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Whereas the activation of MAPKs (mitogen activated kinases) and Rho dependant pathways by GPCR (G protein coupled receptors) has been the subject of many studies, its implication in the signalling of olfactory receptors, which constitute the largest GPCR family, has been far less analysed. Using an in vitro heterologous system, we showed that odorant activated ORs activate SRE containing promoters via the ERK pathway. We also demonstrated that RhoA and Rock kinases but not Rac were involved in ORs-induced SRE/SRF activation and that AP1 was activated, via JNK and p38 MAPKinase. Using real time PCR we found that mOR23, RnI7 and CfOR12A07 induced elevated levels of transcription factors ELK-4, srf, c-fos and c-jun mRNAs whereas mOREG induced an elevated transcription levels of c-fos and c-jun mRNA only. We showed also that odorant activated ORs stimulate the downstream MAPKs and Rho pathways in primary cultures of rat olfactory sensory neurons (OSNs). Similar results were also obtained with OE (olfactory epithelium) extracts prepared from rats exposed to odorants in vivo. Finally, we showed the important role of the AKT and MAPK signalling pathways in OSNs survival. Taken together, these data provide direct evidence that the binding of odorants onto their ORs activates the MAPK and Rho signalling pathways that are involved in OSNs survival events. This suggests that these pathways could be implicated in the regulation of OSNs homeostasis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Vascular fibrosis is a major complication of hypertension and atherosclerosis, yet it is largely untreatable. Natriuretic peptides (NPs) repress fibrogenic activation of vascular smooth muscle cells (VSMCs), but the intracellular mechanism mediating this effect remains undetermined. Here we show that inhibition of RhoA through phosphorylation at Ser188, the site targeted by the NP effector cyclic GMP (cGMP)-dependent protein kinase I (cGK I), is critical to fully exert antifibrotic potential. cGK I(+/-) mouse blood vessels exhibited an attenuated P-RhoA level and concurrently increased RhoA/ROCK signaling. Importantly, cGK I insufficiency caused dynamic recruitment of ROCK into the fibrogenic programs, thereby eliciting exaggerated vascular hypertrophy and fibrosis. Transgenic expression of cGK I-unphosphorylatable RhoA(A188) in VSMCs augmented ROCK activity, vascular hypertrophy, and fibrosis more prominently than did that of wild-type RhoA, consistent with the notion that RhoA(A188) escapes the intrinsic inhibition by cGK I. Additionally, VSMCs expressing RhoA(A188) became refractory to the antifibrotic effects of NPs. Our results identify cGK I-mediated Ser188 phosphorylation of RhoA as a converging node for pro- and antifibrotic signals and may explain how diminished cGMP signaling, commonly associated with vascular malfunction, predisposes individuals to vascular fibrosis.

Lysophosphatidic acid receptors stimulate a Galpha(12/13)/RhoA-dependent gene transcription program involving the serum response factor (SRF) and its coactivator and oncogene, megakaryoblastic leukemia 1 (MKL1). Inhibitors of this pathway could serve as useful biological probes and potential cancer therapeutic agents. Through a transcription-based high-throughput serum response element-luciferase screening assay, we identified two small-molecule inhibitors of this pathway. Mechanistic studies on the more potent CCG-1423 show that it acts downstream of Rho because it blocks SRE.L-driven transcription stimulated by Galpha(12)Q231L, Galpha(13)Q226L, RhoA-G14V, and RhoC-G14V. The ability of CCG-1423 to block transcription activated by MKL1, but not that induced by SRF-VP16 or GAL4-VP16, suggests a mechanism targeting MKL/SRF-dependent transcriptional activation that does not involve alterations in DNA binding. Consistent with its role as a Rho/SRF pathway inhibitor, CCG-1423 displays activity in several in vitro cancer cell functional assays. CCG-1423 potently (<1 mumol/L) inhibits lysophosphatidic acid-induced DNA synthesis in PC-3 prostate cancer cells, and whereas it inhibits the growth of RhoC-overexpressing melanoma lines (A375M2 and SK-Mel-147) at nanomolar concentrations, it is less active on related lines (A375 and SK-Mel-28) that express lower levels of Rho. Similarly, CCG-1423 selectively stimulates apoptosis of the metastasis-prone, RhoC-overexpressing melanoma cell line (A375M2) compared with the parental cell line (A375). CCG-1423 inhibited Rho-dependent invasion by PC-3 prostate cancer cells, whereas it did not affect the Galpha(i)-dependent invasion by the SKOV-3 ovarian cancer cell line. Thus, based on its profile, CCG-1423 is a promising lead compound for the development of novel pharmacologic tools to disrupt transcriptional responses of the Rho pathway in cancer.

p116Rip, originally identified as a binding partner of activated RhoA, is an actin-binding protein that interacts with the regulatory myosin-binding subunit (MBS) of myosin-II phosphatase and is essential for Rho-regulated cytoskeletal contractility. Here, we have examined the role of p116Rip in RhoA-mediated activation of the transcription factor SRF. We show that p116Rip oligomerizes via its C-terminal coiled-coil domain and, when overexpressed, inhibits RhoA-induced SRF activation without affecting RhoA-GTP levels. Mutant forms of p116Rip that fail to oligomerize or bind to MBS are still capable of inhibiting SRF activity. Our results suggest that p116Rip interferes with RhoA-mediated transcription through its ability to disassemble the actomyosin cytoskeleton downstream of RhoA.

Rho GTPases integrate the intracellular signaling in a wide range of cellular processes. Activation of these G proteins is tightly controlled by a number of guanine nucleotide exchange factors (GEFs). In this study, we addressed the functional role of the recently identified p114RhoGEF in in vivo experiments. Activation of endogenous G protein-coupled receptors with lysophosphatidic acid resulted in activation of a transcription factor, serum response element (SRE), that was enhanced by p114RhoGEF. This stimulation was inhibited by the functional scavenger of Gbetagamma subunits, transducin. We have determined that Gbetagamma subunits but not Galpha subunits of heterotrimeric G proteins stimulated p114RhoGEF-dependent SRE activity. Using coimmunoprecipitation assay, we have determined that Gbetagamma subunits interacted with full-length and DH/PH domain of p114RhoGEF. Similarly, Gbetagamma subunits stimulated SRE activity induced by full-length and DH/PH domain of p114RhoGEF. Using in vivo pull-down assays and dominant-negative mutants of Rho GTPases, we have determined that p114RhoGEF activated RhoA and Rac1 but not Cdc42 proteins. Functional significance of RhoA activation was established by the ability of p114RhoGEF to induce actin stress fibers and cell rounding. Functional significance of Rac1 activation was established by the ability of p114RhoGEF to induce production of reactive oxygen species (ROS) followed by activation of NADPH oxidase enzyme complex. In summary, our data showed that the novel guanine nucleotide exchange factor p114RhoGEF regulates the activity of RhoA and Rac1, and that Gbetagamma subunits of heterotrimeric G proteins are activators of p114RhoGEF under physiological conditions. The findings help to explain the integrated effects of LPA and other G-protein receptor-coupled agonists on actin stress fiber formation, cell shape change, and ROS production.