ROCK1 induces dopaminergic nerve cell apoptosis via the activation of Drp1-mediated aberrant mitochondrial fission in Parkinson's disease.

Dopamine deficiency is mainly caused by apoptosis of dopaminergic nerve cells in the substantia nigra of the midbrain and the striatum and is an important pathologic basis of Parkinson's disease (PD). Recent research has shown that dynamin-related protein 1 (Drp1)-mediated aberrant mitochondrial fission plays a crucial role in dopaminergic nerve cell apoptosis. However, the upstream regulatory mechanism remains unclear. Our study showed that Drp1 knockdown inhibited aberrant mitochondrial fission and apoptosis. Importantly, we found that ROCK1 was activated in an MPP+-induced PD cell model and that ROCK1 knockdown and the specific ROCK1 activation inhibitor Y-27632 blocked Drp1-mediated aberrant mitochondrial fission and apoptosis of dopaminergic nerve cells by suppressing Drp1 dephosphorylation/activation. Our in vivo study confirmed that Y-27632 significantly improved symptoms in a PD mouse model by inhibiting Drp1-mediated aberrant mitochondrial fission and apoptosis. Collectively, our findings suggest an important molecular mechanism of PD pathogenesis involving ROCK1-regulated dopaminergic nerve cell apoptosis via the activation of Drp1-induced aberrant mitochondrial fission.

Ginsenoside Rg1 protects rat bone marrow mesenchymal stem cells against ischemia induced apoptosis through miR-494-3p and ROCK-1.

This study aimed to verify the cytoprotective effect of ginsenoside Rg1 in vivo, and to elucidate the mechanism of Rg1 in the ischemic microenvironment. Male rat bone marrow mesenchymal stem cells (rBMSCs) or rBMSCs treated with Rg1 were injected into ischemic region of the arterial embolism hind limb in female rats. Behavioral and histological data, obtained one-week post injection, showed that rBMSCs with Rg1 could improve the survival rate of BMSCs and enhance the therapeutic effects. rBMSCs treated with hypoxia and serum deprivation for 24h (H/SD-rBMSCs) showed the up-regulated expression of ras homolog family member A (RhoA), Rho associated coiled-coil containing protein kinase 1 (ROCK-1), myosin light chain 2 (MLC-2), Bcl2 associated agonist of cell death (Bad) and Bcl2 associated X, apoptosis regulator (Bax); while the expression of miR-148b-3p, miR-148b-5p and miR-494-3p was down-regulated. H/SD with Rg1 treatment (H/SD+Rg1-rBMSCs) inhibited the expression of ROCK-1, MLC-2, Bad and Bax, increased the expression of Bcl-2, miR-494-3p. After ROCK-1 knockout, the expression of Bad and Bax were downregulated and Bcl-2 upregulated, but Rg1 no longer altered their expression. Mir-494-3p functional study established that miR-494-3 mimic downregulated and miR-494-3 inhibitor upregulated ROCK-1 gene expression, Rg1 did not have the ability to change the ROCK gene expression after loss of function of miR-494-3p. Also, the function loss of mir-494-3p promoted apoptosis; otherwise reduced apoptosis. The anti-apoptotic effect of Rg1 disappeared after mir-494-3p loss or gain function. In conclusion, Ginsenoside Rg1 has shown to have protective effects on ischemic-induced rBMSCs apoptosis through mir-494-3p→ROCK-1→Bcl-2 signaling pathway.

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-ß, 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.

Rho kinase proteins regulate global miRNA expression in endothelial cells.

BACKGROUND: Therapeutic targeting of Rho-Associated, Coiled-Coil Containing Protein Kinase (ROCK) signaling for tumor cells and tumor endothelium has shown efficacy in pre-clinical tumors models, and a better understanding of how proteins regulate tumor progression will strengthen our knowledge over disease etiology and treatment of patients with cancer. Recent reports have shown that ROCK activity is critical for the expression of a large number of mRNA transcripts across multiple cell types including endothelial cells. MATERIALS AND METHODS: To examine the effects of ROCK proteins on microRNA (miRNA) expression in tumor-forming endothelial cells, we utilized microarrays to evaluate expression levels of 1088 miRNAs in vascular tumor-forming endothelial cells knocked-down for ROCK1 or ROCK2 or treated with a pharmacological inhibitor of ROCK activity. RESULTS: Microarray analysis demonstrated that inhibiting ROCK activity altered global miRNA expression. We confirmed our findings using qPCR and identified cell-cycle progression, calcium transport, and neurogenesis/synaptogenesis as the most highly overrepresented predicted target gene networks for the identified miRNAs whose expression was altered by ROCK inhibition. CONCLUSION: ROCK signaling induces large-scale changes in global miRNA expression and may lead to a better understanding of how these proteins affect aberrant vascular states.

Dissecting the roles of ROCK isoforms in stress-induced cell detachment.

The homologous Rho kinases, ROCK1 and ROCK2, are involved in stress fiber assembly and cell adhesion and are assumed to be functionally redundant. Using mouse embryonic fibroblasts (MEFs) derived from ROCK1(-/-) and ROCK2(-/-) mice, we have recently reported that they play different roles in regulating doxorubicin-induced stress fiber disassembly and cell detachment: ROCK1 is involved in destabilizing the actin cytoskeleton and cell detachment, whereas ROCK2 is required for stabilizing the actin cytoskeleton and cell adhesion. Here, we present additional insights into the roles of ROCK1 and ROCK2 in regulating stress-induced impairment of cell-matrix and cell-cell adhesion. In response to doxorubicin, ROCK1(-/-) MEFs showed significant preservation of both focal adhesions and adherens junctions, while ROCK2(-/-) MEFs exhibited impaired focal adhesions but preserved adherens junctions compared with the wild-type MEFs. Additionally, inhibition of focal adhesion or adherens junction formations by chemical inhibitors abolished the anti-detachment effects of ROCK1 deletion. Finally, ROCK1(-/-) MEFs, but not ROCK2(-/-) MEFs, also exhibited preserved central stress fibers and reduced cell detachment in response to serum starvation. These results add new insights into a novel mechanism underlying the anti-detachment effects of ROCK1 deletion mediated by reduced peripheral actomyosin contraction and increased actin stabilization to promote cell-cell and cell-matrix adhesion. Our studies further support the differential roles of ROCK isoforms in regulating stress-induced loss of central stress fibers and focal adhesions as well as cell detachment.

Distinct roles for ROCK1 and ROCK2 in the regulation of cell detachment.

This study, using mouse embryonic fibroblast (MEF) cells derived from ROCK1(-/-) and ROCK2(-/-) mice, is designed to dissect roles for ROCK1 and ROCK2 in regulating actin cytoskeleton reorganization induced by doxorubicin, a chemotherapeutic drug. ROCK1(-/-) MEFs exhibited improved actin cytoskeleton stability characterized by attenuated periphery actomyosin ring formation and preserved central stress fibers, associated with decreased myosin light chain 2 (MLC2) phosphorylation but preserved cofilin phosphorylation. These effects resulted in a significant reduction in cell shrinkage, detachment, and predetachment apoptosis. In contrast, ROCK2(-/-) MEFs showed increased periphery membrane folding and impaired cell adhesion, associated with reduced phosphorylation of both MLC2 and cofilin. Treatment with inhibitor of myosin (blebbistatin), inhibitor of actin polymerization (cytochalasin D), and ROCK pan-inhibitor (Y27632) confirmed the contributions of actomyosin contraction and stress fiber instability to stress-induced actin cytoskeleton reorganization. These results support a novel concept that ROCK1 is involved in destabilizing actin cytoskeleton through regulating MLC2 phosphorylation and peripheral actomyosin contraction, whereas ROCK2 is required for stabilizing actin cytoskeleton through regulating cofilin phosphorylation. Consequently, ROCK1 and ROCK2 can be functional different in regulating stress-induced stress fiber disassembly and cell detachment.

Prevention of diabetes-induced arginase activation and vascular dysfunction by Rho kinase (ROCK) knockout.

AIMS: We determined the role of the Rho kinase (ROCK) isoforms in diabetes-induced vascular endothelial dysfunction and enhancement of arginase activity and expression. METHODS AND RESULTS: Studies were performed in aortic tissues from haplo-insufficient (H-I) ROCK1 and ROCK2 mice and wild-type (WT) mice rendered diabetic with streptozotocin and in bovine aortic endothelial cells (BAECs) treated with high glucose (HG, 25 mM). Protein expression of both ROCK isoforms was substantially elevated in aortas of WT mice after 8 weeks of diabetes and in BAECs after 48 h in HG. Impairment of endothelium-dependent vasorelaxation of aortas was observed in diabetic WT mice. However, there was no impairment in aortas of diabetic ROCK1 H-I mice and less impairment in aortas of diabetic ROCK2 H-I mice, compared with non-diabetic mice. These vascular effects were associated with the prevention of diabetes-induced decrease in nitric oxide (NO) production and a rise in arginase activity/expression. Acute treatment with the arginase inhibitor, BEC, improved endothelium-dependent vasorelaxation of aortas of both diabetic WT and ROCK2, but not of ROCK1 mice. CONCLUSION: Partial deletion of either ROCK isoform, but to a greater extent ROCK1, attenuates diabetes-induced vascular endothelial dysfunction by preventing increased arginase activity and expression and reduction in NO production in type 1 diabetes. Limiting ROCK and arginase activity improves vascular function in diabetes.

Rho-associated coiled-coil-containing kinase 2 deficiency in bone marrow-derived cells leads to increased cholesterol efflux and decreased atherosclerosis.

BACKGROUND: Macrophages play a central role in the development of atherosclerosis. However, the signaling pathways that regulate their function are not well understood. The Rho-associated coiled-coil-containing kinases (ROCK1 and ROCK2) are serine-threonine protein kinases that are involved in the regulation of the actin cytoskeleton. Recent studies suggest that ROCK1 in macrophages and bone marrow-derived cells mediates atherogenesis. However, a similar role for ROCK2 in the pathogenesis of atherosclerosis has not been determined. METHODS AND RESULTS: The bone marrows from wild-type, ROCK2(+/-), and ROCK2(-/-) mice were transplanted into irradiated recipient low-density lipoprotein receptor(-/-) mice, and atherosclerosis was induced with a 16-week high-cholesterol diet. Compared with wild-type bone marrow-transplanted mice, ROCK2(+/-) bone marrow-transplanted and ROCK2(-/-) bone marrow-transplanted mice showed substantially less lipid accumulation in the aorta (8.46±1.42% and 9.80±2.34% versus 15.64±1.89%; P<0.01 for both) and decreased atherosclerotic lesions in the subaortic sinus (158.1±44.4 and 330.1±109.5×10(3)µm(2) versus 520.2±125.7×10(3)µm(2); P<0.01 for both). These findings correlated with decreased foam cell formation (2.27±0.57 versus 4.10±0.3; P<0.01) and increased cholesterol efflux (17.65±0.6 versus 9.75±0.8; P<0.05) in ROCK2-deficient mice that are mediated, in part, through the peroxisome proliferator-activated receptor-γ/liver X receptor/ATP-binding cassette transporter A1 pathway in macrophages. CONCLUSIONS: ROCK2 contributes to atherosclerosis, in part, by inhibiting peroxisome proliferator-activated receptor-γ-mediated reverse cholesterol transport in macrophages, which contributes to foam cell formation. These findings suggest that inhibition of ROCK2 in macrophages may have therapeutic benefits in preventing the development of atherosclerosis.

Targeted disruption of outer limiting membrane junctional proteins (Crb1 and ZO-1) increases integration of transplanted photoreceptor precursors into the adult wild-type and degenerating retina.

Diseases culminating in photoreceptor loss are a major cause of untreatable blindness. Transplantation of rod photoreceptors is feasible, provided donor cells are at an appropriate stage of development when transplanted. Nevertheless, the proportion of cells that integrate into the recipient outer nuclear layer (ONL) is low. The outer limiting membrane (OLM), formed by adherens junctions between Müller glia and photoreceptors, may impede transplanted cells from migrating into the recipient ONL. Adaptor proteins such as Crumbs homologue 1 (Crb1) and zona occludins (ZO-1) are essential for localization of the OLM adherens junctions. We investigated whether targeted disruption of these proteins enhances donor cell integration. Transplantation of rod precursors in wild-type mice achieved 949 +/- 141 integrated cells. By contrast, integration is significantly higher when rod precursors are transplanted into Crb1(rd8/rd8) mice, a model of retinitis pigmentosa and Lebers congenital amaurosis that lacks functional CRB1 protein and displays disruption of the OLM (7,819 +/- 1,297; maximum 15,721 cells). We next used small interfering (si)RNA to transiently reduce the expression of ZO-1 and generate a reversible disruption of the OLM. ZO-1 knockdown resulted in similar, significantly improved, integration of transplanted cells in wild-type mice (7,037 +/- 1,293; maximum 11,965 cells). Finally, as the OLM remains largely intact in many retinal disorders, we tested whether transient ZO-1 knockdown increased integration in a model of retinitis pigmentosa, the rho(-/-) mouse; donor cell integration was significantly increased from 313 +/- 58 cells without treatment to 919 +/- 198 cells after ZO-1 knockdown. This study shows that targeted disruption of OLM junctional proteins enhances integration in the wild-type and degenerating retina and may be a useful approach for developing photoreceptor transplantation strategies.

ROCK1 functions as a suppressor of inflammatory cell migration by regulating PTEN phosphorylation and stability.

Rho kinases belong to a family of serine/threonine kinases whose role in recruitment and migration of inflammatory cells is poorly understood. We show that deficiency of ROCK1 results in increased recruitment and migration of macrophages and neutrophils in vitro and in vivo. Enhanced migration resulting from ROCK1 deficiency is observed despite normal expression of ROCK2 and a significant reduction in overall ROCK activity. ROCK1 directly binds PTEN in response to receptor activation and is essential for PTEN phosphorylation and stability. In the absence of ROCK1, PTEN phosphorylation, stability, and its activity are significantly impaired. Consequently, increased activation of downstream targets of PTEN, including PIP3, AKT, GSK-3beta, and cyclin D1, is observed. Our results reveal ROCK1 as a physiologic regulator of PTEN whose function is to repress excessive recruitment of macrophages and neutrophils during acute inflammation.