RGC-32 and diseases: the first 20 years

The response gene to complement (RGC)-32 acts as a cell cycle regulator and mediator of TGF-β effects. However, recent studies have revealed other functions for RGC-32 in diverse processes such as cellular migration, differentiation, and fibrosis. In addition to its induction by complement activation and the C5b-9 terminal complement complex, RGC-32 expression is also stimulated by growth factors, hormones, and cytokines. RGC-32 is induced by TGF-β through Smad3 and RhoA signaling and plays an important role in cell differentiation. In particular, RGC-32 is essential for the differentiation of Th17 cells. RGC-32^−/− mice display an attenuated experimental autoimmune encephalomyelitis phenotype that is accompanied by decreased central nervous system inflammation and reductions in IL-17- and GM-CSF-producing CD4⁺ T cells. Accumulating evidence has drawn attention to the deregulated expression of RGC-32 in human cancers, atherogenesis, metabolic disorders, and autoimmune disease. Furthermore, RGC-32 is a potential therapeutic target in multiple sclerosis and other Th17-mediated autoimmune diseases. A better understanding of the mechanism(s) by which RGC-32 contributes to the pathogenesis of all these diseases will provide new insights into its therapeutic potential.

     

Response gene to complement 32 (RGC-32) expression on M2-polarized and tumor-associated macrophages is M-CSF-dependent and enhanced by tumor-derived IL-4

Response gene to complement 32 (RGC-32) is a cell cycle regulator involved in the proliferation, differentiation and migration of cells and has also been implicated in angiogenesis. Here we show that RGC-32 expression in macrophages is induced by IL-4 and reduced by LPS, indicating a link between RGC-32 expression and M2 polarization. We demonstrated that the increased expression of RGC-32 is characteristic of alternatively activated macrophages, in which this protein suppresses the production of pro-inflammatory cytokine IL-6 and promotes the production of the anti-inflammatory mediator TGF-β. Consistent with in vitro data, tumor-associated macrophages (TAMs) express high levels of RGC-32, and this expression is induced by tumor-derived ascitic fluid in an M-CSF- and/or IL-4-dependent manner. Collectively, these results establish RGC-32 as a marker for M2 macrophage polarization and indicate that this protein is a potential target for cancer immunotherapy, targeting tumor-associated macrophages.     

Epigenetic modifications induced by RGC-32 in colon cancer

First described as a cell cycle activator, RGC-32 is both an activator and a substrate for CDC2. Deregulation of RGC-32 expression has been detected in a wide variety of human cancers. We have now shown that RGC-32 is expressed in precancerous states, and its expression is significantly higher in adenomas than in normal colon tissue. The expression of RGC-32 was higher in advanced stages of colon cancer than in precancerous states or the initial stages of colon cancer. In order to identify the genes that are regulated by RGC-32, we used gene array analysis to investigate the effect of RGC-32 knockdown on gene expression in the SW480 colon cancer cell line. Of the 230 genes that were differentially regulated after RGC-32 knockdown, a group of genes involved in chromatin assembly were the most significantly regulated in these cells: RGC-32 knockdown induced an increase in acetylation of histones H2B lysine 5 (H2BK5), H2BK15, H3K9, H3K18, and H4K8. RGC-32 silencing was also associated with decreased expression of SIRT1 and decreased trimethylation of histone H3K27 (H3K27me3). In addition, RGC-32 knockdown caused a significantly higher percentage of SW480 cells to enter S phase and subsequently G2/M. These data suggest that RGC-32 may contribute to the development of colon cancer by regulating chromatin assembly.     

Response gene to complement 32 is required for C5b-9 induced cell cycle activation in endothelial cells

Proliferation of vascular endothelial cells (EC) and smooth muscle cells (SMC) is a critical event in angiogenesis and atherosclerosis. We previously showed that the C5b-9 assembly during complement activation induces cell cycle in human aortic EC (AEC) and SMC. C5b-9 can induce the expression of Response Gene to Complement (RGC)-32 and over expression of this gene leads to cell cycle activation. Therefore, the present study was carried out to test the requirement of endogenous RGC-32 for the cell cycle activation induced by C5b-9 by knocking-down its expression using siRNA. We identified two RGC-32 siRNAs that can markedly reduce the expression of RGC-32 mRNA in AEC. RGC-32 silencing in these cells abolished DNA synthesis induced by C5b-9 and serum growth factors, indicating the requirement of RGC-32 activity for S-phase entry. RGC-32 siRNA knockdown also significantly reduced the C5b-9 induced CDC2 activation and Akt phosphorylation. CDC2 does not play a role in G1/S transition in HeLa cells stably overexpressing RGC-32. RGC-32 was found to physically associate with Akt and was phosphorylated by Akt in vitro. Mutation of RGC-32 protein at Ser 45 and Ser 47 prevented Akt mediated phosphorylation. In addition, RGC-32 was found to regulate the release of growth factors from AEC. All these data together suggest that cell cycle induction by C5b-9 in AEC is RGC-32 dependent and this is in part through regulation of Akt and growth factor release.     

Overexpression of RGC-32 in colon cancer and other tumors

Tumors often exhibit deregulation of the cell cycle and overexpression of cyclins and cyclin-dependent kinases (CDKs). Response gene to complement (RGC)-32 is a substrate and regulator of CDC2 and its overexpression induces cell cycle activation. We investigated RGC-32 mRNA and protein expression in tumors with special emphasis in colon carcinoma. By using an expression array technique we found that 19% of tumor tissues showed increased RGC-32 mRNA expression over the levels of corresponding normal tissues. On the other hand, an increased RGC-32 protein was found in 70% of colon adenocarcinoma samples tested. In colon carcinomas, two major patterns of RGC-32 immunoreactivity were seen: staining of malignant epithelial cells only in some tumors and RGC-32 reactivity of both malignant epithelia as well as cells in the interstitium in others. Colonic epithelium obtained from normal individuals was consistently negative for RGC-32 protein. Overexpression of RGC-32 protein was found in other tumors including prostate, bladder, breast, lung, and other digestive tract tumors. RGC-32 expression was present in the same malignant epithelial cells that also expressed the proliferation marker Ki-67. Our data suggest that RGC-32 overexpression might be part of the deregulation of the cell cycle that is required for the growth of tumor cells.     

Dual role of Response gene to complement-32 in multiple sclerosis

Response gene to complement (RGC)-32 is a novel molecule that plays an important role in cell proliferation. We investigated the expression of RGC-32 in multiple sclerosis (MS) brain and in peripheral blood mononuclear cells (PBMCs) obtained from patients with relapsing–remitting multiple sclerosis. We found that CD3⁺, CD68⁺, and glial fibrillar acidic protein (GFAP)⁺ cells in MS plaques co-localized with RGC-32. Our results show a statistically significant decrease in RGC-32 mRNA expression in PBMCs during relapses when compared to the levels in stable MS patients. This decrease might be useful in predicting disease activity in patients with relapsing–remitting MS. RGC-32 expression was also correlated with that of FasL mRNA during relapses. FasL mRNA expression was significantly reduced after RGC-32 silencing, indicating a role for RGC-32 in the regulation of FasL expression. In addition, the expression of Akt1, cyclin D1, and IL-21 mRNA was significantly increased during MS relapses when compared to levels in healthy controls. Furthermore, we investigated the role of RGC-32 in TGF-β-induced extracellular matrix expression in astrocytes. Blockage of RGC-32 using small interfering RNA significantly inhibits TGF-β induction of procollagen I, fibronectin and of the reactive astrocyte marker α-smooth muscle actin (α-SMA). Our data suggest that RGC-32 plays a dual role in MS, both as a regulator of T-cells mediated apoptosis and as a promoter of TGF-β-mediated profibrotic effects in astrocytes.     

RGC-32 regulates reactive astrocytosis and extracellular matrix deposition in experimental autoimmune encephalomyelitis

Extracellular matrix (ECM) deposition in active demyelinating multiple sclerosis (MS) lesions may impede axonal regeneration and can modify immune reactions. Response gene to complement (RGC)-32 plays an important role in the mediation of TGF-β downstream effects, but its role in gliosis has not been investigated. To gain more insight into the role played by RGC-32 in gliosis, we investigated its involvement in TGF-β-induced ECM expression and the upregulation of the reactive astrocyte markers α-smooth muscle actin (α-SMA) and nestin. In cultured neonatal rat astrocytes, collagens I, IV, and V, fibronectin, α-SMA, and nestin were significantly induced by TGF-β stimulation, and RGC-32 silencing resulted in a significant reduction in their expression. Using astrocytes isolated from RGC-32 knock-out (KO) mice, we found that the expression of TGF-β-induced collagens I, IV, and V, fibronectin, and α-SMA was significantly reduced in RGC-32 KO mice when compared with wild-type (WT) mice. SIS3 inhibition of Smad3 phosphorylation was also associated with a significant reduction in RGC-32 nuclear translocation and TGF-β-induced collagen I expression. In addition, during experimental autoimmune encephalomyelitis (EAE), RGC-32 KO mouse astrocytes displayed an elongated, bipolar phenotype, resembling immature astrocytes and glial progenitors whereas those from WT mice had a reactive, hypertrophied phenotype. Taken together, our data demonstrate that RGC-32 plays an important role in mediating TGF-β-induced reactive astrogliosis in EAE. Therefore, RGC-32 may represent a new target for therapeutic intervention in MS.     

Role of galectin-3 in the pathogenesis of bladder transitional cell carcinoma

Galectins constitute an evolutionary conserved family that binds to β-galactosides. There is growing evidence that galectins are implicated in essential biological processes such as cellular communication, inflammation, differentiation and apoptosis. Galectin-3 is one of the best-known galectins, which is found in vertebrates. Galectin-3 has been shown to be expressed in some cell lines and plays important roles in several physiological and pathological processes, including cell adhesion, cell activation and chemoattraction, cell cycle, apoptosis, cell growth, and differentiation. Moreover, this galectin is of interest due to its involvement in regulation of cancer. Changes in galectin-3 expression are commonly seen in cancerous and pre-cancerous conditions and galectin-3 may be involved in the regulation of cancer cell activities that contribute to tumourigenesis, cancer progression and metastasis. Finally, galectin-3 seems to be involved in cell events in tumor microenvironment, and therefore it could be considered as a target in transitional cell carcinoma therapies. This review aims to describe recent progress in understanding the role of galectin-3 in cancer biology, with emphasis on bladder tumor progression and metastasis.     

Cytoglobin: a novel potential gene medicine for fibrosis and cancer therapy

Attempts have been made by conventional gene therapy to suppress hepatic fibrogenesis, but potential oncogenic activity may prevent its clinical use. Recently, a novel major approach has been developed for resolution of liver fibrosis and cirrhosis: inactivation of hepatic stellate cells (HSC) using the endogenous expressing gene, which could mediate the homeostatic adaptation of liver. Cytoglobin (Cygb), originally identified in cultured rat HSC, is in a new class of heme containing proteins known as the hexacoordinate globin superfamily. These proteins exhibit peroxidase activity against hydrogen peroxides and lipid hydroperoxides. Considerable attention has been focused on the potential benefits of use of Cygb in fibrosis therapy, as up-regulation of Cygb expression could reduce oxidant stress, suppress HSC differentiation to a myofibroblast-like phenotype and eventually prevent the progress of liver fibrosis. Cygb has also been found to be a candidate tumor suppressor gene that might provide a new option for cancer gene therapy. In this review we systematically analyze the potential of Cygb as a prospective gene medicine for curing fibrosis, cancer, and other diseases such as diabetes. The molecular structure, regulation and subcellular location of Cygb are reviewed as well.     

Engineering stem cells for therapy

The differentiation of a stem cell is dependent on the environmental cues that it receives and can be modulated by the expression of different master regulators or by secreted factors or inducers. The use of genetically modified stem cells to express the required factors can direct differentiation along the requisite pathway. This approach to the engineering of stem cells is important, as control of the pluripotentiality of stem cells is necessary in order to avoid unwanted growth, migration or differentiation to nontarget tissues. The authors provide an overview of the stem cell engineering field, highlighting challenges and solutions, and focusing on recent developments in therapeutic applications in areas such as autoimmunity, CNS lesions, bone and joint diseases, cancer and myocardial infarction.     

RNAi in embryonic stem cells

Embryonic stem (ES) cells are pluripotent cells that can be isolated and grown in vitro from the inner cell mass of blastocysts. Their potential to differentiate into any cell of the body makes them a promising starting material for cell therapy Much progress has been made in recent years to develop ES cell differentiation protocols employing cocktails of certain growth factors or by using cell-type-restricted promoters driving the expression of selection markers or fluorescent proteins. However, little is known about the molecular details underlying the earliest processes of mammalian development. Genetic tools that provide novel insight into these processes would be very helpful to gain a better molecular understanding and to design better differentiation protocols. Recently, RNAi has emerged as a powerful technology to perform loss-of-function studies in mammalian cells. This technology should be ideal to identify and study genes required for ES cell self-renewal and differentiation. Here, we review the recent advances and challenges of RNAi research in ES cells and we provide a perspective on possible applications to enhance our understanding of ES cell self-renewal and early differentiation.     

Reduction of pp32 expression in poorly differentiated pancreatic ductal adenocarcinomas and intraductal papillary mucinous neoplasms with moderate dysplasia.

Nuclear phosphoprotein 32 (pp32) inhibits K-ras induced transformation in experimental models. pp32 mRNA expression correlates with differentiation status in breast and prostate cancers. In this study, we evaluated pp32 protein expression in relation to the differentiation status of pancreatic ductal adenocarcinomas and precursor lesions of the pancreatic cancers. pp32 expression showed strong nuclear staining in normal pancreatic acini and ducts. The intensity of this staining was maintained in pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasms with mild dysplasia, well-differentiated adenocarcinomas, and in a subset of moderately differentiated adenocarcinomas. pp32 staining was absent or reduced in poorly differentiated tumors and in intraductal papillary mucinous neoplasms with moderate dysplasia. We validated pp32 expression by a second technique, immunoblot analysis of lysates from resected pancreatic ductal adenocarcinomas and pancreatic cancer cell lines. The well-differentiated pancreatic cancer cell line HPAC expressed high amounts of pp32, as compared to the poorly differentiated pancreatic cancer cell lines MiaPaCa2, Pl19, and Pl21 cells. Artificial introduction of pp32 expression into a poorly differentiated cell line, MiaPaCa2, caused an increase in G1 arrest compared to control cells. On the basis of this study and previous functional work that shows pp32 can inhibit K-ras transformation, we propose that reduction in pp32 expression levels may be a critical event in the progression of pancreatic tumorigenesis in an aggressive subset of pancreatic ductal adenocarcinomas.     

Transforming growth factor-β signaling in systemic sclerosis

Systemic sclerosis (SSc) is a complex, multiorgan autoimmune disease of unknown etiology. Manifestation of the disease results from an interaction of three key pathologic features including irregularities of the antigen-specific immune system and the non-specific immune system, resulting in autoantibody production, vascular endothelial activation of small blood vessels, and tissue fibrosis as a result of fibroblast dysfunction. Given the heterogeneity of clinical presentation of the disease, a lack of universal models has impeded adequate testing of potential therapies for SSc. Regardless, recent research has elucidated the roles of various ubiquitous molecular mechanisms that contribute to the clinical manifestation of the disease. Transforming growth factor β (TGF-β) has been identified as a regulator of pathological fibrogenesis in SSc. Various processes, including cell growth, apoptosis, cell differentiation, and extracellular matrix synthesis are regulated by TGF-β, a type of cytokine secreted by macrophages and many other cell types. Understanding the essential role TGF-β pathways play in the pathology of systemic sclerosis could provide a potential outlet for treatment and a better understanding of this severe disease.     

叶黄素抑制叔丁基过氧化氢诱导的视网膜神经节细胞凋亡

目的:观察叶黄素(lutein)对叔丁基过氧化氢(t-BHP)处理的视网膜神经节细胞(RGC-5细胞系)的保护效应并探讨其作用机制。方法:用免疫荧光染色检测视网膜神经节特异性蛋白Brn-3和神经微管结合蛋白MAP-2的表达来鉴定RGC-5细胞;将RGC-5细胞随机分为对照组、t-BHP处理组、t-BHP和lutein共同处理组、lutein处理组,培养24 h,MTT实验检测细胞活力;Annexin V-FITC/PI双染流式细胞术检测细胞凋亡;免疫细胞化学技术检测caspase-3蛋白的活化情况;Western blot检测Bcl-2/Bax、cleaved caspase-3、JNK和c-Jun蛋白的变化。结果:MTT实验和流式细胞检测结果显示,lutein能提高t-BHP处理的RGC-5细胞的活力,并降低t-BHP诱导的RGC-5细胞凋亡;免疫荧光结果显示lutein能抑制t-BHP诱导的caspase-3的活化;与对照组比较,t-BHP处理后RGC-5细胞抗凋亡蛋白Bcl-2表达下调(P0.05),Bax/Bcl-2比率升高,cleaved caspase-3表达上调(P0.05),JNK和c-Jun蛋白的磷酸化水平增加(P0.05),t-BHP的上述作用可被lutein部分逆转。结论:Lutein能够降低t-BHP诱导的RGC-5细胞凋亡,其机制与其上调Bcl-2的表达、抑制caspase-3的活化并降低JNK和c-Jun蛋白的磷酸化有关。     

WNT信号通路与肿瘤

WNT信号通路在肿瘤发生中有重要意义,它调节细胞生长、迁移和分化。由于它在众多人类肿瘤的发生中广泛活化,近年来这条通路在肿瘤研究方面受到很多关注。本文将介绍该通路与肿瘤发生的关系,以及目前该通路的研究在抗肿瘤治疗中的应用前景。     

TGF-β1 induces peritoneal fibrosis by activating the Smad2 pathway in mesothelial cells and promotes peritoneal carcinomatosis

Peritoneal dissemination is one of the main causes of death in gastric cancer patients. Our previous study demonstrated that peritoneal fibrosis induced by transforming growth factor-β1 (TGF-β1) may provide a favorable environment for the dissemination of gastric cancer. The role of Smad3 in the development of dermal fibrosis, subcapsular cataract, and peritoneal fibrosis has been reported. However, the potential role of Smad2 in the development of fibrosis is unclear. The objective of this study was to determine the effect of Smad2 in peritoneal fibrosis, induced by TGF-β1, on dissemination of gastric cancer. Here we demonstrate that TGF-β1 significantly stimulated the expression of collagen?III and fibronectin in mesothelial cells through the Smad2 signal transduction pathway, but knockdown of the Smad2 gene by silencing siRNA partially inhibited these effects. This inhibition was associated with a depressed adhesion and invasiveness of gastric cancer cells. We conclude that peritoneal fibrosis induced by TGF-β1 is dependent on Smad2 signaling and may provide a hospitable environment for carcinomatosis.     

Periostin在骨矿化过程中的生物学功能研究进展

Periostin是一种细胞外基质蛋白,最初发现于MC3T3一E1成骨细胞样细胞系,在骨、牙周膜、皮肤及心脏瓣膜等多种组织中均有表达,亦见于人类多种恶性肿瘤及创伤愈合的组织中,参与肿瘤侵袭、转移及皮肤损伤和心肌缺血后重建过程。研究发现,Periostin是骨强度的调控因子之一,在成骨细胞黏附、分化,胶原纤维形成,骨基质的矿化中有调节作用,并能够促进骨组织创伤后的愈合。其表达与应力刺激密切相关,亦受多种转录因子、激素及细胞因子等的调节。基于Periostin的一般特征和表达定位,对Periostin参与骨代谢的研究进展作一综述。     

CTGF基因沉默对肺成纤维细胞增殖及表型转化的影响

近期研究表明,结缔组织生长因子(Connective tissue growth factor,CTGF)参与了肺成纤维细胞表型转化,与肺纤维化的形成关系密切.因此,本研究应用siRNA表达载体介导的RNA干扰(RNA interference,RNAi)技术,将CTGF-siRNA表达质粒稳定转染人肺成纤维细胞MRC-5,并通过定量PCR、Western-blot、细胞生长曲线及群体倍增时间、免疫细胞化学测定等方法检测基因表达、细胞增殖及表型转化等变化.结果显示,与非特异对照Scrambled-siRNA质粒稳定转染的MRC-5细胞或未转染MRC-5细胞相比,CTGF-siRNA表达质粒稳定转染的MRC-5细胞的CTGF表达明显降低;细胞增殖能力明显降低,群体倍增时间延长,由24.63或25.05 h延长至31.14 h(P＜0.05);TGFβ1刺激24 h后,细胞内α-平滑肌肌动蛋白α-smooth muscle actin,α-SMA)、Ⅰ型胶原及纤维连接蛋白(fibronectin,FN)的表达均明显下降.本研究提示,通过RNA干扰使CTGF基因沉默后,在体外可明显抑制MRC-5肺成纤维细胞的增殖、表型转化及细胞外基质的合成,可为基因治疗肺纤维化提供实验依据.