RhoGDIß inhibition via miR-200c/AUF1/SOX2/miR-137 axis contributed to lncRNA MEG3 downregulation-mediated malignant transformation of human bronchial epithelial cells.

Nickel pollution is a recognized factor contributing to lung cancer. Understanding the molecular mechanisms of its carcinogenic effects is crucial for lung cancer prevention and treatment. Our previous research identified the downregulation of a long noncoding RNA, maternally expressed gene 3 (MEG3), as a key factor in transforming human bronchial epithelial cells (HBECs) into malignant cells following nickel exposure. In our study, we found that deletion of MEG3 also reduced the expression of RhoGDIß. Notably, artificially increasing RhoGDIß levels counteracted the malignant transformation caused by MEG3 deletion in HBECs. This indicates that the reduction in RhoGDIß contributes to the transformation of HBECs due to MEG3 deletion. Further exploration revealed that MEG3 downregulation led to enhanced c-Jun activity, which in turn promoted miR-200c transcription. High levels of miR-200c subsequently increased the translation of AUF1 protein, stabilizing SOX2 messenger RNA (mRNA). This stabilization affected the regulation of miR-137, SP-1 protein translation, and the suppression of RhoGDIß mRNA transcription and protein expression, leading to cell transformation. Our study underscores the co-regulation of RhoGDIß expression by long noncoding RNA MEG3, multiple microRNAs (miR-200c and miR-137), and RNA-regulated transcription factors (c-Jun, SOX2, and SP1). This intricate network of molecular events sheds light on the nature of lung tumorigenesis. These novel findings pave the way for developing targeted strategies for the prevention and treatment of human lung cancer based on the MEG3/RhoGDIß pathway.

Complement component C3 and C5b-9 deposition on hypoxia reperfused endothelial cells by non-HLA antibodies against RhoGDI2: A player involved in graft failure?

Antibodies against Rho GDP-dissociation inhibitor 2 (RhoGDI2) are associated with inferior graft survival in transplant patients receiving a kidney from deceased donors. Although this suggests that these antibodies contribute to graft injury because of ischemia, it remains unknown whether they are also pathogenically involved in the process of graft loss. To study this, we firstly analyzed the IgG subclass profile of anti-RhoGDI2 antibodies in kidney transplant recipients, and whether antibody titers change over time or because of acute rejection. Next, we investigated the expression of RhoGDI2 on primary kidney and lung endothelial cells (ECs) upon hypoxia reperfusion. In addition, the complement-fixing properties of anti-RhoGDI2 antibodies were studied using imaging flow cytometry. Anti-RhoGDI2 antibodies in patients are mainly IgG1, and titers remained stable and seemed not be changed because of rejection. Antibodies against RhoGDI2, which surface expression seemed to increase upon hypoxia reperfusion, co-localized with C3 on ECs. Binding of human IgG1 monoclonal anti-RhoGDI2 antibodies as well as patient derived antibodies, resulted in complement activation, suggesting that these antibodies are complement fixing. This study suggested a potential pathogenic role of anti-RhoGDI2 antibodies in kidney graft loss. During ischemia reperfusion, the ability of these antibodies to fix complement could be one of the mechanisms resulting in tissue injury.

RhoGDI2 induced malignant phenotypes of pancreatic cancer cells via regulating Snail expression.

BACKGROUND: Rho GDP dissociation inhibitor 2 (RhoGDI2) has been shown to contribute to the aggressive phenotypes of human cancers, such as tumor metastasis and chemoresistance. OBJECTIVE: This study aimed to assess the effects of RhoGDI2 on tumor progression and chemoresistance in pancreatic cancer cells. METHODS: The expression of RhoGDI2 in pancreatic cancer cells was detected by Western blot analysis. Gain-of-function and loss-of-function approaches were done to examine the malignant phenotypes of the RhoGDI2-expressing or RhoGDI2-depleting cells. The correlation between RhoGDI2 and Snail was also analyzed. RESULTS: Differential expression of RhoGDI2 protein in pancreatic cancer cell lines was identified. Gain-of-function and loss-of-function experiments showed that RhoGDI2 induced the malignant phenotypes of pancreatic cancer cells, including proliferation, migration, invasion, and gemcitabine (GEM) chemoresistance. The upregulation of RhoGDI2 stimulated the expression of Snail, resulting in the altered expression of epithelial marker E-cadherin and mesenchymal marker Vimentin, which were characteristics of the tumorigenic activity of epithelial-mesenchymal transition. The expression of RhoGDI2 and Snail was upregulated in clinical tumor samples, and higher expression of RhoGDI2 or Snail was significantly associated with poor patient survival in pancreatic ductal adenocarcinoma (PDAC). CONCLUSION: The findings indicated that RhoGDI2 promoted GEM resistance and tumor progression in pancreatic cancer and that RhoGDI2 might be a potential therapeutic target in patients with PDAC.

Comprehensive analysis of miRNA-mRNA regulatory network and potential drugs in chronic chagasic cardiomyopathy across human and mouse.

BACKGROUND: Chronic chagasic cardiomyopathy (CCC) is the leading cause of heart failure in Latin America and often causes severe inflammation and fibrosis in the heart. Studies on myocardial function and its molecular mechanisms in patients with Chronic chagasic cardiomyopathy are very limited. In order to understand the development and progression of Chronic chagasic cardiomyopathy and find targets for its diagnosis and treatment, the field needs to better understand the exact molecular mechanisms involved in these processes. METHODS: The mRNA microarray datasets GSE84796 (human) and GSE24088 (mouse) were obtained from the Gene Expression Omnibus (GEO) database. Homologous genes between the two species were identified using the online database mining tool Biomart, followed by differential expression analysis, gene enrichment analysis and protein-protein interaction (PPI) network construction. Cytohubba plug-in of Cytoscape software was used to identify Hub gene, and miRNet was used to construct the corresponding miRNA-mRNA regulatory network. miRNA-related databases: miRDB, Targetscan and miRWalk were used to further evaluate miRNAs in the miRNA-mRNA network. Furthermore, Comparative Toxicogenomics Database (CTD) and L1000 Platform were used to identify hub gene-related drugs. RESULTS: A total of 86 homologous genes were significantly differentially expressed in the two datasets, including 73 genes with high expression and 13 genes with low expression. These differentially expressed genes were mainly enriched in the terms of innate immune response, signal transduction, protein binding, Natural killer cell mediated cytotoxicity, Tuberculosis, Chemokine signaling pathway, Chagas disease and PI3K-Akt signaling pathway. The top 10 hub genes LAPTM5, LCP1, HCLS1, CORO1A, CD48, TYROBP, RAC2, ARHGDIB, FERMT3 and NCF4 were identified from the PPI network. A total of 122 miRNAs were identified to target these hub genes and 30 of them regulated two or more hub genes at the same time. miRDB, Targetscan and miRWalk were further analyzed and screened out hsa-miR-34c-5p, hsa-miR-34a-5p and hsa-miR-16-5p as miRNAs regulating these hub genes. Finally, Progesterone, Flutamide, Nimesulide, Methotrexate and Temozolomide were identified to target these hub genes and might be targeted therapies for Chronic chagasic cardiomyopathy. CONCLUSIONS: In this study, the potential genes associated with Chronic chagasic cardiomyopathy are identified and a miRNA-mRNA regulatory network is constructed. This study explores the molecular mechanisms of Chronic chagasic cardiomyopathy and provides important clues for finding new therapeutic targets.

CRIF1 deficiency suppresses endothelial cell migration via upregulation of RhoGDI2.

Rho GDP-dissociation inhibitor (RhoGDI), a downregulator of Rho family GTPases, prevents nucleotide exchange and membrane association. It is responsible for the activation of Rho GTPases, which regulate a variety of cellular processes, such as migration. Although RhoGDI2 has been identified as a tumor suppressor gene involved in cellular migration and invasion, little is known about its role in vascular endothelial cell (EC) migration. CR6-interacting factor 1 (CRIF1) is a CR6/GADD45-interacting protein with important mitochondrial functions and regulation of cell growth. We examined the expression of RhoGDI2 in CRIF1-deficient human umbilical vein endothelial cells (HUVECs) and its role in cell migration. Expression of RhoGDI2 was found to be considerably higher in CRIF1-deficient HUVECs along with suppression of cell migration. Moreover, the phosphorylation levels of Akt and CREB were decreased in CRIF1-silenced cells. The Akt-CREB signaling pathway was implicated in the changes in endothelial cell migration caused by CRIF1 downregulation. In addition to RhoGDI2, we identified another factor that promotes migration and invasion of ECs. Adrenomedullin2 (ADM2) is an autocrine/paracrine factor that regulates vascular tone and other vascular functions. Endogenous ADM2 levels were elevated in CRIF1-silenced HUVECs with no effect on cell migration. However, siRNA-mediated depletion of RhoGDI2 or exogenous ADM2 administration significantly restored cell migration via the Akt-CREB signaling pathway. In conclusion, RhoGDI2 and ADM2 play important roles in the migration of CRIF1-deficient endothelial cells.

[Disruption of OsRhoGDI2 by CRISPR/Cas9 technology leads to semi-dwarf in rice].

OsRhoGDI2 was isolated as a putative partner of Rho protein family member OsRacD from rice panicles by yeast two-hybrid, but its function remains unknown. In order to identify the function of OsRhoGDI2, OsRhoGDI2 knockout mutants were created by CRISPR/Cas9 technology. The results showed that two different homozygous mutants were obtained in T0 generation, and eight kinds homozygous mutants were identified in T1 generation. Sequence analysis revealed that the base substitution or base deletion occurred near the editing targets of the gene in knockout rice, and it could be expected that the truncated OsRhoGDI2 proteins lacking the RhoGDI conserved domain would be generated. Phenotype analysis showed that the OsRhoGDI2 knockout rice plants were significantly lower than the control plants. Statistical analysis confirmed that the significant decrease of plant height was due to the shortening of the second and third internodes, suggesting that OsRhoGDI2 gene may be related with rice height control.

Systematic investigation of biomarker-like role of ARHGDIB in breast cancer.

BACKGROUND: ARHGDIB, a Rho GDP dissociation inhibitor protein, has been reported playing critical roles in regulation of multiple biological responses. However, whether ARHGDIB serves as a valuable biomarker in cancer is little known so far, especially in breast cancer. OBJECTIVE: In this study, we aimed to investigate the importance of ARHGDIB in breast cancer, including but not limited to biomarker-like role, as well as potential mechanisms. METHODS: Total 100 breast cancer samples and 100 benign breast disease samples were enrolled and underwent detailed pathological assessment and IHC analysis. Human breast cancer cell lines and epithelial cell line were subjected to siRNA-mediated knock-down, RT-qPCR, western blot, MTT staining, cell cycle assay, transwell analysis respectively. RESULTS: We observed the expression of ARHGDIB is significantly higher in human breast cancer tissues compared with the benign tissues. ARHGDIB expression was positively correlated with tumor size, lymph node metastasis and TNM stage in breast cancer patients. Moreover, ARHGDIB depletion decreased proliferation, migration and invasion of breast cancer cells. Furthermore, we found ARHGDIB mediated epithelial-mesenchymal transition, and MMP2 is the key downstream effector of ARHGDIB. CONCLUSIONS: Hence, our results suggested the significance and predictive role of ARHGDIB in breast cancer. High expression of ARHGDIB indicated the poor prognosis for breast cancer patients.

RhoGDI stability is regulated by SUMOylation and ubiquitination via the AT1 receptor and participates in Ang II-induced smooth muscle proliferation and vascular remodeling.

BACKGROUND AND AIMS: The physiological role of Rho-specific guanine nucleotide dissociation inhibitor (RhoGDI) in vascular remodeling remains unknown. We investigated the function of RhoGDI in angiotensin II (Ang II)-induced vascular remodeling in cultured human aortic vascular smooth muscle cells (HA-VSMCs) and in an Ang II-infusion vascular remodeling mouse model. METHODS: For in vitro assays of HA-VSMCs, proliferation was assessed by BrdU and EdU assays and immunofluorescence analysis of ki-67 expression. RhoGDI1 and RhoGDI2 function and expression were assessed by RNAi, Western blotting and real-time RT-PCR. RhoGDI ubiquitination and SUMOylation levels were evaluated by co-immunoprecipitation and Western blotting. The functions of proteosomal-mediated degradation, ubiquitination, SUMOylation and Ang II receptors were assessed using specific inhibitors. To evaluate the in vivo effects of Ang II and RhoGDI, H & E staining, Masson's trichrome staining, and immunostaining were employed. RESULTS: Ang II treatment of HA-VSMCs for 6 or 48 h promoted RhoGDI1 and RhoGDI2 protein degradation and reduced cell proliferation, which was reversed by proteosome inhibition. In contrast, treatment with Ang II for 12 or 24 h induced dose-dependent cell proliferation without affecting RhoGDI expression. RNA interference of either RhoGDI1 or RhoGDI2 blocked proliferation induced by 12 or 24 h treatment of Ang II. Moreover, Ang II-dependent degradation at 6 and 48 h correlated with RhoGDI ubiquitination and inversely correlated with RhoGDI SUMOylation and cell proliferation. Treatment with specific inhibitors suggests that ubiquitin and SUMO competitively bind to RhoGDI1 and RhoGDI2 to reciprocally regulate RhoGDI stability and HA-VSMC proliferation. Furthermore, inhibition of the Ang II receptor 1 (AT1 receptor), but not the Ang II receptor 2, blocked Ang II-dependent RhoGDI stabilization and proliferation at 12 and 24 h. In mice, Ang II infusion increased the intima-media thickness, collagen and myofiber production and VSMC proliferation, and these effects were shown to be dependent on RhoGDI1, RhoGDI2 and AT1 receptor. Ang II infusion exerted no significant effect on RhoGDI1 and RhoGDI2 protein levels, which were decreased after AT1 receptor inhibition. CONCLUSIONS: Together, the results of this study reveal a novel mechanism by which Ang II regulates RhoGDI stability by SUMOylation and ubiquitination via AT1 receptor activation and thus affects VSMC proliferation and vascular remodeling.

NFκB2 p52 stabilizes rhogdiß mRNA by inhibiting AUF1 protein degradation via a miR-145/Sp1/USP8-dependent axis.

Although overexpression of the non-canonical NFκB subunit p52 has been observed in several tumors, the function and mechanism of p52 in bladder cancer (BC) are less well understood. Here, we aimed at understanding the role and mechanism underlying p52 regulation of BC invasion. Human p52 was stably knockdown with shRNA targeting p52 in two bladder cancer cell lines (T24 and UMUC3). Two constitutively expressing constructs, p52 and p100, were stably transfected in to T24 or UMUC3, respectively. The stable transfectants were used to determine function and mechanisms responsible for p52 regulation of BC invasion. We demonstrate that p52 mediates human BC invasion. Knockdown of p52 impaired bladder cancer invasion by reduction of rhogdiß mRNA stability and expression. Positively regulation of rhogdiß mRNA stability was mediated by p52 promoting AUF1 protein degradation, consequently resulting in reduction of AUF1 binding to rhogdiß mRNA. Further studies indicated that AUF1 protein degradation was mediated by upregulating USP8 transcription, which was modulated by its negative regulatory transcription factor Sp1. Moreover, we found that p52 upregulated miR-145, which directly bound to the 3'-UTR of sp1 mRNA, leading to downregulation of Sp1 protein translation. Our results reveal a comprehensive pathway that p52 acts as a positive regulator of BC invasion by initiating a novel miR-145/Sp1/USP8/AUF1/RhoGDIß axis. These findings provide insight into the understanding of p52 in the pathology of human BC invasion and progression, which may be useful information in the development of preventive and therapeutic approaches for using p52 as a potential target.

Adenosine Inhibits Ovarian Cancer Growth Through Regulating RhoGDI2 Protein Expression.

OBJECTIVE: This study aimed to investigate the effect of adenosine (Ado) on the growth of ovarian cancer and to explore the related mechanisms. METHODS: The effect of Ado on the proliferation of A2780 human ovarian cancer cells was examined according to the MTT method. Moreover, the nude mouse model of subcutaneous A2780 xenograft was constructed, and then, Ado and cisplatin were administered intraperitoneally to investigate the effect of Ado on tumor growth in vivo. Immunohistochemistry (IHC) was carried out to study the effect of Ado on the expression of Rho-specific guanine nucleotide dissociation inhibitor 2 (RhoGDI2) in the subcutaneous xenografts. Afterwards, the commercially constructed RhoGDI2 siRNA plasmid was transfected into A2780 cells, and tube formation assay was conducted to determine the effect of down-regulating RhoGDI2 expression on the regulation of angiogenesis in ovarian cancer by Ado. Besides, Western blotting was performed to detect the effect of RhoGDI2 down-regulation on the regulation of matrix metalloproteinase 2 (MMP-2), MMP-9, vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-ß), tumor necrosis factor (TNF-α), and platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31) expression in ovarian cancer cells by Ado. RESULTS: The relative viability of cells subsequent to Ado treatment proved to be both concentration- and time dependent. IHC results showed that Ado evidently enhanced the RhoGDI2 protein expression. In addition, interference with RhoGDI2 outstandingly attenuated the ability of Ado to suppress tumor cell invasion and induce angiogenesis in vitro. Furthermore, molecular mechanism studies indicated that Ado remarkably inhibited the expression of MMP-2, MMP-9, VEGF, TGF-ß, TNF-α, and CD31, while interference with RhoGDI2 restored the expression of the above-mentioned angiogenic factors. CONCLUSION: Ado inhibits the growth of A2780 human ovarian cancer cells through inhibiting tumor cell invasion and angiogenesis in a RhoGDI2-dependent manner.

Unconventional myosin VIIA promotes melanoma progression.

Unconventional myosin VIIA (Myo7a) is an actin-based motor molecule that normally functions in the cochlear hair cells of the inner ear. Mutations of MYO7A/Myo7a have been implicated in inherited deafness in both humans and mice. However, there is limited information about the functions of Myo7a outside of the specialized cells of the ears. Herein, we report a previously unidentified function of Myo7a by demonstrating that it plays an important role in melanoma progression. We found that silencing Myo7a by means of RNAi inhibited melanoma cell growth through upregulation of cell cycle regulator p21 (also known as CDKN1A) and suppressed melanoma cell migration and invasion through downregulation of RhoGDI2 (also known as ARHGDIB) and MMP9. Furthermore, Myo7a depletion suppressed melanoma cell metastases to the lung, kidney and bone in mice. In contrast, overexpression of Myo7a promoted melanoma xenograft growth and lung metastasis. Importantly, Myo7a levels are remarkably elevated in human melanoma patients. Collectively, we demonstrated for the first time that Myo7a is able to function in non-specialized cells, a finding that reveals the complicated disease-related roles of Myo7a, especially in melanomas.

XIAP overexpression promotes bladder cancer invasion in vitro and lung metastasis in vivo via enhancing nucleolin-mediated Rho-GDIß mRNA stability.

Our recent studies demonstrate that X-linked inhibitor of apoptosis protein (XIAP) is essential for regulating colorectal cancer invasion. Here, we discovered that RhoGDIß was a key XIAP downstream effector mediating bladder cancer (BC) invasion in vitro and in vivo. We found that both XIAP and RhoGDIß expressions were consistently elevated in BCs of N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-treated mice in comparison to bladder tissues from vehicle-treated mice and human BCs in comparison to the paired adjacent normal bladder tissues. Knockdown of XIAP attenuated RhoGDIß expression and reduced cancer cell invasion, whereas RhoGDIß expression was attenuated in BBN-treated urothelium of RING-deletion knockin mice. Mechanistically, XIAP stabilized RhoGDIß mRNA by its positively regulating nucleolin mRNA stability via Erks-dependent manner. Moreover, ectopic expression of GFP-RhoGDIß in T24T(shXIAP) cells restored its lung metastasis in nude mice. Our results demonstrate that XIAP-regulated Erks/nucleolin/RhoGDIß axis promoted BC invasion and lung metastasis.

RhoGDIß promotes Sp1/MMP-2 expression and bladder cancer invasion through perturbing miR-200c-targeted JNK2 protein translation.

Our most recent studies demonstrate that RhoGDIß is able to promote human bladder cancer (BC) invasion and metastasis in an X-link inhibitor of apoptosis protein-dependent fashion accompanied by increased levels of matrix metalloproteinase (MMP)-2 protein expression. We also found that RhoGDIß and MMP-2 protein expressions are consistently upregulated in both invasive BC tissues and cell lines. In the present study, we show that knockdown of RhoGDIß inhibited MMP-2 protein expression accompanied by a reduction of invasion in human BC cells, whereas ectopic expression of RhoGDIß upregulated MMP-2 protein expression and promoted invasion as well. The mechanistic studies indicated that MMP-2 was upregulated by RhoGDIß at the transcriptional level by increased specific binding of the transcription factor Sp1 to the mmp-2 promoter region. Further investigation revealed that RhoGDIß overexpression led to downregulation of miR-200c, whereas miR-200c was able directly to target 3'-UTR of jnk2mRNA and attenuated JNK2 protein translation, which resulted in attenuation of Sp1mRNA and protein expression in turn, inhibiting Sp1-dependent mmp-2 transcription. Collectively, our studies demonstrate that RhoGDIß overexpression inhibits miR-200c abundance, which consequently results in increases of JNK2 protein translation, Sp1 expression, mmp-2 transcription, and BC invasion. These findings, together with our previous results showing X-link inhibitor of apoptosis protein mediating mRNA stabilization of both RhoGDIß and mmp-2, reveal the nature of the MMP-2 regulatory network, which leads to MMP-2 overexpression and BC invasion.

Two distinct mTORC2-dependent pathways converge on Rac1 to drive breast cancer metastasis.

BACKGROUND: The importance of the mTOR complex 2 (mTORC2) signaling complex in tumor progression is becoming increasingly recognized. HER2-amplified breast cancers use Rictor/mTORC2 signaling to drive tumor formation, tumor cell survival and resistance to human epidermal growth factor receptor 2 (HER2)-targeted therapy. Cell motility, a key step in the metastatic process, can be activated by mTORC2 in luminal and triple negative breast cancer cell lines, but its role in promoting metastases from HER2-amplified breast cancers is not yet clear. METHODS: Because Rictor is an obligate cofactor of mTORC2, we genetically engineered Rictor ablation or overexpression in mouse and human HER2-amplified breast cancer models for modulation of mTORC2 activity. Signaling through mTORC2-dependent pathways was also manipulated using pharmacological inhibitors of mTOR, Akt, and Rac. Signaling was assessed by western analysis and biochemical pull-down assays specific for Rac-GTP and for active Rac guanine nucleotide exchange factors (GEFs). Metastases were assessed from spontaneous tumors and from intravenously delivered tumor cells. Motility and invasion of cells was assessed using Matrigel-coated transwell assays. RESULTS: We found that Rictor ablation potently impaired, while Rictor overexpression increased, metastasis in spontaneous and intravenously seeded models of HER2-overexpressing breast cancers. Additionally, migration and invasion of HER2-amplified human breast cancer cells was diminished in the absence of Rictor, or upon pharmacological mTOR kinase inhibition. Active Rac1 was required for Rictor-dependent invasion and motility, which rescued invasion/motility in Rictor depleted cells. Rictor/mTORC2-dependent dampening of the endogenous Rac1 inhibitor RhoGDI2, a factor that correlated directly with increased overall survival in HER2-amplified breast cancer patients, promoted Rac1 activity and tumor cell invasion/migration. The mTORC2 substrate Akt did not affect RhoGDI2 dampening, but partially increased Rac1 activity through the Rac-GEF Tiam1, thus partially rescuing cell invasion/motility. The mTORC2 effector protein kinase C (PKC)α did rescue Rictor-mediated RhoGDI2 downregulation, partially rescuing Rac-guanosine triphosphate (GTP) and migration/motility. CONCLUSION: These findings suggest that mTORC2 uses two coordinated pathways to activate cell invasion/motility, both of which converge on Rac1. Akt signaling activates Rac1 through the Rac-GEF Tiam1, while PKC signaling dampens expression of the endogenous Rac1 inhibitor, RhoGDI2.

Increased dermal collagen bundle alignment in systemic sclerosis is associated with a cell migration signature and role of Arhgdib in directed fibroblast migration on aligned ECMs.

Systemic sclerosis (SSc) is a devastating disease affecting the skin and internal organs. Dermal fibrosis manifests early and Modified Rodnan Skin Scores (MRSS) correlate with disease progression. Transcriptomics of SSc skin biopsies suggest the role of the in vivo microenvironment in maintaining the pathological myofibroblasts. Therefore, defining the structural changes in dermal collagen in SSc patients could inform our understanding of fibrosis pathogenesis. Here, we report a method for quantitative whole-slide image analysis of dermal collagen from SSc patients, and our findings of more aligned dermal collagen bundles in diffuse cutaneous SSc (dcSSc) patients. Using the bleomycin-induced mouse model of SSc, we identified a distinct high dermal collagen bundle alignment gene signature, characterized by a concerted upregulation in cell migration, adhesion, and guidance pathways, and downregulation of spindle, replication, and cytokinesis pathways. Furthermore, increased bundle alignment induced a cell migration gene signature in fibroblasts in vitro, and these cells demonstrated increased directed migration on aligned ECM fibers that is dependent on expression of Arhgdib (Rho GDP-dissociation inhibitor 2). Our results indicate that increased cell migration is a cellular response to the increased collagen bundle alignment featured in fibrotic skin. Moreover, many of the cell migration genes identified in our study are shared with human SSc skin and may be new targets for therapeutic intervention.

Therapeutic effects of Euphorbia Pekinensis and Glycyrrhiza glabra on Hepatocellular Carcinoma Ascites Partially Via Regulating the Frk-Arhgdib-Inpp5d-Avpr2-Aqp4 Signal Axis.

To clarify unknown rationalities of herbaceous compatibility of Euphorbia Pekinensis (DJ) and Glycyrrhiza glabra (GC) acting on hepatocellular carcinoma (HCC) ascites, peritoneum transcriptomics profiling of 15 subjects, including normal control (Con), HCC ascites mouse model (Mod), DJ-alone, DJ/GC-synergy and DJ/GC-antagonism treatment groups were performed on OneArray platform, followed by differentially expressed genes (DEGs) screening. DEGs between Mod and Con groups were considered as HCC ascites-related genes, and those among different drug treatment and Mod groups were identified as DJ/GC-combination-related genes. Then, an interaction network of HCC ascites-related gene-DJ/GC combination-related gene-known therapeutic target gene for ascites was constructed. Based on nodes' degree, closeness, betweenness and k-coreness, the Frk-Arhgdib-Inpp5d-Avpr2-Aqp4 axis with highly network topological importance was demonstrated to be a candidate target of DJ/GC combination acting on HCC ascites. Importantly, both qPCR and western blot analyses verified this regulatory effects based on HCC ascites mice in vivo and M-1 collecting duct cells in vitro. Collectively, different combination designs of DJ and GC may lead to synergistic or antagonistic effects on HCC ascites partially via regulating the Frk-Arhgdib-Inpp5d-Avpr2-Aqp4 axis, implying that global gene expression profiling combined with network analysis can offer an effective way to understand pharmacological mechanisms of traditional Chinese medicine prescriptions.

Characterizing isomiR variants within the microRNA-34/449 family.

miR-34/449 microRNAs are conserved regulators of multiciliated cell differentiation. Here, we evidence and characterize expression of two isomiR variant sequences from the miR-34/449 family in human airway epithelial cells. These isomiRs differ from their canonical counterparts miR-34b and miR-449c by one supplemental uridine at their 5'-end, leading to a one-base shift in their seed region. Overexpression of canonical miR-34/449 or 5'-isomiR-34/449 induces distinct gene expression profiles and biological effects. However, some target transcripts and functional activities are shared by both canonical microRNAs and isomiRs. Indeed, both repress important targets that result in cell cycle blockage and Notch pathway inhibition. Our findings suggest that 5'-isomiR-34/449 may represent additional mechanisms by which miR-34/449 family finely controls several pathways to drive multiciliogenesis.

Protective Role of Rho Guanosine Diphosphate Dissociation Inhibitor, Ly-GDI, in Pulmonary Alveolitis.

Growing evidences indicate that Ly-GDI, an inhibitory protein of Rho GTPases, plays an essential role in regulating actin cytoskeletal alteration which is indispensible for the process such as phagocytosis. However, the role of Ly-GDI in inflammation remains largely unknown. In the current study, we found that Ly-GDI expression was significantly decreased in the IgG immune complex-injured lungs. To determine if Ly-GDI might regulate the lung inflammatory response, we constructed adenovirus vectors that could mediate ectopic expression of Ly-GDI (Adeno-Ly-GDI). In vivo mouse lung expression of Ly-GDI resulted in a significant attenuation of IgG immune complex-induced lung injury, which was due to the decreased pulmonary permeability and lung inflammatory cells, especially neutrophil accumulation. Upon IgG immune complex deposition, mice with Ly-GDI over-expression in the lungs produced significant less inflammatory mediators (TNF-α, IL-6, MCP-1, and MIP-1α) in bronchoalveolar lavage fluid when compared control mice receiving airway injection of Adeno-GFP. Mechanically, IgG immune complex-induced NF-κB activity was markedly suppressed by Ly-GDI in both alveolar macrophages and lungs as measured by luciferase assay and electrophoretic mobility shift assay. These findings suggest that Ly-GDI is a critical regulator of inflammatory injury after deposition of IgG immune complexes and that it negatively regulates the lung NF-κB activity.

RhoGDIß Inhibits Bone Morphogenetic Protein 4 (BMP4)-induced Adipocyte Lineage Commitment and Favors Smooth Muscle-like Cell Differentiation.

The integration of signals involved in deciding the fate of mesenchymal stem cells is largely unknown. We used proteomics profiling to identify RhoGDIß, an inhibitor of the small G-protein Rho family, as a component that regulates commitment of C3H10T1/2 mesenchymal stem cells to the adipocyte or smooth muscle cell lineage in response to bone morphogenetic protein 4 (BMP4). RhoGDIß is notably down-regulated during BMP4-induced adipocytic lineage commitment of C3H10T1/2 mesenchymal stem cells, and this involves the cytoskeleton-associated protein lysyl oxidase. Excess RhoGDIß completely prevents BMP4-induced commitment to the adipocyte lineage and simultaneously stimulates smooth muscle cell commitment by suppressing the activation of Rac1. Overexpression of RhoGDIß induces stress fibers of F-actin by a process involving phosphomyosin light chain, indicating that cytoskeletal tension regulated by RhoGDIß contributes to determining adipocyte versus myocyte commitment. Furthermore, the overexpression of RacV12 (constitutively active form of Rac1) totally rescues the inhibition of adipocyte commitment by RhoGDIß, simultaneously preventing formation of the smooth muscle-like phenotype and disrupting the stress fibers in cells overexpressing RhoGDIß. Collectively, these results indicate that RhoGDIß functions as a novel BMP4 signaling target that regulates adipogenesis and myogensis.

Autoantibodies specific to D4GDI modulate Rho GTPase mediated cytoskeleton remodeling and induce autophagy in T lymphocytes.

T lymphocytes from patients with Systemic Lupus Erythematosus (SLE) display multiple abnormalities, including increased cell activation, abnormal cell death by apoptosis and impairment of autophagy pathway. In the present study we report the presence of specific antibodies to D4GDI, a small GTPase family inhibitor, in a significant percentage (46%) of SLE patient sera. We also found a significant association between the presence of these autoantibodies and hematologic manifestations occurring in these patients. Investigating the possible implication of anti-D4GDI autoantibodies in SLE pathogenesis or progression, we found that these antibodies were capable of binding D4GDI expressed at the lymphocyte surface and triggering a series of subcellular events, including Rho GTPase activation. These antibodies were also able to induce autophagy in T cells from both healthy donors and SLE patients, but only those negative to these antibodies. We can conclude that anti-D4GDI autoantibodies could be capable of triggering important responses in T cells such as cytoskeleton remodeling and autophagy pathway and that, in SLE patients, the chronic exposure to these specific autoantibodies could lead to the selection of autophagy-resistant T cell clones contributing to the pathogenesis of the disease.