Rho-associated Coiled-coil Containing Protein Kinase Inhibition Research Articles

Neurodegenerative Disease Associated Pathways in the Brains of Triple Transgenic Alzheimer's Model Mice Are Reversed Following Two Weeks of Peripheral Administration of Fasudil.

The pan Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor fasudil acts as a vasodilator and has been used as a medication for post-cerebral stroke for the past 29 years in Japan and China. More recently, based on the involvement of ROCK inhibition in synaptic function, neuronal survival, and processes associated with neuroinflammation, it has been suggested that the drug may be repurposed for neurodegenerative diseases. Indeed, fasudil has demonstrated preclinical efficacy in many neurodegenerative disease models. To facilitate an understanding of the wider biological processes at play due to ROCK inhibition in the context of neurodegeneration, we performed a global gene expression analysis on the brains of Alzheimer's disease model mice treated with fasudil via peripheral IP injection. We then performed a comparative analysis of the fasudil-driven transcriptional profile with profiles generated from a meta-analysis of multiple neurodegenerative diseases. Our results show that fasudil tends to drive gene expression in a reverse sense to that seen in brains with post-mortem neurodegenerative disease. The results are most striking in terms of pathway enrichment analysis, where pathways perturbed in Alzheimer's and Parkinson's diseases are overwhelmingly driven in the opposite direction by fasudil treatment. Thus, our results bolster the repurposing potential of fasudil by demonstrating an anti-neurodegenerative phenotype in a disease context and highlight the potential of in vivo transcriptional profiling of drug activity.

Y-27632, a ROCK inhibitor, improved laser-induced shock wave (LISW)-induced cochlear synaptopathy in mice

Recently, a pathological condition called cochlear synaptopathy has been clarified, and as a disorder of the auditory nerve synapses that occurs prior to failure of hair cells, it has been recognized as a major cause of sensorineural hearing loss. However, cochlear synaptopathy is untreatable. Inhibition of rho-associated coiled-coil containing protein kinase (ROCK), a serine-threonine protein kinase, has been reported to have neuroprotective and regenerative effects on synaptic pathways in the nervous system, including those in the inner ear. We previously demonstrated the regenerative effect of the ROCK inhibitor, Y-27632, on an excitotoxic cochlear nerve damage model in vitro. In this study, we aimed to validate the effect of ROCK inhibition on mice with cochlear synaptopathy induced by laser-induced shock wave (LISW) in vivo. After the elevation of ROCK1/2 expression in the damaged cochlea was confirmed, we administered Y-27632 locally via the middle ear. The amplitude of wave I in the auditory brainstem response and the number of synapses in the Y-27632-treated cochlea increased significantly. These results clearly demonstrate that ROCK inhibition has a promising clinical application in the treatment of cochlear synaptopathy, which is the major pathology of sensorineural hearing loss.

Regenerative Effect of a ROCK Inhibitor, Y-27632, on Excitotoxic Trauma in an Organotypic Culture of the Cochlea.

In the past, most inner ear diseases were thought to start with the impairment of the sensory epithelium of the cochlea before subsequently progressing to secondary neural degeneration. However, recent studies show that loss of primary synapses accompanied by excitotoxic degeneration of peripheral axons is likely to be the underlying pathology in sensorineural hearing loss. Rho-associated coiled-coil containing protein kinase (ROCK) inhibition has been reported to have neuroprotective and regenerative effects on synaptic pathways. Therefore, we analyzed the effect of ROCK inhibition using Y-27632 in a model of peripheral axonal damage in the spiral ganglion neurons created using the glutamate agonists, N-methyl-D-aspartate (NMDA) and kainic acid, to induce excitotoxic trauma in the explanted cochlea. The number of axons projecting to hair cells in the cochlea treated with Y-27632 was significantly greater than those in the cochlea treated only with NMDA + kainic acid. Furthermore, there was a significant increase in synapses between the spiral ganglion and the inner hair cells in the cochlea treated with Y-27632. The findings of this study suggest that ROCK inhibition could be a potential strategy for the regeneration of peripheral axons in the spiral ganglion and synapse formation in the inner hair cells of a cochlea that has sustained excitotoxic injury, which is one of the primary etiologies of inner ear disease.

Epithelial polarization in 3D matrix requires DDR1 signaling to regulate actomyosin contractility.

Epithelial cells form sheets and tubules in various epithelial organs and establish apicobasal polarity and asymmetric vesicle transport to provide functionality in these structures. However, the molecular mechanisms that allow epithelial cells to establish polarity are not clearly understood. Here, we present evidence that the kinase activity of the receptor tyrosine kinase for collagen, discoidin domain receptor 1 (DDR1), is required for efficient establishment of epithelial polarity, proper asymmetric protein secretion, and execution of morphogenic programs. Lack of DDR1 protein or inhibition of DDR1 kinase activity disturbed tubulogenesis, cystogenesis, and the establishment of epithelial polarity and caused defects in the polarized localization of membrane-type 1 matrix metalloproteinase (MT1-MMP), GP135, primary cilia, laminin, and the Golgi apparatus. Disturbed epithelial polarity and cystogenesis upon DDR1 inhibition was caused by excess ROCK (rho-associated, coiled-coil-containing protein kinase)-driven actomyosin contractility, and pharmacological inhibition of ROCK was sufficient to correct these defects. Our data indicate that a DDR1-ROCK signaling axis is essential for the efficient establishment of epithelial polarity.

RHOA activity in expanding blastocysts is essential to regulate HIPPO-YAP signaling and to maintain the trophectoderm-specific gene expression program in a ROCK/actin filament-independent manner.

What molecular signals are required to maintain the functional trophectoderm (TE) during blastocyst expansion of the late stage of preimplantation development? The activity of ras homology family member A (RHOA) GTPases is necessary to retain the expanded blastocyst cavity and also to sustain the gene expression program specific to TE. At the early stages of preimplantation development, the precursor of the TE lineage is generated through the molecular signals that integrate RHOA, RHO-associated coiled-coil containing protein kinase (ROCK), the apicobasal cell polarity, and the HIPPO-Yes-associated protein (YAP) signaling pathway. By contrast, molecular mechanisms regulating the maintenance of the TE characteristics at the later stage, which is crucial for blastocyst hatching and implantation, are scarcely understood. Expanding mouse blastocysts, obtained from crosses of the F1 (C57BL6 × DBA/2) strain, were exposed to chemical agents that interfere with RHOA, ROCK, or the actin cytoskeleton for up to 8 h, and effects on the blastocyst cavity, HIPPO-YAP signaling, and cell lineage-specific gene expression profiles were examined. Mouse embryos at the embryonic stage E3.5 (expanding blastocysts) and E4.5 (fully expanded blastocysts) were treated with RHOA inhibitor (C3 exoenzyme), ROCK inhibitor (Y27632), or actin filament disruptors (cytochalasin B and latrunculin A). The integrity of the blastocyst cavity was evaluated based on the gross morphology. Effects on HIPPO-YAP signaling were assessed based on the presence of nuclearized YAP protein by immunofluorescence staining and the expression of YAP/TEA domain family member (TEAD) target genes by quantitative RT-PCR (qRT-PCR). The impact of these disruptors on cell lineages was evaluated based on expression of the TE-specific and inner cell mass-specific marker genes by qRT-PCR. The integrity of the apicobasal cell polarity was assessed by localization of protein kinase C zeta (PRKCZ; apical) and scribbled planar cell polarity (SCRIB; basal) proteins by immunofluorescence staining. For comparisons, cultured cell lines, NIH/3T3 (mouse fibroblast) and P19C5 (mouse embryonal carcinoma), were also treated with RHOA inhibitor, ROCK inhibitor, and actin filament disruptors for up to 8 h, and effects on HIPPO-YAP signaling were assessed based on expression of YAP/TEAD target genes by qRT-PCR. Each experiment was repeated using three independent batches of embryos (n = 40-80 per batch) or cell collections. Statistical analyses of data were performed, using one-way ANOVA and two-sample t-test. Inhibition of RHOA deflated the cavity, diminished nuclear YAP (P < 0.01), and down-regulated the YAP/TEAD target and TE-specific marker genes in both E3.5 and E4.5 blastocysts (P < 0.05), indicating that the maintenance of the key TE characteristics is dependent on RHOA activity. However, inhibition of ROCK or disruption of actin filament only deflated the blastocyst cavity, but did not alter HIPPO-YAP signaling or lineage-specific gene expressions, suggesting that the action of RHOA to sustain the TE-specific gene expression program is not mediated by ROCK or the actomyosin cytoskeleton. By contrast, ROCK inhibitor and actin filament disruptors diminished YAP/TEAD target gene expressions in cultured cells to a greater extent than RHOA inhibitor, implicating that the regulation of HIPPO-YAP signaling in expanding blastocysts is distinctly different from that in the cell lines. Furthermore, the apicobasal cell polarity proteins in the expanding blastocyst were mislocalized by ROCK inhibition but not by RHOA inhibition, indicating that cell polarity is not linked to regulation of HIPPO-YAP signaling. Taken together, our study suggests that RHOA activity is essential to maintain the TE lineage in the expanding blastocyst and it regulates HIPPO-YAP signaling and the lineage-specific gene expression program through mechanisms that are independent of ROCK or actomyosin cytoskeleton. Not applicable. This study was conducted using one species, the mouse. Direct translation of the experiments and findings to human fertility preservation and ART requires further investigations. The elucidation of the mechanisms of TE formation is highly pertinent to fertility preservation in women. Our findings may raise awareness among providers of ART that the TE is sensitive to disturbance even in the late stage of blastocyst expansion and that rational approaches should be devised to avoid conditions that may impair the TE and its function. This study was funded by grants from the Ingeborg v.F. McKee Fund of the Hawaii Community Foundation (16ADVC-78882 to V.B.A.), and the National Institutes of Health (P20 GM103457 and R03 HD088839 to V.B.A.). The authors have no conflict of interest to declare.

TNF-stimulated gene 6 promotes formation of hyaluronan–inter-α-inhibitor heavy chain complexes necessary for ozone-induced airway hyperresponsiveness

Exposure to pollutants, such as ozone, exacerbates airway inflammation and hyperresponsiveness (AHR). TNF-stimulated gene 6 (TSG-6) is required to transfer inter-α-inhibitor heavy chains (HC) to hyaluronan (HA), facilitating HA receptor binding. TSG-6 is necessary for AHR in allergic asthma, because it facilitates the development of a pathological HA-HC matrix. However, the role of TSG-6 in acute airway inflammation is not well understood. Here, we hypothesized that TSG-6 is essential for the development of HA- and ozone-induced AHR. TSG-6-/- and TSG-6+/+ mice were exposed to ozone or short-fragment HA (sHA), and AHR was assayed via flexiVent. The AHR response to sHA was evaluated in the isolated tracheal ring assay in tracheal rings from TSG-6-/- or TSG-6+/+, with or without the addition of exogenous TSG-6, and with or without inhibitors of Rho-associated, coiled-coil-containing protein kinase (ROCK), ERK, or PI3K. Smooth-muscle cells from mouse tracheas were assayed in vitro for signaling pathways. We found that TSG-6 deficiency protects against AHR after ozone (in vivo) or sHA (in vitro and in vivo) exposure. Moreover, TSG-6-/- tracheal ring non-responsiveness to sHA was reversed by exogenous TSG-6 addition. sHA rapidly activated RhoA, ERK, and Akt in airway smooth-muscle cells, but only in the presence of TSG-6. Inhibition of ROCK, ERK, or PI3K/Akt blocked sHA/TSG-6-mediated AHR. In conclusion, TSG-6 is necessary for AHR in response to ozone or sHA, in part because it facilitates rapid formation of HA-HC complexes. The sHA/TSG-6 effect is mediated by RhoA, ERK, and PI3K/Akt signaling.

Effects of ripasudil hydrochloride hydrate (K-115), a Rho-kinase inhibitor, on ocular blood flow and ciliary artery smooth muscle contraction in rabbits

Ripasudil, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, is a novel drug for glaucoma in Japan. ROCK inhibition not only reduces intraocular pressure (IOP) but also increases ocular blood flow. We investigated the effects of ripasudil on optic disc blood flow (ODBF) in rabbit eyes and in isolated rabbit ciliary arteries. We measured IOP by tonometry and ODBF by laser speckle flowgraphy (LSFG) in male Dutch rabbits. A single drop (20μL) of 0.8% ripasudil was delivered to the ocular surface after topical application of phenylephrine hydrochloride to reduce the ODBF. The effects of ripasudil on isolated rabbit ciliary artery smooth muscle contractions were measured in vitro with a myograph. Ripasudil inhibited the reduction of ODBF induced by phenylephrine at 30 and 120min after instillation (P<.05). The blood flow change was not significantly correlated with the IOP change. Ripasudil induced a concentration-dependent relaxation in isolated rabbit ciliary arteries precontracted with a high-potassium solution. This relaxation was not mediated through the endothelium-dependent activities of nitric oxide synthase, prostacyclin, or the large-conductance calcium-activated K+ channel as shown by the inability of specific inhibitors of these pathways to block the ripasudil-induced relaxation. Taken together, our results showed that ripasudil not only decreased IOP but also increased ODBF in rabbits. However, the changes in IOP were not correlated with the changes in ODBF. Ripasudil also induced a concentration-dependent relaxation of isolated rabbit ciliary arteries through a NO-independent mechanism. Further investigation of the effect of ripasudil on ODBF is needed.

Substrate Stiffness Influences Doxorubicin-Induced p53 Activation via ROCK2 Expression.

The physical properties of the extracellular matrix (ECM), such as stiffness, are involved in the determination of the characteristics of cancer cells, including chemotherapy sensitivity. Resistance to chemotherapy is often linked to dysfunction of tumor suppressor p53; however, it remains elusive whether the ECM microenvironment interferes with p53 activation in cancer cells. Here, we show that, in MCF-7 breast cancer cells, extracellular stiffness influences p53 activation induced by the antitumor drug doxorubicin. Cell growth inhibition by doxorubicin was increased in response to ECM rigidity in a p53-dependent manner. The expression of Rho-associated coiled coil-containing protein kinase (ROCK) 2, which induces the activation of myosin II, was significantly higher when cells were cultured on stiffer ECM substrates. Knockdown of ROCK2 expression or pharmacological inhibition of ROCK decreased doxorubicin-induced p53 activation. Our results suggest that a soft ECM causes downregulation of ROCK2 expression, which drives resistance to chemotherapy by repressing p53 activation.

Interaction between mDia1 and ROCK in Rho-induced migration and adhesion of human dental pulp cells.

To investigate the effects of mammalian homologue of Drosophila diaphanous-1(mDia1) and Rho-associated coiled-coil-containing protein kinase (ROCK) on the migration and adhesion of dental pulp cells (DPCs). Lysophosphatidic acid (LPA) was used to activate Rho signalling. mDia1 and ROCK were inhibited by short interfering RNA and the specific inhibitor, Y-27632, respectively. The migration of DPCs was assessed using the transwell migration assay and scratch test. Formation of cytoskeleton and focal adhesions(FAs) was observed by confocal laser scanning microscopy. Cell adhesion and spreading assays were performed. Phosphorylation of focal adhesion kinase (FAK) and paxillin was detected by Western blotting, and the bands were analysed using Adobe Photoshop CS5 software. All experiments were performed at least three times, and data were analysed with one-way anova and a post hoc test. LPA-triggered activation of Rho and inhibition of ROCK significantly increased the cell migration rate. Cell migration was inhibited by silencing mDia1. mDia1 silencing and ROCK inhibition suppressed the LPA-induced formation of the cytoskeleton, FA and phosphorylation of FAK and paxillin. Inhibition of ROCK or mDia1 facilitated early cell adhesion and spreading; by contrast, the combined inhibition of ROCK and mDia1 neutralized these effects. mDia1 promoted RhoA-induced migration of DPCs, but ROCK had an opposite effect. Both mDia1 and ROCK participated in cytoskeleton formation and adhesion of DPCs. The interactions between mDia1 and ROCK might influence dental pulp repair by determining the migration and adhesion of DPCs.

Incarvine C suppresses proliferation and vasculogenic mimicry of hepatocellular carcinoma cells via targeting ROCK inhibition.

BackgroundStudies have described vasculogenic mimicry (VM) as an alternative circulatory system to blood vessels in multiple malignant tumor types, including hepatocellular carcinoma (HCC). In the current study, we aimed to seek novel and more efficient treatment strategies by targeting VM and explore the underlying mechanisms in HCC cells.MethodsCell counting kit-8 (CCK-8) assay and colony survival assay were performed to explore the inhibitory effect of incarvine C (IVC) on human cancer cell proliferation. Flow cytometry was performed to analyze the cell cycle distribution after DNA staining and cell apoptosis by the Annexin V-PE and 7-AAD assay. The effect of IVC on Rho-associated, coiled-coil-containing protein kinase (ROCK) was determined by western blotting and stress fiber formation assay. The inhibitory role of IVC on MHCC97H cell VM formation was determined by formation of tubular network structures on Matrigel in vitro, real time-qPCR, confocal microscopy and western blotting techniques.ResultsWe explored an anti-metastatic HCC agent, IVC, derived from traditional Chinese medicinal herbs, and found that IVC dose-dependently inhibited the growth of MHCC97H cells. IVC induced MHCC97H cell cycle arrest at G1 transition, which was associated with cyclin-dependent kinase 2 (CDK-2)/cyclin-E1 degradation and p21/p53 up-regulation. In addition, IVC induced apoptotic death of MHCC97H cells. Furthermore, IVC strongly suppressed the phosphorylation of the ROCK substrate myosin phosphatase target subunit-1 (MYPT-1) and ROCK-mediated actin fiber formation. Finally, IVC inhibited cell-dominant tube formation in vitro, which was accompanied with the down-regulation of VM-key factors as detected by real time-qPCR and immunofluorescence.ConclusionsTaken together, the effective inhibitory effect of IVC on MHCC97H cell proliferation and neovascularization was associated with ROCK inhibition, suggesting that IVC may be a new potential drug candidate for the treatment of HCC.

Mechanisms of favorable effects of Rho kinase inhibition on myocardial remodeling and systolic function after experimental myocardial infarction in the rat

The objective of this study was to determine the molecular mechanisms by which cardiac Rho-associated coiled-coil containing protein kinase (ROCK) activation after myocardial infarction (MI) does intervene in cardiac systolic function decline and remodeling. Simultaneous measurement of different cardiac ROCK target proteins levels, in vivo left ventricular (LV) systolic function, myocardial fibrosis and hypertrophy in rats with MI under ROCK inhibition with fasudil. Seven days after MI, the ventricular mass increased significantly by 5.6% in the MI group and was reduced with fasudil. LV systolic dysfunction improved significantly with fasudil whereas cardiac ROCK activation was reduced to sham levels. The ROCK inhibitor also reduced increased cardiac levels of both ROCK1 and ROCK2 isoforms, cardiomyocyte ROCK2 fluorescence levels and β-myosin heavy chain (MHC) levels in addition to myocardial collagen volume fraction decline. Compared with sham rats, troponin phosphorylation levels after MI were similar and ROCK inhibition reduced them. MI significantly increased phosphorylation levels of extracellular-signal-regulated kinase (ERK) 42 and ERK 44 by twofold and 63%, respectively, whereas in the fasudil-treated MI group these levels were similar to those in the sham group. MI significantly increased phosphorylated levels of the transcription factor GATA-4 and the ROCK inhibitor normalized them. LV systolic dysfunction after MI was strongly associated with cardiac ROCK activation and subsequent phosphorylation of ROCK target proteins that promote ventricular remodeling such as β-MHC and the ERK/GATA-4 pathway. ROCK inhibition with fasudil significantly improved systolic function, diminished myocardial fibrosis and normalized β-MHC and ERK/GATA-4 phosphorylation levels.

ROCK activity and the Gβγ complex mediate chemotactic migration of mouse bone marrow-derived stromal cells.

IntroductionBone marrow-derived stromal cells (BMSCs), also known as mesenchymal stem cells, are the focus of intensive efforts worldwide to elucidate their function and biology. Despite the importance of BMSC migration for their potential therapeutic uses, the mechanisms and signalling governing stem cell migration are still not fully elucidated.MethodsWe investigated and detailed the effects of MCP-1 activation on BMSCs by using inhibitors of G protein-coupled receptor alpha beta (GPCR αβ), ROCK (Rho-associated, coiled-coil containing protein kinase), and PI3 kinase (PI3K). The effects of MCP-1 stimulation on intracellular signalling cascades were characterised by using immunoblotting and immunofluorescence. The effectors of MCP-1-mediated migration were investigated by using migration assays (both two-dimensional and three-dimensional) in combination with inhibitors.ResultsWe established the kinetics of the MCP-1-activated signalling cascade and show that this cascade correlates with cell surface re-localisation of chemokine (C motif) receptor 2 (CCR2) (the MCP-1 receptor) to the cell periphery following MCP-1 stimulation. We show that MCP-1-initiated signalling is dependent on the activation of βγ subunits from the GPCR αβγ complex. In addition, we characterise a novel role for PI3Kγ signalling for the activation of both PAK and ERK following MCP-1 stimulation. We present evidence that the Gβγ complex is responsible for PI3K/Akt, PAK, and ERK signalling induced by MCP-1 in BMSCs. Importantly, we found that, in BMSCs, inhibition of ROCK significantly inhibits MCP-1-induced chemotactic migration, in contrast to previous reports in other systems.ConclusionsOur results indicate differential chemotactic signalling in mouse BMSCs, which has important implications for the translation of in vivo mouse model findings into human trials. We identified novel components and interactions activated by MCP-1-mediated signalling, which are important for stem cell migration. This work has identified additional potential therapeutic targets that could be manipulated to improve BMSC delivery and homing.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0125-y) contains supplementary material, which is available to authorized users.

ROCK inhibition enhances microRNA function by promoting deadenylation of targeted mRNAs via increasing PAIP2 expression.

The reduced expression levels and functional impairment of global miRNAs are related to various human diseases, including cancers. However, relatively little is known about how global miRNA function may be upregulated. Here, we report that global miRNA function can be enhanced by Rho-associated, coiled-coil-containing protein kinase (ROCK) inhibitors. The regulation of miRNA function by ROCK inhibitors is mediated, at least in part, by poly(A)-binding protein-interacting protein 2 (PAIP2), which enhances poly(A)-shortening of miRNA-targeted mRNAs and leads to global upregulation of miRNA function. In the presence of a ROCK inhibitor, PAIP2 expression is enhanced by the transcription factor hepatocyte nuclear factor 4 alpha (HNF4A) through increased ROCK1 nuclear localization and enhanced ROCK1 association with HNF4A. Our data reveal an unexpected role of ROCK1 as a cofactor of HNF4A in enhancing PAIP2 transcription. ROCK inhibitors may be useful for the various pathologies associated with the impairment of global miRNA function.

Osteogenic differentiation regulated by Rho-kinase in periodontal ligament cells

The periodontal ligament is a multifunctional soft connective tissue, which functions not only as a cushion supporting the teeth against occlusal force, but is also a source of osteogenic cells that can regenerate neighboring hard tissues. Periodontal ligament cells (PDL cells) contain heterogeneous cell populations, including osteogenic cell progenitors. However, the precise mechanism underlying the differentiation process remains elusive. Cell differentiation is regulated by the local biochemical and mechanical microenvironment that can modulate gene expression and cell morphology by altering actin cytoskeletal organization mediated by Rho-associated, coiled-coil containing protein kinase (ROCK). To determine its role in PDL cell differentiation, we examined the effects of ROCK on cytoskeletal changes and kinetics of gene expression during osteogenic differentiation. PDL cells were isolated from human periodontal ligament on extracted teeth and cultured in osteogenic medium for 14 days. Y-27632 was used for ROCK inhibition assay. Osteogenic phenotype was determined by monitoring alkaline phosphatase (ALP) activity and calcium deposition by Alizarin Red staining. ROCK-induced cytoskeletal changes were examined by immunofluorescence analysis of F-actin and myosin light chain 2 (MLC2) expression. Real-time PCR was performed to examine the kinetics of osteogenic gene expression. F-actin and phospho-MLC2 were markedly induced during osteogenic differentiation, which coincided with upregulation of ALP activity and mineralization. Subsequent inhibition assay indicated that Y-27632 significantly inhibited F-actin and phospho-MLC2 expression in a dose-dependent manner with concomitant partial reversal of the PDL cell osteogenic phenotype. PCR array analysis of osteogenic gene expression indicated that extracellular matrix genes, such as fibronectin 1, collagen type I and III, and biglycan, were significantly downregulated by Y27632. These findings indicated crucial effects of ROCK in cytoskeletal reorganization and differentiation of PDL cells toward osteogenic cells. ROCK contributes to induction of osteogenic differentiation by synergistic increases in extracellular matrix gene expression in PDL cells.

Regulation of the Actin Cytoskeleton by Rho Kinase Controls Antigen Presentation by CD1d

CD1d molecules are MHC class I-like molecules that present lipid Ags to NKT cells. Although we have previously shown that several different cell signaling molecules can play a role in the control of Ag presentation by CD1d, a defined mechanism by which a cell signaling pathway regulates CD1d function has been unclear. In the current study, we have found that the Rho kinases, Rho-associated, coiled-coil containing protein kinase (ROCK)1 and ROCK2, negatively regulate both human and mouse CD1d-mediated Ag presentation. Inhibition of ROCK pharmacologically, through specific ROCK1 and ROCK2 short hairpin RNA, or by using dendritic cells generated from ROCK1-deficient mice all resulted in enhanced CD1d-mediated Ag presentation compared with controls. ROCK regulates the actin cytoskeleton by phosphorylating LIM kinase, which, in turn, phosphorylates cofilin, prohibiting actin fiber depolymerization. Treatment of APCs with the actin filament depolymerizing agent, cytochalasin D, as well as knockdown of LIM kinase by short hairpin RNA, resulted in enhanced Ag presentation to NKT cells by CD1d, consistent with our ROCK inhibition data. Therefore, our overall results reveal a model whereby CD1d-mediated Ag presentation is negatively regulated by ROCK via its effects on the actin cytoskeleton.

Nogo-66 receptor activation inhibits neurite outgrowth and increases β-amyloid protein secretion of cortical neurons

A Nogo-A to Nogo-66 receptor (NgR) pathway is well known to contribute to the inhibition of the neurite regeneration of adult central nervous system neurons after traumatic injuries. Recent evidence suggests that Nogo-A and NgR are involved in the pathology of Alzheimer's disease (AD), as evidenced by the fact that Nogo-A is overexpressed by hippocampal neurons in patients with AD and is associated with β-amyloid protein (Aβ) deposits in senile plaques. In the present experiments, we investigated the potential role of Nogo-A in both neurite outgrowth and Aβ generation in cortical neurons. Our results showed that activation of NgR not only inhibited neurite outgrowth in cortical neurons by activating the rho-associated coiled coil-containing protein kinase (ROCK) and protein kinase C, but also promoted their Aβ secretion, which was at least in part activated by ROCK. These findings suggest that the overexpression of Nogo-A and the activation of NgR inhibit neurite outgrowth and alter neuronal metabolism, resulting in overproduction and/or release of Aβ, which in turn may trigger the onset and development of AD. Inhibition of ROCK can promote neurite outgrowth and reduce Aβ production of cortical neuron, which suggests that ROCK appears to be a good target for AD therapy.

Effects of Rosuvastatin versus Atorvastatin on Rho-Associated Coiled-Coil Containing Protein Kinase Activity and Endothelial Function in Patients with Atherosclerosis

This randomized, double-blind, parallel-design study compared the short-term effects of rosuvastatin and atorvastatin on serum lipids and markers of inflammation and endothelial function in patients with stable atherosclerosis. Patients received either 10 mg/day rosuvastatin (n = 18) or 20 mg/day atorvastatin (n = 18), orally, for 4 weeks. Serum lipids, high-sensitivity Creactive protein (hsCRP), Rho-associated coiled-coil containing protein kinase (ROCK) activity and flow-mediated dilation (FMD) of the brachial artery were assessed before and after therapy. Both statins produced significant reductions in total cholesterol, low-density lipoprotein cholesterol (LDL-C), triglyceride and hsCRP levels, and significant increases in FMD. Both statins significantly reduced ROCK activity and inhibition was significantly greater with rosuvastatin. There was no correlation between ROCK activity and LDL-C level in either group. There was a significant correlation between ROCK activity and FMD for both statins, but no correlations between FMD and LDL-C or hsCRP levels. Short-term treatment with either rosuvastatin or atorvastatin inhibits ROCK activity independent of cholesterol reduction, and improves endothelium dysfunction in patients with atherosclerosis.

Functional role of rho‐kinase in ameloblast differentiation

During tooth development, inner enamel epithelial (IEE) cells differentiate into enamel-secreting ameloblasts, a polarized and elongated cellular population. The molecular underpinnings of this morphogenesis and cytodifferentiation, however, are not well understood. Here, we show that Rho-associated coiled-coil-containing protein kinase (ROCK) regulates ameloblast differentiation and enamel formation. In mouse incisor organ cultures, inhibition of ROCK, hindered IEE cell elongation and disrupted polarization of differentiated ameloblasts. Expression of enamel matrix proteins, such as amelogenin and ameloblastin, and formation of the terminal band structure of actin and E-cadherin were also perturbed. Cultures of dental epithelial cells revealed that ROCK regulates cell morphology and cell adhesion through localization of actin bundles, E-cadherin, and β-catenin to cell membranes. Moreover, inhibition of ROCK promoted cell proliferation. Small interfering RNA specific for ROCK1 and ROCK2 demonstrated that the ROCK isoforms performed complementary functions in the regulation of actin organization and E-cadherin-mediated cell-cell adhesion. Thus, our results have uncovered a novel role for ROCK in amelogenesis.

Rho kinase inhibition activates the homologous angiotensin-converting enzyme-angiotensin-(1–9) axis in experimental hypertension

Angiotensin II (Ang II) levels depend on renin, angiotensin-converting enzyme (ACE), and on the homologous angiotensin-converting enzyme (ACE2). Increased ACE and Ang II levels are associated with higher Rho kinase activity. However, the relationship between Rho kinase activation and ACE2 in hypertension is unknown. The role of the Rho kinase signaling pathway in both enzymatic activity and aortic gene expression of ACE2 in deoxycorticosterone acetate (DOCA) hypertensive rats was assessed in the present study. Compared with sham animals, Rho kinase activity was higher by 400% (P<0.05) in the aortic wall of the DOCA hypertensive rats. In addition to blood pressure reduction, the specific Rho kinase inhibitor fasudil reduced aortic Rho kinase activity to levels observed in the sham control group and increased ACE2 enzymatic activity (by 83% in plasma and by 52% in the aortic wall, P<0.05), ACE2, and endothelial nitric oxide synthase (eNOS) aortic mRNA levels (by 340 and 40%, respectively, P<0.05) with respect to the untreated hypertensive DOCA rats. Fasudil also increased significantly plasma levels of Ang-(1-9) in normotensive and in the hypertensive rats. Aortic mRNA and protein levels of transforming growth factor-β1 (TGF-β1), plasminogen activator inhibitor 1 (PAI-1), and monocyte chemoattractant protein 1 (MCP-1) were significantly (P<0.05) higher in the untreated DOCA rats and were normalized by fasudil administration. In experimental hypertension, Rho-associated, coiled-coil containing protein kinase (ROCK) inhibition reduces blood pressure and increases ACE2 levels and activity. At the same time, ROCK inhibition reduces angiotensin II and increases Ang-(1-9) plasma levels. Fasudil also increases vascular eNOS mRNA levels and reduces aortic overexpression of the remodeling promotion proteins TGF-β1, PAI-1, and MCP-1. This effect might additionally contribute to the antihypertensive and antiremodeling effects of ROCK inhibition in hypertension.

The Rho Kinase Inhibitor, Fasudil, Inhibits Wound Contraction

Introduction: Scar contracture affects approximately 40 million people worldwide. There are currently no effective preventative treatments. Scar contracture is caused by fibroblast and myofibroblast contractility. Cellular contractility is stimulated by calcium independent activation of the small GTP-ases, Rho A and Rho C, which phosphorylate and activate the downstream kinase Rho-associated coiled coil-containing protein kinase (ROCK). ROCK has recently been found to cause renal, pulmonary, and cardiovascular fibrosis in vivo. This study evaluates the expression profile of RhoA and ROCK in human scar tissue and determines the effects of ROCK inhibition on rodent wound contraction. Methods: Human scar tissue and surrounding normal skin in the same specimen was immunostained for RhoA and ROCK. Full-thickness wounds (2×2cm) were created on the backs of age-matched Wistar-han rats (n=10). Fasudil (30mg/kg/d) or vehicle alone were continuously delivered systemically by subcutaneous implanted osmotic pumps for 21d. Wound contraction was measured by gravitational planimetry and rodents were assessed for signs of toxicity by measuring weight changes and observing behavior. On day 21, rodents were euthanized and wounds were harvested for Mason's trichrome, hematoxylin and eosin (H&E), and picosirius red staining, and immunohistochemistry performed for α-SMA (myofibroblasts), Ki-67 (proliferation) and CD31 (angiogenesis). Results: Human scar tissue showed significantly increased expression of RhoA and ROCK in dermal fibroblasts compared to surrounding normal tissue. Fasudil significantly inhibited wound contraction as compared to controls. Fasudil was non-toxic. H&E, Masson's, and Sirius Red staining was similar between groups. Fasudil did not alter angiogenesis nor proliferation (p>0.05). Conclusion: The increased expression of RhoA and ROCK in human scar tissue suggests an association between protein presence and scar contracture. Fasudil significantly inhibited rodent wound contraction without adversely affecting healing. Fasudil may be a potential drug to prevent scar contractures. Future work will involve a dose response curve of fasudil in rodents and application of Fasudil to the red duroc pig scar model.