Rho-associated Coiled-coil Protein Kinase 1 Research Articles

Activation of the 5-hydroxytryptamine 4 receptor ameliorates tight junction barrier dysfunction in the colon of type 1 diabetic mice.

Hyperglycemia drives dysfunction of the intestinal barrier. 5-Hydroxytryptaine 4 receptor (5-HT 4R) agonists have been considered therapeutics for constipation in clnic. However, the roles of 5-HT 4R activation in mucosa should be fully realized. Here, we investigate the effects of 5-HT 4R activation on diabetes-induced disruption of the tight junction (TJ) barrier in the colon. Not surprisingly, the TJ barrier in diabetic mice with or without 5-HT 4R is tremendously destroyed, as indicated by increased serum fluorescein isothiocyanate (FITC)-dextran and decreased transepithelial electrical resistance (TER). Simultaneously, decreased expressions of TJ proteins are shown in both wild-type (WT) and 5-HT 4R knockout (KO) mice with diabetes. Notably, chronic treatment with intraperitoneal injection of a 5-HT 4R agonist in WT mice with diabetes repairs the TJ barrier and promotes TJ protein expressions, including occludin, claudin-1 and ZO-1, in the colon, whereas a 5-HT 4R agonist does not improve TJ barrier function or TJ protein expressions in 5-HT 4R KO mice with diabetes. Furthermore, stimulation of 5-HT 4R inhibits diabetes-induced upregulation of myosin light chain kinase (MLCK), Rho-associated coiled coil protein kinase 1 (ROCK1), and phosphorylated myosin light chain (p-MLC), which are key molecules that regulate TJ integrity, in the colonic mucosa of WT mice. However, such action induced by a 5-HT 4R agonist is not observed in 5-HT 4R KO mice with diabetes. These findings indicate that 5-HT 4R activation may restore TJ integrity by inhibiting the expressions of MLCK, ROCK1 and p-MLC, improving epithelial barrier function in diabetes.

LncRNA BCYRN1 promotes cell migration and invasion of non-small cell lung cancer via the miR-30b-3p/ROCK1 axis.

Long non-coding RNA brain cytoplasmic RNA 1 (LncRNA BCYRN1) has been proved to participate in the cancer cell metastasis process, including non-small cell lung cancer (NSCLC). However, the underlying molecular mechanisms involved in the BCYRN1-mediated function remain largely unknown. The qRT-PCR analysis was carried out to examine the relative expressions of BCYRN1, microRNA-30b-3p (miR-30b-3p), and Rho-associated coiled-coil protein kinase 1 (ROCK1). ROCK1 protein level was detected via western blot assay. The migrative and invasive abilities of H520 and A549 cells were evaluated via Transwell assay. The relationships between BCYRN1 and miR-30b-3p or ROCK1 and miR-30b-3p were examined by luciferase reporter assay. The expression levels of BCYRN1 and ROCK1 were upregulated in NSCLC tissues and cells, while miR-30b-3p was downregulated. Higher BCYRN1 expression indicated lymph node metastasis and advanced tumor-node-metastasis (TNM) stage of NSCLC patients. Loss of BCYRN1 suppressed cell migration and invasion. More importantly, miR-30b-3p possessed the binding sites with BCYRN1. Besides, BCYRN1 negatively regulated the expression level of miR-30b-3p. Meanwhile, ROCK1 was proven to be directly targeted by miR-30b-3p. In addition, the silencing of miR-30b-3p also weakened the effect of BCYRN1 knockdown on cell migration and invasion. In vivo, BCYRN1 silencing reduced the growth of A549 cells. LncRNA BCYRN1 was involved in the metastasis of NSCLC through modulating the miR-30b-3p/ROCK1 axis.

NANOG improves type I collagen expression in human fetal scleral fibroblasts

Human fetal scleral fibroblasts (HFSFs) are components of the sclera and play important roles in its structure and function. In myopia, scleral remodeling reduces collagen fibers and the sclera begins to thin. NANOG is a key transcription factor essential for pluripotent and self-renewing phenotypes of undifferentiated embryonic stem cells. To determine whether NANOG improves human fetal scleral fibroblast quality and the underlying mechanisms in these cells, we established stable NANOG-overexpressing HFSFs. We studied type I collagen (COL1A 1) and Rho-associated coiled-coil protein kinase 1 (ROCK1) expression in transfected cells. We also investigated POU5F1, SOX2, KLF4, MYC and SALL4 expression in NANOG stably-overexpressed fibroblasts. Our data show that NANOG expression increased proliferation rates in fibroblasts. When compared to controls, expression of COL1A 1 in transfected fibroblasts was increased and the expression of ROCK1 was decreased. Similarly, the expression of POU5F1, SOX2 and KLF4 was downregulated, the expression of MYC was upregulated and there was no significant change in the expression of SALL4 in transfected fibroblasts. Our results suggest that in fibroblasts, NANOG regulates ROCK1 expression and improves COL1A 1 expression to delay scleral remodeling.

Role of inhibiting LIM-kinase2 in improving erectile function through suppression of corporal fibrosis in a rat model of cavernous nerve injury.

We evaluated whether LIM-kinase 2 inhibitor (LIMK2i) could improve erectile function by suppressing corporal fibrosis through the normalization of the Rho-associated coiled-coil protein kinase 1 (ROCK1)/LIMK2/Cofilin pathway in a rat model of cavernous nerve crush injury (CNCI). Sixty 11-week-old male Sprague-Dawley rats were divided equally into five groups: sham surgery (S), CNCI (I), and CNCI treated with low-dose (L), medium-dose (M), and high-dose (H) LIMK2i. The L, M, and H groups were treated with a daily intraperitoneal injection of LIMK2i (2.5, 5.0, and 10.0 mg kg−1 body weight, respectively) for 1 week after surgery. The erectile response was assessed using electrostimulation at 1 week, postoperatively. Penile tissues were processed for Masson's trichrome staining, double immunofluorescence, and Western blot assay. Erectile responses in the H group improved compared with the I group, while the M group showed only partial improvement. A significantly decreased smooth muscle/collagen ratio and an increased content of fibroblasts positive for phospho-LIMK2 were noted in the I group. The M and H groups revealed significant improvements in histological alterations and the dysregulated LIMK2/Cofilin pathway, except for LIMK2 phosphorylation in the M group. The inhibition of LIMK2 did not affect the ROCK1 protein expression. The content of fibroblasts positive for phospho-LIMK2 in the H group returned to the level found in the S group, whereas it did not in the M group. However, the L group did not exhibit such improvements. Our data suggest that the inhibition of LIMK2, particularly with administration of 10.0 mg kg−1 body weight LIMK2i, can improve corporal fibrosis and erectile function by normalizing the LIMK2/Cofilin pathway.

Vitamin D Reduces Oxidative Stress-Induced Procaspase-3/ROCK1 Activation and MP Release by Placental Trophoblasts.

Increased microparticle (MP) shedding by placental trophoblasts contributes to maternal vascular inflammatory response and endothelial dysfunction in preeclampsia. Vitamin D has beneficial effects in pregnancy; however, its effect on trophoblast MP release has not been investigated. To investigate if vitamin D could protect trophoblasts from oxidative stress-induced MP release. Placental trophoblasts were isolated from uncomplicated and preeclamptic placentas. Effects of vitamin D on MP release induced by oxidative stress inducer CoCl2 were studied. Annexin V+ MPs were assessed by flow cytometry. Expression of caveolin-1, endothelial nitric oxide synthase (eNOS), procaspase-3, cleaved caspase-3, and Rho-associated coiled-coil protein kinase 1 (ROCK1) in trophoblasts and trophoblast-derived MPs were determined by Western blot. Trophoblasts from preeclamptic pregnancies released significantly more MPs than cells from uncomplicated pregnancies (P < 0.01). CoCl2-induced increase in MP release was associated with upregulation of caveolin-1 and downregulation of eNOS expression in trophoblasts (P < 0.05), which could be attenuated by 1,25(OH)2D3. Moreover, 1,25(OH)2D3 could also inhibit CoCl2-induced procaspase-3 cleavage and ROCK1 activation in trophoblasts. Consistently, CoCl2-induced upregulation of procaspase-3, cleaved caspase-3, and ROCK1 expression in trophoblast-derived MPs were also reduced in cells treated with 1,25(OH)2D3. Placental trophoblasts from preeclamptic pregnancies released more MP than cells from uncomplicated pregnancies. Oxidative stress-induced increase in MP shedding is associated with upregulation of caveolin-1 and downregulation of eNOS expression in placental trophoblasts. Inhibition of caspase-3 cleavage and ROCK1 activation, together with upregulation of eNOS expression, could be the potential cellular/molecular mechanism(s) of vitamin D protective effects on placental trophoblasts.

Vitamin D suppresses oxidative stress-induced microparticle release by human umbilical vein endothelial cells.

Endothelial microparticle (MP) release was increased in numerous cardiovascular diseases including preeclampsia. Oxidative stress is a potent inducer of endothelial dysfunction. In this study, we aimed to investigate if vitamin D could protect endothelial cells (ECs) from MP release induced by oxidative stress. Endothelial cell (from human umbilical vein) oxidative stress was induced by cultivation of cells under lowered oxygen condition (2%O2) for 48 h and cells cultured under standard condition (21%O2) served as control. 1,25(OH)2D3 was used as bioactive vitamin D. Using annexin-V as a marker of released MP assessed by flow cytometry and cytochrome c reduction assay to measure EC superoxide generation, we found that MP release and superoxide generation were significantly increased when cells were cultured under 2%O2, which could be significantly inhibited by 1,25(OH)2D3. To study the potential mechanisms of 1,25(OH)2D3 protective effects on ECs, EC expression of endothelial nitric oxide synthase (eNOS), p-eNOSSer1177, p-eNOSThr495, caveolin-1, extracellular signal-regulated kinase (ERK), p-ERK, Akt, p-AktSer473, Rho-associated coiled-coil protein kinase 1 (ROCK1), and vitamin D receptor were determined. Microparticle expression of eNOS and caveolin-1 were also determined. We found that under lowered oxygen condition, 1,25(OH)2D3 could upregulate EC eNOS, p-eNOSSer1177, and p-AktSer473 expression, but inhibit cleaved ROCK1 expression. The upregulatory and inhibitory effects induced by 1,25(OH)2D3 were dose dependent. Strikingly, we also found that oxidative stress-induced decrease in ratio of eNOS and caveolin-1 expression in MP could be attenuated when 1,25(OH)2D3 was present in culture. These results suggest that upregulation of eNOSSer1177 and AktSer473 phosphorylation and inhibition of ROCK1 cleavage in EC and modulation of eNOS and caveolin-1 expression in MP could be plausible mechanisms of vitamin D protective effects on ECs.

MiR-135a modulates tendon stem/progenitor cell senescence via suppressing ROCK1

Tendon stem/progenitor cell (TSPC) senescence may lead to age-related tendon disorders and impair tendon regeneration and replacement capacity in humans. However, the mechanisms governing TSPC aging and degeneration remain obscure. Recently, it has been reported that Rho-associated coiled-coil protein kinase 1 (ROCK1) might be a key player in TSPC aging process. miRNAs are also involved in cellular senescence. In this study, whether miRNAs modulate senescence of TSPCs through targeting ROCK1 was evaluated. We found that miR-135a, which directly binds to the 3′-untranslated region of ROCK1, is significantly downregulated in aged compared with young TSPCs. Overexpression of miR-135a in young TSPCs suppresses senescence, promotes proliferation, and induces migration and tenogenic differentiation, whereas suppression of miR-135a in aged TSPCs has the opposite effects. By gain-of-function and loss-of-function studies, we confirmed that ROCK1 mediates the effects of miR-135a in TSPCs. Taken together, our data suggest that miR-135a plays an important role in TSPC senescence via targeting ROCK1.

Gene Expression Profile During Testicular Development in Patients With SRY-negative 46,XX Testicular Disorder of Sex Development

To elucidate alternative pathways in testicular development, we attempted to clarify the genetic characteristics of SRY-negative XX testes. We previously reported 5 cases of SRY-negative 46,XX testicular disorders of sex development and demonstrated that coordinated expression of genes such as SOX9, SOX3, and DAX1 was associated with testicular development. We performed a case-control study between the aforementioned boy with 46,XX testicular disorders of sex development and an age-matched patient with hydrocele testis (46,XY). During their consecutive surgeries, testicular biopsy specimens were obtained. Genes with differential expression compared with XY testis were identified using polymerase chain reaction (PCR)-based subtractive hybridization and sequencing. For validation of differential gene expression, real-time RT-PCR was performed using gene-specific primers. The distribution of candidate proteins in the testicular tissue was clarified by immunohistochemistry in human and rodent specimens. Moreover, in vitro inhibitory assays were performed. We identified 13 upregulated and 7 downregulated genes in XX testis. Among the candidate genes, we focused on ROCK1 (Rho-associated, coiled-coil protein kinase 1) in the upregulated gene group, because high expression in XX testis was validated by real-time RT-PCR. ROCK1 protein was detected in germ cells, Leydig cells, and Sertoli cells by immunohistochemistry. Moreover, the addition of specific ROCK1 inhibitor to Sertoli cells decreased SOX9 gene expression. On the basis of in vitro inhibitory assay, it is suggested that ROCK1 phosphorylates and activates SOX9 in Sertoli cells. Testes formation might be initiated by an alternative signaling pathway attributed to ROCK1, not SRY, activation in XX testes.

Dexamethasone inhibits U937 cell adhesion via the down-regulation of ROCK1 activity.

To explore the effect of dexamethasone (DEX) on monocyte adhesion function and its underlying mechanism. The effects of DEX and fasudil on adhesion of cultured U937 monocytes to human umbilical vein endothelial cells (HUVEC) following stimulation with phorbol myristate acetate (PMA) were studied; Changes in the Rho-associated coiled-coil protein kinase 1 (ROCK1) protein content and activity were evaluated. DEX and fasudil significantly inhibited U937 cell adhesion rates under PMA stimulation and inhibited ROCK1 activity. Mifepristone (RU-486) and cycloheximide (CHX) did not alter these effects of DEX. DEX interferes with the adhesion function of U937 cells through the inhibition of ROCK1 activity.

THE EFFECT OF ROCK1 AND ROCK2 SILENCING BY SHRNA ON APOPTOSIS INDUCED BY HYPOXIA IN RAT CARDIOMYOCYTE

ObjectivesIt is well known that the apoptosis of cardiomyocyte involves the development of some cardiovascular diseases, such as heart failure, coronary heart disease and so on. Down-regulation of ROCK1 (Rho—associated...

Enhanced ROCK1 dependent contractility in fibroblast from chronic obstructive pulmonary disease patients

BackgroundDuring wound healing processes fibroblasts account for wound closure by adopting a contractile phenotype. One disease manifestation of COPD is emphysema which is characterized by destruction of alveolar walls and our hypothesis is that fibroblasts in the COPD lungs differentiate into a more contractile phenotype as a response to the deteriorating environment.MethodsBronchial (central) and parenchymal (distal) fibroblasts were isolated from lung explants from COPD patients (n = 9) (GOLD stage IV) and from biopsies from control subjects and from donor lungs (n = 12). Tissue-derived fibroblasts were assessed for expression of proteins involved in fibroblast contraction by western blotting whereas contraction capacity was measured in three-dimensional collagen gels.ResultsThe basal expression of rho-associated coiled-coil protein kinase 1 (ROCK1) was increased in both centrally and distally derived fibroblasts from COPD patients compared to fibroblasts from control subjects (p < 0.001) and (p < 0.01), respectively. Distally derived fibroblasts from COPD patients had increased contractile capacity compared to control fibroblasts (p < 0.01). The contraction was dependent on ROCK1 activity as the ROCK inhibitor Y27632 dose-dependently blocked contraction in fibroblasts from COPD patients. ROCK1-positive fibroblasts were also identified by immunohistochemistry in the alveolar parenchyma in lung tissue sections from COPD patients.ConclusionsDistally derived fibroblasts from COPD patients have an enhanced contractile phenotype that is dependent on ROCK1 activity. This feature may be of importance for the elastic dynamics of small airways and the parenchyma in late stages of COPD.

Mechanism of dexamethasone inhibiting U937 cell adhesion and phagocytose function

Objective To investigate the mechanism of dexamethasone (Dex) in inhibiting monocyte adhesion and phagocytose function. Methods Under the stimulation of phorbo1-12-myristate-13-acetate (PMA),U937 monocytes cultured in vitro were treated with Dex and Fasudil respectively.The adhesion rate of U937 monocles to human umbilical vein endothelial cells (HUVECs) and their phagocytic ability of India ink were studied.The protein content and activity of rho-associated coiled-coil protein kinase 1 ( ROCK1 ) as well as the effects of mifepristone and cycloheximide on Dex were determined.Results Both DEX and Fasudil could significantly inhibit the adhesion tate and phagocytosis of U937 cells stimulated by PMA and suppressed the activity of ROCK1.While mifepristone and cycloheximide could not alter these effects of DEX. Conclusion DEX interferes with the adhesion and phagocytosis function of U937 cells by inhibiting ROCKI activity. Key words: Dexamethasone; Phagocytose; Non-genomic effect

Communication between Cardiomyocytes and Cardiac Fibroblasts: Mechanistic Role of ROCK1 in Fibrotic Cardiac Remodeling

Study objectiveOur previous study found that Rho associated coiled‐coil protein kinase 1 (ROCK1) was cleaved into an active isoform ROCKΔ1 in failing human hearts. The study is to investigate the pathological consequence of ROCKΔ1 accumulated in heart and the associated molecular mechanism.Methods and ResultsWe generated transgenic mice expressing ROCKΔ1 in cardiomyocytes to mimic human heart disease. Mutant mice developed fibrotic cardiomyopathy with diastolic dysfunction. Transgenic heart displayed the activation of TGFβ1 and NF‐κB signaling. Treatment with Rho kinase inhibitor attenuated the phenotype. Cardiac fibroblasts were differentiated into myofibroblasts by co‐culturing with transgenic cardiomyocytes but not with wild‐type cardiomyocytes. Inhibitors of Rho kinase, TGFβR1 and NF‐κB diminished the process. A new cis‐regulatory element of TGFβ1, the serum response factor‐dependent TGFβ1 regulation, was responsible for Rho kinase‐mediated TGFβ1 signaling activation.ConclusionsThe results demonstrate that ROCKΔ1 is a novel and potent fibrogenic factor. Activation of TGFβ1 and NF‐κB signaling along with a subset of cytokines released from cardiomyocytes induces cardiac fibroblast maturation, facilitates collagen deposition and macrophage infiltration. Both pathways synergistically contribute to Rho kinase‐mediated pathological fibrosis.Supported by NIH R01HL102314.

Mechanism of fibrotic cardiomyopathy in mice expressing truncated Rho‐associated coiled‐coil protein kinase 1

We have previously found that in failing human hearts, Rho-associated coiled-coil protein kinase 1 (ROCK1) is processed by caspase-3 into an active isoform, ROCKΔ1. The purpose of the current investigation was to elucidate the pathological consequences of truncated ROCK1 accumulation in the heart, the associated molecular mechanism of ROCKΔ1-mediated cardiac phenotype, and the molecular signaling between Rho kinase activation in cardiomyocytes and extracellular matrix response. We generated transgenic mice expressing ROCKΔ1 in cardiomyocytes to mimic the situation observed in human heart disease, whereas an additional kinase-deficient mouse was generated as a control. The ROCKΔ1 transgenic mice developed fibrotic cardiomyopathy with diastolic dysfunction. Transgenic hearts displayed activated TGFβ1 and NF-κB signaling and a release of a subset of cytokines and were susceptible to angiotensin II stress. Treatment with a Rho kinase inhibitor attenuated the fibrotic phenotype. Cardiac fibroblasts differentiated into myofibroblasts when cocultured with transgenic cardiomyocytes but not with wild-type cardiomyocytes. Inhibitors of Rho kinase as well as TGFβR1 and NF-κB decreased these effects. The serum response factor-dependent TGFβ1 regulation was shown to be responsible for the Rho kinase-mediated activation of TGFβ1 signaling. We conclude that ROCKΔ1 is a novel fibrotic factor. Activation of TGFβ1 and NF-κB signaling contributes to the Rho kinase-mediated pathological fibrosis.

Activation of Rho-associated coiled-coil protein kinase 1 (ROCK-1) by caspase-3 cleavage plays an essential role in cardiac myocyte apoptosis

Rho-associated coiled-coil protein kinase 1 (ROCK-1) is a direct cleavage substrate of activated caspase-3, which is associated with heart failure. In the course of human heart failure, we found marked cleavage of ROCK-1 resulting in a 130-kDa subspecies, which was absent in normal hearts and in an equivalent cohort of patients with left ventricular assist devices. Murine cardiomyocytes treated with doxorubicin led to enhanced ROCK-1 cleavage and apoptosis, all of which was blocked by a caspase-3 inhibitor. In addition, a bitransgenic mouse model of severe cardiomyopathy, which overexpresses Gq protein and hematopoietic progenitor kinase-/germinal center kinase-like kinase, revealed the robust accumulation of the 130-kDa ROCK-1 cleaved fragment. This constitutively active ROCK-1 subspecies, when expressed in cardiomyocytes, led to caspase-3 activation, indicating a positive feed-forward regulatory loop. ROCK-1-dependent caspase-3 activation was coupled with the activation of PTEN and the subsequent inhibition of protein kinase B (Akt) activity, all of which was attenuated by siRNA directed against ROCK-1 expression. Similarly, ROCK-1-null mice (Rock-1(-/-)) showed a marked reduction in myocyte apoptosis associated with pressure overload. These data suggest an obligatory role for ROCK-1 cleavage in promoting apoptotic signals in myocardial hypertrophy and/or failure.