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Rho Kinase Inhibitor (rho + kinase_inhibitor)
Selected AbstractsLysophosphatidic acid in malignant ascites stimulates migration of human mesenchymal stem cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2008Mi 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] Sphingosine 1-phosphate induces cell contraction via calcium-independent/Rho-dependent pathways in undifferentiated skeletal muscle cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2004L. 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] Rho kinase activates ezrin-radixin-moesin (ERM) proteins and mediates their function in cortical neuron growth, morphology and motility in vitroJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1 2007Matilda A. Haas Abstract The ezrin-radixin-moesin (ERM) family of proteins contribute to cytoskeletal processes underlying many vital cellular functions. Their previously elucidated roles in non-neuronal cells are an indication of their potential importance in CNS neurons. The specific mechanisms of their activation are unknown, but are likely to depend on factors such as the cell type and biological context. For ERM proteins to become active they must be phosphorylated at a specific C-terminal threonine residue. In non-neuronal cells, several kinases, including the Rho GTPase family member Rho kinase, have been identified as capable of phosphorylating the C-terminal threonine. In these experiments we have investigated specifically the potential role of Rho kinase mediated ERM activation in cortical neurons, utilizing a new pharmacologic inhibitor of Rho kinase and quantitative analysis of aspects of neuronal functions potentially mediated by ERM proteins. Rho kinase inhibition significantly suppressed aspects of neuronal development including neurite initiation and outgrowth, as well as growth cone morphology, with a concomitant loss of phosphorylated ERM immunolabeling in areas associated with neuronal growth. The ability of the Rho kinase inhibitor to decrease the amount of pERM protein was shown by immunoblotting. Post-injury responses were negatively affected by Rho kinase inhibition, namely by a significant decrease in the number of regenerative neurites. We investigated a novel role for ERM proteins in neuron migration using a post-injury motility assay, where Rho kinase inhibition resulted in significant and drastic reduction in neuron motility and phosphorylated ERM immunolabeling. Thus, Rho kinase is an important activator of ERMs in mediating specific neuronal functions. © 2006 Wiley-Liss, Inc. [source] Acute and Chronic Alcohol Exposure Impair the Phagocytosis of Apoptotic Cells and Enhance the Pulmonary Inflammatory ResponseALCOHOLISM, Issue 10 2010Darren M. Boé Background:, Alcohol abuse increases the risk for acute respiratory distress syndrome (ARDS). Efferocytosis, the clearance of apoptotic cells, is important in the resolution of inflammation and is regulated by RhoA and rho kinase (ROCK) activation. The effects of alcohol on pulmonary Rho pathway activation and efferocytosis have not been determined. We hypothesize that acute and chronic alcohol exposure impair pulmonary efferocytosis, leading to heightened inflammation during ARDS. Methods:, For in vivo experiments, C57BL/6 mice received either a single intraperitoneal injection of alcohol or chronic ethanol-in-water for 8 weeks prior to intratracheal instillation of apoptotic cells or lipopolysaccharide (LPS). Bronchoalveolar lavage (BAL) was performed for cells counts, calculation of the phagocytic index (PI), and Rho activity measurements. For in vitro studies, primary alveolar macrophages were cultured in alcohol (25,100 mM) and then co-cultured with apoptotic cells. RhoA activity was determined following alcohol exposure, and the PI was determined before and after treatment with the ROCK inhibitor, Y27632. Results:, Acute alcohol exposure was associated with impaired efferocytosis. Following LPS exposure, acute alcohol exposure was also associated with increased BAL neutrophils. Chronic alcohol exposure alone did not alter efferocytosis. However, following exposure to LPS, chronic alcohol exposure was associated with both impaired efferocytosis and increased BAL neutrophils. In vitro alcohol exposure caused a dose-dependent decrease in efferocytosis. Despite the fact that RhoA activity was decreased by alcohol exposure and RhoA inhibition did not alter the effects of alcohol on efferocytosis, treatment with the Rho kinase inhibitor, Y27632, reversed the effects of alcohol on efferocytosis. Conclusions:, Acute alcohol exposure impairs pulmonary efferocytosis, whereas exposure to chronic alcohol is only associated with impaired efferocytosis following LPS-induced lung injury. Both forms of alcohol exposure are associated with increased alveolar neutrophil numbers in response to LPS. The acute effects of alcohol on efferocytosis appear to be mediated, at least in part, by RhoA-independent activation of ROCK. Further studies are needed to dissect the differences between the effects of acute and chronic alcohol exposure on efferocytosis and to determine the effects of alcohol on alternative activators of ROCK. [source] Statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, function as inhibitors of cellular and molecular components involved in type I interferon productionARTHRITIS & RHEUMATISM, Issue 7 2010Hideki Amuro Objective Statins, which are used as cholesterol-lowering agents, have pleiotropic immunomodulatory properties. Although beneficial effects of statins have been reported in autoimmune diseases, the mechanisms of these immunomodulatory effects are still poorly understood. Type I interferons (IFNs) and plasmacytoid dendritic cells (PDCs) represent key molecular and cellular pathogenic components in autoimmune diseases such as systemic lupus erythematosus (SLE). Therefore, PDCs may be a specific target of statins in therapeutic strategies against SLE. This study was undertaken to investigate the immunomodulatory mechanisms of statins that target the IFN response in PDCs. Methods We isolated human blood PDCs by flow cytometry and examined the effects of simvastatin and pitavastatin on PDC activation, IFN, production, and intracellular signaling. Results Statins inhibited IFN, production profoundly and tumor necrosis factor , production modestly in human PDCs in response to Toll-like receptor ligands. The inhibitory effect on IFN, production was reversed by geranylgeranyl pyrophosphate and was mimicked by either geranylgeranyl transferase inhibitor or Rho kinase inhibitor, suggesting that statins exert their inhibitory actions through geranylgeranylated Rho inactivation. Statins inhibited the expression of phosphorylated p38 MAPK and Akt, and the inhibitory effect on the IFN response was through the prevention of nuclear translocation of IFN regulatory factor 7. In addition, statins had an inhibitory effect on both IFN, production by PDCs from SLE patients and SLE serum,induced IFN, production. Conclusion Our findings suggest a specific role of statins in controlling type I IFN production and a therapeutic potential in IFN-related autoimmune diseases such as SLE. [source] Mast cell,derived tryptase inhibits apoptosis of human rheumatoid synovial fibroblasts via rho-mediated signalingARTHRITIS & RHEUMATISM, Issue 4 2010Norifumi Sawamukai Objective An abundance of mast cells are found in the synovium of patients with rheumatoid arthritis (RA). However, the role of mast cells in the pathogenesis of RA remains unclear. This study was undertaken to elucidate a role for mast cells in RA by investigating the antiapoptotic effects of tryptase, a major product of mast cells, on RA synovial fibroblasts (RASFs). Methods RA synovial tissue was obtained from RA patients during joint replacement surgery, and histologic changes in the tissue were examined. The expression of cell surface molecules and apoptotic markers on RASFs were detected by flow cytometry. Rho activation was determined using a pull-down assay. Results Mast cells, bearing both c-Kit and tryptase, accumulated in the sublining area of proliferating synovial tissue from RA patients. Protease-activated receptor 2 (PAR-2), a receptor for tryptase, was expressed on RASFs in the lining area, close to tryptase-positive mast cells in the RA synovium. Fas-mediated apoptosis of RASFs was significantly inhibited, in a dose-dependent manner, by the addition of tryptase, and this effect correlated with increased activation of Rho kinase. Furthermore, Y27632, a Rho kinase inhibitor, reduced the antiapoptotic effect of tryptase on RASFs, suggesting that Rho was responsible for the antiapoptotic effects of tryptase. Conclusion These results demonstrate that tryptase has a strong antiapoptotic effect on RASFs through the activation of Rho. Thus, we propose that the release of tryptase by mast cells leads to the binding of tryptase to PAR-2 on RASFs and inhibits the apoptosis of RASFs via the activation of Rho. Such mechanisms could play a pivotal role in the marked proliferation of RASFs and hyperplasia of synovial tissue seen in RA synovium. [source] RhoA/ROK Pathway Related to the Mechanism of Higher Susceptibility to Spasm in RA Than in IMAJOURNAL OF CARDIAC SURGERY, Issue 6 2009Xia 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] Rho kinase inhibitors reduce neurally evoked contraction of the rat tail artery in vitroBRITISH JOURNAL OF PHARMACOLOGY, Issue 6 2005Melanie Yeoh The effects of Rho kinase inhibitors (Y27632, HA-1077) on contractions to electrical stimulation and to application of phenylephrine, clonidine or ,,, -methylene adenosine 5,-triphosphate (,,, -mATP) were investigated in rat tail artery in vitro. In addition, continuous amperometry and intracellular recording were used to monitor the effects of Y27632 on noradrenaline (NA) release and postjunctional electrical activity, respectively. Y27632 (0.5 and 1 ,M) and HA-1077 (5 ,M) reduced neurally evoked contractions. In contrast, the protein kinase C inhibitor, Ro31-8220 (1 ,M), had little effect on neurally evoked contraction. In the absence and the presence of Y27632 (0.5 ,M), the reduction of neurally evoked contraction produced by the , -adrenoceptor antagonists prazosin (10 nM) and idazoxan (0.1 ,M) was similar. The P2-purinoceptor antagonist, suramin (0.1 mM), had no inhibitory effect on neurally evoked contraction in the absence or the presence of Y27632 (1 ,M). In the presence of Y27632, desensitization of P2X-purinoceptors with ,,, -mATP (10 ,M) increased neurally evoked contractions. Y27632 (1 ,M) and H-1077 (5 ,M) reduced sensitivity to phenylephrine and clonidine. In addition, Y27632 reduced contractions to ,,, -mATP (10 ,M). Y27632 (1 ,M) had no effect on the NA-induced oxidation currents or the purinergic excitatory junction potentials and NA-induced slow depolarizations evoked by electrical stimulation. Rho kinase inhibitors reduce sympathetic nerve-mediated contractions of the tail artery. This effect is mediated at a postjunctional site, most likely by inhibition of Rho kinase-mediated ,Ca2+ sensitization' of the contractile apparatus. British Journal of Pharmacology (2005) 146, 854,861. doi:10.1038/sj.bjp.0706377 [source] | |||||||||||||