joanna Song Junmei ZhouQunfengWu andLiyaPiThis manuscript was written by Liya Pi QunfengWu andMarda Jorgensen All the authors approved the final paper

Acknowledgments

This study is supported by Chris DiMarco InstitutionalResearch Grant from American Cancer Society andK01AA024174 grant from National Institute on AlcoholAbuse and Alcoholism awarded to Liya Pi This study is alsopartly supported by Childrenrsquos Miracle Network researchawarded to Liya Pi


References

[1] T A Roskams N D Theise C Balabaud et al ldquoNomenclatureof the finer branches of the biliary tree canals ductules andductular reactions in human liversrdquo Hepatology vol 39 no 6pp 1739ndash1745 2004

[2] A S H Gouw A D Clouston and N D Theise ldquoDuctularreactions in human liver diversity at the interfacerdquoHepatologyvol 54 no 5 pp 1853ndash1863 2011

[3] M J Williams A D Clouston and S J Forbes ldquoLinksbetween hepatic fibrosis ductular reaction and progenitor cellexpansionrdquo Gastroenterology vol 146 no 2 pp 349ndash356 2014

[4] M Okabe Y Tsukahara M Tanaka et al ldquoPotential hepaticstem cells reside in EpCAM+ cells of normal and injured mouseliverrdquo Development vol 136 no 11 pp 1951ndash1960 2009

[5] G Lanzoni V Cardinale and G Carpino ldquoThe hepaticbiliary and pancreatic network of stemprogenitor cell nichesin humans a new reference frame for disease and regenerationrdquoHepatology vol 64 no 1 pp 277ndash286 2016

[6] K N Lowes B A Brennan G C Yeoh and J K Olynyk ldquoOvalcell numbers in human chronic liver diseases are directly relatedto disease severityrdquoThe American Journal of Pathology vol 154no 2 pp 537ndash541 1999

[7] ADClouston E E PowellM JWalshMMRichardsonA JDemetris and J R Jonsson ldquoFibrosis correlates with a ductularreaction in hepatitis C roles of impaired replication progenitorcells and steatosisrdquoHepatology vol 41 no 4 pp 809ndash818 2005

[8] T Roskams S Q Yang A Koteish et al ldquoOxidative stress andoval cell accumulation in mice and humans with alcoholic andnonalcoholic fatty liver diseaserdquo American Journal of Pathologyvol 163 no 4 pp 1301ndash1311 2003

[9] M M Richardson J R Jonsson E E Powell et al ldquoProgressivefibrosis in nonalcoholic steatohepatitis association with alteredregeneration and a ductular reactionrdquoGastroenterology vol 133no 1 pp 80ndash90 2007

[10] S Y Xiao L Lu and H L Wang ldquoFibrosing cholestatic hep-atitis clinicopathologic spectrum diagnosis and pathogenesisrdquoInternational Journal of Clinical and Experimental Pathologyvol 1 no 1 pp 396ndash402 2008

[11] M J Wood V L Gadd L W Powell G A Ramm and A DClouston ldquoDuctular reaction in hereditary hemochromatosisthe link between hepatocyte senescence and fibrosis progres-sionrdquo Hepatology vol 59 no 3 pp 848ndash857 2014

[12] S Sell ldquoThe role of determined stem-cells in the cellularlineage of hepatocellular carcinomardquo The International Journalof Developmental Biology vol 37 no 1 pp 189ndash201 1993

[13] M Komuta B Spee S Vander Borght et al ldquoClinicopatholog-ical study on cholangiolocellular carcinoma suggesting hepaticprogenitor cell originrdquoHepatology vol 47 no 5 pp 1544ndash15562008

[14] M L Dumble E J Croager G C T Yeoh and E A QuailldquoGeneration and characterization of p53 null transformedhepatic progenitor cells oval cells give rise to hepatocellularcarcinomardquo Carcinogenesis vol 23 no 3 pp 435ndash445 2002

[15] N D Theise J L Yao K Harada et al ldquoHepatic lsquostem cellrsquomalignancies in adults four casesrdquo Histopathology vol 43 no3 pp 263ndash271 2003

[16] V Cardinale R Semeraro A Torrice et al ldquoIntra-hepaticand extra-hepatic cholangiocarcinoma new insight into epi-demiology and risk factorsrdquo World Journal of GastrointestinalOncology vol 2 no 11 pp 407ndash416 2010

[17] O Govaere M Komuta J Berkers et al ldquoKeratin 19 a keyrole player in the invasion of humanhepatocellular carcinomasrdquoGut vol 63 no 4 pp 674ndash685 2014

[18] M Komuta O Govaere V Vandecaveye et al ldquoHistologicaldiversity in cholangiocellular carcinoma reflects the differentcholangiocyte phenotypesrdquoHepatology vol 55 no 6 pp 1876ndash1888 2012

[19] The International Agency for Research on Cancer ldquoWHOclassification of tumours of the digestive system rdquo in IARCWHO Classification of Tumours F T Bosman F Carneiro RH Hruban and N D Theise Eds pp 1ndash418 World HealthOrganization Geneva Switzerland 4th edition 2010

[20] F Zhang X-P ChenW Zhang et al ldquoCombined hepatocellularcholangiocarcinoma originating from hepatic progenitor cellsImmunohistochemical and double-fluorescence immunostain-ing evidencerdquo Histopathology vol 52 no 2 pp 224ndash232 2008

[21] G-W Zuo C D Kohls B-C He et al ldquoThe CCN proteinsimportant signaling mediators in stem cell differentiation andtumorigenesisrdquo Histology and Histopathology vol 25 no 6 pp795ndash806 2010

[22] B Spee G Carpino B A Schotanus et al ldquoCharacterisationof the liver progenitor cell niche in liver diseases potentialinvolvement of Wnt and Notch signallingrdquo Gut vol 59 no 2pp 247ndash257 2010

[23] R Weiskirchen ldquoCCN proteins in normal and injured liverrdquoFrontiers in Bioscience vol 16 no 5 pp 1939ndash1961 2011

[24] Q Jia Q Dong and L Qin ldquoCCN core regulatory proteins inthe microenvironment that affect the metastasis of hepatocellu-lar carcinomardquo Oncotarget vol 7 no 2 pp 1203ndash1214 2016

[25] H Zhang W Li P Huang et al ldquoExpression of CCN familymembers correlates with the clinical features of hepatocellularcarcinomardquoOncology Reports vol 33 no 3 pp 1481ndash1492 2015

[26] P Fickert U Stoger A Fuchsbichler et al ldquoA new xenobiotic-induced mouse model of sclerosing cholangitis and biliaryfibrosisrdquo The American Journal of Pathology vol 171 no 2 pp525ndash536 2007

[27] L Pi P M Robinson M Jorgensen et al ldquoConnective tissuegrowth factor and integrin120572v1205736 a newpair of regulators criticalfor ductular reaction and biliary fibrosis in micerdquo Hepatologyvol 61 no 2 pp 678ndash691 2015

[28] S Tanaka K Sugimachi T Kameyama et al ldquoHumanWISP1va member of the CCN family is associated with invasivecholangiocarcinomardquo Hepatology vol 37 no 5 pp 1122ndash11292003

[29] X Wang M Foster M Al-Dhalimy E Lagasse M Finegoldand M Grompe ldquoThe origin and liver repopulating capacityof murine oval cellsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 1 pp 11881ndash11888 2003

[30] A Gardini B Corti M Fiorentino et al ldquoExpression ofconnective tissue growth factor is a prognostic marker forpatients with intrahepatic cholangiocarcinomardquo Digestive andLiver Disease vol 37 no 4 pp 269ndash274 2005

[31] F Gaunitz D Deichsel K Heise M Werth U Anderegg andR Gebhardt ldquoAn intronic silencer element is responsible forspecific zonal expression of glutamine synthetase in the ratliverrdquo Hepatology vol 41 no 6 pp 1225ndash1232 2005

[32] O A Gressner and A M Gressner ldquoConnective tissue growthfactor a fibrogenicmaster switch in fibrotic liver diseasesrdquo LiverInternational vol 28 no 8 pp 1065ndash1079 2008


[33] G Huang and D R Brigstock ldquoRegulation of hepatic stellatecells by connective tissue growth factorrdquo Frontiers in Biosciencevol 17 pp 2495ndash2507 2012

[34] Z Tong R Chen D S Alt S Kemper B Perbal and D RBrigstock ldquoSusceptibility to liver fibrosis in mice expressinga connective tissue growth factor transgene in hepatocytesrdquoHepatology vol 50 no 3 pp 939ndash947 2009

[35] Y-C Jian J-J Wang S Dong et al ldquoWnt-induced secretedprotein 1CCN4 in liver fibrosis both in vitro and in vivordquoClinical Laboratory vol 60 no 1 pp 29ndash35 2014

[36] X Li Y ChenW Ye et al ldquoBlockade of CCN4 attenuates CCl4-

induced liver fibrosisrdquo Archives of Medical Science vol 11 no 3pp 647ndash653 2015

[37] K-H Kim C-C Chen R I Monzon and L F Lau ldquoMatricel-lular proteinCCN1 promotes regression of liver fibrosis throughinduction of cellular senescence in hepatic myofibroblastsrdquoMolecular and Cellular Biology vol 33 no 10 pp 2078ndash20902013

[38] T Abd El Kader S Kubota D Janune et al ldquoAnti-fibrotic effectof CCN3 accompanied by altered gene expression profile of theCCN familyrdquo Journal of Cell Communication and Signaling vol7 no 1 pp 11ndash18 2013

[39] E Borkham-Kamphorst B T Steffen E Van de Leur et alldquoCCN1CYR61 overexpression in hepatic stellate cells inducesER stress-related apoptosisrdquo Cellular Signalling vol 28 no 1pp 34ndash42 2016

[40] E Borkham-Kamphorst C R van Roeyen E Van de JLear and R Weiskirchen ldquoCCN3NOV small interfering RNAenhances fibrogenic gene expression in primary hepatic stellatecells and cirrhotic fat storing cell line CFSCrdquo Journal of CellCommunication and Signaling vol 6 no 1 pp 11ndash25 2012

[41] K-H Kim C-C Chen G Alpini and L F Lau ldquoCCN1 induceshepatic ductular reaction through integrin 120572v120573

5-mediated acti-

vation of NF-120581Brdquo Journal of Clinical Investigation vol 125 no5 pp 1886ndash1900 2015

[42] L Pi S-H Oh T Shupe and B E Petersen ldquoRole of connectivetissue growth factor in oval cell response during liver regenera-tion after 2-AAFPHx in ratsrdquo Gastroenterology vol 128 no 7pp 2077ndash2088 2005

[43] L Pi X Ding M Jorgensen et al ldquoConnective tissue growthfactor with a novel fibronectin binding site promotes cell adhe-sion and migration during rat oval cell activationrdquo Hepatologyvol 47 no 3 pp 996ndash1004 2008

[44] C C Chen K H Kim and L F Lau ldquoThe matricellularprotein CCN1 suppresses hepatocarcinogenesis by inhibitingcompensatory proliferationrdquoOncogene vol 35 no 10 pp 1314ndash1323 2016

[45] Z-Q Li W Ding S-J Sun et al ldquoCyr61CCN1 is regulatedby wnt120573-catenin signaling and plays an important role in theprogression of hepatocellular carcinomardquo PLoS ONE vol 7 no4 Article ID e35754 2012

[46] M Xiu Y-H Liu D R Brigstock F-H He R-J Zhang andR-P Gao ldquoConnective tissue growth factor is overexpressedin human hepatocellular carcinoma and promotes cell invasionand growthrdquo World Journal of Gastroenterology vol 18 no 47pp 7070ndash7078 2012

[47] S Hirasaki N Koide K Ujike T Shinji and T Tsuji ldquoExpres-sion of Nov CYR61 and CTGF genes in human hepatocellularcarcinomardquo Hepatology Research vol 19 no 3 pp 294ndash3052001

[48] F-E Mo A G Muntean C-C Chen D B Stolz S CWatkins and L F Lau ldquoCYR61 (CCN1) is essential for placental

development and vascular integrityrdquo Molecular and CellularBiology vol 22 no 24 pp 8709ndash8720 2002

[49] S Ivkovic B S Yoon S N Popoff et al ldquoConnective tissuegrowth factor coordinates chondrogenesis and angiogenesisduring skeletal developmentrdquo Development vol 130 no 12 pp2779ndash2791 2003

[50] T Shimoyama S Hiraoka M Takemoto et al ldquoCCN3 inhibitsneointimal hyperplasia through modulation of smooth musclecell growth and migrationrdquo Arteriosclerosis Thrombosis andVascular Biology vol 30 no 4 pp 675ndash682 2010

[51] A Maeda M Ono K Holmbeck et al ldquoWNT1-inducedSecreted Protein-1 (WISP1) a novel regulator of bone turnoverandWnt signalingrdquoThe Journal of Biological Chemistry vol 290no 22 pp 14004ndash14018 2015

[52] S Hann L Kvenvold B N Newby M Hong and M L War-man ldquoA Wisp3 Cre-knockin allele produces efficient recombi-nation in spermatocytes during early prophase of meiosis IrdquoPLoS ONE vol 8 no 9 Article ID e75116 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


recruitment of progenitor cells contributing to liver cancerleading to HCC and intrahepatic CCA [12ndash15] In additionclinical-pathological correlation and risk factors for HCCand intrahepatic CCA support the role of HPC in differentsubtypes of liver cancers Recent evidences indicate that HPCmay be the cell of origin in certain subtypes of CCA suchas cholangiolocarcinoma and the so-called mixed-type CCAor HCC tumors such as CK19+ HCC [13 16ndash18] Lastlycombined hepatocellular cholangiocarcinoma (CHC) a rareform of primary liver cancer that accounts for 04ndash142 ofliver carcinomas [19] exhibits pathological features of HCCand CCA It has been proposed that CHC directly derivesfrom HPC activation and differentiation [20]

Matricellular proteins of the CCN (CYR61CTGFNOV)family are important signal modifiers in stem cell nichesand can modulate multiple signaling pathways mediatedby Notch Wnt and transforming growth factor-120573 (TGF-120573) [3 21 22] The CCN family is composed of six struc-turally conserved secreted proteins CCN1cysteine-rich 61(CYR61) CCN2connective tissue growth factor (CTGF)CCN3nephroblastoma overexpressed (NOV) CCN4Wnt1induced secreted protein-1 (WISP-1) CCN5WISP-2 andCCN6WISP-3 All CCN proteins share a conserved four-modular structure with the exception of CCN5WISP-2which lacks a cysteine knot domain module CCN proteinsexert activity on various types of cells through their broadbinding capabilities to ECM proteins growth factors andcell surface proteins [21] CCN proteins modulate cell-matrixinteraction and mediate cell adhesion migration differenti-ation apoptosis and survival Altered levels of CCN expres-sion or activities contribute to the pathogenesis of many dis-eases that occur when inflammation or tissue injury becomeschronic such as liver fibrosis and liver cancer [21 23ndash25]In order to understand CCN function in HPC activationand CCA development we examined the expression of CCNproteins in mouse HPC induced by 35-diethoxycarbonyl-14-dihydrocollidine (DDC) a biliary toxin that inducesductular reaction and biliary fibrosis [26] The levels ofCCNmRNAswere also quantitativelymeasured fromhumanintrahepatic CAAWithin the family CCN2CTGF exhibitedpredominant expression in both mouse HPC and humanCCA tumors

2 Materials and Methods

21 Human Tissues Human liver tissues were obtainedthrough Shands Hospital according to an approved protocolapproved by the institutional review board at the Universityof FloridaWritten informed consents were obtained from allsubjects Tumor or adjacent nontumor tissues from intrahep-aticCCAcontaining liverswere separated after dissection andsnap-frozen for molecular analyses

22 The DDCMouse Model Transgenic mice carrying Ccn2Ctgf promoter driven GFP (Ctgfp-GFP) were previouslydescribed [27] Wild-type or Ctgfp-GFP mice at 8ndash10 weeksof age were fed with a diet supplemented with 01 DDC(Bio-Serv Frenchtown NJ) to induce cholangitis ductular

reactions and biliary fibrosis All protocols and procedureswere approved by the University of Florida IACUC and werein accordance with National Institutes of Health guidelines

23 Immunohistochemistry Allmouse liver tissueswere fixedovernight in 4 paraformaldehyde to preserve the GFPfluorescence signal Tissues were infiltrated with 20 sucrosebefore being embedded in OCT 6120583m sections were usedin this study GFP staining in combination with EpCAMantibody (eBioscience San Diego CA) or hepatocyte nuclearfactor 4120572 (HNF4120572) antibody (Santa Cruz BiotechnologiesDallas Texas) required antigen retrieval at 37∘C for 10minutes in trypsin (Digest-All 2 Invitrogen Carlsbad CA)Primary antibodies employed were 1 100 rat anti-EpCAM1 100 rabbit anti-HNF4120572 and 1 750 chicken anti-GFP(Aves Labs Tigard OR) Alexa Fluor 488 conjugated goatanti-chicken and Alexa Fluor 594 conjugated donkey anti-rabbit secondary antibodies (Invitrogen Carlsbad CA) wereused at 1 500 for detection Human intrahepatic CCA andHCC tissue arrays were purchased from US Biomax Inc(Rockville MD) Gomorirsquos Trichrome staining for collagenfibers was carried out using aThermo Scientific Chromaviewcollagen blue kit according to themanufacturerrsquos instructions(Richard-Allan Scientific Kalamazoo MI) To evaluate fibro-sis in DDC treated mice liver sections were stained withPicrosirius Red solution (American MasterTech Lodi CA)In addition staining for CK19 CCN2CTGF and 120572-SMArequires antigen retrieval in Trilogy pretreatment solution(Cell Marque Rocklin CA) at 95∘C for 25 minutes Thesections were blocked in 3 horse serum (Vector Laborato-ries) for one hour prior to overnight incubation with anti-CK19 antibody (Abcam) anti-CCN2CTGF antibody (SantaCruz Biotechnologies) and anti-120572SMA antibody (SigmaSt Louis MO) Additional IHC for CCN2CTGF in liversections of human intrahepatic CCA was carried out usinga rabbit antibody (1 200 Abcam) and detected using aVECTASTAINABC-AP kit and Vector Red substrate (VectorLaboratories Burlingame CA)

24 RNA Isolation and Reverse Transcriptase-PolymeraseChain Reaction (RT-PCR) Analysis Total RNAs were ex-tracted using RNeasy Mini kit (Qiagen Valencia CA) fol-lowed by incubation with RQ1 RNase-free DNase (PromegaMadison WI) to remove any genomic DNA contamination2 120583g of total RNA and 50 pmol random hexamer were usedfor Superscript III First-Strand cDNA Synthesis (Invit-rogen) RT-PCR was carried out using 05 120583L cDNAs astemplates 02120583M of each set of primers and REDExtract-N-Amp tissue PCR mixtures (Sigma) Amplification con-ditions consisted of thirty cycles of denaturation at 94∘Cfor 30 sec annealing at 55∘C for 30 sec and extension at72∘C for 30 sec Primer pairs for mouse genes includethe following 51015840-TTTACAGTTGGGCTGGAAGC-31015840 and51015840-CACCGCTCTGAAAGGGATCT-31015840 for Ccn1Cyr61 51015840-GTCTTCACACTGGTGCAGCC-31015840 and 51015840-ACTGGAAGA-CACATTTGGCC-31015840 for Ccn2Ctgf 51015840-CTTGGTGCGGAG-ACACTTTT-31015840 and 51015840-CGCCAGTGTGAGATGGTAAA-31015840 for Ccn3Nov 51015840-TGAGTGCTTGAGCATCCAGA-31015840





3 Results






0 5 10 15 20Days on DDC

18S

EpCAM

Ccn2Ctgf

Ccn1Cyr61

Ccn3Nov

Ccn5Wisp2

Ccn6Wisp3

Ccn4Wisp1v

TNF

-SMA

Collagen 1(I)

(a)

Hamp

ESi

rius R


(b)CCN2CTGF

NL

NT T NL

NT T

NL

NT TNL

NT T

NL

NT T

CCN3NOV

CCN4WISP1v

CCN5WISP2

CCN6WISP3

00

15

CCN1CRY61

NL

NT T

lowast

lowast

lowast

lowast

0

2

4

6

8

Fold

incr

ease

Fold

incr

ease

0

1

2

3

4

5

Fold

incr

ease

0

1

2

3

4

5

Fold

incr

ease

0

1

2

3

Fold

incr

ease

00

15

Fold

incr

ease

(c)




Nor

mal

DD

C

(a)


(b)


CCN2CTGFTrichrome

(a)

CCN2CTGFTrichrome

(b)






CCN2CTGF Merge-SMA

(a)

CCN2CTGFCK19 Merge

(b)

IH-CCA

(c)

CK19CCN2CTGF Merge

(d)

HCC

(e)



4 Discussion








5 Conclusion


Abbreviations







Competing Interests




Acknowledgments



References












































5-mediated acti-



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















3 Results






0 5 10 15 20Days on DDC

18S

EpCAM

Ccn2Ctgf

Ccn1Cyr61

Ccn3Nov

Ccn5Wisp2

Ccn6Wisp3

Ccn4Wisp1v

TNF

-SMA

Collagen 1(I)

(a)

Hamp

ESi

rius R


(b)CCN2CTGF

NL

NT T NL

NT T

NL

NT TNL

NT T

NL

NT T

CCN3NOV

CCN4WISP1v

CCN5WISP2

CCN6WISP3

00

15

CCN1CRY61

NL

NT T

lowast

lowast

lowast

lowast

0

2

4

6

8

Fold

incr

ease

Fold

incr

ease

0

1

2

3

4

5

Fold

incr

ease

0

1

2

3

4

5

Fold

incr

ease

0

1

2

3

Fold

incr

ease

00

15

Fold

incr

ease

(c)




Nor

mal

DD

C

(a)


(b)


CCN2CTGFTrichrome

(a)

CCN2CTGFTrichrome

(b)






CCN2CTGF Merge-SMA

(a)

CCN2CTGFCK19 Merge

(b)

IH-CCA

(c)

CK19CCN2CTGF Merge

(d)

HCC

(e)



4 Discussion








5 Conclusion


Abbreviations







Competing Interests




Acknowledgments



References












































5-mediated acti-



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 5 10 15 20Days on DDC

18S

EpCAM

Ccn2Ctgf

Ccn1Cyr61

Ccn3Nov

Ccn5Wisp2

Ccn6Wisp3

Ccn4Wisp1v

TNF

-SMA

Collagen 1(I)

(a)

Hamp

ESi

rius R


(b)CCN2CTGF

NL

NT T NL

NT T

NL

NT TNL

NT T

NL

NT T

CCN3NOV

CCN4WISP1v

CCN5WISP2

CCN6WISP3

00

15

CCN1CRY61

NL

NT T

lowast

lowast

lowast

lowast

0

2

4

6

8

Fold

incr

ease

Fold

incr

ease

0

1

2

3

4

5

Fold

incr

ease

0

1

2

3

4

5

Fold

incr

ease

0

1

2

3

Fold

incr

ease

00

15

Fold

incr

ease

(c)




Nor

mal

DD

C

(a)


(b)


CCN2CTGFTrichrome

(a)

CCN2CTGFTrichrome

(b)






CCN2CTGF Merge-SMA

(a)

CCN2CTGFCK19 Merge

(b)

IH-CCA

(c)

CK19CCN2CTGF Merge

(d)

HCC

(e)



4 Discussion








5 Conclusion


Abbreviations







Competing Interests




Acknowledgments



References












































5-mediated acti-



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Nor

mal

DD

C

(a)


(b)


CCN2CTGFTrichrome

(a)

CCN2CTGFTrichrome

(b)






CCN2CTGF Merge-SMA

(a)

CCN2CTGFCK19 Merge

(b)

IH-CCA

(c)

CK19CCN2CTGF Merge

(d)

HCC

(e)



4 Discussion








5 Conclusion


Abbreviations







Competing Interests




Acknowledgments



References












































5-mediated acti-



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















CCN2CTGF Merge-SMA

(a)

CCN2CTGFCK19 Merge

(b)

IH-CCA

(c)

CK19CCN2CTGF Merge

(d)

HCC

(e)



4 Discussion








5 Conclusion


Abbreviations







Competing Interests




Acknowledgments



References












































5-mediated acti-



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















5 Conclusion


Abbreviations







Competing Interests




Acknowledgments



References












































5-mediated acti-



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















References












































5-mediated acti-



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























5-mediated acti-



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article members of the cyr61/ctgf/nov protein...

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