role of p38 mapk pathway in bmp4-mediated smad-dependent ... · their co-operation in bmp4-induced...

Biochem. J. (2011) 433, 333–343 (Printed in Great Britain) doi:10.1042/BJ20100404 333

Role of p38 MAPK pathway in BMP4-mediated Smad-dependent prematuresenescence in lung cancer cellsDongmei SU*, Xiue PENG†, Shan ZHU*, Ying HUANG†, Zhixiong DONG*, Yu ZHANG*, Jianchao ZHANG*, Qian LIANG†,Jun LU†1 and Baiqu HUANG*1

*The Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024, People’s Republic of China, and †The Key Laboratory of Molecular Epigenetics of Ministry ofEducation (MOE), Northeast Normal University, Changchun 130024, People’s Republic of China

BMP4 (bone morphogenetic protein 4) is a multifunctionalcytokine known to exert its biological effects through a varietyof signalling pathways. The diverse function of BMP4 appearsto be due to multiple pathways activated by BMP4 itself.Our previous studies have demonstrated that BMP4 is able todrive lung cancer cells into a process of premature senescence;however, the signalling pathways, as well their interplays androles associated with this process, are not well understood. Toaddress these questions, in the present study we investigatedthe signalling and molecular mechanisms underlying the BMP4-induced senescence, and our data demonstrated that p38 MAPK(mitogen-activated protein kinase) and Smad pathways werenecessary for this process. Meanwhile, the ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway, which is required for

senescence, was not activated by BMP4 in the lung cancercell line NCI-H460. We also showed that the BMP4-responsiveR-Smads (receptor-regulated Smads), i.e. Smad1 and Smad5,were necessary for the up-regulation of p16INK4a and p21WAF1/cip1

and for the induction of premature senescence. Furthermore,we found that activation of the p38 MAPK pathway by BMP4was essential for the full activation of transcription potential ofSmad1/5. Overall, the results of the present study implicate acomplex co-operation between p38 MAPK and Smad pathwaysin BMP4-mediated premature senescence.

Key words: bone morphogenetic protein 4 (BMP4), p38mitogen-activated protein kinase (p38 MAPK), prematuresenescence, Smad1, Smad5.

INTRODUCTION

BMPs (bone morphogenetic proteins) are secreted signallingmolecules belonging to the TGFβ (transforming growth factor β)superfamily [1]. They were originally isolated from bone matrix[2]. Although BMPs function as osteogenic factors, they also havepleiotrophic roles in cell growth, differentiation, migration andapoptosis, and are critical in embryogenesis and organogenesis[2,3]. BMPs elicit their effects through activation of type-1 andtype-2 serine/threonine kinase receptors. BMPs and TGFβ/activinreceptor-phosphorylated Smads (R-Smads) oligomerize with thecommon mediator Smad4 (Co-Smad) to regulate gene expressionby binding to DNA upon their nuclear import [4]. BMP receptorsactivate Smad1, Smad5 and Smad8, whereas Smad2 and Smad3are phosphorylated by activin or TGFβ receptors [4,5]. Smad6 andSmad7 have been identified as the inhibitory Smads (I-Smads).I-Smads stably interact with activated type-1 receptors andcompete with R-Smads for activation by the receptors. Smad7inhibits both TGFβ/activin and BMP signalling, whereas Smad6efficiently inhibits BMP signalling, but only weakly inhibitsTGFβ/activin signalling [6,7].

The Smad-independent pathways, such as ERK1/2(extracellular-signal-regulated kinase 1/2), p38 MAPK (mitogen-activated protein kinase) and c-Jun N-terminal kinase, may beactivated following treatment with BMPs in specific cell contexts[8−10]. The cross-talk between the Smad pathway and other

pathways may lead to the diverse function of BMP4. Our previousstudies and other studies have shown that BMP4 was able todrive lung cancer cells into premature senescence or replicativesenescence in vitro [11,12]. However, the molecular events and thesignalling pathways underlying this process are still unclear. p38MAPK and ERK/MAPK pathways have been highlighted to belinked to premature senescence [13−15], implying that they mayparticipate in mediating BMP4-induced premature senescence.

The relative contribution of these different pathways inBMP4-induced premature senescence is poorly understood.The aim of the present study was to identify the roles ofdiverse pathways that are activated by BMP4, as well astheir co-operation in BMP4-induced premature senescence inlung cancer cells. Our results showed that BMP4 was able toactivate the p38 MAPK pathway to mediate senescence in NCI-H460 cells in addition to Smad signalling. However, the ERKsignalling pathway, another senescence-associated signallingpathway, was not activated by BMP4 in the process of BMP4-mediated senescence. Both Smad-dependent and -independentp38 MAPK pathways synergistically contributed in BMP4-triggered premature senescence. Furthermore, the present studyrevealed that the p38 MAPK pathway positively modulated Smadfunction by affecting their transcriptional activation potential.Overall, the results of the present study demonstrate a complexinterplay between Smad and p38 MAPK pathways in BMP4-mediated premature senescence.

Abbreviations used: BMP, bone morphogenetic protein; CE, cytosolic extract; ChIP, chromatin immunopreciptation; CoIP, co-immunoprecipitation; DAPI,4′,6-diamidino-2-phenylindole; DOX, doxycycline; DTT, dithiothreitol; ERK1/2, extracellular-signal-regulated kinase 1/2; FBS, fetal bovine serum IMDM,Iscove’s modified Dulbecco’s medium; MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide;NE, nuclear extract; ONPG, o-nitrophenyl-β-D-galactopyranoside; RNAi, RNA interference; siRNA, small interfering RNA; TGFβ, transforming growthfactor β.

1 Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).

c© The Authors Journal compilation c© 2011 Biochemical Society

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334 D. Su and others

EXPERIMENTAL

Materials

The p16INK4a (hereafter termed p16) promoter reporter (−869to +1 bp from the cap site) ligated to the luciferase reportergene (pGL2 basic; Promega) was provided by Dr E. Hara(Imperial Cancer Research Fund Laboratories, London, U.K.).The pcDNA3-Myc-Smad6 expression plasmid was provided byDr S. Itoh (Department of Experimental Pathology, GraduateSchool of Comprehensive Human Sciences, University ofTsukuba, Japan). The plasmid of p21−luc (2400/+11) was agift from Dr Bert Vogelstein (The Johns Hopkins UniversitySchool of Medicine at Maryland, Baltimore, MD, U.S.A.). pA3-Flag-Smad1 was a gift from Dr C. Laurie (The Oncology andMolecular Endocrinology Research Center, CHUL ResearchCenter and Laval University, Quebec, Canada). pcDNA3.1-Flag-Smad5 was provided by Dr T. Aigner (University of Leipzig,Leipzig, Germany). Specific inhibitors for ERK (U0126) and p38MAPK (SB203580) were obtained from Sigma.

Cell culture, transfection and luciferase reporter assay

NCI-H460 lung cancer cells and the pSTAR-hBMP4 stablytransfected cell line were maintained in IMDM (Iscove’s modifiedDulbecco’s medium) supplemented with 10% FBS (fetal bovineserum), 100 mg/ml penicillin and 100 mg/ml streptomycin ina humidified atmosphere containing 5 % CO2 at 37 ◦C. ThepSTAR/hBMP4 stably transfected cell line was constructed in ourprevious study [12]. The cell line that inducibly expresses BMP4protein by DOX (doxycycline) (Clontech) in both a dose- andtime-dependent manner was maintained in IMDM supplementedwith 10% FBS in the presence of G418 (1000 μg/ml).

For the luciferase reporter assay, 5 × 104 cells were seeded in24-well tissue culture plates for 24 h before they were transientlytransfected with 1 μg of p16 or p21 reporter plasmid and 0.5 μgof indicated constructs or vector alone using the FuGENETM

HD transfection reagent (Roche). The Renilla luciferase controlplasmid pREP7-RLuc was co-transfected at 50 ng/well as aninternal control reporter. At 30 h post-transfection, cells werewashed and lysed in passive lysis buffer (Promega) and thetransfection efficiency was normalized to the paired Renillaluciferase activity using the Dual Luciferase Reporter AssaySystem (Promega) according to the manufacture’s instructions.

Western blot analysis

NCI-H460 cells were harvested after treatments. Cells (1 × 106)were digested and lysed in lysis buffer (50 mM Tris/HCl, 1 %Nonidet P40, 150 mM NaCl, 1 mM EDTA and 1 mM PMSF) for30 min at 4 ◦C. Total cell extracts were separated by SDS/PAGE(12% gels), and then transferred on to PVDF membranes.The membranes were incubated with anti-p16 (Santa CruzBiotechnology, sc-468), anti-p21 (Santa Cruz Biotechnology, sc-756), anti-BMP4 (Santa Cruz Biotechnology, sc-6896), anti-p38(Cell Signaling Technology, #9212), anti-Smad1 (Cell SignalingTechnology, #9743), anti-Smad5 (Cell Signaling Technology,#9517), anti-Smad8 (Santa Cruz Biotechnology, sc-11393),anti-phospho-p38 MAPK (Cell Signaling Technology, #9211),anti-tubulin (Cell Signaling Technology, #3873), anti-laminB(Santa Cruz Biotechnology, sc-6216), anti-phospho-Smad1(Ser463/Ser465)/Smad5(Ser463/Ser465)/Smad8(Ser426/Ser428) (CellSignaling Technology, #9511), anti-phospho-Smad1/Smad5-(Ser463/Ser465) (Cell Signaling Technology, #9516) or anti-β-actin(Sigma, A1978) antibodies. The signals were visualized by usingthe chemiluminescent substrate method with the SuperSignal

West Pico kit provided by Pierce. β-Actin was used as an internalcontrol for normalizing the loading materials.

Cell proliferation and MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazoliumbromide] assay

Cell proliferation was measured by using the MTT assay.Cells were seeded on 96-well plates at a density of 2 × 103

cells/well. After transfection or treatment, cells were incubatedwith 5 mg/ml MTT solution for 4 h. The medium was aspirated,and the formazan product was solubilized with 100 μl of DMSO.Viability was assessed by measuring the absorbance at 492 nmwith a microplate reader.

Cellular immunofluorescence

Cells were fixed in 1% formaldehyde in culture medium for10 min at 37 ◦C and permeabilized with 0.2% Triton X-100 inPBS for 10 min at 4 ◦C. Endogenous Smad1, Smad5 and Smad8were detected with antibodies against Smad1, Smad5 and Smad8respectively and visualized with an FITC-conjugated anti-rabbitIgG secondary antibody. DAPI (4′,6-diamidino-2-phenylindole)was used to stain the nucleus. Immunofluorescence with anti-tubulin was used to stain the cytoplasm. Photographs were takenunder a fluorescence microscope.

Senescence-associated β-galactosidase activity assay andcytochemical staining for SA-β-galactosidase

Cells were lysed in reporter lysis buffer (50 mM Tris/HCl, 1%Nonidet P40, 150 mM NaCl, 1 mM EDTA and 1 mM PMSF). Celllysates containing equal amounts of total protein were diluted inequal volumes of 2× assay buffer comprising 1.33 mg/ml ONPG(o-nitrophenyl-β-D-galactopyranoside), 2 mM MgCl2 and 100 μlof 2-mercaptoethanol in 200 mM phosphate buffer (pH 6.0), andwere incubated at 37 ◦C for 4 h. The absorbance at 420 nm wasmeasured after the addition of an equal volume of 1 M Na2CO3.

Cytochemical staining for SA-β-galactosidase was performedusing a senescence-β-galactosidase staining kit (Cell SignalingTechnology) at pH 6.0. All of the experiments were repeated threetimes, and one of the representative results is shown.

RNAi (RNA interference)

The siRNA (small interfering RNA) targeting sequences ofSmad1, Smad5 and Smad8 were 5′-AACCTGTCATTATTGCT-TACT-3′ [16], 5′-AATTACATCCTGCCGGTGATA-3′ [17]and 5′-AAGTTAAAGAAGAAGAAGGGA-3′ respectively. Thecontrol siRNA sequence was 5′-CGTCAACATGGCTTTCACC-3′. Oligonucleotides that represent small hairpin RNAs targetingthese sequences were designed, synthesized and cloned into thepSliencer4.1-CMV neo vector (Ambion) between BamHI andHindIII sites according to the manufacturer’s instructions.

ChIP (chromatin immunoprecipitation)

The protocol for ChIP has been described previously [12],and an anti-phospho-Smad1(Ser463/Ser465)/Smad5 (Ser463/Ser465)antibody was used. Samples were analysed by PCR. The primersspecific to sequences at the P16 promoter were: P1 sense,5′-CATTCGCTAAGTGCTCGGAGT-3′ and antisense, 5′-CT-GCTCCCCGCCGCCCGCTGCCTG-3′; P2 sense, 5′-TAGGAA-GGTTGTATCGCGGAGG-3′ and antisense, 5′-CAAGGAAG-GAGGACTGGGCTC-3′; P3 sense, 5′-AGACAGCCGTTTT-ACACGCAG-3′ and antisense, 5′-CACCGAGAAATCGAAAT-CACC-3′; and P4 sense, 5′-GCTGAGGCAGGAGAATT-3′


p38 MAPK and Smad pathways in premature senescence 335

and antisense, 5′-TTTGGGATGTCAAGTATG-3′ The primersspecific to sequences at the P21 promoter were: Q1 sense, 5′-GG-TGTCTAGGTGCTCCAGGT-3′ and antisense, 5′-GCACTCTC-CAGGAGGACACA-3′; Q2 sense, 5′-CGTGGTGGTGGTGAG-CTA-3′ and antisense, 5′-CTGTCTGCACCTTCGCTCCT-3′;and Q3 sense, 5′-AATTCCTCTGAAAGCTGACTGCC-3′ andantisense, 5′-AGGTTTACCTGGGGTCTTTAGA-3′.

Fractionation procedure

NEs (nuclear extracts) and CEs (cytosolic extracts) were preparedessentially as follows. Cells were washed and lysed in hypotonicbuffer comprising 1% (w/v) Nonidet P40, 10 mM Hepes/KOH(pH 7.4), 120 mM NaCl, 1 mM DTT (dithiothreitol), 1 mMEDTA, 1 mM sodium orthovanadate, 1 mM PMSF, and 5 μg/mleach of leupeptin and aprotinin at 4 ◦C for 30 min with gentleshaking. Nuclei were collected by centrifugation at 10000 gfor 8 min at 4 ◦C, and the supernatant was saved for CEs. Thepellet was resuspended in high-salt buffer [20 mM Hepes/KOH(pH 7.4), 20 % (v/v) glycerol, 500 mM NaCl, 1.5 mM MgCl2,0.2 mM EDTA, 1 mM DTT, 1 mM sodium orthovanadate, 1 mMPMSF and 5 μg/ml each of leupeptin and aprotinin], and thenincubated at 4 ◦C for 30 min. The lysates were clarified bycentrifugation at 15000 g at 4 ◦C for 10 min. The supernatantwas used as the NE. Equal amounts of protein in CEs and NEswere loaded on to SDS gels for Western detection.

CoIP (co-immunoprecipitation) assay

Total cell extracts from DOX-treated cells were pre-clearedwith salmon sperm DNA/Protein A−agarose beads (Upstate).Rabbit anti-FLAG (Sigma, F7425) antibody was added forimmunoprecipitation. The precipitates were then subjected toSDS/PAGE followed by transfer on to a PVDF membraneand incubation with anti-Sp1 (Santa Cruz Biotechnology, sc-14027), anti-Smad4 (Abcam, ab3219) or anti-FLAG antibody.Samples were detected using the Super Signal West PicoChemiluminescent Substrate (Pierce) detection method followingthe manufacturer’s instructions.

Statistical analysis

A Student’s t test was used to calculate the statistical significanceof the experimental data. The significance level was set as: *P,#P < 0.05; **P, ##P<0.01.

RESULTS

The p38 MAPK pathway, but not the ERK pathway, plays animportant role in mediating BMP4-induced premature senescence

Our previous data showed that BMP4 was able to inducepremature senescence in lung cancer cells [12], but the sig-nalling pathways in BMP4-induced premature senescence remainlargely unknown. p38 MAPK and ERK signalling pathways havebeen suggested to be involved in cellular senescence by variousstimulations [14,18]. We first examined whether the BMP4-induced premature senescence is mediated by these pathwaysusing specific kinase inhibitors. As shown in Figure 1(A),treatment with SB203580, an effective inhibitor of the p38MAPK signalling pathway, partly restored the capacity ofBMP4 in reduction of proliferation of pSTAR-hBMP4 cells,which express BMP4 protein upon DOX induction in botha dose- and time-dependent manner [12]. Meanwhile, our

results also demonstrated that the inhibitor specific for p38MAPK did not affect BMP4 production in pSTAR-hBMP4cells, indicating that p38 MAPK may be the downstreamsignalling molecule of BMP4 (Supplementary Figure S1 athttp://www.BiochemJ.org/bj/433/bj4330333add.htm). Treatmentwith the ERK1/2 inhibitor U0126 did not block the reductionof proliferation by BMP4 treatment. As a control, DMSO didnot alter this reduction effect of BMP4 (Figure 1A). We furthershowed that SB203580, but not U0126, restrained prematuresenescence induced by BMP4 overexpression in pSTAR-hBMP4cells (Figures 1B−1D). In addition, we found that SB203580(20 μM) was able to reduce the expression of p16 and p21,which are important senescence-associated proteins and are up-regulated in the process of BMP4-induced premature senescence(Figure 1E). Meanwhile, no changes in p16 and p21 expressionwere observed upon U0126 (20 μM) treatment (Figure 1E). Thesedata suggest that p38 MAPK, but not the ERK pathway, is involvedin BMP4-induced premature senescence in NCI-H460 lung cancercells.

p38 MAPK and Smad signalling pathways are activated by BMP4

Besides the classical Smad pathway, evidence has implied thatSmad-independent signalling pathways were also activated inBMP signalling in different cellular contexts [8−10]. We wantedto know whether p38 MAPK, Smad and ERK pathways areactivated in the process of BMP4-induced premature senescence.Western blotting analysis of total cell extracts from pSTAR-hBMP4 cells treated with DOX showed activation of the Smadand p38 MAPK pathways (Figures 2A and 2C), whereas the ERKpathway was not activated by BMP4 (Figure 2B). These resultsmay give a reason why the p38 MAPK pathway, but not the ERKpathway, was involved in mediating BMP4-induced senescence.

Both the p38 MAPK and Smad pathways play important roles inmediating BMP4-induced premature senescence

To investigate whether the p38 MAPK and Smad pathwayare required for BMP4-induced premature senescence, weemployed two inhibitors in the experiments, i.e. the Smad6expression plasmid for inhibiting the Smad pathway andSB203580 for inhibiting the p38 MAPK pathway. As shownin Figures 3(A)−3(C), blocking the Smad pathway partlyinterfered with the BMP4-induced premature senescence bySmad6 overexpression, which is known to inhibit BR-Smad(Smad1, Smad5 and Smad8) phosphorylation and nucleartranslocation [7,19]; meanwhile, inhibition of the p38 MAPKpathway by SB203580 only partly restrained BMP4-inducedpremature senescence in pSTAR-hBMP4 cells. However, whenboth Smad and p38 MAPK were blocked, the senescence triggeredby BMP4 was almost completely abrogated. Further investigationusing Western blot analysis showed that the BMP4-induced up-regulation of p16 and p21 was decreased more significantly whenboth Smad and p38 MAPK were inhibited than that when onlyeither one of the pathways was inhibited (Figure 3D). Theseresults indicated that both the p38 MAPK and Smad pathwayswere essential for BMP4-induced premature senescence and theyappeared to co-operate with each other to mediate BMP4-inducedpremature senescence.

Smad1 and Smad5 are required in BMP4-induced prematuresenescence

Our previous results have shown that the Smad pathway played acritical role in mediating BMP4-induced premature senescence



Figure 1 The p38 MAPK signalling pathway participates in mediating BMP4-induced premature senescence in lung cancer cells

A) The p38 MAPK inhibitor SB203580, but not the ERK1/2 inhibitor U0126, was able to antagonize the anti-proliferative effect of BMP4 in lung cancer cells, as revealed by the MTT assay.pSTAR-hBMP4 stably transfected cells were seeded on to 96-well plates at a density of 2 × 103 cells/well, and were treated either with DOX alone or together with SB203580 (10 and 20 μM) or U0126(10 and 20 μM) for 2 days, and the cell viability was assessed by measuring the absorbance at 492 nm with a microplate reader. ∗P < 0.05; ∗∗P < 0.01 compared with the untreated group. #P < 0.05;##P < 0.01 compared with the DOX-treated group (n = 3). (B) BMP4-induced premature senescence was restrained by blocking the p38 MAPK signalling pathway. The SA-β-galactosidase activityassay was performed using ONPG as a substrate at pH 6.0 in pSTAR-hBMP4 cells treated with DOX alone or together with SB203580 (10 and 20 μM) or U0126 (10 and 20 μM) for 6 days.∗P < 0.05; ∗∗P < 0.01 compared with the untreated group. #P < 0.05; ##P < 0.01 compared with the DOX-treated group (n = 3). (C) The p38 MAPK inhibitor, but not the ERK1/2 inhibitor,restrained BMP4-induced premature senescence. SA-β-galactosidase staining was performed on cells cultured for 6 days after the indicated treatments. (D) The average percentage of positivelystained cells, counted based on three independent microscopic fields. ∗P < 0.05; ∗∗P < 0.01 compared with the untreated group. #P < 0.05; ##P < 0.01 compared with the DOX-treated group(n = 3). (E) The p38 MAPK inhibitor, but not the ERK1/2 inhibitor, repressed BMP4-stimulated senescence-associated gene expression (p16 and p21). Lysates were prepared 2 days following DOXtreatment alone or together with inhibitors (SB203580 at 10 and 20 μM; U0126 at 10 and 20 μM), and probed with the antibodies indicated. β-Actin was used as an internal reference control.

[12]. Smad1, Smad5 and Smad8 (termed BR-Smad), are themain cytoplasmic signalling molecules in response to BMPs[20]. It is believed that functional differences among these BR-Smads exist, although details of these differences have not beenestablished so far. In the next experiments, we intended todefine which BR-Smad participates in mediating BMP4-induced

premature senescence. We used an RNAi approach to knockdownSmad1, Smad5 and Smad8 expression individually to assess theireffects. The expression of endogenous Smad1, Smad5 and Smad8proteins was specifically reduced by transfection of specificsiRNAs without affecting the expression of the other Smads asdetermined by Western blotting (Figure 4A). We then showed



Figure 2 BMP4 elevates the phosphorylation levels of Smad1/5/8 and p38MAPK, but not that of ERK1/2

Estimation of the phosphorylation levels of Smad1/5/8, p38 MAPK and ERK1/2 in pSTAR-hBMP4cells treated with DOX for 2 days. Western blotting assays were performed with the antibodiesindicated.

that suppression of endogenous Smad1 and Smad5 expressionrestrained the BMP4-induced senescence in pSTAR-hBMP4cells (Figures 4B−4E), eliciting the critical role of Smad1/5 inmediating BMP4-induced premature senescence, whereas Smad8did not affect this process.

The p38 MAPK pathway is not involved in mediating nucleartranslocation of Smad proteins in response to BMP4

MAPK pathways have been reported to have either positiveor negative roles in mediating the Smad pathway [9,21,22].Activation of MAPK pathways has been shown to inducenuclear translocation of Smad2 [23,24], or, in other cases,to inhibit TGFβ-dependent nuclear translocation [16]. Wetherefore tested whether the p38 MAPK pathway has an effect onBMP4-dependent nuclear translocation of the Smad proteins inpSTAR-hBMP4 cells by using cellular immunofluorescence. Ascan be seen in Figures 5(A) and 5(B), Smad1 and Smad5 proteinswere mainly located in the nucleus in the BMP4-overexpressiongroup (middle column), whereas they were located both in thenucleus and cytoplasm in the control group (left-hand column).In addition, the p38 MAPK signalling inhibitor SB203580 didnot inhibit BMP4-dependent nuclear translocation of Smad1and Smad5 (right-hand column). The effect of p38 MAPKon BMP-4-dependent nuclear translocation of Smad proteinswas also analysed by Western blotting, which confirmed thatBMP4 overexpression prominently promoted Smad proteinnuclear translocation; however, this process was not inhibitedby SB203580 (Figure 5C and 5D). Our further studies showed thatSB203580 did not affect the phosphorylation of Smad1/5 at itsC-terminal region, which was required for Smad protein activationand interacted with Smad4 for nuclear translocation (Figure 5E).These experiments indicate that the p38 MAPK pathway is notinvolved in mediating nuclear translocation of Smad proteins.

p38 MAPK affects Smad1/5-dependent transcriptional activation

Next, we were interested in determining whether the p38 MAPKpathway affects the Smad1/5 transcriptional activity in responseto BMP4. We showed previously that P16 and P21 were BMP4-

responsive target genes and were required for BMP4-inducedpremature senescence [12]. In the present study we show thatBMP4-responsive R-Smads, i.e. Smad1 and Smad5, were able topartly increase the activity of P16 and P21 promoters, whereas thetranscriptional activation by Smad protein was partly restrainedby addition of SB203580, as determined by luciferase reporterassays (Figure 6A) in NCI-H460 cells. Furthermore, we foundthat Smad1/5 exhibited potent transcriptional activation in thepresence of BMP4, which is able to induce phosphorylation andactivation of Smad proteins, but such activation was efficientlyblocked by SB203580 in pSTAR-hBMP4 cells (Figure 6B). Theseresults indicated that the p38 MAPK pathway played an importantrole in mediating the transactivation of Smad1 and Smad5. Sincewe previously discovered that BMP4 was able to up-regulateP16 and P21 expression through promoting the enrichment ofthe downstream transcription factors Smad1/5 at the definedpromoter regions [12], we wanted to know whether the p38MAPK pathway also mediates the occupation of Smad1/5 onP16 and P21 promoters. ChIP assays were performed to detectthe presence of phospho-Smad1/5 at P16 and P21 promotersin the presence of SB203580. We designed a series of ChIPprimers at the P16 promoter, i.e. P1, P2 and P3 at −110,−500 and −800 bp respectively (Figure 6C, left-hand panel),according to the presence of Smad1/5 at different regions of theP16 promoter [12]. P4 locates at the far upstream of the p16promoter (−2600 bp) as a negative control. Likewise, primers forthe P21 gene were also designed (Q1, Q2 and Q3 at −600, −1800and −2600 bp) (Figure 6C, right-hand panel). The ChIP resultsrevealed that the enhanced enrichment of phospho-Smad1/5 at allof these promoter regions of P16 and P21 genes in response toBMP4 was restrained by the addition of SB203580 (Figure 6D).Taken together, these results indicate that the p38 MAPK pathwayis essential for Smad-dependent transcriptional activation inresponse to BMP4 in pSTAR-hBMP4 cells.

Sp1 functions synergistically with Smad1/Smad5 to activatethe P16 and P21 promoters

p38 MAPK can regulate transcription through up-regulationor phosphorylation of several transcription factors such asAP-1 and Sp1 [25,26]. AP-1 and Sp1 have been shown toassociate with Smad3 on up-regulation of collagenase-3 geneand PAI-1 (plasminogen-activator inhibitor-1) in response toTGFβ respectively [27,28]. Thus we examined the possibilityof the co-operation of AP-1 and Sp1 with Smad1/5 on up-regulation of P16 and P21 in response to BMP4. Luciferasereporter assays showed that the promoter activity of P16 wasup-regulated by 4.2-fold and 5-fold upon the co-expression ofSp1 with Smad1 and Smad5 respectively (Figure 7A, left-handpanel). Likewise, the promoter activity of P21 was up-regulatedby 8.6-fold and 9-fold (Figure 7A, right-hand panel). However,there was little change in the promoter activities of P16 andP21 when AP-1 was co-transfected with the Smad1 or Smad5plasmid (Figure 7A), indicating that there was little co-operationof AP-1 and Smad1/Smad5 in the up-regulation of P16 and P21.Further study revealed the up-regulation of promoter activitiesof P16 and P21 by Sp1 with Smad1 and Smad5 was inhibitedby the addition of SB203580 (p38 MAPK inhibitor), suggestingthat Sp1, the downstream protein of p38 MAPK, may associatewith Smad1 or Smad5 to strengthen the Smad1/5-dependenttranscriptional activation (Figure 7B). To validate this assumption,we examined the association of Smad1/5 with Sp1 by using a CoIPassay. Transiently transfected FLAG−Smad1 or FLAG−Smad5expression plasmid was immunoprecipitated from DOX-treatedpSTAR-hBMP4 cells using the anti-FLAG antibody, and the



Figure 3 Roles of Smad and p38 MAPK pathways in BMP4-induced premature senescence

(A) Inhibition of both BR-Smad and p38 MAPK pathways by the Smad6 expression plasmid transfection and SB203580 treatment resumed the proliferation of pSTAR-hBMP4 cells inhibited by BMP4.pSTAR-hBMP4 cells transfected with the Smad6 expression plasmid were treated either with DOX alone or together with the p38 MAPK inhibitor SB203580 (20 μM) for 2 days, and cell proliferationwas assessed by the MTT assay. (B and C) Blockage of both Smad and p38 MAPK pathways completely abrogated BMP4-induced premature senescence. pSTAR-hBMP4 cells were transfectedwith Smad6, and then treated either with DOX alone or together with SB203580 (20 μM) for 6 days. The senescence was examined by SA-β-galactosidase staining and by the SA-β-galactosidaseactivity assay. ∗P < 0.05; ∗∗P < 0.01 compared with the untreated group. #P < 0.05; ##P < 0.01 compared with the DOX-treated group. Data are based on three independent experiments. (D)Blockage of both BR-Smad and p38 MAPK pathways counteracted the up-regulation of p16 and p21 induced by BMP4 as revealed by Western blotting.

precipitated complex was probed with Sp1. The associationbetween Sp1 with Smad1 or Smad5 was greatly increased com-pared with the untreated group, but decreased again when cellswere treated with SB203580 (Figure 7C). The co-Smad Smad4was also detected in the DOX-treated cells, and its presence wasnot affected by inhibition of p38 MAPK activity (Figure 7C).Taken together, these results indicated that p38 MAPK may affectthe Smad1/5-dependent transcriptional activation by mediatingthe co-operation of its downstream factors Sp1 and Smad1/5.

DISCUSSION

BMP4 is a multifunctional cytokine with a plethora of biologicaleffects, many of which cannot be attributed to Smad signallingalone [8−10]. Previous studies have shown that BMP4 activateda number of signalling pathways in addition to the Smadpathway, which may affect the biological outcome eitherpositively or negatively in a cell-context-dependent manner

[9,21,22]. However, the interplay among these pathways havenot so far been fully characterized, especially in terms oftheir contributions to the more complex end point eventssuch as BMP4-mediated senescence and tumour inhibition. Inorder to address this question, we investigated the signallingand molecular mechanisms behind BMP4-induced prematuresenescence in the lung cancer cell line NCI-H460. In thepresent study, experiments using inhibitors specific for variousnon-Smad signalling pathways showed that the p38 MAPKpathway, but not the ERK pathway, contributed to BMP4-mediated growth inhibition and senescence induction in tumourcells (Figures 1A−1E). Furthermore, the results suggested that thefunctional difference between the p38 MAPK and ERK pathwaywas due to the distinctive activation of pathways by BMP4 inNCI-H460 cells; specifically, the p38 MAPK pathway, but notthe ERK pathway, was phosphorylated and activated by BMP4(Figure 2A and 2B). In addition, since inhibitors of the p38 MAPKpathway did not completely account for the effects of BMP4 ongrowth inhibition and senescence induction (Figure 1B−1D), it



Figure 4 Smad1 and Smad5 are required in BMP4-induced premature senescence

(A) Western blots confirming the partial silencing of Smad1, Smad5 and Smad8 by their respective siRNAs; the negative control was an irrelevant siRNA. (B) Knockdown of Smad1 or Smad5,but not Smad8, resumed the proliferation of NCI-H460 cells inhibited by BMP4. pSTAR-hBMP4 cells transfected with Smad1siRNA, Smad5siRNA or Smad8siRNA were treated with DOX, andcell proliferation was determined by the MTT assay. ∗P < 0.05; ∗∗P < 0.01 compared with the untreated group. #P < 0.05; ##P < 0.01 compared with the DOX-treated group (n = 4). (C and D)Knockdown of Smad1 or Smad5 inhibited premature senescence of pSTAR-hBMP4 cells induced by BMP4. pSTAR-hBMP4 cells transfected with Smad1siRNA, Smad5siRNA or Smad8siRNA weretreated with DOX for 6 days, and the senescence was examined by SA-β-galactosidase staining and the SA-β-galactosidase activity assay. ∗P < 0.05; ∗∗P < 0.01 compared with the untreatedgroup. #P < 0.05; ## P < 0.01 compared with the DOX-treated group (n = 3). (E) Knockdown of Smad1 or Smad5 counteracted the BMP4-induced up-regulation of p16 and p21 proteins.

is possible that the p38 MAPK pathway acts co-operatively withthe Smad pathway to participate in BMP4-induced prematuresenescence.

Evidence from the present study demonstrated that both Smadand p38 MAPK signalling contributed to BMP4-induced prema-ture senescence (Figures 3A−3D). To understand the basis of theco-operative effect of Smad and p38 MAPK signalling, we firststudied whether the Smad pathway was mediated by thep38 MAPK pathway, which has been reported to modulateSmad activation in different cellular contexts [29−31]. Ourimmunofluorescence study showed that the p38 MAPK pathwaydid not affect Smad protein nuclear translocation induced byBMP4 (Figures 5A−5D), and this implied that the p38 MAPKpathway may affect the Smad pathway by other means. To test thisassumption, we used the luciferase reporter assay to demonstratethat p38 MAPK did affect Smad1/5-dependent transcriptional

activation, as well as the presence of phospho-Smad1/5 onP16 and P21 promoters (Figures 6A−6D). This conclusionwas consistent with that proposed by Kaminska et al. [31].MAPKs have been reported to affect Smad activity through theirphosphorylation on the C-terminus or other regions [32−34].The phosphorylation status of Smad protein at the C-terminalregion plays an important role in Smad protein activation andinteracted with Smad4 for nuclear translocation [35]. But theirphosphorylation on the C-terminus was not greatly affected byp38 MAPK (Figure 5E). This result may explain why SB203580did not affect the localization of Smad1 and Smad5. Then, ourluciferase reporter assays and CoIP assays demonstrated that p38MAPK played an important role in strengthening the associationof Smad protein and Sp1 (Figure 7). These results indicatedthat p38 MAPK modulated the Smad-dependent transcription byaffecting the co-operation of its downstream transcription factors



Figure 5 The p38 MAPK pathway does not mediate nuclear translocation of Smad proteins in response to BMP4

(A) Treatment of pSTAR-hBMP4 cells with the p38 MAPK pathway inhibitor SB203580 did not interfere with BMP4-dependent nuclear translocation of endogenous Smad1/5. Smad1 and Smad5 werevisualized by immunofluorescence using anti-Smad1 or anti-Smad5 antibodies after cells were treated either with DOX alone or together with SB203580 for 2 days. DAPI was used to stain the nucleus.Immunofluorescence using an anti-tubulin antibody was used to visualize the cytoplasm. (B) Quantitative representation of Smad1 or Smad5 distribution was performed by the evaluation of cellsfrom three independent experiments. ∗P < 0.05; ∗∗P <0.01 compared with the untreated group. #P < 0.05; ##P < 0.01 compared with the DOX-treated group (n = 3). (C) and (D) Western blotsdemonstrating that SB203580 did not inhibit BMP4-dependent nuclear translocation of endogenous Smad1/5. pSTAR-hBMP4 cells were treated with DOX either alone or together with SB203580(20 μM), and were lysed for Western blotting with anti-Smad1 or anti-Smad5 antibodies. Tubulin and laminB, which are the markers for cytoplasm and nucleus respectively were used to distinguishbetween cytoplasm and nucleus. (E) SB203580 did not affect the phosphorylation of Smad1/5 at its C-terminal region. pSTAR-hBMP4 cells were treated with DOX either alone or together withSB203580, and cells were lysed for Western blotting with the phospho-Smad1/5(Ser463/Ser465) antibody.

and Smad protein. Overall, there was a transcriptional synergismbetween Smad and the p38 MAPK pathway during BMP4-mediated premature senescence.

Although the classic intracellular signalling pathways utilizedby BMPs have been shown to involve a specific set ofreceptor-mediated Smad proteins, such as Smad1, Smad5and Smad8 [4,5], the functional differences among these

Smads are unclear. Presumably, the models in which differentBR-Smad proteins work in specific cell types will probably haveto be taken into account. In the present study, we found thatknockdown of Smad1 or Smad5 partly restrained the BMP4-induced premature senescence (Figures 4A−4E), implying thatSmad1 and Smad5 function similarly in mediating BMP4-induced premature senescence in lung cancer cells. However,



Figure 6 p38 MAPK affects the transcriptional activity of Smad1 and Smad5

(A) The p38 MAPK inhibitor SB203580 decreased the P16 and P21 promoter activities up-regulated by Smad1 and Smad5. NCI-H460 cells transfected with p16 or p21 reporter plasmid, togetherwith Smad1 or Smad5 expression plasmid, were treated with SB203580, and lysed for luciferase reporter assay. Luciferase activity was determined 30 h after transfection, and was normalized to theRenilla activity. The pcDNA3.1 plasmid was used as a negative control. ∗P < 0.05; ∗∗P < 0.01 compared with the untreated group (n = 3). (B) The enhanced transactivation of Smad1/5 by BMP4was efficiently blocked by SB203580 (20 μM). pSTAR-hBMP4 cells transfected with the plasmids indicated were treated with DOX either alone or together with SB203580 (20 μM), and cells werelysed for luciferase assay. ∗P < 0.05; ∗∗P < 0.01 compared with the untreated group. #P < 0.05; ##P < 0.01 compared with the DOX-treated group (n = 3). (C) Schematic representation of the5′-flanking regions of the P16 and P21 genes. Lines below (P1−P4 and Q1−Q3) indicate the important regions of the P16 and P21 promoters respectively and were amplified in ChIP assays. (D)Enhanced binding of phospho-Smad1/5/8 at the two promoter regions in response to BMP4 were restrained by the addition of SB203580. pSTAR-hBMP4 cells were treated either with DOX alone ortogether with SB203580 for 2 days, and harvested for the ChIP assay. Samples were immunoprecipitated with anti-phospho-Smad1/5 antibodies. Precipitated DNAs were amplified using PCR.



Figure 7 Sp1 functions co-operatively with Smad1/Smad5 for transcriptional activation

(A) Sp1 synergistically activated the P16 and P21 promoters with Smad1/5. NCI-H460 cells transfected with the P16 or P21 reporter plasmid, together with expression vector for Smad1, Smad5,Sp1, AP-1 or vector alone. Luciferase activity was determined 30 h after transfection, and was normalized to the Renilla activity. ∗P < 0.05; ∗∗P < 0.01 compared with the untreated group (n = 3).(B) The enhanced co-operation between Sp1 and Smad1/5 on transactivation of the P16 or P21 gene by BMP4 was efficiently blocked by SB203580 (20 μM). pSTAR-hBMP4 cells transfected withthe plasmids indicated were treated with DOX either alone or together with SB203580 (20 μM), and lysed for luciferase assay. ∗P < 0.05; ∗∗P < 0.01 compared with the untreated group. #P < 0.05;##P < 0.01 compared with the DOX-treated group (n = 3). (C) The association of Sp1 and Smad proteins upon BMP4 overexpression was mediated by the p38 MAPK pathway. pSTAR-hBMP4 cellstransfected with the FLAG−Smad1 or FLAG−Smad5 expression plasmid were treated either with DOX alone or together with the p38 MAPK inhibitor SB203580 (20 μM) for 2 days. Whole-cellextracts were prepared and immunoprecipitated with the anti-FLAG antibody, and were incubated with Protein A−agarose beads. Immunoprecipitates were subjected to immunoblotting analysis withanti-Sp1, anti-Smad4 and anti-FLAG antibodies. Western blots of the whole-cell extracts were detected with anti-Sp1 and anti-Smad4 antibodies.

knockdown of Smad8 did not restrain the BMP4-inducedpremature senescence (Figures 4B−4E). Furthermore, cellularimmunofluorescence revealed that Smad8 was still retained inthe cytoplasm in BMP4-overexpression cells, indicating thatSmad8 was not able to translocate into the nucleus to mediateBMP4-induced premature senescence (Supplementary Figure S2at http://www.BiochemJ.org/bj/433/bj4330333add.htm). Pangaset al. [36] also found that Smad1 worked in a much more similarway to Smad5 than to Smad8 in tumour inhibition. Presumably,

the functional divergence of Smad1/5 and Smad8 suggests theimportance of uncovering the specific function of the BR-Smadpathway in specific cellular contexts.

Collectively, the results of the present study demonstrate thatboth the Smad and p38 MAPK pathways are activated by BMP4,and this activation is required for induction and persistence ofpremature senescence in lung cancer cells. Furthermore, we alsofound that the p38 MAPK pathway positively modulated the Smadfunction by affecting their transcriptional activation potential.



These results support a novel model in which the co-operationbetween the Smad and p38 MAPK pathways is essential inmediating BMP4-induced premature senescence.

AUTHOR CONTRIBUTION

Dongmei Su designed and performed all of the experimental work. Xiue Peng, Shan Zhu,Ying Huang, Zhixiong Dong, Yu Zhang, Jianchao Zhang and Qian Liang participated inthe experiments and contributed to work. Jun Lu contributed to the design of the study.Baiqu Huang obtained grant support, and designed and supervised the study, and wrotethe manuscript. All authors contributed to the editing of the manuscript.

FUNDING

This work was supported by the National Basic Research Program of China [grant numbers2005CB522404, 2006CB910506]; the Program for Changjiang Scholars and InnovativeResearch Team (PCSIRT) in Universities [grant number IRT0519]; and the National NaturalScience Foundation of China [grant numbers 30771232, 30671184].

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Received 23 April 2010/3 November 2010; accepted 4 November 2010Published as BJ Immediate Publication 4 November 2010, doi:10.1042/BJ20100404


Biochem. J. (2011) 433, 333–343 (Printed in Great Britain) doi:10.1042/BJ20100404

SUPPLEMENTARY ONLINE DATARole of p38 MAPK pathway in BMP4-mediated Smad-dependent prematuresenescence in lung cancer cellsDongmei SU*, Xiue PENG†, Shan ZHU*, Ying HUANG†, Zhixiong DONG*, Yu ZHANG*, Jianchao ZHANG*, Qian LIANG†,Jun LU†1 and Baiqu HUANG*1

*The Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024, People’s Republic of China, and †The Key Laboratory of Molecular Epigenetics of Ministry ofEducation (MOE), Northeast Normal University, Changchun 130024, People’s Republic of China

Figure S1 The p38 MAPK inhibitor does not affect DOX-induced BMP4production in pSTAR-hBMP4 stably transfected cells

Lysates were prepared 2 days following DOX treatment either alone or together with the inhibitor(SB203580, 20 μM), and probed with the anti-BMP4 antibody. β-Actin was used as an internalreference control.

Figure S2 BMP4 does not mediate nuclear translocation of Smad8 proteinsin pSTAR-hBMP4 stably transfected cells

Smad8 were visualized by immunofluorescence using an anti-Smad8 antibody after cells weretreated with DOX for 2 days. DAPI was used to stain the nucleus. Immunofluorescence usinganti-tubulin was used to stain the cytoplasm.

Received 23 April 2010/3 November 2010; accepted 4 November 2010Published as BJ Immediate Publication 4 November 2010, doi:10.1042/BJ20100404

1 Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).


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