Rho Family GTPases RhoA Research Articles

Mechanistic insights into Nav1.7-dependent regulation of rat prostate cancer cell invasiveness revealed by toxin probes and proteomic analysis.

Voltage-gated sodium channels are involved in tumor metastasis, as potentiating or attenuating their activities affects the migration and invasion process of tumor cells. In the present study, we tested the effect of two peptide toxins, JZTX-I and HNTX-III which function as Nav1.7 activator and inhibitor, respectively, on the migration and invasion ability of prostate cancer (PCa) cell line Mat-LyLu. These two peptides showed opposite effects, and subsequently a comparative proteomic analysis characterized 64 differentially expressed membrane proteins from the JZTX-I- and HNTX-III-treated groups. Among these, 15 proteins were down-regulated and 49 proteins were up-regulated in the HNTX-III group. Bioinformatic analysis showed eight proteins are cytoskeleton proteins or related regulators, which might play important roles in the metastasis of Mat-LyLu cells. The altered expressions of four of these proteins, fascin, muskelin, annexin A2, and cofilin-1, were validated by western blot analysis. Further function network analysis of these proteins revealed that the Rho family GTPases RhoA and Rac1 might be of particular importance for the rat PCa cell invasion. Pharmacological data revealed that JZTX-I and HNTX-III could modulate the Rho signaling pathway in a Nav1.7-dependent manner. In summary, this study suggests that the Nav1.7-dependent regulation of Rho GTPase activity plays a vital role in Mat-LyLu cell migration and invasion and provides new insights into the treatment of PCa.

You-Gui pills promote nerve regeneration by regulating netrin1, DCC and Rho family GTPases RhoA, Racl, Cdc42 in C57BL/6 mice with experimental autoimmune encephalomyelitis

Ethnopharmacological relevanceYou-Gui pills (YGPs) are an effective traditional Chinese formula being used clinically for the treatment of multiple sclerosis (MS). Previous studies demonstrated that YGPs exerted the potent neuroprotective effects in murine models of experimental autoimmune encephalomyelitis (EAE), which is an equivalent animal model for multiple sclerosis (MS). However, the mechanism of YGPs functions remained unclear. Aim of this studyThe aim of this study was to evaluate the therapeutic effect of YGPs in MOG35–55-induced EAE mice and to further elucidate the underlying molecular mechanism. Methods: Female C57BL/6 mice were divided into six groups, including the non-treated EAE model, prednisone acetate- and 1.2, 2.4 or 4.8g/kg YGPs-treated EAE groups, and a normal control group. The EAE model was established by injecting the mice subcutaneously with MOG35–55 antigen. The body weights were measured and the neurological functions were scored in each group. The pathology and morphology of the brain and spinal cord was examined. The expression of MAP-2 was detected by immunofluorescent staining. The levels of netrin1, DCC, RhoA, Rac1, and Cdc42 were assayed by immunohistochemistry, qRT-PCR and Western blot on day 40 post-immunization (PI). ResultsYGPs treatments significantly reduced neurological function scores in EAE mice, where the inflammatory infiltration was reduced and the axon and myelin damage in both brain and spinal cord was alleviated. In the brain and spinal cord tissues, YGPs increased the expression of neuronal factors MAP-2, netrin1 and DCC. The expression of Rac1 and Cdc42 were increased, while RhoA was reduced following YGPs treatments. ConclusionOur results demonstrated that YGPs exhibited a neuroprotective effect on promoting nerve regeneration at the brain and spinal cord in EAE mice induced by MOG35–55. Netrin1, DCC and the Rho family GTPases of RhoA, Racl, Cdc42 were involved in mediating the effects of YGPs on nerve regeneration.

Dendritic spine geometry can localize GTPase signaling in neurons.

Dendritic spines are the postsynaptic terminals of most excitatory synapses in the mammalian brain. Learning and memory are associated with long-lasting structural remodeling of dendritic spines through an actin-mediated process regulated by the Rho-family GTPases RhoA, Rac, and Cdc42. These GTPases undergo sustained activation after synaptic stimulation, but whereas Rho activity can spread from the stimulated spine, Cdc42 activity remains localized to the stimulated spine. Because Cdc42 itself diffuses rapidly in and out of the spine, the basis for the retention of Cdc42 activity in the stimulated spine long after synaptic stimulation has ceased is unclear. Here we model the spread of Cdc42 activation at dendritic spines by means of reaction-diffusion equations solved on spine-like geometries. Excitable behavior arising from positive feedback in Cdc42 activation leads to spreading waves of Cdc42 activity. However, because of the very narrow neck of the dendritic spine, wave propagation is halted through a phenomenon we term geometrical wave-pinning. We show that this can account for the localization of Cdc42 activity in the stimulated spine, and, of interest, retention is enhanced by high diffusivity of Cdc42. Our findings are broadly applicable to other instances of signaling in extreme geometries, including filopodia and primary cilia.

A distinct role of RhoB in gastric cancer suppression

Although Rho family GTPases RhoA, RhoB and RhoC share more than 85% amino acid sequence identity, they may play distinct roles in tumor progression. RhoA and RhoC have been suggested to have positive effects on tumor progression, but the role of RhoB in cancer, particularly in gastric cancer, remains unclear. In our study, we have examined the expression levels of these three Rho GTPases in a large panel of specimens from gastric cancer patients by immunohistochemistry. We found that RhoA and RhoC expression were significantly elevated, while RhoB was reduced or absent, in surgically removed gastric cancer tissues when compared to normal gastric tissues. The significant reduction of RhoB expression was confirmed in another group of gastric cancer samples in comparison to the adjacent non-neoplastic tissues. Then we transfected the plasmids containing RhoA, RhoB or RhoC cDNA into two gastric cancer cell lines, SGC7901 and AGS cells, respectively. By overexpression experiments, we found that RhoA promoted the gastric cancer cell proliferation and RhoC stimulated migration and invasion of the cancer cell. RhoB expression, however, significantly inhibited the proliferation, migration and invasion of the gastric cancer cells and also enhanced the chemosensitivity of these cells to anticancer drugs. It appears that RhoB plays an opposing role from that of RhoA and/or RhoC in gastric cancer cells. Our work suggests that RhoB may play a tumor suppressor role and subsequently may have potential implications in future targeted therapy.

Rapid calcium‐dependent reduction of intraendothelial cAMP: a trigger to increase vascular permeability?

Further understanding of vascular endothelial barrier modulation will better enable clinical control of fluid balance and aid drug delivery. Acute inflammatory mediators increase intracellular Ca2+ very rapidly in vascular endothelium, leading to breakdown of the barrier. Investigations both in vivo and in cell culture show that this response is strongly inhibited by increased intracellular cAMP. Ca2+ and cAMP signalling pathways are subject to potential crosstalk through Ca2+-regulated adenylate cyclase 6 (AC6) and Ca2+-dependent phosphodiesterase I as well as interaction at converging downstream effectors which regulate endothelial cell contraction and endothelial junctions. Ca2+-sensitive fluorophores enable rapid optical-based measurement of intracellular Ca2+. However, understanding pathway crosstalk has been hampered by poor temporal resolution of cAMP measurement. Previous studies of cAMP in endothelium used enzyme immunoassays and yielded limited temporal information (Cioffi et al. 2002; Baumer et al. 2008). Fluorescence resonance energy transfer (FRET) has been used previously to determine changes in cAMP concentration but use of the regulatory and catalytic subunits of protein kinase A (PKA) was problematic (Adams et al. 1991). A new technique enables FRET measurement based on a single cAMP binding domain and yields a fast, sensitive indicator that has no catalytic or targeting domains to interfere with cellular processes (Nikolaev et al. 2004). The latter research group has now used their technique to investigate endothelium. The study by Werthmann et al. (2009) in this issue of The Journal of Physiology uses the new FRET assay to measure cAMP in real time using cultured endothelial cells stimulated with thrombin and pre-stimulated with isoproterenol to elevate cAMP. The data clearly demonstrate that the rapid initial Ca2+ rise is closely followed by transient suppression of cAMP concentration. This thrombin-induced fall in cAMP was abolished by prior transfection with siRNA to downregulate AC6, thus directly implicating Ca2+-inhibited AC6 in rapid cAMP regulation. While the study yields important new data on early intracellular signals after thrombin stimulation, questions remain about the link between these mechanisms and endothelial barrier regulation. For example, while both the Ca2+ increase and subsequent cAMP decrease reach respective peaks in seconds, the permeability increase of thrombin-stimulated endothelial monolayers that can be measured (in the absence of pre-stimulation of cAMP) does not peak for about 10 min in vivo and can last an hour or more in cell culture (Baumer et al. 2009) even though the present study suggests cAMP levels return toward pre-thrombin levels in 1.5 min. Several potential pathways lead from Ca2+ and cAMP to modulation of the adhesion and contraction of endothelial cells via both PKA and small GTPases (Adamson et al. 2008). Rho family GTPases RhoA and Rac1 regulate the actin cytoskeleton and endothelial barrier and are potential targets of modulated cAMP pathways; cAMP has been shown to activate Rac1 via activation of Ras family GTPase Rap1 through the GTP exchange factor (GEF) Epac. Further investigations will require measurement of changes in these GTPases under conditions where the time course of cAMP is followed. This will require use of inflammatory agents such as bradykinin and platelet-activating factor that do not stimulate the RhoA pathway in intact microvessels as much as the thrombin response in cell culture (Adamson et al. 2003). Also the role of an increase in cAMP in both cAMP pre-stimulated and non-pre-stimulated cells that followed the initial reduction about 60 s after thrombin application needs evaluation. It is notable that pre-stimulation with isoproterenol or forskolin was required in the experiments of Werthmann et al. to demonstrate a thrombin-induced decrease in cAMP concentration. As elevated cAMP blocks permeability increase, any thrombin-induced change in permeability could not be measured under the same conditions that cAMP concentration was measured in this study. Thus, an important goal will be to determine whether cAMP can be monitored at lower concentrations in the absence of pharmacological stimulation or whether such changes can be measured in vivo, where endothelium may be in a different state perhaps having higher basal cAMP. The recent collaborative study by the two Wurzburg groups using thrombin to stimulate a drop in trans-endothelial resistance may point the way using a variety of cell culture and intact endothelial barriers (Baumer et al. 2009). A focus on signalling pathways, changes in permeability and possible spatial imaging of cAMP will enable further investigation of vascular permeability regulation.

Dysferlin Deficiency Enhances Monocyte Phagocytosis: A Model for the Inflammatory Onset of Limb-Girdle Muscular Dystrophy 2B

Dysferlin deficiency causes limb-girdle muscular dystrophy type 2B (LGMD2B; proximal weakness) and Miyoshi myopathy (distal weakness). Muscle inflammation is often present in dysferlin deficiency, and patients are frequently misdiagnosed as having polymyositis. Because monocytes normally express dysferlin, we hypothesized that monocyte/macrophage dysfunction in dysferlin-deficient patients might contribute to disease onset and progression. We therefore examined phagocytic activity, in the presence and absence of cytokines, in freshly isolated peripheral blood monocytes from LGMD2B patients and in the SJL dysferlin-deficient mouse model. Dysferlin-deficient monocytes showed increased phagocytic activity compared with control cells. siRNA-mediated inhibition of dysferlin expression in the J774 macrophage cell line resulted in significantly enhanced phagocytosis, both at baseline and in response to tumor necrosis factor-alpha. Immunohistochemical analysis revealed positive staining for several mononuclear cell activation markers in LGMD2B human muscle and SJL mouse muscle. SJL muscle showed strong up-regulation of endocytic proteins CIMPR, clathrin, and adaptin-alpha, and LGMD2B muscle exhibited decreased expression of decay accelerating factor, which was not dysferlin-specific. We further showed that expression levels of small Rho family GTPases RhoA, Rac1, and Cdc 42 were increased in dysferlin-deficient murine immune cells compared with control cells. Therefore, we hypothesize that mild myofiber damage in dysferlin-deficient muscle stimulates an inflammatory cascade that may initiate, exacerbate, and possibly perpetuate the underlying myofiber-specific dystrophic process.

Integrin Signaling through Arg Activates p190RhoGAP by Promoting Its Binding to p120RasGAP and Recruitment to the Membrane

The Rho family GTPases RhoA (Rho), Rac1, and Cdc42 are essential effectors of integrin-mediated cell attachment and spreading. Rho activity, which promotes formation of focal adhesions and actin stress fibers, is inhibited upon initial cell attachment to allow sampling of the new adhesive environment. The Abl-related gene (Arg) tyrosine kinase mediates adhesion-dependent inhibition of Rho through phosphorylation and activation of the Rho inhibitor p190RhoGAP-A (p190). p190 phosphorylation promotes its binding to p120RasGAP (p120). Here, we elucidate the mechanism by which p120 binding regulates p190 activation after adhesion. We show that p190 requires its p120-binding domain to undergo Arg-dependent activation in vivo. However, p120 binding does not activate p190RhoGAP activity in vitro. Instead, activation of p190 requires recruitment to the cell periphery. Integrin-mediated adhesion promotes relocalization of p190 and p120 to the cell periphery in wild-type fibroblasts, but not in arg(-/-) fibroblasts. A dominant-negative p120 fragment blocks p190:p120 complex formation, prevents activation of p190 by adhesion, and disrupts the adhesion-dependent recruitment of p190 to the cell periphery. Our results demonstrate that integrin signaling through Arg activates p190 by promoting its association with p120, resulting in recruitment of p190 to the cell periphery where it inhibits Rho.

Leishmania donovani promastigotes induce periphagosomal F-actin accumulation through retention of the GTPase Cdc42

Leishmania donovani promastigotes inhibit phagosome maturation and induce the accumulation of periphagosomal F-actin during the establishment of infection within macrophages. These events are mediated by the surface glycolipid lipophosphoglycan (LPG), but the underlying mechanisms remain to be elucidated. In this study, we addressed the role of the Rho-family GTPases RhoA, Rac1 and Cdc42 in the uptake of L. donovani promastigotes and in the accumulation of periphagosomal F-actin. Confocal microscopy analyses revealed that association of both Rac1 and RhoA to phagosomes containing L. donovani promastigotes was independent of the presence of LPG. In contrast, Cdc42 and proteins required for F-actin assembly (Arp2/3, WASP, Myosin, alpha-actinin) were retained on phagosomes in a LPG-dependent manner. Expression of the RhoA inhibitor C3-transferase blocked the internalization of complement-opsonized promastigotes, whereas the dominant-negative Rac1N17 blocked the uptake of unopsonized promastigotes. The dominant-negative Cdc42N17 inhibited LPG-mediated phagosomal accumulation of F-actin and retention of Arp2/3 and Myosin. Thus, our data suggest that the effect of LPG on the accumulation of periphagosomal F-actin is the consequence of an abnormal retention or activation of Cdc42 at the phagosome.

A Novel Strategy for Specifically Down-regulating Individual Rho GTPase Activity in Tumor Cells

The Rho family GTPases RhoA, RhoB, and RhoC regulate the actin cytoskeleton, cell movement, and cell growth. Unlike Ras, up-regulation or overexpression of these GDP/GTP binding molecular switches, but not activating point mutations, has been associated with human cancer. Although they share over 85% sequence identity, RhoA, RhoB, and RhoC appear to play distinct roles in cell transformation and metastasis. In NIH 3T3 cells, RhoA or RhoB overexpression causes transformation whereas RhoC increases the cell migration rate. To specifically target RhoA, RhoB, or RhoC function, we have generated a set of chimeric molecules by fusing the RhoGAP domain of p190, a GTPase-activating protein that accelerates the intrinsic GTPase activity of all three Rho GTPases, with the C-terminal hypervariable sequences of RhoA, RhoB, or RhoC. The p190-Rho chimeras were active as GTPase-activating proteins toward RhoA in vitro, co-localized with the respective active Rho proteins, and specifically down-regulated Rho protein activities in cells depending on which Rho GTPase sequences were included in the chimeras. In particular, the p190-RhoA-C chimera specifically inhibited RhoA-induced transformation whereas p190-RhoC-C specifically reversed the migration phenotype induced by the active RhoC. In human mammary epithelial-RhoC breast cancer cells, p190-RhoC-C, but not p190-RhoA-C or p190-RhoB-C, reversed the anchorage-independent growth and invasion phenotypes caused by RhoC overexpression. In the highly metastatic A375-M human melanoma cells, p190-RhoC-C specifically reversed migration, and invasion phenotypes attributed to RhoC up-regulation. Thus, we have developed a novel strategy utilizing RhoGAP-Rho chimeras to specifically down-regulate individual Rho activity and demonstrate that this approach may be applied to multiple human tumor cells to reverse the growth and/or invasion phenotypes associated with disregulation of a distinct subtype of Rho GTPase.

Rho-GAP Actin' at NMDA Receptor

Physical changes in synapses of neurons in the brain are thought to allow long-term storage of information and to provide molecular mechanisms that contribute to the processes of learning and memory. Changes in the actin cytoskeleton are associated with receptor-induced structural alterations in dendritic spines of postsynaptic neurons and are regulated by the small GTPases (guanosine triphosphatases) Rac and Rho. Now, in a hunt for proteins that physically interact with the NMDA (N-methyl-D-aspartate) receptor GluRϵ2 subunit, Nakazawa et al. have identified a protein with sequence similarity to GTPase-activating proteins (GAPs) that they call p250GAP. The p250GAP protein showed GAP activity toward the Rho-family GTPases RhoA and Cdc42 both in vitro and in transfected cells. Immunofluorescence microscopy and cell fractionation showed p250GAP to be concentrated in postsynaptic densities and localized near NMDA receptors. In Neuro-2A neuroblastoma cells in culture, overexpression of p250GAP either promoted or inhibited neurite outgrowth, depending on whether serum was or was not present. Treatment of cultures of hippocampal slices from mouse brain with NMDA appeared to cause dephosphorylation of p250GAP and to cause release of p250GAP from synaptic complexes. Such modulation could provide a mechanism for local remodeling of particular synaptic spines in which the NMDA receptor has been activated and might thereby contribute to generation of synaptic plasticity.T. Nakazawa, A. M. Watabe, T. Tezuka, Y. Yoshida, K. Yokoyama, H. Umemori, A. Inoue, S. Okabe, T. Manabe, T. Yamamoto, p250GAP, a novel brain-enriched GTPase-activating protein for Rho family GTPases, is involved in the N-methyl-D-aspartate receptor signaling. Mol. Biol. Cell 14, 2921-2934 (2003). [Abstract] [Full Text]

Rho-GAP Actin' at NMDA Receptor

Physical changes in synapses of neurons in the brain are thought to allow long-term storage of information and to provide molecular mechanisms that contribute to the processes of learning and memory. Changes in the actin cytoskeleton are associated with receptor-induced structural alterations in dendritic spines of postsynaptic neurons and are regulated by the small GTPases (guanosine triphosphatases) Rac and Rho. Now, in a hunt for proteins that physically interact with the NMDA ( N -methyl-D-aspartate) receptor GluRϵ2 subunit, Nakazawa et al. have identified a protein with sequence similarity to GTPase-activating proteins (GAPs) that they call p250GAP. The p250GAP protein showed GAP activity toward the Rho-family GTPases RhoA and Cdc42 both in vitro and in transfected cells. Immunofluorescence microscopy and cell fractionation showed p250GAP to be concentrated in postsynaptic densities and localized near NMDA receptors. In Neuro-2A neuroblastoma cells in culture, overexpression of p250GAP either promoted or inhibited neurite outgrowth, depending on whether serum was or was not present. Treatment of cultures of hippocampal slices from mouse brain with NMDA appeared to cause dephosphorylation of p250GAP and to cause release of p250GAP from synaptic complexes. Such modulation could provide a mechanism for local remodeling of particular synaptic spines in which the NMDA receptor has been activated and might thereby contribute to generation of synaptic plasticity. T. Nakazawa, A. M. Watabe, T. Tezuka, Y. Yoshida, K. Yokoyama, H. Umemori, A. Inoue, S. Okabe, T. Manabe, T. Yamamoto, p250GAP, a novel brain-enriched GTPase-activating protein for Rho family GTPases, is involved in the N -methyl-D-aspartate receptor signaling. Mol. Biol. Cell 14 , 2921-2934 (2003). [Abstract] [Full Text]

Spire contains actin binding domains and is related to ascidian posterior end mark-5.

Spire is a maternal effect locus that affects both the dorsal-ventral and anterior-posterior axes of the Drosophila egg and embryo. It is required for localization of determinants within the developing oocyte to the posterior pole and to the dorsal anterior corner. During mid-oogenesis, spire mutants display premature microtubule-dependent cytoplasmic streaming, a phenotype that can be mimicked by pharmacological disruption of the actin cytoskeleton with cytochalasin D. Spire has been cloned by transposon tagging and is related to posterior end mark-5, a gene from sea squirts that encodes a posteriorly localized mRNA. Spire mRNA is not, however, localized to the posterior pole. SPIRE also contains two domains with similarity to the actin monomer-binding WH2 domain, and we demonstrate that SPIRE binds to actin in the interaction trap system and in vitro. In addition, SPIRE interacts with the rho family GTPases RHOA, RAC1 and CDC42 in the interaction trap system. Thus, our evidence supports the model that SPIRE links rho family signaling to the actin cytoskeleton.

Lipid Products of Phosphoinositide 3-Kinase Interact with Rac1 GTPase and Stimulate GDP Dissociation

A number of reports suggest that under different conditions leading to cytoskeleton reorganization the GTPase Rac1 and possibly RhoA are downstream targets of phosphoinositide 3-kinase (PI 3-kinase). In order to gain more insight into this particular signaling pathway, we have addressed the question of a possible direct interaction of PI 3-kinase products with the Rho family GTPases RhoA, Rac1, and Cdc42. Using recombinant proteins, we found that Rac1 and, to a lesser extent, RhoA but not Cdc42 were capable to selectively bind to phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) in a mixture of crude brain phosphoinositides. Nucleotide-depleted Rac1 was the most efficient, but the GDP- and GTP-bound forms retained significant PtdIns(3,4,5)P3 binding activity. This protein-lipid association involved electrostatic as well as hydrophobic interactions, since both phosphate groups located at specific positions of the inositol ring and fatty-acyl chains were absolutely required. Based on the sequence of Rac1, two potential binding sites were identified, one at the C terminus and one in the extra alpha-helical domain. Deletion of these two domains resulted in a complete loss of binding to PI 3-kinase products. Finally, PtdIns(3, 4,5)P3 strongly stimulated GDP dissociation from Rac1 in a dose-dependent manner. In agreement, data obtained in intact cells suggest that PtdIns(3,4,5)P3 might target Rac1 to peculiar membrane domains, allowing formation of specific clusters containing not only small GTPases but other partners bearing pleckstrin homology domains such as specific exchange factors required for Rac1 and RhoA activation.

The Rho family GTPases RhoA, Racl , and CDC42Hsregulate transcriptional activation by SRF

The c-fos serum response element (SRE) forms a ternary complex with the transcription factors SRF (serum response factor) and TCF (ternary complex factor). By itself, SRF can mediate transcriptional activation induced by serum, lysophosphatidic acid, or intracellular activation of heterotrimeric G proteins. Activated forms of the Rho family GTPases RhoA, Rac1, and CDC42Hs also activate transcription via SRF and act synergistically at the SRE with signals that activate TCF. Functional Rho is required for signaling to SRF by several stimuli, but not by activated CDC42Hs or Rac1. Activation of the SRF-linked signaling pathway does not correlate with activation of the MAP kinases ERK, SAPK/JNK, or MPK2/p38. Functional Rho is required for regulated activity of the c-fos promoter. These results establish SRF as a nuclear target of a novel Rho-mediated signaling pathway.