The Effect of Sustained Inflammation on Hepatic Mevalonate Pathway Results in Hyperglycemia.

Control of plasma glucose level is essential to organismal survival. Sustained inflammation has been implicated in control of glucose homeostasis in cases of infection, obesity, and type 2 diabetes; however, the precise role of inflammation in these complex disease states remains poorly understood. Here, we find that sustained inflammation results in elevated plasma glucose due to increased hepatic glucose production. We find that sustained inflammation suppresses CYP7A1, leading to accumulation of intermediate metabolites at the branch point of the mevalonate pathway. This results in prenylation of RHOC, which is concomitantly induced by inflammatory cytokines. Subsequent activation of RHO-associated protein kinase results in elevated plasma glucose. These findings uncover an unexpected mechanism by which sustained inflammation alters glucose homeostasis.

Elevated expression of wildtype RhoC promotes ErbB2- and Pik3ca-induced mammary tumor formation.

Copy number gains in genes coding for Rho activating exchange factors as well as losses affecting genes coding for RhoGAP proteins are common in breast cancer (BC), suggesting that elevated Rho signaling may play an important role. Extra copies and overexpression of RHOC also occur, although a role for RhoC overexpression in driving tumor formation has not been assessed in vivo. To this end, we report on the development of a Rosa26 (R26)-targeted Cre-conditional RhoC overexpression mouse (R26RhoC). This mouse was crossed to two models for ERBB2/NEU+ breast cancer: one based on expression of an oncogenic ErbB2/Neu cDNA downstream of the endogenous ErbB2 promoter (FloxNeoNeuNT), the other, a metastatic model that is based on high-level expression from MMTV regulatory elements (NIC). RhoC overexpression dramatically enhanced mammary tumor formation in FloxNeoNeuNT mice but showed a more subtle effect in the NIC line, which forms multiple mammary tumors after a very short latency. RhoC overexpression also enhanced mammary tumor formation in an activated Pik3ca model for breast cancer (Pik3caH1047R). The transforming effect of RhoC was associated with epithelial/mesenchymal transition (EMT) in ErbB2/NeuNT and Pik3caH1047R systems. Thus, our study reveals the importance of elevated wildtype Rho protein expression as a driver of breast tumor formation and highlights the significance of Copy Number Abberations that affect Rho signalling.

Investigating Ras homolog gene family member C (RhoC) and Ki67 expression following external beam radiation therapy show increased RhoC expression in relapsing prostate cancer xenografts.

Ras homolog gene family member C (RhoC) is a GTPase involved in cell migration, implicated in epithelial-mesenchymal transition and treatment resistance and metastasis of cancer. For example, RhoC has been shown to be involved in resistance to radiation in cervical carcinoma. Here, the effect of X-ray irradiation on RhoC expression in prostate cancer (PCa) xenografts was investigated in both xenografts in regression and relapse. Male BALB/cAnNRj-Foxn1nu/nu mice were inoculated with 4-6 million LNCaP-FGC cells and established xenografts were irradiated with X-rays (200 kV, 1 Gymin-1), 5, 10 or 15 Gy using a Gulmay Medical X-ray system. Expression of RhoC and Ki67, a known proliferation marker, was investigated in xenografts, given 15 Gy, 7 days (midst response as measured by size) or 3 weeks (relapse) post irradiation. Staining was quantified using the Halo software (v2.3.2089.34) with the Indica Labs - cytonuclear v1.6 algorithm. RhoC and Ki67 staining was divided into weak, medium, and strong staining and the percentage of cells stained, single and dual staining, was quantified. The HALO software was further used to classify the tissue in each section so that analysis of RhoC and Ki67 expression in cancer cells, stroma and necrotic areas could be done separately. The results showed that RhoC expression in cancer and stroma cells was significantly higher in relapsed xenografts than in those in regression. This was not seen for Ki67 staining, where the percentage of stained cells were the same in regressing and relapsing tumors. RhoC could be a useful biomarker to confirm relapse following external beam radiation therapy.

[Trametenolic acid inhibits migration and invasion of hepatocellular carcinoma HepG2.2.15 cells via RhoC/ROCK1 pathway].

This study investigated the effect of trametenolic acid(TA) on the migration and invasion of human hepatocellular carcinoma HepG2.2.15 cells by using Ras homolog gene family member C(RhoC) as the target and probed into the mechanism, aiming to provide a basis for the utilization of TA. The methyl thiazolyl tetrazolium(MTT) assay was employed to examine the proliferation of HepG2.2.15 cells exposed to TA, and scratch and Transwell assays to examine the cell migration and invasion. The pull down assay was employed to determine the impact of TA on RhoC GTPase activity. Western blot was employed to measure the effect of TA on the transport of RhoC from cytoplasm to cell membrane and the expression of RhoC/Rho-associated kinase 1(ROCK1)/myosin light chain(MLC)/matrix metalloprotease 2(MMP2)/MMP9 pathway-related proteins. RhoC was over-expressed by transient transfection of pcDNA3.1-RhoC. The changes of F-actin in the cytoskeleton were detected by Laser confocal microscopy. In addition, the changes of cell migration and invasion, expression of proteins in the RhoC/ROCK1/MLC/MMP2/MMP9 pathway, and RhoC GTPase activity were detected. The subcutaneously transplanted tumor model of BALB/c nude mice and the low-, medium-, and high-dose(40, 80, and 120 mg·kg~(-1), respectively) TA groups were established and sorafenib(20 mg·kg~(-1)) was used as the positive control. The tumor volume and weight in each group were measured, and the expression of related proteins in the tumor tissue was determined by Western blot. The results showed that TA inhibited the proliferation of HepG2.2.15 cells in a concentration-dependent manner, with the IC_(50) of 66.65 and 23.09 µmol·L~(-1) at the time points of 24 and 48 h, respectively. The drug administration groups had small tumors with low mass. The tumor inhibition rates of sorafenib and low-, medium-and high-dose TA were 62.23%, 26.48%, 55.45%, and 62.36%, respectively. TA reduced migrating and invading cells and inhibited RhoC protein expression and RhoC GTPase activity in a concentration-dependent manner, dramatically reducing RhoC and membrane-bound RhoC GTPase. The expression of ROCK1, MLC, p-MLC, MMP2, and MMP9 downstream of RhoC can be significantly inhibited by TA, as confirmed in both in vitro and in vivo experiments. After HepG2.2.15 cells were transfected with pcDNA3.1-RhoC to overexpress RhoC, TA down-regulated the protein levels of RhoC, ROCK1, MLC, p-MLC, MMP2, and MMP9 and decreased the activity of RhoC GTPase, with the inhibition level comparable to that before overexpression. In summary, TA can inhibit the migration and invasion of HepG2.2.15 cells. It can inhibit the RhoC/ROCK1/MLC/MMP2/MMP9 signaling pathway by suppressing RhoC GTPase activity and down-regulating RhoC expression. This study provides a new idea for the development of autophagy modulators targeting HSP90α to block the proliferation and inhibit the invasion and migration of hepatocellular carcinoma cells via multiple targets of active components in traditional Chinese medicines.

Silencing of RhoC induces macrophage M1 polarization to inhibit migration and invasion in colon cancer via regulating the PTEN/FOXO1 pathway.

Ras homologue family member C (RhoC) is an oncogene in diverse types of human cancers, whereas its regulatory mechanisms involving macrophage polarization is rarely investigated. This study is designed to explore the regulatory role of RhoC in colon cancer and the underlying molecular mechanisms involving macrophage polarization. We detected RhoC expression by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, and analysed the biological function of RhoC knockdown in CC cells by the MTT, wound healing and transwell assay. Macrophage polarization-associated markers, genes associated with migration, phosphatase and tensin homologue (PTEN) and forkhead box O (FOXO) were determined by qRT-PCR and western blot. The xenograft tumour mouse model was used to assess the role of RhoC in vivo. RhoC is highly expressed in CC cells. The cell viability, invasion and migration abilities of CC cells were reduced by knockdown of RhoC. RhoC knockdown promoted M1 polarization, inhibited M2 polarization and decreased levels of genes associated with migration (matrix metalloproteinase-2 and matrix metalloproteinase-9). Silencing of RhoC inhibited tumour growth and expression of genes associated with migration in the xenografted model. In addition, silencing of RhoC promoted PTEN/FOXO1 expression, and PTEN inhibitor (SF1670) reversed the inhibitory effects of RhoC silencing. We demonstrated that silencing of RhoC reduced CC cells invasion and migration, and tumour growth by suppressing M2 macrophage polarization via regulating the PTEN/FOXO1 pathway.

Colonoscopy Combined with Me-Nbi to Observe the Canceration Characteristics of Colon Polyps and the Correlation of RHOC Protein Expression.

The current study was carried out to analyze the characteristics of colon polyps canceration observed by colonoscopy combined with ME-NBI (Magnifying Endoscopy combined with Narrow-Band Imaging) and its correlation with RhoC (Ras homolog gene family, member C) protein expression. For this purpose, A total of 300 patients with colorectal polyps and cancerous lesions (192 colorectal polyps and 200 cancerous lesions) who were treated in the digestive endoscopy room of the hospital and underwent colonoscopy were selected, and they were divided into polyp group and malignant lesion according to the diagnosis results. groups, 150 cases in each group. There were 75 patients with non-adenomatous polyps and 75 patients with adenomatous polyps in the polyp group; 75 patients with high-grade neoplasia and cancerous changes in the malignant group. The microvascular structure and surface structure of the lesions were observed by colonoscopy, and the correlation between microvascular morphological characteristics and RhoC protein expression was analyzed. Results showed that the probability of positive RhoC protein expression in the polyp group was significantly lower than that in the malignant transformation group, and the difference was statistically significant (P<0.05). In the malignant transformation group, the positive rate of RhoC expression in mucosal and submucosal superficial infiltration of 150 patients with colon polyp carcinoma was lower than that in submucosal deep infiltration, and the difference was statistically significant (P<0.05). NICE (National Institute for Clinical Excellence) type 2 was diagnosed as colorectal superficial submucosal The sensitivity, specificity, and accuracy of colorectal submucosal invasion were 73.1%, 84.6%, and 83.2%, respectively; the sensitivity, specificity, and accuracy of NICE type 3 in diagnosing colorectal submucosal invasion were 74.6%, 96.8%, and 92.7%, respectively. . Type 2 and type 3 lesions with cancerous features in NICE classification were correlated with the expression of RhoC protein (P<0.05). In conclusion, NICE classification under colonoscopy combined with magnifying colonoscopy has a good effect on colorectal lesions. Differential diagnostic value, RhoC protein is highly expressed in colon cancer and is closely related to the occurrence of colon cancer and the depth of lesion invasion. With the progression of colorectal adenomas, the expression of RhoC protein in the lesions gradually increased.

RhoC in association with TET2/WDR5 regulates cancer stem cells by epigenetically modifying the expression of pluripotency genes.

Emerging evidence illustrates that RhoC has divergent roles in cervical cancer progression where it controls epithelial to mesenchymal transition (EMT), migration, angiogenesis, invasion, tumor growth, and radiation response. Cancer stem cells (CSCs) are the primary cause of recurrence and metastasis and exhibit all of the above phenotypes. It, therefore, becomes imperative to understand if RhoC regulates CSCs in cervical cancer. In this study, cell lines and clinical specimen-based findings demonstrate that RhoC regulates tumor phenotypes such as clonogenicity and anoikis resistance. Accordingly, inhibition of RhoC abrogated these phenotypes. RNA-seq analysis revealed that RhoC over-expression resulted in up-regulation of 27% of the transcriptome. Further, the Infinium MethylationEPIC array showed that RhoC over-expressing cells had a demethylated genome. Studies divulged that RhoC via TET2 signaling regulated the demethylation of the genome. Further investigations comprising ChIP-seq, reporter assays, and mass spectrometry revealed that RhoC associates with WDR5 in the nucleus and regulates the expression of pluripotency genes such as Nanog. Interestingly, clinical specimen-based investigations revealed the existence of a subset of tumor cells marked by RhoC+/Nanog+ expression. Finally, combinatorial inhibition (in vitro) of RhoC and its partners (WDR5 and TET2) resulted in increased sensitization of clinical specimen-derived cells to radiation. These findings collectively reveal a novel role for nuclear RhoC in the epigenetic regulation of Nanog and identify RhoC as a regulator of CSCs. The study nominates RhoC and associated signaling pathways as therapeutic targets.

LACTB suppresses migration and invasion of glioblastoma via downregulating RHOC/Cofilin signaling pathway.

Glioblastoma (GBM) is the most malignant tumor in human brain. High invasiveness of this tumor is the main reason causing treatment failure and recurrence. Previous study has found that LACTB is a novel tumor suppressor in breast cancer. Moreover, the function of LACTB in other tumors and mechanisms involving LACTB were also reported. However, the role and relevant mechanisms of LACTB in GBM invasion remains to be revealed. Our aim is to investigate the role LACTB in GBM migration and invasion. We found that LACTB was downregulated in gliomas compared to normal brain tissues. Overexpression of LACTB suppressed migration and invasion of LN229 and U87 cell lines. Mechanistically, LACTB overexpression downregulated the mesenchymal markers. Moreover, LACTB overexpression downregulated the expression of RHOC and inhibited RHOC/Cofilin signaling pathway. The study suggests that LACTB suppresses migration and invasion of GBM cell lines via downregulating RHOC/Cofilin signaling pathway. These findings suggest that LACTB may be a potential treatment target of GBM.

RNF168 suppresses the cancer stem cell-like traits of nonsmall cell lung cancer cells by mediating RhoC ubiquitination.

The critical roles of E3 ubiquitin ligase RNF168 have been widely revealed in various tumors, however, its roles in lung cancer progression are still confusing. Here, we found that RNF168 expression is positively correlated with the overall survival, first-progression survival, and postprogression survival of lung adenocarcinoma, but not correlated with these survivals of squamous cell carcinoma of lung. Furthermore, it was shown that RNF168 mRNA expression is lowly expressed in lung adenocarcinoma tissues, but highly expressed in squamous cell carcinoma of lung. Functional experiments indicated that RNF168 overexpression significantly suppressed the cancer stem cell (CSC)-like traits of nonsmall cell lung cancer (NSCLC) cells, as characterized by the attenuation of sphere-formation ability, ALDH activity, and the expression of lung CSC markers. Mechanistic studies demonstrated that RNF168 facilitated the ubiquitination of RhoC, which had been considered as a fascinating target for CSCs, and thus promoted RhoC protein degradation. Notably, RNF168 failed to affect the mRNA expression of RhoC and overexpression of RhoC rescued the inhibitory effects of RNF168 overexpression on the CSC-like traits of NSCLC cells. Therefore, this study revealed RNF168 as a novel regulator of RhoC protein in NSCLC cells, this RNF168/RhoC regulatory axis might be a potential target for NSCLC treatment.

Nanobody-Based Quantification of GTP-Bound RHO Conformation Reveals RHOA and RHOC Activation Independent from Their Total Expression in Breast Cancer.

As key regulators of the actin cytoskeleton, RHO GTPase expression and/or activity are deregulated in tumorigenesis and metastatic progression. Nevertheless, the vast majority of experiments supporting this conclusion was conducted on cell lines but not on human tumor samples that were mostly studied at the expression level only. Up to now, the activity of RHO proteins remains poorly investigated in human tumors. In this article, we present the development of a robust nanobody-based ELISA assay, with a high selectivity that allows an accurate quantification of RHO protein GTP-bound state in the nanomolar range (1 nM; 20 µg/L), not only in cell lines after treatment but also in tumor samples. Of note, we present here a fine analysis of RHOA-like and RAC1 active state in tumor samples with the most comprehensive study of RHOA-GTP and RHOC-GTP levels performed on human breast tumor samples. We revealed increased GTP-bound RHOA and RHOC protein activities in tumors compared to normal tissue counterparts, and demonstrated that the RHO active state and RHO expression are two independent parameters among different breast cancer subtypes. Our results further highlight the regulation of RHO protein activation in tumor samples and the relevance of directly studying RHO GTPase activities involvement in molecular pathways.

RNF168 promotes RHOC degradation by ubiquitination to restrain gastric cancer progression via decreasing HDAC1 expression.

Gastric cancer (GC) is the most common cancer worldwide. Although advances in the treatments, the oncogenic mechanisms are still largely unknown. RNF168 (ring-finger nuclear factor 168) is an important regulator of DNA double-strand break (DSB) repair, and its defects have been involved in the pathogenesis of a number of human diseases including cancer. However, its effects on GC are still unclear. In the study, we demonstrated that RNF168 expression was remarkably down-regulated in human GC tissues, and its low expression showed worse overall survival rate in GC patients. Importantly, we here reported that RNF168 directly interacted with Ras homolog gene family member C (RHOC) and induced its ubiquitination to promote RHOC degradation. RHOC exhibited higher expression in human GC tissues, and its knockdown significantly restrained cell proliferation, migration and invasion in GC cell lines. Moreover, RHOC knockdown led to a significant reduction in GC tumor growth in a xenograft mouse model. Additionally, histone deacetylase 1 (HDAC1) was found to be markedly decreased in GC cells with RHOC knockdown. Intriguingly, RHOC suppression-ameliorated proliferative and migratory ability in GC cells were significantly diminished by HDAC1 over-expression. Our in vivo studies finally confirmed that RHOC inhibition dramatically reduced the lung metastasis in nude mice. Collectively, all our results demonstrated that RNF168 directly interacted with RHOC to induce its degradation via promoting its ubiquitination, contributing to the inhibition of cell proliferation and metastasis in GC through decreasing HDAC1. Thus, targeting RNF168/RHOC/HDAC1 axis might be promising to develop effective therapies for GC treatment.

Leukemia-Associated Rho Guanine Nucleotide Exchange Factor and Ras Homolog Family Member C Play a Role in Glioblastoma Cell Invasion and Resistance.

Glioblastoma (GBM) is the most common primary malignant brain cancer in adults. A hallmark of GBM is aggressive invasion of tumor cells into the surrounding normal brain. Both the current standard of care and targeted therapies have largely failed to specifically address this issue. Therefore, identifying key regulators of GBM cell migration and invasion is important. The leukemia-associated Rho guanine nucleotide exchange factor (LARG) has previously been implicated in cell invasion in other tumor types; however, its role in GBM pathobiology remains undefined. Herein, we report that the expression levels of LARG and ras homolog family members C (RhoC), and A (RhoA) increase with glial tumor grade and are highest in GBM. LARG and RhoC protein expression is more prominent in invading cells, whereas RhoA expression is largely restricted to cells in the tumor core. Knockdown of LARG by siRNA inhibits GBM cell migration in vitro and invasion ex vivo in organotypic brain slices. Moreover, siRNA-mediated silencing of RhoC suppresses GBM cell migration in vitro and invasion ex vivo, whereas depletion of RhoA enhances GBM cell migration and invasion, supporting a role for LARG and RhoC in GBM cell migration and invasion. Depletion of LARG increases the sensitivity of GBM cells to temozolomide treatment. Collectively, these results suggest that LARG and RhoC may represent unappreciated targets to inhibit glioma invasion.

Cytotoxic necrotizing factor 1 promotes bladder cancer angiogenesis through activating RhoC.

Uropathogenic Escherichia coli (UPEC), a leading cause of urinary tract infections, is associated with prostate and bladder cancers. Cytotoxic necrotizing factor 1 (CNF1) is a key UPEC toxin; however, its role in bladder cancer is unknown. In the present study, we found CNF1 induced bladder cancer cells to secrete vascular endothelial growth factor (VEGF) through activating Ras homolog family member C (RhoC), leading to subsequent angiogenesis in the bladder cancer microenvironment. We then investigated that CNF1-mediated RhoC activation modulated the stabilization of hypoxia-inducible factor 1α (HIF1α) to upregulate the VEGF. We demonstrated in vitro that active RhoC increased heat shock factor 1 (HSF1) phosphorylation, which induced the heat shock protein 90α (HSP90α) expression, leading to stabilization of HIF1α. Active RhoC elevated HSP90α, HIF1α, VEGF expression, and angiogenesis in the human bladder cancer xenografts. In addition, HSP90α, HIF1α, and VEGF expression were also found positively correlated with the human bladder cancer development. These results provide a potential mechanism through which UPEC contributes to bladder cancer progression, and may provide potential therapeutic targets for bladder cancer.

Autophagy regulates exosome secretion in rat nucleus pulposus cells via the RhoC/ROCK2 pathway.

Our present study investigated whether exosome secretion of nucleus pulposus cells (NPCs) is regulated by autophagy. Different autophagic states of NPCs were induced by rapamycin (Rap), bafilomycin A1 (Baf) and other agents, and it was found that exosomes were secreted in an autophagy-dependent manner. Activation or inhibition of autophagy increased or decreased, respectively, the amount of exosomes that were released into the extracellular space. In addition, in order to confirm that Rap-promoted release of exosomes was mediated by autophagy rather than other pathways, we used autophagy associated gene 5 (ATG5) small-interfering RNA (siRNA) to silence the expression of ATG5 gene, which is indispensable for autophagy. The results showed that siRNA against ATG5 (siATG5) induced an accumulation of intraluminal vesicles (ILVs) in NPCs and a concomitant decrease in the amount of exosomes isolated from supernatant. Ras homolog gene (Rho) and Rho-associated coiled-coil forming protein kinase (ROCK) family molecules are capable of cytoskeletal remodeling and affecting vesicle transport. Therefore, we carried out targeted interventions and evaluated the effects of the RhoC/ROCK2 pathway on the secretion of exosomes within autophagic environment. Knockdown of RhoC and ROCK2 with corresponding siRNA significantly inhibited the secretion of exosomes originating from ILVs in NPCs, even when NPCs were subsequently treated with Rap. Taken together, our findings suggest that autophagy positively regulates expression levels of RhoC and ROCK2, and that the RhoC/ROCK2 pathway exerts a key function on NPCs-derived exosome secretion.

hMENA is a key regulator in endothelin-1/ß-arrestin1-induced invadopodial function and metastatic process.

Aberrant activation of endothelin-1 receptors (ET-1R) elicits pleiotropic effects relevant for tumor progression. The network activated by this receptor might be finely, spatially, and temporarily orchestrated by ß-arrestin1 (ß-arr1)-driven interactome. Here, we identify hMENA, a member of the actin-regulatory protein ENA/VASP family, as an interacting partner of ß-arr1, necessary for invadopodial function downstream of ET-1R in serous ovarian cancer (SOC) progression. ET-1R activation by ET-1 up-regulates expression of hMENA/hMENAΔv6 isoforms through ß-arr1, restricted to mesenchymal-like invasive SOC cells. The interaction of ß-arr1 with hMENA/hMENAΔv6 triggered by ET-1 leads to activation of RhoC and cortactin, recruitment of membrane type 1-matrix metalloprotease, and invadopodia maturation, thereby enhancing cell plasticity, transendothelial migration, and the resulting spread of invasive cells. The treatment with the ET-1R antagonist macitentan impairs the interaction of ß-arr1 with hMENA and inhibits invadopodial maturation and tumor dissemination in SOC orthotopic xenografts. Finally, high ETAR/hMENA/ß-arr1 gene expression signature is associated with a poor prognosis in SOC patients. These data define a pivotal function of hMENA/hMENAΔv6 for ET-1/ß-arr1-induced invadopodial activity and ovarian cancer progression.

Cooperation of G12/13 and Gi proteins via lysophosphatidic acid receptor-2 (LPA2) signaling enhances cancer cell survival to cisplatin.

Lysophosphatidic acid (LPA) through six subtypes of G protein-coupled LPA receptors (LPA1 to LPA6) mediates a variety of cancer cell functions. The aim of this study was to evaluate the cooperative effects of G12/13 and Gi proteins through LPA2 on cancer cell survival to cisplatin (CDDP). In cell survival assay, cells were treated with CDDP every 24 h for 2 days. The long-term CDDP treated (HT-CDDP) cells established from fibrosarcoma HT1080 cells were pretreated with an LPA2 agonist, GRI-977143. The cell survival rate to CDDP of HT-CDDP cells was significantly increased by GRI-977143. The elevated cell survival to CDDP was suppressed by LPA2 knockdown. Since G12/13 protein stimulates Rho-mediated signaling, RhoA and RhoC knockdown cells were generated from HT1080 cells (HT1080-RhoA and HT1080-RhoC cells, respectively). In the presence of GRI-977143, HT1080-RhoA and HT1080-RhoC cells showed the low cell survival rates to CDDP. On the other hand, Gi protein inhibits adenylyl cyclase (AC) activity. Before cell survival assay, cells were treated with a Gi protein inhibitor, pertussis toxin (PTX) for 24 h. The cell survival rate to CDDP of HT1080 cells was significantly reduced by PTX. Furthermore, when HT1080-RhoA and HT1080-RhoC cells were pretreated with PTX, the cell survival rates to CDDP of both cells were markedly inhibited by PTX. The present results suggest that cooperation of G12/13 and Gi proteins activated by LPA2 enhances the cell survival of HT1080 cells treated with CDDP.

YAP and the RhoC regulator ARHGAP18, are required to mediate flow-dependent endothelial cell alignment.

BACKGROUND: Vascular endothelial cell alignment in the direction of flow is an adaptive response that protects against aortic diseases such as atherosclerosis. The RhoGTPases are known to regulate this alignment. We have shown previously that ARHGAP18 in endothelial cells is a negative regulator of RhoC and its expression is essential in flow-mediated alignment. Depletion of ARHGAP18 inhibits alignment and results in the induction of a pro-inflammatory phenotype. In embryogenesis, ARHGAP18 was identified as a downstream effector of the Yes-associated protein, YAP, which regulates cell shape and size. METHODS: We have used siRNA technology to deplete either ARHGAP18 or YAP in human endothelial cells. The in vitro studies were performed under athero-protective, laminar flow conditions. The analysis of YAP activity was also investigated, using high performance confocal imaging, in our ARHGAP18 knockout mutant mice. RESULTS: We show here that loss of ARHGAP18, although decreasing the expression of YAP results in its nuclear localisation consistent with activation. We further show that depletion of YAP itself results in its activation as defined by an in increase in its nuclear localisation and an increase in the YAP target gene, CyR61. Depletion of YAP, similar to that observed for ARHGAP18 depletion, results in loss of endothelial cell alignment under high shear stress mediated flow and also in the activation of NFkB, as determined by p65 nuclear localisation. In contrast, ARHGAP18 overexpression results in upregulation of YAP, its phosphorylation, and a decrease in the YAP target gene Cyr61, consistent with YAP inactivation. Finally, in ARHGAP18 deleted mice, in regions where there is a loss of endothelial cell alignment, a situation associated with a priming of the cells to a pro-inflammatory phenotype, YAP shows nuclear localisation. CONCLUSION: Our results show that YAP is downstream of ARHGAP18 in mature endothelial cells and that this pathway is involved in the athero-protective alignment of endothelial cells under laminar shear stress. ARHGAP18 depletion leads to a disruption of the junctions as seen by loss of VE-Cadherin localisation to these regions and a concomitant localisation of YAP to the nucleus.

miR-382-3p Overexpression attenuates N-methyl-D-aspartate-induced HT22 cell apoptosis via the regulation of the RhoC/ROCK1 signaling pathway.

miR-382-3p can regulate apoptosis through multiple pathways, but the mechanism remains unknown. In this experiment, we explored whether miR-382-3p can modulate the N-methyL-D-aspartate (NMDA)- induced HT22 cell apoptosis by regulating the RhoC/ROCK1 signaling pathway. An excitatory neurotoxicity model of HT22 cells was induced in vitro with 2 mmol/L NMDA. The cells were divided into normal control, NMDA-induced, NMDA + miR-382-3p mimic, and NMDA + miR-382-3p inhibitor groups. The 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) method, Real-time PCR, Western blot, and flow cytometry were performed to investigate the mechanisms. The results found that NMDA can increase the oxidative stress of HT22 cells in a dose-dependent manner, downregulate the expression of miR-382-3p, upregulate the expression of mRNA and protein abundance of ROCK1 and RhoC, increase the expression levels of proapoptotic proteins Bax, Caspase-3, and Caspase-9, increase the apoptosis of HT22 cells, and reduce the activity and survival rate of HT22 cells. Compared with the NMDA-induced group, the miR-382-3p mimic-transfected HT22 cells increased the expression of miR- 382-3p, reduced the expression of the mRNA and protein abundance of ROCK1 and RhoC, inhibited the expression of proapoptotic proteins Bax, Caspase-3, and Caspase-9, reduced the apoptosis of HT22 cells, and increased the activity and survival rate of HT22 cells. The results suggest that increasing the expression of miR-382-3p can inhibit the activity of the RhoC/ROCK1 signaling pathway, reduce the expression of proapoptotic proteins, reduce the oxidative stress and apoptosis of HT22 cells, and increase the activity and survival rate of HT22 cells.

Dendritic cells control fibroblastic reticular network tension and lymph node expansion.

After immunogenic challenge, infiltrating and dividing lymphocytes markedly increase lymph node cellularity, leading to organ expansion. Here we report that the physical elasticity of lymph nodes is maintained in part by podoplanin (PDPN) signalling in stromal fibroblastic reticular cells (FRCs) and its modulation by CLEC-2 expressed on dendritic cells. We show in mouse cells that PDPN induces actomyosin contractility in FRCs via activation of RhoA/C and downstream Rho-associated protein kinase (ROCK). Engagement by CLEC-2 causes PDPN clustering and rapidly uncouples PDPN from RhoA/C activation, relaxing the actomyosin cytoskeleton and permitting FRC stretching. Notably, administration of CLEC-2 protein to immunized mice augments lymph node expansion. In contrast, lymph node expansion is significantly constrained in mice selectively lacking CLEC-2 expression in dendritic cells. Thus, the same dendritic cells that initiate immunity by presenting antigens to T lymphocytes also initiate remodelling of lymph nodes by delivering CLEC-2 to FRCs. CLEC-2 modulation of PDPN signalling permits FRC network stretching and allows for the rapid lymph node expansion--driven by lymphocyte influx and proliferation--that is the critical hallmark of adaptive immunity.

MiR-17 and miR-19 cooperatively promote skeletal muscle cell differentiation.

Skeletal myogenesis is a highly coordinated process that involves cell proliferation, differentiation and fusion controlled by a complex gene regulatory network. The microRNA gene cluster miR-17-92 has been shown to be related to this process; however, the exact role of each cluster member remains unclear. Here, we show that miR-17 and miR-20a could effectively promote the differentiation of both C2C12 myoblasts and primary bovine satellite cells. In contrast, miR-18a might play a negative role in C2C12 cell differentiation, while miR-19 and miR-92a had little influence. Transcriptome and target analyses revealed that miR-17 could act on Ccnd2, Jak1 and Rhoc genes that are critical for cell proliferation and/or fusion. Notably, the addition of miR-19 could reverse the lethal effect of miR-17 and could thus facilitate the maturation of myotubes. Furthermore, by co-injecting the lentiviral shRNAs of miR-17 and miR-19 into mouse tibialis anterior muscles, we demonstrated the wound healing abilities of the two miRNAs. Our findings indicate that in combination with miR-19, miR-17 is a potent inducer of skeletal muscle differentiation.