RhoA Expression Is Controlled by Nitric Oxide through cGMP-dependent Protein Kinase Activation

Abstract

Small G proteins of the Rho family function as tightly regulated molecular switches that govern a wide range of cell functions (1). A large body of evidence has now been obtained regarding the important functions of Rho proteins in the vasculature, and RhoA has been shown to play a major role in vascular processes such as smooth muscle cell contraction, proliferation, and differentiation; endothelial permeability; platelet activation; and leukocyte migration (2-4). The activity of Rho is under the direct control of a large set of other regulatory proteins (1). In the inactive GDP-bound form, RhoA is locked in the cytosol by guanine dissociation inhibitors. The guanine nucleotide exchange factors catalyze the exchange of GDP for GTP to activate RhoA (5). Activation is then turned off by GTPase-activating proteins that hydrolyze GTP to GDP. Therefore, both the relative expression of these proteins (in particular, that of RhoA) and the fraction of active GTP-bound RhoA are key determinants of RhoA protein activity.Data are now accumulating regarding the regulation of the amount of active GTP-bound RhoA. In vascular smooth muscle cells, several agonists of G protein-coupled receptors, including thrombin, thromboxane A 2 , endothelin, carbachol, angiotensin, ␣-adrenergic agonists, sphingolipids, and extracellular nucleotides, stimulate RhoA activity through the activation of guanine nucleotide exchange factors and increases in the fraction of GTP-bound RhoA. This RhoA activation is accompanied by the membrane translocation of GTP-bound RhoA (5-8). On the other hand, the NO, cGMP, and cGMP-dependent kinase (PKG) 1 signaling pathway exerts inhibitory action on RhoA functions in cells stimulated by these G protein-coupled receptor agonists. We have previously demonstrated that PKG phosphorylates RhoA at Ser 188 in vitro and that the effects of PKG activation on actin cytoskeleton are lost in cells expressing the non-phosphorylatable RhoA A188 mutant, suggesting that inhibitory effects of PKG on RhoA-mediated contraction and actin organization are due to phosphorylation of RhoA at Ser 188 (9). This effect involves inhibition of membrane translocation of GTP-bound RhoA. Several additional reports have now confirmed the inhibitory effect of the NO/cGMP/PKG signaling pathway on the RhoA-dependent component of agonist-induced contraction (10 -13). Recently, it has also been shown that PKG inhibits RhoA-mediated serum response element (SRE)-dependent transcription (14). PKG inhibits SRE-dependent transcription induced by serum, constitutively active G␣ 12 or G␣ 13 , constitutively active Rho exchange factor p115 RhoGEF , or constitutively active RhoA L63 . This inhibition is associated with a decrease in the amount of active GTP-bound RhoA in cells stimulated with serum or constitutively active G␣ 12 or G␣ 13 , but not in p115 RhoGEF -or RhoA L63 -expressing cells, suggesting that PKG can act both upstream and downstream of RhoA. The effect on steps downstream of RhoA has been confirmed by showing that SRE-dependent transcrip...

Suppression of guanylyl cyclase (β1 subunit) expression impairs neurite outgrowth and synapse maturation in cultured cerebellar granule cells

“…The involvement of other proteins may explain this effect because cGMP/cGK signaling may control cell shape in different cell models by regulating either the expression or activity of the RhoA GTPase. 38,39 It is believed that RhoA suppresses neurite outgrowth 31 and thus, an increase in RhoA activity in sGCb1depleted cells might also explain the inhibition of neurite outgrowth.…”

RhoA Activation Mediates Phosphatidylinositol 3-Kinase–Dependent Proliferation of Human Vascular Endothelial Cells

“…This study demonstrated that RhoA was rapidly activated and then upregulated at the transcriptional level in response to HLA class I ligation on vascular EC. To our knowledge and as reported in recent publications (23,24), regulation of RhoA at a transcription or protein level is almost unknown. These studies showed an increase of RhoA mRNA associated with enhanced RhoA protein level.…”

Opposing Roles of Nitric Oxide and Rho-Kinase in Lipid Metabolism in Mice

“…These results indicate that lipid-lowering effects of Rho-kinase inhibition are mediated by AMPK phosphorylation but not by eNOS activation. Rho-kinase causes instability of eNOS mRNA (Takemoto et al 2002), whereas NO inhibits RhoA expression and Rho-kinase activity (Sauzeau et al 2003;Kato et al 2012). Moreover, Rho-kinase inhibits phosphorylation of Akt (Wolfrum et al 2004) and AMPK (Noda et al 2014), the up-stream molecules of eNOS.…”

“…Since Rho-kinase inhibited the expression and activity of eNOS (Takemoto et al 2002;Sauzeau et al 2003), we examined the effects of fasudil on eNOS expression and activity in WT mice. As expected, long-term treatment with fasudil significantly enhanced eNOS phosphorylation, expression and activity (as evaluated by phosphorylated/ total eNOS) in the liver of HFD-fed WT mice (Fig.…”

“…Rho-kinase suppresses eNOS expression through destabilization of eNOS mRNA, while NO inhibits RhoA expression and Rho-kinase activity through the cGMP and protein kinase G (PKG) pathway (Sauzeau et al 2003;Kato et al 2012). Moreover, Rho-kinase inhibits Akt (Wolfrum et al 2004) and AMPK (Noda et al 2014), the up-stream molecules of eNOS.…”