Oncogene database

6010

RHO

rhodopsin;RHO;rhodopsin

opsin 2, rod pigment|opsin-2

3q21-q24

protein-coding

Proteins of the Rho family regulate cytoskeletal rearrangements in response to receptor stimulation and are involved in the establishment and maintenance of epithelial cell morphology. We recently showed that Rac is able to downregulate Rho activity and that the reciprocal balance between Rac and Rho activity is a major determinant of cellular morphology and motility in NIH3T3 fibroblasts. Using biochemical pull-down assays, we analyzed the effect of transient and sustained oncogenic Ras signaling on the activation state of Rac and Rho in epithelial MDCK cells. In contrast to the activation of Rac by growth factor-induced Ras signaling, we found that sustained signaling by oncogenic RasV12 permanently downregulates Rac activity, which leads to upregulation of Rho activity and epithelial-mesenchymal transition. Oncogenic Ras decreases Rac activity through sustained Raf/MAP kinase signaling, which causes transcriptional downregulation of Tiam1, an activator of Rac in epithelial cells. Reconstitution of Rac activity by expression of Tiam1 or RacV12 leads to downregulation of Rho activity and restores an epithelial phenotype in mesenchymal RasV12- or RafCAAX-transformed cells. The present data reveal a novel mechanism by which oncogenic Ras is able to interfere with the balance between Rac and Rho activity to achieve morphological transformation of epithelial cells.

mutations in the epidermal growth factor receptor have been identified in several human tumor types, including gliomas. These receptor mutants have deletions in their extracellular ligand-binding domains and are, therefore, no longer regulated by ligand, resulting in constitutive activation of the receptor kinase. These mutants have been proposed to transduce oncogenic signals via ligand-independent signaling pathways. Avian viral homologues of these oncogenic epidermal growth factor receptors exhibit structurally homologous deletions and form tumors in chickens. One such mutant, S3v-ErbB, transforms fibroblasts in vitro, and transformation has been correlated with the formation of a novel tyrosine phosphoprotein complex. V-ErbB-mediated complex formation and transformation have been shown to occur independently of Ras activation. The major aims of this study are to further characterize this ligand-independent v-ErbB oncogenic signaling pathway. Here we show that both v-ErbB-mediated phosphoprotein complex formation and transformation are inhibited by a dominant negative mutant of Rho. This inhibition is specific for dominant negative Rho; dominant negative mutants of Rac and Cdc42 have no effect on transformation or on tyrosine phosphorylation of the phosphoprotein complex. Based on these observations, we propose that S3v-ErbB stimulates a Rho-dependent tyrosine kinase, resulting in complex formation and ultimately oncogenic transformation.

Transformation by oncogenic Ras requires signaling through Rho family proteins including RhoA, but the mechanism(s) whereby oncogenic Ras regulates the activity of RhoA is (are) unknown. We examined the effect of Ras on RhoA activity in NIH 3T3 cells either stably transfected with H-Ras(V12) under control of an inducible promoter or transiently expressing the activated H-Ras. Using a novel method to quantitate enzymatically the GTP bound to Rho, we found that expression of the oncogenic Ras increased Rho activity approximately 2-fold. Increased Rho activity was associated with increased plasma membrane binding of RhoA and decreased activity of the Rho/Ras-regulated p21(WAF1/CIP1) promoter. RhoA activation by oncogenic Ras could be explained by a decrease in cytosolic p190 Rho-GAP activity and translocation of p190 Rho-GAP from the cytosol to a detergent-insoluble cytoskeletal fraction. Pharmacologic inhibition of the Ras/Raf/MEK/ERK pathway prevented Ras-induced activation of RhoA and translocation of p190 Rho-GAP; expression of constitutively active Raf-1 kinase or MEK was sufficient to induce p190 Rho-GAP translocation. We conclude that in NIH 3T3 cells oncogenic Ras activates RhoA through the Raf/MEK/ERK pathway by decreasing the cytosolic activity and changing the subcellular localization of p190 Rho-GAP.

Transformation of fibroblasts by oncogenic Src causes disruption of actin stress fibers and formation of invasive adhesions called podosomes. Because the small GTPase Rho stimulates stress fiber formation, Rho inactivation by Src has been thought to be necessary for stress fiber disruption. However, we show here that Rho[GTP] levels do not decrease after transformation by activated Src. Inactivation of Rho in Src-transformed fibroblasts by dominant negative RhoA or the Rho-specific inhibitor C3 exoenzyme disrupted podosome structure as judged by localization of podosome components F-actin, cortactin, and Fish. Inhibition of Rho strongly inhibited Src-induced proteolytic degradation of the extracellular matrix. Furthermore, development of an in situ Rho[GTP] affinity assay allowed us to detect endogenous Rho[GTP] at podosomes, where it colocalized with F-actin, cortactin, and Fish. Therefore, Rho is not globally inactivated in Src-transformed fibroblasts, but is necessary for the assembly and function of structures implicated in tumor cell invasion.

tumour angiogenesis is mediated by increased levels of vascular endothelial growth factor (VEGF). We have studied the mechanism by which endogenous activation of Rho oncoproteins regulates VEGF expression in COS-7 and NIH3T3 cells. We carried out transient and stable transfection with constitutively activated rhoA, rac1, and cdc42 mutants in COS-7 and NIH3T3 cells, respectively in the absence of external stimuli. Western blot and inmunohistochemistry assays of those cells revealed increased VEGF protein expression. Cotransfection with constitutively activated rhoA, rac1, and cdc42 mutants and a VEGF promoter-reporter construct showed an increase in VEGF promoter transcriptional activity induced by Rho oncoproteins in COS-7 and NIH3T3. c-Jun kinase had been described as a MAPK involved in Rho oncoproteins pathways. Interestingly, we found that c-Jun kinase chemical inhibition as well as transient transactivation assays using dominant negative c-Jun kinase mutant abolished the VEGF promoter transcriptional induction by Rac1 and Cdc42 but not by RhoA. These findings indicate that Rho oncoprotein endogenously activated regulates VEGF expression through a transcriptional mechanism, and that the c-Jun kinase activity is a mediator in the expression of VEGF induced by Rac1 and Cdc42 oncoproteins, but not of that induced by RhoA.

Generation of reactive oxygen species (ROS) by Ras oncogene-induced NADPH oxidase (Nox) 1 is required for Ras transformation phenotypes including anchorage-independent growth, morphological transformation, and tumorigenesity, but the signaling mechanism downstream of Nox1 remains elusive. Rho is known to be a critical regulator of actin stress fiber formation. Nonetheless, Rho was reported to no longer couple to loss of actin stress fibers in Ras-transformed Swiss3T3 cells despite the elevation of Rho activity. In this study, however, we demonstrate that Rho is inactivated in K-Ras-transformed normal rat kidney cells, and that abrogation of Nox1-generated ROS by Nox1 small interference RNAs or diphenyleneiodonium restores Rho activation, suggesting that Nox1-generated oxidants mediate down-regulation of the Rho activity. This down-regulation involves oxidative inactivation of the low molecular weight protein-tyrosine phosphatase by Nox1-generated ROS and a subsequent elevation in the tyrosine-phosphorylated active form of p190RhoGAP, the direct target of the phosphatase. Furthermore, the decreased Rho activity leads to disruption of both actin stress fibers and focal adhesions in Ras-transformed cells. As for Rac1, Rac1 also appears to participate in the down-regulation of Rho via Nox1. Our discovery defines a mediating role of Nox1-redox signaling for Ras oncogene-induced actin cytoskeletal changes.

Urothelial cell carcinoma is the most common type of malignancy found in long-term dialysis patients and kidney transplant recipients in Taiwan. Surgical specimens of tumorous and non-tumorous bladder tissues were collected from 12 patients with bladder cancer. Increased expressions of Ras, RhoA, Akt, PI-3K were demonstrated in the tumors as compared to adjacent control tissues. To understand the impact of Ras over-expression on bladder cancer progression, human bladder cancer TSGH 8301 cells were transfected with Ras DNA. The Ras-transfected cells were then treated with either a PI-3K inhibitor (wortmannin) or Rho kinase inhibitor (Y-27632) and the expressions of Ras, PI-3K, Akt, NF-kappaB, and RhoA were analyzed. Fluorescent phalloidin staining demonstrated more intense F-actin staining in the Ras over-expressed cells than in the control cells, and the intensity of F-actin was inhibited by Y-27632. A gelatin zymography study demonstrated that the MMP-2 and MMP-9 expressions of the Ras-transfected cells were enhanced, and Y-27632 treatment reduced the levels of MMP-2 and MMP-9. Similarly, a wound healing assay revealed that the ability of cell migration was markedly increased by Ras transfection and the healing rate after treatment of Y-27632 was delayed. Our results provide evidence that Ras-induced RhoA and NF-kappaB activation was involved in the invasion/migration of bladder cancer. Through Ras and/or RhoA inhibition, there might be an opportunity for new therapeutic interventions in bladder cancer.

We demonstrate that Rho, a regulator of cytoskeletal actin, is necessary for Ras transformation. A dominant inhibitory Rho gene (RhoBN19) specifically suppressed Rat1 cell focus formation induced by oncogenic Ras but not by Raf. An activated Rho gene (RhoBV14) lacked focus formation activity but augmented the focus formation activity of both oncogenes. NIH3T3 cell lines expressing RhoBV14 grew to higher saturation density and displayed reduced serum and anchorage requirements for growth. We concluded that Rho played a role in cell growth regulation and was required for transformation by oncogenic Ras but not Raf. A model for Ras signal transduction proposing separate Rho-dependent and Raf-dependent pathways is discussed.