Rho GTPase activity modulates Wnt3a/beta-catenin signaling.

Wnt proteins constitute a family of secreted signaling molecules that regulate highly conserved pathways essential for development and, when aberrantly activated, drive oncogenesis in a number of human cancers. A key feature of the most widely studied Wnt signaling cascade is the stabilization of cytosolic beta-catenin, resulting in beta-catenin nuclear translocation and transcriptional activation of multiple target genes. In addition to this canonical, beta-catenin-dependent pathway, Wnt3A has also been shown to stimulate RhoA GTPase. While the importance of activated Rho to non-canonical Wnt signaling is well appreciated, the potential contribution of Wnt3A-stimulated RhoA to canonical beta-catenin-dependent transcription has not been examined and is the focus of this study. We find that activated Rho is required for Wnt3A-stimulated osteoblastic differentiation in C3H10T1/2 mesenchymal stem cells, a biological phenomenon mediated by stabilized beta-catenin. Using expression microarrays and real-time RT-PCR analysis, we show that Wnt3A-stimulated transcription of a subset of target genes is Rho-dependent, indicating that full induction of these Wnt targets requires both beta-catenin and Rho activation. Significantly, neither beta-catenin stabilization nor nuclear translocation stimulated by Wnt3A is affected by inhibition or activation of RhoA. These findings identify Rho activation as a critical element of the canonical Wnt3A-stimulated, beta-catenin-dependent transcriptional program.

Endothelial nitric oxide synthase inhibits G12/13 and rho-kinase activated by the angiotensin II type-1 receptor: implication in vascular migration.

BACKGROUND: Although, endothelial nitric oxide (NO) synthase (eNOS) is believed to antagonize vascular remodeling induced by the angiotensin II (AngII) type-1 receptor, the exact signaling mechanism remains unclear. METHODS AND RESULTS: By expressing eNOS to vascular smooth muscle cells (VSMCs) via adenovirus, we investigated a signal transduction mechanism of the eNOS gene transfer in preventing vascular remodeling induced by AngII. We found marked inhibition of AngII-induced Rho/Rho-kinase activation and subsequent VSMC migration by eNOS gene transfer whereas G(q)-dependent transactivation of the epidermal growth factor receptor by AngII remains intact. This could be explained by the specific inhibition of G(12/13) activation by eNOS-mediated G(12/13) phosphorylation. CONCLUSIONS: The eNOS/NO cascade specifically targets the Rho/Rho-kinase system via inhibition of G(12/13) to prevent vascular migration induced by AngII, representing a novel signal cross-talk in cardiovascular protection by NO.

Distinct roles of protease-activated receptors in signal transduction regulation of endothelial nitric oxide synthase.

Protease-activated receptors (PARs), such as PAR1 and PAR2, have been implicated in the regulation of endothelial NO production. We hypothesized that PAR1 and PAR2 distinctly regulate the activity of endothelial NO synthase through the selective phosphorylation of a positive regulatory site, Ser(1179), and a negative regulatory site, Thr(497), in bovine aortic endothelial cells. A selective PAR1 ligand, TFLLR, stimulated the phosphorylation of endothelial NO synthase at Thr(497). It had a minimal effect on Ser(1179) phosphorylation. In contrast, a selective PAR2 ligand, SLIGRL, stimulated the phosphorylation of Ser(1179) with no noticeable effect on Thr(497). Thrombin has been shown to transactivate PAR2 through PAR1. Thus, thrombin, as well as a peptide mimicking the PAR1 tethered ligand, TRAP, stimulated phosphorylation of both sites. Also, thrombin and SLIGRL, but not TFLLR, stimulated cGMP production. A G(q) inhibitor blocked thrombin- and SLIGRL-induced Ser(1179) phosphorylation, whereas it enhanced thrombin-induced Thr(497) phosphorylation. In contrast, a G(12/13) inhibitor blocked thrombin- and TFLLR-induced Thr(497) phosphorylation, whereas it enhanced the Ser(1179) phosphorylation. Although a Rho-kinase inhibitor, Y27632, blocked the Thr(497) phosphorylation, other inhibitors that targeted Rho-kinase failed to block TFLLR-induced Thr(497) phosphorylation. These data suggest that PAR1 and PAR2 distinctly regulate endothelial NO synthase phosphorylation and activity through G(12/13) and G(q), respectively, delineating the novel signaling pathways by which the proteases act on protease-activated receptors to potentially modulate endothelial functions.

The regulator of G protein signaling domain of axin selectively interacts with Galpha12 but not Galpha13.

Axin, a negative regulator of the Wnt signaling pathway, contains a canonical regulator of G protein signaling (RGS) core domain. Herein, we demonstrate both in vitro and in cells that this domain interacts with the alpha subunit of the heterotrimeric G protein G12 but not with the closely related Galpha13 or with several other heterotrimeric G proteins. Axin preferentially binds the activated form of Galpha12, a behavior consistent with other RGS proteins. However, unlike other RGS proteins, that of axin (axinRGS) does not affect intrinsic GTP hydrolysis by Galpha12. Despite its inability to act as a GTPase-activating protein, we demonstrate that in cells, axinRGS can compete for Galpha12 binding with the RGS domain of p115RhoGEF, a known G12-interacting protein that links G12 signaling to activation of the small G protein Rho. Moreover, ectopic expression of axinRGS specifically inhibits Galpha12-directed activation of the Rho pathway in MDA-MB 231 breast cancer cells. These findings establish that the RGS domain of axin is able to directly interact with the alpha subunit of heterotrimeric G protein G12 and provide a unique tool to interdict Galpha12-mediated signaling processes.

A role for the G12 family of heterotrimeric G proteins in prostate cancer invasion.

Many studies have suggested a role for the members of the G12 family of heterotrimeric G proteins (Galpha12 and Galpha13) in oncogenesis and tumor cell growth. However, few studies have examined G12 signaling in actual human cancers. In this study, we examined the role of G12 signaling in prostate cancer. We found that expression of the G12 proteins is significantly elevated in prostate cancer. Interestingly, expression of the activated forms of Galpha12 or Galpha13 in the PC3 and DU145 prostate cancer cell lines did not promote cancer cell growth. Instead, expression of the activated forms of Galpha12 or Galpha13 in these cell lines induced cell invasion through the activation of the RhoA family of G proteins. Furthermore, inhibition of G12 signaling by expression of the RGS domain of the p115-Rho-specific guanine nucleotide exchange factor (p115-RGS) in the PC3 and DU145 cell lines did not reduce cancer cell growth. However, inhibition of G12 signaling with p115-RGS in these cell lines blocked thrombin- and thromboxane A2-stimulated cell invasion. These observations identify the G12 family proteins as important regulators of prostate cancer invasion and suggest that these proteins may be targeted to limit invasion- and metastasis-induced prostate cancer patient mortality.

Selective uncoupling of G alpha 12 from Rho-mediated signaling.

The heterotrimeric G protein G(12) has been implicated in such cellular regulatory processes as cytoskeletal rearrangement, cell-cell adhesion, and oncogenic transformation. Although the activated alpha-subunit of G(12) has been shown to interact directly with a number of protein effectors, the roles of many of these protein-protein interactions in G(12)-mediated cell physiology are poorly understood. To begin dissecting the specific cellular pathways engaged upon G(12) activation, we produced a series of substitution mutants in the regions of Galpha(12) predicted to play a role in effector binding. Here we report the identification and characterization of an altered form of Galpha(12) that is functionally uncoupled from signaling through the monomeric G protein Rho, a protein known to propagate several Galpha(12)-mediated signals. This mutant of Galpha(12) fails to bind the Rho-specific guanine nucleotide exchange factors p115RhoGEF and LARG (leukemia-associated RhoGEF), fails to stimulate Rho-dependent transcriptional activation, and fails to trigger activation of RhoA and the Rho-mediated cellular responses of cell rounding and c-jun N-terminal kinase activation. Importantly, this mutant of Galpha(12) retains coupling to the effector protein E-cadherin, as evidenced by its ability both to bind E-cadherin in vitro and to disrupt E-cadherin-mediated cell-cell adhesion. Furthermore, this mutant retains the ability to trigger beta-catenin release from the cytoplasmic domain of cadherin. This identification of a variant of Galpha(12) that is selectively uncoupled from one signaling pathway while retaining signaling capacity through a separate pathway will facilitate investigations into the mechanisms through which G(12) proteins mediate diverse biological responses.