RhoA

Distribution by Scientific Domains
Distribution within Life Sciences

Kinds of RhoA

  • active rhoa

  • Terms modified by RhoA

  • rhoa activation
  • rhoa activity

  • Selected Abstracts


    Changes in proximal femoral mineral geometry precede the onset of radiographic hip osteoarthritis: The study of osteoporotic fractures

    ARTHRITIS & RHEUMATISM, Issue 7 2009
    M. K. Javaid
    Objective Radiographic hip osteoarthritis (RHOA) is associated with increased hip areal bone mineral density (aBMD). This study was undertaken to examine whether femoral geometry is associated with RHOA independent of aBMD. Methods Participants in the Study of Osteoporotic Fractures in whom pelvic radiographs had been obtained at visits 1 and 5 (mean 8.3 years apart) and hip dual x-ray absorptiometry (DXA) had been performed (2 years after baseline) were included. Prevalent and incident RHOA phenotypes were defined as composite (osteophytes and joint space narrowing [JSN]), atrophic (JSN without osteophytes), or osteophytic (femoral osteophytes without JSN). Analogous definitions of progression were based on minimum joint space and total osteophyte score. Hip DXA scans were assessed using the Hip Structural Analysis program to derive geometric measures, including femoral neck length, width, and centroid position. Relative risks and 95% confidence intervals for prevalent, incident, and progressive RHOA per SD increase in geometric measure were estimated in a hip-based analysis using multinomial logistic regression with adjustment for age, body mass index, knee height, and total hip aBMD. Results In 5,245 women (mean age 72.6 years), a wider femoral neck with a more medial centroid position was associated with prevalent and incident osteophytic and composite RHOA phenotypes (P < 0.05). Increased neck width and centroid position were associated with osteophyte progression (both P < 0.05). No significant geometric associations with atrophic RHOA were found. Conclusion Differences in proximal femoral bone geometry and spatial distribution of bone mass occur early in hip OA and predict prevalent, incident, and progressive osteophytic and composite phenotypes, but not the atrophic phenotype. These bone differences may reflect responses to loading occurring early in the natural history of RHOA. [source]


    Defining incident radiographic hip osteoarthritis for epidemiologic studies in women

    ARTHRITIS & RHEUMATISM, Issue 4 2009
    Nigel K. Arden
    Objective To evaluate definitions of radiographic hip osteoarthritis (RHOA) for use in longitudinal epidemiologic studies of disease incidence in women. Methods We studied 5,839 women from the Study of Osteoporotic Fractures who had had serial pelvic radiographs obtained (mean of 8.3 years apart) and who were followed up (mean followup 7.1 years from the time of the second radiograph) for evaluation of clinical outcomes. Definitions of RHOA were assessed for construct validity (association with symptoms and signs at the time of the second radiograph) and predictive validity (association with total hip replacement [THR] and signs and symptoms a mean of 7.1 years later). Odds ratios (ORs) and 95% confidence intervals were calculated to assess the strength of association using logistic regression. Results The cumulative incidence of RHOA ranged from 2.2% to 11.7%. All definitions displayed significant construct validity; the most consistent was found for composite definitions that required the concurrent presence of 2 or more individual radiographic features and definitions based on stringent criteria for joint space narrowing. All definitions except minimum joint space ,2.5 mm displayed consistent predictive validity. Composite definitions had the strongest associations with THR (OR 10.5,18.5) and hip pain (OR 2.6,2.9). The hips identified as having OA by each definition varied, with especially small overlap between findings using definitions based on osteophytes and those using definitions based on joint space narrowing alone. Conclusion Most definitions of incident RHOA display good construct and predictive validity. Composite definitions have the best overall performance, and definitions requiring the presence of both osteophytes (in particular, femoral osteophytes) and joint space narrowing would be recommended for most epidemiologic and genetic studies. [source]


    The adaptive responses in several mediators linked with hypertrophy and atrophy of skeletal muscle after lower limb unloading in humans

    ACTA PHYSIOLOGICA, Issue 2 2009
    K. Sakuma
    Abstract Aim:, To determine the adaptive changes in several molecules regulating muscle hypertrophy and atrophy after unloading, we examined whether unilateral lower limb suspension changes the mRNA and protein levels of SRF-linked (RhoA, RhoGDI, STARS and SRF), myostatin-linked (myostatin, Smad2, Smad3 and FLRG) and Foxo-linked (P-Akt, Foxo1, Foxo3a and Atrogin-1) mediators. Methods:, A single lower limb of each of eight healthy men was suspended for 20 days. Biopsy specimens were obtained from the vastus lateralis muscle pre- and post-suspension. Results:, The volume of the vastus lateralis muscle was significantly decreased after unloading. The amount of RhoA, RhoGDI or SRF protein in the muscle was not significantly changed post-suspension. An RT-PCR semiquantitative analysis showed increased levels of myostatin mRNA but not Smad2, Smad3 or FLRG mRNA. Unloading did not elicit significant changes in the amount of p-Smad3 or myostatin protein in the muscle. The amount of p-Akt protein was markedly reduced in the unloaded muscle. Lower limb suspension did not influence the expression pattern of Foxo1, Foxo3a or Atrogin-1. Conclusion:, Unloading inducing a mild degree of muscle atrophy may decrease p-Akt and increase myostatin but not SRF-linked mediators. [source]


    Simulated microgravity activates MAPK pathways in fibroblasts cultured on microgrooved surface topography

    CYTOSKELETON, Issue 2 2008
    W. A. Loesberg
    Abstract This study evaluated in vitro the differences in morphological behaviour between fibroblast cultured on smooth and microgrooved substrata (groove depth: 0.5 ,m, width: 1 ,m), which were subjected to simulated microgravity. The aim of the study was to clarify which of these parameters was more dominant to determine cell behaviour. Morphological characteristics were investigated using scanning electron microscopy and fluorescence microscopy in order to obtain qualitative information on cell alignment. Expression of collagen type I, and ,1-, ,1-, ,3-integrin were investigated by QPCR. Finally, immunoblotting was applied to visualise MAPK signalling pathways. Microscopy and image analysis showed that the fibroblasts aligned along the groove direction on all textured surfaces. On the smooth substrata, cells had spread out in a random fashion. The alignment of cells cultured on grooved surfaces under simulated microgravity, after 48 h of culturing appeared similar to those cultured at 1g, although cell shape was different. Analysis of variance proved that all main parameters: topography, gravity force, and time were significant. In addition, gene levels were reduced by simulated microgravity particularly those of ,3-integrin and collagen, however alpha-1 and beta-1 integrin levels were up-regulated. ERK1/2 was reduced in RPM, however, JNK/SAPK and p38 remained active. The members of the small GTPases family were stimulated under microgravity, particularly RhoA and Cdc42. The results are in agreement that application of microgravity to fibroblasts promotes a change in their morphological appearance and their expression of cell-substratum proteins through the MAPK intracellular signalling pathways. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source]


    An actin-stabilizing peptide conjugate deduced from the major outer sheath protein of the bacterium Treponema denticola

    CYTOSKELETON, Issue 9 2007
    Mohsen Amin
    Abstract A synthetic peptide conjugated to bovine serum albumin, P34BSA, based on a 10-mer in the deduced amino acid sequence of the major outer sheath protein of Treponema denticola, was found to stabilize actin filaments of fibroblasts. Pretreatment of cells with P34BSA inhibited the actin disruption induced by cytochalasin D and latrunculin B. P34BSA was taken up by the cells and localized among actin filaments. P34BSA bound actin from fibroblast lysates, and cell exposure to P34BSA led to the activation of RhoA, a key regulator of actin filament assembly in fibroblasts. Exposure of fibroblasts to P34BSA retarded their migration on a collagen substratum. P34BSA also inhibited chemotaxis of murine neutrophils. Our findings with a novel peptide conjugate imply that bacterial proteins known to perturb the cytoskeleton represent a rich source of molecular models upon which to design synthetic reagents for modulating actin-dependent cellular functions. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source]


    Maspin controls mammary tumor cell migration through inhibiting Rac1 and Cdc42, but not the RhoA GTPase

    CYTOSKELETON, Issue 5 2007
    Heidi Y. Shi
    Abstract Rac1 and Cdc42 are members of the Rho family of small GTPases that play essential roles in diverse cellular functions, including cell migration. The activities of these Rho family proteins are controlled by growth factor receptor activation and cell-ECM interactions. Here, we show that maspin, a well-documented tumor suppressor gene, also controls cell motility through inhibiting Rac1/Cdc42 activity. Using the GST-PAK and GST-Rho binding protein pull-down assays for GTP-bound Rac1, Cdc42, and RhoA, we showed that treatment of MDA-MB-231 tumor cells with recombinant maspin for a short time period significantly inhibited the activity of Rac1 and Cdc42, but not RhoA. The reactive site loop (RSL) within maspin protein is the functional domain involved in the inhibition. Maspin mutants with the RSL deleted or a point mutation in the RSL region lost their inhibitory activity. We further examined the ability of maspin to inhibit Rac1- and Cdc42-mediated signaling pathways and transcription factors. Treatment of MDA-MB-231 cells with maspin led to the inhibition of JNK kinase activity as assayed by immuno-kinase assays. In addition, the AP-1 transcription activity downstream of JNK kinase pathway was also reduced. Together, we have identified Rac1 and Cdc42 as the downstream targets that mediate the inhibition of mammary tumor cell migration by maspin. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source]


    Myosin-mediated cytoskeleton contraction and Rho GTPases regulate laminin-5 matrix assembly

    CYTOSKELETON, Issue 2 2004
    Gregory W. deHart
    Abstract Laminin-5 is a major structural element of epithelial tissue basement membranes. In the matrix of cultured epithelial cells, laminin-5 is arranged into intricate patterns. Here we tested a hypothesis that myosin II-mediated actin contraction is necessary for the proper assembly of a laminin-5 matrix by cultured SCC12 epithelial cells. To do so, the cells were treated with ML-7, a myosin II light chain kinase inhibitor, or Y-27632, an inhibitor of Rho-kinase (ROCK), both of which block actomyosin contraction. Under these conditions, laminin-5 shows an aberrant localization in dense patches at the cell periphery. Since ROCK activity is regulated by the small GTPase Rho, this suggests that members of the Rho family of GTPases may also be important for laminin-5 matrix assembly by SCC12 cells. We confirmed this hypothesis since SCC12 cells expressing mutant proteins that inhibit RhoA, Rac, and Cdc42 assemble the same aberrant laminin-5 protein arrays as drug-treated cells. We have also evaluated the organization of the laminin-5 receptors ,3,1 and ,6,4 integrin and hemidesmosome proteins in ML-7- and Y-27632-treated cells or in cells in which RhoA, Rac, and Cdc42 activity were inhibited. In all instances, ,3,1 and ,6,4 integrin heterodimers, as well as hemidesmosome proteins, localize precisely with laminin-5 in the matrix of the cells. In summary, our results provide evidence that myosin II-mediated actin contraction and the activity of Rho GTPases are necessary for the proper organization of a laminin-5 matrix and localization of hemidesmosome protein arrays in epithelial cells. Cell Motil. Cytoskeleton 57:107,117, 2004. © 2004 Wiley-Liss, Inc. [source]


    Blocking Dishevelled signaling in the noncanonical Wnt pathway in sea urchins disrupts endoderm formation and spiculogenesis, but not secondary mesoderm formation

    DEVELOPMENTAL DYNAMICS, Issue 7 2009
    Christine A. Byrum
    Abstract Dishevelled (Dsh) is a phosphoprotein key to beta-catenin dependent (canonical) and beta-catenin independent (noncanonical) Wnt signaling. Whereas canonical Wnt signaling has been intensively studied in sea urchin development, little is known about other Wnt pathways. To examine roles of these beta-catenin independent pathways in embryogenesis, we used Dsh-DEP, a deletion construct blocking planar cell polarity (PCP) and Wnt/Ca2+ signaling. Embryos overexpressing Dsh-DEP failed to gastrulate or undergo skeletogenesis, but produced pigment cells. Although early mesodermal gene expression was largely unperturbed, embryos exhibited reduced expression of genes regulating endoderm specification and differentiation. Overexpressing activated beta-catenin failed to rescue Dsh-DEP embryos, indicating that Dsh-DEP blocks endoderm formation downstream of initial canonical Wnt signaling. Because Dsh-DEP-like constructs block PCP signaling in other metazoans, and disrupting RhoA or Fz 5/8 in echinoids blocks subsets of the Dsh-DEP phenotypes, our data suggest that noncanonical Wnt signaling is crucial for sea urchin endoderm formation and skeletogenesis. Developmental Dynamics 238:1649,1665, 2009. © 2009 Wiley-Liss, Inc. [source]


    Dynamic expression patterns of RhoV/Chp and RhoU/Wrch during chicken embryonic development

    DEVELOPMENTAL DYNAMICS, Issue 4 2008
    Cécile Notarnicola
    Abstract Rho GTPases play central roles in the control of cell adhesion and migration, cell cycle progression, growth, and differentiation. However, although most of our knowledge of Rho GTPase function comes from the study of the three classic Rho GTPases RhoA, Rac1, and Cdc42, recent studies have begun to explore the expression, regulation, and function of some of the lesser-known members of the Rho GTPase family. In the present study, we cloned the avian orthologues of RhoV (or Chp for Cdc42 homologous protein) and RhoU (or Wrch - 1 for Wnt-regulated Cdc42 homolog-1) and examined their expression patterns by in situ hybridization analysis both during early chick embryogenesis and later on, during gastrointestinal tract development. Our data show that both GTPases are detected in the primitive streak, the somites, the neural crest cells, and the gastrointestinal tract with distinct territories and/or temporal expression windows. Although both proteins are 90% identical, our results indicate that cRhoV and cRhoU are distinctly expressed during chicken embryonic development. Developmental Dynamics 237:1165,1171, 2008. © 2008 Wiley-Liss, Inc. [source]


    Myosin-II negatively regulates minor process extension and the temporal development of neuronal polarity

    DEVELOPMENTAL NEUROBIOLOGY, Issue 5 2009
    K.M. Kollins
    Abstract The earliest stage in the development of neuronal polarity is characterized by extension of undifferentiated "minor processes" (MPs), which subsequently differentiate into the axon and dendrites. We investigated the role of the myosin II motor protein in MP extension using forebrain and hippocampal neuron cultures. Chronic treatment of neurons with the myosin II ATPase inhibitor blebbistatin increased MP length, which was also seen in myosin IIB knockouts. Through live-cell imaging, we demonstrate that myosin II inhibition triggers rapid minor process extension to a maximum length range. Myosin II activity is determined by phosphorylation of its regulatory light chains (rMLC) and mediated by myosin light chain kinase (MLCK) or RhoA-kinase (ROCK). Pharmacological inhibition of MLCK or ROCK increased MP length moderately, with combined inhibition of these kinases resulting in an additive increase in MP length similar to the effect of direct inhibition of myosin II. Selective inhibition of RhoA signaling upstream of ROCK, with cell-permeable C3 transferase, increased both the length and number of MPs. To determine whether myosin II affected development of neuronal polarity, MP differentiation was examined in cultures treated with direct or indirect myosin II inhibitors. Significantly, inhibition of myosin II, MLCK, or ROCK accelerated the development of neuronal polarity. Increased myosin II activity, through constitutively active MLCK or RhoA, decreased both the length and number of MPs and, consequently, delayed or abolished the development of neuronal polarity. Together, these data indicate that myosin II negatively regulates MP extension, and the developmental time course for axonogenesis. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009 [source]


    RhoA/ROCK and Cdc42 regulate cell-cell contact and N-cadherin protein level during neurodetermination of P19 embryonal stem cells

    DEVELOPMENTAL NEUROBIOLOGY, Issue 3 2004
    Isabel Laplante
    Abstract RhoGTPases regulate actin-based signaling cascades and cellular contacts. In neurogenesis, their action modulates cell migration, neuritogenesis, and synaptogenesis. Murine P19 embryonal stem cells differentiate to neurons upon aggregation in the presence of retinoic acid, and we previously showed that RhoA and Cdc42 RhoGTPases are sequentially up-regulated during neuroinduction, suggesting a role at this very early developmental stage. In this work, incubation of differentiating P19 cells with C3 toxin resulted in decreased aggregate cohesion and cadherin protein level. In contrast, C3 effects were not observed in cells overexpressing recombinant dominant active RhoA. On the other hand, C3 did not affect cadherin in uninduced cells and their postmitotic neuronal derivatives, respectively expressing E- and N-cadherin. RhoA is thus influential on cell aggregation and cadherin expression during a sensitive time window that corresponds to the switch of E- to N-cadherin. Cell treatment with Y27632 inhibitor of Rho-associated-kinase ROCK, or advanced overexpression of Cdc42 by gene transfer of a constitutively active form of the protein reproduced C3 effects. RhoA-antisense RNA also reduced cadherin level and the size of cell aggregates, and increased the generation of fibroblast-like cells relative to neurons following neuroinduction. Colchicin, a microtubule disrupter, but not cytochalasin B actin poison, importantly decreased cadherin in neurodifferentiating cells. Overall, our results indicate that the RhoA/ROCK pathway regulates cadherin protein level and cell-cell interactions during neurodetermination, with an impact on the efficiency of the process. The effect on cadherin seems to involve microtubules. The importance of correct timing of RhoA and Cdc42 functional expression in neurogenesis is also raised. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 289,307, 2004 [source]


    Neuroprotective signal transduction in model motor neurons exposed to thrombin: G-protein modulation effects on neurite outgrowth, Ca2+ mobilization, and apoptosis ,

    DEVELOPMENTAL NEUROBIOLOGY, Issue 2 2001
    Irina V. Smirnova
    Abstract Thrombin, the ultimate protease in the blood coagulation cascade, mediates its known cellular effects by unique proteolytic activation of G-protein-coupled protease-activated receptors (PARs), such as PAR1, PAR3, and PAR4, and a "tethered ligand" mechanism. PAR1 is variably expressed in subpopulations of neurons and largely determines thrombin's effects on morphology, calcium mobilization, and caspase-mediated apoptosis. In spinal cord motoneurons, PAR1 expression correlates with transient thrombin-mediated [Ca2+]i flux, receptor cleavage, and elevation of rest [Ca2+]i activating intracellular proteases. At nanomolar concentrations, thrombin retracts neurites via PAR1 activation of the monomeric, 21 kDa Ras G-protein RhoA, which is also involved in neuroprotection at lower thrombin concentrations. Such results suggest potential downstream targets for thrombin's injurious effects. Consequently, we employed several G-protein-specific modulators prior to thrombin exposure in an attempt to uncouple both heterotrimeric and monomeric G-proteins from motoneuronal PAR1. Cholera toxin, stimulating Gs, and lovastatin, which blocks isoprenylation of Rho, reduced thrombin-induced calcium mobilization. In contrast, pertussis toxin and mastoparan, inhibiting or stimulating Go/Gi, were found to exacerbate thrombin action. Effects on neuronal rounding and apoptosis were also detected, suggesting therapeutic utility may result from interference with downstream components of thrombin signaling pathways in human motor neuron disorders, and possibly other neurodegenerative diseases. Published 2001 John Wiley & Sons, Inc. J Neurobiol 48: 87,100, 2001 [source]


    Zoledronate has an antitumor effect and induces actin rearrangement in dexamethasone-resistant myeloma cells

    EUROPEAN JOURNAL OF HAEMATOLOGY, Issue 5 2007
    Masayuki Koizumi
    Abstract New strategies are needed to overcome the resistance of multiple myeloma (MM) to dexamethasone (Dex). Several recent in vitro studies demonstrated the antitumor effect of nitrogen-containing amino-bisphosphonates (N-BPs) in various tumor cell lines. Inhibition of the prenylation of small G proteins is assumed to be one of the principal mechanisms by which N-BPs exert their effects. There have been few reports on N-BP treatment of MM cells that are resistant to Dex. Additionally, it is not known how small G proteins are altered in N-BP-treated MM cells. In this study, we evaluated the effect of the most potent N-BP, zoledronate (ZOL), on a Dex-resistant human MM cell subline (Dex-R) that we established from the well-documented RPMI8226 cell line. ZOL reduced the viability and induced apoptosis of Dex-R cells. Some of the ZOL-treated RPMI8226 cells and ZOL-treated Dex-R cells were elongated; however, elongated cells were not seen among the Dex-treated RPMI8226 cells. Furthermore, we found that portions of the small G proteins, Rho and Rap1A, were unprenylated in the ZOL-treated MM cells. Geranylgeraniol reduced the above-mentioned ZOL-induced effects. These findings suggest that ZOL may be beneficial for the treatment of Dex-resistant MM by suppressing the processing of RhoA and Rap1A. [source]


    The RhoA- and CDC42-specific exchange factor Dbs promotes expansion of immature thymocytes and deletion of double-positive and single-positive thymocytes

    EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 3 2004

    Abstract Specific members of the Rho family of GTPases exert unique influences on thymocyte proliferation, differentiation and deletion. Dbs is a guanine nucleotide exchange factor which is expressed throughout thymocyte development and is able to activate the Rho family GTPases CDC42, RhoA and RhoG. Transgenic mice expressing an activated form of Dbs had increased numbers of double-negative thymocytes. The Dbs transgene promoted expansion of double-negative thymocytes in the absence of pre-TCR, but had no effect on pre-TCR-dependent differentiation of double-negative thymocytes into double-positive thymocytes. Transgenic double-positive thymocytes were proliferative in vivo, but were also susceptible to apoptosis in vivo and in vitro. The transgenic single-positive thymocytes had attenuated proliferative responses following TCR ligation, and were depleted rather than expanded during culture in the presence of anti-CD3. When expressing a positively selectable TCR, transgenic double-positive thymocytes were increased in number and activated, but the output of single-positive thymocytes was reduced. Transgenic double-positive thymocytes were acutely sensitive to deletion by TCR ligation in vivo. These results indicate that activation of Dbs has the potential to promote proliferation throughout thymocyte development, but also sensitizes double-positive and single-positive thymocytes to deletion. [source]


    Characterization of the expression of PDZ-RhoGEF, LARG and G,12/G,13 proteins in the murine nervous system

    EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2002
    R. Kuner
    Small GTPases of the Rho-family, like Rho, Rac and Cdc42, are involved in neuronal morphogenesis by regulating growth cone morphology or dendritic spine formation. G-proteins of the G12 -family, G12 and G13, couple G-protein-coupled receptors (GPCRs) to the activation of RhoA. Recently, two novel Rho-specific guanine nucleotide exchange factors (RhoGEFs), PDZ-RhoGEF and LARG, have been identified to interact with the activated ,-subunits of G12/G13 and are thus believed to mediate GPCR-induced Rho activation. Although studies in neuronal cell lines have shown that G12/G13 and PDZ-RhoGEF mediate GPCR-induced neurite retraction, the role, as well as the expression of this signalling pathway, in intact brain has not been adequately studied. In the present study, we have characterized systematically the expression of G,12, G,13, PDZ-RhoGEF and LARG in various murine tissues as well as their subcellular localization in the central and peripheral nervous systems. By performing immunohistochemistry, using polyclonal antibodies raised against the above proteins, we observed that G,12, G,13 and their RhoGEF-effectors are distributed widely in the mammalian nervous system. Moreover, these proteins localize to distinct morphological compartments within neurons. While LARG and G,12 were mainly found in somata of the neurons, PDZ-RhoGEF and G,13 were predominantly localized in the neuropil of central neurons. Interestingly, PDZ-RhoGEF is a neural-specific protein, whereas LARG is nearly ubiqoutous. Our data provide evidence that the G12/13,RhoGEF-mediated pathway is present throughout the adult brain and may be involved in regulation of neuronal morphogenesis and function via GPCRs. [source]


    Inhibition of Rac1 decreases the severity of pancreatitis and pancreatitis-associated lung injury in mice

    EXPERIMENTAL PHYSIOLOGY, Issue 10 2008
    Marcelo G. Binker
    Pancreatitis is a disease with high morbidity and mortality. In vitro experiments on pancreatic acini showed that supramaximal but not submaximal cholecystokinin (CCK) stimulation induces effects in the acinar cell that can be correlated with acinar morphological changes observed in the in vivo experimental model of cerulein-induced pancreatitis. The GTPase Rac1 was previously reported to be involved in CCK-evoked amylase release from pancreatic acinar cells. Here, we demonstrate that pretreatment with the Rac1 inhibitor NSC23766 (100 ,m, 2 h) effectively blocked Rac1 translocation and activation in CCK-stimulated pancreatic acini, without affecting activation of its closely related GTPase, RhoA. This specific Rac1 inhibition decreased supramaximal (10 nM) CCK-stimulated acinar amylase release (27.% reduction), which seems to be connected to the reduction observed in serum amylase (46.6% reduction) and lipase levels (46.1% reduction) from cerulein-treated mice receiving NSC23766 (100 nmol h,1). The lack of Rac1 activation also reduced formation of reactive oxygen species (ROS; 20.8% reduction) and lactate dehydrogenase release (LDH; 24.3% reduction), but did not alter calcium signaling or trypsinogen activation in 10 nM CCK-stimulated acini. In the in vivo model, the cerulein-treated mice receiving NSC23766 also presented a decrease in both pancreatic and lung histopathological scores (reduction in oedema, 32.4 and 66.4%; haemorrhage, 48.3 and 60.2%; and leukocyte infiltrate, 53.5 and 43.6%, respectively; reduction in pancreatic necrosis, 65.6%) and inflammatory parameters [reduction in myeloperoxidase, 52.2 and 38.9%; nuclear factor ,B (p65), 61.3 and 48.6%; and nuclear factor ,B (p50), 46.9 and 44.9%, respectively], together with lower serum levels for inflammatory (TNF-,, 40.4% reduction) and cellular damage metabolites (LDH, 52.7% reduction). Collectively, these results suggest that pharmacological Rac1 inhibition ameliorates the severity of pancreatitis and pancreatitis-associated lung injury through the reduction of pancreatic acinar damage induced by pathological digestive enzyme secretion and overproduction of ROS. [source]


    RhoA, encoding a Rho GTPase, is associated with smoking initiation

    GENES, BRAIN AND BEHAVIOR, Issue 8 2007
    X. Chen
    We used microarray analysis of acute nicotine responses in mouse brain to choose rationale candidates for human association studies on tobacco smoking and nicotine dependence (ND). Microarray studies on the time,course of acute response to nicotine in mouse brain identified 95 genes regulated in ventral tegmental area. Among these, 30 genes were part of a gene network, with functions relevant to neural plasticity. On this basis and their known roles in drug abuse or synaptic plasticity, we chose the genes RhoA and Ywhag as candidates for human association studies. A synteny search identified human orthologs and we investigated their role in tobacco smoking and ND in a human case,control association study. We genotyped five and three single nucleotide polymorphisms from the RhoA and Ywhag genes, respectively. Both single marker and haplotype analyses were negative for the Ywhag gene. For the RhoA gene, rs2878298 showed highly significant genotypic association with both smoking initiation (SI) and ND (P = 0.00005 for SI and P = 0.0007 for ND). In the allelic analyses, rs2878298 was only significant for SI. In the multimarker haplotype analyses, significant association with SI was found for the RhoA gene (empirical global P values ranged from 9 × 10,5 to 10,5). In all multimarker combinations analyzed, with or without inclusion of the single most significant marker rs2878298, identical risk and protective haplotypes were identified. Our results indicated that the RhoA gene is likely involved in initiation of tobacco smoking and ND. Replication and future model system studies will be needed to validate the role of RhoA gene in SI and ND. [source]


    Proteomic profiling reveals the prognostic value of adenomatous polyposis coli,end-binding protein 1 in hepatocellular carcinoma,

    HEPATOLOGY, Issue 6 2008
    Tatsuya Orimo
    Histological differentiation is a major pathological parameter associated with poor prognosis in patients with hepatocellular carcinoma (HCC) and the molecular signature underlying HCC differentiation may involve key proteins potentially affecting the malignant characters of HCC. To develop prognostic biomarkers for HCC, we examined the global protein expression profiles of 45 surgically resected tissues, including 27 HCCs with different degree of histological differentiation, 11 adjacent nontumor tissues, and seven normal liver tissues. Unsupervised classification grouped the 45 samples according to their histological classification based on the protein expression profiles created by laser microdissection and two-dimensional difference gel electrophoresis (2D-DIGE). Statistical analysis and mass spectrometry identified 26 proteins with differential expression, of which 14 were functionally linked to c-Myc, AP-1, HIF1A, hepatocyte nuclear factor 4 alpha, or the Ras superfamily (RhoA, CDC42, and Rac1). Among the proteins identified, we focused on APC-binding protein EB1 (EB1) because it was dominantly expressed in poorly differentiated HCCs, which generally correlate with the poor prognosis in patients with HCC. In addition, EB1 is controlled by c-Myc, RhoA, and CDC42, which have all been linked to HCC malignancy. Immunohistochemistry in a further 145 HCC cases revealed that EB1 significantly correlated with the degree of histological differentiation (P < 0.001), and univariate and multivariate analyses indicated that EB1 is an independent prognostic factor for recurrence (hazard ratio, 2.740; 95% confidence interval, 1.771,4.239; P < 0.001) and survival (hazard ratio, 2.256; 95% confidence interval, 1.337,3.807; P = 0.002) of patients with HCC after curative surgery. Conclusion: Proteomic profiling revealed the molecular signature behind the progression of HCC, and the prognostic value of EB1 in HCC. (HEPATOLOGY 2008;48:1851-1863.) [source]


    Dominant-negative Rac increases both inherent and ionizing radiation-induced cell migration in C6 rat glioma cells

    INTERNATIONAL JOURNAL OF CANCER, Issue 8 2006
    So-Young Hwang
    Abstract Rho-like GTPases, including Cdc42, Rac1 and RhoA, regulate distinct actin cytoskeleton changes required for cell adhesion, migration and invasion. In the present study, we examined the role of Rac signaling in inherent migration, as well as radiation-induced migration, of rat glioma cells. Stable overexpression of dominant-negative Rac1N17 in a C6 rat glioma cell line (C6-RacN17) promoted cell migration, and ionizing radiation further increased this migration. Migration was accompanied by decreased expression of the focal adhesion molecules FAK and paxillin. Focal contacts and actin stress fibers were also reduced in C6-RacN17 cells. Downstream effectors of Rac include JNK and p38 MAP kinases. Irradiation transiently activated p38, JNK and ERK1/2 MAP kinases in C6-RacN17 cells, while p38 and JNK were constitutively activated in C6 control cells. Blocking JNK activity with JNK inhibitor SP600125 inhibited migration, suggesting that the JNK pathway may regulate radiation-induced, as well as inherent, migration of C6-RacN17 cells. Additionally, the radiation-induced migration increase was also inhibited by SB203580, a specific inhibitor of p38 MAP kinase. However, PD98059, a MEK kinase 1 inhibitor, failed to influence migration. This is the first evidence that suppression of Rac signaling may be involved in invasion or metastasis of glioma cells before and/or after radiotherapy. These data further suggest that radiotherapy for malignant glioma needs to be used with caution because of the potential for therapy-induced cell migration or invasion and that pharmacological inhibition of cell migration and invasion through targeting the Rac signaling pathway may represent a new approach for improving the therapeutic efficacy of radiotherapy for malignant glioma. © 2005 Wiley-Liss, Inc. [source]


    RhoA/ROK Pathway Related to the Mechanism of Higher Susceptibility to Spasm in RA Than in IMA

    JOURNAL OF CARDIAC SURGERY, Issue 6 2009
    Xia Kun M.D.
    Methods: RA, IMA, and GSV that would otherwise have been discarded were collected from 25 patients who underwent coronary artery bypass grafting. Eleven matched rings of RA, IMA, and GSV were used to evaluate the vasodilatory properties of 10,7,10,4 mol/l of fasudil, a Rho-kinase inhibitor, by using in vitro organ chambers. Another 14 matched RA, IMA, and GSV were used to demonstrate the immunohistochemistry (IHC) of RhoA and mRNA of RhoA and Rho kinase. Results: The maximal vasodilation of RA to fasudil was significantly greater than IMA. RhoA protein IHC staining was different in IMA, RA, and GSV (RA > GSV >IMA). The expression of RhoA and Rho kinase mRNA in the RA was significantly greater than in the IMA. Conclusions: The expression of RhoA/Rho kinase mRNA and protein and function in the RA were significantly stronger than in the IMA, suggesting that RhoA/Rho kinase pathway may be one mechanism by which RA is more susceptible to spasm than IMA. Rho kinase inhibitors can be effective drug candidates to prevent and treat vasospasm. [source]


    Expression of RhoA by inflammatory macrophages and T cells in rat experimental autoimmune neuritis

    JOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 1 2007
    Zhiren Zhang
    Abstract RhoA is one of the best-studied members of Rho GTPases. Experimental autoimmune neuritis (EAN), which is characterized by infiltration of T cells and macrophages into the peripheral nervous system, is an autoantigen-specific T-cell-mediated animal model of human Guillain-Barr, Syndrome. In this study, RhoA expression has been investigated in the dorsal/ventral roots of EAN rats by immunohistochemistry. A significant accumulation of RhoA+ cells was observed on Day 12, with a maximum around Day 15, correlating to the clinical severity of EAN. In dorsal/ventral roots of EAN, RhoA+ cells were seen in perivascular areas but also in the parenchyma. Furthermore, double-labelling experiments showed that the major cellular sources of RhoA were reactive macrophages and T cells. In conclusion, this is the first demonstration of the presence of RhoA in the dorsal/ventral roots of EAN. The time courses and cellular sources of RhoA together with the functions of RhoA indicate that RhoA may function to facilitate macrophage and T-cell infiltration in EAN and therefore could be a potential therapeutic target. [source]


    Reversible translocation of p115-RhoGEF by G12/13 -coupled receptors

    JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2008
    Bruno H. Meyer
    Abstract G protein-coupled receptors (GPCRs) are important targets for medicinal agents. Four different G protein families, Gs, Gi, Gq, and G12, engage in their linkage to activation of receptor-specific signal transduction pathways. G12 proteins were more recently studied, and upon activation by GPCRs they mediate activation of RhoGTPase guanine nucleotide exchange factors (RhoGEFs), which in turn activate the small GTPase RhoA. RhoA is involved in many cellular and physiological aspects, and a dysfunction of the G12/13 -Rho pathway can lead to hypertension, cardiovascular diseases, stroke, impaired wound healing and immune cell functions, cancer progression and metastasis, or asthma. In this study, regulator of G protein signaling (RGS) domain-containing RhoGEFs were tagged with enhanced green fluorescent protein (EGFP) to detect their subcellular localization and translocation upon receptor activation. Constitutively active G,12 and G,13 mutants induced redistribution of these RhoGEFs from the cytosol to the plasma membrane. Furthermore, a pronounced and rapid translocation of p115-RhoGEF from the cytosol to the plasma membrane was observed upon activation of several G12/13 -coupled GPCRs in a cell type-independent fashion. Plasma membrane translocation of p115-RhoGEF stimulated by a GPCR agonist could be completely and rapidly reversed by subsequent application of an antagonist for the respective GPCR, that is, p115-RhoGEF relocated back to the cytosol. The translocation of RhoGEF by G12/13 -linked GPCRs can be quantified and therefore used for pharmacological studies of the pathway, and to discover active compounds in a G12/13 -related disease context. J. Cell. Biochem. 104: 1660,1670, 2008. © 2008 Wiley-Liss, Inc. [source]


    Lysophosphatidic acid in malignant ascites stimulates migration of human mesenchymal stem cells

    JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2008
    Mi Jeong Lee
    Abstract Lysophosphatidic acid (LPA) is elevated in ascites of ovarian cancer patients and is involved in growth and invasion of ovarian cancer cells. Accumulating evidence suggests a pivotal role of mesenchymal stem cells (MSCs) or stromal cells in tumorigenesis. In the present study, we demonstrated that ascites from ovarian cancer patients and LPA increased migration of human MSCs. The migration of MSCs induced by LPA and malignant ascites was completely abrogated by pretreatment with Ki16425, an antagonist of LPA receptors, and by silencing of endogenous LPA1, but not LPA2, with small interference RNA, suggesting a key role of LPA played in the malignant ascites-induced migration. LPA induced activation of ERK through pertussis toxin-sensitive manner, and pretreatment of MSCs with U0126, a MEK inhibitor, or pertussis toxin attenuated the LPA-induced migration. Moreover, LPA induced activation of RhoA in MSCs, and pretreatment of the cells with Y27632, a Rho kinase inhibitor, markedly inhibited the LPA-induced migration. In addition, LPA and malignant ascites increased intracellular concentration of calcium in MSCs, and Ki16425 completely inhibited the elevation of intracellular calcium. These results suggest that LPA is a crucial component of the malignant ascites which induce the migration of MSCs and elevation of intracellular calcium. J. Cell. Biochem. 104: 499,510, 2008. © 2007 Wiley-Liss, Inc. [source]


    Carboxy-terminal fragment of osteogenic growth peptide regulates myeloid differentiation through RhoA

    JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2004
    Letizia Mattii
    Abstract The carboxy-terminal fragment of osteogenic growth peptide, OGP(10,14), is a pentapeptide with bone anabolic effects and hematopoietic activity. The latter activity appears to be largely enhanced by specific growth factors. To study the direct activity of OGP(10,14) on myeloid cells, we tested the pentapeptide proliferating/differentiating effects in HL60 cell line. In this cell line, OGP(10,14) significantly inhibited cell proliferation, and enhanced myeloperoxidase (MPO) activity and nitroblue tetrazolium reducing ability. Moreover, it induced cytoskeleton remodeling and small GTP-binding protein RhoA activation. RhoA, which is known to be involved in HL60 differentiation, mediated these effects as shown by using its specific inhibitor, C3. Treatment with GM-CSF had a comparable OGP(10,14) activity on proliferation, MPO expression, and RhoA activation. Further studies on cell proliferation and RhoA activation proved enhanced activity by association of the two factors. These results strongly suggest that OGP(10,14) acts directly on HL60 cells by activating RhoA signaling although other possibilities cannot be ruled out. © 2004 Wiley-Liss, Inc. [source]


    Sphingosine 1-phosphate induces cell contraction via calcium-independent/Rho-dependent pathways in undifferentiated skeletal muscle cells

    JOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2004
    L. Formigli
    We have previously shown that sphingosine 1-phosphate (S1P) can induce intracellular Ca2+ mobilization and cell contraction in C2C12 myoblasts and that the two phenomena are temporally unrelated. Although Ca2+ -independent mechanisms of cell contraction have been the focus of numerous studies on Ca2+ sensitization of smooth muscle, comparatively less studies have focused on the role that these mechanisms play in the regulation of skeletal muscle contractility. Phosphorylation and activation of myosin by Rho-dependent kinase mediate most of Ca2+ -independent contractile responses. In the present study, we examined the potential role of Rho/Rho-kinase cascade activation in S1P-induced C2C12 cell contraction. First, we showed that depletion of Ca2+, by pre-treatment with BAPTA, did not affect S1P-induced myoblastic contractility, whereas it abolished S1P-induced Ca2+ transients. These results correlated with the absence of troponin C and with the immature cytoskeletal organization of these cells. Experimental evidence demonstrating the involvement of Rho pathway in S1P-stimulated myoblast contraction included: the activation/translocation of RhoA to the membrane in response to agonist-stimulation in cells depleted of Ca2+ and the inhibition of dynamic changes of the actin cytoskeleton in cells where Rho functions had been inhibited either by overexpression of RhoGDI, a physiological inhibitor of GDP dissociation from Rho proteins, or by pretreatment with Y-27632, a specific Rho kinase inhibitor. Contribution of protein kinase C in this cytoskeletal rearrangement was also evaluated. However, the pretreatment with Gö6976 or rottlerin, specific inhibitors of PKC, and PKC,, respectively, failed to inhibit the agonist-induced myoblastic contraction. Single particle tracking of G-actin fluorescent probe was performed to statistically evaluate actin cytoskeletal dynamics in response to S1P. Stimulation with S1P was also able to increase the phosphorylation level of myosin light chain II. In conclusion, our results strongly suggest that Ca2+ -independent/Rho-Rho kinase-dependent pathways may exert an important role in S1P-induced myoblastic cell contraction. J. Cell. Physiol. 198: 1,11, 2004© 2003 Wiley-Liss, Inc. [source]


    RhoE stimulates neurite-like outgrowth in PC12 cells through inhibition of the RhoA/ROCK-I signalling

    JOURNAL OF NEUROCHEMISTRY, Issue 4 2010
    Raquel Talens-Visconti
    J. Neurochem. (2010) 112, 1074,1087. Abstract Neurite formation involves coordinated changes between the actin cytoskeleton and the microtubule network. Rho GTPases are clearly implicated in several aspects of neuronal development and function. Indeed, RhoA is a negative regulator of neurite outgrowth and its effector Rho-kinase mediates the Rho-driven neurite retraction. Considering that RhoE/round protein (Rnd3) acts antagonistically to RhoA and it is also able to bind and inhibit rho kinase-I (p160ROCK) , ROCK-I, it is tempting to speculate a role of RhoE in neurite formation. We show for the first time that, in the absence of nerve growth factor (NGF), RhoE induces neurite-like outgrowth. Our results demonstrate that over-expression of RhoE decreases the activity of RhoA and reduces the expression of both ROCK-I and the phosphorylated myosin light chain phosphatase (MLCPp). Conversely, over-expression of either active RhoA or ROCK-I abolishes the RhoE-promoted neurite outgrowth, suggesting that RhoE induces neurite-like formation through inhibition of the RhoA/ROCK-I signalling. We also show that Rac and Cdc42 have a role in RhoE-induced neurite outgrowth. Finally, the present data further indicate that RhoE may be involved in the NGF-induced neurite outgrowth in PC12 cells, as depletion of RhoE by siRNA reduces the neurite formation induced by NGF. These findings provide new insights into the molecular mechanism implicated in neuronal development and may provide novel therapeutic targets in neurodegenerative disorders. [source]


    LIM-only protein 4 interacts directly with the repulsive guidance molecule A receptor Neogenin

    JOURNAL OF NEUROCHEMISTRY, Issue 2 2008
    Gregor Schaffar
    Abstract Repulsive guidance molecule A (RGM A) was recently described as a potent inhibitor of neuroregeneration in a rat spinal cord injury model. The receptor mediating RGM A's repulsive activity was shown to be Neogenin, a member of the Deleted in Colorectal Cancer (DCC) family of netrin receptors. Binding of RGM A to Neogenin induces activation of the small GTPase RhoA and of its effector Rho-kinase by an unknown mechanism. Here we show, that the cytoplasmic tail of Neogenin interacts directly with the transcriptional coactivator LIM domain only 4 (LMO4) in human SH-SY5Y cells, human Ntera neurons, and in embryonic rat cortical neurons. RGM A binding to Neogenin but not binding of Netrin-1, induces release of LMO4 from Neogenin. Down-regulation of LMO4 neutralizes the repulsive activity of RGM A in neuronal cell lines and embryonic rat cortical neurons and prevents RhoA activation. These results show for the first time that an interaction of Neogenin with LMO4 is involved in the RGM A , Neogenin signal transduction pathway for RhoA activation. [source]


    Regulation of dendritic spine morphology by an NMDA receptor-associated Rho GTPase-activating protein, p250GAP

    JOURNAL OF NEUROCHEMISTRY, Issue 4 2008
    Takanobu Nakazawa
    Abstract The NMDA receptor regulates spine morphological plasticity by modulating Rho GTPases. However, the molecular mechanisms for NMDA receptor-mediated regulation of Rho GTPases remain elusive. In this study, we show that p250GAP, an NMDA receptor-associated RhoGAP, regulates spine morphogenesis by modulating RhoA activity. Knock-down of p250GAP increased spine width and elevated the endogenous RhoA activity in primary hippocampal neurons. The increased spine width by p250GAP knock-down was suppressed by the expression of a dominant-negative form of RhoA. Furthermore, p250GAP is involved in NMDA receptor-mediated RhoA activation. In response to NMDA receptor activation, exogenously expressed green fluorescent protein (GFP)-tagged p250GAP was redistributed. Thus, these data suggest that p250GAP plays an important role in NMDA receptor-mediated regulation of RhoA activity leading to spine morphological plasticity. [source]


    The cyclic GMP-protein kinase G pathway regulates cytoskeleton dynamics and motility in astrocytes

    JOURNAL OF NEUROCHEMISTRY, Issue 1 2007
    Mariela Susana Borán
    Abstract We have previously demonstrated that inflammatory compounds that increase nitric oxide (NO) synthase expression have a biphasic effect on the level of the NO messenger cGMP in astrocytes. In this work, we demonstrate that NO-dependent cGMP formation is involved in the morphological change induced by lipopolysaccharide (LPS) in cultured rat cerebellar astroglia. In agreement with this, dibutyryl-cGMP, a permeable cGMP analogue, and atrial natriuretic peptide, a ligand for particulate guanylyl cyclase, are both able to induce process elongation and branching in astrocytes resulting from a rapid, reversible and concentration-dependent redistribution of glial fibrillary acidic protein (GFAP) and actin filaments without significant change in protein levels. These effects are also observed in astrocytes co-cultured with neurons. The cytoskeleton rearrangement induced by cGMP is prevented by the specific protein kinase G inhibitor Rp-8Br-PET-cGMPS and involves downstream inhibition of RhoA GTPase since is not observed in cells transfected with constitutively active RhoA. Furthermore, dibutyryl-cGMP prevents RhoA-membrane association, a step necessary for its interaction with effectors. Stimulation of the cGMP-protein kinase G pathway also leads to increased astrocyte migration in an in vitro scratch-wound assay resulting in accelerated wound closure, as seen in reactive gliosis following brain injury. These results indicate that cGMP-mediated pathways may regulate physio-pathologically relevant responses in astroglial cells. [source]


    Implication of Rho-associated kinase in the elevation of extracellular dopamine levels and its related behaviors induced by methamphetamine in rats

    JOURNAL OF NEUROCHEMISTRY, Issue 2 2003
    Minoru Narita
    Abstract A growing body of evidence suggests that several protein kinases are involved in the expression of pharmacological actions induced by a psychostimulant methamphetamine. The present study was designed to investigate the role of the Rho/Rho-associated kinase (ROCK)-dependent pathway in the expression of the increase in extracellular levels of dopamine in the nucleus accumbens and its related behaviors induced by methamphetamine in rats. Methamphetamine (1 mg/kg, subcutaneously) produced a substantial increase in extracellular levels of dopamine in the nucleus accumbens, with a progressive augmentation of dopamine-related behaviors including rearing and sniffing. Methamphetamine also induced the decrease in levels of its major metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA). Both the increase in extracellular levels of dopamine and the induction of dopamine-related behaviors by methamphetamine were significantly suppressed by pretreatment with an intranucleus accumbens injection of a selective ROCK inhibitor Y-27632. In contrast, Y-27632 had no effect on the decrease in levels of DOPAC and HVA induced by methamphetamine. Under these conditions, there were no changes in protein levels of membrane-bound RhoA in the nucleus accumbens following methamphetamine treatment. It is of interest to note that the microinjection of Y-27632 into the nucleus accumbens failed to suppress the increases in extracellular levels of dopamine, DOPAC, and HVA in the nucleus accumbens induced by subcutaneous injection of a prototype of µ-opioid receptor agonist morphine (10 mg/kg). Furthermore, perfusion of a selective blocker of voltage-dependent Na+ channels, tetrodotoxin (TTx) into the rat nucleus accumbens did not affect the increase in extracellular levels of dopamine in the rat nucleus accumbens by methamphetamine, whereas the morphine-induced dopamine elevation was eliminated by this application of TTx. The extracellular level of dopamine in the nucleus accumbens was also increased by perfusion of a selective dopamine re-uptake inhibitor 1-[2-[bis(4-fluorophenyl)methoxy]-4-(3-phenylpropyl)piperazine (GBR-12909) in the nucleus accumbens. This effect was not affected by pretreatment with intranucleus accumbens injection of Y-27632. These findings provide first evidence that Rho/ROCK pathway in the nucleus accumbens may contribute to the increase in extracellular levels of dopamine in the nucleus accumbens evoked by a single subcutaneous injection of methamphetamine. In contrast, this pathway is not essential for the increased level of dopamine in this region induced by morphine, providing further evidence for the different mechanisms of dopamine release by methamphetamine and morphine in rats. [source]