HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 24, 21325-21331, June 14, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/24/21325    most recent M201608200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ghosh, S. K. Articles by Rao, G. N. Search for Related Content PubMed PubMed Citation Articles by Ghosh, S. K. Articles by Rao, G. N. Social Bookmarking                      What's this?

ATF-1 Mediates Protease-activated Receptor-1 but Not Receptor Tyrosine Kinase-induced DNA Synthesis in Vascular Smooth Muscle Cells^*

Salil K. Ghosh^§, Laxmisilpa Gadiparthi^§, Zhao-Zhu Zeng, Manjula Bhanoori, Carmen Tellez^¶, Menashe Bar-Eli^¶, and Gadiparthi N. Rao

From the  Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee 38163 and the ^¶ Department of Cancer Biology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

Received for publication, February 16, 2002, and in revised form, March 22, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Previously we have demonstrated that activation of p38 mitogen-activated protein kinase (MAPK) and induction of DNA synthesis in response to receptor tyrosine kinase (RTK) and G protein-coupled receptor (GPCR) agonists require NADH/NADPH-like oxidase activity in vascular smooth muscle cells (VSMC). Here we tested the role of p38 MAPK in RTK and GPCR agonist-induced DNA synthesis in VSMC. Platelet-derived growth factor (PDGF)-BB and thrombin (RTK and GPCR agonists, respectively) activated p38 MAPK in a time-dependent manner in VSMC. Inhibition of p38 MAPK led to a 50% decrease in the DNA synthesis induced by thrombin but not PDGF-BB. ATF-1 was found to be the predominant member of the cyclic AMP response element (CRE)-DNA complex formed in VSMC in response to PDGF-BB and thrombin, and both agonists induced its phosphorylation. Regardless of this, inhibition of p38 MAPK reduced only thrombin- but not PDGF-BB-induced ATF-1 phosphorylation. Similarly, inhibition of p38 MAPK caused a 50% decrease in thrombin- but not PDGF-BB-induced CRE promoter-dependent transcription. Ectopic expression of an inhibitory anti-ATF-1 single-chain antibody fragment, ScFv, significantly interfered with DNA synthesis induced by thrombin but not PDGF-BB. Together, these results suggest the following conclusions. 1) Both RTK and GPCR agonists activate p38 MAPK and induce CRE promoter-dependent transcription; 2) both RTK and GPCR agonists induce ATF-1 phosphorylation, and ATF-1 is a predominant member in the CRE-DNA complexes formed in response to these agents; and 3) p38 MAPK-dependent ATF-1 phosphorylation and CRE promoter-mediated transcription are associated with GPCR agonist-induced VSMC growth.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Increased vascular smooth muscle cell growth is a contributing factor in the pathogenesis of atherosclerosis and restenosis (1). Increased levels of mitogens such as platelet-derived growth factor (PDGF)¹ and fibroblast growth factor (FGF) were reported in atherosclerotic arteries compared with normal (1-4). These mitogens modulate VSMC growth both in an autocrine and paracrine manner in VSMC (1). In addition, in vitro studies have shown that a variety of receptor tyrosine kinase (RTK) and G protein-coupled receptor (GPCR) agonists and of oxidants are potent mitogens to VSMC (5-10), suggesting that many of the molecules that are produced at the site of arterial injury by various cell types can account for the increased VSMC growth during the formation of these lesions. Towards understanding the role of specific mitogens in the induction of VSMC growth during the formation of these lesions, several investigators have used neutralizing antibodies and antisense oligonucleotides (4, 7, 10, 11). Use of neutralizing antibodies against PDGF or FGF resulted only in partial inhibition of VSMC growth and lesion progression (4, 10). Similarly, the use of antisense oligonucleotides against a molecule that appears to be critical in the mitogenic signaling events, such as c-Myc, resulted only in partial inhibition of VSMC growth and lesion progression (11). Thus, inhibition of individual mitogens or proto-oncogenes has so far resulted in minimal reduction in VSMC growth and lesion progression in animal models of atherosclerosis and restenosis.

Identification of molecules that are critical in the signaling pathways of several mitogens may provide more successful targets. Mitogen-activated protein kinases (MAPK) are a group of serine/threonine kinases that are ubiquitously expressed (12-14). These are grouped primarily into three major categories, namely 1) extracellular signal-regulated kinases, 2) Jun N-terminal kinases, and 3) p38 MAPKs (13, 14). Furthermore, differences were observed in the responsiveness of different groups of MAPKs to various external stimuli. Specifically, extracellular signal-regulated kinases have been reported to respond preferentially to agents that induce cell growth and differentiation (15-20), whereas Jun N-terminal kinases and p38 MAPK have been reported to be potently activated by cellular stressors and cytokines (21-24). Despite these differences in responsiveness, cross-talk between different groups of MAPK has been observed in mediating cellular responses to certain agonists (25). In addition, activation of one or all three groups of MAPKs are involved in the regulation of activities of various transcriptional factors, including activator protein 1 (AP-1) and nuclear factor B (NFB) (12). Previously we have demonstrated that activation of p38 MAPK and induction of DNA synthesis in response to RTK and GPCR agonists require NADH/NADPH-like oxidase activity in VSMC (26). The purpose of the present investigation was to study the role of p38 MAPK in the induction of growth by both RTK and GPCR agonists in VSMC. We show that 1) both RTK and GPCR agonists activate p38 MAPK in VSMC; 2) although activation of p38 MAPK is not required for PDGF-BB-induced DNA synthesis, it is involved in thrombin-induced DNA synthesis; 3) thrombin- but not PDGF-BB-induced p38 MAPK activation is required for ATF-1 phosphorylation and CRE promoter-dependent transcription; 4) thrombin-stimulated p38 MAPK-dependent ATF-1 phosphorylation and CRE promoter-mediated transcription are associated with growth in VSMC; and 5) disruption of ATF-1 activity by intracellular expression of an inhibitory anti-ATF-1 single-chain antibody fragment, ScFv, interfered with only thrombin but not PDGF-BB-induced DNA synthesis in VSMC. Together, these findings reveal that ATF-1 plays a role in thrombin- but not PDGF-BB-induced VSMC growth.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reagents-- Acetyl-coenzyme A, aprotinin, dithiothreitol, HEPES, leupeptin, phenylmethylsulfonyl fluoride, poly(dI-dC), sodium orthovanadate, sodium deoxycholate, and thrombin were purchased from Sigma. Recombinant human PDGF-BB was bought from R&D Systems Inc. (Minneapolis, MN). Anti-c-Fos (SC-052), anti-Fos-B (SC-048), anti-Fra-1 (SC-183), anti-Jun-B (SC-73), anti-Jun-D (SC-74), anti-ATF-1 (SC-270), anti-ATF-2 (SC-187), and anti-cAMP response element-binding protein-1 (CREB-1) (SC-271) antibodies and consensus oligonucleotides for AP-1 (5'-CGCTTGATGACTCAGCCGGAA-3') (SC-2501) and CRE (5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3') (SC-2504) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Phosphospecific anti-CREB (9191S) and anti-p38 MAPK (9211S) antibodies were obtained from Cell Signaling Technology (Beverly, MA). SB203580 was bought from Calbiochem Corp. (San Diego, CA). T4 polynucleotide kinase was procured from Invitrogen. pCRE-LUC plasmid was from Stratagene (La Jolla, CA). Luciferase assay kit was bought from Promega (Madison, WI). [-³²P]ATP (3000 Ci/mmol) and [³H]thymidine (20 Ci/mmol) were obtained from PerkinElmer Life Sciences. D-Threo-[dichloroacetyl-1-¹⁴C]chloramphenicol (58 mCi/mmol) was purchased from Amersham Biosciences.

Cell Culture-- VSMC were isolated from the thoracic aortas of 200-300-g male Sprague-Dawley rats by enzymatic dissociation as described previously (5). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin, and 100 µg/ml streptomycin. Cultures were maintained at 37 °C in a humidified 95% air and 5% CO₂ atmosphere. Cells were growth-arrested by incubating in DMEM containing 0.1% (v/v) FBS for 72 h and used to perform the experiments unless otherwise stated.

DNA Synthesis-- VSMC with and without appropriate treatments were pulse-labeled with 1 µCi/ml [³H]thymidine for the indicated times. After labeling, cells were washed with cold PBS, trypsinized, and collected by centrifugation. The cell pellet was suspended in cold 10% (w/v) trichloroacetic acid and vortexed vigorously to lyse cells. After standing on ice for 20 min, the cell lysis mixture was passed through a glass fiber filter (GF/C, Whatman). The filter was washed once with cold 5% trichloroacetic acid and once with cold 70% (v/v) ethanol. The filter was dried and placed in a liquid scintillation vial containing the scintillant fluid, and the radioactivity was measured in a liquid scintillation counter (LS 5000TA, Beckman).

Electrophoretic Mobility Shift Assay-- Nuclear extracts were prepared from VSMC that were subjected to appropriate treatments or left untreated as described previously (26). Protein-DNA complexes were formed by incubating 5 µg of nuclear protein in a total volume of 20 µl consisting of 15 mM HEPES, pH 7.9, 3 mM Tris-HCl, pH 7.9, 60 mM KCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 4.5 µg of bovine serum albumin, 2 µg of poly(dI-dC), 15% glycerol, and 100,000 cpm of ³²P-labeled oligonucleotide probe for 30 min on ice. The protein-DNA complexes were then resolved by electrophoresis on a 4% polyacrylamide gel using 1× Tris-glycine-EDTA buffer (25 mM Tris-HCl, pH 8.5, 200 mM glycine, 0.1 mM EDTA). Double-stranded oligonucleotides were labeled with [-³²P]ATP using T4 polynucleotide kinase kit following the supplier's protocol (Invitrogen).

Generation of ScFv-Anti-ATF-1-- The construction of ScFv-anti-ATF-1 has been described previously (27). The plasmid pRSV-KCREB was kindly provided by Dr. Richard H. Goodman (Oregon Health Science Center, Portland, OR).

Transient Transfection and Reporter Gene Assay-- VSMC were plated evenly onto 60-mm dishes the day before transfection and grown in DMEM containing 10% (v/v) heat-inactivated FBS, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were transfected with appropriate plasmid DNA (20 µg/60-mm dish) using calcium phosphate precipitation as described previously (26). Cells were washed with PBS 16 h after transfection and incubated in DMEM containing 0.1% (v/v) FBS for 36 h at 37 °C. Cells were then treated with and without PDGF-BB (20 ng/ml) or thrombin (0.1 unit/ml) for the indicated times, and cell lysates were prepared. Cell lysates normalized for protein were assayed for either CAT activity using [¹⁴C]chloramphenicol and acetyl-coenzyme A as substrates or luciferase activity using Luciferase assay system (Promega) and a Turner luminometer (TD-20/20).

Western Blot Analysis-- After appropriate treatments, VSMC were rinsed with cold phosphate-buffered saline (PBS) and frozen immediately in liquid nitrogen. Cells were lysed by thawing in 250 µl of lysis buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, 100 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mM sodium orthovanadate) and scraped into 1.5-ml Eppendorf tubes. After standing on ice for 20 min the cell lysates were cleared by centrifugation at 12,000 rpm for 15 min at 4 °C. The protein content of the supernatants was determined using Micro BCA^TM protein assay reagent kit (Pierce). Cell lysates containing equal amounts of protein were resolved by electrophoresis on 0.1% SDS and 10% polyacrylamide gels. The proteins were transferred electrophoretically to a nitrocellulose membrane (Hybond, Amersham Biosciences). After blocking in 10 mM Tris-HCl buffer, pH 8.0, containing 150 mM sodium chloride, 0.1% Tween 20, and 5% (w/v) nonfat dry milk, the membrane was treated with appropriate primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The antigen-antibody complexes were detected using a chemiluminescence reagent kit (Amersham Biosciences).

Statistics-- All the experiments were repeated at least three times with similar results. Data on luciferase activity and [³H]thymidine incorporation were presented as mean ± S.D., and the treatment effects were analyzed by Student's t test. p values < 0.05 were considered to be statistically significant. In the case of CAT assay, EMSA, and Western blot analysis, one representative set of data is shown.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Previously we have demonstrated that activation of p38 MAPK and induction of DNA synthesis in response to RTK and GPCR agonists require NADH/NADPH-like oxidase activity in VSMC (26). Here we have tested the role of p38 MAPK in RTK and GPCR agonist-induced VSMC growth. Lysates of VSMC that were treated with and without PDGF-BB (20 ng/ml) or thrombin (0.1 unit/ml) were analyzed by Western blotting for phosphorylated p38 MAPK using its phosphospecific antibodies. Both PDGF-BB and thrombin activated p38 MAPK in VSMC in a time-dependent manner with a maximum effect of 10-fold at 30 min and declining thereafter (Fig. 1A). To confirm these observations, lysates of VSMC that were treated with and without PDGF-BB (20 ng/ml) or thrombin (0.1 unit/ml) for 30 min were immunoprecipitated with anti-p38 MAPK antibodies and the immunocomplexes were assayed for p38 MAPK activity using recombinant ATF-2 and [-³²P]ATP as substrates in the presence and absence of SB203580 (10 µM), a potent inhibitor of p38 MAPK. As expected, both PDGF-BB and thrombin increased p38 MAPK activity, and it was completely inhibited by SB203580 (Fig. 1B). To learn whether p38 MAPK plays a role in PDGF-BB- and thrombin-induced VSMC growth, we tested the effect of SB203580 on DNA synthesis induced by these agonists. Quiescent VSMC were treated with and without PDGF-BB (20 ng/ml) or thrombin (0.1 unit/ml) in the presence and absence of SB203580 (10 µM), and DNA synthesis was measured by [³H]thymidine incorporation into trichloroacetic acid-precipitable material. PDGF-BB and thrombin stimulated VSMC DNA synthesis 3-5-fold, and SB203580 inhibited 50% of the thrombin-induced, but not PDGF-BB-induced, DNA synthesis (Fig. 2).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   PDGF-BB and thrombin activate p38 MAPK in VSMC. Panel A, equal amounts of protein from VSMC that were treated for the indicated times with PDGF-BB (20 ng/ml) or thrombin (0.1 unit/ml) or left untreated were analyzed by Western blotting for p38 MAPK using its phosphospecific antibodies. Panel B, equal amounts of protein from VSMC that were treated with PDGF-BB (20 ng/ml) or thrombin (0.1 unit/ml) for 30 min or left untreated were immunoprecipitated with anti-p38 MAPK antibodies, and the immunocomplexes were divided into two halves and subjected to immunocomplex kinase assay in the presence and absence of SB203580 (10 µM) using [-32P]ATP and recombinant ATF-2 protein as substrates. After termination of the reaction, the assay mixture was separated by electrophoresis on 0.1% SDS, 12% acrylamide gel and subjected to autoradiography.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Effect of SB203580 on PDGF-BB- and thrombin-induced DNA synthesis in VSMC. Quiescent VSMC were treated with and without PDGF-BB (20 ng/ml) or thrombin (0.1 unit/ml) in the presence and absence of SB203580 (10 µM) for 24 h, and DNA synthesis was measured by pulse-labeling cells with 1 µCi/ml [3H]thymidine for the last 2 h of the 24-h incubation period and counting the trichloroacetic acid-precipitable radioactivity. *, p < 0.01 versus control; **, p < 0.01 versus thrombin treatment.

Earlier studies from other laboratories have reported that p38 MAPK plays a role in c-Fos gene induction in response to cytokines and ultraviolet B light in some cell types (28-30). Therefore, to understand the mechanism by which p38 MAPK participates in thrombin-induced DNA synthesis, we studied its role in PDGF-BB- and thrombin-induced expression of the Fos and Jun family proteins. Lysates of VSMC that were treated with and without PDGF-BB (20 ng/ml) or thrombin (0.1 unit/ml) in the presence and absence of SB203580 (10 µM) for 2 h were analyzed for Fos and Jun family proteins using their specific antibodies. PDGF-BB and thrombin induced expression of c-Fos, Fra-1, c-Jun, and Jun-B by 10- to more than 100-fold (Fig. 3). Interestingly, Fos-B expression was induced more potently (>100-fold) by thrombin than by PDGF-BB. Other RTK agonists, such as epidermal growth factor (50 ng/ml) and basic FGF (50 ng/ml), also failed to induce the expression of Fos-B (data not shown), indicating that GPCR agonists specifically induce the expression of this proto-oncogene product. Jun-D is expressed constitutively in VSMC, and its levels were increased 3-fold in response to PDGF-BB and thrombin. SB203580, while having no effect on c-Fos, Fos-B, and c-Jun induction, inhibited Fra-1 expression induced by both PDGF-BB and thrombin and Jun-B and Jun-D expression induced by thrombin only. The Fos and Jun family proteins form the transcriptional factor, AP-1, which has been shown to play an important role in cell proliferation, differentiation, and apoptosis (31). Because inhibition of p38 MAPK depleted the levels of Jun-B and Jun-D in thrombin-treated cells, we suspected a role for AP-1 in p38 MAPK-dependent thrombin-induced growth in these cells. To address this we first determined the composition of AP-1 in PDGF-BB and thrombin-treated VSMC. Quiescent VSMC were treated with and without PDGF-BB (20 ng/ml) or thrombin (0.1 unit/ml) for 2 h, and nuclear proteins were isolated. Equal amounts of nuclear protein from control and agonist-treated VSMC were analyzed for AP-1-DNA binding activity by EMSA, using a ³²P-labeled AP-1 consensus oligonucleotide probe. AP-1-DNA binding activity was increased by both PDGF-BB and thrombin compared with control (Fig. 4A). Supershift EMSA for each of the Fos and Jun family proteins showed the presence of c-Fos, Fos-B, Fra-1, Jun-B, and Jun-D in the AP-1 complexes formed in response to both PDGF-BB and thrombin, although their levels differed between the two treatments (Fig. 4, B-D, F, and G). Fra-1, Jun-B, and Jun-D were present in the AP-1 complexes formed in response to both PDGF-BB and thrombin. Fos-B was present more abundantly in thrombin-induced AP-1 complexes. Consistent with the Western blot analyses, inhibition of p38 MAPK by SB203580 resulted in reduced amounts of Fra-1 and Jun-B in the AP-1 complexes formed in response to PDGF-BB and thrombin. Previously, we have reported that Jun-B forms the majority of the AP-1 complex in VSMC in response to PDGF-BB and thrombin and in concert with c-Fos drives AP-1-responsive reporter gene expression (26). Because SB203580 significantly reduced the expression of Jun-B and Fra-1 and thereby their levels in AP-1 complexes, we wanted to examine whether these decreases have any impact on PDGF-BB- and thrombin-induced AP-1-responsive reporter gene expression. VSMC were transiently transfected with an AP-1-responsive reporter plasmid, pCOLL-CAT, growth-arrested, treated with and without PDGF-BB (20 ng/ml) or thrombin (0.1 unit/ml) in the presence and absence of SB203580 for 4 h and cell lysates prepared. Cell lysates containing equal amounts of protein were assayed for CAT activity using coenzyme A and [¹⁴C]chloramphenicol as substrates. As shown in Fig. 5, both PDGF-BB and thrombin induced AP-1-responsive CAT expression in VSMC by 1.8-2.4-fold. Inhibition of p38 MAPK, while causing a 50% reduction in thrombin-induced DNA synthesis, attenuated only the basal but not the PDGF-BB- or thrombin-induced AP-1-responsive reporter gene expression. This result suggests that p38 MAPK mediates thrombin-induced VSMC growth independent of its effects on AP-1.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Effect of SB203580 on PDGF-BB- and thrombin-induced expression of the Fos and Jun family proteins in VSMC. Equal amounts of protein from VSMC that were treated with and without PDGF-BB (20 ng/ml) or thrombin (0.1 unit/ml) in the presence and absence of SB203580 (10 µM) for 2 h were analyzed by Western blotting for the Fos and Jun family proteins using their specific antibodies.

View larger version (38K): [in this window] [in a new window]   Fig. 4.   Effect of SB203580 on PDGF-BB- and thrombin-induced AP-1-DNA binding activity and its composition. Panel A, equal amounts of nuclear protein from VSMC that were treated with and without PDGF-BB (20 ng/ml) or thrombin (0.1 unit/ml) in the presence and absence of SB203580 (10 µM) for 2 h were incubated with 100,000 cpm of 32P-labeled AP-1 consensus oligonucleotide probe, and the protein-DNA complexes were separated by PAGE and subjected to autoradiography. In panels B-G, all conditions were the same except that antibodies to the indicated Fos and Jun family proteins were added to the protein-DNA complexes and incubation continued for an additional 2 h and the complexes were separated by PAGE and subjected to autoradiography.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Effect of SB203580 on PDGF-BB- and thrombin-induced AP-1-dependent reporter gene expression in VSMC. VSMC that were transfected with an AP-1-dependent reporter gene plasmid were growth-arrested and treated with and without PDGF-BB (20 ng/ml) or thrombin (0.1 unit/ml) in the presence and absence of SB203580 (10 µM) for 4 h, and cell lysates were prepared. Cell lysates were assayed for CAT activity using acetyl-coenzyme A and [14C]chloramphenicol as substrates, and the substrate-products were separated by TLC and subjected to autoradiography.

Earlier studies have reported that MAPKs via phosphorylating CREB/ATF1 play a role in CRE-dependent gene expression (32, 33). To test the role of p38 MAPK in CRE-dependent gene expression and its role in VSMC growth, we next studied the effect of PDGF-BB and thrombin on CRE activity. Quiescent VSMC were treated with and without PDGF-BB (20 ng/ml) or thrombin (0.1 unit/ml) in the presence and absence of SB203580 (10 µM) for 2 h, and nuclear proteins were prepared and analyzed by EMSA and supershift EMSA for CRE-DNA binding activity and CRE constituents, respectively. Both PDGF-BB and thrombin induced CRE-DNA binding activity, and SB203580 inhibited ~60% of the thrombin-induced, but not PDGF-BB-induced, CRE-DNA binding activity (Fig. 6). Supershift EMSA using anti-ATF-1 antibodies revealed that ATF-1 constitutes most of the PDGF-BB- and thrombin-induced CRE-DNA complexes. Use of anti-CREB-1 or anti-ATF-2 antibodies neither abolished nor caused a supershift of the CRE-DNA binding activity (data not shown). However, the lack of effect of anti-CREB-1 or anti-ATF-2 antibodies to cause a supershift or abolish the CRE-DNA binding activity does not rule out the presence of these transcriptional factors in the complexes. To confirm CRE-dependent transactivation, VSMC were transfected with a CRE-LUC reporter plasmid, growth-arrested, treated with and without PDGF-BB (20 ng/ml) or thrombin (0.1 unit/ml) in the presence and absence of SB203580 (10 µM) for 4 h, and cell lysates prepared. Cell lysates from control and each treatment were assayed for luciferase activity. Both PDGF-BB and thrombin induced CRE-dependent luciferase activity (Fig. 7). SB203580 blocked only the thrombin-increased luciferase activity by ~50%. To delineate the mechanism underlying this, quiescent VSMC were treated with and without PDGF-BB (20 ng/ml) or thrombin (0.1 unit/ml) in the presence and absence of SB203580 (10 µM) for 30 min and cell lysates were prepared. Equal amounts of protein from control and each treatment were analyzed for the phosphorylation state of CREB-1 and ATF-1 using a phosphospecific antibody that recognizes both the phosphoproteins. Both PDGF-BB and thrombin induced phosphorylation of CREB-1 and ATF-1 (Fig. 8). PDGF-BB and thrombin had no significant effect on the levels of CREB-1 or ATF-1, suggesting that the increase in the phosphorylation of these transcriptional factors was not because of an increase in their levels (data not shown). SB203580, while inhibiting both PDGF-BB- and thrombin-induced CREB-1 phosphorylation, blocked 50% of thrombin-induced ATF-1 phosphorylation only. To obtain additional evidence for the role of ATF-1 in thrombin-induced DNA synthesis, ATF-1 and CREB-1 activities were down-regulated by intracellular expression of an inhibitory anti-ATF-1 single-chain antibody fragment (ScFv) or a dominant negative mutant of CREB-1, respectively, growth-arrested, treated with and without PDGF-BB (20 ng/ml) or thrombin (0.1 unit/ml) for 24 h, and DNA synthesis measured by [³H]thymidine incorporation. Ectopic expression of ScFv but not the dominant negative mutant of CREB-1 attenuated the DNA synthesis induced by thrombin (Fig. 9). DNA synthesis induced by PDGF-BB was not inhibited.

View larger version (80K): [in this window] [in a new window]   Fig. 6.   ATF-1 forms the majority of the CRE-DNA complex in response to PDGF-BB and thrombin in VSMC. Equal amounts of nuclear protein from VSMC that were treated with and without PDGF-BB (20 ng/ml) or thrombin (0.1 unit/ml) in the presence and absence of SB203580 (10 µM) for 2 h were incubated with 100,000 cpm of 32P-labeled CRE consensus oligonucleotide probe with and without anti-ATF-1 antibodies, and the protein-DNA complexes were separated by PAGE and subjected to autoradiography.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Effect of SB203580 on PDGF-BB- and thrombin-induced CRE-dependent reporter gene expression in VSMC. VSMC were transfected with a CRE-dependent luciferase reporter gene plasmid, growth-arrested, treated with and without PDGF-BB (20 ng/ml) or thrombin (0.1 unit/ml) in the presence and absence of SB203580 (10 µM) for 4 h, and cell lysates prepared. Cell lysates were assayed for luciferase activity. *, p < 0.01 versus control; **, p < 0.01 versus thrombin treatment.

View larger version (23K): [in this window] [in a new window]   Fig. 8.   SB203580 blocks thrombin- but not PDGF-BB-induced phosphorylation of ATF-1 in VSMC. Equal amounts of protein from VSMC that were treated with and without PDGF-BB (20 ng/ml) or thrombin (0.1 unit/ml) in the presence and absence of SB203580 (10 µM) for 30 min were analyzed by Western blotting for CREB-1 and ATF-1 using a phosphospecific antibody that recognizes both phospho-CREB-1 and phospho-ATF-1.

View larger version (23K): [in this window] [in a new window]   Fig. 9.   Endogenous expression of an inhibitory anti-ATF-1 ScFv reduces thrombin- but not PDGF-BB-induced DNA synthesis in VSMC. VSMC were transfected with mock or ScFv anti-ATF-1 or KCREB plasmids, growth-arrested, and treated with and without PDGF-BB (20 ng/ml) or thrombin (0.1 unit/ml) for 24 h, and DNA synthesis was measured by pulse-labeling cells with 1 µCi/ml [3H]thymidine for the last 2 h of the 24-h incubation period and counting the trichloroacetic acid-precipitable radioactivity. *, p < 0.01 versus control; **, p < 0.01 versus thrombin treatment.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The important findings of the present study are as follows. 1) p38 MAPK plays a selective role in GPCR agonist-induced DNA synthesis in VSMC, and 2) phosphorylation of ATF-1 appears to be the likely mechanism for the selectivity of p38 MAPK. A role for p38 MAPK in cell growth has been reported previously (34). The present study, however, shows that p38 MAPK plays a differential role in the induction of VSMC growth by RTK and GPCR agonists. The most intriguing finding of the present study is the phosphorylation of ATF-1, a transcriptional factor that belongs to the leucine zipper family of proteins (35, 36). ATF-1, like CREB, binds to a consensus motif 5'-TGACGTCA-3' in the promoter region of genes and activates transcription. These transcriptional factors contain a kinase-inducible transactivation domain that possesses consensus phosphorylation sites for several kinases including protein kinase A (37). In fact, several kinases, including calcium calmodulin kinase IV (38) and p90 ribosomal S6 kinase 2 (39), have been reported to phosphorylate and activate CREB. Although CREB/ATF-1 family proteins have been extensively studied for their role in cAMP and Ca²⁺-responsive gene regulation, their role in cell growth is less clear. The absence of ATF-1 in normal melanocytes and its detection in metastatic melanoma cells, however, suggest that this transcriptional factor may be involved in the regulation of cell growth (40). The most convincing evidence for the role of ATF-1 and its related protein CREB in cell growth comes from depletion studies (27, 41, 42). Ectopic expression of single-chain antibody fragment of ATF-1 or dominant negative mutant of CREB-1 inhibited the tumor growth and metastasis of human melanoma cells and the survival of these cells, respectively (27, 41).

CREB/ATF-1 transcription factors form homodimers or heterodimers with members of the Jun family proteins, and they preferentially bind to CRE (31, 43). Despite the presence of both CREB-1 and ATF-1 in VSMC, only ATF-1 was found in the CRE-DNA complexes formed in response to PDGF-BB and thrombin in these cells. This result implies that ATF-1 exists as either homodimers or heterodimers with other transcriptional factors, such as Jun-B or Jun-D. Although both PDGF-BB and thrombin stimulated ATF-1 phosphorylation, only thrombin-induced phosphorylation was sensitive to p38 MAPK. This finding also suggests that ATF-1 phosphorylation induced by PDGF-BB is independent of p38 MAPK. Regardless of the mechanisms of its phosphorylation, ATF-1 is present both in PDGF-BB- and thrombin-induced CRE-DNA complexes. If it was involved in growth induced by both agonists, then inhibition of its activity via ectopic expression of its single-chain antibody should have interfered with the DNA synthesis induced by both PDGF-BB and thrombin and not the latter only. It is possible that, although it is the predominant component in PDGF-BB- and thrombin-induced CRE-DNA complexes, it may exist as heterodimers with different Jun family proteins in response to PDGF-BB and thrombin and that the ATF-1 complex in GPCR agonist-treated cells is involved in growth induction.

PDGF-BB and thrombin also stimulated the phosphorylation of CREB-1 in a p38 MAPK-dependent manner. CREB-1 phosphorylation by PDGF-BB and thrombin, although mediated by p38 MAPK, appears not to be on the path to growth stimulation. The lack of a role for p38 MAPK-dependent CREB-1 phosphorylation in the induction of growth was further confirmed by the inability of its dominant negative mutant to suppress either PDGF-BB- or thrombin-induced DNA synthesis in VSMC. It was recently reported that CREB plays an important role in VSMC differentiation-specific gene regulation (44). This study further showed that CREB is a negative regulator of VSMC growth. The present findings are consistent with these observations.

CREB/ATF-1 act as survival factors in metastatic melanoma cells (27, 41, 42). In addition, the expression of the thrombin receptor (protease-activated receptor-1) is directly correlated with the metastatic ability of human melanoma cells (46, 47). Our observation that thrombin-induced p38 MAPK-dependent ATF-1 phosphorylation is involved in DNA synthesis may provide a mechanism by which thrombin receptor (protease-activated receptor-1) contributes to the growth of VSMC and tumor cells. In summary, in the present study we demonstrate for the first time that p38 MAPK plays a selective role in GPCR agonist-induced VSMC DNA synthesis via phosphorylating and activating ATF-1.

	ACKNOWLEDGEMENT

We thank Dr. Michael Karin for his generosity in providing us with pCOLL-CAT plasmid.

	Addendum

While this paper was under review, a study from other laboratories reported that p38 MAPK plays a role in angiotensin II- and 12(S)-hydroxyeicosatetraenoic acid-induced hypertrophy in VSMC via a mechanism involving CREB (45). Thus, the above study and ours have independently demonstrated a role for p38 MAPK in GPCR agonist-induced VSMC growth involving CREB/ATF-1 transcriptional factors.

	FOOTNOTES

^* This work was supported in part by grants from the National Institutes of Health (to G. N. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Both authors contributed equally to this work.

To whom correspondence should be addressed: Dept. of Physiology, University of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163. Tel.: 901-448-7321; Fax: 901-448-7126; E-mail: grao@physio1.utmem.edu.

Published, JBC Papers in Press, March 29, 2002, DOI 10.1074/jbc.M201608200

	ABBREVIATIONS

The abbreviations used are: PDGF, platelet-derived growth factor; AP-1, activator protein-1; ATF-1, activating transcription factor-1; CRE, cyclic AMP response element; CREB, cyclic AMP response element-binding protein; DMEM, Dulbecco's modified Eagle's medium; EMSA, electrophoretic mobility shift assay; FBS, fetal bovine serum; FGF, fibroblast growth factor; GPCR, G protein-coupled receptor; p38 MAPK, p38 mitogen-activated protein kinase; RTK, receptor tyrosine kinase; VSMC, vascular smooth muscle cell(s).

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				H. Cao, N. Dronadula, F. Rizvi, Q. Li, K. Srivastava, W. T. Gerthoffer, and G. N. Rao Novel Role for STAT-5B in the Regulation of Hsp27-FGF-2 Axis Facilitating Thrombin-Induced Vascular Smooth Muscle Cell Growth and Motility Circ. Res., April 14, 2006; 98(7): 913 - 922. [Abstract] [Full Text] [PDF]

				N. Dronadula, F. Rizvi, E. Blaskova, Q. Li, and G. N. Rao Involvement of cAMP-response element binding protein-1 in arachidonic acid-induced vascular smooth muscle cell motility J. Lipid Res., April 1, 2006; 47(4): 767 - 777. [Abstract] [Full Text] [PDF]

				H. Cao, N. Dronadula, and G. N. Rao Thrombin induces expression of FGF-2 via activation of PI3K-Akt-Fra-1 signaling axis leading to DNA synthesis and motility in vascular smooth muscle cells Am J Physiol Cell Physiol, January 1, 2006; 290(1): C172 - C182. [Abstract] [Full Text] [PDF]

				M. Fujiwara, E. Jin, M. Ghazizadeh, and O. Kawanami Activation of PAR4 Induces a Distinct Actin Fiber Formation via p38 MAPK in Human Lung Endothelial Cells J. Histochem. Cytochem., September 1, 2005; 53(9): 1121 - 1129. [Abstract] [Full Text] [PDF]

				M. Steinhoff, J. Buddenkotte, V. Shpacovitch, A. Rattenholl, C. Moormann, N. Vergnolle, T. A. Luger, and M. D. Hollenberg Proteinase-Activated Receptors: Transducers of Proteinase-Mediated Signaling in Inflammation and Immune Response Endocr. Rev., February 1, 2005; 26(1): 1 - 43. [Abstract] [Full Text] [PDF]

				C. R. Yellaturu and G. N. Rao A Requirement for Calcium-independent Phospholipase A2 in Thrombin-induced Arachidonic Acid Release and Growth in Vascular Smooth Muscle Cells J. Biol. Chem., October 31, 2003; 278(44): 43831 - 43837. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 277/24/21325    most recent M201608200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar