Rho-associated protein kinase is involved in establishing the cardiomyocyte contractile phenotype

We have shown that Y-27632, an inhibitor of Rho-associated kinase (ROCK), delays sarcomere assembly in cultured rat neonatal cardiomyocytes pretreated with angiotensin II. Y-27632 affects the beat rate of cardiomyocytes, but this effect is only observed at high cell density and, therefore, seems to be related to impairment of cell-cell interactions. Consistent with this suggestion, ROCK2 is localized in myofibrillar Z-disks of human myocardium as well as in intercalated disks. We propose that ROCK participates in maturation of the myocardial contractile system through phosphorylation of its molecular targets in Z-disks and in intercalated disks.     

Rho-kinase in human neutrophils: a role in signalling for myosin light chain phosphorylation and cell migration

The role of a Rho-associated coiled-coil forming kinase in migration of neutrophils has been investigated. Rho-associated coiled-coil forming kinase I was expressed in human neutrophils. Chemotactic peptide led to a Rho-associated coiled-coil forming kinase-dependent increase in phosphorylation of myosin light chain. This was determined with the help of an antibody directed against serine 19-phosphorylated myosin light chain and an inhibitor of Rho-associated coiled-coil forming kinase (Y-27632). Y-27632 suppressed myosin light chain phosphorylation and chemotactic peptide-induced development of cell polarity and locomotion with similar potency (ED50 0.5-1.1 microM). The data strongly suggest that a Rho-associated coiled-coil forming kinase isoform, activated in human neutrophils exposed to chemotactic peptide, is important for motile functions of these cells.     

Tumor Necrosis Factor Disrupts Claudin-5 Endothelial Tight Junction Barriers in Two Distinct NF-κB-Dependent Phases

Capillary leak in severe sepsis involves disruption of endothelial cell tight junctions. We modeled this process by TNF treatment of cultured human dermal microvascular endothelial cell (HDMEC) monolayers, which unlike human umbilical vein endothelial cells form claudin-5-dependent tight junctions and a high-resistance permeability barrier. Continuous monitoring with electrical cell-substrate impedance sensing revealed that TNF disrupts tight junction-dependent HDMEC barriers in discrete steps: an ~5% increase in transendothelial electrical resistance over 40 minutes; a decrease to ~10% below basal levels over 2 hours (phase 1 leak); an interphase plateau of 1 hour; and a major fall in transendothelial electrical resistance to < 70% of basal levels by 8–10 hours (phase 2 leak), with EC₅₀ values of TNF for phase 1 and 2 leak of ~30 and ~150 pg/ml, respectively. TNF leak is reversible and independent of cell death. Leak correlates with disruption of continuous claudin-5 immunofluorescence staining, myosin light chain phosphorylation and loss of claudin-5 co-localization with cortical actin. All these responses require NF-κB signaling, shown by inhibition with Bay 11 or overexpression of IκB super-repressor, and are blocked by H-1152 or Y-27632, selective inhibitors of Rho-associated kinase that do not block other NF-κB-dependent responses. siRNA combined knockdown of Rho-associated kinase-1 and -2 also prevents myosin light chain phosphorylation, loss of claudin-5/actin co-localization, claudin-5 reorganization and reduces phase 1 leak. However, unlike H-1152 and Y-27632, combined Rho-associated kinase-1/2 siRNA knockdown does not reduce the magnitude of phase 2 leak, suggesting that H-1152 and Y-27632 have targets beyond Rho-associated kinases that regulate endothelial barrier function. We conclude that TNF disrupts TJs in HDMECs in two distinct NF-κB-dependent steps, the first involving Rho-associated kinase and the second likely to involve an as yet unidentified but structurally related protein kinase(s).     

Intrathecal administration of Y-27632, a specific rho-associated kinase inhibitor, for rat neoplastic meningitis

The small GTP-binding protein Rho and its target Rho-associated kinase trigger an intracellular signaling cascade that controls actin cytoskeleton and plays an essential role in cell motility and adhesion. A specific Rho-associated kinase inhibitor, Y-27632, has been reported to inhibit cancer invasion. Clinically, disseminated tumor cells in the cerebrospinal fluid invade the intraparenchymal region, damaging the brain and nerves, resulting in fatal brain stem dysfunction, despite intrathecal chemotherapy. To expand therapeutic options for this devastating neoplastic meningitis, we evaluated the potential use of intrathecal Y-27632 administration by employing Walker 256 cells, a rat mammary cancer cell line. Y-27632 dose-dependently inhibited chemotactic and invasive activity of Walker 256 cells. Y-27632 also inhibited the phosphorylation level of regulatory myosin light chain in vitro, but the effect was temporary and was considerably diminished within 16 hours. Y-27632 induced striking morphologic changes in Walker 256 cells, as evidenced by decreased cell-matrix adhesion in culture dishes and three-dimensional collagen I gels, and slightly inhibited colony formation in soft agar. Nevertheless, this drug treatment did not affect Walker 256 cell growth rate. We were able to administer continuous delivery of this inhibitor using an osmotic pump and maintaining drug concentration of 10 mumol/L within the brain. Importantly, this concentration of Y-27632 showed minimal neurotoxicity both in vitro and in vivo. We found that an intrathecal therapy, combining 5-fluoro-2'-deoxyuridine with Y-27632, significantly increased the survival time of rats bearing meningeal carcinomatosis in comparison with animals treated with 5-fluoro-2'-deoxyuridine alone. Taken together, our findings indicate that continuous intrathecal administration of Y-27632 could be a promising therapeutic method when combined with chemotherapy for treating human neoplastic meningitis.     

ROCK and PRK-2 mediate the inhibitory effect of Y-27632 on polyglutamine aggregation

Polyglutamine expansion in huntingtin (Htt) and the androgen receptor (AR) causes untreatable neurodegenerative diseases. Y-27632, a therapeutic lead, reduces Htt and AR aggregation in cultured cells, and Htt-induced neurodegeneration in Drosophila. Y-27632 inhibits both Rho-associated kinases ROCK and PRK-2, making its precise intracellular target uncertain. Over-expression of either kinase increases Htt and AR aggregation. Three ROCK inhibitors (Y-27632, HA-1077, and H-1152P), and a specific ROCK inhibitory peptide reduce polyglutamine protein aggregation, as does knockdown of ROCK or PRK-2 by RNAi. RNAi also indicates that each kinase is required for the inhibitory effects of Y-27632 to manifest fully. These two actin regulatory kinases are thus involved in polyglutamine aggregation, and their simultaneous inhibition may be an important therapeutic goal.     

The stimulatory effect of ROCK inhibitor on bovine corneal endothelial cells

Reagents which can promote the proliferation, adhesion and migration of cultured corneal endothelial cells (CECs) will be helpful for the treatment of reduced visual acuity due to CECs deficiency. The objectives of this study were to investigate the potential use of an inhibitor of Rho-associated protein kinase (ROCK), Y-27632, to cultured bovine corneal endothelial cells (B-CECs) and evaluated its effects on the proliferation, adhesion and migration of B-CECs. The proliferation of cultured B-CECs was moderately enhanced by 10 μM Y-27632. Y-27632 induced fibroblast-like morphological changes in the cultured B-CECs and normal cell morphology could recover after Y-27632 removal. In addition, Y-27632 was found to significantly enhance the adhesion and migration of B-CECs. Furthermore, the hanging drop aggregation assay showed that Y-27632 promoted B-CECs to form cellular networks and sheets, which proliferated along the liquid–air interface and migrated to the surface of the lid of dish. Our study demonstrated that Y-27632 is a potentially powerful reagent which can enhance the proliferation of cultured B-CECs. Y-27632 will be useful in CEC injection therapy and topical application for CEC deficiency.     

心肌细胞缝隙连接重塑与心律失常

缝隙连接是相邻心肌细胞间电、化学偶联的通道,亦是心室肌成为功能性合胞体的重要结构.心肌有缝隙连接蛋白(connexin,CX) 40、43与45的表达,心室肌主要表达CX43.CX43形成的缝隙连接大部分呈点状分布于闰盘部位,心肌细胞膜侧面分布极少.心肌缺血-再灌注、肥厚、衰竭、高胆同醇与糖尿病条件下,心肌细胞缝隙连接...     

A ROCK inhibitor permits survival of dissociated human embryonic stem cells

Poor survival of human embryonic stem (hES) cells after cell dissociation is an obstacle to research, hindering manipulations such as subcloning. Here we show that application of a selective Rho-associated kinase (ROCK) inhibitor, Y-27632, to hES cells markedly diminishes dissociation-induced apoptosis, increases cloning efficiency (from approximately 1% to approximately 27%) and facilitates subcloning after gene transfer. Furthermore, dissociated hES cells treated with Y-27632 are protected from apoptosis even in serum-free suspension (SFEB) culture and form floating aggregates. We demonstrate that the protective ability of Y-27632 enables SFEB-cultured hES cells to survive and differentiate into Bf1(+) cortical and basal telencephalic progenitors, as do SFEB-cultured mouse ES cells.     

Unusual splicing events result in distinct Xin isoforms that associate differentially with filamin c and Mena/VASP

Filamin c is the predominantly expressed filamin isoform in striated muscles. It is localized in myofibrillar Z-discs, where it binds FATZ and myotilin, and in myotendinous junctions and intercalated discs. Here, we identify Xin, the protein encoded by the human gene 'cardiomyopathy associated 1' (CMYA1) as filamin c binding partner at these specialized structures where the ends of myofibrils are attached to the sarcolemma. Xin directly binds the EVH1 domain proteins Mena and VASP. In the adult heart, Xin and Mena/VASP colocalize with filamin c in intercalated discs. In cultured cardiomyocytes, the proteins also localize in the nonstriated part of myofibrils, where sarcomeres are assembled and an extensive reorganization of the actin cytoskeleton occurs. Unusual intraexonic splicing events result in the existence of three Xin isoforms that associate differentially with its ligands. The identification of the complex filamin c-Xin-Mena/VASP provides a first glance on the role of Xin in the molecular mechanisms involved in developmental and adaptive remodeling of the actin cytoskeleton during cardiac morphogenesis and sarcomere assembly.     

ROCK inhibitor Y-27632 prevents porcine oocyte maturation

The inhibitor Y-27632 is a specific selective inhibitor of Rho-associated protein kinases (ROCKs), which are downstream effectors of Rho guanosine triphosphatease (GTPases) and regulate Rho-associated cellular functions, including actin cytoskeletal organization. Little is known regarding the effects of Y-27632 on mammalian oocyte maturation. In the present study, we investigated the effects of Y-27632 on porcine oocyte meiosis and possible regulatory mechanisms of ROCK during porcine oocyte maturation. We found that ROCK accumulated not only at spindles, but also at the cortex in porcine oocytes. Y-27632 treatment reduced ROCK expression, and inhibited porcine oocyte meiotic maturation, which might be because of the impairment of actin expression and actin-related spindle positioning. Y-27632 treatment also disrupted the formation of actin cap and cortical granule-free domain, which further confirmed a spindle positioning failure. Thus, Y-27632 has significant effects on the meiotic competence of mammalian oocytes by reducing ROCK expression, and the regulation is related to its effects on actin-mediated spindle positioning.     

ROCK mediates thrombin's endothelial barrier dysfunction

Thrombin-induced endothelial monolayer hyperpermeability is thought toresult from increased F-actin stress fiber-related contractile tension,a process regulated by the small GTP-binding protein Rho. We testedwhether this process was dependent on the Rho-associated proteinkinase, ROCK, using a specific ROCK inhibitor, Y-27632. The effects ofY-27632 on thrombin-induced myosin light chain phosphorylation (MLCP)and tyrosine phosphorylation of p125 focal adhesion kinase(p125^FAK) and paxillin were measured by Western blotting.F-actin organization and content were analyzed by digital imaging, andendothelial monolayer permeability was measured in bovine pulmonaryartery endothelial cell (EC) monolayers using a size-selectivepermeability assay. Y-27632 enhanced EC monolayer barrier function dueto a decline in small-pore number that was associated with increased ECsurface area, reduced F-actin content, and reorganization of F-actin to-catenin-containing cell-cell adherens junctions. Although Y-27632prevented thrombin-induced MLCP, stress fiber formation, and theincreased phosphotyrosine content of paxillin and p125^FAK,it attenuated but did not prevent the thrombin-induced formation oflarge paracellular holes. These data indicate that thrombin-induced stress fiber formation is ROCK dependent. In contrast, thrombin-induced paracellular hole formation occurs in a ROCK-independent manner, whereas thrombin-induced monolayer hyperpermeability appears to bepartially ROCK dependent.

     

Deactivation of ROCK-II by Y-27632 enhances basolateral pancreatic enzyme secretion and acute pancreatitis induced by CCK analogues

In isolated rat pancreatic acini, protein expression of RhoA and Rho-associated kinase, ROCK-II, and the formation of immunocomplex of RhoA with ROCK-II were enhanced by CCK-8, carbachol, and the phorbol ester TPA. The ROCK-specific inhibitor, Y-27632, did not alter basal amylase secretion, whereas it potentiated CCK-stimulated pancreatic enzyme secretion in vitro. During caerulein-induced pancreatitis occurring in mice in vivo, Y-27632 enhanced serum amylase levels and the formation of interstitial edema and vacuolization at 12-18h after the first injection of caerulein. Y-27632 in turn inhibited the recovery of protein expression of ROCK-II at 18h after the first caerulein injection. These results suggest that RhoA and ROCK-II assemble normal CCK-stimulated pancreatic enzyme secretion and prevent caerulein-induced acute pancreatitis.     

The Synergistic Enhancement of Cloning Efficiency in Individualized Human Pluripotent Stem Cells by Peroxisome Proliferative-activated Receptor-γ (PPARγ) Activation and Rho-associated Kinase (ROCK) Inhibition

Although human pluripotent stem cells (hPSCs) provide valuable sources for regenerative medicine, their applicability is dependent on obtaining both suitable up-scaled and cost effective cultures. The Rho-associated kinase (ROCK) inhibitor Y-27632 permits hPSC survival upon dissociation; however, cloning efficiency is often still low. Here we have shown that pioglitazone, a selective peroxisome proliferative-activated receptor-γ agonist, along with Y-27632 synergistically diminished dissociation-induced apoptosis and increased cloning efficiency (2–3-fold versus Y-27632) without affecting pluripotency of hPSCs. Pioglitazone exerted its positive effect by inhibition of glycogen synthase kinase (GSK3) activity and enhancement of membranous β-catenin and E-cadherin proteins. These effects were reversed by GW-9662, an irreversible peroxisome proliferative-activated receptor-γ antagonist. This novel setting provided a step toward hPSC manipulation and its biomedical applications.     

Platelet shape change is mediated by both calcium-dependent and -independent signaling pathways. Role of p160 Rho-associated coiled-coil-containing protein kinase in platelet shape change.

Platelets undergo shape change upon activation with agonists. During shape change, disc-shaped platelets turn into spiculated spheres with protruding filopodia. When agonist-induced cytosolic Ca(2+) increases were prevented using the cytosolic Ca(2+) chelator, 5, 5'-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (5, 5'-dimethyl-BAPTA), platelets still underwent shape change, although the onset was delayed and the initial rate was dramatically decreased. In the absence of cytosolic Ca(2+), agonist-stimulated myosin light chain phosphorylation was significantly inhibited. The myosin light chain was maximally phosphorylated at 2 s in control platelets compared with 30 s in 5,5'-dimethyl-BAPTA-treated platelets. ADP, thrombin, or U46619-induced Ca(2+)-independent platelet shape change was significantly reduced by staurosporine, a nonselective kinase inhibitor, by the selective p160 Rho-associated coiled-coil-containing protein kinase inhibitor Y-27632, or by HA 1077. Both Y-27632 and HA 1077 reduced peak levels of ADP-induced platelet shape change and myosin light chain phosphorylation in control platelets. In 5,5'-dimethyl-BAPTA-treated platelets, Y-27632 and HA 1077 completely abolished both ADP-induced platelet shape change and myosin light chain phosphorylation. Our results indicate that Ca(2+)/calmodulin-stimulated myosin light chain kinase and p160 Rho-associated coiled-coil-containing protein kinase independently contribute to myosin light chain phosphorylation and platelet shape change, through Ca(2+)-sensitive and Ca(2+)-insensitive pathways, respectively.     

Y-27632, an Inhibitor of Rho-Associated Kinases, Prevents Tyrosine Phosphorylation of Focal Adhesion Kinase and Paxillin Induced by Bombesin: Dissociation from Tyrosine Phosphorylation of p130

A rapid increase in tyrosine phosphorylation of focal adhesion kinase (FAK), paxillin, and Crk-associated substrate (CAS) are prominent early events triggered by many G protein-coupled receptors (GPCRs), but the mechanisms involved remain unclear. Here, we examined whether the Rho-associated protein serine/threonine kinase family (ROCK) is a critical Rho effector in the pathway that links GPCR activation to the tyrosine phosphorylation of FAK, CAS, and paxillin. Treatment of Swiss 3T3 cells with Y-27632, a preferential inhibitor of ROCK, dramatically inhibited the formation of actin stress fibers, the assembly of focal contacts, and the increase in tyrosine phosphorylation of FAK and paxillin induced by bombesin in these cells. Surprisingly, we found that treatment with Y-27632 did not produce any detectable effect on bombesin-elicited CAS tyrosine phosphorylation even at the highest concentrations of Y-27632 tested. HA-1077, a preferential inhibitor of ROCK activity structurally unrelated to Y-27632, also attenuated the increase in the tyrosine phosphorylation of FAK and paxillin but did not affect the tyrosine phosphorylation of CAS induced by bombesin in Swiss 3T3 cells. The results demonstrate that ROCK-dependent tyrosine phosphorylation of FAK and paxillin can be dissociated from a ROCK-independent pathway leading to tyrosine phosphorylation of CAS.     

Y-27632, an inhibitor of Rho-associated kinases, prevents tyrosine phosphorylation of focal adhesion kinase and paxillin induced by bombesin: dissociation from tyrosine phosphorylation of p130(CAS)

A rapid increase in tyrosine phosphorylation of focal adhesion kinase (FAK), paxillin, and Crk-associated substrate (CAS) are prominent early events triggered by many G protein-coupled receptors (GPCRs), but the mechanisms involved remain unclear. Here, we examined whether the Rho-associated protein serine/threonine kinase family (ROCK) is a critical Rho effector in the pathway that links GPCR activation to the tyrosine phosphorylation of FAK, CAS, and paxillin. Treatment of Swiss 3T3 cells with Y-27632, a preferential inhibitor of ROCK, dramatically inhibited the formation of actin stress fibers, the assembly of focal contacts, and the increase in tyrosine phosphorylation of FAK and paxillin induced by bombesin in these cells. Surprisingly, we found that treatment with Y-27632 did not produce any detectable effect on bombesin-elicited CAS tyrosine phosphorylation even at the highest concentrations of Y-27632 tested. HA-1077, a preferential inhibitor of ROCK activity structurally unrelated to Y-27632, also attenuated the increase in the tyrosine phosphorylation of FAK and paxillin but did not affect the tyrosine phosphorylation of CAS induced by bombesin in Swiss 3T3 cells. The results demonstrate that ROCK-dependent tyrosine phosphorylation of FAK and paxillin can be dissociated from a ROCK-independent pathway leading to tyrosine phosphorylation of CAS.     

Phosphorylation of profilin by ROCK1 regulates polyglutamine aggregation

Y-27632, an inhibitor of the Rho-associated kinase ROCK, is a therapeutic lead for Huntington disease (HD). The downstream targets that mediate its inhibitory effects on huntingtin (Htt) aggregation and toxicity are unknown. We have identified profilin, a small actin-binding factor that also interacts with Htt, as being a direct target of the ROCK1 isoform. The overexpression of profilin reduces the aggregation of polyglutamine-expanded Htt and androgen receptor (AR) peptides. This requires profilin's G-actin binding activity and its direct interaction with Htt, which are both inhibited by the ROCK1-mediated phosphorylation of profilin at Ser-137. Y-27632 blocks the phosphorylation of profilin in HEK293 cells and primary neurons, which maintains profilin in an active state. The knockdown of profilin blocks the inhibitory effect of Y-27632 on both AR and Htt aggregation. A signaling pathway from ROCK1 to profilin thus controls polyglutamine protein aggregation and is targeted by a promising therapeutic lead for HD.     

Rho kinase inhibitors stimulate the migration of human cultured osteoblastic cells by regulating actomyosin activity

We investigated the effects of Rho-associated kinase (ROCK) on migration and cytoskeletal organization in primary human osteoblasts and Saos-2 human osteosarcoma cells. Both cell types were exposed to two different ROCK inhibitors, Y-27632 and HA-1077. In the improved motility assay used in the present study, Y-27632 and HA-1077 significantly increased the migration of both osteoblasts and osteosarcoma cells on plastic in a dose-dependent and reversible manner. Fluorescent images showed that cells of both types cultured with Y-27632 or HA-1077 exhibited a stellate appearance, with poor assembly of stress fibers and focal contacts. Western blotting showed that ROCK inhibitors reduced myosin light chain (MLC) phosphorylation within 5 min without affecting overall myosin light-chain protein levels. Inhibition of ROCK activity is thought to enhance the migration of human osteoblasts through reorganization of the actin cytoskeleton and regulation of myosin activity. ROCK inhibitors may be potentially useful as anabolic agents to enhance the biocompatibility of bone and joint prostheses.     

Rho inhibition decreases TNF-induced endothelial MAPK activation and monolayer permeability.

Our laboratory previously demonstrated that MAPK activation is an important signal during cytokine-induced endothelial permeability (Nwariaku FE, Liu Z, Terada L, Duffy S, Sarosi G, and Turnage R. Shock 18: 82-85, 2002). Because GTP-binding proteins have been implicated in MAPK activation, we now hypothesize that the GTP-binding protein Rho is a mediator of TNF-induced MAPK activation and increased endothelial permeability. Transmonolayer permeability was assessed in human lung microvascular cells by measuring transmonolayer electrical resistance. MAPK activity was assessed by using a phospho-specific immunoprecipitation kinase assay and by comparing Western blots for phospho-MAPK with total MAPK. MAPK inhibitors used were SB-202190 and PD-098059, whereas Clostridium botulinum C3 transferase was used as a Rho inactivator. Rho-associated coiled-coil kinase was inhibited with Y-27632. TNF increased pulmonary endothelial permeability in vitro and caused a rapid, sustained increase in endothelial p38 and extracellular signal-regulated kinase MAPK activity. Inhibition of p38 and extracellular signal-regulated kinase MAPK with SB-202190 and PD-098059, respectively, decreased TNF-induced endothelial permeability. C3 transferase attenuated TNF-induced MAPK activation and blocked TNF-induced endothelial permeability. Finally, inhibition of Rho-associated coiled-coil kinase with Y-27632 prevented both MAPK activation and TNF-induced decreases in transmonolayer resistance. Rho acts upstream of mitogen-activated protein kinases in mediating TNF-induced pulmonary endothelial leak.     

Rho-Associated Protein Kinases Play an Important Role in the Differentiation of Rat Adipose-Derived Stromal Cells into Cardiomyocytes In Vitro

Adipose-derived stromal cells (ADSCs) represent a readily available abundant supply of mesenchymal stem cells and have the ability to differentiate into cardiomyocytes in mice and human, making ADSCs a promising source of cardiomyocytes for transplantation. However, there has been no report of differentiation of rat ADSCs into cardiomyocytes. In addition, signaling pathways in the differentiation process from ADSCs to cardiomyocytes are unknown. In this study, we first demonstrated that rat ADSCs spontaneously differentiated into cardiomyocytes in vitro, when cultured on a complete medium formulation MethoCult GF M3534. These differentiated cells possessed cardiomyocyte phenotype and expressed cardiac markers. Moreover, these cells showed open excitation-contracting coupling and Ca²⁺ transient and contracted spontaneously. The role of Rho-associated protein kinases (ROCKs) in the differentiation process was then studied by using ROCK-specific inhibitor Y-27632 and ROCK siRNAs. These agents changed the arrangement of cytoskeleton and diminished appearance of cardiomyocyte phenotype, accompanied by inhibition of c-Jun N-terminal kinase (JNK) phosphorylation and promotion of Akt phosphorylation. Collectively, this is the first study to demonstrate that rat ADSCs could spontaneously differentiate into cardiomyocytes in vitro and ROCKs play an important role in the differentiation of ADSCs into beating cardiomyocytes in conjunction of the PI3K/Akt pathway and the JNK pathway.