RGC-32 mediates proinflammatory and profibrotic pathways in immune-mediated kidney disease.

Systemic lupus erythematosus is an autoimmune disease that results in immune-mediated damage to kidneys and other organs. We investigated the role of response gene to complement-32 (RGC-32), a proinflammatory and profibrotic mediator induced by TGFß and C5b-9, in nephrotoxic nephritis (NTN), an experimental model that mimics human lupus nephritis. Proteinuria, loss of renal function and kidney histopathology were attenuated in RGC-32 KO NTN mice. RGC-32 KO NTN mice displayed downregulation of the CCL20/CCR6 and CXCL9/CXCR3 ligand/receptor pairs resulting in decreased renal recruitment of IL-17+ and IFNγ+ cells and subsequent decrease in the influx of innate immune cells. RGC-32 deficiency attenuated renal fibrosis as demonstrated by decreased deposition of collagen I, III and fibronectin. Thus, RGC-32 is a unique mediator shared by the Th17 and Th1 dependent proinflammatory and profibrotic pathways and a potential novel therapeutic target in the treatment of immune complex mediated glomerulonephritis such as lupus nephritis.

Regulator of Cell Cycle Protein (RGCC/RGC-32) Protects against Pulmonary Fibrosis.

Some previous studies in tissue fibrosis have suggested a profibrotic contribution from elevated expression of a protein termed either RGCC (regulator of cell cycle) or RGC-32 (response gene to complement 32 protein). Our analysis of public gene expression datasets, by contrast, revealed a consistent decrease in RGCC mRNA levels in association with pulmonary fibrosis. Consistent with this observation, we found that stimulating primary adult human lung fibroblasts with transforming growth factor (TGF)-ß in cell cultures elevated collagen expression and simultaneously attenuated RGCC mRNA and protein levels. Moreover, overexpression of RGCC in cultured lung fibroblasts attenuated the stimulating effect of TGF-ß on collagen levels. Similar to humans with pulmonary fibrosis, the levels of RGCC were also decreased in vivo in lung tissues of wild-type mice challenged with bleomycin in both acute and chronic models. Mice with constitutive RGCC gene deletion accumulated more collagen in their lungs in response to chronic bleomycin challenge than did wild-type mice. RNA-Seq analyses of lung fibroblasts revealed that RGCC overexpression alone had a modest transcriptomic effect, but in combination with TGF-ß stimulation, induced notable transcriptomic changes that negated the effects of TGF-ß, including on extracellular matrix-related genes. At the level of intracellular signaling, RGCC overexpression delayed early TGF-ß-induced Smad2/3 phosphorylation, elevated the expression of total and phosphorylated antifibrotic mediator STAT1, and attenuated the expression of a profibrotic mediator STAT3. We conclude that RGCC plays a protective role in pulmonary fibrosis and that its decline permits collagen accumulation. Restoration of RGCC expression may have therapeutic potential in pulmonary fibrosis.

Response gene to complement-32 promotes cell survival via the NF-κB pathway in non-small-cell lung cancer.

Response gene to complement (RGC)-32 regulates the cell cycle in response to complement activation. The present study demonstrated that the expression level of RGC-32 is higher in human non-small-cell lung cancer (NSCLC) tissues compared with health controls. Overexpressing RGC-32 induced p65 nucleus translocation, significantly increased nuclear p65 levels and promoted the proliferation of A549 cells. Knockdown of RGC-32 by short hairpin RNA decreased the expression level of nuclear p65 and inhibited cell proliferation. The increase in cell proliferation induced by RGC32 could be abolished by the NF-κB inhibitor pyrrolidine dithiocarbamate. Mechanistic studies indicated that RGC32 mediated NF-κB downstream genes, including vascular cell adhesion protein 1, interleukin-6, cyclin dependent kinase inhibitor 2C, testin and vascular endothelial growth factor A. In summary, the present study demonstrated a novel role of RGC-32 in the progression of NSCLC via the NF-κB pathway and p65. Therefore, RGC-32 could be a potential therapeutic target for NSCLC.

RGC-32 Regulates Generation of Reactive Astrocytes in Experimental Autoimmune Encephalomyelitis.

Astrocytes are increasingly recognized as critical contributors to multiple sclerosis pathogenesis. We have previously shown that lack of Response Gene to Complement 32 (RGC-32) alters astrocyte morphology in the spinal cord at the peak of experimental autoimmune encephalomyelitis (EAE), suggesting a role for RGC-32 in astrocyte differentiation. In this study, we analyzed the expression and distribution of astrocytes and astrocyte progenitors by immunohistochemistry in spinal cords of wild-type (WT) and RGC-32-knockout (KO) mice with EAE and of normal adult mice. Our analysis showed that during acute EAE, WT astrocytes had a reactive morphology and increased GFAP expression, whereas RGC-32 KO astrocytes had a morphology similar to that of radial glia and an increased expression of progenitor markers such as vimentin and fatty acid binding protein 7 (FABP7). In control mice, GFAP expression and astrocyte density were also significantly higher in the WT group, whereas the number of vimentin and FABP7-positive radial glia was significantly higher in the RGC-32 KO group. In vitro studies on cultured neonatal astrocytes from WT and RGC-32 KO mice showed that RGC-32 regulates a complex array of molecular networks pertaining to signal transduction, growth factor expression and secretion, and extracellular matrix (ECM) remodeling. Among the most differentially expressed factors were insulin-like growth factor 1 (IGF1), insulin-like growth factor binding proteins (IGFBPs), and connective tissue growth factor (CTGF); their expression was downregulated in RGC-32-depleted astrocytes. The nuclear translocation of STAT3, a transcription factor critical for astrogliogenesis and driving glial scar formation, was also impaired after RGC-32 silencing. Taken together, these data suggest that RGC-32 is an important regulator of astrocyte differentiation during EAE and that in the absence of RGC-32, astrocytes are unable to fully mature and become reactive astrocytes.

Response Gene to Complement 32 in Vascular Diseases.

Response gene to complement 32 (RGC32) is a protein that was identified in rat oligodendrocytes after complement activation. It is expressed in most of the organs and tissues, such as brain, placenta, heart, and the liver. Functionally, RGC32 is involved in various physiological and pathological processes, including cell proliferation, differentiation, fibrosis, metabolic disease, and cancer. Emerging evidences support the roles of RGC32 in vascular diseases. RGC32 promotes injury-induced vascular neointima formation by mediating smooth muscle cell (SMC) proliferation and migration. Moreover, RGC32 mediates endothelial cell activation and facilitates atherosclerosis development. Its involvement in macrophage phagocytosis and activation as well as T-lymphocyte cell cycle activation also suggests that RGC32 is important for the development and progression of inflammatory vascular diseases. In this mini-review, we provide an overview on the roles of RGC32 in regulating functions of SMCs, endothelial cells, and immune cells, and discuss their contributions to vascular diseases.

Response Gene to Complement-32 Promotes the Imbalance of Treg/Th17 in Patients with Dilated Cardiomyopathy.

BACKGROUND/AIMS: The imbalance of Treg/Th17 cells plays important role in the pathogenesis of dilated cardiomyopathy (DCM). Response gene to complement (RGC)-32 is a cell cycle regulator that plays an important role in cell proliferation. We evaluated whether the upregulation of RGC-32 was implicated in the homeostasis of Treg/Th17 cells in DCM. METHODS: The levels of plasma RGC-32, IL-17 and TGF-ß1, and the frequencies of circulating CD4+ RGC-32+ T cells, Th17 and Treg cells in patients with DCM were determined by Cytokine-specific sandwich ELISA and the flow cytometer (FCM), respectively. RESULTS: A significant elevation of plasma RGC-32 in patients with DCM compared with healthy control (HC) subjects was observed. This upregulation was associated with an increase in frequency of Th17 and a decrease in frequency of Treg cells. To further assessed the role of RGC-32, we investigated the effects of RGC-32 up- or down-regulation on frequencies of Th17 and Treg cells in peripheral blood mononuclear cells (PBMCs) from subjects. Importantly, overexpression of RGC-32 was accompanied by an augmentation of Th17 and a reduction of Treg expression. CONCLUSION: In summary, our study demonstrated the up-regulation of RGC-32 contributed to the imbalance of Treg/Th17 cells in patients with DCM.

Hypoxia-inducible factor 1α participates in hypoxia-induced epithelial-mesenchymal transition via response gene to complement 32.

The aim of the present study was to explore the function of response gene to complement 32 (RGC-32) in hypoxia-induced epithelial-mesenchymal transition (EMT) in pancreatic cancer. Three kinds of hypoxia-inducible factor 1α (HIF-1α) small interfering (si)RNA were synthesized and the different effects on the expression of HIF-1α were detected by western blotting. In human pancreatic cancer BxPC-3 cells, HIF-1α levels were diminished using siRNA transfection or HIF-1α inhibitor pretreatment, and the expression levels of RGC-32 and EMT-associated proteins were analyzed using reverse transcription-quantitative polymerase chain reaction and western blotting. Subsequently, the protein levels of epithelial marker, E-cadherin, and mesenchymal marker, vimentin, were determined by western blotting. Results demonstrated that HIF-1α-Homo-488 siRNA and HIF-1α-Homo-1216 siRNA diminished the protein level of HIF-1α. Compared with normoxia, hypoxia induced the levels of HIF-1α, RGC-32, N-cadherin and vimentin, but suppressed the expression of E-cadherin and cytokeratins. The inhibition of HIF-1α by HIF-1α-Homo-1216 siRNA transfection or HIF-1α inhibitor repressed hypoxia-induced HIF-1α, RGC-32, N-cadherin and vimentin, but increased the expression of E-cadherin and cytokeratins. When RGC-32 was knocked down, hypoxia-induced vimentin was suppressed; however, hypoxia-suppressed N-cadherin was released. In conclusion, the present results demonstrated that hypoxia induced the expression of HIF-1α to activate the levels of RGC-32, in turn to regulate the expression EMT-associated proteins for EMT. These findings revealed the function of RGC-32 in hypoxia-induced EMT and may have identified a novel link between HIF-1α and EMT for pancreatic cancer therapy.

Response gene to complement 32 regulates the G2/M phase checkpoint during renal tubular epithelial cell repair.

BACKGROUND: The aim of this study was to evaluate the influence of RGC-32 (response gene to complement 32) on cell cycle progression in renal tubular epithelial cell injury. METHODS: NRK-52E cells with overexpressed or silenced RGC-32 were constructed via transient transfection with RGC-32 expression plasmid and RGC-32 siRNA plasmid, and the cell cycle distribution was determined. The expression levels of fibrosis factors, including smooth muscle action (α-SMA), fibronectin (FN) and E-cadherin, were assessed in cells with silenced RGC-32. RESULTS: The cells were injured via TNF-α treatment, and the injury was detectable by the enhanced expression of neutrophil gelatinase-associated lipocalin (NGAL). RGC-32 expression also increased significantly. The number of cells at G2/M phase increased dramatically in RGC-32 silenced cells, indicating that RGC-32 silencing induced G2/M arrest. In addition, after treatment with TNF-α, the NRK-52E cells with silenced RGC-32 showed significantly increased expression of α-SMA and FN, but decreased expression of E-cadherin. CONCLUSIONS: The results of this study suggest that RGC-32 probably has an important impact on the repair process of renal tubular epithelial cells in vitro by regulating the G2/M phase checkpoint, cell fibrosis and cell adhesion. However, the exact mechanism needs to be further elucidated.

The expression of response gene to complement 32 on renal ischemia reperfusion injury in rat.

To investigate the expression of response gene to complement 32 (RGC32) in rat with acute kidney injury (AKI) and to explore the role of RGC32 in renal injury and repair induced by ischemia reperfusion. Rats were randomly divided into two groups, including sham operation group (n = 48) and acute ischemia reperfusion injury (IRI) group (n = 48). Rats were sacrificed following reperfusion 2 h, 6 h, 24 h, 48 h, 72 h, 1 week (w), 2 w, and 4 w. The distribution and expression of RGC32 in renal tissue were observed by means of immunohistochemistry. The mean density of the images detected by Image-Pro Plus 6 was designated as the representative RGC32 expression levels. Meanwhile, RGC32 mRNA expression was measured by qPCR. RGC32 mainly expressed in cytoplasm of proximal tubular epithelial cells. However, RGC32 did not express in renal interstitium and vessels. The expression levels of RGC32 measured by immunohistochemistry at different reperfusion time were 0.0168 ± 0.0029, 0.0156 ± 0.0021, 0.0065 ± 0.0013, 0.0075 ± 0.0013, 0.0096 ± 0.0014, 0.0132 ± 0.0016, 0.0169 ± 0.0014, 0.0179 ± 0.0022, respectively. Compared with the sham group, the level of RGC32 expression in IRI group was significant lower at 24 h, 48 h, 72 h after IRI (p < 0.05). The expression levels of RGC32 mRNA at different reperfusion time measured by qPCR were corroborated the immunohistochemistry finding. The in vitro experiments show the expression of α-SMA and extracellular matrix expression increased signification when the RGC32 was silenced. Our data showed that the RGC32 expression in AKI rat decreased significantly reduces with different reperfusion time and performs a time-dependent manner. RGC32 may play an important role in the pathogenesis of AKI following IRI and repair in rat.

RGC-32 Deficiency Protects against Hepatic Steatosis by Reducing Lipogenesis.

Hepatic steatosis is associated with insulin resistance and metabolic syndrome because of increased hepatic triglyceride content. We have reported previously that deficiency of response gene to complement 32 (RGC-32) prevents high-fat diet (HFD)-induced obesity and insulin resistance in mice. This study was conducted to determine the role of RGC-32 in the regulation of hepatic steatosis. We observed that hepatic RGC-32 was induced dramatically by both HFD challenge and ethanol administration. RGC-32 knockout (RGC32(-/-)) mice were resistant to HFD- and ethanol-induced hepatic steatosis. The hepatic triglyceride content of RGC32(-/-) mice was decreased significantly compared with WT controls even under normal chow conditions. Moreover, RGC-32 deficiency decreased the expression of lipogenesis-related genes, sterol regulatory element binding protein 1c (SREBP-1c), fatty acid synthase, and stearoyl-CoA desaturase 1 (SCD1). RGC-32 deficiency also decreased SCD1 activity, as indicated by decreased desaturase indices of the liver and serum. Mechanistically, insulin and ethanol induced RGC-32 expression through the NF-κB signaling pathway, which, in turn, increased SCD1 expression in a SREBP-1c-dependent manner. RGC-32 also promoted SREBP-1c expression through activating liver X receptor. These results demonstrate that RGC-32 contributes to the development of hepatic steatosis by facilitating de novo lipogenesis through activating liver X receptor, leading to the induction of SREBP-1c and its target genes. Therefore, RGC-32 may be a potential novel drug target for the treatment of hepatic steatosis and its related diseases.

RGC32 deficiency protects against high-fat diet-induced obesity and insulin resistance in mice.

Obesity is an important independent risk factor for type 2 diabetes, cardiovascular diseases and many other chronic diseases. Adipose tissue inflammation is a critical link between obesity and insulin resistance and type 2 diabetes and a contributor to disease susceptibility and progression. The objective of this study was to determine the role of response gene to complement 32 (RGC32) in the development of obesity and insulin resistance. WT and RGC32 knockout (Rgc32(-/-) (Rgcc)) mice were fed normal chow or high-fat diet (HFD) for 12 weeks. Metabolic, biochemical, and histologic analyses were performed. 3T3-L1 preadipocytes were used to study the role of RGC32 in adipocytes in vitro. Rgc32(-/-) mice fed with HFD exhibited a lean phenotype with reduced epididymal fat weight compared with WT controls. Blood biochemical analysis and insulin tolerance test showed that RGC32 deficiency improved HFD-induced dyslipidemia and insulin resistance. Although it had no effect on adipocyte differentiation, RGC32 deficiency ameliorated adipose tissue and systemic inflammation. Moreover, Rgc32(-/-) induced browning of adipose tissues and increased energy expenditure. Our data indicated that RGC32 plays an important role in diet-induced obesity and insulin resistance, and thus it may serve as a potential novel drug target for developing therapeutics to treat obesity and metabolic disorders.

Response gene to complement 32 (RGC-32) in endothelial cells is induced by glucose and helpful to maintain glucose homeostasis.

Endothelium dysfunction has been understood primarily in terms of abnormal vasomotor function, which plays an important role in the pathogenesis of diabetes and chronic diabetic complications. However, it has not been fully studied that the endothelium may regulate metabolism itself. The response gene to complement 32 (RGC-32) has be considered as an angiogenic inhibitor in the context of endothelial cells. We found that RGC-32 was induced by high fat diet in vivo and by glucose or insulin in endothelial cells, and then we set out to investigate the role of endothelial RGC-32 in metabolism. DNA array analysis and qPCR results showed that glutamine-fructose-6-phosphate aminotransferase [isomerizing] 1 (GFPT1), solute carrier family 2 (facilitated glucose transporter), member 12 (SLC2A12, GLUT12) and glucagon-like peptide 2 receptor (GLP2R) may be among possible glucose metabolism related downstream genes of RGC-32. Additionally, in the mice with endothelial specific over-expressed RGC-32, the disposal of carbohydrate was improved without changing insulin sensitivity when mice were faced with high fat diet challenges. Taken together, our findings suggest that RGC-32 in the endothelial cells regulates glucose metabolism related genes and subsequent helps to maintain the homeostasis of blood glucose.

Response gene to complement 32 protein promotes macrophage phagocytosis via activation of protein kinase C pathway.

Macrophage phagocytosis plays an important role in host defense. The molecular mechanism, especially factors regulating the phagocytosis, however, is not completely understood. In the present study, we found that response gene to complement 32 (RGC-32) is an important regulator of phagocytosis. Although RGC-32 is induced and abundantly expressed in macrophage during monocyte-macrophage differentiation, RGC-32 appears not to be important for this process because RGC-32-deficient bone marrow progenitor can normally differentiate to macrophage. However, both peritoneal macrophages and bone marrow-derived macrophages with RGC-32 deficiency exhibit significant defects in phagocytosis, whereas RGC-32-overexpressed macrophages show increased phagocytosis. Mechanistically, RGC-32 is recruited to macrophage membrane where it promotes F-actin assembly and the formation of phagocytic cups. RGC-32 knock-out impairs F-actin assembly. RGC-32 appears to interact with PKC to regulate PKC-induced phosphorylation of F-actin cross-linking protein myristoylated alanine-rich protein kinase C substrate. Taken together, our results demonstrate for the first time that RGC-32 is a novel membrane regulator for macrophage phagocytosis.

Response gene to complement 32 deficiency causes impaired placental angiogenesis in mice.

AIMS: The objectives of this study are to determine the role of response gene to complement 32 (RGC-32) in the placental angiogenesis during pregnancy and explore the underlying mechanisms. METHODS AND RESULTS: RGC-32-deficient (RGC32(-/-)) mice were generated from C57BL/6 embryonic stem cells with deletion of exon 2 and 3 of the RGC-32 gene. Most of the RGC32(-/-) mice can survive. However, their body sizes were much smaller compared with their wild-type littermates when they were born. By examining the embryo development and placentas at 16.5 days post-coitum, we found that RGC32(-/-) embryos and foetal placentas were significantly smaller than the wild-type. Further analysis showed that the labyrinth zone of RGC32(-/-) placenta was smaller with defective angiogenesis. Mechanistically, vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and placental growth factor (PlGF) were significantly down-regulated in RGC32(-/-) placentas, suggesting that VEGFR2 and PlGF may mediate RGC-32 function in placental angiogenesis. Indeed, knockdown of RGC-32 by shRNA inhibited VEGF-induced endothelial cell proliferation, migration, and tube formation while blocking VEGFR2 expression. RGC-32 appeared to regulate VEGFR2 expression via activation of NF-kB. Moreover, RGC-32 regulated trophoblasts proliferation via control of PlGF expression. CONCLUSION: Absence of RGC-32 caused foetal growth restriction through interrupting the placental angiogenesis, which was due to the decrease in VEGFR2 expression through the NF-kB-dependent pathway in endothelial cells and PlGF expression in trophoblasts.

Expressions of RGC-32 and E-cadherin in pancreatic cancer and their clinicopathological significance / 中华胰腺病杂志

Objective To investigate the expressions of RGC-32 and E-cadherin in pancreatic cancer and analyze their clinicopathological significance and the correlation with each other.Methods SP immunohistochemistry was used to detect the expressions of RGC-32 and E-cadherin in 42 cases of pancreatic cancer tissues,12 cases of chronic pancreatitis tissues and 8 cases of normal pancreatic tissues.Results The positive staining for RGC-32 was predominantly observed in the cytoplasm of pancreatic acinar cells.The positive staining for E-cadherin was mainly observed in the cytomembrane of normal pancreatic and chronic pancreatitis acinar cells,but aberrant expression ( cytoplasm expression and ( or ) weaker expression) could be found in pancreatic cancer cells.The positive expression rate of RGC-32 and aberrant expression rate of E-cadherin were 78.6％ (33/42) and 54.8％ (23/42),respectively,in pancreatic cancer tissues,which were significantly higher than those in normal pancreatic tissues [37.5％ (3/8) and 0] and chronic pancreatitis [41.7％ (5/12)and 8.3％ (1/12) with statisticai significance,P ＜0.05].The expression of RG C-32 in pancreatic cancer was associated with lymph node metastasis and TNM staging (P =0.016,0.025,respectively),but not with age,gender and differentiation degree ( P =0.831,1.000,0.629,respectively).The aberrant expression of E-cadherin was associated with differentiation degree,lymph node metastasis and TNM staging ( P =0.024,0.004,0.004,respectively),but not with age and gender ( P =0.970,1.000,respectively).A significantly positive correlation was found between positive expression rate of RGC-32 and aberrant expression rate of E-cadherin (r =0.458,P ＜0.01 ).Conclusions Both positive expression rate of RGC-32 and aberrant expression rate of E-cadherin are up-regulated significantly in pancreatic cancer tissues and RGC-32 may be involved in the invasion and metastasis of pancreatic cancer by regulating epithelial mesenchymal transition.