Deleted in liver cancer 1 suppresses the growth of prostate cancer cells through inhibiting Rho-associated protein kinase pathway

Objective:Deleted in liver cancer 1(DLC1)is a GTPase-activating protein that is reported as a suppressor in certain human cancers.However,the detailed biological function of DLC1 is still unclear in human prostate cancer(PCa).In the present study,we aimed to explore the function of DLC1 in PCa cells.Methods:Silencing and overexpression of DLC1 were induced in an androgen-sensitive PCa cell line(LNCaP)using RNA interference and lentiviral vector transduction.The Cell Counting Kit-8 assay was performed to determine cell proliferation.The cell cycle was examined by performing a propidium iodide staining assay.Results:Our results indicated that DLC1 overexpression markedly suppressed the proliferation and cell cycle progression of LNCaP cells.Moreover,DLC1 expression was negatively correlated with Rho-associated protein kinase(ROCK)expression in LNCaP cells.Importantly,this study showed that the ROCK inhibitor Y27632 restored the function of DLC1 in LNCaP cells and reduced the tumorigenicity of LNCaP cells in vivo.Conclusion:Our results indicated that DLC1 overexpression markedly suppressed the proliferation and cell cycle progression of PCa cells and negatively correlated with ROCK expression in PCa cells and tissue.

Rho-associated protein kinase modulates neurite extension by regulating microtubule remodeling and vinculin distribution

Rho-associated protein kinase is an essential regulator of cytoskeletal dynamics during the process of neurite extension. However, whether Rho kinase regulates microtubule remodeling or the distri- bution of adhesive proteins to mediate neurite outgrowth remains unclear. By specifically modulat- ing Rho kinase activity with pharmacological agents, we studied the morpho-dynamics of neurite outgrowth. We found that lysophosphatidic acid, an activator of Rho kinase, inhibited neurite out- growth, which could be reversed by Y-27632, an inhibitor of Rho kinase. Meanwhile, reorganization of microtubules was noticed during these processes, as indicated by their significant changes in the soma and growth cone. In addition, exposure to lysophosphatidic acid led to a decreased mem- brane distribution of vinculin, a focal adhesion protein in neurons, whereas Y-27632 recruited vin- culin to the membrane. Taken together, our data suggest that Rho kinase regulates rat hippocampal neurite growth and microtubule formation via a mechanism associated with the redistribution of vinculin.

Downregulation of rho-associated protein kinase 1 by mi R-124 in colorectal cancer

AIM: To investigate the roles and interactions of rhoassociatedprotein kinase (ROCK)1 and miR-124 inhuman colorectal cancer (CRC).METHODS: Expression of ROCK1 protein wasexamined by Western blotting, and quantitativereverse transcriptase PCR was performed to measureexpression of ROCK1 mRNA and miR-124. Two cancercell lines were transfected with pre-miR-124 (mimic)and anti-miR-124 (inhibitor) and the effects onROCK1 protein and mRNA expression were observed.In addition, cell proliferation was assessed via a5-ethynyl-2′ deoxyuridine assay. Soft agar formationassay, and cell migration and invasion assays wereused to determine the effect of survivin on thetransformation and invasion activity of CRC cells.RESULTS: miR-124 was significantly downregulated inCRC compared to normal specimens (0.603 ± 0.092 vs1.147 ± 0.286, P = 0.016) and in metastatic comparedto nonmetastatic CRC specimens (0.416 ± 0.047 vs0.696 ± 0.089, P = 0.020). Expression of miR-124 wassignificantly associated with CRC metastasis, tumor Tand N stages, and tumor grade (all P < 0.05). ROCK1protein was significantly increased in CRC comparedto normal tissues (1.896 ± 0.258 vs 0.866 ± 0.136,P = 0.026), whereas ROCK1 mRNA expression wasunaltered (2.613 ± 0.251 vs 2.325 ± 0.246). miR-124and ROCK1 were inversely expressed in CRC tissuesand cell lines. ROCK1 mRNA was unaltered in cellstransfected with miR-124 mimic and miR-124 inhibitor,compared to normal controls. There was a significantreduction in ROCK1 protein in cells transfected withmiR-124 mimic and a significant increase in cells transfected with miR-124 inhibitor (P s < 0.05).Transformation and invasion of cells transfectedwith miR-124 inhibitor were significantly increasedcompared to those in normal controls (P < 0.05). Cellstransfected with miR-124 inhibitor showed increasedcell proliferation.CONCLUSION: miR-124 promotes hyperplasia andcontributes to invasion of CRC cells, but downregulatesROCK1. ROCK1 and miR-124 may play important rolesin CRC.

Inhibition of RhoA/Rho-kinase pathway suppresses the expression of extracellular matrix induced by CTGF or TGF-β in ARPE-19

AIM:To investigate the role of Rho-associated protein kinase (ROCK) inhibitor, Y27632, in mediating the production of extracellular matrix (ECM) components including fibronectin, matrix metallo-proteinase-2 (MMP-2) and type I collagen as induced by connective tissue growth factor(CTGF) or transforming growth factor-β (TGF-β) in a human retinal pigment epithelial cell line, ARPE-19. METHODS:The effect of Y27632 on the CTGF or TGF-β induced phenotype in ARPE-19 cells was measured with immunocytochemistry as the change in F-actin. ARPE-19 cells were treated with CTGF (1, 10, 100ng/mL)and TGF-β (10ng/mL) in serum free media, and analyzed for fibronectin, laminin, and MMP-2 and type I collagen by RT-qPCR and immunocytochemistry. Cells were also pretreated with an ROCK inhibitor, Y27632, to analyze the signaling contributing to ECM production. ·RESULTS:Treatment of ARPE-19 cells in culture with TGF-β or CTGF induced an ECM change from a cobblestone morphology to a more elongated swirl pattern indicating a mesenchymal phenotype. RT-qPCR analysis and different gene expression analysis demonstrated an upregulation in expression of genes associated with cytoskeletal structure and motility. CTGFor TGF-β significantly increased expression of fibronectin mRNA (P =0.006, P =0.003 respectively), laminin mRNA (P =0.006, P =0.005), MMP-2 mRNA (P =0.006, P =0.001), COL1A1 mRNA (P =0.001, P =0.001), COL1A2 mRNA (P = 0.001, P =0.001). Preincubation of ARPE-19 with Y27632 (10mmol/L) significantly prevented CTGF or TGF-β induced fibronectin (P=0.005, P=0.003 respectively), MMP-2 (P = 0.003, P =0.002), COL1A1 (P =0.006, P =0.003), and COL1A2 (P =0.006, P =0.004) gene expression, but not laminin (P =0.375, P =0.516). CONCLUSION:Our study demonstrated that both TGF-β and CTGF upregulate the expression of ECM components including fibronectin, laminin, MMP-2 and type I collagen by activating the RhoA/ROCK signaling pathway. During this process, ARPE-19 cells were shown to change from an epithelial to a mesenchymal phenotype in vitro. Y27632, a ROCK inhibitor, inhibited the transcription of fibronectin, MMP-2 and type I collagen, but not laminin. The data from our work suggest a role for CTGF as a profibrotic mediator. Inhibiting the RhoA/ROCK pathway represents a potential target to prevent the fibrosis of retinal pigment epithelial (RPE) cells. This might lead to a novel therapeutic approach to preventing the onset of early proliferative vitreoretinopathy(PVR).

Effect of manipulation on cartilage in rats with knee osteoarthritis based on the Rho-associated protein kinase/LIM kinase 1/Cofilin signaling pathways

OBJECTIVE:To investigate the effect of manipulation treatment on knee osteoarthritis rats and the effect on Rho-associated protein kinase(ROCK)/LIM-kinase1(LIMK1)/Cofilin signaling pathway.METHOD:Fifty Specific pathogen Free Sprague-Dawley rats were randomly divided into five groups(n=8 each):blank group,model group,manipulation group,celecoxib group,and manipulation combined with celecoxib group(MC group).The osteoarthritis model was established by injecting 0.2 m L 4%papain into the articular disc of the rats.After successfully establishing the model,we treated the manipulation group with pushing manipulation using one-finger-meditation to the Neixiyan(EX-LE4),Waixiyan(EX-LE5),Xuehai(SP10),Liangqiu(ST34),and Zusanli(ST36)acupoints for 10 min each time.Also,the celecoxib group was gavaged with 24 mg·kg^(-1)·d^(-1 )celecoxib,while the MC group was treated using both of these two methods.After four weeks,the cartilage of the right femur was removed for hematoxylin-eosin staining of the cartilage tissue.The expressions of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in serum were observed using the enzyme-linked immunosorbent assay.Besides,we detected the expressions of ROCK,LIMK1,Phospho-LIM-kinase1(Phospho-LIMK1),Cofilin,and Phospho-Cofilin by Western blot.RESULTS:Compared to the model group,the manipulation group,celecoxib group,and MC group all exhibited superior results concerning pathological morphologic changes of cartilage,as observed by hematoxylin-eosin staining and calculated using the Mankin score.Besides,in contrast to the blank group,the model group exhibited elevated serum levels of IL-1βand TNF-α(P<0.01),while the expression of ROCK,LIMK1,Phospho-LIMK1,Cofilin,and Phospho-Cofilin in cartilage were all higher(P<0.01).Also,the serum levels of IL-1βand TNF-αin each treatment group were lower(P<0.01)than in the model group.Moreover,there were lower expressions of ROCK,LIMK1,Phospho-LIMK1,Cofilin,and Phospho-Cofilin in cartilage in the manipulation group and the MC group(P<0.01).Compared with the model group,the expression of ROCK,LIMK1,PhosphoLIMK1,Cofilin,and Phospho-Cofilin in cartilage in the celecoxib group were not statistically different(P>0.05).CONCLUSION:In this study,we established that manipulation has a better curative effect than celecoxib.Manipulation inhibits the development of cytoskeleton damage in cartilage and slows articular degeneration by regulating the expression of related proteins in the cytoskeletal signaling pathway.

Desensitization of G-protein-coupled receptors induces vascular hypocontractility in response to norepinephrine in the mesenteric arteries of cirrhotic patients and rats

BACKGROUND: The increased β-arrestin-2 and its combination with G-protein-coupled receptors (GPCRs) lead to GPCRs desensitization. The latter may be responsible for decreased contractile reactivity in the mesenteric arteries of cirrhotic patients and rats. The present study is to investigate the machinery changes of α-adrenergic receptors and G proteins and their roles in the contractility of mesenteric arteries of cirrhotic patients and animal models. METHODS: Patients with cirrhosis due to hepatitis B and cirrhotic rats induced by CCl 4 were studied. Mesenteric artery contractility in response to norepinephrine was determined by a vessel perfusion system. The contractile effect of G protein-coupled receptor kinase-2 (GRK-2) inhibitor on the mesenteric artery was evaluated. The protein expression of the α 1 adrenergic receptor, G proteins, β-arrestin-2, GRK-2 as well as the activity of Rho associated coiled-coil forming protein kinase-1 (ROCK-1) were measured by Western blot. In addition, the interaction of α 1 adrenergic receptor with β-arrestin-2 was assessed by co-immunoprecipitation. RESULTS: The portal vein pressure of cirrhotic patients and rats was significantly higher than that of controls. The doseresponse curve to norepinephrine in mesenteric arteriole was shifted to the right, and EC 50 was significantly increased in cirrhotic patients and rats. There were no significant differences in the expressions of the α 1 adrenergic receptor and G proteins in the cirrhotic group compared with the controls. However, the protein expressions of GRK-2 and β-arrestin-2 were significantly elevated in cirrhotic patients and rats compared with those of the controls. The interaction of the α 1 adrenergic receptor and β-arrestin-2 was significantly aggravated. This interaction was significantly reversed by GRK-2 inhibitor. Both the protein expression and activity of ROCK-1 were significantly decreased in the mesenteric artery in patients with cirrhosis compared with those of the controls, and this phenomenon was not shown in the cirrhotic rats. Norepinephrine significantly increased the activity of ROCK-1 in normal rats but not in cirrhotic ones. Norepinephrine significantly increased ROCK-1 activity in cirrhotic rats when GRK-2 inhibitor was used. CONCLUSIONS: β-arrestin-2 expression and its interaction with GPCRs are significantly upregulated in the mesenteric arteries in patients and rats with cirrhosis. These upregulations result in GPCR desensitization, G-protein dysfunction and ROCK inhibition. These may explain the decreased contractility of the mesenteric artery in response to vasoconstrictors.

Regulation of enolase activation to promote neural protection and regeneration in spinal cord injury

Spinal cord injury(SCI)is a debilitating condition characterized by damage to the spinal cord resulting in loss of function,mobility,and sensation with no U.S.Food and Drug Administration-approved cure.Enolase,a multifunctional glycolytic enzyme upregulated after SCI,promotes pro-and anti-inflammatory events and regulates functional recovery in SCI.Enolase is normally expressed in the cytosol,but the expression is upregulated at the cell surface following cellular injury,promoting glial cell activation and signal transduction pathway activation.SCI-induced microglia activation triggers pro-inflammatory mediators at the injury site,activating other immune cells and metabolic events,i.e.,Rho-associated kinase,contributing to the neuroinflammation found in SCI.Enolase surface expression also activates cathepsin X,resulting in cleavage of the C-terminal end of neuron-specific enolase(NSE)and non-neuronal enolase(NNE).Fully functional enolase is necessary as NSE/NNE C-terminal proteins activate many neurotrophic processes,i.e.,the plasminogen activation system,phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B,and mitogen-activated protein kinase/extracellular signal-regulated kinase.Studies here suggest an enolase inhibitor,ENOblock,attenuates the activation of Rho-associated kinase,which may decrease glial cell activation and promote functional recovery following SCI.Also,ENOblock inhibits cathepsin X,which may help prevent the cleavage of the neurotrophic C-terminal protein allowing full plasminogen activation and phosphatidylinositol-4,5-bisphosphate 3-kinase/mitogen-activated protein kinase activity.The combined NSE/cathepsin X inhibition may serve as a potential therapeutic strategy for preventing neuroinflammation/degeneration and promoting neural cell regeneration and recovery following SCI.The role of cell membrane-expressed enolase and associated metabolic events should be investigated to determine if the same strategies can be applied to other neurodegenerative diseases.Hence,this review discusses the importance of enolase activation and inhibition as a potential therapeutic target following SCI to promote neuronal survival and regeneration.

Axonal growth inhibitors and their receptors in spinal cord injury:from biology to clinical translation

Axonal growth inhibitors are released during traumatic injuries to the adult mammalian central nervous system, including after spinal cord injury. These molecules accumulate at the injury site and form a highly inhibitory environment for axonal regeneration. Among these inhibitory molecules, myelinassociated inhibitors, including neurite outgrowth inhibitor A, oligodendrocyte myelin glycoprotein, myelin-associated glycoprotein, chondroitin sulfate proteoglycans and repulsive guidance molecule A are of particular importance. Due to their inhibitory nature, they represent exciting molecular targets to study axonal inhibition and regeneration after central injuries. These molecules are mainly produced by neurons, oligodendrocytes, and astrocytes within the scar and in its immediate vicinity. They exert their effects by binding to specific receptors, localized in the membranes of neurons. Receptors for these inhibitory cues include Nogo receptor 1, leucine-rich repeat, and Ig domain containing 1 and p75 neurotrophin receptor/tumor necrosis factor receptor superfamily member 19(that form a receptor complex that binds all myelin-associated inhibitors), and also paired immunoglobulin-like receptor B. Chondroitin sulfate proteoglycans and repulsive guidance molecule A bind to Nogo receptor 1, Nogo receptor 3, receptor protein tyrosine phosphatase σ and leucocyte common antigen related phosphatase, and neogenin, respectively. Once activated, these receptors initiate downstream signaling pathways, the most common amongst them being the Rho A/ROCK signaling pathway. These signaling cascades result in actin depolymerization, neurite outgrowth inhibition, and failure to regenerate after spinal cord injury. Currently, there are no approved pharmacological treatments to overcome spinal cord injuries other than physical rehabilitation and management of the array of symptoms brought on by spinal cord injuries. However, several novel therapies aiming to modulate these inhibitory proteins and/or their receptors are under investigation in ongoing clinical trials. Investigation has also been demonstrating that combinatorial therapies of growth inhibitors with other therapies, such as growth factors or stem-cell therapies, produce stronger results and their potential application in the clinics opens new venues in spinal cord injury treatment.

Upregulation of miR-345-5p suppresses cell growth of lung adenocarcinoma by regulating ras homolog family member A(RhoA)and Rho/Rho associated protein kinase(Rho/ROCK)pathway

Background:Microribose nucleic acids(miRNAs)are implicated in the progression of lung adenocarcinoma.MicroRNA-345-5p(miR-345-5p)is a recently identified anti-oncogene in some human cancers,but its functional role and possible molecular mechanism in lung adenocarcinoma remain unknown.This study aimed to identify the biological function and underlying mechanism of miR-345-5p in lung adenocarcinoma cells.Methods:In this study,lung adenocarcinoma tissues and adjacent tissues were collected in the First Affiliated Hospital of Anhui Medical University between April 2016 and February 2017.The expression of miR-345-5p and ras homolog family member A(RhoA)in lung adenocarcinoma tissues and human lung adenocarcinoma cell lines(A549,H1650,PC-9,and H441)was detected by reverse transcription quantitative polymerase chain reaction analysis.Functional assays including colony formation,flow cytometry analysis,wound healing,and transwell assays were performed to assess the proliferation,apoptosis,migration,and invasion of lung adenocarcinoma cells.In addition,RNA pulldown and luciferase reporter assays were conducted to evaluate the relationship between miR-345-5p and RhoA.Difference between the two groups was analyzed with Student’st test,while that among multiple groups was analyzed with one-way analysis of variance.Results:MiR-345-5p expression displayed lower level in lung adenocarcinoma tissues(0.241±0.095vs.1.000±0.233,t=19.247,P<0.001)and cell lines(F=56.992,P<0.001)than control tissues and cells.Functional experiments demonstrated that upregulation of miR-345-5p inhibited the malignant phenotypes of lung adenocarcinoma cells via suppressing cell proliferation,migration,invasion,and facilitating cell apoptosis.Additionally,RhoA was verified to be the downstream target of miR-345-5p.Expression of RhoA was downregulated by overexpression of miR-345-5p in PC-9(0.321±0.047vs.1.000±0.127,t=8.536,P<0.001)and H1650(0.398±0.054vs.1.000±0.156,t=4.429,P=0.011)cells.Rescue assays revealed that overexpression of RhoA rescued the suppressive effects of miR-345-5p upregulation on proliferation,migration,and invasion of lung adenocarcinoma cells.Further,miR-345-5p was found to regulate the Rho/Rho-associated protein kinase(ROCK)signaling pathway by downregulation of RhoA in lung adenocarcinoma cells.Conclusions:MiR-345-5p plays a tumor suppressor role in lung adenocarcinoma cells by downregulating RhoA to inactivate the Rho/ROCK pathway.

The role of the Rho/ROCK signaling pathway in inhibiting axonal regeneration in the central nervous system

The Rho/Rho-associated coiled-coil containing protein kinase（Rho/ROCK） pathway is a major signaling pathway in the central nervous system, transducing inhibitory signals to block regeneration. After central nervous system damage, the main cause of impaired regeneration is the presence of factors that strongly inhibit regeneration in the surrounding microenvironment. These factors signal through the Rho/ROCK signaling pathway to inhibit regeneration. Therefore, a thorough understanding of the Rho/ROCK signaling pathway is crucial for advancing studies on regeneration and repair of the injured central nervous system.

Rho/ROCK signaling in motility and metastasis of gastric cancer

Gastric cancer is one of the most frequent and lethal malignancies worldwide because of high frequency of metastasis. Tumor cell motility and invasion play fundamental roles in cancer metastasis. Recent studies have revealed that the Rho/Rho-associated protein kinases(ROCK) pathway plays a critical role in the regulation of cancer cell motility and invasion. In addition,the Rho/ROCK pathway plays important roles in invasion and metastasis on the basis of its predominant function of cell cytoskeletal regulation in gastric cancer. According to the current understanding of tumor motility,there are two modes of tumor cell movement:mesenchymal and amoeboid. In addition,cancer cell movement can be interchangeable between the mesenchymal and amoeboid movements under certain conditions. Control of cell motility through the actin cytoskeleton creates the potential for regulating tumor cell metastasis. In this review we discuss Rho GTPases and ROCK signaling and describe the mechanisms of Rho/ROCK activity with regard to motility and metastasis in gastric cancer.In addition,we provide an insight of the therapeutic potential of targeting the Rho/ROCK pathway.