Targeting matrix stiffness-induced activation of retinal pigment epithelial cells through the RhoA/YAP pathway ameliorates proliferative vitreoretinopathy.

The purpose of this study was to investigate whether excessive extracellular matrix (ECM) deposition-induced mechanical matrix stiffness plays a key role in promoting retinal pigment epithelial (RPE) cell activation and the subsequent development of proliferative vitreoretinopathy (PVR). Human ARPE-19 cells were cultured on either 50 kappa (stiff) or 0.5 kappa (soft) gel-coated coverslips. Reverse and knockdown experiments were carried out to establish a model of matrix stiffness-induced activation in ARPE-19 cells in vitro. A PVR mouse model was established by the intravitreal injection of dispase. The effects of RhoA/YAP signalling blockade on matrix stiffness-induced ARPE-19 cell activation and PVR-induced retinal fibrosis were determined by using a combination of the Yes-associated protein (YAP) inhibitor verteporfin and the RhoA inhibitor C3 exoenzyme. Matrix stiffness stimulated YAP nuclear translocation and expression in ARPE-19 cells. The effect of YAP activation was dependent on F-actin cytoskeleton polymerization and RhoA activity, forming the RhoA/YAP signalling pathway. Upstream pharmacological blockade of RhoA by C3 exoenzyme or downstream blockade of YAP by verteporfin reduced the invasion, migration, and MMP expression of ARPE-19 cells and collagen gel contraction. Furthermore, blockade of RhoA/YAP signalling reduced PVR-induced retinal fibrogenesis and inhibited the TGF-ß/Smad pathway in vivo. RhoA/YAP signalling modulates matrix stiffness-induced activation of ARPE-19 cells. Targeting this signalling pathway could alleviate PVR-induced retinal fibrosis and suggests attractive novel therapeutic strategies for intervening in the progression of PVR.

The impact of culture dimensionality on behavioral epigenetic memory contributing to pluripotent state of iPS cells.

Three-dimensional (3D) culture platforms have been explored to establish physiologically relevant cell culture environment and permit expansion scalability; however, little is known about the mechanisms underlying the regulation of pluripotency of human induced pluripotent stem cells (hiPSCs). This study elucidated epigenetic modifications contributing to pluripotency of hiPSCs in response to 3D culture. Unlike two-dimensional (2D) monolayer cultures, 3D cultured cells aggregated with each other to form ball-like aggregates. 2D cultured cells expressed elevated levels of Rac1 and RhoA; however, Rac1 level was significantly lower while RhoA level was persisted in 3D aggregates. Compared with 2D monolayers, the 3D aggregates also exhibited significantly lower myosin phosphorylation. Histone methylation analysis revealed remarkable H3K4me3 upregulation and H3K27me3 maintenance throughout the duration of 3D culture; in addition, we observed the existence of naïve pluripotency signatures in cells grown in 3D culture. These results demonstrated that hiPSCs adapted to 3D culture through alteration of the Rho-Rho kinase-phospho-myosin pathway, influencing the epigenetic modifications and transcriptional expression of pluripotency-associated factors. These results may help design culture environments for stable and high-quality hiPSCs.

RhoA/ROCK pathway mediates the effect of oestrogen on regulating epithelial-mesenchymal transition and proliferation in endometriosis.

Endometriosis is a benign gynaecological disease appearing with pelvic pain, rising dysmenorrhoea and infertility seriously impacting on 10% of reproductive-age females. This research attempts to demonstrate the function and molecular mechanism of RhoA/ROCK pathway on epithelial-mesenchymal transition (EMT) and proliferation in endometriosis. The expression of Rho family was abnormally changed in endometriotic lesions; in particular, RhoA and ROCK1/2 were significantly elevated. Overexpression of RhoA in human eutopic endometrial epithelial cells (eutopic EECs) enhanced the cell mobility, epithelial-mesenchymal transition (EMT) and proliferation, and RhoA knockdown exhibited the opposite function. Oestrogen up-regulated the RhoA activity and expression of RhoA and ROCK1/2. RhoA overexpression reinforced the effect of oestrogen on promoting EMT and proliferation, and RhoA knockdown impaired the effect of oestrogen. oestrogen receptor α (ERα) was involved with the regulation of oestrogen on EMT and proliferation and up-regulated RhoA activity and expression of RhoA and ROCK1/2. The function of ERα was modulated by the change in RhoA expression. Furthermore, phosphorylated ERK that was enhanced by oestrogen and ERα promoted the protein expression of RhoA/ROCK pathway. Endometriosis mouse model revealed that oestrogen enhanced the size and weight of endometriotic lesions. The expression of RhoA and phosphorylated ERK in mouse endometriotic lesions was significantly elevated by oestrogen. We conclude that abnormal activated RhoA/ROCK pathway in endometriosis is responsible for the function of oestrogen/ERα/ERK signalling, which promoted EMT and proliferation and resulted in the development of endometriosis.

Acetyl-CoA carboxylase inhibition regulates microtubule dynamics and intracellular transport in cystic fibrosis epithelial cells.

The use of high-dose ibuprofen as an anti-inflammatory therapy in cystic fibrosis (CF) has been shown to be an effective intervention although use is limited due to potential adverse events. Identifying the mechanism of ibuprofen efficacy would aid in the development of new therapies that avoid these adverse events. Previous findings demonstrated that ibuprofen treatment restores the regulation of microtubule dynamics in CF epithelial cells through a 5'-adenosine monophosphate-activated protein kinase (AMPK)-dependent mechanism. The goal of this study is to define the AMPK pathway that leads to microtubule regulation. Here, it is identified that inhibition of acetyl-CoA carboxylase (ACC) is the key step in mediating the AMPK effect. ACC inhibition with 5-(tetradecyloxy)-2-furoic acid (TOFA) increases microtubule reformation rates in cultured and primary CF epithelial cells to wild-type (WT) rates. TOFA treatment also restores microtubule-dependent distribution of cholesterol and Rab7-positive organelles, as well as reduces expression of the proinflammatory signaling molecule RhoA to WT levels. ACC activation with citrate replicates these CF phenotypes in WT cells further supporting the role of AMPK signaling through ACC as a key mediator in CF cell signaling. It is concluded that ACC inhibition is the key step in the efficacy of AMPK activation at the cellular level and could represent a novel site of therapeutic intervention to address inflammation in CF.

The Role of the RhoA/ROCK Signaling Pathway in Mechanical Strain-Induced Scleral Myofibroblast Differentiation.

Purpose: Biomechanical properties changes and α-smooth muscle actin (α-SMA) overexpression are involved in myopia scleral remodeling. However, interactions between altered tissue biomechanics and cellular signaling that sustain scleral remodeling have not been well defined. We determine the mechanisms of mechanotransduction in the regulation of α-SMA expression during myopia scleral remodeling. Methods: Guinea pigs were used to establish a form-deprivation myopia (FDM) model. Protein profiles in myopic sclera were examined using tandem mass spectrometry. Ras homolog gene family member A (RhoA) and α-SMA expressions were confirmed using quantitative (q) RT-PCR and Western blotting. Scleral fibroblasts were cultured and subjected to 4% cyclic strain. Levels of RhoA, rho-associated protein kinase-2 (ROCK2), myocardin-related transcription factor-A (MRTF-A), serum response factor (SRF), and α-SMA were determined by qRT-PCR and Western blotting in groups with or without the RhoA siRNA or ROCK inhibitor Y27632. MRTF-A and α-SMA were evaluated by confocal immunofluorescent microscopy and myofibroblasts were enumerated using flow cytometry. Results: mRNA and protein levels of RhoA and α-SMA were significantly increased in the FDM eyes after 4 weeks of form-deprivation treatment. The 4% static strain increased expressions of RhoA, ROCK2, MRTF-A, SRF, and α-SMA as well as nuclear translocalization of MRTF-A in scleral fibroblasts compared to those without strain stimulation. Additionally, the percentage of myofibroblasts increased after strain stimulation. Conversely, inhibition of RhoA or ROCK2 reversed the strain-induced α-SMA expression and myofibroblast ratio. Conclusions: Mechanical strain activated RhoA signaling and scleral myofibroblast differentiation. Strain also mediated myofibroblast differentiation via the RhoA/ROCK2-MRTF-A/SRF pathway. These findings provided evidence for a mechanical strain-induced RhoA/ROCK2 pathway that may contribute to myopia scleral remodeling.

YAP and MRTF-A, transcriptional co-activators of RhoA-mediated gene expression, are critical for glioblastoma tumorigenicity.

The role of YAP (Yes-associated protein 1) and MRTF-A (myocardin-related transcription factor A), two transcriptional co-activators regulated downstream of GPCRs (G protein-coupled receptors) and RhoA, in the growth of glioblastoma cells and in vivo glioblastoma multiforme (GBM) tumor development was explored using human glioblastoma cell lines and tumor-initiating cells derived from patient-derived xenografts (PDX). Knockdown of these co-activators in GSC-23 PDX cells using short hairpin RNA significantly attenuated in vitro self-renewal capability assessed by limiting dilution, oncogene expression, and neurosphere formation. Orthotopic xenografts of the MRTF-A and YAP knockdown PDX cells formed significantly smaller tumors and were of lower morbidity than wild-type cells. In vitro studies used PDX and 1321N1 glioblastoma cells to examine functional responses to sphingosine 1-phosphate (S1P), a GPCR agonist that activates RhoA signaling, demonstrated that YAP signaling was required for cell migration and invasion, whereas MRTF-A was required for cell adhesion; both YAP and MRTF-A were required for proliferation. Gene expression analysis by RNA-sequencing of S1P-treated MRTF-A or YAP knockout cells identified 44 genes that were induced through RhoA and highly dependent on YAP, MRTF-A, or both. Knockdown of F3 (tissue factor (TF)), a target gene regulated selectively through YAP, blocked cell invasion and migration, whereas knockdown of HBEGF (heparin-binding epidermal growth factor-like growth factor), a gene selectively induced through MRTF-A, prevented cell adhesion in response to S1P. Proliferation was sensitive to knockdown of target genes regulated through either or both YAP and MRTF-A. Expression of TF and HBEGF was also selectively decreased in tumors from PDX cells lacking YAP or MRTF-A, indicating that these transcriptional pathways are regulated in preclinical GBM models and suggesting that their activation through GPCRs and RhoA contributes to growth and maintenance of human GBM.

Long non-coding RNA LOC554202 promotes laryngeal squamous cell carcinoma progression through regulating miR-31.

Laryngeal squamous cell carcinoma (LSCC) is one aggressive malignancy and accounts for 20% of all head and neck cancer. However, the role of LOC554202 in human LSCC remains unknown. The expression level of LOC554202 and miR-31 was detected in the LSCC tiussues by using qRT-PCR. Cell growth was measured by CCK-8 assay. Flow cytometry and matrigel-coated membrane was used to detect for cell cycle and invasion respectively. We indicated that lncRNA LOC554202 expression was overexpressed in LSCC tissues compared with the paired adjacent samples and higher LOC554202 expression was associated with the advanced stage. In addition, we demonstrated that the expression level of miR-31 was downregulated in LSCC tissues compared to the paired adjacent samples and lower miR-31 expression was correlated with the advanced stage. Moreover, the expression of miR-31 was negatively correlated with the expression of LOC554202 in LSCC tissues. Ectopic expression of LOC554202 promoted LSCC cell growth, cell cyle and cell invasion and overexpression of miR-31 inhibited LSCC cell growth, cell cyle and cell invasion. Elevated expression of LOC554202 suppressed miR-31 expression and promoted RhoA expression in LSCC cell, which was a direct target gene of miR-31. Furthermore, LOC554202 increased LSCC cell growth, cell cyle and cell invasion through suppressing miR-31 expression. These results suggested that LOC554202 acted as an oncogene in the development of LSCC.

Cobalt (II) ions and nanoparticles induce macrophage retention by ROS-mediated down-regulation of RhoA expression.

Histological assessments of synovial tissues from patients with failed CoCr alloy hip prostheses demonstrate extensive infiltration and accumulation of macrophages, often loaded with large quantities of particulate debris. The resulting adverse reaction to metal debris (ARMD) frequently leads to early joint revision. Inflammatory response starts with the recruitment of immune cells and requires the egress of macrophages from the inflamed site for resolution of the reaction. Metal ions (Co2+ and Cr3+) have been shown to stimulate the migration of T lymphocytes but their effects on macrophages motility are still poorly understood. To elucidate this, we studied in vitro and in vivo macrophage migration during exposure to cobalt and chromium ions and nanoparticles. We found that cobalt but not chromium significantly reduces macrophage motility. This involves increase in cell spreading, formation of intracellular podosome-type adhesion structures and enhanced cell adhesion to the extracellular matrix (ECM). The formation of podosomes was also associated with the production and activation of matrix metalloproteinase-9 (MMP9) and enhanced ECM degradation. We showed that these were driven by the down-regulation of RhoA signalling through the generation of reactive oxygen species (ROS). These novel findings reveal the key mechanisms driving the wear/corrosion metallic byproducts-induced inflammatory response at non-toxic concentrations. STATEMENT OF SIGNIFICANCE: Adverse tissue responses to metal wear and corrosion products from CoCr alloy implants remain a great challenge to surgeons and patients. Macrophages are the key regulators of these adverse responses to the ions and debris generated. We demonstrated that cobalt, rather than chromium, causes macrophage retention by restructuring the cytoskeleton and inhibiting cell migration via ROS production that affects Rho Family GTPase. This distinctive effect of cobalt on macrophage behaviour can help us understand the pathogenesis of ARMD and the cellular response to cobalt based alloys, which provide useful information for future implant design and biocompatibility testing.

Anti-nerve growth factor antibody improves airway hyperresponsiveness by down-regulating RhoA.

BACKGROUND: The pathogenesis of asthma is complex and continues to be considered as a challenging subject. Some studies have shown that nerve growth factor (NGF) participates in the pathogenesis of asthma, but the mechanism of airway contraction caused by NGF is still unclear. OBJECTIVE: Our aim was to discuss the effect of anti-NGF antibody on RhoA expression, and further explore the role of NGF in airway hyperresponsiveness (AHR). METHODS: Thirty female BALB/c mice were divided into three groups randomly: control group (group C, n = 10), asthma group (group A, n = 10) and anti-NGF antibody intervention group (group N, n = 10). The asthmatic mice were stimulated by OVA suspension, the intervention mice were given nasal instillation of anti-NGF antibody before the stimulation. Airway responsiveness, eosinophils, IL-13, IFN-γ were measured. The protein expression and mRNA level of NGF and RhoA were detected by immunohistochemical and Real Time-PCR (RT-PCR) analyses. RESULTS: Airway responsiveness, eosinophils and IL-13 levels in group A were significantly increased compare with the other groups, and significantly decreased in group N than those in group A. IFN-γ level was significantly reduced in group A and increased in group N. Immunohistochemistry and RT-PCR analyses showed that the protein expression and mRNA level of NGF and RhoA were significantly increased in group A and significantly decreased in group N. CONCLUSION: NGF participates in the pathogenesis of asthma in mice. Anti-NGF antibody can inhibit airway inflammation and alleviate AHR by down-regulating the protein expression and mRNA level of RhoA.

TGF-ß Stimulates Expression of Chondroitin Polymerizing Factor in Nucleus Pulposus Cells Through the Smad3, RhoA/ROCK1, and MAPK Signaling Pathways.

The enzyme chondroitin polymerizing factor (ChPF) is primarily involved in extension of the chondroitin sulfate backbone required for the synthesis of sulfated glycosaminoglycan (sGAG). Transforming growth factor beta (TGF-ß) upregulates sGAG synthesis in nucleus pulposus cells; however, the mechanisms mediating this induction are incompletely understood. Our study demonstrated that ChPF expression was negatively correlated with the grade of degenerative intervertebral disc disease. Treatment of nucleus pulposus cells with TGF-ß induced ChPF expression and enhanced Smad2/3, RhoA/ROCK activation, and the JNK, p38, and ERK1/2 MAPK signaling pathways. Selective inhibitors of Smad2/3, RhoA or ROCK1/2, and knockdown of Smad3 and ROCK1 attenuated ChPF expression and sGAG synthesis induced by TGF-ß. In addition, we showed that RhoA/ROCK1 signaling upregulated ChPF via activation of the JNK pathway but not the p38 and ERK1/2 signaling pathways. Moreover, inhibitors of JNK, p38 and ERK1/2 activity also blocked ChPF expression and sGAG synthesis induced by TGF-ß in a Smad3-independent manner. Collectively, our data suggest that TGF-ß stimulated the expression of ChPF and sGAG synthesis in nucleus pulposus cells through Smad3, RhoA/ROCK1 and the three MAPK signaling pathways. J. Cell. Biochem. 119: 566-579, 2018. © 2017 Wiley Periodicals, Inc.

MicroRNA-200b suppresses the invasion and migration of hepatocellular carcinoma by downregulating RhoA and circRNA_000839.

MicroRNAs could mediate the targeted coding gene and the targeted non-coding RNA to form endogenous competition, which have an important regulatory role in tumorigenesis of many types of cancer, including hepatocellular carcinoma. The goal of this study was to characterize the role of miR-200b in the pathogenesis of hepatocellular carcinoma. We identified miR-200b that was predicted to regulate RhoA and circ_000839. Our data establish that miR-200b is expressed at a relatively low level in hepatocellular carcinoma ( p < 0.001). RhoA and circ_000839 are expressed at a relatively high level in hepatocellular carcinoma ( p < 0.001, respectively). Our mechanistic data indicate that RhoA is a direct target of miR-200b ( p < 0.001), binding of which affects the expression of invasion and migration in hepatocellular carcinoma cell lines ( p < 0.05). And correlation analysis showed that miR-200b was inversely correlated with RhoA and circ_000839 ( p = 0.012, p = 0.002, respectively), while RhoA was positively correlated with circ_000839 ( p < 0.001). Taken together, our data suggest that miR-200b could mediate RhoA gene and circ_000839 to form endogenous competition. And this is a direction for the association study of miR-200b and RhoA in the future.

RhoA/ROCK pathway inhibition by fasudil suppresses the vasculogenic mimicry of U2OS osteosarcoma cells in vitro.

GTPase RhoA and its downstream Rho-associated coiled-coil-containing protein kinases (ROCKs) are frequently overexpressed in human cancers. Inhibition of the RhoA/ROCK pathway blocks angiogenesis mediated by the vascular endothelial growth factor, which led us to investigate the role of this pathway in vasculogenic mimicry (VM) - a process by which aggressive cancer cells form vessel-like structures that provide adequate blood supply for tumor growth. We showed that the expression of RhoA and its effector kinases ROCK1/2 was much higher in human osteosarcoma (OS) tissues and the human OS cell line U2OS than in nontumorous tissues and cell line hFOB 1.19 using western blot analysis and real-time PCR. Inhibition of the RhoA/ROCK signaling pathway by the pharmacological inhibitor fasudil reduced vascular-like channels of U2OS cells in Matrigel. Furthermore, we used rhodamine-phalloidin immunofluorescence, wound healing assay, and transwell migration assay to examine the effect of fasudil on tumor cell plasticity and motility, both of which play key roles in VM formation. Finally, we explored the underlying mechanisms of fasudil-induced VM destruction. In this context, we showed that the RhoA/ROCK signaling pathway is a novel regulator in VM of U2OS OS cells and suggest that fasudil in conjunction with established treatments may present a novel therapeutic strategy for OS.

Anti-cancer effect of danshen and dihydroisotanshinone I on prostate cancer: targeting the crosstalk between macrophages and cancer cells via inhibition of the STAT3/CCL2 signaling pathway.

Danshen (Salvia miltiorrhiza Bunge) is widely used in traditional Chinese medicine. In our study, the in vivo protective effect of danshen in prostate cancer patients was validated through data from the National Health Insurance Research Database in Taiwan. In vitro, we discovered that dihydroisotanshinone I (DT), a bioactive compound present in danshen, can inhibit the migration of both androgen-dependent and androgen-independent prostate cancer cells. In addition, we noted that DT substantially inhibited the migratory ability of prostate cancer cells in both a macrophage-conditioned medium and macrophage/prostate cancer coculture medium. Mechanistically, DT both diminished the ability of prostate cancer cells to recruit macrophages and reduced the secretion of chemokine (C-C motif) ligand 2 (CCL2) from both macrophages and prostate cancer cells in a dose-dependent manner. Moreover, DT inhibited the protein expression of p-STAT3 and decreased the translocation of STAT3 into nuclear chromatin. DT also suppressed the expression of tumor epithelial-mesenchymal transition genes, including RhoA and SNAI1. In conclusion, danshen can prolong the survival rate of prostate cancer patients in Taiwan. Furthermore, DT can inhibit the migration of prostate cancer cells by interrupting the crosstalk between prostate cancer cells and macrophages via the inhibition of the CCL2/STAT3 axis. These results may provide the basis for a new therapeutic approach toward the treatment of prostate cancer progression.

Inhibition of KLF5-Myo9b-RhoA Pathway-Mediated Podosome Formation in Macrophages Ameliorates Abdominal Aortic Aneurysm.

RATIONALE: Abdominal aortic aneurysms (AAAs) are characterized by pathological remodeling of the aortic wall. Although both increased Krüppel-like factor 5 (KLF5) expression and macrophage infiltration have been implicated in vascular remodeling, the role of KLF5 in macrophage infiltration and AAA formation remains unclear. OBJECTIVE: To determine the role of KLF5 in AAA formation and macrophage infiltration into AAAs. METHODS AND RESULTS: KLF5 expression was significantly increased in human AAA tissues and in 2 mouse models of experimental AAA. Moreover, in myeloid-specific Klf5 knockout mice (myeKlf5-/- mice), macrophage infiltration, medial smooth muscle cell loss, elastin degradation, and AAA formation were markedly decreased. In cell migration and time-lapse imaging analyses, the migration of murine myeKlf5-/- macrophages was impaired, and in luciferase reporter assays, KLF5 activated Myo9b (myosin IXB) transcription by direct binding to the Myo9b promoter. In subsequent coimmunostaining studies, Myo9b was colocalized with filamentous actin, cortactin, vinculin, and Tks5 in the podosomes of phorbol 12,13-dibutyrate-treated macrophages, indicating that Myo9b participates in podosome formation. Gain- and loss-of-function experiments showed that KLF5 promoted podosome formation in macrophages by upregulating Myo9b expression. Furthermore, RhoA-GTP levels increased after KLF5 knockdown in macrophages, suggesting that KLF5 lies upstream of RhoA signaling. Finally, Myo9b expression was increased in human AAA tissues, located in macrophages, and positively correlated with AAA size. CONCLUSIONS: These data are the first to indicate that KLF5-dependent regulation of Myo9b/RhoA is required for podosome formation and macrophage migration during AAA formation, warranting consideration of the KLF5-Myo9b-RhoA pathway as a therapeutic target for AAA treatment.

Effect of curcumin and paclitaxel on breast carcinogenesis.

Global cancer burden increased to 14.1 million new cases in 2012; and breast cancer is the most common cancer in women worldwide, with nearly 1.7 million new cases diagnosed in 2012. Curcumin is the major bioactive ingredient extracted from the rhizome of the plant Curcuma longa (turmeric). Paclitaxel is a microtubule-stabilizing agent originally isolated from the bark of Taxus brevifolia. Curcumin and paclitaxel were evaluated with two human breast cancer cell lines as the luminal MCF-7 and the basal-like MDA-MB-231 that are either positive or negative for hormonal receptors estrogen receptor, progesterone receptor and HER2, respectively. Results indicated that curcumin combined with paclitaxel decreased c-Ha-Ras, Rho-A, p53 and Bcl-xL gene expression in comparison to control and substances alone in MCF-7 cell line. These two substances alone and combined decreased gene expression of Bcl-2 and NF-κB. However, CCND1 increased when both substances were combined in MCF-7 cells. Such substances decreased Bcl-2 and increased Bax protein expression. However, curcumin alone decreased IκBα and Stat-3 gene expression. Paclitaxel alone and combined increased IκBα and Stat-3. Curcumin alone and combined with paclitaxel increased p53, Bid, caspase-3, caspase-8 and Bax gene expression in MDA-MB-231, whereas Bcl-xL decreased such expression in MDA-MB-231 cells. When paclitaxel and curcumin were combined the expression of Bcl-2 protein was decreased. However, either substance alone and combined increased Bax protein expression corroborating the apoptotic effect of these substances. It can be concluded that curcumin may be of considerable value in synergistic therapy of breast cancer reducing the associated toxicity with use of drugs.

RhoA/rock signaling mediates peroxynitrite-induced functional impairment of Rat coronary vessels.

BACKGROUND: Diabetes-induced vascular dysfunction may arise from reduced nitric oxide (NO) availability, following interaction with superoxide to form peroxynitrite. Peroxynitrite can induce formation of 3-nitrotyrosine-modified proteins. RhoA/ROCK signaling is also involved in diabetes-induced vascular dysfunction. The study aimed to investigate possible links between Rho/ROCK signaling, hyperglycemia, and peroxynitrite in small coronary arteries. METHODS: Rat small coronary arteries were exposed to normal (NG; 5.5 mM) or high (HG; 23 mM) D-glucose. Vascular ring constriction to 3 mM 4-aminopyridine and dilation to 1 µM forskolin were measured. Protein expression (immunohistochemistry and western blot), mRNA expression (real-time PCR), and protein activity (luminescence-based G-LISA and kinase activity spectroscopy assays) of RhoA, ROCK1, and ROCK2 were determined. RESULTS: Vascular ring constriction and dilation were smaller in the HG group than in the NG group (P < 0.05); inhibition of RhoA or ROCK partially reversed the effects of HG. Peroxynitrite impaired vascular ring constriction/dilation; this was partially reversed by inhibition of RhoA or ROCK. Protein and mRNA expressions of RhoA, ROCK1, and ROCK2 were higher under HG than NG (P < 0.05). This HG-induced upregulation was attenuated by inhibition of RhoA or ROCK (P < 0.05). HG increased RhoA, ROCK1, and ROCK2 activity (P < 0.05). Peroxynitrite also enhanced RhoA, ROCK1, and ROCK2 activity; these actions were partially inhibited by 100 µM urate (peroxynitrite scavenger). Exogenous peroxynitrite had no effect on the expression of the voltage-dependent K+ channels 1.2 and 1.5. CONCLUSIONS: Peroxynitrite-induced coronary vascular dysfunction may be mediated, at least in part, through increased expressions and activities of RhoA, ROCK1, and ROCK2.

Arhgef1 negatively regulates neurite outgrowth through activation of RhoA signaling pathways.

Neurite outgrowth is essential for the establishment of functional neuronal connections during brain development. This study identifies that Arhgef1 is predominantly expressed in early neuronal developmental stages and negatively regulates neurite outgrowth. Knockdown of Arhgef1 in either Neuro-2a cells or primary cortical neurons leads to excess growth of neurites, whereas overexpression of Arhgef1 prominently restricts neurite formation. Arhgef1 strongly activates RhoA activity while concomitantly inhibits Rac1 and Cdc42 activities. Pharmacological blockade of RhoA activity restores normal neurite outgrowth in Arhgef1-overexpressed neurons. Importantly, Arhgef1 promotes F-actin polymerization in neurons, probably through inhibiting the activity of the actin-depolymerizing factor cofilin. Collectively, these findings reveal that Arhgef1 functions as a negative regulator of neurite outgrowth through regulating RhoA-cofilin pathway and actin dynamics.

p120 Catenin-Mediated Stabilization of E-Cadherin Is Essential for Primitive Endoderm Specification.

E-cadherin-mediated cell-cell adhesion is critical for naive pluripotency of cultured mouse embryonic stem cells (mESCs). E-cadherin-depleted mESC fail to downregulate their pluripotency program and are unable to initiate lineage commitment. To further explore the roles of cell adhesion molecules during mESC differentiation, we focused on p120 catenin (p120ctn). Although one key function of p120ctn is to stabilize and regulate cadherin-mediated cell-cell adhesion, it has many additional functions, including regulation of transcription and Rho GTPase activity. Here, we investigated the role of mouse p120ctn in early embryogenesis, mESC pluripotency and early fate determination. In contrast to the E-cadherin-null phenotype, p120ctn-null mESCs remained pluripotent, but their in vitro differentiation was incomplete. In particular, they failed to form cystic embryoid bodies and showed defects in primitive endoderm formation. To pinpoint the underlying mechanism, we undertook a structure-function approach. Rescue of p120ctn-null mESCs with different p120ctn wild-type and mutant expression constructs revealed that the long N-terminal domain of p120ctn and its regulatory domain for RhoA were dispensable, whereas its armadillo domain and interaction with E-cadherin were crucial for primitive endoderm formation. We conclude that p120ctn is not only an adaptor and regulator of E-cadherin, but is also indispensable for proper lineage commitment.

Rapamycin inhibits epithelial-to-mesenchymal transition of peritoneal mesothelium cells through regulation of Rho GTPases.

Epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) is a key process of peritoneal fibrosis. Rapamycin has been previously shown to inhibit EMT of PMCs and prevent peritoneal fibrosis. In this study, we investigated the undefined molecular mechanisms by which rapamycin inhibits EMT of PMCs. To define the protective effect of rapamycin, we initially used a rat PD model which was daily infused with 20 mL of 4.25% high glucose (HG) dialysis solution for 6 weeks to induce fibrosis. The HG rats showed decreased ultrafiltration volume and obvious fibroproliferative response, with markedly increased peritoneal thickness and higher expression of α-smooth muscle actin (α-SMA) and transforming growth factor-ß1. Rapamycin significantly ameliorated those pathological changes. Next, we treated rat PMCs with HG to induce EMT and/or rapamycin for indicated time. Rapamycin significantly inhibited HG-induced EMT, which manifests as increased expression of α-SMA, fibronectin, and collagen I, decreased expression of E-cadherin, and increased mobility. HG increased the phosphorylation of PI3K, Akt, and mTOR. Importantly, rapamycin inhibits the RhoA, Rac1, and Cdc42 activated by HG. Moreover, rapamycin repaired the pattern of F-actin distribution induced by HG, reducing the formation of stress fiber, focal adhesion, lamellipodia, and filopodia. Thus, rapamycin shows an obvious protective effect on HG-induced EMT, by inhibiting the activation of Rho GTPases (RhoA, Rac1, and Cdc42).

ARHGAP10, downregulated in ovarian cancer, suppresses tumorigenicity of ovarian cancer cells.

Rho GTPase-activating proteins (RhoGAPs) are implicated in the development and progression of ovarian cancer. ARHGAP10 is a member of RhoGAP proteins and inactivates Cdc42 by converting GTP-bound form to GDP-bound form. Here, we aimed to evaluate ARHGAP10 expression profile and functions in ovarian cancer. The decreased expression of ARHGAP10 was found in 77.3% (58/75) of ovarian cancer tissues, compared with their non-tumorous counterparts. Furthermore, overall survival in ovarian cancer patients with higher expression of ARHGAP10 was longer than those with lower expression. Ectopic expression of ARHGAP10 in two ovarian cancer cell lines with lower expression of ARHGAP10 (A2780 and HO-8910) dramatically suppressed cell proliferation in vitro. In nude mice, its stable overexpression significantly inhibited the tumorigenicity of A2780 cells. We further demonstrated that overexpression of ARHGAP10 significantly inhibited cell adhesion, migration and invasion, resulted in cell arrest in G1 phase of cell cycle and a significant increase of apoptosis. Moreover, ARHGAP10 interacted with Cdc42 and overexpression of ARHGAP10 inhibited the activity of Cdc42 in A2780 cells. Gene set enrichment analysis on The Cancer Genome Atlas dataset showed that KEGG cell cycle, replication and base excision repair (BER) pathways were correlatively with the ARHGAP10 expression, which was further confirmed in ovarian cancer cells by western blotting. Hence, ARHGAP10 may serve as a tumor suppressor through inactivating Cdc42, as well as inhibiting cell cycle, replication and BER pathways. Our data suggest an important role of ARHGAP10 in the molecular etiology of cancer and implicate the potential application of ARHGAP10 in cancer therapy.