RhoA Modulates Smad Signaling during Transforming Growth Factor-β-induced Smooth Muscle Differentiation

Abstract

We recently reported that transforming growth factor (TGF)-␤ induced the neural crest stem cell line Monc-1 to differentiate into a spindle-like contractile smooth muscle cell (SMC) phenotype and that Smad signaling played an important role in this phenomenon. In addition to Smad signaling, other pathways such as mitogenactivated protein kinase (MAPK), phosphoinositol-3 kinase, and RhoA have also been shown to mediate TGF-␤ actions. The objectives of this study were to examine whether these signaling pathways contribute to TGF-␤-induced SMC development and to test whether Smad signaling cross-talks with other pathway(s) during SMC differentiation induced by TGF-␤. We demonstrate here that RhoA signaling is critical to TGF-␤-induced SMC differentiation. RhoA kinase (ROCK) inhibitor Y27632 significantly blocks the expression of multiple SMC markers such as smooth muscle ␣-actin, SM22␣, and calponin in TGF-␤-treated Monc-1 cells. In addition, Y27632 reversed the cell morphology and abolished the contractility of TGF-␤-treated cells. RhoA signaling was activated as early as 5 min following TGF-␤ addition. Dominant negative RhoA blocked nuclear translocation of Smad2 and Smad3 because of the inhibition of phosphorylation of both Smads and inhibited Smaddependent SBE promoter activity, whereas constitutively active RhoA significantly enhanced SBE promoter activity. Consistent with these results, C3 exotoxin, an inhibitor of RhoA activation, significantly attenuated SBE promoter activity and inhibited Smad nuclear translocation. Taken together, these data point to a new role for RhoA as a modulator of Smad activation while regulating TGF-␤-induced SMC differentiation.

TGF-β-mediated activation of RhoA signalling is required for efficient V12HaRas and V600EBRAF transformation

“…TGF-b may activate RhoA signalling in a biphasic manner involving a rapid activation within minutes of ligand addition followed by a second, potentially Smad-dependent wave of activation after hours of TGF-b stimulation (Bhowmick et al, 2001(Bhowmick et al, , 2003Shen et al, 2001;Edlund et al, 2002;Vardouli et al, 2005;Chen et al, 2006). Rapid TGF-bdriven activation of RhoA has been described in Swiss-3T3 and mouse embryonic fibroblast (MEFs) (Shen et al, 2001;Vardouli et al, 2005), and we first investigated whether this also takes place in NIH3T3 cells.…”

“…TGF-b can activate RhoA signalling in several cell types (Bhowmick et al, 2001(Bhowmick et al, , 2003Shen et al, 2001;Edlund et al, 2002;Vardouli et al, 2005;Chen et al, 2006). Rho GTPases are molecular switches that are inactive when GDP bound and active when GTP bound, where they propagate their signals through interaction with numerous downstream signalling effectors (Jaffe and Hall, 2005).…”

Smad3-mediated Myocardin Silencing

“…Confocal Microscopy-10T1/2 cells were treated with vehicle or TGF-␤ for 24 h. The cells were fixed and incubated with rabbit anti-Smad3 and mouse anti-Nkx2.5 antibodies (Zymed Laboratories Inc. and Santa Cruz Biotechnology), followed by incubation with different fluorescent dye-conjugated secondary goat anti-rabbit or anti-mouse IgG as described previously (32). The co-localization of Smad3 with Nkx2.5 was observed with confocal microscopy.…”

“…Cells were then transiently transfected (in triplicate) with Lipofectamine LTX according to the manufacturer's recommendation. Luciferase assay was performed as described previously (26,32).…”

Transcriptional Profiling Reveals Crosstalk Between Mesenchymal Stem Cells and Endothelial Cells Promoting Prevascularization by Reciprocal Mechanisms

“…6D), indicating that RhoA/ROCK might also participate in the coculture-induced early myogenic differentiation of HMSCs. RhoA/ROCK signaling can be activated by various external signals, including TGF-b1 and TGF-b3 [43,44], which were significantly upregulated in both co-MSCs and co-HUVECs (Supplementary Fig. S7).…”