Response Gene to Complement 32, a Novel Regulator for Transforming Growth Factor-β-induced Smooth Muscle Differentiation of Neural Crest Cells*

Abstract

We previously developed a robust in vitro model system for vascular smooth muscle cell (VSMC) differentiation from neural crest cell line Monc-1 upon transforming growth factor-β (TGF-β) induction. Further studies demonstrated that both Smad and RhoA signaling are critical for TGF-β-induced VSMC development. To identify downstream targets, we performed Affymetrix cDNA array analysis of Monc-1 cells and identified a gene named response gene to complement 32 (RGC-32) to be important for the VSMC differentiation. RGC-32 expression was increased 5-fold after 2 h and 50-fold after 24 h of TGF-β induction. Knockdown of RGC-32 expression in Monc-1 cells by small interfering RNA significantly inhibited the expression of multiple smooth muscle marker genes, including SM α-actin (α-SMA), SM22α, and calponin. Of importance, the inhibition of RGC-32 expression correlated with the reduction of α-SMA while not inhibiting smooth muscle-unrelated c-fos gene expression, suggesting that RGC-32 is an important protein factor for VSMC differentiation from neural crest cells. Moreover, RGC-32 overexpression significantly enhanced TGF-β-induced α-SMA, SM22α, and SM myosin heavy chain promoter activities in both Monc-1 and C3H10T1/2 cells. The induction of VSMC gene promoters by RGC-32 appears to be CArG-dependent. These data suggest that RGC-32 controls VSMC differentiation by regulating marker gene transcription in a CArG-dependent manner. Further studies revealed that both Smad and RhoA signaling are important for RGC-32 activation.