Inhibition of Rho-kinase induces alphaB-crystallin expression in lens epithelial cells

The small heat shock protein, alphaB-crystallin, has been shown to interact with actin and intermediate filament proteins. However, little is known regarding the cellular mechanisms regulating such interactions. In this study, we explored the role of the Rho/Rho-kinase pathway in alphaB-crystallin distribution and expression in porcine lens epithelial cells. alphaB-crystallin was distributed uniformly throughout the cytoplasm and did not exhibit any unique redistribution in response to actin depolymerization induced by Rho/Rho-kinase inhibitors (C3-exoenzyme or Y-27632) or by overexpression of the dominant negative mutant of Rho-kinase (DNRK) in porcine lens epithelial cells. Interestingly, alphaB-crystallin levels markedly increased in lens epithelial cells treated with the inhibitors of Rho/Rho-kinase proteins (lovastatin, Y-27632 or DNRK) while a protein kinase C inhibitor (GF109203x) was found to have no effect. Further, Y-27632 showed a dose (2-50 microM) response effect on alphaB-crystallin induction. Nocodazole, a microtubule-depolymerizing agent, elicited an increase in alphaB-crystallin levels but latrunculin, an actin depolymerizing agent, did not show any significant effect. Pretreatment with cycloheximide or genistein blocked the Rho-kinase inhibitor-induced increase in alphaB-crystallin protein levels. Rho-kinase inhibitor-induced increases in alphaB-crystallin levels were found to be associated with activation of P38 mitogen-activated protein kinase (MAPK). These results suggest that Rho/Rho-kinase negatively regulates alphaB-crystallin expression, and this response appears to be dependent on tyrosine-protein kinase and P38 MAPK function. Finally, alphaB-crystallin induction appears to be better correlated with the direct inhibition of Rho/Rho-kinase than with actin depolymerization per se.     

Rho-kinase mediates TNF-α-induced MCP-1 expression via p38 MAPK signaling pathway in mesangial cells

Macrophage accumulation has been implicated in the pathogenesis of inflammatory glomerular disease. Monocyte chemoattractant protein-1 (MCP-1) plays a central role in recruiting monocytes to the glomeruli. Tumor necrosis factor-α (TNF-α) has been shown to induce MCP-1 expression in mesangial cells, although the precise mechanisms remain unclear. We previously demonstrated that RhoA and its effector, Rho-kinase (Rho-associated coiled-coil containing protein kinase, ROCK), are involved in the pathogenesis of diabetic nephropathy. However, its role in MCP-1 induction by TNF-α has not been elucidated. In the present study, we investigated whether the Rho/Rho-kinase signaling pathway regulates the TNF-α-mediated induction of MCP-1 in mesangial cells. Exposure of mouse mesangial cells (MES-13) to TNF-α resulted in an increase of MCP-1 expression (by RT-PCR) and secretion into the medium (by ELISA). Pull down and Western blot analysis revealed that TNF-α activated RhoA and Rho-kinase. Based on these observations, we speculated that the Rho/Rho-kinase signaling pathway may be involved in MCP-1 induction by TNF-α. In agreement with this concept, Y-27632, a specific Rho-kinase inhibitor, attenuated TNF-α-mediated induction of MCP-1. We demonstrated that Y-27632 inhibited TNF-α-mediated monocyte migration and attenuated TNF-α-mediated p38 MAPK activation. Based on these data we infer that Y-27632 inhibits TNF-α-induced MCP-1 expression, secretion and function through inhibition of Rho-kinase and p38 MAPK activity. Our study suggests that Rho/Rho-kinase is an important therapeutic target of monocyte recruitment and accumulation within the glomerulus in inflammatory renal disease.     

Thrombin induces MCP-1 expression through Rho-kinase and subsequent p38MAPK/NF-κB signaling pathway activation in vascular endothelial cells

Thrombin has been shown to increase expression of chemokines such as monocyte chemoattractant protein 1 (MCP-1) in endothelial cells, leading to the development of atherosclerosis. However, the precise mechanism of this induction remains unknown. In the present study, we investigated whether the small G protein RhoA, and its effector, Rho-kinase are involved in MCP-1 induction by thrombin in endothelial cells. Y-27632, a specific Rho-kinase inhibitor, potently inhibited MCP-1 induction by thrombin. Y-27632 significantly decreased the chemotactic activity of thrombin-stimulated supernatants of endothelial cells on monocytes. Importantly, fasudil, a specific Rho-kinase inhibitor, attenuated MCP-1 gene expression in the aorta of db/db mice. Y-27632 attenuated thrombin-mediated phosphorylation of p38MAPK and p65, indicating that Rho-kinase mediates thrombin-induced MCP-1 expression through p38MAPK and NF-κB activation. Our findings demonstrate that the Rho/Rho-kinase signaling pathway plays a critical role in thrombin-mediated MCP-1 expression and function, and suggest that Rho/Rho-kinase may be an important target in the development of new therapeutic strategies for atherosclerosis.     

Attenuation of acute hypoxic pulmonary vasoconstriction and hypoxic pulmonary hypertension in mice by inhibition of Rho-kinase

RhoA GTPase mediates a variety of cellular responses, including activation of the contractile apparatus, growth, and gene expression. Acute hypoxia activates RhoA and, in turn, its downstream effector, Rho-kinase, and previous studies in rats have suggested a role for Rho/Rho-kinase signaling in both acute and chronically hypoxic pulmonary vasoconstriction. We therefore hypothesized that activation of Rho/Rho-kinase in the pulmonary circulation of mice contributes to acute hypoxic pulmonary vasoconstriction and chronic hypoxia-induced pulmonary hypertension and vascular remodeling. In isolated, salt solution-perfused mouse lungs, acute administration of the Rho-kinase inhibitor Y-27632 (1 x 10(-5) M) attenuated hypoxic vasoconstriction as well as that due to angiotensin II and KCl. Chronic treatment with Y-27632 (30 mg x kg(-1) x day(-1)) via subcutaneous osmotic pump decreased right ventricular systolic pressure, right ventricular hypertrophy, and neomuscularization of the distal pulmonary vasculature in mice exposed to hypobaric hypoxia for 14 days. Analysis of a small number of proximal pulmonary arteries suggested that Y-27632 treatment reduced the level of phospho-CPI-17, a Rho-kinase target, in hypoxic lungs. We also found that endothelial nitric oxide synthase protein in hypoxic lungs was augmented by Y-27632, suggesting that enhanced nitric oxide production might have played a role in the Y-27632-induced attenuation of chronically hypoxic pulmonary hypertension. In conclusion, Rho/Rho-kinase activation is important in the effects of both acute and chronic hypoxia on the pulmonary circulation of mice, possibly by contributing to both vasoconstriction and vascular remodeling.     

Rho-kinase inhibitor retards migration and in vivo dissemination of human prostate cancer cells

The Rho-kinase inhibitor, Y-27632, inhibited in vitro chemotactic migration to bone marrow fibroblast conditioned media and metastatic growth in immune-compromised mice of highly invasive human prostatic cancer (PC3) cells. Y-27632 also reduced myosin light chain phosphorylation and markedly altered the morphology of cells that developed numerous processes containing microtubules. A strikingly different, rounded phenotype was induced by an inhibitor of myosin light chain kinase, ML9. The M(110-130) subunit of the myosin phosphatase that is regulated by Rho-kinase was present in PC3 cells that contained significantly more RhoA than the less invasive, LNCaP cells. Y-27632 also inhibited angiogenesis as measured by endothelial cell tube formation on Matrigel. We conclude that invasiveness of human prostate cancer is facilitated by the Rho/Rho-kinase pathway, and exploration of selective Rho-kinase inhibitors for limiting invasive progress of prostate cancer is warranted.     

Role of oxidative stress, endoplasmic reticulum stress, and c-Jun N-terminal kinase in pancreatic beta-cell dysfunction and insulin resistance

Type 2 diabetes is the most prevalent and serious metabolic disease affecting people all over the world. Pancreatic beta-cell dysfunction and insulin resistance are the hallmark of type 2 diabetes. Normal beta-cells can compensate for insulin resistance by increasing insulin secretion and/or beta-cell mass, but insufficient compensation leads to the onset of glucose intolerance. Once hyperglycemia becomes apparent, beta-cell function gradually deteriorates and insulin resistance aggravates. Under diabetic conditions, oxidative stress and endoplasmic reticulum stress are induced in various tissues, leading to activation of the c-Jun N-terminal kinase pathway. The activation of c-Jun N-terminal kinase suppresses insulin biosynthesis and interferes with insulin action. Indeed, suppression of c-Jun N-terminal kinase in diabetic mice improves insulin resistance and ameliorates glucose tolerance. Thus, the c-Jun N-terminal kinase pathway plays a central role in pathogenesis of type 2 diabetes and could be a potential target for diabetes therapy.     

Inhibition of Rho/Rho-kinase signaling downregulates plasminogen activator inhibitor-1 synthesis in cultured human monocytes

Increased production of plasminogen activator inhibitor-1 (PAI-1) in plaques plays a role in the pathogenesis of atherosclerosis. This study was conducted to investigate the effect of blockade of Rho/Rho-kinase signaling on the synthesis of PAI-1 in cultured human peripheral blood monocytes. HMG-CoA reductase inhibitors (statins) and inhibitors of Rho and Rho-kinase were added to monocyte cultures. The levels of PAI antigen and mRNA were determined by Western blotting and RT-PCR, respectively, and PAI-1 expression was assessed by immunohistochemistry. We performed pull-down assays to determine the activity of Rho by measuring the GTP-bound form of Rho A. In unstimulated and lipopolysaccharide (LPS)-stimulated cultured monocytes, statins reduced the levels of PAI-1 antigen and mRNA. The suppressive effects of statins on PAI-1 synthesis were reversed by geranylgeranylpyrophosphate (GGPP) and were mimicked by C3 exoenzyme. Immunohistochemistry confirmed the role of lipid modification by GGPP in suppressive effect of statins in PAI-1 synthesis. Pull-down assays demonstrated that statins decreased the levels of the GTP-bound form of Rho A. Our findings suggest that statins decrease the activity of Rho by inhibiting geranylgeranylation. Moreover, Rho-kinase inhibitors, Y-27632 and fasudil, suppressed the synthesis of PAI-1 in this culture system. We show that inhibition of Rho/Rho-kinase signaling downregulates the synthesis of PAI-1 in human monocytes.     

Role of oxidative stress, endoplasmic reticulum stress, and c-Jun N-terminal kinase in pancreatic beta-cell dysfunction and insulin resistance

Type 2 diabetes is the most prevalent and serious metabolic disease affecting people all over the world. Pancreatic beta-cell dysfunction and insulin resistance are the hallmark of type 2 diabetes. Normal beta-cells can compensate for insulin resistance by increasing insulin secretion and/or beta-cell mass, but insufficient compensation leads to the onset of glucose intolerance. Once hyperglycemia becomes apparent, beta-cell function gradually deteriorates and insulin resistance aggravates. Under diabetic conditions, oxidative stress and endoplasmic reticulum stress are induced in various tissues, leading to activation of the c-Jun N-terminal kinase pathway. The activation of c-Jun N-terminal kinase suppresses insulin biosynthesis and interferes with insulin action. Indeed, suppression of c-Jun N-terminal kinase in diabetic mice improves insulin resistance and ameliorates glucose tolerance. Thus, the c-Jun N-terminal kinase pathway plays a central role in pathogenesis of type 2 diabetes and could be a potential target for diabetes therapy.     

Activation of Rho signaling contributes to lysophosphatidic acid-induced contraction of intact ileal smooth muscle of guinea-pig

To elucidate the possible role of Rho A/Rho-kinase on lysophosphatidic acid (LPA)-induced contraction in intact guinea-pig ileal smooth muscle, we examined effects of pretreatment with a specific inhibitor of Rho-kinase (Y-27632) on the LPA-induced contraction and MLC20 phosphorylation. In addition, we investigated whether LPA actually elicits an activation of Rho A by studying subcellular distribution of Rho A in unstimulated and stimulated smooth muscles by LPA. LPA induced a less intense, but sustained, contraction compared with ACh, and was accompanied by significant increases in MLC20 phosphorylation. The effects of LPA on tension and MLC20 phosphorylation were inhibited by Y-27632. The ACh-induced contraction, but not increases in MLC20 phosphorylation, was partially inhibited by Y-27632. High K+-induced contraction was unaffected by the inhibitor. LPA stimulated translocation of Rho A from the cytosol to the membrane fraction of the muscle. Translocation of Rho A was also induced by ACh and high K+. These results suggest that LPA-induced contraction of intact ileal smooth muscle is dominated through activation of Rho A and Rho-kinase and subsequent increases in MLC20 phosphorylation.     

Wnt-signaling mediates the anti-adipogenic action of lysophosphatidic acid through cross talking with the Rho/Rho associated kinase (ROCK) pathway

Lysophosphatidic acid (LPA) is a bioactive phospholipid with a diverse range of biological activities including the modulation of adipogenesis. Treatment of 3T3-L1 cells and 3T3F44A cells with LPA inhibits adipogenesis and reduces expression of PPARγ through activation of RhoGTPase and its downstream Rho associated kinase (ROCK). The mechanism of suppression of PPARγ expression by Rho/ROCK is poorly understood. By treating the differentiating 3T3-L1 cells with various combinations of LPA and ROCK inhibitors, Y-27632 and fasudil, we observed that LPA treatment resulted in attenuation of adipogenesis and a significant reduction in PPARγ mRNA as early as 3?d post-induction. LPA treatment also resulted in significant but delayed upregulation of components of the canonical Wnt signaling, namely Wnt10b mRNA, β-catenin protein, and mRNA expression of β-catenin target genes, detectable at day 7, but not day 3. Treatment of the 3T3-L1 cells with ROCK inhibitors Y-27632 and fasudil revealed a tonic activation of β-catenin/target genes by ROCK. This study identified the existence of a novel cross talk between the Rho/ROCK pathway and the Wnt-signaling pathway. The LPA/Rho/ROCK pathway inhibits expression of PPARγ and adipogenesis in part through a delayed activation of the canonical Wnt-signaling pathway based on increased Wnt10b expression and β-catenin induction.     

Rapid stimulation of tyrosine phosphorylation signals downstream of G-protein-coupled receptors for thromboxane A2 in human platelets

Signals ensuing from trimeric G-protein-coupled receptors synergize to induce platelet activation. At low doses, the thromboxane A2 analogue U46619 does not activate integrin alphaIIbbeta3 or trigger platelet aggregation, but it induces shape changes. In the present study, we addressed whether low doses of U46619 trigger tyrosine phosphorylation independently of integrin alphaIIbbeta3 activation and ADP secretion, and synergize with adrenaline (epinephrine) to induce aggregation in acetylsalicylic acid (aspirin)-treated platelets. Low doses of U46619 triggered tyrosine phosphorylation of different proteins, including FAK (focal adhesion kinase), Src and Syk, independently of signals ensuing from integrin alphaIIbbeta3 or ADP receptors engaged by secreted ADP. The G(12/13)-mediated Rho/Rho-kinase pathway was also increased by low doses of U46619; however, this pathway was not upstream of tyrosine phosphorylation, because this occurred in the presence of the Rho-kinase inhibitor Y-27632. Although low doses of U46619 or adrenaline alone were unable to trigger platelet aggregation and integrin alphaIIbbeta3 activation, the combination of the two stimuli effectively induced these responses. PP2, a tyrosine kinase inhibitor, and Y-27632 inhibited platelet activation induced by low doses of U46619 plus adrenaline and, when used in combination, totally suppressed this platelet response. In addition, the two inhibitors selectively blocked tyrosine kinases and the Rho/Rho-kinase pathway respectively. These findings suggest that both tyrosine phosphorylation and the Rho/Rho-kinase pathway are required to activate platelet aggregation via G(12/13) plus G(z) signalling.     

Involvement of Rho/Rho-kinase signalling in the contractile activity and acetylcholine release in the mouse gastric fundus

In this study effects of Rho kinase inhibitors have been examined on the mouse gastric fundal smooth muscle reactivity and neurotransmitter (acetylcholine) release. Two Rho-kinase inhibitors, Y-27632 and fasudil (HA-1077), conspicuously suppressed the contractile responses to carbachol (CCh) and KCl as well as electrical field stimulation (EFS, 40 V, 0.5 ms, and 20 s). pEC(50) value for CCh and EC(50) value for KCl were 6.68+/-0.15 M and 10.4+/-2.8 mM, respectively. EFS induced reproducible contraction (38.3+/-4.75 mN/g tissue) which was almost abolished and potentiated in the presence of atropine (10(-6)M) and eserine (10(-6)M), respectively. The Rho-kinase inhibitors relaxed the fundic strips preconstricted by submaximal concentration of CCh or KCl in a concentration dependent manner. With CCh-elicited contraction, the pEC(50) values of Y-27632 and fasudil were 5.45+/-0.14 and 5.11+/-0.14 M, respectively (p>0.05). However, the pEC(50) values for Y-27632 and fasudil on KCl-induced tone were 6.09+/-0.1 and 5.35+/-0.06 M (p<0.001), respectively. Moreover, [3H]acetylcholine ([3H]ACh) release upon EFS from the gastric fundus was measured and it was found that Y-27632 (10(-4)M) significantly impaired the release. At 3 Hz the radioactivity ratio obtained after and before EFS (S(2)/S(1) ratio) was 0.88+/-0.03 in control but 0.63+/-0.08 in the presence of 10(-4)M Y-27632 (p<0.05). These results suggest that Rho kinase inhibitors can not only relax the gastric fundus but also modulate CCh, cholinergic nerve stimulation, and KCl-induced contraction. Furthermore, Rho/Rho kinase signalling may play a role in the neurotransmitter (ACh) release in the mouse gastric fundus.     

Evaluation of the Rho A/Rho-kinase pathway in the uterus of the rat model of polycystic ovary syndrome

The aim of this study was to investigate the expression of RhoA/Rho-kinase in the uterus and the effect of Rho-kinase inhibitors on uterine contractions of dehydroepiandrosterone (DHEA) induced polycystic ovary syndrome (PCOS) rats. Forty-four female Sprague-Dawley (21 days old) rats divided into three groups: The control group (n?=?14, any procedure was not performed), vehicle group (n?=?14, 0.2?ml of sesame oil, subcutaneous injection, 20 days) and PCOS group (n?=?16, DHEA 6?mg/100?g in 0.2?ml of sesame oil, subcutaneous injection, 20 days). The myometrium thickness and uterine wet weight were assessed. The mRNA and protein expressions of Rho A, the effect of Rho-kinase inhibitors (fasudil and Y-27632) on KCl, carbachol, and PGF2α induced contractions were evaluated in the uterus. In the PCOS group, the myometrium thickness and uterine wet weight significantly increased compared to the control group and vehicle group. The mRNA expression level and the immunoreactive score of Rho A, ROCK 1, ROCK 2 were similar in all groups. In the PCOS group, KCl, carbachol, and PGF2α induced uterine contractions significantly increased compared to the control group and vehicle group. Fasudil and Y-27632 significantly inhibited KCl, carbachol, and PGF2α induced uterine contractions in all groups. In conclusion, the expression of Rho A, ROCK 1, ROCK 2 not changed although myometrium thickness, uterine wet weight and the contractile responses of uterus increased in the PCOS group. The results suggest that the Rho-kinase inhibitors effectively suppressed increased contractions in the PCOS group they might be potential therapeutic agents.     

Improved erectile function after Rho-kinase inhibition in a rat castrate model of erectile dysfunction

Androgens are reported to act as strong modulators of erectile function influencing both nitric oxide and vasoconstrictor signaling. Castration results in a depressed erectile response that is associated with a loss of nitric oxide production and increased responsiveness to constrictive agents. The increased vasoconstrictor response may be a result of an active RhoA/Rho-kinase signaling pathway. We report here results of studies designed to test the hypothesis that inhibition of the Rho-kinase pathway restores erectile function in a castrate model by relaxing the smooth muscle. Mean arterial (MAP) and corpus cavernosal (CCP) pressures were monitored during intracavernosal injection of the Rho-kinase inhibitor Y-27632. Castration reduced the maximal erectile response (CCP/MAP) by 33%, and testosterone replacement restored the response (intact, 0.736 +/- 0.040; castrate, 0.492 +/- 0.022; testosterone, 0.681 +/- 0.073). Injection of Y-27632 increased CCP in all experimental groups; it also left shifted the voltage response curve and increased the maximal CCP/MAP response (intact, 0.753 +/- 0.091; castrate, 0.782 +/- 0.081; testosterone treated, 0.894 +/- 0.033). Y-27632 dose dependently relaxed phenylephrine-stimulated cavernosal tissues. Cavernosal tissues showed increased RhoA and Rho-kinase protein levels after castration. Our data support the hypothesis that an active Rho/Rho-kinase pathway contributes to the reduced erectile response after castration due to an upregulation of RhoA/Rho-kinase protein levels and that inhibition of this pathway may serve as an effective treatment for erectile dysfunction.     

A multi-parameter, high-content, high-throughput screening platform to identify natural compounds that modulate insulin and Pdx1 expression

Diabetes is a devastating disease that is ultimately caused by the malfunction or loss of insulin-producing pancreatic beta-cells. Drugs capable of inducing the development of new beta-cells or improving the function or survival of existing beta-cells could conceivably cure this disease. We report a novel high-throughput screening platform that exploits multi-parameter high-content analysis to determine the effect of compounds on beta-cell survival, as well as the promoter activity of two key beta-cell genes, insulin and pdx1. Dispersed human pancreatic islets and MIN6 beta-cells were infected with a dual reporter lentivirus containing both eGFP driven by the insulin promoter and mRFP driven by the pdx1 promoter. B-score statistical transformation was used to correct systemic row and column biases. Using this approach and 5 replicate screens, we identified 7 extracts that reproducibly changed insulin and/or pdx1 promoter activity from a library of 1319 marine invertebrate extracts. The ability of compounds purified from these extracts to significantly modulate insulin mRNA levels was confirmed with real-time PCR. Insulin secretion was analyzed by RIA. Follow-up studies focused on two lead compounds, one that stimulates insulin gene expression and one that inhibits insulin gene expression. Thus, we demonstrate that multi-parameter, high-content screening can identify novel regulators of beta-cell gene expression, such as bivittoside D. This work represents an important step towards the development of drugs to increase insulin expression in diabetes and during in vitro differentiation of beta-cell replacements.     

Oxidative stress and the JNK pathway are involved in the development of type 1 and type 2 diabetes

Failure of pancreatic beta-cells is the common characteristic of type 1 and type 2 diabetes. Type 1 diabetes mellitus is induced by destruction of pancreatic beta-cells which is mediated by an autoimmune mechanism and consequent inflammatory process. Various inflammatory cytokines and oxidative stress are produced during this process, which has been proposed to play an important role in mediating beta-cell destruction. The JNK pathway is also activated by such cytokines and oxidative stress, and is involved in beta-cell destruction. Type 2 diabetes is the most prevalent and serious metabolic disease, and beta-cell dysfunction and insulin resistance are the hallmark of type 2 diabetes. Under diabetic conditions, chronic hyperglycemia gradually deteriorates beta-cell function and aggravates insulin resistance. This process is called "glucose toxicity". Under such conditions, oxidative stress is provoked and the JNK pathway is activated, which is likely involved in pancreatic beta-cells dysfunction and insulin resistance. In addition, oxidative stress and activation of the JNK pathway are also involved in the progression of atherosclerosis which is often observed under diabetic conditions. Taken together, it is likely that oxidative stress and subsequent activation of the JNK pathway are involved in the pathogenesis of type 1 and type 2 diabetes.     

Mechanisms of NOS2 regulation by Rho GTPase signaling in airway epithelial cells

The aberrant dysregulation of the inducible form of nitric oxide synthase (NOS2) is thought to play a role in many inflammatory disorders including cystic fibrosis (CF). The complex regulation of NOS2 expression is the subject of intense investigation, and one intriguing regulatory pathway known to influence NOS2 expression is the Rho GTPase cascade. We examined NOS2 regulation in response to inflammatory cytokines in a human alveolar epithelial cell line treated with inhibitors of different upstream and downstream components of the Rho GTPase pathway to better define potential signaling mechanisms. Statin-mediated 3-hydroxy-3-methylglutaryl-CoA reductase inhibition increased cytokine-dependent activation of the NOS2 promoter, reversible by the addition of geranylgeranyl pyrphosphate. However, inhibition of Rho-associated kinase (ROCK) with Y-27632 resulted in a decrease in NOS2 promoter activity, yet an increase in NOS2 mRNA and protein levels. Our results suggest that prenylation events influence NOS2 promoter activity independently of the Rho GTPase pathway and that Rho GTPase signaling mediated through ROCK suppresses NOS2 production downstream of promoter function at the message and protein level.