Rho GDP Dissociation Inhibitor Beta Research Articles

Differential functions of RhoGDIβ in malignant transformation and progression of urothelial cell following N-butyl-N-(4-hydmoxybutyl) nitrosamine exposure

BackgroundFunctional role of Rho GDP-dissociation inhibitor beta (RhoGDIβ) in tumor biology appears to be contradictory across various studies. Thus, the exploration of the molecular mechanisms underlying the differential functions of this protein in urinary bladder carcinogenesis is highly significant in the field. Here, RhoGDIβ expression patterns, biological functions, and mechanisms leading to transformation and progression of human urothelial cells (UROtsa cells) were evaluated following varying lengths of exposure to the bladder carcinogen N-butyl-N-(4-hydmoxybutyl) nitrosamine (BBN).ResultsIt was seen that compared to expression in vehicle-treated control cells, RhoGDIβ protein expression was downregulated after 2-month of BBN exposure, but upregulated after 6-month of exposure. Assessments of cell function showed that RhoGDIβ inhibited UROtsa cell growth in cells with BBN for 2-month exposure, whereas it promoted the invasion of cells treated with BBN for 6 months. Mechanistic studies revealed that 2-month of BBN exposure markedly attenuated DNMT3a abundance, and this led to reduced miR-219a promoter methylation, increased miR-219a binding to the RhoGDIβ mRNA 3’UTR, and reduced RhoGDIβ protein translation. While after 6-mo of BBN treatment, the cells showed increased PP2A/JNK/C-Jun axis phosphorylation and this in turn mediated overall RhoGDIβ mRNA transcription and protein expression as well as invasion.ConclusionsThese findings indicate that RhoGDIβ is likely to inhibit the transformation of human urothelial cells during the early phase of BBN exposure, whereas it promotes invasion of the transformed/progressed urothelial cells in the late stage of BBN exposure. The studies also suggest that RhoGDIβ may be a useful biomarker for evaluating the progression of human bladder cancers.

PD-L1 enhances migration and invasion of trophoblasts by upregulating ARHGDIB via transcription factor PU.1

As the main constituent cells of the human placenta, trophoblasts proliferate, differentiate, and invade the uterine endometrium via a series of processes, which are regulated exquisitely through intercellular signaling mediated by hormones, cytokines, and growth factors. Programmed cell death ligand 1 (PD-L1) is a biomarker of the response to immune checkpoint inhibitors and can regulate maternal-fetal immune tolerance during pregnancy progression. Recently, it was found that PD-L1 may regulate obstetric complications by affecting the function of trophoblasts. Therefore, we examined the expression and localization of PD-L1 in the human placenta and observed the effects of PD-L1 on trophoblasts migration and invasion in both the trophoblasts line HTR-8/SVneo and an extravillous explant culture model. Finally, we explored the molecular mechanisms underlying PD-L1-regulated trophoblasts migration and invasion through RNA sequencing and bioinformatics analysis. Our data showed that PD-L1 was mainly expressed in syncytiotrophoblasts and that its protein levels increased with gestational age. Interestingly, the protein expression of PD-L1 was significantly decreased in placentas from pregnancies with preeclampsia compared with normal placentas. Importantly, the migration and invasion abilities of trophoblasts were significantly changed after knockdown or overexpression of PD-L1 in HTR-8/SVneo cells and an extravillous explant culture model, which was partially mediated through the transcription factor PU.1 (encoded by Spi1)-regulated Rho GDP-dissociation inhibitor beta (ARHGDIB) expression. These results suggested that PD-L1 was highly involved in the regulation of trophoblasts migration and invasion, providing a potential target for the diagnosis and treatment of placenta-derived pregnancy disorders.

FHL1 Overexpression as A Inhibitor of Lung Cancer Cell Invasion via Increasing RhoGDIß mRNA Expression.

Objective Four and a half Lin-11, Isl-1, Mac-3 (LIM) protein 1 (FHL1) is one of the FHL protein family, which is regarded as a tumor suppressor in the multiple malignant tumors. In this study, we aimed to explore the regulatory effects and mechanisms of FHL1 on lung cancer cell invasion. Materials and Methods In this experimental study, bioinformatics analysis of FHL1 transcripts in human lung adenocarcinomas of TCGA database was performed. Quantitative real-time polymerasechain reaction (PCR) was performed to detect FHL1 mRNA expression in 15 paired human lung cancer tissues and their adjacent normal lung tissues, or lung cancer cell lines (A549 and H1299) in comparison with human bronchial epithelial cell line (Beas- 2B). Moreover, western blot was used to analyze FHL1 and rho GDP-dissociation inhibitor beta (RhoGDIβ) protein expression in the indicated cell lines. Also, transwell assays were employed to measure the migrated, and invaded of indicated cell lines. Results FHL1 transcripts were downregulated in the human lung adenocarcinoma. The impaired FHL1 transcripts were positively correlated with advanced tumor node metastasis (TNM) stage. Moreover, as compared to the adjacent normal lung tissues, FHL1 mRNA was low expressed in 15 paired human lung cancer tissues than their adjacent normal lung tissues. Besides, FHL1 mRNA and protein expression were also reduced in H1299 and A549 cell lines in comparison with Beas-2B cell line. Overexpressed FHL1 protein inhibited the invasive ability of H1299 and A549 cell lines. Mechanically, FHL1 protein overexpression increased the RhoGDIβ protein and mRNA abundance, while knockdown of RhoGDIβ protein, completely restored the invasion ability of A549 (Flag-FHL1) cell line. ConclusionOur findings indicated that as a key FHL1 downstream regulator, RhoGDIβ is in charge of FHL1 inhibiting lung cancer cell invasion abilities, providing a critical insight into understanding the role of FHL1 for lung cancer development.

Anti-D4GDI antibodies activate platelets in vitro: a possible link with thrombocytopenia in primary antiphospholipid syndrome

BackgroundThrombocytopenia is a manifestation associated with primary antiphospholipid syndrome (PAPS), and many studies have stressed the leading role played by platelets in the pathogenesis of antiphospholipid syndrome (APS). Platelets are highly specialized cells, and their activation involves a series of rapid rearrangements of the actin cytoskeleton. Recently, we described the presence of autoantibodies against D4GDI (Rho GDP dissociation inhibitor beta, ARHGDIB) in the serum of a large subset of SLE patients, and we observed that anti-D4GDI antibodies activated the cytoskeleton remodeling of lymphocytes by inhibiting D4GDI and allowing the upregulation of Rho GTPases, such as Rac1. Proteomic and transcriptomic studies indicate that D4GDI is very abundant in platelets, and small GTPases of the RHO family are critical regulators of actin dynamics in platelets.MethodsWe enrolled 38 PAPS patients, 15 patients carrying only antiphospholipid antibodies without clinical criteria of APS (aPL carriers) and 20 normal healthy subjects. Sera were stored at − 20 °C to perform an ELISA test to evaluate the presence of anti-D4GDI antibodies. Then, we purified autoantibodies anti-D4GDI from patient sera. These antibodies were used to conduct in vitro studies on platelet activation.ResultsWe identified anti-D4GDI antibodies in sera from 18/38 (47%) patients with PAPS, in sera from 2/15(13%) aPL carriers, but in no sera from normal healthy subjects. Our in vitro results showed a significant 30% increase in the activation of integrin αIIbβ3 upon stimulation of platelets from healthy donors preincubated with the antibody anti-D4GDI purified from the serum of APS patients.ConclusionsIn conclusion, we show here that antibodies anti-D4GDI are present in the sera of PAPS patients and can prime platelet activation, explaining, at least in part, the pro-thrombotic state and the thrombocytopenia of PAPS patients. These findings may lead to improved diagnosis and treatment of APS.

Rho GDP dissociation inhibitor beta ARHGDIB in renal cell cancer.

474 Background: Rho GDP dissociation inhibitor 2 (ARHGDIB) is an important mediator of cellular signaling. The expression of ARHGDIB correlates with tumor growth and metastasis in a variety of non genitourinary cancers, however the role of ARHGDIB in renal cell cancer (RCC) has not yet been evaluated. Methods: Tissue samples from 106 patients undergoing surgery for RCC were obtained. The expression of ARHGDIB mRNA in normal kidney tissue and in corresponding cancer tissue was analyzed by means of quantitative real time PCR. Differences in mRNA expression levels were assessed using paired two-sample tests. Associations of relative mRNA expression levels and clinicopathological parameters were statistically analyzed using an univariate logistic regression model. Relative mRNA expression levels in healthy renal tissue compared to cancerous tissue from the same kidney was assessed using a paired t-test. Results: When comparing 74 tissues from kidney tumors with adjacent histologically normal appearing paired tissues, mRNA expression of ARHGDIB was significantly higher in the tumor tissue (p < 0.001). Paired analysis did not only show significantly higher mRNA expression levels for ARHGDIB over all RCC but also for the subgroup with clear cell RCC (ccRCC). The mRNA expression of ARHGDIB was also more pronounced in ccRCC when compared with papillary RCC (p < 0.001). When looking at clinicopathological parameters in univariate logistic regression analysis ccRCC was significantly associated with nodal involvement (p = 0.03) and also with tumor grade (p = 0.05). For all RCC there was no association with clinicopathological parameters. A bivariate Cox regression model, adjusted for metastatic status (p = 0.001), tumor diameter (p = 0.043), state of advanced disease (p = 0.030) and lymph node metastasis (p = 0.006) identified ARHGDIB mRNA expression as a candidate positive prognosticator for RFS. Conclusions: Increased ARHGDIB mRNA expression is significantly associated with RCC tissues. Higher relative expression observed within tumor tissues represents a candidate prognosticator for better RFS of patients.

Rho GDP dissociation inhibitor beta promotes cell proliferation and invasion by modulating the AKT pathway in hepatocellular carcinoma.

Rho GDP dissociation inhibitor (GDI) beta, (RhoGDI2), has been identified as a proto-oncogene that is upregulated in human cancers, but the role of RhoGDI2 in hepatocellular carcinoma (HCC) remains unclear. In the present study, we investigated the RhoGDI2 expression level in HCC tissues and the function of RhoGDI2 in HCC cell growth and metastasis. We examined the RhoGDI2 mRNA expression level in 64 sets of HCC tissue and their adjacent nontumor tissue counterparts using quantitative real-time polymerase chain reaction. In vitro proliferation and invasion assays were conducted to determine the effect of RhoGDI2 on the ability of HCC cells to proliferate and invade, respectively. Western blot analysis was conducted to examine expression levels of RhoGDI2p-AKT, MMP-2, and MMP-9 in HCC cells. RhoGDI2 mRNA was significantly overexpressed in the HCC specimens compared with the nonneoplastic liver specimens, and the RhoGDI2 mRNA and protein levels were higher in the HCC cell lines, especially the highly metastatic cell lines 97L and 97H. To further investigate the role that RhoGDI2 plays in HCC, we overexpressed RhoGDI2 using a lentivirus-mediated overexpression technique in two HCC cell lines (Huh7 and 7721) that endogenously express a low level of RhoGDI2. Stable cells overexpressing RhoGDI2 demonstrated a significant increase in cell proliferation and invasion. Furthermore, our additional findings indicated that RhoGDI2-mediated cellular invasion requires the PI3K/Akt signaling-dependent expression of matrix metalloproteinases (MMPs). Our findings suggest that RhoGDI2 plays an important role in HCC growth and invasion and should be considered a novel HCC therapeutic target candidate.

Cox-2 is a target gene of Rho GDP dissociation inhibitor beta in breast cancer cells

Cox-2 is a target gene of Rho GDP dissociation inhibitor beta in breast cancer cells

Differential protein expression in mouse splenic mononuclear cells treated with polysaccharides from spores of Ganoderma lucidum

Polysaccharides were one of the main components of Ganoderma lucidum, a medicinal mushroom well known for its immuno-modulation effects. In the present study, we demonstrated that polysaccharides extracted from Ganoderma lucidum spores (GL-SP) could stimulate splenic mononuclear cells (MNCs) proliferation and cytokine production. To identify the possible cellular targets of GL-SP in MNCs, two-dimensional gel electrophoresis (2-DE)-based comparative proteomics was performed and proteins altered in expressional level after GL-SP treatment were identified by MALDI-TOF MS/MS. Ten proteins with >2-fold increase or decrease expression in GL-SP-treated MNCs compared with control were found and further identified by MALDI-TOF MS/MS analysis and database searching. In the GL-SP-treated MNCs, there were increases in the expression of myosin regulatory light chain 2-A, Rho GDP dissociation inhibitor beta, T-cell-specific GTPase, phosphatidylinositol transfer protein alpha, and decreases in the expression of apoptosis-associated speck-like protein containing a CARD, 14-3-3 tau, beta-actin, tubulin alpha 2, copine I, and gamma-actin. The results of the present study help to provide insight into the immuno-modulating activities of GL-SP.

Identification of nasopharyngeal carcinoma antigens that induce humoral immune response by proteomic analysis

A proteomics-based approach has been used to identify proteins that commonly elicit a humoral immune response in nasopharyngeal carcinoma (NPC). Sera from 19 newly diagnosed NPC patients and 19 healthy individuals were analyzed for IgG autoantibodies against NPC proteins resolved by 2-DE. Protein spots that exhibited selective reactivity with sera from NPC patients were identified by MS. Among nine identified proteins, cytokeratin 19 (CK19), Erb3 binding protein (EBP1), and Rho GDP dissociation inhibitor-beta (Rho-GDI-2) induced autoantibodies in more than 36.8% of NPC patients but not in healthy individuals. Furthermore, Western blot analysis and immunohistochemical staining were performed to determine the expression and localization of CK19, EBP1, and Rho-GDI-2 in NPC and normal nasopharyngeal mucosal tissues. Up-regulated CK19 and EBP1, but not Rho-GDI-2, were observed in NPC vs. normal tissue. Subcellular localization of the three proteins in NPC tissue was same as that in the normal tissue. Thus, overexpression of CK19 and EBP1 may be one of the mechanisms for their autoantibody development in NPC. To validate the findings of a proteomic analysis, occurrence of autoantibodies against these three proteins was detected by immunoprecipitation and Western blot analysis in additional 30 NPC patients, 23 other types of cancer patients and 20 healthy individuals. Results showed that frequency of autoantibodies against CK19, EBP1 and Rho-GDI-2 in NPC patients was significantly higher than that in other types of cancer patients and healthy individuals. We conclude that CK19, EBP1 and Rho-GDI-2 may have utility in NPC screening and diagnosis.

A Glutathione S-Transferase π-Activated Prodrug Causes Kinase Activation Concurrent with S-Glutathionylation of Proteins

Nitric oxide (NO) is an endogenous, diffusible, transcellular messenger shown to affect regulatory and signaling pathways with impact on cell survival. Exposure to NO can impart direct post-translational modifications on target proteins such as nitration and/or nitrosylation. As an alternative, after interaction with oxygen, superoxide, glutathione, or certain metals, NO can lead to S-glutathionylation, a post-translational modification potentially critical to signaling pathways. A novel glutathione S-transferase pi (GSTpi)-activated pro-drug, O(2)-[2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl]1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO), liberates NO and elicits toxicity in vitro and in vivo. We now show that PABA/NO induces nitrosative stress, resulting in undetectable nitrosylation, limited nitration, and high levels of S-glutathionylation. After a single pharmacologically relevant dose of PABA/NO, S-glutathionylation occurs rapidly (<5 min) and is sustained for approximately 7 h, implying a half-life for the deglutathionylation process of approximately 3 h. Two-dimensional SDS-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody to S-glutathionylated residues indicated that numerous proteins were S-glutathionylated. Subsequent matrix-assisted laser desorption ionization/time of flight analysis identified 10 proteins, including beta-lactate dehydrogenase, Rho GDP dissociation inhibitor beta, ATP synthase, elongation factor 2, protein disulfide isomerase, nucleophosmin-1, chaperonin, actin, protein tyrosine phosphatase 1B (PTP1B), and glucosidase II. In addition, we showed that sustained S-glutathionylation was temporally concurrent with drug-induced activation of the stress kinases, known to be linked with cell death pathways. This is consistent with the fact that PABA/NO induces S-glutathionylation and inactivation of PTP1B, one phosphatase that can participate in deactivation of kinases. These effects were consistent with the presence of intracellular PABA/NO or metabolites, because cells overexpressing MRP1 were less sensitive to the drug and had reduced levels of S-glutathionylated proteins.

Holistic differential analysis of embryo‐induced alterations in the proteome of bovine endometrium in the preattachment period

During the peri-implantation period, molecular signaling between embryo and endometrium (layer of tissue lining the uterus lumen) is supposed to be crucial for the maintenance of pregnancy. To investigate embryo-induced alterations in the proteome of bovine endometrium in the preattachment period (day 18), we used monozygotic cattle twins (generated by embryo splitting) as a model eliminating genetic variability as a source for proteome differences. One of the twins was pregnant after the transfer of two in vitro produced blastocysts, while the corresponding twin received a sham-transfer and served as a nonpregnant control. The two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) analysis of the endometrium samples of three twin pairs (pregnant/nonpregnant) revealed four proteins with significantly higher abundance (p < 10(-9)) in each sample derived from the pregnant animals: Rho GDP dissociation inhibitor beta; 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD); soluble NADP(+)-dependent isocitrate dehydrogenase 1; and acyl-CoA-binding protein. To verify the accuracy of the 2-D DIGE quantification, the abundances of 20 alpha-HSD were quantified by a targeted cleavable isotope-coded affinity tag (ICAT) approach. The mass spectrometry-based ICAT quantification matched perfectly the results obtained by 2-D DIGE quantification, demonstrating the accuracy of our data. These results demonstrate that our model (monozygotic twins) in combination with the appropriate analytical tools is particularly suitable for the detection of the proteins involved in the embryo-maternal interactions.

Overexpression of autocrine motility factor in metastatic tumor cells: possible association with augmented expression of KIF3A and GDI-β

Autocrine motility factor (AMF), which is identical to phosphohexose isomerase (PHI)/glucose-6-phosphate isomerase (GPI) , a ubiquitous enzyme essential for glycolysis, neuroleukin (NLK), a neurotrophic growth factor, and maturation factor (MF) mediating the differentiation of human myeloid cells, enhances the motility and metastatic ability of tumor cells. AMF/PHI activity is elevated in the serum or urine in patients with malignant tumors. Here, we constructed an amf/phi/nlk/mf gene using adenovirus vector and transfected into two tumor cell lines. Overexpression of AMF/PHI/NLK/MF enhanced AMF secretion into the culture media in both tumor cell lines. However, upregulation of motility and metastatic ability was found only in metastatic fibrosarcoma cells expressing an AMF receptor, gp78, and was not found in gp78-undetectable osteosarcoma cells. Thus, not only serum AMF activity but also gp78-expression in tumor cells may be required for metastasis-related motility induction. With the use of microarray analyses, we detected two augmented genes, rho GDP dissociation inhibitor beta and kinesin motor 3A, as well as AMF itself. The RNA message and protein expression of these two molecules was confirmed to be upregulated, suggesting a possible association with AMF-induced signaling for cell motility and metastasis.