RhoA modulates Smad signaling during transforming growth factor-beta-induced smooth muscle differentiation.
We recently reported that transforming growth factor (TGF)-beta 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 mitogen-activated protein kinase (MAPK), phosphoinositol-3 kinase, and RhoA have also been shown to mediate TGF-beta actions. The objectives of this study were to examine whether these signaling pathways contribute to TGF-beta-induced SMC development and to test whether Smad signaling cross-talks with other pathway(s) during SMC differentiation induced by TGF-beta. We demonstrate here that RhoA signaling is critical to TGF-beta-induced SMC differentiation. RhoA kinase (ROCK) inhibitor Y27632 significantly blocks the expression of multiple SMC markers such as smooth muscle alpha-actin, SM22alpha, and calponin in TGF-beta-treated Monc-1 cells. In addition, Y27632 reversed the cell morphology and abolished the contractility of TGF-beta-treated cells. RhoA signaling was activated as early as 5 min following TGF-beta addition. Dominant negative RhoA blocked nuclear translocation of Smad2 and Smad3 because of the inhibition of phosphorylation of both Smads and inhibited Smad-dependent 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-beta-induced SMC differentiation.
Chen
Shiyou
S
Department of Cell Biology, Georgetown University Medical School, Washington, DC 20057, USA. sc229@Georgetown.edu
Crawford
Michelle
M
Day
Regina M
RM
Briones
Victorino R
VR
Leader
Jennifer E
JE
Jose
Pedro A
PA
Lechleider
Robert J
RJ
D000577:Amides / Q000494:pharmacology; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D051379:Mice; D008809:Mice, Inbred C3H; D009125:Muscle Relaxants, Central / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D011725:Pyridines / Q000494:pharmacology; D051899:Smad2 Protein / Q000502:physiology*; D051900:Smad3 Protein / Q000502:physiology*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D000577:Amides; D009125:Muscle Relaxants, Central; D011725:Pyridines; D051899:Smad2 Protein; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta; C108830:Y 27632; D020742:rhoA GTP-Binding Protein