Transforming Growth Factor‐β Regulates RGC‐32 Transcription During Smooth Muscle Differentiation From Neural Crest Cells

Abstract

Previous studies demonstrated that transforming growth factor-β (TGF-β) induced vascular smooth muscle cell (VSMC) differentiation from neural crest stem cell line Monc-1. We recently identified a gene, namely response gene to complement 32 (RGC-32) to be critical for TGF-β-induced VSMC differentiation. RGC-32 was upregulated by TGF-β but how TGF-β controls RGC-32 expression remains to be determined. The purpose of this study was to identify the molecular mechanism of TGF-β regulation on RGC-32 gene transcription. A Smad binding site and a polyomavirus enhancer activator (PEA3) binding site is located in the 5′ upstream regulatory region of RGC-32 gene. Mutation of Smad site significantly inhibited RGC-32 promoter activity. Double mutations of Smad and PEA3 sites completely abolished the activity although PEA3 mutation alone appeared to have no effect. By performing gel mobility shift assay (EMSA), competitive EMSA, supershift assay as well as transfection and luciferase assay, we found that Smad2 but not Smad3 significantly upregulated RGC-32 promoter activity. PEA3 alone did not exhibit a significant effect, but PEA3 synergized with Smad2 in upregulating RGC-32 promoter activity. These results were further confirmed by small RNA interference experiments. Taken together, these data demonstrate that Smad2 and PEA3 synergistically regulate TGF-β induction of RGC-32 transcription during VSMC differentiation from neural crest cells.