Abstract:

ABSTRACT Objective：To research the molecular mechanisms of Smad3 regulation RGC-32 transcription in TGF-β1 induced Epithelial-mesenchymal Transition on NRK-52E Cells. Methodology: The NRK-52E cells were cultured and transiently transfected in vitro. Luciferase assays were performed to determine whether the SBE plays roles in TGF-β1-induced RGC-32 promoter activation. Electrophoretic mobility shift assays (EMSA) were selected to determine which proteins are the formations of TGF-β1-induced nuclear complex. Results: (1) The Smad binding element (SBE) was important for TGF-β1-induced RGC-32 transcriptional activation. The SBE mutation structure significantly inhibited RGC-32 promoter activity. (2) The SBE was essential for the interaction of TGF-β1-induced nuclear proteins with RGC-32 promoter. It was found that the TGF-β-induced interaction between nuclear factors and RGC-32 promoter in EMSA, and the wild-type DNA containing SBE site as competitors abolished the formation of TGF-β-induced complex. (3) Smad2/3 was the composition of TGF-β1-inducible transcription factor complex bound to RGC-32 promoter as recognized by their specific antibodies in EMSA. (4) Smad2 and Smad3 expression plasmids were co-transfected individually into NRK-52E cells, and then Smad3 significantly increased the RGC-32 promoter activity. This suggests that Smad3, but not Smad2, be presented in the TGF-β1-inducible complex formed by nuclear proteins with RGC-32 promoter. Conclusion: These results illuminated that Smad3 may be combined with RGC-32 promoter region SBE and play an important role in transcriptional regulation in TGF-β-induced renal tubular EMT of NRK-52E cells.

Key words: Response gene to complement 32, Epithelial-mesenchymal transition, Transforming growth factor-β1, Smads, Transcriptional regulation