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Induction of Platelet-derived Growth Factor B-chain Expression by Transforming Growth Factor-β Involves Transactivation by Smads

2000; Elsevier BV; Volume: 275; Issue: 22 Linguagem: Inglês

10.1074/jbc.275.22.16709

1083-351X

Lisa Taylor, Levon M. Khachigian,

Platelet Disorders and Treatments

Transforming growth factor-β (TGF-β) regulates a diverse array of biological processes, such as proliferation, differentiation, extracellular matrix production, and apoptosis. In cultured vascular endothelial cells, TGF-β induces the expression of platelet-derived growth factor (PDGF) B-chain, a mitogen and chemoattractant, at the level of transcription. The molecular mechanism(s) underlying this process are not presently understood. In this study, we performed serial 5′ deletion and transient transfection analysis to define a region in the PDGF-B promoter mediating inducible responsiveness to TGF-β. This region contains an atypical nucleotide recognition element for the Smad family of transcriptional regulators. Electrophoretic mobility shift analysis revealed that nuclear proteins bound to this site in a transient and specific manner. Supershift studies demonstrated the physical association of Smad4 with the promoter. Overexpression of Smad4 activated the PDGF-B promoter and superinduced PDGF-B promoter-dependent expression in cells exposed to TGF-β. Moreover, simultaneous cotransfection of Smad3 and Smad4 activated the PDGF-B promoter. This effect was attenuated when Smad4 was substituted with its dominant negative counterpart. Mutation of the −81CAGA−78 motif in the PDGF-B promoter abrogated Smad-inducible promoter-dependent expression. Overexpression of Smad2 and Smad3 transactivated the PDGF-B promoter in a synergistic manner. These findings demonstrate the existence of a novel, functional binding element in the proximal region of the PDGF-B promoter mediating responsiveness to TGF-β. Transforming growth factor-β (TGF-β) regulates a diverse array of biological processes, such as proliferation, differentiation, extracellular matrix production, and apoptosis. In cultured vascular endothelial cells, TGF-β induces the expression of platelet-derived growth factor (PDGF) B-chain, a mitogen and chemoattractant, at the level of transcription. The molecular mechanism(s) underlying this process are not presently understood. In this study, we performed serial 5′ deletion and transient transfection analysis to define a region in the PDGF-B promoter mediating inducible responsiveness to TGF-β. This region contains an atypical nucleotide recognition element for the Smad family of transcriptional regulators. Electrophoretic mobility shift analysis revealed that nuclear proteins bound to this site in a transient and specific manner. Supershift studies demonstrated the physical association of Smad4 with the promoter. Overexpression of Smad4 activated the PDGF-B promoter and superinduced PDGF-B promoter-dependent expression in cells exposed to TGF-β. Moreover, simultaneous cotransfection of Smad3 and Smad4 activated the PDGF-B promoter. This effect was attenuated when Smad4 was substituted with its dominant negative counterpart. Mutation of the −81CAGA−78 motif in the PDGF-B promoter abrogated Smad-inducible promoter-dependent expression. Overexpression of Smad2 and Smad3 transactivated the PDGF-B promoter in a synergistic manner. These findings demonstrate the existence of a novel, functional binding element in the proximal region of the PDGF-B promoter mediating responsiveness to TGF-β. Transforming growth factor-β (TGF-β) 1The abbreviations used are: TGF-β, transforming growth factor-β; Egr-1, early growth response factor-1; EMSA, electrophoretic mobility shift analysis; PDGF, platelet-derived growth factor; PDGF-B, platelet-derived growth factor B-chain; PBS, phosphate-buffered saline; DTT, dithiothreitol; CMV, cytomegalovirus. 1The abbreviations used are: TGF-β, transforming growth factor-β; Egr-1, early growth response factor-1; EMSA, electrophoretic mobility shift analysis; PDGF, platelet-derived growth factor; PDGF-B, platelet-derived growth factor B-chain; PBS, phosphate-buffered saline; DTT, dithiothreitol; CMV, cytomegalovirus. regulates a wide variety of biological processes, including proliferation, differentiation, extracellular matrix production, and apoptosis (1.Heldin C.-H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3326) Google Scholar). These diverse biological effects are mediated by the activity of cell surface, heterotrimeric serine/threonine kinase receptors, type I and type II. Upon ligand binding, the type II receptor phosphorylates the type I receptor activating a specific set of recently discovered positive and negative regulatory transcription factors known as Smads. The Smad family, mammalian homologues of the Drosophila geneMad (2.Kretzschmar M. Massague J. Curr. Opin. Gen. Dev. 1998; 8: 103-111Crossref PubMed Scopus (429) Google Scholar, 3.Nakao A. Imamura T. Souchelnytskyi S. Kawabata M. Ishisaki A. Oeda E. Tamaki K. Hanai J. Heldin C.-H. Miyazono K. ten Dijke P. EMBO J. 1997; 16: 5353-5362Crossref PubMed Scopus (905) Google Scholar), translocate to the nucleus after activation and modulate gene expression. Smad1, Smad2, Smad3, Smad5, and Smad8, the receptor-associated Smads, are direct substrates of the type I receptor. Smad4, a common mediator Smad, is not directly phosphorylated by the receptor. Rather, it physically associates with other Smads in the cytoplasm prior to nuclear translocation as a complex. Smad6 and Smad7 are "anti-Smads" that have been found to inhibit TGF-β-induced Smad-dependent activation. A role for Smad family members in TGF-β signal transduction has been defined in a large number of promoters, including the human plasminogen activator inhibitor-1 (3.Nakao A. Imamura T. Souchelnytskyi S. Kawabata M. Ishisaki A. Oeda E. Tamaki K. Hanai J. Heldin C.-H. Miyazono K. ten Dijke P. EMBO J. 1997; 16: 5353-5362Crossref PubMed Scopus (905) Google Scholar, 4.Song C.-Z. Siok T.E. Gelehrter T.D. J. Biol. Chem. 1998; 273: 29287-29290Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 5.Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.-M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1578) Google Scholar), JunB (6.Jonk L.J.C. Itoh S. Heldin C.-H. ten Dijke P. Kruijer W. J. Biol. Chem. 1998; 273: 21145-21152Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar), and p21/WAF1/Cip1 (7.Moustakas A. Kardassis D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6733-6738Crossref PubMed Scopus (321) Google Scholar), by interactions directly with DNA and/or other transcriptional regulators. Platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant for cells of mesenchymal origin. It consists of two chains, A and B, held together in homodimeric or heterodimeric configuration by disulfide bonds. The three isoforms of PDGF (AA, BB, and AB) modulate biological responsiveness by interacting specifically with two (α and β) cell surface tyrosine kinase receptors; α binds the A-chain and the B-chain, whereas β binds only to the B-chain. The PDGF ligand receptor system has strongly been implicated to play roles in a large number of normal and pathologic settings, such as embryonic development, wound healing embryo, tumorigenesis, and atherosclerosis. For example, PDGF-A and PDGF-B mRNA and protein are present in human atherosclerotic tissue (8.Barrett T.B. Benditt E.P. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1099-1103Crossref PubMed Scopus (135) Google Scholar, 9.Ross R. Masuda J. Raines E.W. Gown A.M. Katsuda S. Sasahara M. Malden L.T. Masuko H. Sato H. Science. 1990; 248: 1009-1012Crossref PubMed Scopus (471) Google Scholar, 10.Rekhter M. Gordon D. Circ. Res. 1994; 75: 410-417Crossref PubMed Scopus (53) Google Scholar) and in human coronary arteries after percutaneous transluminal coronary angioplasty (11.Ueda M. Becker A.E. Kasayuki N. Kojima A. Morita Y. Tanaka S. Am. J. Pathol. 1996; 149: 831-843PubMed Google Scholar). In rat arteries, PDGF-A, PDGF-B, and the α-receptor are activated in endothelium following mechanical injury to the vessel wall (12.Lindner V. Reidy M.A. Am. J. Pathol. 1995; 146: 1488-1497PubMed Google Scholar). Infusion of recombinant PDGF-BB stimulates migration and intimal thickening (13.Jawien A. Bowen-Pope D.F. Lindner V. Schwartz S.M. Clowes A.W. J. Clin. Invest. 1992; 89: 507-511Crossref PubMed Scopus (590) Google Scholar). Moreover, PDGF-B gene transfer into pig arteries stimulates intimal hyperplasia (14.Nabel E.G. Yang Z. Liptay S. San H. Gordon D. Haudenschild C.C. Nabel G.J. J. Clin. Invest. 1993; 91: 1822-1829Crossref PubMed Scopus (215) Google Scholar, 15.Pompili V.J. Gordon D. San H. Yang Z. Muller D.W.M. Nabel G.J. Nabel E.G. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 2254-2264Crossref PubMed Scopus (40) Google Scholar). The PDGF B-chain gene (or c-sis proto-oncogene) resides on chromosome 22 (22q12.3-q13.1) (16.Swan D.C. McBride O.W. Robbins K.C. Keithley D.A. Reddy E.P. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 4691-4695Crossref PubMed Scopus (126) Google Scholar, 17.Bartram C.R. De Klein A. Hagemeijer A. Grosveld G. Heisterkamp N. Groffen J. Blood. 1984; 63: 223-225Crossref PubMed Google Scholar) and spans approximately 24 kilobases of genomic DNA. The PDGF-B promoter is transcriptionally activated in a wide variety of normal and transformed cell types, including vascular endothelial cells (18.Resnick N. Collins T. Atkinson W. Bonthron D.T. Dewey Jr., C.F. Gimbrone Jr., M.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4591-4595Crossref PubMed Scopus (448) Google Scholar, 19.Khachigian L.M. Fries J.W.U. Benz M.W. Bonthron D.T. Collins T. J. Biol. Chem. 1994; 269: 22647-22656Abstract Full Text PDF PubMed Google Scholar, 20.Khachigian L.M. Resnick N. Gimbrone Jr., M.A. Collins T. J. Clin. Invest. 1995; 96: 1169-1175Crossref PubMed Scopus (294) Google Scholar, 21.Khachigian L.M. Lindner V. Williams A.J. Collins T. Science. 1996; 271: 1427-1431Crossref PubMed Scopus (476) Google Scholar), megakaryocytes (22.Pech M. Rao C.D. Robbins K.C. Aaronson S.A. Mol. Cell. Biol. 1989; 9: 396-405Crossref PubMed Scopus (50) Google Scholar, 23.Jin H.-M. Brady M.L. Fahl W.E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7563-7567Crossref PubMed Scopus (29) Google Scholar), fibroblasts (22.Pech M. Rao C.D. Robbins K.C. Aaronson S.A. Mol. Cell. Biol. 1989; 9: 396-405Crossref PubMed Scopus (50) Google Scholar), Jurkat T-cells (24.Trejo S.R. Fahl W.E. Ratner L. J. Biol. Chem. 1996; 271: 14584-14590Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), glioblastoma cells (22.Pech M. Rao C.D. Robbins K.C. Aaronson S.A. Mol. Cell. Biol. 1989; 9: 396-405Crossref PubMed Scopus (50) Google Scholar), fibrosarcoma cells (22.Pech M. Rao C.D. Robbins K.C. Aaronson S.A. Mol. Cell. Biol. 1989; 9: 396-405Crossref PubMed Scopus (50) Google Scholar), and osteosarcoma cells (25.Jin H.-M. Robinson D.F. Liang Y. Fahl W.E. J. Biol. Chem. 1994; 269: 28648-28654Abstract Full Text PDF PubMed Google Scholar, 26.Liang Y. Robinson D.F. Dennig J. Suske G. Fahl W.E. J. Biol. Chem. 1996; 271: 11792-11797Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Since the structure of the PDGF-B transcriptional unit was first defined (27.Rao C.D. Igarashi H. Chiu I.-M. Robbins K.C. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2392-2396Crossref PubMed Scopus (79) Google Scholar), a number of transcription factors with the ability to modulate PDGF-B promoter-dependent expression have been identified. Sp1 (19.Khachigian L.M. Fries J.W.U. Benz M.W. Bonthron D.T. Collins T. J. Biol. Chem. 1994; 269: 22647-22656Abstract Full Text PDF PubMed Google Scholar, 24.Trejo S.R. Fahl W.E. Ratner L. J. Biol. Chem. 1996; 271: 14584-14590Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 26.Liang Y. Robinson D.F. Dennig J. Suske G. Fahl W.E. J. Biol. Chem. 1996; 271: 11792-11797Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), Sp3 (24.Trejo S.R. Fahl W.E. Ratner L. J. Biol. Chem. 1996; 271: 14584-14590Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 26.Liang Y. Robinson D.F. Dennig J. Suske G. Fahl W.E. J. Biol. Chem. 1996; 271: 11792-11797Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), and early growth response factor-1 (Egr-1) (21.Khachigian L.M. Lindner V. Williams A.J. Collins T. Science. 1996; 271: 1427-1431Crossref PubMed Scopus (476) Google Scholar) bind to overlapping nucleotide recognition elements in the PDGF-B promoter near the TATA box. In vivo footprinting demonstrates that this region is occupied in genomic DNA (28.Dirks R.P.H. Jansen H.J. van Gerven B. Onnekink C. Bloemers H.P.J. Nucleic Acids Res. 1995; 23: 1119-1126Crossref PubMed Scopus (17) Google Scholar). Nuclear factor-κB (20.Khachigian L.M. Resnick N. Gimbrone Jr., M.A. Collins T. J. Clin. Invest. 1995; 96: 1169-1175Crossref PubMed Scopus (294) Google Scholar) interacts with the shear stress response element (18.Resnick N. Collins T. Atkinson W. Bonthron D.T. Dewey Jr., C.F. Gimbrone Jr., M.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4591-4595Crossref PubMed Scopus (448) Google Scholar) located upstream. The PDGF-B promoter is also negatively regulated by the zinc finger protein, ZNF174, which interacts with the transcriptional start site (29.Williams A.J. Khachigian L.M. Shows T. Collins T. J. Biol. Chem. 1995; 270: 22143-22152Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Steady-state PDGF-B mRNA expression is increased in microvascular endothelial cells exposed to TGF-β (30.Daniel T.O. Gibbs V.C. Milfay D.F. Williams L.T. J. Biol. Chem. 1987; 262: 11893-11896Abstract Full Text PDF PubMed Google Scholar). Nuclear run-on studies determined that this is regulated at the level of transcription (31.Kavanaugh W.M. Harsh G.R., IV Starksen N.F. Rocco C.M. Williams L.T. J. Biol. Chem. 1988; 263: 8470-8472Abstract Full Text PDF PubMed Google Scholar). The molecular mechanism(s) mediating the induction of PDGF-B by TGF-β have not yet been elucidated. Because Smads play a regulatory role in TGF-β signaling, in this paper we investigated whether this family of transcriptional regulators control TGF-β induction of PDGF-B. Here, we used deletion analysis to define a region in the proximal PDGF-B promoter mediating responsiveness to TGF-β. Nuclear proteins from endothelial cells exposed to TGF-β bind to this region in an inducible and specific manner. Transfection studies demonstrate that the PDGF-B promoter is activated by a number of Smad factors, individually and synergistically. Dominant negative mutants of Smad4 blocked Smad and TGF-β induction of the PDGF-B promoter. These findings demonstrate the involvement of Smads in the regulation of PDGF-B expression by TGF-β. Bovine aortic endothelial cells were use in experiments between passages 4 and 8. The cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum supplemented with 50 μg/ml streptomycin and 50 IU/ml penicillin, at 37 °C in a 5% CO2 incubator. At 50–60% confluency, the cells were transfected with the indicated amount of plasmid DNA and 2 μg of theRenilla luciferase reporter plasmid, pRL-TK (Promega) using the modified calcium phosphate precipitation procedure (32.Khachigian L.M. Williams A.J. Collins T. J. Biol. Chem. 1995; 270: 27679-27686Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). After incubation overnight at 37 °C at 3% CO2, the cells were washed twice with PBS, pH 7.4, and incubated with 0.5% fetal bovine serum/Dulbecco's modified Eagle's medium for 24 h at 37 °C and 5% CO2 before exposure to 10 ng/ml of TGF-β (Promega). The cells were incubated for a further 24 h before harvesting by washing twice with PBS, pH 7.4, and scraping in 1 ml of passive lysis buffer (Promega). The suspension was freeze/thawed and spun, and the supernatant used to determine luciferase activity using the Dual Luciferase Kit (Promega) according to the manufacturer's directions. The Firefly luciferase activity was determined using a TD-20/20 Luminometer (Turner Designs) and normalized againstRenilla activity. Nuclear extracts were prepared from 100-mm dishes. Growth-arrested endothelial cells were incubated with 10 ng/ml of TGF-β for the times indicated and harvested. Cells were washed twice with PBS, pH 7.4, and centrifuged at 1300 rpm for 15 min at 4 °C. The cell pellet was resuspended in 100 μl of ice-cold Solution A (10 mm Hepes, pH 7.9, 1.5 mm MgCl2, 10 mm KCl, 0.5% Nonidet P-40, 1 mm DTT, 0.5 mm phenylmethylsulfonyl fluoride, 4 μg/ml aprotinin, and 10 μg/ml leupeptin) and incubated on ice for 5 min. The suspension was microfuged at 13,000 rpm, and the cytosolic fraction was removed. The nuclei were resuspended and lysed in 20 μl of ice-cold Solution C (20 mm Hepes, pH 7.9, 1.5 mm MgCl2, 420 mm NaCl, 0.2 mm EDTA, 1 mm DTT, 0.5 mmphenylmethylsulfonyl fluoride, 4 μg/ml aprotinin, and 10 μg/ml leupeptin) by gentle shaking for 20 min at 4 °C. Cell debris was removed by centrifugation at 13,000 rpm for 1 min, and the nuclear extract was combined 1:1 with ice-cold Solution D (20 mmHepes, pH 7.9, 100 mm KCl, 0.2 mm EDTA, 20% glycerol, 1 mm DTT, 0.5 mm phenylmethylsulfonyl fluoride, 4 μg/ml aprotinin, and 10 μg/ml leupeptin). Nuclear extracts were snap frozen on dry ice and stored at −80 °C until use. Binding conditions were essentially as described (33.Vindevoghel L. Kon A. Lechleider R.J. Uitto J. Roberts A. Mauvel A. J. Biol. Chem. 1998; 273: 13053-13057Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). In general, 4–6 μg of nuclear extract and 150,000 cpm of 32P-labeled double-stranded oligonucleotide were incubated in 20 μl containing 10 mmHepes-KOH, pH 7.9, 40 mm KCl, 0.4 mm DTT, 0.4 mm EDTA, 4% glycerol, and 1 μg of poly(dI-dC) for 30 min on ice. In supershift studies, 4 μg of affinity-purified antibody (Santa Cruz Biotechnology) was incubated with the binding mixture at 4 °C overnight prior to the addition of probe. Bound complexes were separated from free probe by nondenaturing (5%) polyacrylamide gel electrophoresis in 0.5× TBE running buffer at 200 V (constant voltage). The gels were vacuum dried and exposed to Hyperfilm-MP (Amersham Pharmacia Biotech) overnight at −80 °C. Transfected cells were washed twice with ice-cold PBS, pH 7.4, before being lysed on ice in 1× RIPA 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, 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 DTT) and boiled for 3 min with 0.5 m iodoacetamide added before loading on an 8% SDS-polyacrylamide gel with a 4% stacking gel. Gels were blotted onto Immobilon-P (polyvinylidine difluoride) membranes (Millipore, Bedford, MA) for 1 h at 100 V, dried, and blocked overnight at 4 °C in 5% skim milk powder, 0.05% Tween 20 and PBS, pH 7.4. Three 15-min washes with 0.05% Tween-PBS were made prior to 1 h of incubation with the primary antibody (1:1000 in 1% bovine serum albumin, 0.05% Tween-PBS) at 22 °C. The membrane blots were washed again before 1 h of incubation at 22 °C with rabbit immunoglobulin secondary antibody (1:1000 in 1% bovine serum albumin, 0.05% Tween-PBS) conjugated with horseradish peroxidase (DAKO). The blots were washed again before chemiluminescence detection according to the manufacturer's protocol (NEN Life Science Products). Previous investigations have determined that TGF-β can induce PDGF-B expression in vascular endothelial cells at the level of transcription (31.Kavanaugh W.M. Harsh G.R., IV Starksen N.F. Rocco C.M. Williams L.T. J. Biol. Chem. 1988; 263: 8470-8472Abstract Full Text PDF PubMed Google Scholar, 34.Starksen N.F. Harsh G.R. Gibbs V.C. Williams L.T. J. Biol. Chem. 1987; 262: 14381-14384Abstract Full Text PDF PubMed Google Scholar). To understand the molecular basis for this effect, we performed transient transfection analysis using a nested series of Firefly luciferase-based reporter constructs bearing various sized fragments of the PDGF-B promoter. The endothelial cells were cotransfected with a Renilla luciferase-based expression vector to correct for transfection efficiency. PDGF-B promoter-dependent reporter activity increased in cells transfected with constructs RA-luc, NA-luc, and SDA-luc (26.Liang Y. Robinson D.F. Dennig J. Suske G. Fahl W.E. J. Biol. Chem. 1996; 271: 11792-11797Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), whose 5′ end points extend 398, 251, and 103 base pairs upstream of the transcriptional start site (27.Rao C.D. Igarashi H. Chiu I.-M. Robbins K.C. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2392-2396Crossref PubMed Scopus (79) Google Scholar) (Fig.1 A). However, TGF-β-inducible reporter activity was no longer observed in transfectants harboring construct SPA-luc, whose expression is driven by 64 base pairs of the PDGF-B promoter (Fig. 1 A) and retains binding sites for Sp1, Sp3, and Egr-1 (19.Khachigian L.M. Fries J.W.U. 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 (476) Google Scholar, 26.Liang Y. Robinson D.F. Dennig J. Suske G. Fahl W.E. J. Biol. Chem. 1996; 271: 11792-11797Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). These findings suggested that these transcription factors may not play a direct regulatory role in TGF-β-inducible PDGF-B promoter-dependent expression. Rather, TGF-β-activation is mediated by the region located between nucleotides −103 and −64 in the PDGF-B promoter, which, interestingly, bears a putative atypical nucleotide recognition element for the Smad family of transcription factors, whose core motif is 5′-CAGA-3′ (5.Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.-M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1578) Google Scholar) (TableI). TGF-β activation of the PDGF-B promoter-reporter construct SDA-luc (Fig. 1 A), which contains a single CAGA motif (−81/−78), compares favorably with TGF-β activation of construct (CAGA)6-TK-luc (Fig.1 B), which contains six copies of the AGCCAGACA element located at position −730 in the plasminogen activator inhibitor-1 promoter (5.Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.-M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1578) Google Scholar).Table I5′-CAGA-3′ motifs in various promoters activated by TGF-βGeneSpeciesSequencePAI-1 (5)HumanAGCCAGACAAGACAGACAAGACAGACAJunB (6)MurineTCTCAGACAα2(I)Collagen (54)HumanATGCAGACAPDGF-BHuman−84GGCCAGAAG−76 Open table in a new tab To explore the mechanisms underlying PDGF-B promoter activation by TGF-β, we performed electrophoretic mobility shift analysis (EMSA) with 32P-Oligo-93/-63, a double-stranded oligonucleotide spanning this putative Smad-binding site in the PDGF-B promoter. Using nuclear extracts of growth-quiescent cells, several distinct nucleoprotein complexes were detected (Fig.2, lane 2). Within 30 min of incubation with TGF-β, a single inducible complex was observed (Fig.2, lane 3, indicated by arrow) that remained elevated even after exposure to TGF-β for 3 h, but not 4 h (Fig. 2). To demonstrate that nuclear protein(s) interact with this region in a specific fashion, we performed oligonucleotide competition experiments in EMSA. A 75-fold molar excess of unlabeled Oligo-93/-63 virtually abrogated the appearance of the inducible complex (Fig.3 A, indicated byarrow). In contrast, the same fold molar excess of an irrelevant oligonucleotide LMSSREm3 (20.Khachigian L.M. Resnick N. Gimbrone Jr., M.A. Collins T. J. Clin. Invest. 1995; 96: 1169-1175Crossref PubMed Scopus (294) Google Scholar) had no effect on the formation of this complex (Fig. 3 A). Similarly, when the CAGA sequence in Oligo-93/-63 was deleted to produce Oligo-93/-63Δ2, a 75-fold molar excess of the latter oligonucleotide failed to interfere with nucleoprotein complex formation (Fig. 3 A), nor could it directly support formation of the TGF-β-inducible complex (Fig.3 B, indicated by arrow). We performed supershift analysis to shed light on the identity of the nuclear proteins interacting with 32P-Oligo-93/-63. Inclusion of polyclonal antibodies specifically targeting Smad4 in EMSA produced a distinct supershift (Fig. 4,right panel, lane 3, indicated by S), which was not observed when an identical amount of polyclonal antibodies raised against Egr-1 were used (Fig. 4, right panel, lane 4). To ensure that the Smad4 shift was not the product of a nonspecific interaction between the antibody and the radiolabeled probe, nuclear extract was omitted from the EMSA reaction. This strategy did not produce a supershift with either antibody (Fig.4, left panel, lanes 2 and 3). The inability of Smad4 antibodies to affect the intensity of nucleoprotein complex or effect a complete supershift has previously been observed by other investigators in EMSA (4.Song C.-Z. Siok T.E. Gelehrter T.D. J. Biol. Chem. 1998; 273: 29287-29290Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 5.Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.-M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1578) Google Scholar, 6.Jonk L.J.C. Itoh S. Heldin C.-H. ten Dijke P. Kruijer W. J. Biol. Chem. 1998; 273: 21145-21152Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar) and may be a reflection of the low affinity or conformation specificity of the antibody. The specific nature of the supershift nevertheless indicates the involvement of Smad4. These findings demonstrate the presence of Smad4 in endothelial nuclei and its physical association with the PDGF-B promoter. To determine whether Smad4 plays a role in TGF-β-inducible PDGF-B promoter-dependent gene expression, we performed transient transfection analysis using cytomegalovirus (CMV) promoter-driven expression vectors generating wild type and dominant negative forms of Smad4. TGF-β induced PDGF-B promoter-dependent luciferase activity in cells transfected with the empty expression vector (Fig.5 A, compare firstand second columns). Overexpression of wild type Smad4 (CMV-Smad4) activated the PDGF-B promoter (Fig. 5 A,third column, and Fig. 7,third column), which further increased upon exposure of the transfectants to TGF-β (Fig. 5 A, sixth column). In contrast, in cells transfected with the dominant negative Smad4 expression vector, DPC4(1–514), the capacity of TGF-β to augment Smad4-inducible PDGF-B promoter expression was inhibited (Fig.5 A, seventh column). These findings demonstrate the capacity of Smad4 to transactivate the PDGF-B promoter, alone and in concert with exposure to TGF-β. Renilla luciferase activity generated by the normalizing plasmid pRL-TK was independent of Smad cotransfection or exposure to TGF-β (data not shown), indicating specificity. The comparable increase in TGF-β- and Smad-inducible PDGF-B promoter-dependent expression (Fig. 5 A,second and third columns) is consistent with previous findings by different groups in other promoters (6.Jonk L.J.C. Itoh S. Heldin C.-H. ten Dijke P. Kruijer W. J. Biol. Chem. 1998; 273: 21145-21152Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar, 35.Feng X.H. Zhang Y. Wu R.Y. Derynk R. Genes Dev. 1998; 12: 2153-2163Crossref PubMed Scopus (448) Google Scholar, 36.Hua X. Liu X. Ansari D.O. Lodish H.F. Genes Dev. 1998; 12: 3084-3095Crossref PubMed Scopus (258) Google Scholar). That Smad protein was generated in endothelial cells following transfection was confirmed by Western immunoblot analysis (Fig.5 B).Figure 7Smad3 transactivates the PDGF-B promoter alone and together with Smad4. Endothelial cells were transfected with 10 μg of SDA-luc, 2 μg of pRL-TK, and 3 μg (black bars) of pcDNA3, CMV-Smad3, or Smad4 or a total of 6 μg (open bars) of pcDNA3, pCMV5, CMV-Smad3/CMV-Smad4, or CMV-Smad3/DPC4(1–514). The cells were growth-arrested after transfection and harvested after 24 h prior to determination of luciferase activity by luminometry.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To demonstrate the functional significance of the CAGA-binding site in the proximal PDGF-B promoter, we mutated the PDGF-B−81CAGA−78 in Oligo-93/-63 to generate Oligo-93/-63TTTT. A 100-fold molar excess of Oligo-93/-63TTTT failed to block the formation of the TGF-β-inducible nucleoprotein complex using the 32P-labeled wild type PDGF-B promoter fragment (compare Fig. 6 A with Fig. 3 A). Introduction of this mutation into SDA-luc generated SDAm7-luc. Cotransfection experiments revealed that the intact−81CAGA−78 element is crucial for Smad3-inducible PDGF-B promoter-dependent expression (Fig.6 B). We next mobilized six copies of the−83GCCAGAAG−76 motif into the polylinker region of pGL3-prom, driven by the SV40 promoter, in efforts to establish portability of the element and demonstrate greater transactivation upon cotransfection with Smad3. However, the heterologous promoter bearing this sequence in either the forward or the reverse orientation failed to generate any additional reporter activity (data not shown). These findings, which are not entirely unexpected, suggest that additional cis-acting elements in the PDGF-B promoter are required; these may be absent, disrupted, or mis-spaced in the heterologous construct. Previous studies have found that Smad proteins functionally cooperate over nucleotide elements in the promoters of a number of different genes (2.Kretzschmar M. Massague J. Curr. Opin. Gen. Dev. 1998; 8: 103-111Crossref PubMed Scopus (429) Google Scholar, 3.Nakao A. Imamura T. Souchelnytskyi S. Kawabata M. Ishisaki A. Oeda E. Tamaki K. Hanai J. Heldin C.-H. Miyazono K. ten Dijke P. EMBO J. 1997; 16: 5353-5362Crossref PubMed Scopus (905) Google Scholar, 37.Zhang Y. Musci T. Derynck R. Curr. Biol. 1997; 7: 270-276Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Having demonstrated a functional role for Smad4 in the regulation of PDGF-B promoter activity, we next determined whether this Smad member collaborates with other Smads to modulate PDGF-B promoter-dependent expression. Transient transfection studies revealed that Smad3, like Smad4, activates the PDGF-B promoter in endothelial cells (Fig. 7,second column). Simultaneous transfection of Smad3 and Smad4 further activated the PDGF-B promoter (Fig. 7, sixth column). This effect was markedly attenuated if Smad4 was substituted by its dominant negative counterpart (Fig. 7, seventh column). We explored whether Smad2, like Smad3 and Smad4, activates the PDGF-B promoter. Overexpression of Smad2 alone induced PDGF-B promoter-dependent reporter gene expression (Fig.8 A, second column). Coexpression of both Smad2 and Smad3 resulted in synergistic activation (Fig. 8 A, fifth column). In contrast, Smad2, Smad3, and Smad4 were not able to activate the shorter PDGF-B promoter-reporter construct, SPA-luc (Fig. 8 B), thus demonstrating that Smad activation of the PDGF-B promoter is sequence-specific. Physical and cooperative interactions between these Smad proteins have previously been demonstrated by other investigators (3.Nakao A. Imamura T. Souchelnytskyi S. Kawabata M. Ishisaki A. Oeda E. Tamaki K. Hanai J. Heldin C.-H. Miyazono K. ten Dijke P. EMBO J. 1997; 16: 5353-5362Crossref PubMed Scopus (905) Google Scholar, 38.Lagna G. Hata A. Hemmati-Brivanlou A. Massague J. Nature. 1996; 383: 832-836Crossref PubMed Scopus (808) Google Scholar). In this paper, we demonstrate that TGF-β induction of the PDGF-B promoter in vascular endothelial cells is mediated by the Smad family of transcriptional regulators. Serial 5′ deletion and transient transfection analysis defined a region in the PDGF-B promoter mediating inducible responsiveness to TGF-β. This region contains an atypical nucleotide recognition element for the Smad family that preserves the 5′-CAGA-3′ motif. EMSA revealed that nuclear proteins bound to this site in an inducible, transient, and specific manner. This nucleoprotein complex was detected within 30 min of exposure to TGF-β, consistent with rapid Smad translocation from the cytoplasm to the nucleus and its interaction with the PAI-1 promoter (4.Song C.-Z. Siok T.E. Gelehrter T.D. J. Biol. Chem. 1998; 273: 29287-29290Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 5.Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.-M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1578) Google Scholar). Formation of the TGF-β-inducible complex was blocked by a molar excess of unlabeled Oligo-93/-63 but was unaffected by an oligonucleotide bearing a deletion of the−81CAGA−78 motif (Oligo-93/-63Δ2) or one in which the −81CAGA−78 motif was mutated to−81TTTT−78 (Oligo-93/-63TTTT). Overexpression of Smad4 activated the PDGF-B promoter and superinduced PDGF-B promoter-dependent expression in endothelial cells exposed to TGF-β. Simultaneous expression of Smad3 and Smad4, or Smad2 and Smad3, activated the PDGF-B promoter. Mutation of the−81CAGA−78 motif in the PDGF-B promoter (SDAm7-Luc) abrogated Smad-inducible reporter gene expression. These findings demonstrate the existence of a novel Smad-binding element in the PDGF-B promoter. TGF-β activation of the PDGF-B promoter was blocked by overexpression of cDNA encoding a truncated, dominant negative form of Smad4 (amino acids 1–514). Conversely, exogenous wild type Smad4 augmented TGF-β-inducible PDGF-B expression, testing the fidelity of Smad4 activation of PDGF-B in a manner consistent with TGF-β induction of the PAI-1 promoter (35.Feng X.H. Zhang Y. Wu R.Y. Derynk R. Genes Dev. 1998; 12: 2153-2163Crossref PubMed Scopus (448) Google Scholar, 38.Lagna G. Hata A. Hemmati-Brivanlou A. Massague J. Nature. 1996; 383: 832-836Crossref PubMed Scopus (808) Google Scholar). The dominant negative approach has been used previously by other groups to define the involvement of Smad4 in signal transduction stimulated by TGF-β. For example, TGF-β-activation of the p21/WAF/Cip1 promoter in HepG2 cells is repressed by dominant negative Smad4 (7.Moustakas A. Kardassis D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6733-6738Crossref PubMed Scopus (321) Google Scholar). Similarly, overexpression of Smad4 lacking its carboxyl terminus attenuates TGF-β-inducible PAI-1 promoter-dependent expression to levels comparable with those in cells expressing truncated forms of the type I and II receptors (39.Zhang Y. Feng X.-H. Wu R.-Y. Derynck R. Nature. 1996; 383: 168-172Crossref PubMed Scopus (759) Google Scholar). Additionally, the Smad4 mutant also inhibited additive Smad3 and Smad4 activation. These data demonstrate that Smad4 is necessary and sufficient for TGF-β induction of the PDGF-B promoter. It is becoming increasingly clear that Smad proteins can undergo combinatorial interactions with a diverse array of transcription factors other than themselves. For example, Smad proteins functionally interact with Sp1 in the regulation of the p21/WAF/Cip1 promoter (7.Moustakas A. Kardassis D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6733-6738Crossref PubMed Scopus (321) Google Scholar), NF-κB in the type VII collagen promoter (40.Kon A. Vindevoghel L. Kouba D.J. Fujimura Y. Uitto J. Mauviel A. Oncogene. 1999; 18: 1837-1844Crossref PubMed Scopus (60) Google Scholar), AP1 in the c-Jun promoter (41.Wong C. Rougier-Chapman E.M. Frederick J.P. Datto M.B. Liberati N.T. Li J.M. Wang X.F. Mol. Cell. Biol. 1999; 19: 1821-1830Crossref PubMed Scopus (227) Google Scholar), and CBP/p300 (35.Feng X.H. Zhang Y. Wu R.Y. Derynk R. Genes Dev. 1998; 12: 2153-2163Crossref PubMed Scopus (448) Google Scholar) or transcription factor μE3 (TFE3) (36.Hua X. Liu X. Ansari D.O. Lodish H.F. Genes Dev. 1998; 12: 3084-3095Crossref PubMed Scopus (258) Google Scholar) in the PAI-1 promoter. Smads also functionally interact with CREB-binding protein (42.Topper J.N. DiChiara M.R. Brown J.D. Williams A.J. Falb D. Collins T. Gimbrone Jr., M.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9506-9511Crossref PubMed Scopus (154) Google Scholar). 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Atherosclerosis. 1998; 139: 31-39Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). That the TGF-β-inducible gene, βig-h3, is overexpressed in atherosclerotic and restenotic lesions suggests that TGF-β may indeed be capable of modulating gene expression in the vessel wall (53.O'Brien E.R. Bennett K.L. Garvin M.R. Zderic T.W. Hinohara T. Simpson J.B. Kimura T. Nobuyoshi M. Mizgala H. Purchio A. Schwartz S.M. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 576-584Crossref PubMed Scopus (93) Google Scholar). Whether Smads are activated by arterial injury has not yet been demonstrated. Still, it is tempting to speculate that this family of transcriptional mediators may play an important mechanistic role in the vascular response to injury by regulating the inducible expression of at least one growth factor by TGF-β. We thank Dr. William E. Fahl (University of Wisconsin Medical School, Madison) for the generous gift of PDGF-B-luciferase constructs and Drs. Peter ten Dijke (Ludwig Institute of Cancer Research, Uppsala, Sweden), Rik Derynck (UCSF, San Francisco) for Smad expression vectors, and Dr. Jean-Michel Gauthier (Laboratoire Glaxo-Wellcome, France) for construct (CAGA)6-TK-luc.