RhoA-Rho Kinase Research Articles

Dual spatio-temporal regulation of axon growth and microtubule dynamics by RhoA signaling pathways.

RhoA plays a crucial role in neuronal polarization, where its action restraining axon outgrowth has been thoroughly studied. We now report that RhoA has not only an inhibitory but also a stimulatory effect on axon development depending on when and where exerts its action and the downstream effectors involved. In cultured hippocampal neurons, FRET imaging revealed that RhoA activity selectively localized in growth cones of undifferentiated neurites, whereas in developing axons it displayed a biphasic pattern, being low in nascent axons and high in elongating ones. RhoA-Rho kinase (ROCK) signaling prevented axon initiation but had no effect on elongation, whereas formin inhibition reduced axon extension without significantly altering initial outgrowth. In addition, RhoA-mDia signaling promoted axon elongation by stimulating growth cone microtubule stability and assembly, as opposed to RhoA-ROCK signaling, which restrained growth cone microtubule assembly and protrusion.

RhoA rescues cardiac senescence by regulating Parkin-mediated mitophagy

Heart failure is one of the leading causes of death worldwide. RhoA, a small GTPase, governs actin dynamics in various tissue and cell types, including cardiomyocytes; however, its involvement in cardiac function has not been fully elucidated. Here, we generated cardiomyocyte-specific RhoA conditional knockout (cKO) mice, which demonstrated a significantly shorter lifespan with left ventricular dilation and severely impaired ejection fraction. We found that the cardiac tissues of the cKO mice exhibited structural disorganization with fibrosis and also exhibited enhanced senescence compared with control mice. In addition, we show that cardiomyocyte mitochondria were structurally abnormal in the aged cKO hearts. Clearance of damaged mitochondria by mitophagy was remarkably inhibited in both cKO cardiomyocytes and RhoA-knockdown HL-1 cultured cardiomyocytes. In RhoA-depleted cardiomyocytes, we reveal that the expression of Parkin, an E3 ubiquitin ligase that plays a crucial role in mitophagy, was reduced, and expression of N-Myc, a negative regulator of Parkin, was increased. We further reveal that the RhoA-Rho kinase axis induced N-Myc phosphorylation, which led to N-Myc degradation and Parkin upregulation. Re-expression of Parkin in RhoA-depleted cardiomyocytes restored mitophagy, reduced mitochondrial damage, attenuated cardiomyocyte senescence, and rescued cardiac function both invitro and invivo. Finally, we found that patients with idiopathic dilated cardiomyopathy without causal mutations for dilated cardiomyopathy showed reduced cardiac expression of RhoA and Parkin. These results suggest that RhoA promotes Parkin-mediated mitophagy as an indispensable mechanism contributing to cardioprotection in the aging heart.

Sphingosine 1-phosphate receptor type 2 positively regulates interleukin (IL)-4/IL-13-induced STAT6 phosphorylation

Previous reports have demonstrated that sphingosine 1-phosphate receptor type 2 (S1P2) is involved in the activation of signal transducer and activator of transcription (STAT) 6. Additionally, the major signaling pathway of S1P2 is the Rho-Rho kinase pathway. In this study, we examined the role of S1P2 in STAT6 activation in a macrophage (Mφ) model using THP-1 cells differentiated with phorbol 12-myristate 13-acetate (PMA). We established S1P2knockout THP-1 cells using the CRISPR-Cas9 gene editing system. The PMA-treated S1P2knockout THP-1 Mφs showed decreases in IL-4/IL-13-induced phosphorylation of Janus-activated kinase (JAK) 1, JAK2, and STAT6 as well as mRNA expression of the M2 marker ARG1 compared with wild-type THP-1 Mφs. Pretreatment of PMA-treated THP-1 Mφs with the S1P2 antagonist JTE-013, the Rho inhibitor Rhosin or the Rho kinase inhibitor Y27632 inhibited the IL-4/IL-13-induced increase in STAT6 phosphorylation. The expressions of suppressor of cytokine signaling 3 in the S1P2knockout THP-1 Mφs were higher than those in wild-type THP-1 Mφs. In addition, the protein tyrosine phosphatase inhibitor vanadate enhanced IL-4-induced STAT6 phosphorylation in the S1P2knockout THP-1 Mφs, suggesting that S1P2-Rho-Rho kinase inhibited the negative regulation of STAT6. These results suggest that the S1P2-Rho-Rho kinase pathway is necessary for full activation of STAT6 by IL-4/IL-13 in Mφs.

Estrogen Enhances the Microvascular Reactivity Through RhoA-ROCK Pathway in Female Mice During Hemorrhagic Shock.

Vascular hypo-reactivity plays a critical role inducing organ injury during hemorrhagic shock. 17β-estradiol (E2) can induce vasodilation to increase blood flow in various vascular beds. This study observed whether E2 can restore vascular hypo-reactivity induced by hemorrhagic shock, and whether E2 effects are associated with RhoA-Rho kinase (ROCK)-myosin light chain kinase phosphatase (MLCP) pathway. The hemorrhagic shock model (40 ± 2 mm Hg for 1 h, resuscitation for 4 h) was established in ovary intact sham operation (OVI), ovariectomized (OVX), and OVX plus E2 supplement female mice. Intestinal microvascular loop was used to assess blood flow in vivo, mRNA expression and vascular reactivity in vitro. Hemorrhagic shock significantly reduced norepinephrine microvascular reactivity. Decreased microvascular reactivity was exacerbated by OVX and reversed by E2 supplement. U-46619 (RhoA agonist) increased microvascular reactivity, and C3 transferase (an ADP ribosyl transferase that selectively induces RhoA ribosylation) or Y-27632 (ROCK inhibitor) inhibited sham mice microvascular reactivity. Similarly, U-46619 increased microvascular reactivity in OVI and OVX mice following hemorrhagic shock, which was abolished by Y-27632 or concomitant incubation of okadaic acid (OA) (MLCP inhibitor) and Y-27632. In OVX plus E2 supplement mice with hemorrhagic shock, Y-27632 inhibited microvascular reactivity, which was abolished by concomitant U-46619 application. Lastly, hemorrhagic shock remarkably decreased intestinal loop blood flow, RhoA and ROCK mRNA expressions in vascular tissues in OVX females, but not in OVI females, which were reversed by E2 supplement. These results indicate that estrogen improves microvascular reactivity during hemorrhagic shock, and RhoA-ROCK signaling pathway may mediate E2 effects.

Mechanism of α1-Adrenergic Receptor-Induced Increased Contraction of Rat Mesenteric Artery in Aging Hypertension Rats

Introduction: Vasoconstriction is triggered by an increase in intracellular-free calcium concentration. Growing evidence indicates that contraction is also regulated by calcium-independent mechanisms involving RhoA-Rho kinase (ROCK), protein kinase C (PKC), and so on. In this study, we studied the changes of vascular reactivity as well as the underlying signaling pathways in aging spontaneously hypertensive rats (SHRs). Methods: The artery tension induced by α1-adrenergic receptor activator (α1-AR) phenylephrine (PE) was measured in the absence or presence of myosin light chain kinase (MLCK), PKC, and ROCK inhibitors. The α1-AR, PKC, ROCK, phosphorylation of myosin light chain (MLC), and PKC-potentiated phosphatase inhibitors of 17 kDa (CPI-17) of rat mesenteric arteries were analyzed at the mRNA level or protein level. Results: The vascular tension measurements showed that there was a significant increase in the mesenteric artery contraction induced by PE in old SHR. MLCK inhibitor ML-7 can similarly inhibit PE-induced vasoconstriction. PKC inhibitor GF109203X has the weakest inhibitory effect on PE-induced contraction in old SHR. At the presence of ROCK inhibitor H1152, PE-induced contraction was significantly reduced in young Wistar-Kyoto (WKY) rats, but this phenomenon disappeared in other rats. Furthermore, in old SHR the protein expression of α1-AR decreased and phosphorylation of MLC and CPI-17 were upregulated and MLC phosphatase (MLCP) activity was significantly lower. The expressions of PKC were upregulated in SHR and old rats. In addition, the expression of ROCK-1 was decreased and ROCK-2 was significantly upregulated with age in SHR. Conclusion: In aging hypertension, the expression/activity of PKC or ROCK-2/CPI-17 excessively increased, MLCP activity decreased and MLC phosphorylation enhanced, leading to increased α1-AR-induced vasoconstriction.

Anti-apoptotic efficacy of Qingnao Yizhi formula in hypoxia/reoxygenation primary cortical neurons through the promotion of synaptic plasticity by modulating the NogoA-Nogo receptor/Rho-Rho kinase signaling pathway.

To evaluate the anti-apoptotic efficacy of Qingnao Yizhi formula (，QNYZ) in cultured cerebral cortical neuronal cells (CNCs) and the regulation of the NogoA-Nogo receptor (NgR)/Rho-Rho kinase (ROCK) signaling pathway. Primary cultured CNCs were randomly divided into the following groups: normal control group (N-C), hypoxia-reoxygenation group (H/R), high-dose QNYZ group (Q-H), low-dose QNYZ group (Q-L) butylphthalide (NBP) group, and Y-27632 (a selective ROCK transduction pathway inhibiter) group. Except those in the N-C group, CNCs were placed in hypoxic conditions for 24 h and then in reoxygenation conditions for 24 h. Cell media was changed every 48 h, and various assays were performed on the 7th day. Cell viability was evaluated by measuring mitochondrial dehydrogenase activity, using a CCK-8 assay, in triplicate. Synapsin (SYN) protein concentrations were evaluated by enzyme-linked immunosorbent assay. NogoA and RhoA protein expression were evaluated through Western blotting. The gene expression of NogoA, NgR, RhoA, and ROCK was evaluated by reverse transcription-polymerase chain reaction. Cell apoptosis was measured using a terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay. Compared with the N-C group, the cell viability of the H/R group decreased significantly (P < 0.05). The cell viability values for the Q-H and Q-L groups increased compared with that for the H/R group, and the difference was significant for the Q-H group (P < 0.05). The NogoA and RhoA protein levels and the NogoA, NgR, RhoA, and ROCK mRNA expression levels increased in the H/R group, compared with the N-C group, and decreased significantly in the Q-H and Q-L groups (P < 0.05) and in the Y-27632 group (P < 0.05) compared with the H/R group. The SYN levels in the Q-H, Q-L, and NBP groups significantly increased compared with that in the H/R group (P < 0.05). Compared with the H/R group, the numbers of apoptotic cells in the Q-H, Q-L, and NBP groups significantly decreased (P < 0.05). The presented study demonstrated that QNYZ exerted anti-apoptotic effects on H/R-induced CNCs, possibly through the modulation of the NogoA-NgR/Rho-ROCK signaling pathway and the promotion of synaptic plasticity in H/R CNCs.

Inhibiting Th1/2 cells influences hepatic capillarization by adjusting sinusoidal endothelial fenestrae through Rho-ROCK-myosin pathway.

CD4+ T cells are considered to be vital in chronic liver diseases, but their exact roles in hepatic capillarization, the typical characteristic of liver fibrosis, are poorly understood. This study aimed to assess the roles of typical subtype of CD4+ T cells, named T helper 1 (Th1) and Th2 cells in liver fibrosis. Taking advantage of well established fibrotic rat model, we conducted in vitro and in vivo experiments to explore the interactions between liver sinusoidal endothelial cells (LSECs) and Th1/2 cells; meanwhile we evaluated the degree of hepatic capillarization when inhibiting these interactions with inhibitory antibodies. Our results showed that prohibiting interactions between Th2 cells and LSECs caused the restoration of fenestrae, increased cytokine level of Th1 cells and reduction of hepatic capillarization; inhibiting the interaction between Th1 cells and LSECs produced the opposite effects. Moreover, increased Rho and myosin light chain phosphorylation were observed when Th1 cells were inhibited with the corresponding inhibitory antibody; Th2 cell inhibition yielded the opposite results. This study indicated that Th1/2 cells steer the capillarization process in different directions and this effect is probably mediated by the Rho-Rho kinase (ROCK)-myosin signaling pathway.

Effects of ROCK Inhibitors on Apoptosis of Corneal Endothelial Cells in CMV-Positive Posner-Schlossman Syndrome Patients.

PurposeTo examine the role of aqueous tumor necrosis factor α (TNF-α)–RhoA–Rho kinase (ROCK) signaling in cytomegalovirus (CMV)-induced apoptosis and the barrier function of cultured human corneal endothelial cells (hCECs) in CMV-positive Posner–Schlossman syndrome (CMV+/PSS) patients.MethodsAqueous levels of TNF-α, IL-8, IL-10, and several other cytokines in 19 CMV+/PSS patients and 20 healthy control subjects were quantitated using a multiplex assay. The expression of active RhoA in hCECs post-CMV infection was determined using western blotting (WB). The expression levels of TNF-α and nuclear factor kappa B (NF-κB) in CMV-infected hCECs were examined by immunocytochemistry (ICC) and WB with and without ROCK inhibitors. The apoptotic rate and barrier integrity in CMV-infected hCECs were also examined.ResultsThe expression levels of TNF-α, monocyte chemoattractant protein-1 (MCP-1), IL-8, and IL-10 were upregulated in the aqueous humor of CMV+/PSS patients, and among these upregulated cytokines aqueous TNF-α was negatively correlated with the number of corneal endothelial cells. In CMV-infected hCECs, upregulation of TNF-α and NF-κB was determined by WB and ICC. In hCECs, CMV infection induced apoptosis and significantly impaired cell–cell contacts, effects that were attenuated by treatment with a ROCK inhibitor.ConclusionsAqueous TNF-α was upregulated in CMV+/PSS patients, which may have triggered corneal endothelial cell loss. Modulation of TNF-α, including its downstream Rho–ROCK signaling, could serve as a novel treatment modality for corneal endothelial cell loss in CMV+/PSS patients.

NK2 receptor-mediated detrusor muscle contraction involves Gq/11-dependent activation of voltage-dependent Ca2+ channels and the RhoA-Rho kinase pathway.

Tachykinins (TKs) are involved in both the physiological regulation of urinary bladder functions and development of overactive bladder syndrome. The aim of the present study was to investigate the signal transduction pathways of TKs in the detrusor muscle to provide potential pharmacological targets for the treatment of bladder dysfunctions related to enhanced TK production. Contraction force, intracellular Ca2+ concentration, and RhoA activity were measured in the mouse urinary bladder smooth muscle (UBSM). TKs and the NK2 receptor (NK2R)-specific agonist [β-Ala8]-NKA(4-10) evoked contraction, which was inhibited by the NKR2 antagonist MEN10376. In Gαq/11-deficient mice, [β-Ala8]-NKA(4-10)-induced contraction and the intracellular Ca2+ concentration increase were abolished. Although Gq/11 proteins are linked principally to phospholipase Cβ and inositol trisphosphate-mediated Ca2+ release from intracellular stores, we found that phospholipase Cβ inhibition and sarcoplasmic reticulum Ca2+ depletion failed to have any effect on contraction induced by [β-Ala8]-NKA(4-10). In contrast, lack of extracellular Ca2+ or blockade of voltage-dependent Ca2+ channels (VDCCs) suppressed contraction. Furthermore, [β-Ala8]-NKA(4-10) increased RhoA activity in the UBSM in a Gq/11-dependent manner and inhibition of Rho kinase with Y-27632 decreased contraction force, whereas the combination of Y-27632 with either VDCC blockade or depletion of extracellular Ca2+ resulted in complete inhibition of [β-Ala8]-NKA(4-10)-induced contractions. In summary, our results indicate that NK2Rs are linked exclusively to Gq/11 proteins in the UBSM and that the intracellular signaling involves the simultaneous activation of VDCC and the RhoA-Rho kinase pathway. These findings may help to identify potential therapeutic targets of bladder dysfunctions related to upregulation of TKs.

Calcium-Dependent Src Phosphorylation and Reactive Oxygen Species Generation Are Implicated in the Activation of Human Platelet Induced by Thromboxane A2 Analogs.

The thromboxane (TX) A2 elicits TP-dependent different platelet responses. Low amounts activate Src kinases and the Rho–Rho kinase pathway independently of integrin αIIbβ3 and ADP secretion and synergize with epinephrine to induce aggregation. Aim of the present study was to investigate the role Src kinases and the interplay with calcium signals in reactive oxygen species (ROS) generation in the activatory pathways engaged by TXA2 in human platelets. All the experiments were performed in vitro or ex vivo. Washed platelets were stimulated with 50–1000 nM U46619 and/or 10 μM epinephrine in the presence of acetylsalicylic acid and the ADP scavenger apyrase. The effects of the ROS scavenger EUK-134, NADPH oxidase (NOX) inhibitor apocynin, Src kinase inhibitor PP2 and calcium chelator BAPTA were tested. Intracellular calcium and ROS generation were measured. Platelet rich plasma from patients treated with dasatinib was used to confirm the data obtained in vitro. We observed that 50 nM U46619 plus epinephrine increase intracellular calcium similarly to 1000 nM U46619. ROS generation was blunted by the NOX inhibitor apocynin. BAPTA inhibited ROS generation in resting and activated platelets. Phosphorylation of Src and MLC proteins were not significantly affected by antioxidants agents. BAPTA and antioxidants reduced P-Selectin expression, activation of integrin αIIbβ3and platelet aggregation. TXA2-induced increase in intracellular calcium is required for Src phosphorylation and ROS generation. NADPH oxidase is the source of ROS in TX stimulated platelets. The proposed model helps explain why an incomplete inhibition of TP receptor results in residual platelet activation, and define new targets for antiplatelet treatment.

Vascular smooth muscle contraction in hypertension.

Hypertension is a major risk factor for many common chronic diseases, such as heart failure, myocardial infarction, stroke, vascular dementia, and chronic kidney disease. Pathophysiological mechanisms contributing to the development of hypertension include increased vascular resistance, determined in large part by reduced vascular diameter due to increased vascular contraction and arterial remodelling. These processes are regulated by complex-interacting systems such as the renin-angiotensin-aldosterone system, sympathetic nervous system, immune activation, and oxidative stress, which influence vascular smooth muscle function. Vascular smooth muscle cells are highly plastic and in pathological conditions undergo phenotypic changes from a contractile to a proliferative state. Vascular smooth muscle contraction is triggered by an increase in intracellular free calcium concentration ([Ca2+]i), promoting actin–myosin cross-bridge formation. Growing evidence indicates that contraction is also regulated by calcium-independent mechanisms involving RhoA-Rho kinase, protein Kinase C and mitogen-activated protein kinase signalling, reactive oxygen species, and reorganization of the actin cytoskeleton. Activation of immune/inflammatory pathways and non-coding RNAs are also emerging as important regulators of vascular function. Vascular smooth muscle cell [Ca2+]i not only determines the contractile state but also influences activity of many calcium-dependent transcription factors and proteins thereby impacting the cellular phenotype and function. Perturbations in vascular smooth muscle cell signalling and altered function influence vascular reactivity and tone, important determinants of vascular resistance and blood pressure. Here, we discuss mechanisms regulating vascular reactivity and contraction in physiological and pathophysiological conditions and highlight some new advances in the field, focusing specifically on hypertension.

Abstract TP402: The Cerebral Cavernous Malformation Disease Causing Gene Krit1 Participates in Epithelial Barrier Maintenance and Regulation

Background: Cerebral cavernous malformation (CCM) patients develop mulberry-like microvessels in the brain. These lesions can bleed, causing clinical symptoms. Germline mutations of CCM genes have been identified in familial form of CCM, and decreased endothelial CCM protein expression leads to RhoA-Rho kinase (ROCK) dependent actin cytoskeleton reorganization and increased endothelial permeability. It is not known if CCM mutations, which occur in all cells of a familial CCM patient, can impact barrier function of other body cavity lining cells, such as epithelial cells. Understanding how CCM protein function in epithelia may help us identify risks of CCM patients to epithelium-related diseases. Aim: To determine if and how the CCM gene KRIT1/CCM1 affects epithelial barrier maintenance and regulation. Methods: KRIT1 knockdown (KD) intestinal epithelial Caco-2 cells were generated by stably transfecting an anti-KRIT1 siRNA expressing plasmid. Epithelial barrier function, measured as transepithelial resistance (TER) and size selective cation permeability was determined by current clamp. Protein expression and localization was determined by western blot and immunofluorescent microscopy, respectively. Results: KRIT1 KD intestinal epithelial Caco-2 cell monolayers have decreased TER and selectively increased permeability to small cations, along with decreased claudin-1expression. ROCK inhibition (Y-27632) and myosin motor inhibition (blebbistatin) both decreased TER in control Caco-2 monolayers, but not in KRIT1 KD monolayers, indicating KRIT1 is important for myosin regulation of epithelial barrier function. Tumor necrosis factor (TNF), a cytokine that decreases epithelial barrier function through inducing myosin contraction, caused more severe barrier loss in KRIT1 KD monolayers relative to control monolayers, which is blocked by pan-caspase inhibition (zVAD), indicating KRIT1 loss promotes TNF-induced barrier loss through inducing cell death. Conclusion: KRIT1 regulates epithelial barrier by affecting tight junction protein expression and impacting myosin- and apoptosis- dependent processes. These changes in epithelial function may increase familial CCM patients’ susceptibility to intestinal and other diseases.

Symptômes du bas appareil urinaire secondaire à une hyperplasie bénigne de prostate et dysfonction érectile : une revue systématique de la littérature

To provide a systematic review of epidemiological data regarding the association between erectile dysfunction (ED) and lower urinary tract symptoms (LUTS) in men. A research has been conducted on the Medline database using the keywords: ("erectile dysfunction" or "sexual dysfunction") and ("benign prostatic hyperplasia" or "lower urinary tract symptoms"). The eligibility of studies was defined using the PICOS method in accordance with the PRISMA statement. Cross-sectional studies and prospective cohorts assessing the association between LUTS and ED in the primary care setting or in general practice (i.e. exclusion of patients seen in outpatient urology or andrology) were included. Among 898reports assessed, seven studies were included in this systematic review (whole cohort: 1,196,393men). There were five cross-sectional studies and two prospective cohorts. The whole seven studies reported an association between LUTS and ED (range of odds-ratio: 1.52-4.03). Four common pathogenic mechanisms were found in the literature, all of them being somewhat related with metabolic syndrome and cardiovascular risk factors: reduced nitric oxide (NO) pathway signalling, increased RhoA-Rho kinase signalling, autonomic nervous system hyperactivity and pelvic atherosclerosis. The main limitations of this review were: a possible publication bias, the relatively low number of included studies and the lack of assessment of potential confounders such as factors related to sexual partner. The close epidemiological and pathogenic links between LUTS and ED have given rise to a new nosological entity: the erectile urogenital dysfunction, which should be assessed globally with special considerations to frequently associated comorbidities such as metabolic syndrome and cardiovascular risk factors.

Dissociative role for dorsal hippocampus in mediating heroin self‐administration and relapse through CDK5 and RhoB signaling revealed by proteomic analysis

Addiction is characterized by drug craving, compulsive drug taking and relapse, which is attributed to aberrant neuroadaptation in brain regions implicated in drug addiction, induced by changes in gene and protein expression in these regions after chronic drug exposure. Accumulating evidence suggests that the dorsal hippocampus (DH) plays an important role in mediating drug-seeking and drug-taking behavior and relapse. However, the molecular mechanisms underlying these effects of the DH are unclear. In the present study, we employed a label-free quantitative proteomic approach to analyze the proteins altered in the DH of heroin self-administering rats. A total of 4015 proteins were quantified with high confidence, and 361 proteins showed significant differences compared with the saline control group. Among them, cyclin-dependent kinase 5 (CDK5) and ras homolog family member B (RhoB) were up-regulated in rats with a history of extended access to heroin. Functionally, inhibition of CDK5 in the DH enhanced heroin self-administration, indicating that CDK5 signaling in the DH acts as a homeostatic compensatory mechanism to limit heroin-taking behavior, whereas blockade of the Rho-Rho kinase (ROCK) pathway attenuated context-induced heroin relapse, indicating that RhoB signaling in the DH is required for the retrieval (recall) of addiction memory. Our findings suggest that manipulation of CDK5 signaling in the DH may be essential in determining vulnerability to opiate taking, whereas manipulation of RhoB signaling in the DH may be essential in determining vulnerability to relapse. Overall, the present study suggests that the DH can exert dissociative effects on heroin addiction through CDK5 and RhoB signaling.

Attenuation of obesity-induced insulin resistance in mice with heterozygous deletion of ROCK2.

Obesity-associated insulin resistance is a major risk factor for the development of type 2 diabetes, cardiovascular disease and non-alcoholic liver disease. Over-activation of the RhoA-Rho kinase (ROCK) pathway has been implicated in the development of obesity-induced insulin resistance, but the relative contribution of ROCK2 has not been elucidated. This was investigated in the present study. Male ROCK2+/- mice and their wild-type (WT) littermate controls were fed normal chow or a high fat diet (HFD) for 18 weeks. Glucose and insulin tolerance tests were conducted 8 and 16 weeks after the start of feeding. At termination, isoform-specific ROCK activity and insulin signaling were evaluated in epididymal adipose tissue. Adipocyte size was assessed morphometrically, while adipose tissue production of PPARγ was determined by western blotting, and inflammatory cytokines were evaluated by RT-PCR and immunofluorescence. The decrease in systemic insulin sensitivity and glucose tolerance produced by high fat feeding was attenuated in ROCK2+/- mice. There was no reduction in food intake, body weight or epididymal fat pad weight in HFD-ROCK2+/- mice. However, the increase in adipocyte size detected in HFD-WT mice was attenuated in HFD-ROCK2+/- mice. The increase in adipose tissue ROCK2 activity produced by high fat feeding in WT mice was also prevented in ROCK2+/- mice, and this was accompanied by improved insulin-induced phosphorylation of Akt. The expression of both isoforms of PPARγ was increased in adipose tissue from HFD-ROCK2+/- mice, while adipocyte hypertrophy and production of inflammatory cytokines were reduced compared with HFD-WT mice. These data suggest that activation of ROCK2 in adipose tissue contributes to obesity-induced insulin resistance. This may result in part from suppression of PPARγ expression, leading to adipocyte hypertrophy and an increase in inflammatory cytokine production. ROCK2 may be a suitable target to improve insulin sensitivity in obesity.

Interleukin-10 limits increased blood pressure and vascular RhoA/Rho-kinase signaling in angiotensin II-infused mice

Interleukin-10 (IL-10) is a multi-functional cytokine with potent anti-inflammatory properties. We hypothesized that IL-10 limits increased RhoA/Rho-kinase signaling and vascular reactivity in arteries from angiotensin II (Ang II) hypertensive mice. Wild type (WT) and IL-10 knockout ((-/-)) mice were infused with Ang II (90ng/min) for 14days. Additionally, WT mice were infused with Ang II and simultaneously infused with exogenous IL-10 (0.5ηg/min, 14days). Aortic rings were mounted in a myograph and concentration-response curve to phenylephrine (PE) were evaluated. After Ang II infusion, blood pressure responses, but not maximal contraction to PE, was greater in IL-10(-/-) mice, compared to WT. Rho-kinase inhibition (Y-27632; 10μM) resulted in a more evident reduction of PE-induced contraction in WT hypertensive mice, when compared to IL-10(-/-) hypertensive mice. IL-10 exogenous infusion prevented the blood pressure increase in Ang II-infused WT mice. The augmented PE-contraction observed in aorta from WT mice infused with Ang II was also prevented by exogenous infusion of IL-10. Additionally, Rho-kinase inhibition (Y-27632; 10μM) abolished the differences in the contractile response to PE between these groups. These results demonstrate that IL-10 counteracts both the pressoric activity of Ang II as well as vascular dysfunction associated with hypertension, partially, modulating the RhoA-Rho kinase pathway. Strategies to enhance IL-10 levels during hypertension may enhance the benefits provided by regular treatments.

Radial Glial Cell–Neuron Interaction Directs Axon Formation at the Opposite Side of the Neuron from the Contact Site

How extracellular cues direct axon-dendrite polarization in mouse developing neurons is not fully understood. Here, we report that the radial glial cell (RGC)-cortical neuron interaction directs axon formation at the opposite side of the neuron from the contact site. N-cadherin accumulates at the contact site between the RGC and cortical neuron. Inhibition of the N-cadherin-mediated adhesion decreases this oriented axon formation in vitro, and disrupts the axon-dendrite polarization in vivo. Furthermore, the RGC-neuron interaction induces the polarized distribution of active RhoA at the contacting neurite and active Rac1 at the opposite neurite. Inhibition of Rho-Rho-kinase signaling in a neuron impairs the oriented axon formation in vitro, and prevents axon-dendrite polarization in vivo. Collectively, these results suggest that the N-cadherin-mediated radial glia-neuron interaction determines the contacting neurite as the leading process for radial glia-guided neuronal migration and directs axon formation to the opposite side acting through the Rho family GTPases.

Inhibition of prostaglandin E2 receptor EP3 mitigates thrombin-induced brain injury

Prostaglandin E2 EP3 receptor is the only prostaglandin E2 receptor that couples to multiple G-proteins, but its role in thrombin-induced brain injury is unclear. In the present study, we exposed mouse hippocampal slice cultures to thrombin in vitro and injected mice with intrastriatal thrombin in vivo to investigate the role of EP3 receptor in thrombin-induced brain injury and explore its underlying cellular and molecular mechanisms. In vitro, EP3 receptor inhibition reduced thrombin-induced hippocampal CA1 cell death. In vivo, EP3 receptor was expressed in astrocytes and microglia in the perilesional region. EP3 receptor inhibition reduced lesion volume, neurologic deficit, cell death, matrix metalloproteinase-9 activity, neutrophil infiltration, and the number of CD68+ microglia, but increased the number of Ym-1+ M2 microglia. RhoA-Rho kinase levels were increased after thrombin injection and were decreased by EP3 receptor inhibition. In mice that received an intrastriatal injection of autologous arterial blood, inhibition of thrombin activity with hirudin decreased RhoA expression compared with that in vehicle-treated mice. However, EP3 receptor activation reversed this effect of hirudin. These findings show that prostaglandin E2 EP3 receptor contributes to thrombin-induced brain damage via Rho-Rho kinase–mediated cytotoxicity and proinflammatory responses.

FSD-C10: A more promising novel ROCK inhibitor than Fasudil for treatment of CNS autoimmunity.

Rho-Rho kinase (Rho-ROCK) triggers an intracellular signalling cascade that regulates cell survival, death, adhesion, migration, neurite outgrowth and retraction and influences the generation and development of several neurological disorders. Although Fasudil, a ROCK inhibitor, effectively suppressed encephalomyelitis (EAE), certain side effects may limit its clinical use. A novel and efficient ROCK inhibitor, FSD-C10, has been explored. In the present study, we present chemical synthesis and structure of FSD-C10, as well as the relationship between compound concentration and ROCK inhibition. We compared the inhibitory efficiency of ROCKI and ROCK II, the cell cytotoxicity, neurite outgrowth and dendritic formation, neurotrophic factors and vasodilation between Fasudil and FSD-C10. The results demonstrated that FSD-C10, like Fasudil, induced neurite outgrowth of neurons and dendritic formation of BV-2 microglia and enhanced the production of neurotrophic factor brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3). However, the cell cytotoxicity and vasodilation of FSD-C10 were relatively small compared with Fasudil. Although Fasudil inhibited both ROCK I and ROCK II, FSD-C10 more selectively suppressed ROCK II, but not ROCK I, which may be related to vasodilation insensitivity and animal mortality. Thus, FSD-C10 may be a safer and more promising novel ROCK inhibitor than Fasudil for the treatment of several neurological disorders.

Apolipoprotein E3 Inhibits Rho to Regulate the Mechanosensitive Expression of Cox2

Apolipoprotein E3 (apoE3) is thought to protect against atherosclerosis by enhancing reverse cholesterol transport. However, apoE3 also has cholesterol-independent effects that contribute to its anti-atherogenic properties. These include altering extracellular matrix protein synthesis and inhibiting vascular smooth muscle cell proliferation. Both of these cholesterol-independent effects result from an apoE3-mediated induction of cyclooxygenase-2 (Cox2). Nevertheless, how apoE3 regulates Cox2 remains unknown. Here, we show that apoE3 inhibits the activation of Rho, which reduces the formation of actin stress fibers and focal adhesions and results in cellular softening. Inhibition of Rho-Rho kinase signaling or direct cellular softening recapitulates the effect of apoE3 on Cox2 expression while a constitutively active Rho mutant overrides the apoE3 effect on both intracellular stiffness and Cox2. Thus, our results describe a previously unidentified mechanism by which an atheroprotective apolipoprotein uses Rho to control cellular mechanics and Cox2.