Response Gene To Complement 32 Research Articles

Membrane associated RGC‐32 promoted macrophage phagocytosis via direct and specific activation of novel PKC pathway (802.24)

Phagocytosis of bacterial pathogens by macrophages requires the activation of protein kinase C (PKC), a family of protein serine/threonine kinases. Previous publications have demonstrated that both classical and novel PKCs were required for macrophage phagocytosis. However recruitment of classical PKCs or novel PKCs or both is macrophage linage specific, which prompt us to hyposize that there exist PKC isozyme specific regulators in macrophage. Here we found that response gene to complement 32 (RGC32) is a direct and specific activator for novel PKC pathway in macrophage phagocytosis. RGC32 was induced and specifically expressed in macrophage lineages in differentiation. While bone marrow progenitor cells from RGC32 knockout mice did not have significant defects in macrophage differentiation, both peritoneal macrophages (PMs) and bone marrow derived macrophages (BMDMs) presented dramatic defects in their phagocytosis activities. Moreover, macrophages over expressed RGC32 showed promoted phagocytosis activity. During phagocytosis, RGC32 was recruited to cell membrane and co‐localized with F‐actin to forming phagocytic cups and macrophages from RGC32 knockout mice showed impaired F‐actin assembling and phagocytic cup formation. Mechanistically, RGC32 participated in phagocytosis through activation of PKC induced alanine‐rich C‐kinase substrate (MARCKS) phosphorylation. Recombinant RGC32 increased PKC activity in vitro. Knocking down RGC32 in macrophage suppressed MARCKS phosphorylation while over expression RGC32 increased MARCKS phosphorylation. Interestingly, RGC32 directly interacted with novel but not classical PKC isozymes and RGC32 induced MARCKS can only be suppressed by novel PKC inhibitor. Finally, RGC32 knockout mice showed defects in pathogens clearance to peritoneal bacterial infection. In summary, for the first time, we identified RGC32 as an membrane associated novel PKC activator during macrophage phagocytosis.

Response gene to complement 32 deficiency protects against high‐fat diet‐induced insulin resistance and obesity with decreased hepatic lipogenesis in mice (767.7)

The objective of this study was to determine the effects of response gene to complement 32 (RGC‐32) on the development of obesity and insulin resistance in mice. Mice with RGC‐32 deficiency (RGC32‐/‐) gained less body weight with reduced epididymal fat and liver weight compared to the wild‐type animals after a 12‐week high‐fat diet. Biochemical analysis of blood, as well as glucose and insulin tolerance tests showed that RGC32‐/‐ improved insulin sensitivity and lowered serum triacylglycerol and low‐density lipoprotein/very‐low‐density lipoprotein cholesterol concentration. Hepatic lipid accumulation was also decreased in RGC32‐/‐ mice. Additional analysis revealed that lipogenic genes sterol regulatory element (SRE) binding protein (SREBP)‐1c, fatty acid synthase, and stearoyl‐CoA desaturase‐1 (SCD1) were decreased in the livers of RGC32‐/‐ mice. Mechanistic studies using mouse primary hepatocytes showed that SCD1 regulation by RGC‐32 was SREBP‐1c‐dependent since deletion or mutation of SRE abrogated SCD1 promoter activity mediated by RGC‐32. These data demonstrate that RGC‐32 induces SREBP‐1c‐dependent hepatic lipogenesis, which is essential for the high‐fat diet‐induced obesity and insulin resistance.

Abstract 089: Response Gene To Complement 32 Is Involved In Glucose Metabolism Disorder In Endothelial Cells And Mediated By P53

Response gene to complement 32 (RGC-32) was considered as an apoptosis-promoting factor. To explore the novel functions of RGC-32, a DNA array analysis was performed in Human Microvascular Endothelial Cells (HMEC) treated with SiRNA, which showed that glucose metabolism was one of most dramatically changed biological process. qPCR analysis also proved that many glucose metabolism related genes were changed dramatically in RGC32 o/e HMEC, one of which was Insulin-like growth factor binding protein 4(IGFBP4). We propose that RGC-32 may be a potential factor involved in the cycle of endothelial glucose metabolism besides glucose induced apoptosis. Then we found that expression of RGC-32 was dramatically increased in mice hearts and adipose tissue after high fat diet treatment, as well as in HMEC after high glucose (30mM) treatment. In high fat diet treated endothelial specific RGC-32 transgenic mice (VE-Cad/RGC-32), decreases in area under the curve (AUC) of glucose tolerance test were observed when compared to their wild type littermates. Upregulated P53 and downregulated IGFBP4 mRNA expression level were also observed in the heart of VE-Cad/RGC-32 mice.Furthermore, we found that P53 was upregulated in RGC-32 o/e HMEC and blocking of p53 was able to decrease the RGC-32-related induction of apoptotic cells. We also observed that RGC-32 can not only decrease IGFBP4 but also reverse the decrease of IGFBP4 in high glucose or insulin condition. Interestingly, after treated the cells with pifithrin-a, a specific P53 inhibitor, decreased IGFBP4 by RGC-32 was rescued. These results demonstrate that RGC-32 may have some kind of connections with glucose metabolism besides its well-known apoptosis-promoting characters and P53 may be a mediator of RGC-32 related glucose metabolism disorder as well as apoptosis.These findings will provide some clues for novel insights into the functions of RGC-32.

Overexpression of response gene to complement 32 (RGC32) promotes cell invasion and induces epithelial–mesenchymal transition in lung cancer cells via the NF-κB signaling pathway

Response gene to complement 32 (RGC32) is a novel cellular protein that has been reported to be expressed aberrantly in multiple types of human tumors. However, the role of RGC32 in cancer is still controversial, and the molecular mechanisms by which RGC32 contributes to the development of cancer remain largely unknown. In the present study, we constructed a recombinant expression vector pCDNA3.1-RGC32 and transfected it into human lung cancer A549 cells. Stable transformanted cells were identified by real-time PCR and Western blot analysis. Functional analysis showed that forced overexpression of RGC32 increased invasive and migration capacities of lung cancer cells in vitro, and induced the acquisition of epithelial-mesenchymal transition (EMT) phenotype, as demonstrated by the spindle-like morphology, downregulation of E-cadherin, and upregulation of Vimentin, Fibronectin, Snail and Slug. Also, overexpression of RGC32 increased expression and activities of matrix metalloproteinase (MMP)-2 and MMP-9 in A549 cells. Furthermore, the downregulation of E-cadherin induced by RGC32 was remarkably attenuated by nuclear factor-κB (NF-κB) inhibitor BAY 11-7028 and small interfering RNA targeting NF-κB p65, suggesting a role of the NF-κB signaling pathway in RGC32-induced EMT. Taken together, our data suggest that RGC32 promotes cell migration and invasion and induces EMT in lung cancer cells via the NF-κB signaling pathway.

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

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. 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. 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.

Abstract 545: 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 RGC-32 gene. Most of the RGC32-/- mice can survive. However, their body sizes were much smaller compared to 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 fetal placentas were significantly smaller than the wild-type. Further analysis showed that the labyrinth zone of RGC32-/- fetal placenta was smaller with defective angiogenesis. Mechanistically, vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and placental growth factor (PlGF), but not other angiogenic factors, were significantly downregulated in RGC32-/- placentas, suggesting that VEGFR2 and PlGF may mediate RGC-32 function in placenta angiogenesis. Indeed, knockdown of RGC-32 by shRNA inhibited VEGF-induced endothelial cell proliferation, migration, and tube formation while blocking VEGFR2 expression. Knockdown of RGC-32 also inhibited trophoblast proliferation while blocking the expression of PlGF. Conclusion Absence of RGC-32 caused intrauterine growth restriction through interrupting the placental angiogenesis, which was due to the decreased expressions of VEGFR2 in endothelial cells and PlGF in trophoblast cells.

Role of C5b-9 complement complex and response gene to complement-32 (RGC-32) in cancer

Complement system activation plays an important role in both innate and acquired immunity, with the activation of complement and the subsequent formation of C5b-9 terminal complement complex on cell membranes inducing target cell death. Recognition of this role for C5b-9 leads to the assumption that C5b-9 might play an antitumor role. However, sublytic C5b-9 induces cell cycle progression by activating signal transduction pathways and transcription factors in cancer cells, indicating a role in tumor promotion for this complement complex. The induction of the cell cycle by C5b-9 is dependent upon the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/FOXO1 and ERK1 pathways in a Gi protein-dependent manner. C5b-9 also induces response gene to complement (RGC)-32, a gene that plays a role in cell cycle promotion through activation of Akt and the CDC2 kinase. RGC-32 is expressed by tumor cells and plays a dual role in cancers, in that it has both a tumor suppressor role and tumor-promoting activity. Thus, through the activation of tumor cells, the C5b-9-mediated induction of the cell cycle plays an important role in tumor proliferation and oncogenesis.

Response Gene to Complement 32 Is Essential for Fibroblast Activation in Renal Fibrosis

Response gene to complement 32 (RGC-32) is a downstream target of transforming growth factor-β (TGF-β). TGF-β is known to play a pathogenic role in renal fibrosis. In this study, we investigated RGC-32 function in renal fibrosis following unilateral ureteral obstruction (UUO) in mice, a model of progressive tubulointerstitial fibrosis. RGC-32 is normally expressed only in blood vessels of mouse kidney. However, UUO induces RGC-32 expression in renal interstitial cells at the early stage of kidney injury, suggesting that RGC-32 is involved in interstitial fibroblast activation. Indeed, expression of smooth muscle α-actin (α-SMA), an indicator of fibroblast activation, is limited to the interstitial cells at the early stage, and became apparent later in both interstitial and tubular cells. RGC-32 knockdown by shRNA significantly inhibits UUO-induced renal structural damage, α-SMA expression and collagen deposition, suggesting that RGC-32 is essential for the onset of renal interstitial fibrosis. In vitro studies indicate that RGC-32 mediates TGF-β-induced fibroblast activation. Mechanistically, RGC-32 interacts with Smad3 and enhances Smad3 binding to the Smad binding element in α-SMA promoter as demonstrated by DNA affinity assay. In the chromatin setting, Smad3, but not Smad2, binds to α-SMA promoter in fibroblasts. RGC-32 appears to be essential for Smad3 interaction with the promoters of fibroblast activation-related genes in vivo. Functionally, RGC-32 is crucial for Smad3-mediated α-SMA promoter activity. Taken together, we identify RGC-32 as a novel fibrogenic factor contributing to the pathogenesis of renal fibrosis through fibroblast activation.

Expression of response gene to complement-32 in renal tissue of children with immunoglobulin A nephropathy

This study aimed to investigate the expression and significance of response gene to complement-32 (RGC-32) in renal tissue of children with immunoglobulin A nephropathy (IgAN). Forty-five patients diagnosed as having IgAN by renal biopsy were enrolled. The expression of RGC-32, α-smooth muscle actin (α-SMA) and transforming growth factor-β(1) (TGF-β(1)) was observed by immunohistostaining. The relationshis between the expression of RGC-32, α-SMA, TGF-β1, degree of renal pathological lesions in IgAN and clinical index were assessed by Spearman correlation. Immunohistostaining analysis showed that RGC-32 protein was present in epithelial cells of renal tubules in normal and IgAN renal tissues. With more severe renal pathological lesions, the expression of RGC-32 in IgAN was increased. The expression of RGC-32 was positively correlated with that of α-SMA, TGF-β(1) and the degree of renal pathological lesions in children with IgAN (p < 0.05), but had no relationship with serum creatinine, urinary N-acetyl-β-d-glucosaminidase/creatinine, urinary microalbuminuria/creatinine, urinary microimmunoglobulin/creatinine or urinary α(1)-microglobulin/creatinine ratio (p > 0.05). Expression of RGC-32 can reflect the degree of renal pathological lesions in IgAN. RGC-32 may participate in the renal tubulointerstitial lesions in children with IgAN, especially in epithelial -mesenchymal transition induced by TGF-β(1).

Response Gene to Complement 32 Promotes Vascular Lesion Formation Through Stimulation of Smooth Muscle Cell Proliferation and Migration

The objectives of this study were to determine the role of response gene to complement 32 (RGC-32) in vascular lesion formation after experimental angioplasty and to explore the underlying mechanisms. Using a rat carotid artery balloon-injury model, we documented for the first time that neointima formation was closely associated with a significantly increased expression of RGC-32 protein. Short hairpin RNA knockdown of RGC-32 via adenovirus-mediated gene delivery dramatically inhibited the lesion formation by 62% as compared with control groups 14 days after injury. Conversely, RGC-32 overexpression significantly promoted the neointima formation by 33%. Gain- and loss-of-function studies in primary culture of rat aortic smooth muscle cells (RASMCs) indicated that RGC-32 is essential for both the proliferation and migration of RASMCs. RGC-32 induced RASMC proliferation by enhancing p34(CDC2) activity. RGC-32 stimulated the migration of RASMC by inducing focal adhesion contact and stress fiber formation. These effects were caused by the enhanced rho kinase II-α activity due to RGC-32-induced downregulation of Rad GTPase. RGC-32 plays an important role in vascular lesion formation following vascular injury. Increased RGC-32 expression in vascular injury appears to be a novel mechanism underlying the migration and proliferation of vascular smooth muscle cells. Therefore, targeting RGC-32 is a potential therapeutic strategy for the prevention of vascular remodeling in proliferative vascular diseases.

Smad2 and PEA3 cooperatively regulate transcription of response gene to complement 32 in TGF-β-induced smooth muscle cell differentiation of neural crest cells.

Response gene to complement 32 (RGC-32) is activated by transforming growth factor- β (TGF-β) and plays an important role in smooth muscle cell (SMC) differentiation from neural crest Monc-1 cells. The molecular mechanism governing TGF-β activation of RGC-32, however, remains to be determined. The present studies indicate that TGF-β regulates RGC-32 gene transcription. Sequence analysis revealed a Smad binding element (SBE) located in the region from -1344 to -1337 bp upstream of the transcription start site of RGC-32 gene. A polyomavirus enhancer activator (PEA3) binding site is adjacent to the SBE. Mutation at either SBE or PEA3 site significantly inhibited RGC-32 promoter activity. Mutations at both sites completely abolished TGF-β-induced promoter activity. Biochemically, TGF-β stimulated recruitment of Smad2, Smad4, and PEA3 to the RGC-32 promoter, as revealed by gel shift and chromatin immunoprecipitation analyses. Functionally, Smad2, but not Smad3, activated RGC-32 promoter. PEA3 appeared to enhance Smad2 activity. In agreement with their function, Smad2, but not Smad3, physically interacted with PEA3. In TGF-β-induced SMC differentiation of Monc-1 cells, knockdown of Smad2 by short hairpin RNA resulted in downregulation of RGC-32 and SMC marker genes. The downregulation of SMC markers, however, was rescued by exogenously introduced RGC-32. These results demonstrate that Smad2 regulation of RGC-32 transcription is essential for SMC differentiation from neural crest cells.

Connections in chronic kidney disease: connexin 43 and connexin 37 interaction

the prevalence of chronic kidney disease is rising sharply worldwide and affects 13.1% of the population in the US ([3][1]). Patients with chronic kidney disease represent a population not only at risk of progression to end-organ failure but also at higher risk for cardiovascular diseases ([22][2

Response gene to complement 32 interacts with Smad3 to promote epithelial-mesenchymal transition of human renal tubular cells

Previous studies demonstrate that response gene to complement 32 (RGC-32) mediates transforming growth factor-β(1)-induced epithelial-mesenchymal transition (EMT) of human renal proximal tubular cells. However, the mechanisms underlying RGC-32 function remain largely unknown. In the present study, we found that RGC-32 function in EMT is associated with Smad3. Coexpression of RGC-32 and Smad3, but not Smad2, induces a higher mesenchymal marker α-smooth muscle actin (α-SMA) protein expression as compared with RGC-32 or Smad3 alone, while knockdown of Smad3 using short hairpin interfering RNA blocks RGC-32-induced α-SMA expression. These data suggest that RGC-32 interacts with Smad3, but not Smad2, in the regulation of EMT. In addition to α-SMA, RGC-32 and Smad3 also synergistically activate the expression of extracellular matrix protein fibronectin and downregulate the epithelial marker E-cadherin. RGC-32 colocalizes with Smad3 in the nuclei of renal proximal tubular cells. Coimmunoprecipitation assays showed that Smad3, but not Smad2, physically interacts with RGC-32 in renal proximal tubular cells. Mechanistically, RGC-32 and Smad3 coordinate the induction of EMT by regulating the EMT regulators Slug and Snail. Taken together, our data demonstrate for the first time that RGC-32 interacts with Smad3 to mediate the EMT of human renal proximal tubular cells.

Promoter methylation of the RGC32 gene in nonsmall cell lung cancer

Lung cancer is the leading cause of cancer-related deaths worldwide. Epigenetic inactivation of certain genes by aberrant promoter methylation is recognized as a crucial component in the initiation and progression of lung cancer. Response gene to complement 32 (RGC32) has been identified as a cell cycle regulator induced by activation of complements; however, its role in carcinogenesis is still controversial. The authors examined the methylation status in the promoter region of RGC32 gene in nonsmall cell lung cancers (NSCLCs) using a methylation-specific PCR and correlated the results with clinicopathological features. RGC32 methylation was found in 45 of 173 NSCLCs (26.0%) and was related to the gene expression. RGC32 methylation was more frequent in females than in males (P<0.05). RGC32 methylation was not significantly associated with the prognosis of patients; however, when the patients were categorized by TP53 mutational status, the effect of RGC32 methylation on prognosis was significantly different between those with and without TP53 mutations (P = .005 [test for homogeneity]). Specifically, RGC32 methylation was associated with significantly worse survival in the cases with wild-type TP53, whereas it exhibited a better survival outcome in the cases with TP53 mutations. The current findings suggest that methylation-associated down-regulation of RGC32 plays an important role in the pathogenesis of NSCLC, particularly in females. However, further studies with a large number of cases are needed to confirm the authors' findings.

Progress of response gene to complement-32

Response gene to complement-32(RGC-32)as one of important response genes to complement,involves in many biological functions. Recent studies found that many factors could induce the expression of RGC-32 such as membrane attack complex,cortical hormones,growth factors, luteinizing hormone and transforming growth factor-β and so on. The expression products of RGC-32 play a key role in cell proliferation, differentiation, inflammation, tumor metastasis and other processes, thus to participate in the occurrence of many diseases' development processes. However, it has not be clarified in its exact cell biological function, subcellular localization, gene regulation mechanism. This article will make a brief review on gene structure, functions and roles in cell biology as well as the relationship between human diseases for RGC-32. Key words: RGC-32; Membrane attack complex; Proliferation and differentiation; Transforming growth factor-β; Renal tubular epithelial-mesenchymal transition

Response gene to complement 32 in renal fibrosis

Previous studies have shown that response gene to complement 32 (RGC‐32) mediates TGF‐β‐induced epithelial‐mesenchymal transition of human renal proximal tubule cells. The current study is to determine if RGC‐32 is involved in the pathogenesis of renal fibrosis using unilateral ureteral obstruction (UUO) mouse model. RGC‐32 is normally expressed only in blood vessels of mouse kidney. After 5 days of UUO, RGC‐32 is strongly expressed in kidney interstitial cells and weakly in tubular cells, suggesting that UUO induces RGC‐32 expression. α‐SMA staining is limited to the interstitial cells at this early stage of renal fibrosis, suggesting that α‐SMA‐expressing myofibroblasts are derived from interstitial cells. Blockade of RGC‐32 by perfusion of RGC‐32 shRNA into kidney with UUO inhibited the expression of α‐SMA in the interstitium. In vitro studies showed that TGF‐β induced fibroblast activation while inducing RGC‐32 expression. Knockdown of RGC‐32 in fibroblasts blocked the expression of myofibroblast markers α‐SMA, collagen and fibronectin. Our data demonstrate that RGC‐32 plays an important role in the onset of renal fibrosis. (Funding source: NIH R01HL093429)

Response Gene to Complement 32, a Novel Hypoxia-Regulated Angiogenic Inhibitor

Response gene to complement 32 (RGC-32) is induced by activation of complement and regulates cell proliferation. To determine the mechanism of RGC-32 in angiogenesis, we examined the role of RGC-32 in hypoxia-related endothelial cell function. Hypoxia/ischemia is able to stimulate both angiogenesis and apoptosis. Hypoxia-inducible factor-1/vascular endothelial growth factor is a key transcriptional regulatory pathway for angiogenesis during hypoxia. We demonstrated that the increased RGC-32 expression by hypoxia was via hypoxia-inducible factor-1/vascular endothelial growth factor induction in cultured endothelial cells. However, overexpression of RGC-32 reduced the proliferation and migration and destabilized vascular structure formation in vitro and inhibited angiogenesis in Matrigel assays in vivo. Silencing RGC-32 had an opposing, stimulatory effect. RGC-32 also stimulated apoptosis as shown by the increased apoptotic cells and caspase-3 cleavage. Mechanistic studies revealed that the effect of RGC-32 on the antiangiogenic response was via attenuating fibroblast growth factor 2 expression and further inhibiting expression of cyclin E without affecting vascular endothelial growth factor and fibroblast growth factor 2 signaling in endothelial cells. In the mouse hind-limb ischemia model, RGC-32 inhibited capillary density with a significant attenuation in blood flow. Additionally, treatment with RGC-32 in the xenograft tumor model resulted in reduced growth of blood vessels that is consistent with reduced colon tumor size. We provide the first direct evidence for RGC-32 as a hypoxia-inducible gene and antiangiogenic factor in endothelial cells. These data suggest that RGC-32 plays an important homeostatic role in that it contributes to differentiating the pathways for vascular endothelial growth factor and fibroblast growth factor 2 in angiogenesis and provides a new target for ischemic disorder and tumor therapies.

RGC-32 Mediates Transforming Growth Factor-β-induced Epithelial-Mesenchymal Transition in Human Renal Proximal Tubular Cells

Epithelial-mesenchymal transition (EMT) occurs in several disease states, including renal fibrosis and carcinogenesis. Myofibroblasts produced from EMT of renal tubular cells are responsible for the deposition of extracellular matrix components in a large portion of renal interstitial fibrosis. Transforming growth factor-beta (TGF-beta) plays an essential role in the EMT of renal tubular cells, but the molecular mechanism governing this process remains largely unknown. In this study, we found that RGC-32 (response gene to complement 32) is critical for TGF-beta-induced EMT of human renal proximal tubular cells (HPTCs). RGC-32 is not normally expressed in the HPTCs. However, TGF-beta stimulation markedly activates RGC-32 while inducing an EMT, as shown by the induction of smooth muscle alpha-actin (alpha-SMA) and extracellular matrix proteins collagen I and fibronectin, as well as the reduction of epithelial marker E-cadherin. TGF-beta function is mediated by several signaling pathways, but RGC-32 expression in HPTCs appears to be mainly regulated by Smad. Functionally, RGC-32 appears to mediate TGF-beta-induced EMT of HPTCs. Blockage of RGC-32 using short hairpin interfering RNA significantly inhibits TGF-beta induction of myofibroblast marker gene alpha-SMA while repressing the expression of E-cadherin. In contrast, overexpression of RGC-32 induces alpha-SMA expression while restoring E-cadherin. RGC-32 also inhibits the expression of another adherens junction protein, N-cadherin, suggesting that RGC-32 alone induces the phenotypic conversion of renal epithelial cells to myofibroblasts. Additional studies show that RGC-32 stimulates the production of extracellular matrix components fibronectin and collagen I. Mechanistically, RGC-32 induces EMT via the activation of other transcription factors such as Snail and Slug. RGC-32 knockdown inhibits the expression of Snail and Slug during TGF-beta-induced EMT. Taken together, our data demonstrate for the first time that RGC-32 plays a critical role in TGF-beta-induced EMT of renal tubular cells.

Response Gene to Complement 32, a Novel Regulator for Transforming Growth Factor-β-induced Smooth Muscle Differentiation of Neural Crest Cells

We previously developed a robust in vitro model system for vascular smooth muscle cell (VSMC) differentiation from neural crest cell line Monc-1 upon transforming growth factor-beta (TGF-beta) induction. Further studies demonstrated that both Smad and RhoA signaling are critical for TGF-beta-induced VSMC development. To identify downstream targets, we performed Affymetrix cDNA array analysis of Monc-1 cells and identified a gene named response gene to complement 32 (RGC-32) to be important for the VSMC differentiation. RGC-32 expression was increased 5-fold after 2 h and 50-fold after 24 h of TGF-beta induction. Knockdown of RGC-32 expression in Monc-1 cells by small interfering RNA significantly inhibited the expression of multiple smooth muscle marker genes, including SM alpha-actin (alpha-SMA), SM22alpha, and calponin. Of importance, the inhibition of RGC-32 expression correlated with the reduction of alpha-SMA while not inhibiting smooth muscle-unrelated c-fos gene expression, suggesting that RGC-32 is an important protein factor for VSMC differentiation from neural crest cells. Moreover, RGC-32 overexpression significantly enhanced TGF-beta-induced alpha-SMA, SM22alpha, and SM myosin heavy chain promoter activities in both Monc-1 and C3H10T1/2 cells. The induction of VSMC gene promoters by RGC-32 appears to be CArG-dependent. These data suggest that RGC-32 controls VSMC differentiation by regulating marker gene transcription in a CArG-dependent manner. Further studies revealed that both Smad and RhoA signaling are important for RGC-32 activation.

Transforming Growth Factor‐β Regulates RGC‐32 Transcription During Smooth Muscle Differentiation From Neural Crest Cells

Previous studies demonstrated that transforming growth factor-β (TGF-β) induced vascular smooth muscle cell (VSMC) differentiation from neural crest stem cell line Monc-1. We recently identified a gene, namely response gene to complement 32 (RGC-32) to be critical for TGF-β-induced VSMC differentiation. RGC-32 was upregulated by TGF-β but how TGF-β controls RGC-32 expression remains to be determined. The purpose of this study was to identify the molecular mechanism of TGF-β regulation on RGC-32 gene transcription. A Smad binding site and a polyomavirus enhancer activator (PEA3) binding site is located in the 5′ upstream regulatory region of RGC-32 gene. Mutation of Smad site significantly inhibited RGC-32 promoter activity. Double mutations of Smad and PEA3 sites completely abolished the activity although PEA3 mutation alone appeared to have no effect. By performing gel mobility shift assay (EMSA), competitive EMSA, supershift assay as well as transfection and luciferase assay, we found that Smad2 but not Smad3 significantly upregulated RGC-32 promoter activity. PEA3 alone did not exhibit a significant effect, but PEA3 synergized with Smad2 in upregulating RGC-32 promoter activity. These results were further confirmed by small RNA interference experiments. Taken together, these data demonstrate that Smad2 and PEA3 synergistically regulate TGF-β induction of RGC-32 transcription during VSMC differentiation from neural crest cells.