Abstract
Transforming growth factor-beta (TGF-beta) regulates a diverse array of biological processes, such as proliferation, differentiation, extracellular matrix production, and apoptosis. In cultured vascular endothelial cells, TGF-beta 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-beta. 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-beta. 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 (-81)CAGA(-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-beta.
Highlights
Transforming growth factor- (TGF-)1 regulates a wide variety of biological processes, including proliferation, differentiation, extracellular matrix production, and apoptosis [1]
TGF--inducible reporter activity was no longer observed in transfectants harboring construct SPA-luc, whose expression is driven by 64 base pairs of the Platelet-derived growth factor (PDGF)-B promoter (Fig. 1A) and retains binding sites for Sp1, Sp3, and early growth response factor-1 (Egr-1) [19, 21, 26]
These findings suggested that these transcription factors may not play a direct regulatory role in TGF--inducible PDGF-B promoter-dependent expression
Summary
Transient Transfection and Reporter Gene Expression—Bovine aortic endothelial cells were use in experiments between passages 4 and 8. 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- (Promega). 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 mM Hepes, pH 7.9, 100 mM KCl, 0.2 mM EDTA, 20% glycerol, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 4 g/ml aprotinin, and 10 g/ml leupeptin). Western Blot Analysis—Transfected cells were washed twice with ice-cold PBS, pH 7.4, before being lysed on ice in 1ϫ 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 g/ml leupeptin, and 1% aprotinin). The blots were washed again before chemiluminescence detection according to the manufacturer’s protocol (NEN Life Science Products)
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