Ras Homolog Gene Family Member Research Articles

Role of an adaptor protein human germinal center-associated lymphoma (HGAL) in cell signaling and lymphomagenesis

Human germinal center (GC)-associated lymphoma (HGAL) is a multi-domain adaptor protein expressed in GC B lymphocytes, T follicular helper (Tfh) cells and lymphomas derived from these cells. HGAL expression is an independent predictor of longer survival of diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin’s lymphoma (HL) patients. HGAL regulates B cell receptor (BCR) signaling and immunological synapse formation by binding to either the downstream effectors [e.g., spleen tyrosine kinase (Syk)] or other signaling regulators [e.g., growth factor receptor-bound protein 2 (Grb2)]. HGAL regulates the cytoskeleton that reshapes B cell morphology during BCR signaling and cell motility by at least two molecular mechanisms: enhanced Ras homolog gene family member A (RhoA) signaling and inhibition of myosin-actin translocation. These effects on the cytoskeleton decrease lymphoma dissemination in animal models and contribute to decreased lymphoma dissemination in patients. The latter may contribute to the association of HGAL protein expression with longer survival of patients with DLBCL and HL tumors. The ability to regulate multiple and distinct functions simultaneously in B cells implies that the HGAL protein level is tightly regulated. It was demonstrated that HGAL can be regulated by PR/SET domain 1 (PRDM1)/B lymphocyte-induced maturation protein-1 (BLIMP1) and interleukin-4 (IL-4) at the transcription level, by microRNA-155 (miR-155) at the post-transcriptional level, and by F-box protein 10 (FBXO10) at the post-translational level. Constitutive enforced expression of HGAL at physiological levels leads to lymphoid hyperplasia and DLBCL in mice. Future studies need to focus on identifying HGAL interactome, dissecting its interaction network, and understanding HGAL spatiotemporal signaling in live cells in physiological conditions. Further, the recent demonstration of HGAL expression in Tfh cells requires the determination of its function in these cells. These studies will contribute to new insights into the biology of these cellular subsets and how immune dysregulation contributes to lymphomagenesis.

The RhoA-ROCK1/ROCK2 Pathway Exacerbates Inflammatory Signaling in Immortalized and Primary Microglia.

Neuroinflammation is a unifying factor among all acute central nervous system (CNS) injuries and chronic neurodegenerative disorders. Here, we used immortalized microglial (IMG) cells and primary microglia (PMg) to understand the roles of the GTPase Ras homolog gene family member A (RhoA) and its downstream targets Rho-associated coiled-coil-containing protein kinases 1 and 2 (ROCK1 and ROCK2) in neuroinflammation. We used a pan-kinase inhibitor (Y27632) and a ROCK1- and ROCK2-specific inhibitor (RKI1447) to mitigate a lipopolysaccharide (LPS) challenge. In both the IMG cells and PMg, each drug significantly inhibited pro-inflammatory protein production detected in media (TNF-α, IL-6, KC/GRO, and IL-12p70). In the IMG cells, this resulted from the inhibition of NF-κB nuclear translocation and the blocking of neuroinflammatory gene transcription (iNOS, TNF-α, and IL-6). Additionally, we demonstrated the ability of both compounds to block the dephosphorylation and activation of cofilin. In the IMG cells, RhoA activation with Nogo-P4 or narciclasine (Narc) exacerbated the inflammatory response to the LPS challenge. We utilized a siRNA approach to differentiate ROCK1 and ROCK2 activity during the LPS challenges and showed that the blockade of both proteins may mediate the anti-inflammatory effects of Y27632 and RKI1447. Using previously published data, we show that genes in the RhoA/ROCK signaling cascade are highly upregulated in the neurodegenerative microglia (MGnD) from APP/PS-1 transgenic Alzheimer's disease (AD) mice. In addition to illuminating the specific roles of RhoA/ROCK signaling in neuroinflammation, we demonstrate the utility of using IMG cells as a model for primary microglia in cellular studies.

Rhubarb Anthraquinones Ameliorates Inflammatory Lung Injury by Enhancing Alveolar Epithelium Tight Junction Proteins through RhoA/ROCK1 Signalling

Background: Leakage of the alveolar epithelial barrier can lead to pulmonary oedema, leading to organ dysfunction. Objectives: Combination anthraquinone (CA) and free anthraquinone (FA) were extracted from Rhubarb, and the prevention and therapeutic effects of Rhubarb anthraquinones (RA) on lipopolysaccharide (LPS)-induced lung injury were investigated. Materials and Methods: The CA and FA were extracted from Rhubarb by water solvent extraction and ethanol solvent extraction. The extracted RA content was determined using spectrophotometry and high-performance liquid chromatography (HPLC). A mouse model of inflammatory lung injury was established by LPS induction to study the mechanism of RA activity. Inflammatory factors were measured using an enzyme-linked immunosorbent assay. Changes in alveolar epithelial leakage were assessed using a permeability assay. Changes in lung injury were evaluated by histopathological and ultrastructural observations. Tight junction (TJ) markers and Ras homolog gene family member A (RhoA)/Rho-associated protein kinase 1 (ROCK1) pathway-related proteins were measured using immunohistochemistry and Western blotting. As an agonist of RhoA/Rock signalling, U46619 was used to further verify whether RA acts through this pathway. Results: RA could inhibit pulmonary inflammation by decreasing the level of inflammatory factors. The alveolar epithelial permeability was reduced, and the pathological injury of the lung tissue was alleviated after administration with RA. Furthermore, the expression of TJ proteins was up regulated and RhoA/ROCK1 signalling was inhibited in the presence of RA. The effects of RA on TJ proteins were partially reversed by U46619. Conclusion: RA effectively protects mice against inflammatory lung injury by enhancing alveolar epithelial TJ via RhoA/ROCK1 signalling.

The malignancy suppression and ferroptosis facilitation of BCL6 in gastric cancer mediated by FZD7 repression are strengthened by RNF180/RhoC pathway

BackgroundB-cell lymphoma 6 (BCL6) is a transcription repressor that plays a tumor suppressor or promoting role in various tumors. However, its function and molecular mechanism in gastric cancer (GC) remain unclear. Ferroptosis, a novel programmed cell death, is closely related to tumor development. In this research, we aimed to explore the role and mechanism of BCL6 in malignant progression and ferroptosis of gastric cancer.MethodsFirstly, BCL6 was identified as an important biomarker that attenuated the proliferation and metastasis of GC through tumor microarrays and confirmed in GC cell lines. RNA sequence was performed to explore the downstream genes of BCL6. The underlying mechanisms were further investigated by ChIP, dual luciferase reporter assays and rescue experiments. Cell death, lipid peroxidation, MDA and Fe2+ level were detected to determine the effect of BCL6 on ferroptosis and the mechanism was revealed. CHX, MG132 treatment and rescue experiments were used to explore the upstream regulatory mechanism of BCL6.ResultsHere we showed that BCL6 expression was significantly decreased in GC tissues, and patients with low BCL6 expression showed more malignant clinical features and poor prognosis. The upregulation of BCL6 may significantly inhibited the proliferation and metastasis of GC cells in vitro and in vivo. In addition, we found that BCL6 directly binds and transcriptionally represses Wnt receptor Frizzled 7 (FZD7) to inhibit the proliferation, metastasis of GC cells. We also found that BCL6 promoted lipid peroxidation, MDA and Fe2+ level to facilitate ferroptosis of GC cells by FZD7/β-catenin/TP63/GPX4 pathway. Furthermore, the expression and function of BCL6 in GC were regulated by the ring finger protein 180 (RNF180)/ras homolog gene family member C (RhoC) pathway, which had been elucidated to be involved in significantly mediating the proliferation and metastasis of GC cells.ConclusionsIn summary, BCL6 should be considered a potential intermediate tumor suppressor to inhibit the malignant progression and induce ferroptosis, which might be a promising molecular biomarker for further mechanistic investigation of GC.

PKCε inhibition prevents ischemia‑induced dendritic spine impairment in cultured primary neurons.

Brain ischemia is an independent risk factor for Alzheimer's disease (AD); however, the mechanisms underlining ischemic stroke and AD remain unclear. The present study aimed to investigate the function of the ε isoform of protein kinase C (PKCε) in brain ischemia-induced dendritic spine dysfunction to elucidate how brain ischemia causes AD. In the present study, primary hippocampus and cortical neurons were cultured while an oxygen-glucose deprivation (OGD) model was used to simulate brain ischemia. In the OGD cell model, in vitro kinase activity assay was performed to investigate whether the PKCε kinase activity changed after OGD treatment. Confocal microscopy was performed to investigate whether inhibiting PKCε kinase activity protects dendritic spine morphology and function. G-LISA was used to investigate whether small GTPases worked downstream of PKCε. The results showed that PKCε kinase activity was significantly increased following OGD treatment in primary neurons, leading to dendritic spine dysfunction. Pre-treatment with PKCε-inhibiting peptide, which blocks PKCε activity, significantly rescued dendritic spine function following OGD treatment. Furthermore, PKCε could activate Ras homolog gene family member A (RhoA) as a downstream molecule, which mediated OGD-induced dendritic spine morphology changes and caused dendritic spine dysfunction. In conclusion, the present study demonstrated that the PKCε/RhoA signalling pathway is a novel mechanism mediating brain ischemia-induced dendritic spine dysfunction. Developing therapeutic targets for this pathway may protect against and prevent brain ischemia-induced cognitive impairment and AD.

Role of cyclooxygenase pathways in bowel fibrotic remodelling in a murine model of experimental colitis.

Gut fibrosis occurs under chronic inflammation. This study examined the effects of different cyclooxygenase (COX) inhibitors on fibrosis in the inflamed colon. Colitis was induced by 2,4-dinitrobenzenesulfonic acid (DNBS) in albino male Sprague-Dawley rats. After 6, 12 and 18 days, macroscopic and microscopic damage, collagen and elastic fibre content were examined. At day 6, pro-fibrotic factors (collagen I and III, hydroxyproline, fibronectin, matrix metalloproteinase-2 and -9), transforming growth factor-beta (TGF-β) signalling [TGF-β, Ras homolog gene family member A (RhoA), phosphorylated small mother against decapentaplegic (pSMAD)-2 and -6] and peristalsis were assessed, and the effects of indomethacin, SC-560 or celecoxib were tested. Six days after DNBS administration, significant histopathological signs of fibrotic remodelling were observed in rats. At day 6, pro-fibrotic factors were up-regulated and colonic peristalsis was altered. COX inhibitors reversed the histochemical, molecular and functional changes in the fibrotic colon. COX inhibition reduced TGF-β expression, SMAD2 phosphorylation and RhoA, and increased SMAD6 expression. Colonic fibrosis is associated with altered bowel motility and induction of profibrotic factors driven by TGF-β signalling. COX-1 and COX-2 inhibition counteracts this fibrotic remodelling by the modulation of TGF-β/SMAD signalling, mainly via SMAD6 induction and reduction in SMAD2 phosphorylation.

Prognostic prediction of lung adenocarcinoma by integrative analysis of RHOH expression and methylation.

The development of epigenetics holds great promise for diagnosis and treatment of lung adenocarcinoma (LUAD). The purpose of this work was to analyze the correlation between Ras Homolog Gene Family Member H (RHOH) expression and methylation in patients with LUAD and its association with survival. Data related to gene expression, DNA methylation, and clinical features of LUAD from The Cancer Genome Atlas (TCGA) database were analyzed. A total of 50 patients were included in verification group. The methylation level of RHOH in verification group was detected by bisulfite amplicon sequencing. The RHOH methylation level in TCGA cohort was significantly and negatively correlated with its expression level (Cor = -0.5, p = 2.687e-33). Patients with hypermethylation and low expression of RHOH had significantly worse prognosis than patients with hypomethylation and low expression of RHOH (TCGA: p = 0.004; validation cohort: p = 0.006, HR: 4.740, 95% CI: 1.567-14.340). Our research revealed that RHOH may prove to be a new potential prognostic predictor for LUAD patients.

RHOA Therapeutic Targeting in Hematological Cancers.

Primarily identified as an important regulator of cytoskeletal dynamics, the small GTPase Ras homolog gene family member A (RHOA) has been implicated in the transduction of signals regulating a broad range of cellular functions such as cell survival, migration, adhesion and proliferation. Deregulated activity of RHOA has been linked to the growth, progression and metastasis of various cancer types. Recent cancer genome-wide sequencing studies have unveiled both RHOA gain and loss-of-function mutations in primary leukemia/lymphoma, suggesting that this GTPase may exert tumor-promoting or tumor-suppressive functions depending on the cellular context. Based on these observations, RHOA signaling represents an attractive therapeutic target for the development of selective anticancer strategies. In this review, we will summarize the molecular mechanisms underlying RHOA GTPase functions in immune regulation and in the development of hematological neoplasms and will discuss the current strategies aimed at modulating RHOA functions in these diseases.

Upregulation of KLHL17 promotes the proliferation and migration of non-small cell lung cancer by activating the Ras/MAPK signaling pathway

Analysis of the Gene Expression Profiling Interactive Analysis (GEPIA) database revealed that Kelch-like 17 (KLHL17) is overexpressed in non-small cell lung cancer (NSCLC) including adenocarcinoma (ADC) and squamous cell carcinoma (SCC). We therefore explored the role of KLHL17 in the development and progression of NSCLC. Immunohistochemistry and western blotting showed that KLHL17 expression was significantly higher in the tumor tissues from 173 patients with NSCLC, compared with the corresponding non-neoplastic tissue. In addition, upregulated KLHL17 expression was positively correlated with tumor size, lymph node metastasis and tumor node metastasis (TNM) stage, and affected the overall survival (OS) of patients with NSCLC. Consistent with clinical samples, in vitro studies demonstrated that KLHL17 expression was higher in various cell lines of NSCLC (A549, H1299, H460 and SK cells) as compared to normal human bronchial epithelial cells (HBE cells). Overexpression of KLHL17 in the cell lines of NSCLC with KLHL17-Flag plasmid promoted the proliferation and migration of tumor cells, which was associated with elevated activation of Rat sarcoma/Mitogen-activated protein kinases (Ras/MAPK) signaling and increased expression of cyclin D1, cyclin D-dependent kinases 4 (CDK4), matrix metalloproteinase 2 (MMP2) and Ras homolog gene family member A (RhoA). In contrast, knockdown of KLHL17 in the cell lines of NSCLC using KLHL17 small interfering RNA suppressed the proliferation and migration of tumor cells, in association with reduced activation of Ras/MAPK signaling and decreased expression of cyclin D1, CDK4, MMP2 and RhoA. Moreover, treatment of tumor cells with Ras inhibitor salirasib prevented KLHL17-induced Ras/MAPK activity as well as tumor proliferation and migration. These results suggest that upregulated KLHL17 in NSCLC promotes the proliferation and migration of tumor by activating Ras/MAPK signaling pathway. Therefore, KLHL17 may be a novel therapeutic target for the treatment of NSCLC.Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and is associated with limited treatment options. This study elucidates the role of KLHL17 in the development and progression of NSCLC using clinical samples and NSCLC cell lines. The results show that upregulated KLHL17 in NSCLC promotes the proliferation and migration of tumor by activating Ras/MAPK signaling pathway, and suggest that KLHL17 may be a novel therapeutic target for the treatment of NSCLC.

Expression of epithelial–mesenchymal transition markers in squamous cell carcinoma of the uterine cervix: A cross-sectional study

Background: Cervical cancer is the fourth most common cancer in women worldwide. Epithelial– mesenchymal transition (EMT) is a phenomenon related to carcinogenesis, which is characterized by morphological changes from neoplastic epithelial cells to mesenchymal cells, resulting in increased motility and invasiveness of neoplastic cells. To observe the expression of three EMT markers (cytokeratin 19 [CK1]), vimentin, and Ras homolog gene family member C [RhoC]) in individuals with a normal cervix, patients with high-grade squamous intraepithelial lesion (HSIL), and patients with squamous cell carcinoma (SCC) and determine the association with the histological grade and clinical/radiological stage of cervical cancer. Methods: Seventy participants were included in the study: 10 individuals with a normal cervix, 30 patients with HSIL, and 30 patients with newly diagnosed SCC. Immunohistochemistry for CK19, vimentin, and RhoC was performed in all groups. The association of the expressions of the three markers between the groups was analyzed. P values of < 0.005 were considered statistically significant. Results: The mean age of the normal cervix, HSIL, and SCC groups was 46.2 ± 16.12, 49.10 ± 10.13, and 56.27 ± 9.29 years, respectively. CK19 showed a weak basal cell positivity in 80% of the individuals with a normal cervix. Meanwhile, 93.3% and 100% of the patients with HSIL and SCC, respectively, were positive for CK19; the association of the expressions between the groups was insignificant. Vimentin was negative in the normal cervix group and positive in the HSIL (33.3%) and SCC (73.3%) groups; the association of the expressions between the groups was significant. RhoC was positive in the normal cervix, HSIL, and SCC groups (10%, 20%, and 83.33%, respectively), and the association of the expressions between them was significant. There was no significant association found between the expression of vimentin or RhoC and the histopathological grade or FIGO stage. Conclusion: An increased expression of CK19 in patients with HSIL and SCC of the cervix highlights the role of basal cells in cervical carcinogenesis. The expression of vimentin and RhoC in individuals with a normal cervix and patients with HSIL and SCC indicates EMT.

GPX4 Alleviates Diabetes Mellitus-Induced Erectile Dysfunction by Inhibiting Ferroptosis.

Pharmacological therapy of diabetes mellitus-induced erectile dysfunction (DMED) is intractable owig to the poor response to phosphodiesterase type 5 inhibitors (PDE5i). The surge in the number of diabetic patients makes it extremely urgent to find a novel therapy for DMED. Ferroptosis is a recently discovered form of cell death evoked by lipid peroxidation and is related to several diabetic complications. GPX4, an important phospholipid hydroperoxidase, can alleviate ferroptosis and maintain redox balance via reducing lipid peroxides. However, whether GPX4 can be a prospective target of DMED needs to be determined. Fifty rats were randomly divided into control group, DMED group, DMED + negative control group (DMED + NC group), DMED + low-dose group (1 × 106 infectious units), and DMED + high-dose group (2 × 106 infectious units). Erectile function was assessed 4 weeks after intracavernous injection of GPX4 or negative control lentivirus. The penile shafts were collected for subsequent molecular biological and histological analysis. The results demonstrated that erectile function of the rats in DMED and DMED + NC groups was extremely impaired and was improved in a dose-dependent manner with GPX4 lentivirus (GPX4-LV) injection. Additionally, upregulation of the ACSL4-LPCAT3-LOX pathway, iron overload, oxidative stress, fibrosis, and decreased endothelial and smooth muscle cell numbers were observed in the corpus cavernosum of DMED group. Meanwhile, the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway was inhibited, and the Ras homolog gene family member A (RhoA)/Rho-associated protein kinase (ROCK) pathway was promoted in DMED rats. The above histologic alterations and related molecular changes were alleviated after GPX4-LV injection. The results revealed that GPX4 improved erectile function by modulating ferroptosis during DMED progression. This finding is of paramount significance in deciphering the molecular mechanism of hyperglycemia-induced ferroptosis, thereby providing a prospective target for preventing the development of DMED.

Microtopography “not” macrostructure affects the osteogenic differentiation of human mesenchymal stem cells through RHOA and LATS1

Three-dimensional (3D) biomaterials provide a tridimensional living environment for human mesenchymal stem cells (hMSC). However, the regulation mechanism of biomaterials’ macro and micro structure on cell differentiation is not clear. Here, we used selective laser melting (SLM) technology to fabricate 3D biomaterials with different macro and micro structure characteristics. Results showed that the different morphology of hMSCs is not the determinant of their differentiation stat, which might be influenced by the macrostructure of biomaterials. And the effect of microstructure of the biomaterial on osteogenic differentiation of hMSCs was more remarkable than that of the macrostructure ones. The microstructure affected the nuclear localization of Yes-associated protein (YAP) by interfering with the balance between Ras homolog gene family member A (RHOA) and large tumor suppressor 1 (LATS1), thereby influencing the osteogenic differentiation of hMSCs. Precise gene knockout experiments also proved that the mediation of the development and regeneration of cranial bones in mice was caused by the balance of RHOA and LATS1. In addition, we used SLM to fabricate titanium-based implant surface with microstructures to achieve better bone regeneration and implant osseointegration.

DNA methyltransferase 1 (DNMT1) suppresses mitophagy and aggravates heart failure via the microRNA-152-3p/ETS1/RhoH axis

AbstractDNA methyltransferase 1 (DNMT1) shows close link with heart disease. This study aimed to define the role DNMT1 plays in heart failure and determine the underlying mechanism. Expression of microRNA (miR)-152-3p, DNMT1, E26 transformation specific-1 (ETS1) and ras homolog gene family member H (RhoH) was determined by RT-qPCR and/or western blot analysis. The interaction between miR-152-3p and ETS1 was predicted and verified. Methylation of the miR-152-3p promoter region was assessed using methylation-specific PCR. H9c2 cells were chosen for in vitro assays to examine the regulatory role of DNMT1 in autophagy and mitophagy with respect to miR-152-3p/ETS1/RhoH. Doxorubicin (DOX)-induced rat models of heart failure were employed for in vivo validation. DNMT1 expression was upregulated in the heart tissues of DOX-induced rats, where it showed an inverse correlation with miR-152-3p expression. Moreover, DNMT1 was shown to enhance methylation of the miR-152-3p promoter region and suppress its expression, leading to inhibition of mitophagy in H9c2 cells. In addition, DNMT1 enhanced expression of ETS1, which further elevated RhoH expression. Moreover, ETS1-elevated RhoH reduced cell viability and promoted autophagy and mitophagy in H9c2 cells upon treatment with DOX. Next, in vivo results demonstrated that depletion of DNMT1 protected rats from heart failure in a miR-152-3p/ETS1/RhoH-dependent manner. Overall, these findings indicate that DNMT1 may inhibit expression of miR-152-3p by promoting the methylation of miR-152-3p and enhancing the expression of ETS1, thereby inducing RHOH transcriptional activation and inhibiting mitochondrial autophagy, ultimately promoting the development of heart failure.

Laminin-511 Activates the Human Induced Pluripotent Stem Cell Survival via α6β1 Integrin-Fyn-RhoA-ROCK Signaling.

In human induced pluripotent stem cells (hiPSCs), laminin-511/α6β1 integrin interacts with E-cadherin, an intercellular adhesion molecule, to induce the activation of the phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathway. The interaction between laminin-511/α6β1 integrin and E-cadherin, an intercellular adhesion molecule, results in protection against apoptosis through the proto-oncogene tyrosine-protein kinase Fyn(Fyn)-RhoA-ROCK signaling pathway and the Ras homolog gene family member A (RhoA)/Rho kinase (ROCK) signaling pathway (the major pathway for cell death). In this article, the impact of laminin-511 on hiPSC on α6β1 integrin-Fyn-RhoA-ROCK signaling is discussed and explored along with validation experiments. PIK3CA mRNA (mean [standard deviation {SD}]: iMatrix-511, 1.00 [0.61]; collagen+MFGE8, 0.023 [0.02]; **P < 0.01; n = 6) and PIK3R1 mRNA (mean [SD]: iMatrix-511, 1.00 [0.79]; collagen+MFGE8, 0.040 [0.06]; *P < 0.05; n = 6) were upregulated by iMatrix-511 resulting from an increased expression of Integrin α6 mRNA (mean [SD]: iMatrix-511, 1.00 [0.42]; collagen+MFGE8, 0.23 [0.05]; **P < 0.01; n = 6). The iMatrix-511 increased the expression of p120-Catenin mRNA (mean [SD]: iMatrix-511, 1.00 [0.71]; collagen+MFGE8, 0.025 [0.03]; **P < 0.01; n = 6) and RAC1 mRNA (mean [SD]: iMatrix-511, 1.00 [0.28]; collagen+MFGE8, 0.39 [0.15]; **P < 0.01; n = 6) by increasing the expression of E-cadherin mRNA (mean [SD]: iMatrix-511, 1.00 [0.38]; collagen+MFGE8, 0.16 [0.11]; **P < 0.01; n = 6). As a result, iMatrix-511 increased the expression of P190 RhoGAP (GTPase-activating proteins) mRNA, such as ARHGAP1 mRNA (mean [SD]: iMatrix-511, 1.00 [0.57]; collagen+MFGE8, 0.032 [0.03]; **P < 0.01; n = 6), ARHGAP4 mRNA (mean [SD]: iMatrix-511, 1.00 [0.56]; collagen+MFGE8, 0.039 [0.049]; **P < 0.01; n = 6), and ARHGAP5 mRNA (mean [SD]: iMatrix-511, 1.00 [0.39]; collagen+MFGE8, 0.063 [0.043]; **P < 0.01; n = 6). Western blotting showed that phospho-Rac1 remained in the cytoplasm and phospho-Fyn showed nuclear transition in iPSCs cultured on iMatrix-511. Proteome analysis showed that PI3K signaling was enhanced and cytoskeletal actin was activated in iPSCs cultured on iMatrix-511. In conclusion, laminin-511 strongly activated the cell survival by promoting α6β1 integrin-Fyn-RhoA-ROCK signaling in hiPSCs.

Selenoprotein K Is Essential for the Migration and Phagocytosis of Immature Dendritic Cells.

Selenoprotein K (SELENOK) is an endoplasmic reticulum stress (ERS)-regulated protein required for the calcium (Ca2+) flux-mediated migration of T cells and neutrophils, and the migration and phagocytosis of macrophages and microglia. However, the effect of SELENOK on the regulation of the immune function of dendritic cells (DCs), including immature DCs (imDCs) and mature DCs (mDCs), is still unclear. In this study, imDCs prepared from SELENOK knockout mice were used to evaluate the effect of SELENOK on the migration and phagocytosis of imDCs. The results showed that ERS-induced downregulation of imDCs phenotypic markers led to a reduction in Ras homolog gene family member A (RhoA)-dependent migration and enhanced Ca2+/CD205-mediated phagocytosis. SELENOK deficiency-induced upregulation of selenoprotein S (SELENOS) attenuated ERS levels in imDCs. An increase in Ca2+ levels resulted in increased migration and decreased phagocytosis with or without ERS conditions. The migration was RhoA-dependent, and Ca2+ or CD205 was associated with regulating phagocytosis in imDCs. Our study found that SELENOK is required for imDC migration and phagocytosis.

RhoA Signaling in Neurodegenerative Diseases.

Ras homolog gene family member A (RhoA) is a small GTPase of the Rho family involved in regulating multiple signal transduction pathways that influence a diverse range of cellular functions. RhoA and many of its downstream effector proteins are highly expressed in the nervous system, implying an important role for RhoA signaling in neurons and glial cells. Indeed, emerging evidence points toward a role of aberrant RhoA signaling in neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, and amyotrophic lateral sclerosis. In this review, we summarize the current knowledge of RhoA regulation and downstream cellular functions with an emphasis on the role of RhoA signaling in neurodegenerative diseases and the therapeutic potential of RhoA inhibition in neurodegeneration.

Acetyl-L-carnitine improves erectile function in bilateral cavernous nerve injury rats via promoting cavernous nerve regeneration.

Neurogenic erectile dysfunction (NED) caused by cavernous nerve (CN) injury is a typical complication after pelvic surgery, which lacks efficient treatments. Acetyl-L-carnitine (ALCAR) has been proven to promote nerve repair. To investigate the effect and potential mechanism of ALCAR in the treatment of NED. Thirty-two rats were randomly divided into bilateral CN injury (BCNI) group, BCNI + lower-dose ALCAR (50mg/kg/day) group, BCNI + higher-dose (100mg/kg/day) group, and sham-operated group. Erectile function was assessed 14 days after daily intraperitoneal injection of ALCAR or placebo. The penile tissues were gathered for subsequent histological and molecular biological analysis. Rat Schwann cell (SC) line S16 was used to verify the mechanism of ALCAR in vitro. We found that the erectile function of the rats in the BCNI group was severely impaired, which was improved considerably in both BCNI+ALCAR-LD and BCNI+ALCAR-HD groups. Also, we observed decreased smooth muscle and increased collagen content in the corpus cavernosum in the BCNI group. The expressions of fibrosis markers transforming growth factor-beta (TGF-β), connective tissue growth factor (CTGF), and Smad 2/3 were significantly up-regulated in the BCNI group. The above changes were alleviated after the administration of lower and higher-dose ALCAR. Meanwhile, the nitric oxide (NO)/cyclic guanosine monophosphate pathway (cGMP) was promoted and the Ras homolog gene family member A (RhoA)/Rho-associated protein kinase (ROCK) pathway was inhibited in the corpus cavernosum of BCNI rats after ALCAR treatment, accompanied by increased neuronal nitric oxide synthase (nNOS) and down-regulated tyrosine hydroxylase (TH). In vitro, ALCAR promoted the migration and proliferation of SC and increased the expression of 22-kD peripheral myelin protein and nerve growth factor (NGF). Further, rats treated with ALCAR had high expression of ATF3 and S100 in the distal nerve tissues of the CN extrusion site. ALCAR could promote nerve repair and regeneration, inhibit penile fibrosis, and improve penile erection by promoting the proliferation and migration of SC and the secretion of NGF. Our study confirms that ALCAR may be a potential treatment strategy for NED.

Repressed miR-34a Expression Dictates the Cell Fate to Corneal Endothelium Failure.

PurposeTo reveal the mechanism triggering the functional disparity between degenerated and non-degenerated corneal endothelium cells in the water efflux from corneal stroma to the anterior chamber.MethodsThe varied levels of the microRNA (miR)-34, miR-378, and miR-146 family in human corneal endothelium and cultured cells thereof were investigated using 3D-Gene Human miRNA Oligo Chips. Concomitantly, CD44, p53, c-Myc, matrix metalloprotease (MMP)-2 expression, and Ras homolog gene family member A (Rho A) activity was correlated to the expression intensities of these microRNAs, partly complemented with their altered expression levels with the transfection of the corresponding mimics and inhibitors. The levels of miRs were further associated with intracellular pH (pHi) and mitochondrial energy homeostasis.ResultsP53-inducible miR-34a/b repressed CD44 expression, and CD44 was repressed with the elevated c-Myc. The repressed miR-34a activated the CD44 downstream factors Rho A and MMP-2. MiR-34a mimics downregulated pHi, inducing the skewing of mitochondrial respiration to oxidative phosphorylation. The oxidative stress (H2O2) induced on human corneal endothelial cells, which repressed miR-34a/b expression, may account for the impaired signaling cascade to mitochondrial metabolic homeostasis necessary for an efficient water efflux from the corneal stroma.ConclusionsThe upregulated expression of CD44, through repressed miR-34a/b by reactive oxygen species and elevated c-Myc by oxidative stress, may impair mitochondrial metabolic homeostasis, leading to human corneal endothelial failure.

SKF38393 prevents high glucose (HG)-induced endothelial dysfunction by inhibiting the effects of HG on cystathionine γ-lyase/hydrogen sulfide activity and via a RhoA/ROCK1 pathway.

Endothelial dysfunction plays a crucial role in diabetic vascular complications. A decrease in hydrogen sulfide (H2S) levels is increasingly becoming a vital factor contributing to high glucose (HG)-induced endothelial dysfunction. Dopamine D1-like receptors (DR1) activation has important physiological functions in the cardiovascular system. H2S decreases the dysfunction of vascular endothelial cells. However, no studies have reported whether DR1 protects the function of vascular endothelial cells by regulating H2S levels. The present study aimed to determine whether DR1 regulates the levels of endogenous H2S, which exerts protective effects against HG-induced injury of human umbilical vein endothelial cells (HUVECs) via Ras homolog gene family member A (RhoA)/Rho-associated coiled-coil containing kinase 1 (ROCK1) signalling. HUVECs were exposed to HG (30 mM) or normal glucose (5.5 mM) after different treatments. Cell viability, proliferation and migration were measured by Cell Counting Kit-8, EdU cell proliferation assay, transwell assay and wound healing assay, respectively. H2S probe (7-Azido-4-Methylcoumarin) was used to detect levels of H2S. The intracellular calcium concentration ([Ca2+]i) were measured using Fluo-4 AM. The protein expressions were quantified by Western blot. We found that HG decreased the expression of DR1 and cystathionine γ-lyase (CSE) and H2S production. The DR1 agonist SKF38393 significantly increased DR1 and CSE expression and H2S production, whereas NaHS (a H2S donor) only increased CSE expression and H2S production but had no effect on DR1 expression. Meanwhile, SKF38393 further increased the [Ca2+]i induced by HG. In addition, HG reduced cell viability and the expression of Cyclin D1 and proliferating cell nuclear antigen and increased the expression of p21C⁢i⁢p/W⁢A⁢F-1, collagen I, collagen III, matrix metalloproteinase 9, osteopontin and α-smooth muscle actin and the activity of phosphorylated RhoA and ROCK1. SKF38393 and NaHS reversed these effects of HG. PPG (a CSE inhibitor) abolished the beneficial effect of SKF38393. These effects of SKF38393 were similar to those of Y-27632 (a ROCK inhibitor). Taken together, our results suggest that DR1 activation upregulates the CSE/H2S pathway by increasing the [Ca2+]i, which protects endothelial cells from HG-induced injury by inhibiting the RhoA/ROCK1 pathway.

Flavonoids of Coreopsis tinctoria Nutt alleviate the oxidative stress and inflammation of glomerular mesangial cells in diabetic nephropathy via RhoA/ROCK signaling

• FC improved glucolipid metabolic disorder, abnormal renal function, and renal pathological injury in db/db DN mice. • FC could exert a protective effect on DN by regulating oxidative stress mediated by RhoA/ROCK signaling both in vivo and in vitro. • FC down-regulated inflammatory responses by NF-κB/TGF-β1/Smad signaling both in vivo and in vitro. Diabetic nephropathy (DN) is one of the serious diabetic microvascular complications, and oxidative stress and inflammation may play a vital role in its initiation and development. Previous studies indicated that Coreopsis tinctoria Nutt had anti-inflammatory and antioxidant activities. This study aimed to examine the protective effects of flavonoids from Coreopsis tinctoria Nutt (FC) on DN. In vivo, db/db mice were treated with FC (150, 300 mg/kg), FC150 + Metformin. DN model in vivo of high glucose and palmitic acid-induced glomerular mesangial cell (HBZY-1) was established and treated with FC . The results showed that treatment of FC, especially in the dose of 150 mg/kg and FC150 mg/kg plus metformin significantly improved glucolipid metabolic disorder, abnormal renal function, and pathological renal injury in db/db mice. Both in vivo and in vitro , FC intervention reduced the levels of renal oxidative stress-related factors, inflammatory cytokines, and fibrotic factors. Additionally, FC down-regulated the expressions of proteins related to Ras homolog gene family member (RhoA)/Rho-associated protein kinase (ROCK), nuclear factor kappa-B (NF-κB), and transforming growth factor (TGF-β)/Smad signaling pathways. In conclusion, FC might protect against diabetic renal injury by inhibiting renal oxidation, inflammation, and fibrosis via RhoA/ROCK and NF-κB/TGF-β/Smad signalings.