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An Essential Role of the Jak-2/STAT-3/Cytosolic Phospholipase A2 Axis in Platelet-derived Growth Factor BB-induced Vascular Smooth Muscle Cell Motility

2004; Elsevier BV; Volume: 279; Issue: 44 Linguagem: Inglês

10.1074/jbc.m406922200

ISSN

1083-351X

Autores

Indira Neeli, Zhimin Liu, Nagadhara Dronadula, Zhongmin Alex, Gadiparthi N. Rao,

Tópico(s)

Atherosclerosis and Cardiovascular Diseases

Resumo

Platelet-derived growth factor-BB (PDGF-BB) is a potent motogen for vascular smooth muscle cells (VSMCs). To understand its motogenic signaling events, we have studied the role of the Janus-activated kinase/signal transducers and activators of transcription (Jak/STAT) pathway and cytosolic phospholipase A2 (cPLA2). PDGF-BB stimulated tyrosine phosphorylation of Jak-2 and STAT-3 in a time-dependent manner in VSMCs. In addition, AG490 and Jak-2KEpRK5, a selective pharmacological inhibitor and a dominant negative mutant, respectively, of Jak-2, attenuated PDGF-BB-induced STAT-3 tyrosine phosphorylation and its DNA binding and reporter gene activities. PDGF-BB induced VSMC motility in a dose-dependent manner with a maximum effect at 10 ng/ml. Dominant negative mutant-dependent suppression of Jak-2 and STAT-3 blocked PDGF-BB-induced VSMC motility. PDGF-BB induced the expression of cPLA2 in a Jak-2/STAT-3-dependent manner, and pharmacological inhibitors of cPLA2 prevented PDGFBB-induced VSMC motility. Furthermore, either exogenous addition of arachidonic acid or forced expression of cPLA2 rescued PDGF-BB-induced VSMC motility from inhibition by blockade of Jak-2 and STAT-3 activation. Together, these results for the first time show that PDGF-BB-induced VSMC motility requires activation of the Jak-2/STAT-3/cPLA2 signaling axis. Platelet-derived growth factor-BB (PDGF-BB) is a potent motogen for vascular smooth muscle cells (VSMCs). To understand its motogenic signaling events, we have studied the role of the Janus-activated kinase/signal transducers and activators of transcription (Jak/STAT) pathway and cytosolic phospholipase A2 (cPLA2). PDGF-BB stimulated tyrosine phosphorylation of Jak-2 and STAT-3 in a time-dependent manner in VSMCs. In addition, AG490 and Jak-2KEpRK5, a selective pharmacological inhibitor and a dominant negative mutant, respectively, of Jak-2, attenuated PDGF-BB-induced STAT-3 tyrosine phosphorylation and its DNA binding and reporter gene activities. PDGF-BB induced VSMC motility in a dose-dependent manner with a maximum effect at 10 ng/ml. Dominant negative mutant-dependent suppression of Jak-2 and STAT-3 blocked PDGF-BB-induced VSMC motility. PDGF-BB induced the expression of cPLA2 in a Jak-2/STAT-3-dependent manner, and pharmacological inhibitors of cPLA2 prevented PDGFBB-induced VSMC motility. Furthermore, either exogenous addition of arachidonic acid or forced expression of cPLA2 rescued PDGF-BB-induced VSMC motility from inhibition by blockade of Jak-2 and STAT-3 activation. Together, these results for the first time show that PDGF-BB-induced VSMC motility requires activation of the Jak-2/STAT-3/cPLA2 signaling axis. Inflammation at the site of vascular injury is believed to be an initiative event in the pathogenesis of vessel wall diseases (1Ross R. N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19370) Google Scholar, 2Hansson G.K. Libby P. Schonbeck U. Yan Z.Q. Circ. Res. 2002; 91: 281-291Crossref PubMed Scopus (862) Google Scholar). The dysfunctional endothelial cells and inflammatory cells at the site of vascular injury produce a large number of molecules with a broad spectrum of biological activities (1Ross R. N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19370) Google Scholar, 2Hansson G.K. Libby P. Schonbeck U. Yan Z.Q. Circ. Res. 2002; 91: 281-291Crossref PubMed Scopus (862) Google Scholar, 3Berk B.C. Physiol. Rev. 2001; 81: 999-1030Crossref PubMed Scopus (336) Google Scholar). 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Biol. Chem. 1996; 271: 6758-6765Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). Recently, we have reported that activation of the Jak/STAT pathway is required for PDGF-BB-induced cPLA2 expression and proliferation in VSMCs (39Yellaturu C.R. Rao G.N. J. Biol. Chem. 2003; 278: 9986-9992Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). PDGF-BB is also a potent motogen for VSMCs and is involved in the migration of VSMCs from media to intima leading to neointima formation (4Jawien A. Bowen-Pope D.F. Lindner V. Schwartz S.M. Clowes A.W. J. Clin. Investig. 1992; 89: 507-511Crossref PubMed Scopus (594) Google Scholar, 5Kalmes A. Vesti B.R. Daum G. Abraham J.A. Clowes A.W. Circ. Res. 2000; 87: 92-98Crossref PubMed Scopus (167) Google Scholar). To understand the molecular events of PDGF-BB-induced motility in VSMCs, we now have studied the role of Jak-2/STAT-3 and cPLA2. Here, we report for the first time that PDGF-BB induces VSMC motility via activation of Jak-2/STAT-3, which targets the induction of expression of cPLA2. Reagents—Aprotinin, dithiothreitol, phenylmethylsulfonyl fluoride, sodium orthovanadate, sodium deoxycholate, leupeptin, and HEPES were purchased from Sigma. AG490 was obtained from Calbiochem. Arachidonic acid was purchased from Cayman Chemicals (Ann Arbor, MI). Anti-cPLA2 antibodies (2832) and phospho-specific anti-STAT-3 (9131S) antibodies were procured from Cell Signaling Technology (Beverly, MA). Phospho-specific anti-Jak-2 (44-426Z) antibodies were from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-Jak-2 (SC-294) and anti-STAT-3 (SC-482) antibodies and consensus STAT-3 oligonucleotide (5′-GATCCTTCTGGGAATTCCTAGATC-3′, 3′-CTAGGAAGACCCTTAAGGATCTAG-5′) (SC-2571) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). T4 polynucleotide kinase was purchased from Promega (Madison, WI). [γ-32P]ATP (3000 Ci/mmol) was obtained from PerkinElmer Life Sciences. Cell Culture—VSMCs were isolated from the thoracic aortae of male Sprague-Dawley rats by enzymatic dissociation as described earlier (40Rao G.N. Katki K.A. Madamanchi N.R. Wu Y. Birrer M.J. J. Biol. Chem. 1999; 274: 6003-6010Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 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% calf serum for 72 h and were used to perform the experiments unless otherwise stated. Cell Motility—VSMC motility was measured by cell-wounding assay (41Zhuang D. Ceacareanu A.C. Lin Y. Ceacareanu B. Dixit M. Chapman K.E. Waters C.M. Rao G.N. Hassid A. Am. J. Physiol. 2004; 286: H2103-H2112Google Scholar). Quiescent confluent monolayers of VSMCs were wounded with a sterile pipette tip to generate a cell-free gap of ∼1 mm in width, and the wound location in the culture dish was marked. Cells were washed, and fresh serum-free DMEM was added and photographed to record the wound width at 0 h. To prevent replicative DNA synthesis, hydroxyurea was added to the medium to a final concentration of 5 mm just before the addition of agonist. Twenty-four h after the appropriate treatments, photographs were taken again at the marked wound location. Cell migration was measured using the NIH Image 1.62 program, and the cell motility was expressed as distance migrated in μm units. To test the effect of dominant negative mutants of Jak-2 and STAT-3 on PDGFBB-induced motility, cells were first transfected with Jak-2KEpRK5 or FS3DM for 48 h and quiesced before they were subjected to agonist-induced motility. Electrophoretic Mobility Shift Assay (EMSA)—Nuclear extracts were prepared from treated or untreated VSMCs as described previously (40Rao G.N. Katki K.A. Madamanchi N.R. Wu Y. Birrer M.J. J. Biol. Chem. 1999; 274: 6003-6010Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The protein content of the nuclear extracts was determined using a Micro BCA™ protein assay reagent kit (Pierce). 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 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 [γ-32P]ATP using the T4 polynucleotide kinase kit (Promega) following the supplier's protocol. Western Blot Analysis—After appropriate treatments, VSMCs were rinsed with cold phosphate-buffered saline and frozen immediately in liquid nitrogen. Cells were lysed by thawing in 250 μl of lysis buffer (phosphate-buffered saline, 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 extracts were cleared by centrifugation at 12,000 rpm for 20 min at 4 °C. Cell extracts containing an equal amount 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 horse-radish peroxidase-conjugated secondary antibodies. The antigen-antibody complexes were detected using a chemiluminescence reagent kit (Amersham Biosciences). Transient Transfection and CAT Assay—VSMCs were plated evenly onto 100-mm dishes and grown in DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. At 50–80% confluence, the medium was replaced with DMEM containing 0.1% calf serum, and cells were transfected with pSIE-CAT plasmid using LipofectAMINE Plus reagent according to the manufacturer's instructions (Invitrogen). Thirty h after transfection, VSMCs were treated with and without PDGF-BB (20 ng/ml) in the presence and absence of AG490 (25 μm) for 4 h, and cell extracts were prepared. Wherever the effect of dominant negative STAT-3 mutant was tested on agonist-induced CAT activity, cells were co-transfected with FS3DM and pSIE-CAT plasmids. VSMC extracts were normalized for protein and assayed for CAT activity using [14C]chloramphenicol and acetyl coenzyme A as substrates. The substrate and products were extracted with ethyl acetate, separated by thin layer chromatography, and subjected to autoradiography. Statistics—All of the experiments were repeated three times with similar results. Data are presented as mean ± S.D. The treatment effects were analyzed by Student's t test. p values of <0.05 were considered to be statistically significant. In the case of CAT activity, EMSA, and Western blot analysis, one representative set of data is shown. To understand the signaling events underlying PDGF-BB-induced motility in VSMCs, we have studied the role of the Jak/STAT pathway. Quiescent VSMCs were treated with and without PDGF-BB (20 ng/ml) for various times, and cell extracts were prepared. An equal amount of protein from the control and each treatment was analyzed by Western blotting for tyrosine phosphorylation of Jak-2 and STAT-3 using their phospho-specific antibodies. PDGF-BB stimulated tyrosine phosphorylation of both Jak-2 and STAT-3 in a time-dependent manner with a maximum effect of about 20-fold at 10 min and reaching basal levels by 4 h (Fig. 1). Jaks phosphorylate STATs on tyrosine residues and activate them, although other mechanisms were also reported to be involved in the activation of these transcriptional factors (42Nguyen H. Ramana C.V. Bayes J. Stark G.R. J. Biol. Chem. 2001; 276: 33361-33368Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 43Sachsenmaier C. Sadowski H.B. Cooper J.A. Oncogene. 1999; 18: 3583-3592Crossref PubMed Scopus (55) Google Scholar). To find whether PDGFBB-stimulated STAT-3 tyrosine phosphorylation is mediated by Jak-2, we tested the effect of AG490, a potent and specific inhibitor of Jak-2 (44Meydan N. Grunberger T. Dadi H. Shahar M. Arpaia E. Lapidot Z. Leeder J.S. Freedman M. Cohen A. Gazit A. Levitzki A. Roifman C.M. Nature. 1996; 379: 645-646Crossref PubMed Scopus (848) Google Scholar). AG490 (25 μm) significantly inhibited PDGF-BB-stimulated tyrosine phosphorylation of STAT-3 (Fig. 2, upper left panel). To confirm the pharmacological effect of AG490 on PDGF-BB-induced STAT-3 phosphorylation, we next tested the effect of dominant negative Jak-2 (Jak-2KEpRK5). Forced expression of Jak-2KEpRK5 also reduced PDGF-BB-induced STAT-3 phosphorylation by about 50% (Fig. 2, upper right and lower panels).Fig. 2AG490 and Jak-2KEpRK5, a specific pharmacological inhibitor and a dominant negative mutant, respectively, of Jak-2, attenuate PDGF-BB-induced STAT-3 tyrosine phosphorylation. Quiescent VSMCs were treated with and without PDGF-BB (20 ng/ml) in the presence and absence of AG490 (25 μm) for the indicated times, and cell extracts were prepared. An equal amount of protein from the control and each treatment was analyzed by Western blotting for pSTAT-3 using its phospho-specific antibodies. To test the effect of Jak-2KEpRK5 on STAT-3 tyrosine phosphorylation, VSMCs were transfected first with and without Jak-2KEpRK5 and quiesced. Cells were then treated with and without PDGF-BB (20 ng/ml) for 10 min, and cell extracts were prepared. An equal amount of protein from the control and each treatment was analyzed for pSTAT-3 as described above. As a loading control, the blots were reprobed with anti-STAT-3 antibodies. The bar graph represents the quantitative data of three independent experiments on the effect of dominant negative Jak-2 (DnJak-2) on PDGF-BB-induced STAT-3 tyrosine phosphorylation. *, p < 0.01 versus control; **, p < 0.01 versus PDGF-BB treatment alone.View Large Image Figure ViewerDownload (PPT) Upon tyrosine phosphorylation, STATs undergo either homo- or heterodimerization and translocate to the nucleus, where they bind (in this case STAT-3) to their consensus DNA binding sequence present in the promoter regions of genes and induce transcription (13Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5062) Google Scholar, 45Ehret G.B. Reichenbach P. Schindler U. Horvath C.M. Fritz S. Nabholz M. Bucher P. J. Biol. Chem. 2001; 276: 6675-6688Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). To find whether tyrosine phosphorylation of STAT-3 correlates with an increase in its transcriptional activation, its DNA binding activity was measured. Quiescent VSMCs were treated with and without PDGF-BB (20 ng/ml) for 2 h, and nuclear extracts were prepared. Equal amounts of nuclear protein from the control and each treatment were analyzed by EMSA for STAT-3-DNA binding activity using 32P-labeled consensus STAT-3 oligonucleotide as a probe. PDGF-BB increased STAT-3-DNA binding activity by 4-fold, and it was inhibited by AG490 (Fig. 3A, left panel). Forced expression of either dominant negative Jak-2 (Jak-2KEpRK5) or STAT-3 (FS3DM) also substantially reduced PDGF-BB-induced STAT-3-DNA binding activity (Fig. 3A, right panel). To confirm that increased STAT-3-DNA binding activity leads to an increase in its transactivation activity, VSMCs were transiently transfected with a STAT-3-dependent reporter plasmid, pSIE-CAT, quiesced, and treated with and without PDGF-BB (20 ng/ml) for 4 h, and cell extracts were prepared. Cell extracts normalized for protein were assayed for CAT activity. PDGF-BB induced STAT-3-dependent CAT activity by 3-fold, and AG490 substantially inhibited this response (Fig. 3B, left panel). FS3DM also blocked PDGF-BB-induced STAT-3-dependent CAT activity (Fig. 3B, right panel). These findings are consistent with our previous observations on PDGF-BB activation of the Jak/STAT pathway (39Yellaturu C.R. Rao G.N. J. Biol. Chem. 2003; 278: 9986-9992Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). To understand the role of the Jak/STAT pathway in PDGF-BB-induced VSMC motility, we first studied a dose-response effect of PDGF-BB. A cell-free gap was generated in a monolayer of quiescent VSMCs as described under “Materials and Methods.” Cells were then treated with and without various doses of PDGF-BB for 24 h, and cell motility was measured. As shown in Fig. 4A, PDGF-BB induced VSMC motility in a dose-dependent manner. Maximum VSMC motility was observed in response to 10 ng/ml PDGF-BB. We now examined the effect of Jak-2KEpRK5 and FS3DM. Forced expression of either Jak-2KEpRK5 or FS3DM significantly prevented PDGF-BB-induced VSMC motility (Fig. 4B).Fig. 4PDGF-BB induces VSMC motility in a Jak-2/STAT-3-dependent manner.A, a cell-free gap was made in a monolayer of quiescent VSMCs and treated with and without various doses of PDGF-BB for 24 h, and cell motility was measured using the NIH Image 1.62 program. B, VSMCs were transfected with and without Jak-2KEpRK5 (DnJAK-2) or FS3DM, the dominant negative mutants of Jak-2 and STAT-3, respectively, and quiesced prior to testing their responsiveness to PDGF-BB-induced motility. *, p < 0.01 versus control; **, p < 0.01 versus PDGF-BB treatment alone.View Large Image Figure ViewerDownload (PPT) It was reported that arachidonic acid and its eicosanoid metabolites play an important role in cell migration (46Ott V.L. Cambier J.C. Kappler J. Marrack P. Swanson B.J. Nat. Immunol. 2003; 10: 974-981Crossref Scopus (241) Google Scholar, 47Honig S.M. Fu S. Mao X. Yopp A. Gunn M.D. Randolph G.J. Bromberg J.S. J. Clin. Investig. 2003; 111: 627-637Crossref PubMed Scopus (122) Google Scholar, 48Stockton R.A. Jacobson B.S. Mol. Biol. Cell. 2001; 12: 1937-1956Crossref PubMed Scopus (51) Google Scholar, 49Maddox J.F. Colgan S.P. Clish C.B. Petasis N.A. Fokin V.V. Serhan C.N. FASEB J. 1998; 12: 487-494Crossref PubMed Scopus (90) Google Scholar). In addition, arachidonic acid, the precursor for eicosanoids, and other mitogenically active lipids such as phosphatidic acid have been shown to activate GTPases via inhibition of GTPase-activating proteins (29Golubic M. Tanaka K. Dobrowolski S. Wood D. Tsai M.H. Marshall M. Tamanoi F. Stacey D.W. EMBO J. 1991; 10: 2897-2903Crossref PubMed Scopus (50) Google Scholar, 30Tsai M.H. Roudebush M. Dobrowolski S. Yu C.L. Gibbs J.B. Stacey D.W. Mol. Cell. Biol. 1991; 11: 2785-2793Crossref PubMed Scopus (43) Google Scholar, 50Tsai M.H. Yu C.L. Wei F.S. Stacey D.W. Science. 1989; 243: 522-526Crossref PubMed Scopus (231) Google Scholar, 51Han J.W. McCormick F. Macara I.G. Science. 1991; 252: 576-579Crossref PubMed Scopus (85) Google Scholar). GTPases play an essential role in cell proliferation and migration (52Etienne S. Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3875) Google Scholar). 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Chem. 1996; 271: 6758-6765Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar), we envisioned that Jak-STAT signaling may be mediating cell migration via induction of expression of cPLA2. To test this possibility, quiescent VSMCs were treated with and without PDGF-BB (20 ng/ml) for various times, and cell extracts were prepared. An equal amount of protein from control and PDGF-BB-treated cells was analyzed by Western blotting for cPLA2 using its specific antibodies. PDGF-BB induced cPLA2 expression in a time-dependent manner with 2- and 3-fold increases at 8 and 16 h of treatment, respectively (Fig. 5A). PDGF-BB also induced the expression of cPLA2 mRNA by 4- and 5-fold at 8 and 16 h of treatment, respectively, as measured by Northern blot analysis using 32P-labeled rat cPLA2 cDNA probe. 2N. Dronadula et al., unpublished observations. To understand whether the Jak/STAT pathway plays a role in PDGF-BB-induced expression of cPLA2, we next studied the effect of AG490. AG490 completely inhibited PDGF-BB-induced cPLA2 expression (Fig. 5B). To obtain additional evidence on the role of the Jak/STAT pathway in PDGF-BB-induced cPLA2 expression, we tested the effect of dominant negative Jak-2 and STAT-3 mutants, Jak-2KEpRK5 and FS3DM, respectively (17Wu Y.Y. Bradshaw R.A. J. Biol. Chem. 2000; 275: 2147-2156Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 53Feng J. Witthuhn B.A. Matsuda T. Kohlhuber F. Kerr I.M. Ihle J.N. Mol. Cell. Biol. 1997; 17: 2497-2501Crossref PubMed Scopus (279) Google Scholar). As shown in Fig. 5B, forced expression of Jak-2KEpRK5 or FS3DM also blocked PDGF-BB-induced expression of cPLA2. Next we examined the role of cPLA2 in PDGF-BB-induced VSMC motility using a pharmacological approach. Use of methyl arachidonyl fluorophosphonate (MAFP) and palmitoyl trifluoromethyl ketone (PACOCF3), two structurally different and specific inhibitors of cPLA2 (38Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 6758-6765Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar, 54Ackermann E.J. Conde-Frieboes K. Dennis E.A. J. Biol. Chem. 1995; 270: 445-450Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar), significantly blocked PDGF-BB-induced VSMC motility (Fig. 6). If Jak-2/STAT-3 signaling mediates PDGF-BB-induced VSMC motility via induction of expression of cPLA2, then one would expect that exogenous addition of arachidonic acid would rescue PDGFBB-induced VSMC motility from inhibition by blockade of Jak-2/STAT-3 activation. To test this, a cell-free gap was made in a monolayer of quiescent VSMCs that were forced to express FS3DM and treated with and without PDGF-BB (20 ng/ml) in the presence and absence of exogenously added arachidonic acid (5 μm) for 24 h, and cell motility was measured. As shown in Fig. 7, arachidonic acid rescued PDGF-B-induced VSMC motility from inhibition by FS3DM. Exogenous arachidonic acid (5 μm) also surpassed the inhibitory effect of AG490 on PDGF-BB-induced VSMC motility (Fig. 7). To confirm this result further, VSMCs were co-transfected with dominant negative Jak-2 (Jak-2KEpRK5) or STAT-3 (FS3DM) along with and without an expression plasmid for rat cPLA2 (pcDNA3-cPLA2), quiesced, and treated with and without PDGF-BB (20 ng/ml) for 24 h, and cell motility was measured. Forced expression of rat cPLA2 rescued PDGF-BB-induced VSMC motility from inhibition by both the dominant negative Jak-2 and STAT-3 (Fig. 8).Fig. 6MAFP and PACOCF3, selective inhibitors of cPLA2, reduce PDGF-BB-induced VSMC motility. A cell-free gap was made in a monolayer of quiescent VSMCs and treated with and without PDGF-BB (20 ng/ml) in the presence and absence of MAFP (10 μm) or PACOCF3 (10 μm) for 24 h, and cell motility was measured using the NIH Image 1.62 program. *, p < 0.01 versus control; **, p < 0.01 versus PDGF-BB treatment alone.View Large Image Figure ViewerDownload (PPT)Fig. 7Arachidonic acid rescues PDGF-BB-induced VSMC motility from inhibition by AG490 and a dominant negative mutant of STAT-3, FS3DM. A cell-free gap was made in a monolayer of quiescent VSMCs and treated with and without PDGF-BB (20 ng/ml) in the presence and absence of AG490 (μm), and cell motility was measured using the NIH Image 1.62 program. Wherever indicated, arachidonic acid (AA) was added to a final concentration of 5 μm simultaneously with PDGF-BB. When the effect of arachidonic acid was tested on rescue of PDGF-BB-induced VSMC motility from inhibition by FS3DM, cells were transfected first with this plasmid DNA and quiesced before they were subjected to the indicated treatments. *, p < 0.01 versus control; **, p < 0.05 versus PDGF-BB treatment alone; ***, p < 0.01 versus AG490 + PDGF-BB or FS3DM + PDGF-BB.View Large Image Figure ViewerDownload (PPT)Fig. 8Forced expression of cPLA2 rescues PDGF-BB-induced VSMC motility from inhibition by dominant negative mutants of Jak-2 and STAT-3. VSMCs were co-transfected with dominant negative Jak-2 (Jak-2KEpRK5) (DnJak-2) or dominant negative STAT-3 (FS3DM) (DnSTAT-3) along with and without an expression plasmid for rat cPLA2 (pcDNA3-cPLA2) and were quiesced. These cells then were treated with and without PDGF-BB (20 ng/ml) for 24 h, and cell motility was measured using the NIH Image 1.62 program as described above in the legend of Fig. 7. *, p < 0.01 versus control; **, p < 0.05 versus PDGF-BB treatment alone; ***, p < 0.05 versus Jak-2KEpRK5 + PDGF-BB or FS3DM + PDGF-BB.View Large Image Figure ViewerDownload (PPT) The important finding of the present study is that PDGF-BB, a receptor tyrosine kinase agonist, induces VSMC motility via Jak-2/STAT-3-dependent induction of expression of cPLA2. This conclusion is supported by the following observations. 1) PDGF-BB stimulated tyrosine phosphorylation of Jak-2 and STAT-3 in a time-dependent manner. 2) The tyrosine phosphorylation of STAT-3 and its DNA binding and reporter gene activities induced by PDGF-BB are mediated by Jak-2 as its inhibition by pharmacological and dominant negative mutant approaches suppressed the activation of STAT-3. 3) Jak-2-dependent STAT-3 activation is required for VSMC motility induced by PDGF-BB as dominant negative mutants of these signaling molecules prevented the VSMC motility in response to this agonist. 4) PDGF-BB-induced cPLA2 expression is sensitive to inhibition by dominant negative mutants of Jak-2 and STAT-3. 5) Pharmacological inhibition of cPLA2 blocked PDGFBB-induced VSMC motility. 6) Exogenous addition of arachidonic acid rescued PDGF-BB-induced VSMC motility from inhibition by blockade of Jak-2 and STAT-3 activation. 7) Forced expression of rat cPLA2 also overcame the inhibitory effect of dominant negative Jak-2 and STAT-3 on PDGF-BB-induced VSMC motility. In addition to its role in cell proliferation and differentiation (15Fukuda T. Ohtani T. Yoshida Y. Shirogane T. Nishida K. Nakajima K. Hibi M. Hirano T. EMBO J. 1998; 17: 6670-6677Crossref PubMed Scopus (215) Google Scholar, 16Nosaka T. Kawashima T. Misawa K. Ikuta K. Mui A.L. Kitamura T. EMBO J. 1999; 18: 4754-4765Crossref PubMed Scopus (438) Google Scholar, 17Wu Y.Y. Bradshaw R.A. J. Biol. Chem. 2000; 275: 2147-2156Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), emerging evidence indicates the involvement of the Jak/STAT pathway in the regulation of cell migration (55Silver D.L. Montell D.J. Cell. 2001; 107: 831-841Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 56Yahata Y. Shirakata Y. Tokumaru S. Yamasaki K. Sayama K. Hanakawa Y. Detmar M. Hashimoto K. J. Biol. Chem. 2003; 278: 40026-40031Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). 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Nature. 2002; 420: 629-635Crossref PubMed Scopus (3875) Google Scholar). Based on these observations it is presumable that Jak-2/STAT-3-mediated cPLA2 expression may be involved in a sustained arachidonic acid release producing eicosanoids, which in turn promote VSMC chemotaxis. A large body of evidence indicates that cPLA2 mediates the release of arachidonic acid and, thereby, eicosanoid production in response to a variety of bioactive agents (18Piomelli D. Curr. Opin. Cell Biol. 1993; 5: 274-280Crossref PubMed Scopus (256) Google Scholar, 19Dethlefsen S.M. Shepro D. D'Amore P.A. Exp. Cell Res. 1994; 212: 262-273Crossref PubMed Scopus (80) Google Scholar, 20Gronich J. Konieczkowski M. Gelb M.H. Nemenoff R.A. Sedor J.R. J. Clin. Investig. 1994; 93: 1224-1233Crossref PubMed Scopus (107) Google Scholar, 21Rao G.N. Lassegue B. Alexander R.W. Griendling K.K. Biochem. J. 1994; 299: 197-201Crossref PubMed Scopus (95) Google Scholar, 22Rao G.N. Runge M.S. Alexander R.W. Biochim. Biophys. 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Plasma membrane protrusion, retraction, and contraction forces that are essential for cell motility involve membrane phospholipid remodeling. In this regard, it is tempting to speculate that Jak-2/STAT-3-dependent cPLA2 expression may be involved in membrane phospholipid remodeling during cell motility. Future studies are required to test the role of the Jak/STAT/cPLA2 signaling axis in PDGF-BB-induced cytoskeleton and membrane phospholipid remodeling. In summary, the present study demonstrates for the first time that Jak-2/STAT-3 mediates PDGF-BB-induced motility in VSMCs via targeting the induction of expression of cPLA2. We thank Drs. James N. Ihle, Ralph A. Bradshaw, and Howard Young for providing Jak-2KEpRK5, pFS3DM, and pSIE-CAT plasmids, respectively. We also are grateful to Dr. Chandrahasa R. Yellaturu for help with the CAT assays and with the preparation of figures.

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