Biochemical Journal

Research article

Role of p38 MAPK pathway in BMP4-mediated Smad-dependent premature senescence in lung cancer cells

Dongmei Su, Xiue Peng, Shan Zhu, Ying Huang, Zhixiong Dong, Yu Zhang, Jianchao Zhang, Qian Liang, Jun Lu, Baiqu Huang

Abstract

BMP4 (bone morphogenetic protein 4) is a multifunctional cytokine known to exert its biological effects through a variety of signalling pathways. The diverse function of BMP4 appears to be due to multiple pathways activated by BMP4 itself. Our previous studies have demonstrated that BMP4 is able to drive lung cancer cells into a process of premature senescence; however, the signalling pathways, as well their interplays and roles associated with this process, are not well understood. To address these questions, in the present study we investigated the signalling and molecular mechanisms underlying the BMP4-induced senescence, and our data demonstrated that p38 MAPK (mitogen-activated protein kinase) and Smad pathways were necessary 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 cancer cell line NCI-H460. We also showed that the BMP4-responsive R-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 BMP4 was essential for the full activation of transcription potential of Smad1/5. Overall, the results of the present study implicate a complex co-operation between p38 MAPK and Smad pathways in BMP4-mediated premature senescence.

  • bone morphogenetic protein 4 (BMP4)
  • p38 mitogen-activated protein kinase (p38 MAPK)
  • premature senescence
  • Smad1
  • Smad5

INTRODUCTION

BMPs (bone morphogenetic proteins) are secreted signalling molecules 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 have pleiotrophic roles in cell growth, differentiation, migration and apoptosis, and are critical in embryogenesis and organogenesis [2,3]. BMPs elicit their effects through activation of type-1 and type-2 serine/threonine kinase receptors. BMPs and TGFβ/activin receptor-phosphorylated Smads (R-Smads) oligomerize with the common mediator Smad4 (Co-Smad) to regulate gene expression by binding to DNA upon their nuclear import [4]. BMP receptors activate Smad1, Smad5 and Smad8, whereas Smad2 and Smad3 are phosphorylated by activin or TGFβ receptors [4,5]. Smad6 and Smad7 have been identified as the inhibitory Smads (I-Smads). I-Smads stably interact with activated type-1 receptors and compete with R-Smads for activation by the receptors. Smad7 inhibits both TGFβ/activin and BMP signalling, whereas Smad6 efficiently inhibits BMP signalling, but only weakly inhibits TGFβ/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 be activated following treatment with BMPs in specific cell contexts [810]. The cross-talk between the Smad pathway and other pathways may lead to the diverse function of BMP4. Our previous studies and other studies have shown that BMP4 was able to drive lung cancer cells into premature senescence or replicative senescence in vitro [11,12]. However, the molecular events and the signalling pathways underlying this process are still unclear. p38 MAPK and ERK/MAPK pathways have been highlighted to be linked to premature senescence [1315], implying that they may participate in mediating BMP4-induced premature senescence.

The relative contribution of these different pathways in BMP4-induced premature senescence is poorly understood. The aim of the present study was to identify the roles of diverse pathways that are activated by BMP4, as well as their co-operation in BMP4-induced premature senescence in lung cancer cells. Our results showed that BMP4 was able to activate the p38 MAPK pathway to mediate senescence in NCI-H460 cells in addition to Smad signalling. However, the ERK signalling pathway, another senescence-associated signalling pathway, was not activated by BMP4 in the process of BMP4-mediated senescence. Both Smad-dependent and -independent p38 MAPK pathways synergistically contributed in BMP4-triggered premature senescence. Furthermore, the present study revealed that the p38 MAPK pathway positively modulated Smad function by affecting their transcriptional activation potential. Overall, the results of the present study demonstrate a complex interplay between Smad and p38 MAPK pathways in BMP4-mediated premature senescence.

EXPERIMENTAL

Materials

The p16INK4a (hereafter termed p16) promoter reporter (−869 to +1 bp from the cap site) ligated to the luciferase reporter gene (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 by Dr S. Itoh (Department of Experimental Pathology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Japan). The plasmid of p21–luc (2400/+11) was a gift from Dr Bert Vogelstein (The Johns Hopkins University School of Medicine at Maryland, Baltimore, MD, U.S.A.). pA3-Flag-Smad1 was a gift from Dr C. Laurie (The Oncology and Molecular Endocrinology Research Center, CHUL Research Center and Laval University, Québec, Canada). pcDNA3.1-Flag-Smad5 was provided by Dr T. Aigner (University of Leipzig, Leipzig, Germany). Specific inhibitors for ERK (U0126) and p38 MAPK (SB203580) were obtained from Sigma.

Cell culture, transfection and luciferase reporter assay

NCI-H460 lung cancer cells and the pSTAR-hBMP4 stably transfected cell line were maintained in IMDM (Iscove's modified Dulbecco's medium) supplemented with 10% FBS (fetal bovine serum), 100 mg/ml penicillin and 100 mg/ml streptomycin in a humidified atmosphere containing 5% CO2 at 37 °C. The pSTAR/hBMP4 stably transfected cell line was constructed in our previous study [12]. The cell line that inducibly expresses BMP4 protein by DOX (doxycycline) (Clontech) in both a dose- and time-dependent manner was maintained in IMDM supplemented with 10% FBS in the presence of G418 (1000 μg/ml).

For the luciferase reporter assay, 5×104 cells were seeded in 24-well tissue culture plates for 24 h before they were transiently transfected with 1 μg of p16 or p21 reporter plasmid and 0.5 μg of indicated constructs or vector alone using the FuGENE™ HD transfection reagent (Roche). The Renilla luciferase control plasmid pREP7-RLuc was co-transfected at 50 ng/well as an internal control reporter. At 30 h post-transfection, cells were washed and lysed in passive lysis buffer (Promega) and the transfection efficiency was normalized to the paired Renilla luciferase activity using the Dual Luciferase Reporter Assay System (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) for 30 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 Cruz Biotechnology, 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 Signaling Technology, #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) (Cell Signaling Technology, #9511), anti-phospho-Smad1/Smad5(Ser463/Ser465) (Cell Signaling Technology, #9516) or anti-β-actin (Sigma, A1978) antibodies. The signals were visualized by using the chemiluminescent substrate method with the SuperSignal West Pico kit provided by Pierce. β-Actin was used as an internal control for normalizing the loading materials.

Cell proliferation and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] 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 incubated with 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 nm with a microplate reader.

Cellular immunofluorescence

Cells were fixed in 1% formaldehyde in culture medium for 10 min at 37 °C and permeabilized with 0.2% Triton X-100 in PBS for 10 min at 4 °C. Endogenous Smad1, Smad5 and Smad8 were detected with antibodies against Smad1, Smad5 and Smad8 respectively and visualized with an FITC-conjugated anti-rabbit IgG 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 taken under a fluorescence microscope.

Senescence-associated β-galactosidase activity assay and cytochemical 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). Cell lysates containing equal amounts of total protein were diluted in equal volumes of 2× assay buffer comprising 1.33 mg/ml ONPG (o-nitrophenyl-β-D-galactopyranoside), 2 mM MgCl2 and 100 μl of 2-mercaptoethanol in 200 mM phosphate buffer (pH 6.0), and were incubated at 37 °C for 4 h. The absorbance at 420 nm was measured after the addition of an equal volume of 1 M Na2CO3.

Cytochemical staining for SA-β-galactosidase was performed using a senescence-β-galactosidase staining kit (Cell Signaling Technology) at pH 6.0. All of the experiments were repeated three times, and one of the representative results is shown.

RNAi (RNA interference)

The siRNA (small interfering RNA) targeting sequences of Smad1, Smad5 and Smad8 were 5′-AACCTGTCATTATTGCTTACT-3′ [16], 5′-AATTACATCCTGCCGGTGATA-3′ [17] and 5′-AAGTTAAAGAAGAAGAAGGGA-3′ respectively. The control siRNA sequence was 5′-CGTCAACATGGCTTTCACC-3′. Oligonucleotides that represent small hairpin RNAs targeting these sequences were designed, synthesized and cloned into the pSliencer4.1-CMV neo vector (Ambion) between BamHI and HindIII 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 primers specific to sequences at the P16 promoter were: P1 sense, 5′-CATTCGCTAAGTGCTCGGAGT-3′ and antisense, 5′-CTGCTCCCCGCCGCCCGCTGCCTG-3′; P2 sense, 5′-TAGGAAGGTTGTATCGCGGAGG-3′ and antisense, 5′-CAAGGAAGGAGGACTGGGCTC-3′; P3 sense, 5′-AGACAGCCGTTTTACACGCAG-3′ and antisense, 5′-CACCGAGAAATCGAAATCACC-3′; and P4 sense, 5′-GCTGAGGCAGGAGAATT-3′ and antisense, 5′-TTTGGGATGTCAAGTATG-3′ The primers specific to sequences at the P21 promoter were: Q1 sense, 5′-GGTGTCTAGGTGCTCCAGGT-3′ and antisense, 5′-GCACTCTCCAGGAGGACACA-3′; Q2 sense, 5′-CGTGGTGGTGGTGAGCTA-3′ and antisense, 5′-CTGTCTGCACCTTCGCTCCT-3′; and Q3 sense, 5′-AATTCCTCTGAAAGCTGACTGCC-3′ and antisense, 5′-AGGTTTACCTGGGGTCTTTAGA-3′.

Fractionation procedure

NEs (nuclear extracts) and CEs (cytosolic extracts) were prepared essentially as follows. Cells were washed and lysed in hypotonic buffer comprising 1% (w/v) Nonidet P40, 10 mM Hepes/KOH (pH 7.4), 120 mM NaCl, 1 mM DTT (dithiothreitol), 1 mM EDTA, 1 mM sodium orthovanadate, 1 mM PMSF, and 5 μg/ml each of leupeptin and aprotinin at 4 °C for 30 min with gentle shaking. Nuclei were collected by centrifugation at 10000 g for 8 min at 4 °C, and the supernatant was saved for CEs. The pellet 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 mM PMSF and 5 μg/ml each of leupeptin and aprotinin], and then incubated at 4 °C for 30 min. The lysates were clarified by centrifugation at 15000 g at 4 °C for 10 min. The supernatant was used as the NE. Equal amounts of protein in CEs and NEs were loaded on to SDS gels for Western detection.

CoIP (co-immunoprecipitation) assay

Total cell extracts from DOX-treated cells were pre-cleared with salmon sperm DNA/Protein A–agarose beads (Upstate). Rabbit anti-FLAG (Sigma, F7425) antibody was added for immunoprecipitation. The precipitates were then subjected to SDS/PAGE followed by transfer on to a PVDF membrane and 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 Pico Chemiluminescent Substrate (Pierce) detection method following the manufacturer's instructions.

Statistical analysis

A Student's t test was used to calculate the statistical significance of 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 an important role in mediating BMP4-induced premature senescence

Our previous data showed that BMP4 was able to induce premature senescence in lung cancer cells [12], but the signalling pathways in BMP4-induced premature senescence remain largely unknown. p38 MAPK and ERK signalling pathways have been suggested to be involved in cellular senescence by various stimulations [14,18]. We first examined whether the BMP4-induced premature senescence is mediated by these pathways using specific kinase inhibitors. As shown in Figure 1(A), treatment with SB203580, an effective inhibitor of the p38 MAPK signalling pathway, partly restored the capacity of BMP4 in reduction of proliferation of pSTAR-hBMP4 cells, which express BMP4 protein upon DOX induction in both a dose- and time-dependent manner [12]. Meanwhile, our results also demonstrated that the inhibitor specific for p38 MAPK did not affect BMP4 production in pSTAR-hBMP4 cells, indicating that p38 MAPK may be the downstream signalling molecule of BMP4 (Supplementary Figure S1 at http://www.BiochemJ.org/bj/433/bj4330333add.htm). Treatment with the ERK1/2 inhibitor U0126 did not block the reduction of proliferation by BMP4 treatment. As a control, DMSO did not alter this reduction effect of BMP4 (Figure 1A). We further showed that SB203580, but not U0126, restrained premature senescence induced by BMP4 overexpression in pSTAR-hBMP4 cells (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 expression were observed upon U0126 (20 μM) treatment (Figure 1E). These data suggest that p38 MAPK, but not the ERK pathway, is involved in BMP4-induced premature senescence in NCI-H460 lung cancer cells.

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 activity assay 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 positively stained 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 DOX treatment 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.

p38 MAPK and Smad signalling pathways are activated by BMP4

Besides the classical Smad pathway, evidence has implied that Smad-independent signalling pathways were also activated in BMP signalling in different cellular contexts [810]. We wanted to know whether p38 MAPK, Smad and ERK pathways are activated 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 Smad and p38 MAPK pathways (Figures 2A and 2C), whereas the ERK pathway was not activated by BMP4 (Figure 2B). These results may give a reason why the p38 MAPK pathway, but not the ERK pathway, was involved in mediating BMP4-induced senescence.

Figure 2 BMP4 elevates the phosphorylation levels of Smad1/5/8 and p38 MAPK, but not that of ERK1/2

Estimation of the phosphorylation levels of Smad1/5/8, p38 MAPK and ERK1/2 in pSTAR-hBMP4 cells treated with DOX for 2 days. Western blotting assays were performed with the antibodies indicated.

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

To investigate whether the p38 MAPK and Smad pathway are required for BMP4-induced premature senescence, we employed two inhibitors in the experiments, i.e. the Smad6 expression plasmid for inhibiting the Smad pathway and SB203580 for inhibiting the p38 MAPK pathway. As shown in Figures 3(A)–3(C), blocking the Smad pathway partly interfered with the BMP4-induced premature senescence by Smad6 overexpression, which is known to inhibit BR-Smad (Smad1, Smad5 and Smad8) phosphorylation and nuclear translocation [7,19]; meanwhile, inhibition of the p38 MAPK pathway by SB203580 only partly restrained BMP4-induced premature senescence in pSTAR-hBMP4 cells. However, when both Smad and p38 MAPK were blocked, the senescence triggered by BMP4 was almost completely abrogated. Further investigation using Western blot analysis showed that the BMP4-induced up-regulation of p16 and p21 was decreased more significantly when both Smad and p38 MAPK were inhibited than that when only either one of the pathways was inhibited (Figure 3D). These results indicated that both the p38 MAPK and Smad pathways were essential for BMP4-induced premature senescence and they appeared to co-operate with each other to mediate BMP4-induced premature senescence.

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 proliferation was 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 transfected with 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-galactosidase activity 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.

Smad1 and Smad5 are required in BMP4-induced premature senescence

Our previous results have shown that the Smad pathway played a critical role in mediating BMP4-induced premature senescence [12]. Smad1, Smad5 and Smad8 (termed BR-Smad), are the main 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 been established so far. In the next experiments, we intended to define which BR-Smad participates in mediating BMP4-induced premature senescence. We used an RNAi approach to knockdown Smad1, Smad5 and Smad8 expression individually to assess their effects. The expression of endogenous Smad1, Smad5 and Smad8 proteins was specifically reduced by transfection of specific siRNAs without affecting the expression of the other Smads as determined by Western blotting (Figure 4A). We then showed that suppression of endogenous Smad1 and Smad5 expression restrained the BMP4-induced senescence in pSTAR-hBMP4 cells (Figures 4B–4E), eliciting the critical role of Smad1/5 in mediating BMP4-induced premature senescence, whereas Smad8 did not affect this process.

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, and cell 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 were treated 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 untreated group. #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.

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

MAPK pathways have been reported to have either positive or negative roles in mediating the Smad pathway [9,21,22]. Activation of MAPK pathways has been shown to induce nuclear translocation of Smad2 [23,24], or, in other cases, to inhibit TGFβ-dependent nuclear translocation [16]. We therefore tested whether the p38 MAPK pathway has an effect on BMP4-dependent nuclear translocation of the Smad proteins in pSTAR-hBMP4 cells by using cellular immunofluorescence. As can be seen in Figures 5(A) and 5(B), Smad1 and Smad5 proteins were mainly located in the nucleus in the BMP4-overexpression group (middle column), whereas they were located both in the nucleus and cytoplasm in the control group (left-hand column). In addition, the p38 MAPK signalling inhibitor SB203580 did not inhibit BMP4-dependent nuclear translocation of Smad1 and Smad5 (right-hand column). The effect of p38 MAPK on BMP-4-dependent nuclear translocation of Smad proteins was also analysed by Western blotting, which confirmed that BMP4 overexpression prominently promoted Smad protein nuclear translocation; however, this process was not inhibited by SB203580 (Figure 5C and 5D). Our further studies showed that SB203580 did not affect the phosphorylation of Smad1/5 at its C-terminal region, which was required for Smad protein activation and interacted with Smad4 for nuclear translocation (Figure 5E). These experiments indicate that the p38 MAPK pathway is not involved in mediating nuclear translocation of Smad proteins.

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 were visualized 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 cells from 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 blots demonstrating 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 distinguish between 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 with SB203580, and cells were lysed for Western blotting with the phospho-Smad1/5(Ser463/Ser465) antibody.

p38 MAPK affects Smad1/5-dependent transcriptional activation

Next, we were interested in determining whether the p38 MAPK pathway affects the Smad1/5 transcriptional activity in response to BMP4. We showed previously that P16 and P21 were BMP4-responsive target genes and were required for BMP4-induced premature senescence [12]. In the present study we show that BMP4-responsive R-Smads, i.e. Smad1 and Smad5, were able to partly increase the activity of P16 and P21 promoters, whereas the transcriptional activation by Smad protein was partly restrained by addition of SB203580, as determined by luciferase reporter assays (Figure 6A) in NCI-H460 cells. Furthermore, we found that Smad1/5 exhibited potent transcriptional activation in the presence of BMP4, which is able to induce phosphorylation and activation of Smad proteins, but such activation was efficiently blocked by SB203580 in pSTAR-hBMP4 cells (Figure 6B). These results indicated that the p38 MAPK pathway played an important role in mediating the transactivation of Smad1 and Smad5. Since we previously discovered that BMP4 was able to up-regulate P16 and P21 expression through promoting the enrichment of the downstream transcription factors Smad1/5 at the defined promoter regions [12], we wanted to know whether the p38 MAPK pathway also mediates the occupation of Smad1/5 on P16 and P21 promoters. ChIP assays were performed to detect the presence of phospho-Smad1/5 at P16 and P21 promoters in the presence of SB203580. We designed a series of ChIP primers 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 the P16 promoter [12]. P4 locates at the far upstream of the p16 promoter (−2600 bp) as a negative control. Likewise, primers for the P21 gene were also designed (Q1, Q2 and Q3 at −600, −1800 and −2600 bp) (Figure 6C, right-hand panel). The ChIP results revealed that the enhanced enrichment of phospho-Smad1/5 at all of these promoter regions of P16 and P21 genes in response to BMP4 was restrained by the addition of SB203580 (Figure 6D). Taken together, these results indicate that the p38 MAPK pathway is essential for Smad-dependent transcriptional activation in response to BMP4 in pSTAR-hBMP4 cells.

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, together with 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 the Renilla 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 BMP4 was 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 were 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) Schematic representation of the 5′-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 or together 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.

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

p38 MAPK can regulate transcription through up-regulation or phosphorylation of several transcription factors such as AP-1 and Sp1 [25,26]. AP-1 and Sp1 have been shown to associate with Smad3 on up-regulation of collagenase-3 gene and PAI-1 (plasminogen-activator inhibitor-1) in response to TGFβ respectively [27,28]. Thus we examined the possibility of the co-operation of AP-1 and Sp1 with Smad1/5 on up-regulation of P16 and P21 in response to BMP4. Luciferase reporter assays showed that the promoter activity of P16 was up-regulated by 4.2-fold and 5-fold upon the co-expression of Sp1 with Smad1 and Smad5 respectively (Figure 7A, left-hand panel). Likewise, the promoter activity of P21 was up-regulated by 8.6-fold and 9-fold (Figure 7A, right-hand panel). However, there was little change in the promoter activities of P16 and P21 when AP-1 was co-transfected with the Smad1 or Smad5 plasmid (Figure 7A), indicating that there was little co-operation of AP-1 and Smad1/Smad5 in the up-regulation of P16 and P21. Further study revealed the up-regulation of promoter activities of P16 and P21 by Sp1 with Smad1 and Smad5 was inhibited by the addition of SB203580 (p38 MAPK inhibitor), suggesting that Sp1, the downstream protein of p38 MAPK, may associate with Smad1 or Smad5 to strengthen the Smad1/5-dependent transcriptional activation (Figure 7B). To validate this assumption, we examined the association of Smad1/5 with Sp1 by using a CoIP assay. Transiently transfected FLAG–Smad1 or FLAG–Smad5 expression plasmid was immunoprecipitated from DOX-treated pSTAR-hBMP4 cells using the anti-FLAG antibody, and the precipitated complex was probed with Sp1. The association between Sp1 with Smad1 or Smad5 was greatly increased compared with the untreated group, but decreased again when cells were treated with SB203580 (Figure 7C). The co-Smad Smad4 was also detected in the DOX-treated cells, and its presence was not affected by inhibition of p38 MAPK activity (Figure 7C). Taken together, these results indicated that p38 MAPK may affect the Smad1/5-dependent transcriptional activation by mediating the co-operation of its downstream factors Sp1 and Smad1/5.

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 with the 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 cells transfected 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-cell extracts were prepared and immunoprecipitated with the anti-FLAG antibody, and were incubated with Protein A–agarose beads. Immunoprecipitates were subjected to immunoblotting analysis with anti-Sp1, anti-Smad4 and anti-FLAG antibodies. Western blots of the whole-cell extracts were detected with anti-Sp1 and anti-Smad4 antibodies.

DISCUSSION

BMP4 is a multifunctional cytokine with a plethora of biological effects, many of which cannot be attributed to Smad signalling alone [810]. Previous studies have shown that BMP4 activated a number of signalling pathways in addition to the Smad pathway, which may affect the biological outcome either positively or negatively in a cell-context-dependent manner [9,21,22]. However, the interplay among these pathways have not so far been fully characterized, especially in terms of their contributions to the more complex end point events such as BMP4-mediated senescence and tumour inhibition. In order to address this question, we investigated the signalling and molecular mechanisms behind BMP4-induced premature senescence in the lung cancer cell line NCI-H460. In the present study, experiments using inhibitors specific for various non-Smad signalling pathways showed that the p38 MAPK pathway, but not the ERK pathway, contributed to BMP4-mediated growth inhibition and senescence induction in tumour cells (Figures 1A–1E). Furthermore, the results suggested that the functional difference between the p38 MAPK and ERK pathway was due to the distinctive activation of pathways by BMP4 in NCI-H460 cells; specifically, the p38 MAPK pathway, but not the ERK pathway, was phosphorylated and activated by BMP4 (Figure 2A and 2B). In addition, since inhibitors of the p38 MAPK pathway did not completely account for the effects of BMP4 on growth inhibition and senescence induction (Figure 1B–1D), it is possible that the p38 MAPK pathway acts co-operatively with the Smad pathway to participate in BMP4-induced premature senescence.

Evidence from the present study demonstrated that both Smad and p38 MAPK signalling contributed to BMP4-induced premature senescence (Figures 3A–3D). To understand the basis of the co-operative effect of Smad and p38 MAPK signalling, we first studied whether the Smad pathway was mediated by the p38 MAPK pathway, which has been reported to modulate Smad activation in different cellular contexts [2931]. Our immunofluorescence study showed that the p38 MAPK pathway did not affect Smad protein nuclear translocation induced by BMP4 (Figures 5A–5D), and this implied that the p38 MAPK pathway may affect the Smad pathway by other means. To test this assumption, we used the luciferase reporter assay to demonstrate that p38 MAPK did affect Smad1/5-dependent transcriptional activation, as well as the presence of phospho-Smad1/5 on P16 and P21 promoters (Figures 6A–6D). This conclusion was consistent with that proposed by Kaminska et al. [31]. MAPKs have been reported to affect Smad activity through their phosphorylation on the C-terminus or other regions [3234]. The phosphorylation status of Smad protein at the C-terminal region plays an important role in Smad protein activation and interacted with Smad4 for nuclear translocation [35]. But their phosphorylation on the C-terminus was not greatly affected by p38 MAPK (Figure 5E). This result may explain why SB203580 did not affect the localization of Smad1 and Smad5. Then, our luciferase reporter assays and CoIP assays demonstrated that p38 MAPK played an important role in strengthening the association of Smad protein and Sp1 (Figure 7). These results indicated that p38 MAPK modulated the Smad-dependent transcription by affecting the co-operation of its downstream transcription factors and Smad protein. Overall, there was a transcriptional synergism between Smad and the p38 MAPK pathway during BMP4-mediated premature senescence.

Although the classic intracellular signalling pathways utilized by BMPs have been shown to involve a specific set of receptor-mediated Smad proteins, such as Smad1, Smad5 and Smad8 [4,5], the functional differences among these Smads are unclear. Presumably, the models in which different BR-Smad proteins work in specific cell types will probably have to be taken into account. In the present study, we found that knockdown of Smad1 or Smad5 partly restrained the BMP4-induced premature senescence (Figures 4A–4E), implying that Smad1 and Smad5 function similarly in mediating BMP4-induced premature senescence in lung cancer cells. However, knockdown of Smad8 did not restrain the BMP4-induced premature senescence (Figures 4B–4E). Furthermore, cellular immunofluorescence revealed that Smad8 was still retained in the cytoplasm in BMP4-overexpression cells, indicating that Smad8 was not able to translocate into the nucleus to mediate BMP4-induced premature senescence (Supplementary Figure S2 at http://www.BiochemJ.org/bj/433/bj4330333add.htm). Pangas et al. [36] also found that Smad1 worked in a much more similar way to Smad5 than to Smad8 in tumour inhibition. Presumably, the functional divergence of Smad1/5 and Smad8 suggests the importance of uncovering the specific function of the BR-Smad pathway in specific cellular contexts.

Collectively, the results of the present study demonstrate that both the Smad and p38 MAPK pathways are activated by BMP4, and this activation is required for induction and persistence of premature senescence in lung cancer cells. Furthermore, we also found that the p38 MAPK pathway positively modulated the Smad function by affecting their transcriptional activation potential. These results support a novel model in which the co-operation between the Smad and p38 MAPK pathways is essential in mediating 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 in the 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 wrote the manuscript. All authors contributed to the editing of the manuscript.

FUNDING

This work was supported by the National Basic Research Program of China [grant numbers 2005CB522404, 2006CB910506]; the Program for Changjiang Scholars and Innovative Research Team (PCSIRT) in Universities [grant number IRT0519]; and the National Natural Science Foundation of China [grant numbers 30771232, 30671184].

Abbreviations: 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 growth factor β

References

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