Rho-associated Kinase 2 Research Articles

Selective ROCK2 inhibitor down-regulates pro-inflammatory T cell responses via shifting Th17/Treg balance (IRM6P.711)

Abstract Targeting pro-inflammatory cytokines is a promising approach to the treatment of certain autoimmune diseases. However, the signaling mechanisms involved in the initiation and effector phases of auto-aggressive pathways are still an enigma. Rho-associated kinase 2 (ROCK2) regulates the secretion of inflammatory cytokines interleukin (IL)-21 and IL-17 and the development of autoimmunity in mice. Our data from a phase 1 clinical trial demonstrates that oral administration of KD025, a selective ROCK2 inhibitor, to healthy human subjects significantly down-regulates the ability of T cells to secrete IL-21 and IL-17, but not interferon (IFN)-g in response to TCR stimulation in vitro. Pharmacological inhibition with KD025 or siRNA-mediated inhibition of ROCK2, but not ROCK1, leads to down-regulation of STAT3 phosphorylation, interferon regulatory factor 4 (IRF4) and steroid receptor-type nuclear receptor RORgt protein levels in T cells derived from healthy subjects or rheumatoid arthritis (RA) patients. Simultaneously, ROCK2 inhibition induces phosphorylation of STAT5 and SMAD2/3, and subsequently increases the percentage of Foxp3+ T cells. Thus, ROCK2 signaling appears to be instrumental in regulating the balance between pro-inflammatory and regulatory T cell subsets in man. Targeted inhibition of ROCK2 may therefore have a role in the treatment of autoimmune disease with disturbed immune homeostasis.

Effect of electroacupuncture on ROCK1 and ROCK2 expression in rat peri-infarct cortex after focal cerebral infarction

Objective To observe the effect of electroacupuncture(EA)intervention on the expression of Rho-associated kinase 1(ROCK1)and Rho-associated kinase 2(ROCK2)in rat peri-infarct cortex after middle cerebral artery occlusion(MCAO),so as to study the underlying mechanism by which EA improves cerebral ischemia.Methods Totally 40male Sprague Dawley(SD)rats were equally randomized into four groups,control group,sham-operation group,model group and EA group.MACO in the model group and EA group was successfully established by an improved Longaprocedure.EA was given 90 min after resuscitation for the EA group,once a day for 14days.The modified neurological severity scores(mNSS)of rats in each group were determined on the 1st day,3rd day,7th day and the 14th day after operation.Immunohistochemistry and Western blotting analysis were used to detect the expression of ROCK1and ROCK2in the brain 14dafter operation.Results Neural dysfunction was not observed in the control group and sham-operation group.The values of mNSS were significantly different between model group and EA group at 7d,14dafter operation(P0.05).Both immunohistochemical staining and Western blotting analysis indicated that the expression levels of ROCK1and ROCK2were up-regulated in model group,while those in EA group were significantly less than those in the model group(P0.05).Conclusion The expression of ROCK1and ROCK2is up-regulated in rat cortex after focal cerebral infarction,and the up-regulation can be prevented by EA intervention,which might be one of the mechanisms by which EA promotes the recovery of neurological dysfunction after cerebral infarction.

Kamolonol suppresses angiotensin II-induced stress fiber formation and cellular hypertrophy through inhibition of Rho-associated kinase 2 activity

Kamolonol (7-[[(1R,2R,4R,4aS,5R,8aS)-4-hydroxy-1,2,4a,5-tetramethyl-6-oxo-3,4,5,7,8,8a-hexahydro-2H-naphthalen-1-yl]methoxy]chromen-2-one) is a sesquiterpene coumarin and an active component of gum extracts from Ferulaassafoetida. The aim of this study was to investigate the anti-fibrotic and anti-cellular hypertrophic effects of kamolonol, and further to explore its possible mechanism. Kamolonol (3–30μM) significantly inhibited stress fiber formation induced by angiotensin II (Ang II) in rat heart-derived H9c2 cells. Furthermore, kamolonol (3–30μM) showed a potent inhibitory effect on Ang II-induced cellular hypertrophy in H9c2 cells. Next, a Rho-associated kinase (ROCK) activity was measured because actin stress fiber formation and/or cellular hypertrophy are usually induced by the activation of ROCK. Rho-associated kinase 2 (ROCK2) studies using a time-resolved fluorescence resonance energy transfer (TR-FRET) showed that kamolonol possesses a potent ROCK2 inhibitory activity with IC50 values of 2.27μM, and has an ATP-competitive inhibitory mode. In validation study, pretreatment of kamolonol (3–30μM) for 2h decreased the Ang II-induced phosphorylation of myosin phosphatase 1 (MYPT1) and myosin light chain 2 (MLC2). Taken together, these results indicate that kamolonol suppresses Ang II-induced stress fiber formation and cellular hypertrophy, and propose that one mechanism underlying these anti-fibrotic and anti-cellular hypertrophic effects involves inhibition of the ROCK-MLC pathway.

Effects of Progesterone and Medroxyprogesterone on Actin Remodeling and Neuronal Spine Formation

Sex steroids are important regulators of neuronal cell morphology, and this is critical for gender differences in brain function and dysfunction. Neuronal morphology is controlled by multiprotein complexes including moesin (a member of the ezrin/radixin/moesin family), focal adhesion kinase (FAK), or the Wiskott-Aldrich syndrome protein-family verprolin homologous (WAVE1) protein, controlling dynamic remodeling of the cytoskeleton and cell membrane. We investigated the actions of natural progesterone (P) and of the synthetic progestin medroxyprogesterone acetate (MPA) on actin remodeling, focal adhesion complex formation, and actin branching in rat cortical neurons. Treatment with P and, to a lesser extent, MPA, increases the number and density of dendritic spines. P increases the phosphorylation of moesin, FAK, and WAVE1, and their redistribution toward cell membrane sites where spines are formed. Signaling to moesin is achieved by PR via a Gα/Gβ-dependent signaling to the small GTPase Ras homolog gene family, member A and its related kinase, Rho-associated kinase-2. In parallel, WAVE1 recruitment is triggered by a Gαi/Gβ-dependent signaling of PR to c-Src, FAK, and Rac1 GTPase. Rac1 recruits cyclin-dependent kinase-5, which phosphorylates WAVE1. Silencing of moesin, FAK, or WAVE1 abrogates the increase in dendritic spines induced by progesterone. In all applications, MPA is found to act similar to P, albeit with a lower efficacy. In conclusion, our findings indicate that the control of actin polymerization and branching and focal adhesion complex formation via moesin, FAK, and WAVE1 is a key function of progesterone receptor in neurons, which may be relevant for the regulation of dendritic spine turnover and neuronal plasticity.

Pivotal Role of Rho-Associated Kinase 2 in Generating the Intrinsic Circadian Rhythm of Vascular Contractility

The circadian variation in the incidence of cardiovascular events may be attributable to the circadian changes in vascular contractility. The circadian rhythm of vascular contractility is determined by the interplay between the central and peripheral clocks. However, the molecular mechanism of the vascular intrinsic clock that generates the circadian rhythm of vascular contractility still remains largely unknown. The agonist-induced phosphorylation of myosin light chain in cultured smooth muscle cells synchronized by dexamethasone pulse treatment exhibited an apparent circadian oscillation, with a 25.4-hour cycle length. The pharmacological inhibition and knockdown of Rho-associated kinase 2 (ROCK2) abolished the circadian rhythm of myosin light chain phosphorylation. The expression and activity of ROCK2 exhibited a circadian rhythm in phase with that of myosin light chain phosphorylation. A clock gene, RORα, activated the promoter of the ROCK2 gene, whereas its knockdown abolished the rhythmic expression of ROCK2. In the mouse aorta, ROCK2 expression exhibited the circadian oscillation, with a peak at Zeitgeber time 0/24 and a nadir at Zeitgeber time 12. The myofilament Ca(2+) sensitization induced by GTPγS and U46619, a thromboxane A2 analog, at Zeitgeber time 0/24 was greater than that seen at Zeitgeber time 12. The circadian rhythm of ROCK2 expression and myofilament Ca(2+) sensitivity was abolished in staggerer mutant mice, which lack a functional RORα. ROCK2 plays a pivotal role in generating the intrinsic circadian rhythm of vascular contractility by receiving a cue from RORα. The ROCK2-mediated intrinsic rhythm of vascular contractility may underlie the diurnal variation of the incidence of cardiovascular diseases.

Extra-Nuclear Signalling of Estrogen Receptor to Breast Cancer Cytoskeletal Remodelling, Migration and Invasion

BackgroundEstrogen is an established enhancer of breast cancer development, but less is known on its effect on local progression or metastasis. We studied the effect of estrogen receptor recruitment on actin cytoskeleton remodeling and breast cancer cell movement and invasion. Moreover, we characterized the signaling steps through which these actions are enacted.Methodology/Principal FindingsIn estrogen receptor (ER) positive T47-D breast cancer cells ER activation with 17β-estradiol induces rapid and dynamic actin cytoskeleton remodeling with the formation of specialized cell membrane structures like ruffles and pseudopodia. These effects depend on the rapid recruitment of the actin-binding protein moesin. Moesin activation by estradiol depends on the interaction of ERα with the G protein Gα13, which results in the recruitment of the small GTPase RhoA and in the subsequent activation of its downstream effector Rho-associated kinase-2 (ROCK-2). ROCK-2 is responsible for moesin phosphorylation. The Gα13/RhoA/ROCK/moesin cascade is necessary for the cytoskeletal remodeling and for the enhancement of breast cancer cell horizontal migration and invasion of three-dimensional matrices induced by estrogen. In addition, human samples of normal breast tissue, fibroadenomas and invasive ductal carcinomas show that the expression of wild-type moesin as well as of its active form is deranged in cancers, with increased protein amounts and a loss of association with the cell membrane.Conclusions/SignificanceThese results provide an original mechanism through which estrogen can facilitate breast cancer local and distant progression, identifying the extra-nuclear Gα13/RhoA/ROCK/moesin signaling cascade as a target of ERα in breast cancer cells. This information helps to understand the effects of estrogen on breast cancer metastasis and may provide new targets for therapeutic interventions.

A Major Haplotype Block at the Rho-Associated Kinase 2 Locus Is Associated with a Lower Risk of Hypertension in a Recessive Manner: The HYPGENE Study

The goals of our study were to evaluate the haplotype pattern at the Rho-associated kinase 2 (ROCK2) locus and prospectively test the association between the ROCK2 haplotype-tagging single-nucleotide polymorphisms (tagSNPs) with hypertension for verified incident hypertensive patients (n=607) and healthy, normotensive controls (n=586) in a HYPGENE study. Rho-associated kinases (ROCKs) play a central role in signaling pathways that are involved in vascular smooth muscle contraction and endothelial nitric oxide availability. Using a set of stringent criteria (minor allele frequency>or=0.05, pairwise r2>or=0.95), we identified 18 tagSNPs from the 109 SNPs available in the HapMap Caucasian data set. TagSNPs were genotyped using the Illumina BeadStation platform. The 18 tagSNPs consisted of two linkage disequilibrium (LD) blocks. A haplotype defined by four SNPs (rs965665, rs10178332, rs6755196, rs10929732) in LD block 2 was recessively associated with a lower risk of hypertension (p=0.003). Homozygotes for the minor alleles had an 85% lower risk of hypertension than carriers of the common allele. The associations were independent of baseline age, cardiorespiratory fitness, body mass index, sex, and follow-up time. The LD block 2 spans about 137 kb of genomic DNA at the 5'-end of the ROCK2 locus and covers exons encoding the kinase domain of the protein. Our data strongly suggest that a major haplotype block at the ROCK2 locus is recessively associated with a lower risk of hypertension. Identification of functional mutation(s) could thus help in the development of ROCK2-specific treatments.