Rac1 Gene Expression Research Articles

Circulating RAC1 contributed to steroid-sensitive nephrotic syndrome: Mendelian randomization, single-cell RNA-sequencing, proteomic, and experimental evidence

Objectives The small GTPase Rac1 (RAC1) has been linked to podocyte disorders and steroid-sensitive nephrotic syndrome (SSNS). The aim of this study was to explore and validate the potential causal association between circulating RAC1 and SSNS. Methods The association between circulating RAC1 and SSNS at both gene expression and proteomic levels was investigated using Mendelian randomization analysis, and further validated by single-cell RNA-sequencing, proteomic analysis, and experimental studies. The genetic instruments comprised cis-expression quantitative trait loci (cis-eQTLs) associated with RAC1 gene expression and protein QTLs correlated with plasma RAC1 protein levels. Causal associations were estimated utilizing the inverse variance weighted and MR-PRESSO methods. Validation of RAC1 expression was conducted through single-cell RNA-sequencing of peripheral blood mononuclear cells from patients with SSNS and healthy controls. Proteomic analysis was performed among patients with minimal change nephrotic syndrome. Experimental validation was conducted using a puromycin aminonucleoside (PAN)-induced nephrosis model. Results Increased expression of RAC1 was associated with a higher risk of SSNS (gene expression level: odds ratio [OR], 1.53; 95% confidence interval [CI], 1.02–2.28; protein level: OR, 1.82; 95% CI, 1.05–3.17). The results of MR-PRESSO were consistent (gene expression level: OR, 1.49; 95% CI, 1.17–1.92; protein level: OR, 1.81; 95% CI, 1.16–2.85). Single-cell RNA sequencing and proteomic analysis confirmed elevated RAC1 expression in patients with SSNS compared to healthy controls. Experimental data further supported increased RAC1 expression in PAN-induced nephropathy. Conclusions Increased expression of RAC1 might be causally associated with SSNS, suggesting that targeting RAC1 might represent a potential therapeutic strategy for SSNS.

Identification and characterization of RAC1-related immune and prognostic subtypes of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a malignant tumor with significant variability in prognosis among patients. Ras-related C3 botulinum toxin substrate 1 (RAC1) is a key focus in the area of cancer research. However, the molecular mechanisms of RAC1 in HCC remain incompletely elucidated. In this study, bioinformatics analysis was used, and public databases were used to obtain information about HCC cases. The samples were categorized into two groups of high and low expression based on the expression level of RAC1 gene. The limma package was used to calculate the differentially expressed genes between the two groups, and univariate Cox regression analysis was used to screen the prognostic related factors. Consensus clustering analysis was performed using the ConsensusClusterPlus package to identify molecular subtypes of HCC patients. Immune cell infiltration and ESTIMATE scores were assessed using the single sample gene set enrichment analysis and ESTIMATE algorithms. The sensitivity of different isoforms to chemotherapeutic agents was predicted by the oncoPredict package. Finally, we also performed cell function experiments to validate the biological role of RAC1 in vitro. Initially, we classified patients into high and low expression groups based on RAC1 gene expression levels and identified 195 up-regulated genes and 107 down-regulated genes. Through univariate Cox regression analysis, we screened out 169 prognosis-related factors. Furthermore, HCC patients were categorized into two subtypes. Subsequently, Kaplan-Meier survival curves showed that there was a significant difference in prognosis between the two molecular subtypes. Further analysis indicated substantial differences in gene expression levels and TIDE scores between two molecular subtypes. Moreover, these two subtypes exhibited varying sensitivity to chemotherapy drugs, as evidenced by differences in IC50 values. In addition, we found that the silence of RAC1 could effectively inhibit the migration and invasion of HCC cells in vitro. This study sheds light on the molecular intricacies of RAC1 in HCC and identifies patient populations that may benefit from immunotherapeutic interventions, with potential implications for tailored treatment strategies.

Curcuma longa extract inhibits migration by reducing MMP-9 and Rac-1 expression in highly metastatic breast cancer cells.

Highly metastatic breast cancer is a population of cancer cells that has metastasized to other organs in the body leading to apoptosis resistance. It was reported that MDAMB-231 cells contain lower levels of reactive oxygen species associated with metastatic capability. Curcuma longa (CL) possesses cytotoxic effects in several cancer cells including metastatic breast cancer cells. This study aimed to investigate the effect of CL-inhibited cell migration in highly metastatic breast cancer MDAMB-231 cells. CL was extracted under maceration with methanol. The cytotoxic effect on single and combination treatment of CL was assessed through the MTT assay. Migration analysis was evaluated using scratch wound healing assay, MMP-9 expression by gelatine zymography, Rac-1, and MMP-9 gene expression using Real-Time Quantitative Reverse transcription polymerase chain reaction (qRT-PCR). The apoptosis induction was analyzed through Bax gene expression and Bcl-2 protein expression. We found that CL inhibits the growth of MDAMB-231 cells, induces Bax gene expression, and suppresses Bcl-2 expression in a dose-dependent manner. Moreover, cancer cell migration was suppressed by the presence of CL. qRT-PCR and gelatine zymography assay showed that CL downregulates Rac-1 and MMP-9 gene expression. CL could inhibit the growth and migration of highly metastatic breast cancer cells by reducing the Rac-1 gene expression and regulating apoptosis protein expression.

Expression Analysis of the Small GTP-Binding Protein Rac in Pterygium

To determine the roles of small GTP-binding proteins Rac1, Rac2, and Rac3 expression in pterygial tissue and to compare these expressions with normal conjunctival tissue. Seventy-eight patients with primary pterygium were enrolled. Healthy conjunctival graft specimens obtained during pterygium surgery were used as control tissue. The real-time polymerase chain reaction method on the BioMark HD dynamic array system was utilized in genomic mRNA for the gene expression analysis. Protein expressions were analyzed using western blot and immunohistochemical methods. RAC1, RAC2, and RAC3 gene expressions in pterygial tissues were not markedly elevated when compared to the control specimens (p>0.05). As a very low level of RAC1 gene expression was observed, further protein expression analysis was performed for the Rac2 and Rac3 proteins. Western blot and immunohistochemical analysis of Rac2 and Rac3 protein expression revealed no significant differences between pterygial and healthy tissues (p>0.05). This is the first study to identify the contribution of Rac proteins in pterygium. Our results indicate that the small GTP-binding protein Rac may not be involved in pterygium pathogenesis.

RAC1, a Potential Diagnostic and Prognostic Marker for Diffuse Large B Cell Lymphoma

The gene changes for diagnosis and prognosis of diffuse large B cell lymphoma (DLBCL) still remain unclear. RAC1 was reported to be asso;ciated with the B cell receptor signal pathway, but its relations with DLBCL have not yet been systematically explored. In this study, we have conducted molecular, bioinformatics and clinical analyses by using publicly available data from The Cancer Genome Atlas (TCGA). Wilcoxon signed-rank test and logistic regression were performed to evaluate the association between RAC1 and clinical features in patients. Kaplan-Meier and Cox regression methods were used to examine the impacts of RAC1 expression level on overall survival, and a nomogram was performed to illustrate the correlation between RAC1 and the risk of DLBCL. Our results revealed that the expression level of RAC1 in DLBCL was higher than that in normal tissues or lymphadenitis. High-level expression of RAC1 was significantly associated with clinical stage, as well as being an independent factor affecting overall survival. RAC1 was negatively correlated with Bruton's tyrosine kinase (BTK). The association between RAC1 gene expression and the risk of DLBCL was presented in a nomogram. In conclusion, RAC1 expression patterns may be used to predict the development and prognosis of DLBCL.

P.167: Gene Expression and Function in Pig Islets From One-day-old Pigs

Introduction: Islet transplantation is a budding alternative treatment for type 1 diabetics. A potential solution to the limited number of human pancreas donors is pig islet xenotransplantation. Limited study has been performed on pig islet cell signaling pathways, which may differ from the mechanisms seen in humans. We have identified molecules involved in the insulin secretion pathway, including the glucose transporter GLUT2, GTPase RAC1, which is involved in F-actin remodeling in islet cells, and SNAP25, a protein involved in insulin release. We evaluated these molecules across different days of culture in neonatal pig islets. We also quantified the gene expression of common cadherins associated with cell-cell adhesion and islet function as determined by insulin secretion. Method: Islets from 1-day-old pigs (n=3) were isolated and samples collected on days 0, 3, 5 and 7 of culture to quantify GLUT2, SNAP25, RAC1, E-, N- and VE-cadherin gene expression by qRT-PCR. Immunofluorescence staining was performed to localize protein expression. Static glucose stimulation insulin secretion assays (GSIS) and ELISA were used to quantify the ability of these islets to secrete insulin under conditions containing low glucose, high glucose and high glucose with KCl. Kruskal-Wallis testing was completed with GraphPad Prism software. Results: Qualitatively, the islets developed visible membranes and had fewer contaminating exocrine cells as they moved from 0 to 7 days in culture. E-cadherin, SNAP25 and GLUT2 all peaked on day 5 of culture, correlating with when islets formed a distinct spherical structure in-vitro. N-cadherin consistently rose, peaking at day 7 of culture. VE-cadherin was highest at day 0 and decreased with a linear trend. RAC1 gene expression decreased from day 0, remaining stable between days 5 and 7. P-values are indicated in Figure 1. Preliminary immunofluorescence staining showed co-localization of SNAP25 and insulin at all-time points. The amount of insulin secreted in low vs high glucose settings was similar without KCl, and as expected, the amount of insulin secreted when islets were cultured in high glucose with KCl was greater. Kruskal-Wallis testing demonstrated significant differences amongst GSIS groups (Figure 2). Discussion/Conclusion: Our data demonstrates variation in the gene expression of our molecules of interest across 7 days of culture. Our immunofluorescence results demonstrate that SNAP25 colocalized with insulin, suggesting their proximity in islet cells. Our results also demonstrate the limited ability of glucose to stimulate islets from 1-day-old pigs at 7 days of culture. This is different than results seen previously in our lab suggesting islets from 7-day-old pigs at 7 days of culture secrete greater amounts of insulin when exposed to high glucose conditions. These findings may be important for the development of pig islets in vitro and warrant further investigation.Royal College of Physicians and Surgeons of Canada (RCPSC) - Clinician Investigator Program. Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant. Canadian Institutes of Health Research (CIHR) - Canadian Graduate Scholarships - Master’s Program. Walter H. John’s Graduate Fellowship (University of Alberta). Alberta Graduate Excellence Scholarship (AGES).

Assessment of Alveolar Macrophage Dysfunction Using an in vitro Model of Acute Respiratory Distress Syndrome.

Background: Impaired alveolar macrophage (AM) efferocytosis may contribute to acute respiratory distress syndrome (ARDS) pathogenesis; however, studies are limited by the difficulty in obtaining primary AMs from patients with ARDS. Our objective was to determine whether an in vitro model of ARDS can recapitulate the same AM functional defect observed in vivo and be used to further investigate pathophysiological mechanisms.Methods: AMs were isolated from the lung tissue of patients undergoing lobectomy and then treated with pooled bronchoalveolar lavage (BAL) fluid previously collected from patients with ARDS. AM phenotype and effector functions (efferocytosis and phagocytosis) were assessed by flow cytometry. Rac1 gene expression was assessed using quantitative real-time PCR.Results: ARDS BAL treatment of AMs decreased efferocytosis (p = 0.0006) and Rac1 gene expression (p = 0.016); however, bacterial phagocytosis was preserved. Expression of AM efferocytosis receptors MerTK (p = 0.015) and CD206 (p = 0.006) increased, whereas expression of the antiefferocytosis receptor SIRPα decreased following ARDS BAL treatment (p = 0.036). Rho-associated kinase (ROCK) inhibition partially restored AM efferocytosis in an in vitro model of ARDS (p = 0.009).Conclusions: Treatment of lung resection tissue AMs with ARDS BAL fluid induces impairment in efferocytosis similar to that observed in patients with ARDS. However, AM phagocytosis is preserved following ARDS BAL treatment. This specific impairment in AM efferocytosis can be partially restored by inhibition of ROCK. This in vitro model of ARDS is a useful tool to investigate the mechanisms by which the inflammatory alveolar microenvironment of ARDS induces AM dysfunction.

Downregulation of Rac1/PAK1/LIMK1/cofilin signaling pathway in colon cancer SW620 cells treated with Chlorin e6 photodynamic therapy.

Colorectal cancer is one of the most common gastrointestinal malignancies. Photodynamic therapy (PDT) is a novel and non-invasive treatment for tumors as PDT features small trauma, good applicability, andaccurate targeting. PDT may also be a potential treatment for colon cancer as itmay may induce suppressive effects on metastatic potential.. However, the molecular mechanism of the Chlorin e6 Photodynamic therapy (Ce6-PDT) inhibiting the migration of human colon cancer SW620 cells remains unclear. Scratch wound healing assay, scanning electron microscope, MTT, immunofluorescence and laser confocal technique were used to investigate the suppressive effects of Ce6-PDT on the SW620 cells migration, pseudopodia, viability and the actin cytoskeleton. The effect of Ce6-PDT on actin-Filaments and signaling molecules of the Rac1/PAK1/LIMK1/cofilin signaling pathway in SW620 cells were examined by western blot analysis. RNA interference (RNAi) technology was used to establish siRNA-Rac1/SW620 cells. The combined effects of Ce6-PDT and RNAi on colon cancer SW620 cells was investigated by the same technology and methods mentioned above to clarify the signal transduction effect of Rac1/PAK1/LIMK1/cofilin signaling pathway in Ce6-PDT caused inhibition of SW620 cell migration. The healing and migration rate of the SW620 cells was significantly reduced and the cell pseudopodia were reduced or disappeared by Ce6-PDT. The Immunofluorescence and western blot analysis results showed that Ce6-PDT destroy microfilament's original structure and significantly downregulated F-actin protein expression. The Rac1/PAK1/LIMK1/cofilin signaling pathway was downregulated by Ce6-PDT. Furthermore, the RNAi significantly strengthened the effect of Ce6-PDT on colon cancer SW620 cells migration. Actin cytoskeleton and protrusions of SW620 cells correlate with its migration ability. Ce6-PDT suppresses SW620 cells migration by downregulating the Rac1/PAK1/LIMK1/cofilin signaling pathway, and its suppressive effect was enhanced by knocking down Rac1 gene expression.

Clostridioides difficile Toxin A-Induced Wnt/β-Catenin Pathway Inhibition Is Mediated by Rac1 Glucosylation.

Clostridioides difficile toxin A (TcdA) has been shown to inhibit cellular Wnt signaling, the major driving force behind the proliferation of epithelial cells in colonic crypts, likely through the inhibition of β-catenin nuclear translocation. Herein, we aimed to advance the understanding of this mechanism by replicating the findings in vivo and by investigating the specific role of Rac1, a member of the Rho GTPase family, on the inhibition of the Wnt-induced β-catenin nuclear translocation triggered by TcdA. To investigate the effects of TcdA on the Wnt/β-catenin pathway in vivo, we injected the ileal loops of C57BL/6 mice with TcdA [phosphate-buffered saline (PBS) as the control] to induce C. difficile disease-like ileitis. After 4 h post-injection, we obtained ileum tissue samples to assess Wnt signaling activation and cell proliferation through Western blotting, immunohistochemistry, and qPCR. To assess the role of Rac1 on Wnt signaling inhibition by TcdA, we transfected rat intestinal epithelial cells (IEC-6) with either a constitutively active Rac1 plasmid (pcDNA3-EGFP-Rac1-Q61L) or an empty vector, which served as the control. We incubated these cells with Wnt3a-conditioned medium (Wnt3a-CM) to induce Wnt/β-catenin pathway activation, and then challenged the cells with TcdA. We assessed Wnt signaling activation in vitro with TOP/FOPflash luciferase assays, determined nuclear β-catenin translocation by immunofluorescence, measured cyclin D1 protein expression by Western blotting, and quantified cell proliferation by Ki67 immunostaining. In vivo, TcdA decreased β-catenin, cyclin D1, and cMYC expression and inhibited the translocation of β-catenin into the nucleus in the ileum epithelial cells. In addition, TcdA suppressed cell proliferation and increased Wnt3a expression, but did not alter Rac1 gene expression in the ileum tissue. In vitro, constitutively active Rac1 prevented Wnt signaling inhibition by enabling the β-catenin nuclear translocation that had been blocked by TcdA. Our results show that TcdA inhibits Wnt/β-catenin pathway in vivo and demonstrate that this inhibition is likely caused by a Rac1-mediated mechanism.

RAC1 expression and role in IL-1β production and oxidative stress generation in familial Mediterranean fever (FMF) patients

Familial Mediterranean fever (FMF) is a recessively inherited autoinflammatory disorder. The caspase-1-dependent cytokine, IL-1β, plays an important role in FMF pathogenesis, and RAC1 protein has been recently involved in IL-1β secretion. This study aims to investigate RAC1 expression and role in IL-1β and caspase-1 production and oxidative stress generation in FMF. The study included 25 FMF patients (nine during attack and remission, and 16 during remission only), and 25 controls. RAC1 expression levels were analyzed by real-time PCR. Ex vivo production of caspase-1, IL-1β, IL-6 and markers of oxidative stress (malondialdehyde, catalase, and glutathione system) were evaluated respectively in supernatants of patients' and controls' PBMC and PMN cultures, in the presence and absence of RAC1 inhibitor. RAC1 gene expression and IL-1β levels were increased in patients in crises compared to those in remission or controls. RAC1 expression levels were correlated with MEFV genotypes, patients carrying the M694V/M694V genotype having a two-fold increase in the expression levels compared to those carrying other genotypes. Caspase-1 levels were higher in LPS-induced PBMC of patients in remission than controls. Spontaneous and LPS-induced IL-1β production were comparable in patients in remission and controls, whereas LPS-induced IL-6 production was enhanced in patients, compared to controls. RAC1 inhibition resulted in a decrease in caspase-1 and IL-1β, but not IL-6, levels. Malondialdehyde levels produced by LPS-stimulated PMNs were increased in patients in remission compared to those in controls, but decreased following RAC1 inhibition. Catalase and GSH activities were reduced in unstimulated PMN culture supernatants of patients in remission compared to controls and were increased in the presence of RAC1 inhibitor. These results show the involvement of RAC1 in the inflammatory process of FMF by enhancing IL-1β production, through caspase-1 activation, and generating oxidative stress, even during asymptomatic periods.

Abstract MIP-061: RAC 1 AND CDC42 GTPASES AS REGULATORS OF OVARIAN CANCER PROGRESSION AND NICHE INTERACTIONS

Abstract PURPOSE OF THE STUDY: The Ras-homologous (Rho) family GTPases Rac1 and Cdc42 contribute to metastatic dissemination through regulation of actin reorganization, cell motility, cell-cell and cell-extracellular matrix adhesion. The purpose of the study was to investigate the contributions of these GTPases in ovarian cancer progression. EXPERIMENTAL PROCEDURES: GTPase target expression and activity was determined by analysis of The Cancer Genome Atlas (TCGA), immunohistochemistry of ovarian cancer tissues and enzymatic assays of patient samples. The effects Rac1 and Cdc42 expression were investigated by comparing cell behaviors in vitro and in vivo in engineered cell lines and use of inhibitors of Rac1, Cdc42 or dual inhibition by the R-enantiomer of ketorolac. SUMMARY OF THE DATA: Expression of the constitutively active Rac1 splice variant Rac1b and elevation of Cdc42 protein were detected in advanced ovarian cancer specimens thus providing the first evidence for dysregulation of these GTPase targets in ovarian cancer. To confirm the importance of Rac1, RNA-seq gene expression data from 305 Stage III or IV ovarian cancer samples were downloaded from The Cancer Genome Atlas (TCGA) and evaluated for RAC1 gene expression. The samples with the highest Rac1 RNA levels had significantly worse (p=0.039) age adjusted survival than the patients with the lowest Rac1 levels. Additional analyses identified more than 4,000 genes that were differentially expressed (FDR corrected p <0.05) between these patient groups. Gene Ontology analysis revealed several highly enriched biological processes including: extracellular matrix organization (67 genes p=1.60E-25), multicellular organismal catabolic processes (29 genes p=4.30E-22), regulation of epithelial cell proliferation (30 genes p=6.10E-09 and regulation of cell migration (48 genes p=1.50E-08). Relevance of the pathway analyses is illustrated by experimental evidence that increased GTPase expression leads to enhanced homing and engraftment in the omental niche; suggesting that elevated activity detected in patients may contribute to disease progression. Conversely, dual inhibition of Rac1 and Cdc42 activity by R-ketorolac inhibits ovarian patient-derived tumor cell adhesion and invasion, omental homing and tumor implantation in a mouse xenograft model. CONCLUSIONS: These findings indicate that high expression of Rac1 is associated with late stage disease and/or poor patient survival and with genes involved in extracellular matrix, cell proliferation and cell migration. These functions of Rac1 were confirmed in experimental models and inhibition of protein activity provided anti-tumor benefit in vivo. Together, these data suggest that Rac1 and/or Cdc42 function as drivers of ovarian cancer and may represent novel therapeutic targets to improve ovarian cancer outcome. Citation Format: Hudson LG, Kenney SR, Rivera, M., Weisend, J. Luo, L., Ness, S., Gillette, JM, Wandinger-Ness A. RAC 1 AND CDC42 GTPASES AS REGULATORS OF OVARIAN CANCER PROGRESSION AND NICHE INTERACTIONS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-061.

Effect of Quercetin on RAC1 Gene Expression as a Marker of Metastasis in Cervical Cancer Cells

Background:: Cancer metastasis is the most important cause of cancer death and different treatment strategies have targeted on preventing the occurrence of metastasis. Quercetin is a flavonoid and widely used as an antioxidant and recent studies have discovered its anticancer and antiproliferative capabilities. Rac1 is protein known to involve in tumor invasion and metastases. Objectives:: In this study, we investigated the effect of quercetin on expression of Rac1 protein. Materials and Methods:: In this experimental study, HeLa cells were treated with quercetin at 2 concentrations (20 and 40 μM) for 24 hours and then cell lysate was assessed by Western blotting analysis to investigate the impact of quercetin on expression of Rac1 protein. Results:: Quercetin significantly decreased Rac1 expression of HeLa cells assessed by western blotting, in dose-dependent manners (P < 0.05). Conclusions:: This study showed that quercetin decreased Rac1 expression as a marker of metastasis in cervical cancer cells. Therefore, the quercetin may provide an effective new strategy to reduce of tumor metastasis.

GroEL1, a Heat Shock Protein 60 of Chlamydia pneumoniae, Induces Lectin-Like Oxidized Low-Density Lipoprotein Receptor 1 Expression in Endothelial Cells and Enhances Atherogenesis in Hypercholesterolemic Rabbits

Lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) plays a major role in oxidized low-density lipoprotein-induced vascular inflammation. Chlamydia pneumoniae has been found in atherosclerotic lesions and is related to atherosclerotic pathogenesis, although its specific mechanism remains unknown. This study was conducted to investigate the mechanisms of LOX-1 expression in GroEL1 (a heat shock protein from C. pneumoniae)-administered human coronary artery endothelial cells (HCAECs) and atherogenesis in hypercholesterolemic rabbits. We demonstrated that in the hypercholesterolemic rabbit model, GroEL1 administration enhanced fatty streak and macrophage infiltration in atherosclerotic lesions, which may be mediated by elevated LOX-1 expression. In in vitro study using HCAECs, stimulation with GroEL1 increased TLR4 and LOX-1 expression. Increased LOX-1 expression was downregulated by Akt activation and PI3K-mediated endothelial NO synthase activation. PI3K inhibitor and NO synthase inhibitor induced LOX-1 mRNA production, whereas the NO donor ameliorated the increasing effect of LOX-1 mRNA in GroEL1-stimulated HCAECs. LOX-1 expression was regulated by NADPH oxidase, which mediates reactive oxygen species production and intracellular MAPK signaling pathway in GroEL1-stimulated HCAECs. Treatment with polyethylene-glycol-conjugated superoxide dismutase, apocynin, or diphenylene iodonium significantly decreased GroEL1-induced LOX-1 expression, as did the knockdown of Rac1 gene expression by RNA interference. In conclusion, the GroEL1 protein may induce LOX-1 expression in endothelial cells and atherogenesis in hypercholesterolemic rabbits. The elevated level of LOX-1 in vitro may be mediated by the PI3K-Akt signaling pathway, endothelial NO synthase activation, NADPH oxidase-mediated reactive oxygen species production, and MAPK activation in GroEL1-stimulated HCAECs. The GroEL1 protein of C. pneumoniae may contribute to vascular inflammation and cardiovascular disorders.

Inhibition of hypoxia-induced retinal neovascularization in mice with short hairpin RNA targeting Rac1, possibly via blockading redox signaling

NADPH oxidase-derived reactive oxygen species are involved in angiogenesis in vitro and regulated by ras-related C3 botulinum toxin substrate 1 (Rac1). This study has employed vector-based short hairpin RNA targeting Rac1 (Rac1–shRNA) to investigate the inhibitory effect on hypoxia-induced retinal neovascularization (RN) in vivo and the underlying mechanism. pSUPER–Rac1–shRNA was intravitreally injected into the mouse model of oxygen-induced retinopathy. RN was evaluated by FITC–dextran angiography and quantitated histologically. Expressions of Rac1, nuclear factor kappa B (NF-κB) subunit p65, hypoxia-inducible factor-1 alpha (HIF-1α), and vascular endothelial growth factor (VEGF) were determined by real-time quantitative RT-PCR and western blotting. After intravitreal administration of pSUPER–Rac1–shRNA, retinal Rac1 gene expression was reduced by 72% at postnatal day 17 (P17). Retinal flat mount and quantification of the neovascular nuclei demonstrated that RN was significantly inhibited. Meanwhile, the expression levels of NF-κB and HIF-1α, the redox-dependent transcription factors, were significantly downregulated. HIF-1α and its downstream gene VEGF were found to be significantly decreased at both transcriptional and translational levels. Our findings not only suggest that Rac1 may be involved in the process of RN in mouse oxygen-induced retinopathy via regulating the redox signaling, but may also provide a novel therapeutic target for hypoxia-induced retinal neovascular diseases.

Investigation of the effects and mechanisms in exogenous Rac1 gene expression on HT1080fibrosarcoma cell invasion across collagen barrier

Objective To investigate the effects and relevant mechanisms of exogenous Rac1 gene expression on HT1080 fibrosarcoma cell invasion across collagen barrier. Methods HT1080 fibrosarcoma cell lines that stably expressed transfected dominant negative [Rac1V12N17(HN)], constitutively active Rac1 [Rac1V12(HV)] and vector(HW) respectively were used. Structure of actin cytoskeleton was stained with Texas Red-conjugated phalloidin to show the morphological characters of the cells cultured in 3D medium containing collagen protein. Assay of cell invasion across collagen barrier was performed on a thin layer of collagen gel covered the membrane of transwell chamber, and two kinds of protease inhibitor were used to observe their effects on above-mentioned invasive assay. Gelatin substrate zymography were used to detect secreted MMP activity in the medium of cells cultured in 3D matrix. Results HT1080 cells stably expressing Racl mutant exhibited distinct morphological and invasive properties, and the increased invasive ability could be eradicated after using MMP inhibitor. Exogenous Rac1 gene expression on HT1080 fibrosarcoma cell could facilitate the activation of MMP-2 secreted in the medium of either collagen or fibrin 3D cell culture system.Conclusion The stable expression of exogenous Rac1 gene in HT1080 fibrosarcoma cells could induce aggregation of actin fiber and promote invasivc property. The enhancement of MMP-2 activation by exogenous Rac1 gene expression may be one of the relevant mechanisms. Key words: Gene, Rac1; Fibrosarcoma; Matrix metalloproteinase-2; Collagen

The Role of Human Antigen R, an RNA-binding Protein, in Mediating the Stabilization of Toll-Like Receptor 4 mRNA Induced by Endotoxin

Lipopolysaccharide (LPS) interacts with toll-like receptor 4 (TLR4) and induces proliferation of vascular smooth muscle cells (VSMCs) which plays a causal role in atherogenesis. The role of TLR4 expression and regulation in LPS-stimulated VSMCs remains unclear. TLR4 mRNAs often contain AU-rich elements (AREs) in their 3' untranslated regions (3'UTR) which have a high affinity for RNA-binding proteins. It is not know whether the RNA-binding protein, human antigen R (HuR), regulates TLR4 expression in human aortic smooth muscle cells (HASMCs). Stimulation of HASMCs with LPS significantly increased the cytosolic HuR level in vitro. Immunoprecipitation and RT-PCR demonstrated that LPS markedly increased the interaction of HuR and 3'UTR of TLR4 mRNA. The reporter plasmid, which contains the 3'UTR of TLR4 mRNA, significantly increased luciferase reporter gene expression in LPS-induced HASMCs. These data suggest that the 3'UTR of TLR4 mRNA confers LPS responsiveness and that HuR modulates 3'UTR-mediated gene expression. Knock-down of HuR inhibited LPS-induced TLR4 mRNA stability in HASMCs and luciferase reporter gene expression in CMV-Luciferase-TLR4 3'UTR-transfected HASMCs. In addition, inhibition of NADPH oxidase activity by diphenylene iodonium, knock-down of Rac1 gene expression by siRNA, and decrease of p38 MAPK activity by SB203580 significantly decreased the cytosolic HuR level, which mediates TLR4 mRNA stability. Activation of NADPH oxidase and the MAPK-signaling pathway contribute to HuR-mediated stabilization of TLR4 mRNA induced by LPS in HASMCs. In the balloon injured rabbit aorta model, systemic inflammation induced by LPS caused intimal hyperplasia and increased TLR4 and HuR expression.