Rho-associated Coiled Coil-forming Protein Kinase Inhibitor Research Articles

Fasudil, a ROCK inhibitor, prevents neuropathic pain in Minamata disease model rats

Methylmercury (MeHg), an environmental toxicant, is known to cause sensory impairment by inducing neurodegeneration of sensory nervous systems. However, in recent years, it has been revealed that neuropathic pain occurs in the chronic phase of MeHg poisoning, that is, in current Minamata disease patients. Our recent study using Minamata disease model rats demonstrated that MeHg-mediated neurodegeneration in the sensory nervous system may induce inflammatory microglia production in the dorsal horn of the spinal cord and subsequent somatosensory cortical rewiring, leading to neuropathic pain. We hypothesized that inhibition of the Rho-associated coiled coil-forming protein kinase (ROCK) pathway could prevent MeHg-induced neuropathic pain because the ROCK pathway is known to be involved in inducing the production of inflammatory microglia. Here, we showed for the first time that Fasudil, a ROCK inhibitor, can prevent neuropathic pain in Minamata disease model rats. In this model, Fasudil significantly suppressed nerve injury-induced inflammatory microglia production in the dorsal horn of the spinal cord and prevented subsequent somatosensory cortical rewiring. These results suggest that the ROCK pathway is involved in the onset and development of neuropathic pain in the chronic phase of Minamata disease, and that its inhibition is effective in pain prevention.

Data on differentially expressed proteins in rock inhibitor-treated human trabecular meshwork cells using SWATH-based proteomics.

Rho-associated coiled coil-forming protein kinase (ROCK) inhibitors represent a novel class of anti-glaucoma drugs because of their ocular hypotensive effects. However, the underlying mechanisms responsible for lowering intraocular pressure (IOP) are not completely clear. The protein profile changes in primary human trabecular meshwork (TM) cells after two days treatment with a ROCK inhibitor were studied using label-free SWATH acquisition. These results provided significant data of key protein candidates underlying the effect of ROCK inhibitor. Using the sensitive label-free mass spectrometry approach with data-independent acquisition (SWATH-MS), we established a comprehensive TM proteome library. All raw data generated from IDA and SWATH acquisitions were uploaded and published in the Peptide Atlas public repository (http://www.peptideatlas.org/) for general release (Data ID PASS01254).

The Rho Kinase Isoforms ROCK1 and ROCK2 Each Contribute to the Development of Experimental Pulmonary Fibrosis.

Pulmonary fibrosis is thought to result from dysregulated wound repair after repetitive lung injury. Many cellular responses to injury involve rearrangements of the actin cytoskeleton mediated by the two isoforms of the Rho-associated coiled-coil-forming protein kinase (ROCK), ROCK1 and ROCK2. In addition, profibrotic mediators such as transforming growth factor-β, thrombin, and lysophosphatidic acid act through receptors that activate ROCK. Inhibition of ROCK activation may be a potent therapeutic strategy for human pulmonary fibrosis. Pharmacological inhibition of ROCK using nonselective ROCK inhibitors has been shown to prevent fibrosis in animal models; however, the specific roles of each ROCK isoform are poorly understood. Furthermore, the pleiotropic effects of this kinase have raised concerns about on-target adverse effects of ROCK inhibition such as hypotension. Selective inhibition of one isoform might be a better-tolerated strategy. In the present study, we used a genetic approach to determine the roles of ROCK1 and ROCK2 in a mouse model of bleomycin-induced pulmonary fibrosis. Using ROCK1- or ROCK2-haploinsufficient mice, we found that reduced expression of either ROCK1 or ROCK2 was sufficient to protect them from bleomycin-induced pulmonary fibrosis. In addition, we found that both isoforms contribute to the profibrotic responses of epithelial cells, endothelial cells, and fibroblasts. Interestingly, ROCK1- and ROCK2-haploinsufficient mice exhibited similar protection from bleomycin-induced vascular leak, myofibroblast differentiation, and fibrosis; however, ROCK1-haploinsufficient mice demonstrated greater attenuation of epithelial cell apoptosis. These findings suggest that selective inhibition of either ROCK isoform has the potential to be an effective therapeutic strategy for pulmonary fibrosis.

Study on promotion of bone cancer pain via rho associated coiled-coil forming protein kinase2/LIM domain kinase/Cofilin signaling by negative regulation of microRNA in mice

Objective To observe microRNA (miR-93) regulation in bone cancer pain(BCP) and explore the underlying mechanisms of miR-93 and Rho associated coiled-coil forming protein kinase (ROCK) signaling affecting the biological behaviors of BCP in mice. Methods Male mice were randomly divided into Control group, Sham group, bone cancer pain (BCP group), BCP+PBS group and BCP+Y-27632(ROCK inhibitor) group(n=13). The paw mechanical threshold and paw thermal latency were used to evaluate the behavioral changes on 1 day before surgery and 1, 3, 5, 7, 10, 14 after surgery. L4-L6 dorsal root ganglions were removed on postoperative day 14, followed by Real-time PCR to measure the expression of miR-93. The ROCK2 promoter activity regulated by miR-93 was evaluated by a dual luciferase reporter assay. Western blotting was used to evaluate the protein expression level of ROCK2, phospho-LIM domain kinase (LIMK) and phospho-Cofilin. Results Following intrafemoral carcinoma inoculation, robust mechanical allodynia[Sham group(6.81±1.04) g, BCP group(5.12±0.47) g]and thermal hyperalgesia[Sham group(11.75±1.46) s,BCP group (7.61±1.07) s]in C57BL/6J mice were respectively developed on day 5 and 7, and the symptoms grow progressively with time (P<0.05). The expression level miR-93 is decreased in a time dependent way in BCP group with progressively increased pain. However, the ROCK2 expression level was increased (P<0.05), and subsequently activated the LIMK/Cofilin pathways. Dual luciferase reporter assay demonstrated that miR-93 directly targeted the 3'-UTR of the ROCK2 gene, resulting in inhibition of the post-transcriptional translation of ROCK2. ROCK inhibitor significantly up-regulated paw withdrawal threshold[BCP+PBS group(3.43±0.89) g, BCP+Y-27632 group (5.74±1.02) g], paw withdrawal latency[BCP+PBS group (5.48±1.03) s, BCP+Y-27632 group (9.82±1.24) s]and reduced the protein expression level of ROCK2, phospho-LIMK and phospho-Cofilin (P<0.05). Conclusions Reduced expression of miR-93 can promote BCP through activating ROCK2/LIMK/cofilin pathway. Therefore, miR-93 or ROCK inhibitor may be novel targets for BCP. Key words: Bone cancer pain; Analgesia; MicroRNA-93; Rho associated coiled-coil forming protein kinase2/LIM domain kinase/Cofilin signaling

The Rho kinases: critical mediators of multiple profibrotic processes and rational targets for new therapies for pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by progressive lung scarring, short median survival, and limited therapeutic options, creating great need for new pharmacologic therapies. IPF is thought to result from repetitive environmental injury to the lung epithelium, in the context of aberrant host wound healing responses. Tissue responses to injury fundamentally involve reorganization of the actin cytoskeleton of participating cells, including epithelial cells, fibroblasts, endothelial cells, and macrophages. Actin filament assembly and actomyosin contraction are directed by the Rho-associated coiled-coil forming protein kinase (ROCK) family of serine/threonine kinases (ROCK1 and ROCK2). As would therefore be expected, lung ROCK activation has been demonstrated in humans with IPF and in animal models of this disease. ROCK inhibitors can prevent fibrosis in these models, and more importantly, induce the regression of already established fibrosis. Here we review ROCK structure and function, upstream activators and downstream targets of ROCKs in pulmonary fibrosis, contributions of ROCKs to profibrotic cellular responses to lung injury, ROCK inhibitors and their efficacy in animal models of pulmonary fibrosis, and potential toxicities of ROCK inhibitors in humans, as well as involvement of ROCKs in fibrosis in other organs. As we discuss, ROCK activation is required for multiple profibrotic responses, in the lung and multiple other organs, suggesting ROCK participation in fundamental pathways that contribute to the pathogenesis of a broad array of fibrotic diseases. Multiple lines of evidence therefore indicate that ROCK inhibition has great potential to be a powerful therapeutic tool in the treatment of fibrosis, both in the lung and beyond.

Increase in cell motility by carbon ion irradiation via the Rho signaling pathway and its inhibition by the ROCK inhibitor Y-27632 in lung adenocarcinoma A549 cells

This study aimed to investigate the effect of carbon ion (C-ion) irradiation on cell motility through the ras homolog gene family member (Rho) signaling pathway in the human lung adenocarcinoma cell line A549. Cell motility was assessed by a wound-healing assay, and the formation of cell protrusions was evaluated by F-actin staining. Cell viability was examined by the WST-1 assay. The expression of myosin light chain 2 (MLC2) and the phosphorylation of MLC2 at Ser19 (P-MLC2-S19) were analyzed by Western blot. At 48 h after irradiation, the wound-healing assay demonstrated that migration was significantly greater in cells irradiated with C-ion (2 or 8 Gy) than in unirradiated cells. Similarly, F-actin staining showed that the formation of protrusions was significantly increased in cells irradiated with C-ion (2 or 8 Gy) compared with unirradiated cells. The observed increase in cell motility due to C-ion irradiation was similar to that observed due to X-ray irradiation. Western-blot analysis showed that C-ion irradiation (8 Gy) increased P-MLC2-S19 expression compared with in unirradiated controls, while total MLC2 expression was unchanged. Exposure to a non-toxic concentration of Y-27632, a specific inhibitor of Rho-associated coiled-coil-forming protein kinase (ROCK), reduced the expression of P-MLC2-S19 after C-ion irradiation (8 Gy), resulting in a significant reduction in migration. These data suggest that C-ion irradiation increases cell motility in A549 cells via the Rho signaling pathway and that ROCK inhibition reduces that effect.

Attenuation of Lung Ischemia-Reperfusion Injury by Rho-Associated Kinase Inhibition in a Rat Model of Lung Transplantation

A signaling pathway of the small GTPase Rho and Rho-associated coiled-coil-forming protein kinase (ROCK), regulates the contraction of endothelial cells. We studied the effects of Y-27632, a specific ROCK inhibitor, to clarify the role of Rho/ROCK in the pathogenesis of ischemia-reperfusion lung injury in a rat model of single-lung transplantation (LTX). We flushed 5 donor rat lungs with Euro-Collins solution, and 5 donor lungs with Euro-Collins + Y-27632, 0.03 mg/ml, and preserved the lungs for 6 h at 4°C before reperfusion for 4 h. The 5 rat recipients of Y-27632-treated lungs also received a 10-mg/kg bolus of Y-27632 i.p. 30 min before reperfusion. Pretreatment of the donor lungs and recipient rats with Y-27632 prominently suppressed the post-LTX edema, while the permeability index was only slightly decreased. The (1) numbers of neutrophils and macrophages, and (2) tumor necrosis factor (TNF)-α concentration, were significantly lower in the bronchoalveolar lavage fluid of treated than untreated lungs. Y-27632 (1) inhibited the migration of inflammatory cells into the alveolar space, (2) decreased the production of TNF-α, and (3) attenuated the edema after LTX. Endothelial Rho and ROCK may play an important role in the pathogenesis of post-LTX injury.

Fasudil Inhibits the Proliferation and Contractility and Induces Cell Cycle Arrest and Apoptosis of Human Endometriotic Stromal Cells: A Promising Agent for the Treatment of Endometriosis

During the development of endometriotic lesions, excess fibrosis may lead to scarring and to the alterations of tissue function that are the characteristic features of this disease. Enhanced extracellular matrix contractility of endometriotic stromal cells (ECSC) mediated by the mevalonate-Ras homology (Rho)/Rho-associated coiled-coil-forming protein kinase (ROCK) pathway has been shown to contribute to the pathogenesis of endometriosis. To assess the use of fasudil, a selective ROCK inhibitor, for the medical treatment of endometriosis-associated fibrosis, the effects of this agent on the cell proliferation, apoptosis, cell cycle, morphology, cell density, and contractility of ECSC were investigated. The effects of fasudil on the expression of contractility-related, apoptosis-related, and cell cycle-related molecules in ECSC were also evaluated. Fasudil significantly inhibited the proliferation and contractility of ECSC and induced the cell cycle arrest in the G2/M phase and apoptosis of these cells. Morphological observation revealed the suppression of ECSC attachment to collagen fibers and decrease of cell density by fasudil. The expression of α-smooth muscle actin, RhoA, ROCK-I, and ROCK-II proteins was inhibited by fasudil administration. The expression of the antiapoptotic factors, Bcl-2 and Bcl-X(L), in two-dimensional cultured ECSC were down-regulated by the addition of fasudil, whereas, the expression of p16(INK4a) and p21(Waf1/Cip1) was up-regulated by the addition of fasudil. The present findings suggest that fasudil is a promising agent for the treatment of endometriosis. The inhibition of cell proliferation, contractility, and myofibroblastic differentiation, the attenuation of attachment to collagen fibers, the decrease of cell density, and the induction of cell cycle arrest and apoptosis of ECSC are involved in the active mechanisms of fasudil.

Effects of Rho-associated protein kinase inhibitors Y-27632 and Y-39983 on isolated rabbit ciliary arteries

In normotensive eyes, reduced ocular blood flow can lead to glaucoma pathogenesis. Drugs that reduce intraocular pressure (IOP) often cause vasodilation of the ciliary arteries and improve blood flow to the eye. A novel class of drugs called Rho-associated coiled coil-forming protein kinase (ROCK) inhibitors can lower IOP. Therefore, we tested the ability of two ROCK inhibitors, Y-27632 and Y39983, to relax rabbit ciliary arteries. We measured in vitro ciliary artery smooth muscle contractions by isometric tension recordings and changes of intracellular free calcium concentration ([Ca(2+)](i)) by fluorescence photometry. Both Y-27632 and Y-39983 induced a concentration-dependent relaxation in rabbit ciliary arteries precontracted with a high-potassium (high-K) solution. The amplitude of relaxation induced by Y-27632 and Y-39983 was not affected by either 100 μM N (G)-nitro-L: -arginine methyl ester (L: -NAME) or 10 μM indomethacin. In Ca(2+)-free solution, Y-27632 and Y-39983 significantly inhibited the transient contraction of ciliary arteries induced by 10 μM histamine. However, neither Y-27632 nor Y-39983 affected the elevation of [Ca(2+)](i) induced by high-K solution and histamine. We concluded that Y-27632 and Y-39983 relaxed isolated rabbit ciliary artery segments in vitro. The mechanism of relaxation was not dependent on endothelial-derived factors such as nitric oxide (NO) or prostacyclin, nor was it dependent on changes in intracellular Ca(2+) concentration.

7-Chloro-6-piperidin-1-yl-quinoline-5,8-dione (PT-262), a novel ROCK inhibitor blocks cytoskeleton function and cell migration

The 5,8-quinolinediones are precursors for producing multiple types of bioactive products. In this study, we investigated a new compound derived from 5,8-quinolinediones, 7-chloro-6-piperidin-1-yl-quinoline-5,8-dione (designated as PT-262), which markedly induced cytoskeleton remodeling and migration inhibition in lung carcinoma cells. Comparison with various cytoskeleton inhibitors, including paclitaxel, colchicine and phallacidin, the cell morphology following treatment with PT-262 was similar to phallacidin on the cell elongation and abnormal actin polymerization. However, PT-262 did not directly bind to actin filaments. ROCK (Rho-associated coiled-coil forming protein kinase) is a downstream effector of RhoA to mediate the phosphorylation of myosin light chain (MLC) and cytoskeleton reorganization. The RhoA–ROCK–MLC pathway has been shown to promote cancer cell migration and metastasis. Interestingly, PT-262 was more effective on inhibiting ROCK kinase activities than specific ROCK inhibitors Y-27632 and H-1152. PT-262 induced cytoskeleton remodeling and migration inhibition in A549 lung carcinoma cells. The total MLC and phosphorylated MLC proteins and stress fibers were blocked after treatment with PT-262. Nonetheless, the RhoA protein and GTPase activity were not altered by PT-262. A computational model suggests that PT-262 interacts with the ATP-binding site of ROCK protein. Together, these findings demonstrate that PT-262 is a new ROCK inhibitor.

Effects of Fasudil, a Rho-Associated Protein Kinase Inhibitor, on Optic Nerve Head Blood Flow in Rabbits

To investigate the effects of fasudil, a Rho-associated coiled coil-forming protein kinase (ROCK) inhibitor, on normal or impaired optic nerve head (ONH) blood flow in a rabbit model. ONH blood flow was measured by laser speckle flowgraphy. Changes in ONH blood flow were examined during a continuous intravenous infusion of fasudil with and without the application of N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor. Effects of topical fasudil on ONH blood flow were investigated in normal eyes or models of ocular circulation impairment induced by the application of endothelin (ET)-1. Visual-evoked potentials (VEPs) and morphologic and histologic changes were also analyzed in the ET-1-injected eyes. A continuous intravenous infusion of fasudil had no significant effect on normal ONH blood flow, yet it prevented or improved the ONH blood flow impairment induced by the intravenous injection of L-NAME. Repeated intravitreal injections of ET-1 twice a week for 4 weeks decreased the ONH blood flow, prolonged the VEPs' implicit time, enlarged the optic cup, and decreased retinal ganglion cells. Multiple doses of topical fasudil ameliorated the ONH impairments caused by ET-1. These results show that systemic or topical fasudil suppresses impairment of ONH blood flow, function, and morphology induced by L-NAME or ET-1. A ROCK inhibitor can be useful for the treatment of impaired ONH blood flow.

Inhibition of the Rho/ROCK pathway prevents neuronal degeneration in vitro and in vivo following methylmercury exposure

Methylmercury (MeHg) is an environmental neurotoxicant which induces neuropathological changes in both the central nervous and peripheral sensory nervous systems. Our recent study demonstrated that down-regulation of Ras-related C3 botulinum toxin substrate 1 (Rac1), which is known to promote neuritic extension, preceded MeHg-induced damage in cultured cortical neurons, suggesting that MeHg-mediated axonal degeneration is due to the disturbance of neuritic extension. Therefore we hypothesized that MeHg-induced axonal degeneration might be caused by neuritic extension/retraction incoordination. This idea brought our attention to the Ras homolog gene (Rho)/Rho-associated coiled coil-forming protein kinase (ROCK) pathway because it has been known to be associated with the development of axon and apoptotic neuronal cell death. Here we show that inhibition of the Rho/ROCK pathway prevents MeHg-intoxication both in vitro and in vivo. A Rho inhibitor, C3 toxin, and 2 ROCK inhibitors, Fasudil and Y-27632, significantly protected against MeHg-induced axonal degeneration and apoptotic neuronal cell death in cultured cortical neuronal cells exposed to 100 nM MeHg for 3 days. Furthermore, Fasudil partially prevented the loss of large pale neurons in dorsal root ganglia, axonal degeneration in dorsal spinal root nerves, and vacuolar degeneration in the dorsal columns of the spinal cord in MeHg-intoxicated model rats (20 ppm MeHg in drinking water for 28 days). Hind limb crossing sign, a characteristic MeHg-intoxicated sign, was significantly suppressed in this model. The results suggest that inhibition of the Rho/ROCK pathway rescues MeHg-mediated neuritic extension/retraction incoordination and is effective for the prevention of MeHg-induced axonal degeneration and apoptotic neuronal cell death.

Intraocular Pressure–Lowering Effects and Safety of Topical Administration of a Selective ROCK Inhibitor, SNJ-1656, in Healthy Volunteers

To investigate the effects and safety of topical administration of an ophthalmic solution of a selective Rho-associated coiled coil-forming protein kinase (ROCK) inhibitor, SNJ-1656, 0.003% to 0.1%, in healthy male adult volunteers. Randomized, double-masked, group-comparison, phase 1 clinical study. In the initial single-instillation trial, 45 healthy volunteers were randomly subdivided into 5 groups and treated with SNJ-1656 in concentrations of 0.003%, 0.01%, 0.03%, 0.05%, and 0.1% in stepwise fashion. In the repeated-instillation trial, 36 healthy volunteers were assigned to receive SNJ-1656 ophthalmic solution at the following concentrations and dosages: 0.05% once daily, 0.1% once daily, 0.05% twice daily, or 0.1% twice daily. In our studies, the administration of the solution and subsequent examinations (including intraocular pressure [IOP] measurements) were performed in a double-masked fashion. After single instillation of placebo or SNJ-1656, in concentrations of 0.003%, 0.01%, 0.03%, 0.05%, and 0.1%, the changes in IOP from the baseline were -0.91, -1.18, -1.48, - 2.20 (P = .04 vs placebo), -1.48, and -1.98 mm Hg, respectively, at 2 hours, and -0.63,-0.95, -1.79, -2.26 (P = .01 vs placebo), -1.95, and -3.00 mm Hg (P < .001 vs placebo) respectively, at 4 hours. Significant IOP reductions after repeated instillation were also found. On slitlamp examination during the trial, there were no significant adverse findings except hyperemia of the bulbar and palpebral conjunctiva after instillation. This clinical study demonstrated that SNJ-1656 is a safe topical agent effective in reducing IOP in human eyes.

The role of specific ROCK inhibitor in the prevention of experimental PVR in rabbits

To determine the effect of Y27632, a specific inhibitor of p160 Rho-associated coiled-coil forming protein kinase (ROCK), on experimental rabbit PVR. Cultured rabbit retinal pigment epithelial cells were used in the experiments. The effects of Y27632 on RPE alpha-SMA (smooth muscle actin) stress fiber formation were studied by immuno-fluorescent staining. An in vitro type I collagen gel contraction assay and MTT assay were used to detect the effect of Y27632 on RPE cell contractile force and proliferation. Cultured 6 th passage rabbit RPE cells were injected intravitreally to induce the PVR model and then followed injection of 50 micromol/L of Y27632. The presence of tractional retinal detachment (TRD) was assessed to evaluate the effect of this agent in vivo. Electroretinography and histological studies were performed after intravitreal injection of Y27632 into untreated eyes to evaluate toxicity. The results showed that Y27632 disrupted SMA stress fiber formation in the cultured RPE cells and impaired contractile force generated by RPE cells (t = 16.212, P < 0.01). Development of TRD was significantly reduced (P < 0.01) with 50 micromol/L of Y27632. No obvious histological or retinal functional damage was found in the Y27632-treated group. p160 ROCK specific inhibitor Y27632 decreased contractile force generated by RPE cells and attenuated PVR without significant side effect in rabbit. This reagent could be potential therapeutically method in the treatment of PVR.

The Neurofibromatosis Type 1 Gene Product Neurofibromin Enhances Cell Motility by Regulating Actin Filament Dynamics via the Rho-ROCK-LIMK2-Cofilin Pathway

Neurofibromin is a neurofibromatosis type 1 (NF1) tumor suppressor gene product with a domain that acts as a GTPase-activating protein and functions, in part, as a negative regulator of Ras. Loss of neurofibromin expression in NF1 patients is associated with elevated Ras activity and increased cell proliferation, predisposing to a variety of tumors of the peripheral and central nervous systems. We show here, using the small interfering RNA (siRNA) technique, that neurofibromin dynamically regulates actin cytoskeletal reorganization, followed by enhanced cell motility and gross cell aggregation in Matrigel matrix. NF1 siRNA induces characteristic morphological changes, such as excessive actin stress fiber formation, with elevated negative phosphorylation levels of cofilin, which regulates actin cytoskeletal reorganization by depolymerizing and severing actin filaments. We found that the elevated phosphorylation of cofilin in neurofibromin-depleted cells is promoted by activation of a Rho-ROCK-LIMK2 pathway, which requires Ras activation but is not transduced through three major Ras-mediated downstream pathways via Raf, phosphatidylinositol 3-kinase, and RalGEF. In addition, the exogenous expression of the NF1-GTPase-activating protein-related domain suppressed the NF1 siRNA-induced phenotypes. Neurofibromin was demonstrated to play a significant role in the machinery regulating cell proliferation and in actin cytoskeletal reorganization, which affects cell motility and adhesion. These findings may explain, in part, the mechanism of multiple neurofibroma formation in NF1 patients.

Contribution of ROCK in Contraction of Trabecular Meshwork: Proposed Mechanism for Regulating Aqueous Outflow in Monkey and Human Eyes

Aqueous outflow in the conventional outflow pathway is regulated by the contraction and relaxation of the ciliary muscle (CM) and the trabecular meshwork (TM). Rho-associated coiled coil-forming protein kinase (ROCK) is thought to regulate actomyosin-based contractility in many types of cells by phosphorylation of ROCK substrates. In animal models, ROCK inhibitor Y-39983 relaxed CM and TM and decreased intraocular pressure (IOP). Thus, ROCK is implicated in the regulation of aqueous outflow and IOP. However, the site of action of ROCK in monkey and man is unknown. In the present communication, RT-PCR analysis of monkey tissues showed higher levels of mRNAs for ROCK and ROCK substrates in TM compared to CM. Human TM also showed higher levels of mRNAs for ROCK and ROCK substrates compared to CM. Differences between TM and CM in human were not as high as in monkey. ROCK inhibitor Y-39983 led to a dose-dependent relaxation of carbachol-induced, contracted TM from monkey. In contrast, Y-39983 was only slightly effective in relaxing CM. Our results suggested that TM was one of the major sites for regulating IOP by ROCK. ROCK inhibitor Y-39983 might be a candidate drug for lowering IOP by increasing conventional outflow and producing fewer side effects on accommodation and miosis.

Effect of Wf-536, a novel ROCK inhibitor, against metastasis of B16 melanoma.

Rho-associated coiled-coil-forming protein kinase (ROCK) is pivotally involved in invasion by tumor cells and their evolution to metastasis. We have developed a novel inhibitor of ROCK, Wf-536 [(+)-(R)-4-(1-aminoethyl)-N-(4-pyridyl) benzamide monohydrochloride]. In the present study, we investigated its effect on in vitro invasion and in vivo pulmonary metastasis of B16 melanoma. The following were evaluated: the anti-invasive effect of Wf-536 against the motility of mouse B16BL6 melanoma cells through a culture insert layered with reconstituted basement membrane (Matrigel); the cytotoxic effect of Wf-536 in the same cell line; the antimetastatic effect of Wf-536, administered by osmotic pump, on spontaneous pulmonary metastasis following subcutaneous injection of B16BL6 melanoma in mice; and the inhibitory effect of orally administered Wf-536, alone or in combination with the antineoplastic drug paclitaxel, on pulmonary metastasis of intravenously injected B16F10 melanoma in mice. Wf-536 inhibited in vitro invasion by B16BL6 cells significantly and in a concentration-dependent manner and displayed an anti-invasive effect under conditions of both chemotaxis and chemokinesis. No cytotoxic effect was observed at any of the concentrations used. In vivo, Wf-536 administration suppressed tumor colony formation on the lung surface in a dose-dependent manner (0.3-3 mg/kg per day), with a metastasis inhibition rate of 95% at 3 mg/kg per day. In experimental metastasis of B16F10 melanoma, oral administration of Wf-536 significantly decreased tumor colony formation in the lung, with an inhibition rate of 41% at 3 mg/kg per day. The inhibition rate of paclitaxel (5 mg/kg per day) was 27%. The combination of Wf-536 and paclitaxel produced a synergistic effect on B16F10 metastasis and a 68% inhibition rate. Wf-536 administration at the doses used did not alter body weight, blood pressure or the health of treated animals as compared to vehicle-treated controls. The results suggest that Wf-536 is a potentially valuable drug for preventing tumor metastasis both in monotherapy and in combination with an antineoplastic drug.

Small Guanosine Triphospatase RhoA and Rho-Associated Kinase as Regulators of Trophoblast Migration

The small guanosine triphosphatase Rho controls cell adhesion and motility through reorganization of the actin cyto-skeleton and regulation of actomyosin contractility. Among the putative target molecules of Rho, a Rho-associated coiled coil-forming protein kinase (ROCK) is thought to participate in Rho-mediated cell adhesion and motility. In the present study, we explored the expression and function of RhoA and ROCK in human trophoblast cells. The colocalization of RhoA, cytokeratin 8/18, and cytokeratin 7 in some cells located in the decidual stromal region indicated that extravillous trophoblast cells expressed RhoA. In double staining for RhoA and ROCK in human chorionic villi, RhoA staining was strongly positive in the cytoplasm of cytotrophoblasts, whereas ROCK stained in the cytoplasm of cytotrophoblasts and syncytiotrophoblasts. Both RhoA and ROCK were stained in cytoplasma of cultured human cytotrophoblast. Cultured human trophoblast cells contained actin stress fibers that were lost after treatment with C3, an exoenzyme produced by Clostridium botulinum. Y-27632, a selective ROCK inhibitor, suppressed RhoA-induced formation of actin stress fibers and formation of focal contact in trophoblast cells. The trophoblast reacquired actin stress fibers and focal contact after withdrawal of Y-27632. Cultured human cytotrophoblast cells from 7-9 wk of gestation migrated into a fibronectin-coated membrane. Both C3 exoenzyme and Y-27632 inhibited cytotrophoblast migration in a dose-dependent manner. In conclusion, cyto-trophoblasts express RhoA and ROCK in their cytoplasm, and RhoA-ROCK is involved in their assembly of actin stress fibers. Suppression of RhoA-ROCK reduces trophoblast migration. These findings suggest that RhoA-ROCK signaling is a key regulator of trophoblast cell migration.

Y-27632 prevents tubulointerstitial fibrosis in mouse kidneys with unilateral ureteral obstruction

The small GTPase Rho is involved in cell-to-substratum adhesion and cell contraction. These actions of Rho mediated by downstream Rho effectors such as Rho-associated coiled-coil forming protein kinase (ROCK) may be partly responsible for the progression of renal interstitial fibrosis. The anti-fibrosis effects of Y-27632, a specific ROCK inhibitor, were studied both in vivo (unilateral ureteral obstruction; UUO) and in vitro. To investigate the therapeutic efficacy of Y-27632 in UUO kidneys, smooth muscle alpha actin (SMalphaA) expression, macrophage infiltration and fibrosis in the obstructed kidneys were studied. SMalphaA, transforming growth factor beta (TGF-beta), alpha1 (I) collagen, osteopontin, macrophage chemoattractant peptide-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1) gene expression were examined by Northern blotting. To elucidate the mechanism linking the Rho-ROCK pathway with renal fibrosis, the effects of Y-27632 on in vitro cell proliferation and cell migration were studied. In vivo analysis showed that Y-27632 suppressed SMalphaA expression, macrophage infiltration and interstitial fibrosis, and that Y-27632 suppressed SMalphaA, TGF-beta and alpha1 (I) collagen mRNA expression. In vitro analysis showed that Y-27632 did not suppress proliferation of renal fibroblasts but suppressed migration of macrophages. The Rho-ROCK system may play an important role in the development of tissue fibrosis, and the Rho-ROCK signaling pathway may be a new therapeutic target for preventing interstitial fibrosis in progressive renal disease.

The functional role of rho and rho-associated coiled-coil forming protein kinase in eotaxin signaling of eosinophils.

The CC chemokine eotaxin plays a pivotal role in local accumulation of eosinophils. Very little is known about the eotaxin signaling in eosinophils except the activation of the mitogen-activated protein (MAP) kinase family. The p21 G protein Rho and its substrate Rho-associated coiled-coil forming protein kinase (ROCK) regulate the formation of stress fibers and focal adhesions. In the present study, we studied the functional relevance of Rho and ROCK in eosinophils using the ROCK inhibitor (Y-27632) and exoenzyme C3, a specific Rho inhibitor. Eotaxin stimulates activation of Rho A and ROCK II in eosinophils. Exoenzyme C3 almost completely inhibited the ROCK activity, indicating that ROCK is downstream of Rho. We then examined the role of Rho and ROCK in eosinophil chemotaxis. The eotaxin-induced eosinophil chemotaxis was significantly inhibited by exoenzyme C3 or Y-27632. Because extracellular signal-regulated kinase (ERK)1/2 and p38 MAP kinases are activated by eotaxin and are critical for eosinophil chemotaxis, we investigated whether Rho and ROCK are upstream of these MAP kinases. C3 partially inhibited eotaxin-induced phosphorylation of ERK1/2 but not p38. In contrast, neither ERK1/2 nor p38 phosphorylation was abrogated by Y-27632. Both C3 and Y-27632 reduced reactive oxygen species production from eosinophils. We conclude that both Rho and ROCK are important for eosinophil chemotaxis and reactive oxygen species production. There is a dichotomy of downstream signaling pathways of Rho, namely, Rho-ROCK and Rho-ERK pathways. Taken together, eosinophil chemotaxis is regulated by multiple signaling pathways that involve at least ROCK, ERK, and p38 MAP kinase.