Cdc42 small GTPase is a novel regulator of the fibrogenic activation of human intestinal myofibroblasts

Abstract

Cell division cycle 42 (Cdc42) is a member of the Rho family of small GTPases, which plays crucial roles in regulating cytoskeletal remodeling, and membrane trafficking. While previous studies implicated Cdc42 in controlling intestinal epithelial homeostasis, the involvement of this small GTPase in the process of intestinal fibrogenesis remains unexplored. Our study was designed to determine whether Cdc42 regulates the fibrogenic activation of intestinal myofibroblasts in vitro. The study was conducted using a CCD-18Co normal human colonic fibroblast cell line, and primary human intestinal myofibroblasts (HIMF) isolated from Crohns disease (CD) patients. CCD-18Co and HIMF cells were stimulated by transforming growth factor-{beta}1 (TGF-{beta}1). Cdc42 was inhibited either genetically, using siRNA-mediated knockdown, or pharmacologically using specific Cdc42 inhibitors, ML141 and CASIN. Genetic and pharmacologic inhibition of Cdc42 markedly reduced TGF-{beta}1 induced expression of the major contractile cytoskeletal proteins, -smooth muscle actin, calponin 1 and L-caldesmon. Furthermore, Cdc42 inhibition significantly attenuated expression of key extracellular matrix (ECM) proteins, fibronectin and collagen I, in activated CCD-18Co cells and HIMF. Interestingly, decreased expression of contractile and ECM proteins in Cdc42-depleted myofibroblasts was not due to downregulation of the TGF-{beta}1 signaling, decreased mRNA transcription or increased lysosomal or proteasomal degradation of these proteins. Such suppressed pro-fibrotic activation of Cdc42-deficient CCD-18Co cells and HIMF involved a selective inhibition of protein translation due to inactivation of the AKT-mammalian target of rapamycin (mTOR) signaling module. These findings highlight Cdc42 as a key regulator of intestinal fibrosis that controls mTOR activation to enhance ECM production and contractile actomyosin cytoskeleton in intestinal myofibroblasts. In briefZafar et al. unravel a novel role of Cdc42 small GTPase in fibrogenic activation of human intestinal myofibroblasts. Genetic and pharmacologic inhibition of Cdc42 markedly reduced TGF-{beta} induced expression of contractile cytoskeletal proteins and extracellular matrix proteins in activated myofibroblasts. The mechanisms underlying such profibrotic activity of Cdc42 involve regulation of de novo protein translation via activation of the AKT-mTOR signaling module. HighlightsO_LICdc42 regulates TGF-{beta} dependent activation of human intestinal myofibroblasts C_LIO_LIProfibrotic activity of Cdc42 involves regulation of de novo protein translation C_LIO_LICdc42 regulates myofibroblast activation via AKT-mTOR signaling pathway C_LIO_LITargeting Cdc42-Akt-mTOR signaling may facilitate the development of novel antifibrotic therapies C_LI