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Currently there is critical need for the identification of reliable biomarkers to help guide clinical management of multiple sclerosis (MS) patients. We investigated the combined roles of Response Gene to Complement 32 (RGC-32), FasL, CDC2, AKT, and IL-21 as possible biomarkers of relapse and response to glatiramer acetate (GA) treatment in relapsing-remitting MS (RRMS) patients. Over the course of 2 years, a cohort of 15 GA-treated RRMS patients was clinically monitored and peripheral blood mononuclear cells (PBMCs) were collected at 0, 3, 6, and 12 months. Target gene mRNA expression was measured in patients' isolated PBMCs by real-time qRT-PCR. Compared to stable MS patients, those with acute relapses exhibited decreased expression of RGC-32 (p<0.0001) and FasL (p<0.0001), increased expression of IL-21 (p=0.04), but no change in CDC2 or AKT. Compared to non-responders, responders to GA treatment showed increased expression of RGC-32 (p<0.0001) and FasL (p<0.0001), and decreased expression of IL-21 (p=0.02). Receiver operating characteristic (ROC) analysis was used to assess the predictive accuracy of each putative biomarker. The probability of accurately detecting relapse was 90% for RGC-32, 88% for FasL, and 75% for IL-21. The probability of accurately detecting response to GA was 85% for RGC-32, 90% for FasL, and 85% for IL-21. Our data suggest that RGC-32, FasL, and IL-21 could serve as potential biomarkers for the detection of MS relapse and response to GA therapy., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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 (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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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., (Copyright 2009 Elsevier Inc. All rights reserved.)

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.

The role of response gene to complement (RGC)-32 as a cell cycle regulator has been attributed to its ability to activate cdc2 kinases and to induce S-phase entry and mitosis. However, recent studies revealed novel functions for RGC-32 in diverse processes such as cellular differentiation, inflammation, and fibrosis. Besides responding to C5b-9 stimulation, RGC-32 expression is also induced by growth factors, hormones, and cytokines. Transforming growth factor beta activates RGC-32 through Smad and RhoA signaling, thus initiating smooth muscle cell differentiation. Accumulating evidence has drawn attention to the deregulated expression of RGC-32 in human malignancies, hyper-immunoglobulin E syndrome, and fibrosis. RCG-32 expression is up-regulated in cutaneous T cell lymphoma and colon, ovarian, and breast cancer, but down-regulated in invasive prostate cancer, multiple myeloma, and drug-resistant glioblastoma. A better understanding of the mechanism by which RGC-32 contributes to the pathogenesis of these diseases will provide new insights into its therapeutic potential. In this review we provide an overview of this field and discuss the most recent research on RGC-32.