Fibroblast Growth Factor-2 Represses Platelet-derived Growth Factor Receptor-α (PDGFR-α) Transcription via ERK1/2-dependent Sp1 Phosphorylation and an Atypical cis-Acting Element in the Proximal PDGFR-α Promoter

Platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant for vascular smooth muscle cells (SMCs) whose biological activity is mediated via its high affinity interaction with specific cell surface receptors. The molecular mechanisms governing the expression of PDGF receptor-α (PDGFR-α) are poorly understood. Here we demonstrate that PDGFR-α protein and transcriptional regulation in SMCs is under the positive regulatory influence of the zinc finger nuclear protein, Sp1. Electrophoretic mobility shift, competition, and supershift analysis revealed the existence of an atypical G-rich Sp1-binding element located in the PDGFR-α promoter –61 to –52 bp upstream of the transcriptional start site. Mutation of this sequence ablated endogenous Sp1 binding and activation of the PDGFR-α promoter. PDGFR-α transcription, mRNA, and protein expression were repressed in SMCs exposed to fibroblast growth factor-2 (FGF-2). This inhibition was rescued by the blockade of extracellular signal-regulated kinase-1/2 (ERK1/2). FGF-2 repression of PDGFR-α transcription was abrogated upon mutation of this Sp1-response element. FGF-2 stimulated Sp1 phosphorylation in an ERK1/2- but not p38-dependent manner, the growth factor enhancing Sp1 interaction with the PDGFR-α promoter. Mutation of residues Thr453 and Thr739 in Sp1 (amino acids phosphorylated by ERK) blocked FGF-2 repression of PDGFR-α transcription. These findings, taken together, demonstrate that FGF-2 stimulates ERK1/2-dependent Sp1 phosphorylation, thereby repressing PDGFR-α transcription via the –61/–52 element in the PDGFR-α promoter. Phosphorylation triggered by FGF-2 switches Sp1 from an activator to a repressor of PDGFR-α transcription, a finding previously unreported in any Sp1-dependent gene. Platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant for vascular smooth muscle cells (SMCs) whose biological activity is mediated via its high affinity interaction with specific cell surface receptors. The molecular mechanisms governing the expression of PDGF receptor-α (PDGFR-α) are poorly understood. Here we demonstrate that PDGFR-α protein and transcriptional regulation in SMCs is under the positive regulatory influence of the zinc finger nuclear protein, Sp1. Electrophoretic mobility shift, competition, and supershift analysis revealed the existence of an atypical G-rich Sp1-binding element located in the PDGFR-α promoter –61 to –52 bp upstream of the transcriptional start site. Mutation of this sequence ablated endogenous Sp1 binding and activation of the PDGFR-α promoter. PDGFR-α transcription, mRNA, and protein expression were repressed in SMCs exposed to fibroblast growth factor-2 (FGF-2). This inhibition was rescued by the blockade of extracellular signal-regulated kinase-1/2 (ERK1/2). FGF-2 repression of PDGFR-α transcription was abrogated upon mutation of this Sp1-response element. FGF-2 stimulated Sp1 phosphorylation in an ERK1/2- but not p38-dependent manner, the growth factor enhancing Sp1 interaction with the PDGFR-α promoter. Mutation of residues Thr453 and Thr739 in Sp1 (amino acids phosphorylated by ERK) blocked FGF-2 repression of PDGFR-α transcription. These findings, taken together, demonstrate that FGF-2 stimulates ERK1/2-dependent Sp1 phosphorylation, thereby repressing PDGFR-α transcription via the –61/–52 element in the PDGFR-α promoter. Phosphorylation triggered by FGF-2 switches Sp1 from an activator to a repressor of PDGFR-α transcription, a finding previously unreported in any Sp1-dependent gene. Platelet-derived growth factor (PDGF) 1The abbreviations used are: PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; FGF-2, fibroblast growth factor-2; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; MEK, MAP kinase/ERK kinase; SMC, smooth muscle cell; C/EBP, CCAAT/enhancer-binding proteins; PBS, phosphate-buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; CMV, cytomegalovirus; Luc, luciferase. is a family of potent mitogenic and chemotactic proteins (1.Heldin C.H. Westermark B. Physiol. Reviews. 1999; 79: 1283-1300Crossref PubMed Scopus (1996) Google Scholar) whose biological activity is mediated via high affinity interactions with specific protein-tyrosine kinase cell surface receptors, PDGFR-α and -β. PDGFs are secreted as disulfide-linked bivalent ligands that induce receptor dimerization, trans-phosphorylation, and the activation of signal transduction cascades (2.van der Geer P. Hunter T. Annu. Rev. Cell Biol. 1994; 10: 251-337Crossref PubMed Scopus (1252) Google Scholar). PDGFR-α and -β have different ligand binding capabilities for their ligands. PDGFR-α binds PDGF-A, PDGF-B, and PDGF-C, whereas PDGFR-β binds PDGF-B and PDGF-D (3.Betsholtz C Karlsson L Lindahl P. BioEssays. 2001; 23: 494-507Crossref PubMed Scopus (297) Google Scholar). Assembly of the receptor dimer combinations (PDGFR-αα, -αβ, -ββ) is dependent on the isoform of the dimeric ligand (3.Betsholtz C Karlsson L Lindahl P. BioEssays. 2001; 23: 494-507Crossref PubMed Scopus (297) Google Scholar). Consequently, PDGF-AA causes αα receptor complex formation, PDGF-AB induces αα or αβ, and PDGF-BB stimulates αα, αβ, or ββ (4.Heldin C.H. EMBO J. 1992; 11: 4251-4259Crossref PubMed Scopus (318) Google Scholar, 5.Eriksson A. Siegbahn A. Westermark B. Heldin C.H. Claesson-Welsh L. EMBO J. 1992; 11: 543-550Crossref PubMed Scopus (225) Google Scholar, 6.Heidaran M.A. Beeler J.F. Yu C.H. Ishibashi T. LaRochelle W.J. Pierce J.H. Aaronson S.A. J. Biol. Chem. 1993; 268: 9287-9295Abstract Full Text PDF PubMed Google Scholar, 7.Wang C. Stiles C.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7061-7065Crossref PubMed Scopus (28) Google Scholar, 8.Vassbotn F.S. Havnen O.K. Heldin C.H. Holmsen H. J. Biol. Chem. 1994; 269: 13874-13879Abstract Full Text PDF PubMed Google Scholar). PDGF and fibroblast growth factor-2 (FGF-2) have long been recognized to play a regulatory role in vascular pathologies such as atherosclerosis and postangioplasty restenosis involving smooth muscle cell (SMC) growth. Both these growth factors and their receptors are expressed in SMCs. SMC replication and matrix deposition lead to lesion formation and thickening of the artery wall (9.Dzau V.J. Gibbons G.H. Morishita R. Pratt R.E. Hypertension. 1994; 23: 1132-1140Crossref PubMed Scopus (82) Google Scholar). Conversely, SMC apoptosis can weaken the atherosclerotic plaque and lead to its rupture (10.Bennett M.R. Boyle J.J. Atherosclerosis. 1998; 138: 3-9Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 11.Rossig L. Dimmeler S. Zeiher A.M. Basic Res. Cardiol. 2001; 96: 11-22Crossref PubMed Scopus (136) Google Scholar). Interestingly, as the plaque develops from early to late stage, FGF-2 levels decrease (12.Hughes S.E. Crossman D. Hall P.A. Cardiovasc. Res. 1993; 27: 1214-1219Crossref PubMed Scopus (99) Google Scholar), whereas PDGFR-α levels increase (13.Irvine C.D. George S.J. Sheffield E. Johnson J.L. Davies A.H. Lamont P.M. Cardiovasc. Res. 2000; 8: 121-129Google Scholar). Beyond this, however, an interrelationship between FGF-2 and PDGFR-α has not been established. The molecular mechanisms governing the transcription of PDGFR-α are poorly understood. The PDGFR-α gene lacks a typical TATA box but contains GATA motifs and a CCAAT box. It also contains potential sites for AP-1, AP-2, Oct-1, and Oct-2 transcription factors (14.Kawagishi J. Kumabe T. Yoshimoto T. Tamamoto T. Genomics. 1995; 30: 224-232Crossref PubMed Scopus (41) Google Scholar). CCAAT/enhancer-binding proteins C/EBP-β and C/EBP-δ bind to the PDGFR-α promoter and modulate its activity both positively and negatively (15.Fukuoka T. Kitami Y. Okura T. Hiwada K. J. Biol. Chem. 1999; 274: 25576-25582Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 16.Kitami Y. Fukuoka T. Hiwada K. Inagami T. Circ. Res. 1999; 84: 64-73Crossref PubMed Scopus (31) Google Scholar). For example, IL-1β can induce PDGFR-α mRNA expression via induction of C/EBP-δ (15.Fukuoka T. Kitami Y. Okura T. Hiwada K. J. Biol. Chem. 1999; 274: 25576-25582Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 16.Kitami Y. Fukuoka T. Hiwada K. Inagami T. Circ. Res. 1999; 84: 64-73Crossref PubMed Scopus (31) Google Scholar). PDGFR-α is under the positive transcriptional influence of NF-κB (17.Lindroos P.M. Rice A.B. Wang Y.Z. Bonner J.C. J. Immunol. 1998; 161: 3464-3468PubMed Google Scholar). The proto-oncogene product Cbl negatively regulates PDGFR-α by enhancing ubiquitination and degradation (18.Miyake S. Mullane-Robinson K.P. Lill N.L. Douillard P. Band H. J. Biol. Chem. 1999; 274: 16619-16628Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Deletion of PDGFR-α in mice produces severe cardiovascular abnormalities among many other defects, leading to embryonic death at day 8–16 (19.Li X. Ponten A. Aase K. Karlsson L. Abramsson A. Uutela M. Backstrom G. Bostrom H. Li H. Soriano P. Betsholtz C. Heldin C. Alitalo K. Ostman A. Eriksson U. Nat. Cell Biol. 2000; 2: 302-309Crossref PubMed Scopus (508) Google Scholar). Our previous studies have demonstrated that the zinc finger transcription factor Sp1 regulates transcription of the PDGF-A and PDGF-B genes in SMCs and in other vascular cell types (20.Khachigian L.M. Fries J.W. Benz M.W. Bonthron D.T. Collins T. J. Biol. Chem. 1994; 269: 22647-22656Abstract Full Text PDF PubMed Google Scholar, 21.Khachigian L.M. Lindner V. Williams A.J. Collins T. Science. 1996; 271: 1427-1431Crossref PubMed Scopus (478) Google Scholar, 22.Khachigian L.M. Williams A.J. Collins T. J. Biol. Chem. 1995; 270: 27679-27686Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). Here we show that PDGFR-α expression in SMCs is under the positive regulatory influence of Sp1, via an atypical recognition element located –61/–52 bp upstream of the transcriptional start site. This site mediates FGF-2 repression of PDGFR-α expression in an ERK1/2-dependent manner. Phosphorylation of Sp1 upon exposure to FGF-2 switches Sp1 from an activator to a repressor of PDGFR-α transcription. Cell Culture—WKY12-22 rat aortic SMCs were cultured in Waymouth's MB752/1 medium (Invitrogen) supplemented with 10% fetal calf serum, 30 μg/ml l-glutamine, 10 units/ml penicillin, and 10 μg/ml streptomycin at 37 °C and 5% CO2. The cells were passaged every 3–4 days in 75-cm2 flasks. Plasmid Constructs—The plasmid pLuc-a2 (23.Kitami Y. Inui H. Uno S. Inagami T. J. Clin. Invest. 1995; 96: 558-567Crossref PubMed Scopus (45) Google Scholar), containing the promoter region of platelet-derived growth factor receptor-α, was kindly donated by Dr Yutaka Kitami from Ehime University School of Medicine, Ehime, Japan. pLuc-a2.Sp1m3 and CMV-Sp1.mThr453/mThr739 were produced using the QuikChange® XL site-directed mutagenesis kit (Stratagene) in accordance with the manufacturer's instructions. Thr453 was converted to Ala453, and Thr739 was changed to Ala739. The primers are as follows: Sp1m3 forward, 5′-TTTATTTTGAAGAGACCATTTTTTTTTTCTTCATTTCCTGACAGCT-3′; Sp1m3 reverse, 5′ AGCTGTCAGGAAATGAAGAAAAAAAAAATGGTCTCTTCAAAATAAA-3′. CMV-Sp1.mThr453/mThr739 mutant was constructed by performing two mutagenesis reactions, each followed by sequencing, to obtain the double mutant. The primers are as follows: Sp1mThr453 forward, 5′-CCCATCATCATCCGGGCACCAACAGTGGGG-3′; Sp1mThr453 reverse, 5′-CCCCGCTGTTGGTGCCCGGATGATGATGGG-3′; Sp1mThr739 forward, 5′-GGCAGTGGCACTGCCGCTCCTTCAFCCCTTATT-3′; Sp1mThr739 reverse, 5′-AATAAGGGCTGAAGGAGCGGCAGTGGCACTGCC-3′. Transient Transfections—For reporter gene analysis, WKY12-22 cells were transfected with 10 μg of pLuc-a2, pLuc-a2.Sp1m3, or CMV-Sp1.mThr453/mThr739. Cells were also transfected with 0.5 μg of pRL-TK to correct for transfection efficiency. Firefly luciferase activity was normalized to Renilla. Transient transfections were performed using FuGENE6 (Roche Applied Science) where 3 μl of FuGENE6/μg of transfected DNA was added, and the transfection mix was made up to 1 ml with serum-free medium. After incubation at 22 °C for 10 min, the DNA/FuGENE6 mixture was added to cells containing 10 ml of complete medium. Twenty-four h after transfection, cell lysates were prepared for assessment of luciferase activity where the dual luciferase assay reporter system (Promega DLR™) was performed on a manual luminometer (model TD-20/20 Turner Designs, Quantum Science). mRNA Expression Analysis—Cells were washed two times with ice-cold PBS and harvested with 4.5 ml of TRIzol reagent (Invitrogen) in accordance with the manufacturer's instructions. cDNA synthesis from total RNA was performed using Superscript II reverse transcriptase (Promega, WI) in accordance with the manufacturer's instructions. Thermal cycling conditions as follows: PDGFR-α 94 °C for 10 s, 62 °C for 30 s, 72 °C for 1.5 min for 40 cycles; GAPDH 94 °C for 30 s, 60 °C for 30 s, 68 °C for 2 min for 18, 24, and 28 cycles. The PDGFR-α primers are: 5′-AGATAGCTTCATGAGCCGAC-3′ (forward) and 5′-GGAACAGGGTCAATGTCTGG-3′ (reverse); the GAPDH primers are: 5′-ACCACAGTCCATGCCATCAC-3′ (forward) and 5′-TCCACCACCCTGTTGCTGTA-3′ (reverse). Western Immunoblot Analysis—Transfected cells were washed two times with ice-cold PBS before being lysed on ice in 1× radioimmune precipitation assay buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS) containing protease inhibitors (2 mm phenylmethylsulfonyl fluoride (PMSF), 5 mm EDTA, 10 μg/ml leupeptin, and 1% aprotinin). Cell lysates were collected after centrifugation at 14,000 rpm for 20 min at 4 °C, and the protein concentration was determined by BCA protein assay (Pierce). Lysates containing 10 μg of protein were prepared in SDS sample buffer (50 mm Tris, pH 6.8, 10% glycerol, 2% SDS, 0.01% bromphenol blue, and 30 mm dithiothreitol (DTT)) and boiled for 5 min with 0.5 m iodoacetamide added before loading onto an 8% SDS-polyacrylamide gel. Gels were blotted onto Immobilon-P (polyvinylidene difluoride) membranes (Millipore, Bedford, MA) and blocked overnight at 4 °C in 5% skim milk powder, 0.05% Tween 20, and PBS. Sp1 was detected using rabbit polyclonal antibodies (Santa Cruz Biotechnology) and chemiluminescence detection (PerkinElmer Life Sciences) according to the manufacturer's instructions. Preparation of Nuclear Extracts—SMC monolayers were washed two times with ice-cold PBS, pH 7.4, and then scraped into 10 ml of cold PBS. The cells were pelleted by centrifugation at 250 × g for 10 min at 4 °C. Cells were lysed by the addition of ice-cold hypotonic solution (Buffer A) consisting of 10 mm HEPES, pH 8.0, 1.5 mm MgCl2, 10 mm KCl, 0.5 mm DTT, 200 mm sucrose, 0.5% Nonidet P-40, 0.5 mm PMSF, 1 μg/ml aprotinin. The suspension was recentrifuged, and the nuclei were lysed in an ice-cold solution (Buffer C) consisting of 20 mm HEPES, pH 8, 100 mm KCl, 0.2 mm EDTA, glycerol, 1 mm DTT, 0.5 mm PMSF, 1 μg/ml leupeptin, 1 μg/ml aprotinin. The nuclear was with an of mm HEPES, pH 8.0, 100 mm KCl, 0.2 mm EDTA, glycerol, 1 mm DTT, 0.5 mm PMSF, 1 μg/ml leupeptin, 1 μg/ml aprotinin). Electrophoretic for were performed in 20 μl of 10 mm Tris-HCl, mm MgCl2, 1 mm EDTA, 1 mm DTT, 5% glycerol, 1 mm PMSF, 1 μg of DNA and 3 μg of nuclear by Pierce protein The was for min at 22 °C. supershift 2 μl of the polyclonal (Santa Cruz Biotechnology) was with the binding mix for 10 min before the addition of the were from by loading onto a and at for The were at °C and to overnight at °C. Sp1 PDGFR-α and previous demonstrated a positive regulatory role for Sp1 in the transcriptional regulation of PDGF L.M. Williams A.J. Collins T. J. Biol. Chem. 1995; 270: 27679-27686Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar) and (20.Khachigian L.M. Fries J.W. Benz M.W. Bonthron D.T. Collins T. J. Biol. Chem. 1994; 269: 22647-22656Abstract Full Text PDF PubMed Google Scholar, 21.Khachigian L.M. Lindner V. Williams A.J. Collins T. Science. 1996; 271: 1427-1431Crossref PubMed Scopus (478) Google Scholar) via in the promoter of these the PDGF-A and however, for Sp1 not in the PDGFR-α promoter. Sp1 PDGFR-α expression has not been in any cell levels of PDGFR-α protein in vascular SMCs h after transfection with the Sp1 expression Western analysis revealed that Sp1 produced a of molecular was in cells transfected with the these at the of transcription, we SMCs with and pLuc-a2, a Firefly reporter by of PDGFR-α promoter (23.Kitami Y. Inui H. Uno S. Inagami T. J. Clin. Invest. 1995; 96: 558-567Crossref PubMed Scopus (45) Google Scholar). The cells were also transfected with the to correct for transfection efficiency. luciferase activity h after transfection revealed induction of PDGFR-α transcription by Sp1 Sp1-binding in the as a Sp1-response region of the PDGFR-α promoter not a Sp1-binding a 10 was at to the transcriptional start site this site an interaction with Sp1, we performed an mobility assay using SMC nuclear and a whose sequence bp in the PDGFR-α promoter. This produced a of whose was demonstrated by their in the of a of The of an containing the site to the of these mutation of the to in the abrogated complex formation PDGFR-α of with antibodies to Sp1 and and the of and whereas antibodies to endogenous Sp1 protein with this atypical Sp1 element in the PDGFR-α promoter in a Sp1-binding element in the PDGFR-α promoter with endogenous Sp1. This an mobility assay using the region PDGFR-α of the PDGFR-α promoter or the mutant and SMC nuclear analysis with Sp1 antibodies and and of and whereas antibodies have the of this atypical Sp1-binding element in the of of the PDGFR-α the mutation was into pLuc-a2 analysis with or in SMCs with mutant pLuc-a2.Sp1m3 abrogated Sp1 induction of the PDGFR-α promoter These demonstrate the existence of a Sp1-response element in the region of the PDGFR-α promoter. FGF-2 PDGFR-α mRNA, and levels decrease in the as levels of FGF-2 increase (12.Hughes S.E. Crossman D. Hall P.A. Cardiovasc. Res. 1993; 27: 1214-1219Crossref PubMed Scopus (99) Google Scholar, C.D. George S.J. Sheffield E. Johnson J.L. Davies A.H. Lamont P.M. Cardiovasc. Res. 2000; 8: 121-129Google Scholar). that FGF-2 negatively influence PDGFR-α a growth factor can the expression of the receptor of has not been demonstrated activity in SMCs transfected with pLuc-a2 was by FGF-2 in a h analysis these FGF-2 repressed endogenous PDGFR-α mRNA expression h after exposure of the cells to the growth factor GAPDH levels demonstrated sample loading Western analysis revealed FGF-2 of PDGFR-α protein after and h of incubation with FGF-2 These that FGF-2 PDGFR-α gene expression at the of mRNA and of PDGFR-α is in the FGF-2 of PDGFR-α is rescued with Western analysis using total cell of SMCs exposed to FGF-2 for or h is Cells were with or for 1 h to the addition of in the the the PDGFR-α promoter from repression by activity was h of Firefly luciferase activity was normalized to Renilla. S.E. The are of two or FGF-2 of PDGFR-α Expression on but MAP and have demonstrated that a used by FGF-2 in SMCs is the extracellular signal-regulated kinase-1/2 G. D. C. C. P. J. PubMed Scopus Google Scholar, M. A. H. K. Res. Google Scholar, J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, F.S. L.M. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). that FGF-2 repression of PDGFR-α the Western analysis revealed that FGF-2 repression of PDGFR-α protein expression was rescued by the at and h the kinase to modulate FGF-2 of PDGFR-α expression of FGF-2 inhibition by the but not the was also demonstrated at the of transcription in transfection analysis using pLuc-a2 FGF-2 of PDGFR-α via Sp1 studies have that activation of can and the interaction of Sp1 with the of genes J. PubMed Scopus Google Scholar). For example, of Sp1 its interaction with the gene promoter and growth gene expression J.L. M. A. Res. 1999; PubMed Scopus Google Scholar). Sp1 phosphorylation mediates repression of gene expression is not FGF-2 F.S. L.M. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) and FGF-2 repression of the PDGFR-α promoter we that FGF-2 inhibition of PDGFR-α is mediated via of Sp1. to this we exposed SMCs transfected with pLuc-a2.Sp1m3 to FGF-2 and luciferase activity after h FGF-2 of PDGFR-α promoter activity was abrogated by the mutation in the Sp1-binding site This Sp1-response element mediates FGF-2 repression of the PDGFR-α promoter. the of FGF-2 at the of Sp1 FGF-2 Sp1 Phosphorylation in an ERK1/2-dependent Sp1 with the PDGFR-α of Sp1 its molecular the and can by Y. L.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L.M. Res. 2001; PubMed Google Scholar). Western analysis for Sp1 using nuclear from SMCs with FGF-2 for and h demonstrated levels of Sp1 phosphorylation of from cells with calf the of this transcription whereas incubation with These for the that FGF-2 stimulates Sp1 demonstrate FGF-2 stimulates Sp1 phosphorylation via SMCs were with to the addition of analysis after Western for Sp1 revealed that FGF-2 phosphorylation of Sp1 was blocked by the the influence on the of FGF-2 to Sp1 Electrophoretic mobility assay with the PDGFR-α revealed the formation of an complex h of incubation with FGF-2 analysis with Sp1 antibodies the and whereas antibodies to was that two specific residues and in Sp1 J. J. G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). these acids in Sp1 to demonstrate FGF-2 of PDGFR-α is dependent upon these Transient transfection analysis in SMCs transfected with the double mutant exposed to FGF-2 for h revealed that FGF-2 not PDGFR-α transcription These findings, taken together, demonstrate that FGF-2 stimulates ERK1/2-dependent Sp1 phosphorylation, thereby repressing PDGFR-α transcription via the –61/–52 element in the PDGFR-α promoter. The of Thr453 and Thr739 in Sp1 is to FGF-2 inhibition of PDGFR-α transcription. ERK1/2-dependent phosphorylation triggered by FGF-2 switches Sp1 from an activator to a repressor of PDGFR-α transcription. The transcriptional mechanisms PDGFR-α expression are poorly understood. this we demonstrate that PDGFR-α transcription and protein expression are by Sp1, binds to an atypical G-rich binding element located at –61/–52 bp in the PDGFR-α promoter. Interestingly, this element also mediates FGF-2 inhibition of PDGFR-α is by mutation of the element or blockade of but not Mutation of residues Thr453 and Thr739 in Sp1 that are phosphorylated by FGF-2 repression of PDGFR-α transcription. Sp1 Sp1 phosphorylation by FGF-2 its interaction with the PDGFR-α its activity via this element. phosphorylation switches Sp1 from an activator to a repressor of PDGFR-α transcription. This is the of Sp1 phosphorylation by It also the of a growth factor to the expression of the receptor of as has been for the growth receptor with PDGF C. and R. Cell Res. Scholar) and FGF-2 C. H. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). It is to that the between the expression of FGF-2 and PDGFR-α in the complex of a mediated by the phosphorylation of Sp1 and this recognition element in the PDGFR-α promoter.