RGC-32 Promotes Th17 Cell Differentiation and Enhances Experimental Autoimmune Encephalomyelitis.

Th17 cells play a critical role in autoimmune diseases, including multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Response gene to complement (RGC)-32 is a cell cycle regulator and a downstream target of TGF-ß that mediates its profibrotic activity. In this study, we report that RGC-32 is preferentially upregulated during Th17 cell differentiation. RGC-32-/- mice have normal Th1, Th2, and regulatory T cell differentiation but show defective Th17 differentiation in vitro. The impaired Th17 differentiation is associated with defects in IFN regulatory factor 4, B cell-activating transcription factor, retinoic acid-related orphan receptor γt, and SMAD2 activation. In vivo, RGC-32-/- mice display an attenuated experimental autoimmune encephalomyelitis phenotype accompanied by decreased CNS inflammation and reduced frequency of IL-17- and GM-CSF-producing CD4+ T cells. Collectively, our results identify RGC-32 as a novel regulator of Th17 cell differentiation in vitro and in vivo and suggest that RGC-32 is a potential therapeutic target in multiple sclerosis and other Th17-mediated autoimmune diseases.

SIRT1 as a potential biomarker of response to treatment with glatiramer acetate in multiple sclerosis.

SIRT1, a NAD dependent histone and protein deacetylase, is a member of the histone deacetylase class III family. We previously showed that SIRT1 mRNA expression is significantly lower in peripheral blood mononuclear cells (PBMCs) of multiple sclerosis (MS) patients during relapses than in stable patients. We have now investigated SIRT1 as a possible biomarker to predict relapse as well as responsiveness to glatiramer acetate (GA) treatment in relapsing-remitting MS (RRMS) patients. Over the course of 2years, a cohort of 15 GA-treated RRMS patients were clinically monitored using the Expanded Disability Status Scale and assessed for MS relapses. Blood samples collected from MS patients were analyzed for levels of SIRT1 and histone H3 lysine 9 (H3K9) acetylation and dimethylation. During relapses, MS patients had a lower expression of SIRT1 mRNA than did stable MS patients. In addition, there was a significant decrease in H3K9 dimethylation (H3K9me2) during relapses in MS patients when compared to stable patients (p=0.01). Responders to GA treatment had significantly higher SIRT1 mRNA (p=0.01) and H3K9me2 levels than did non-responders (p=0.018). Receiver operating characteristic analysis was used to assess the predictive power of SIRT1 and H3K9me2 as putative biomarkers: for SIRT1 mRNA, the predictive value for responsiveness to GA treatment was 70% (p=0.04) and for H3K9me2 was 71% (p=0.03). Our data suggest that SIRT1 and H3K9me2 could serve as potential biomarkers for evaluating patients' responsiveness to GA therapy in order to help guide treatment decisions in MS.

RGC-32 as a potential biomarker of relapse and response to treatment with glatiramer acetate in multiple sclerosis.

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.

RGC-32 is a novel regulator of the T-lymphocyte cell cycle.

We have previously shown that RGC-32 is involved in cell cycle regulation in vitro. To define the in vivo role of RGC-32, we generated RGC-32 knockout mice. These mice developed normally and did not spontaneously develop overt tumors. To assess the effect of RGC-32 deficiency on cell cycle activation in T cells, we determined the proliferative rates of CD4(+) and CD8(+) T cells from the spleens of RGC-32(-/-) mice, as compared to wild-type (WT, RGC-32(+/+)) control mice. After stimulation with anti-CD3/anti-CD28, CD4(+) T cells from RGC-32(-/-) mice displayed a significant increase in [(3)H]-thymidine incorporation when compared to WT mice. In addition, both CD4(+) and CD8(+) T cells from RGC-32(-/-) mice displayed a significant increase in the proportion of proliferating Ki67(+) cells, indicating that in T cells, RGC-32 has an inhibitory effect on cell cycle activation induced by T-cell receptor/CD28 engagement. Furthermore, Akt and FOXO1 phosphorylation induced in stimulated CD4(+) T-cells from RGC-32(-/-) mice were significantly higher, indicating that RGC-32 inhibits cell cycle activation by suppressing FOXO1 activation. We also found that IL-2 mRNA and protein expression were significantly increased in RGC-32(-/-) CD4(+) T cells when compared to RGC-32(+/+) CD4(+) T cells. In addition, the effect of RGC-32 on the cell cycle and IL-2 expression was inhibited by pretreatment of the samples with LY294002, indicating a role for phosphatidylinositol 3-kinase (PI3K). Thus, RGC-32 is involved in controlling the cell cycle of T cells in vivo, and this effect is mediated by IL-2 in a PI3K-dependent fashion.

SIRT1 is decreased during relapses in patients with multiple sclerosis.

SIRT1 is a member of the histone deacetylase (HDAC) class III family of proteins and is an NAD-dependent histone and protein deacetylase. SIRT1 can induce chromatin silencing through the deacetylation of histones and can modulate cell survival by regulating the transcriptional activities. We investigated the expression of SIRT1 in multiple sclerosis (MS) brains and in peripheral blood mononuclear cells (PBMCs) obtained from patients with relapsing-remitting multiple sclerosis. We found that SIRT1 was expressed by a significant number of cells in both acute and chronic active lesions. We also found that CD4(+), CD68(+), oligodendrocytes (OLG), and glial fibrillar acidic protein (GFAP)(+) cells in MS plaques co-localized with SIRT1. Our results show a statistically significant decrease in SIRT1 mRNA and protein expression in PBMCs during relapses when compared to the levels in controls and stable MS patients. On the other hand, HDAC3 expression was not significantly changed during relapses in MS patients. SIRT1 expression correlated with that of histone H3 lysine 9 acetylation (H3K9ac) and methylation (H3K9me2). SIRT1 mRNA expression was significantly reduced after RGC-32 silencing, indicating a role for RGC-32 in the regulation of SIRT1 expression. Furthermore, we investigated the role of SIRT1 in the expression of FasL and found a significant increase in FasL expression and apoptosis after inhibition of SIRT1 expression. Our data suggest that SIRT1 may represent a biomarker of relapses and a potential new target for therapeutic intervention in MS.

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-ß-induced extracellular matrix expression in astrocytes. Blockage of RGC-32 using small interfering RNA significantly inhibits TGF-ß 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-ß-mediated profibrotic effects in astrocytes.

C5b-9-activated, K(v)1.3 channels mediate oligodendrocyte cell cycle activation and dedifferentiation.

Voltage-gated potassium (K(v)) channels play an important role in the regulation of growth factor-induced cell proliferation. We have previously shown that cell cycle activation is induced in oligodendrocytes (OLGs) by complement C5b-9, but the role of K(v) channels in these cells had not been investigated. Differentiated OLGs were found to express K(v)1.4 channels, but little K(v)1.3. Exposure of OLGs to C5b-9 modulated K(v)1.3 functional channels and increased protein expression, whereas C5b6 had no effect. Pretreatment with the recombinant scorpion toxin rOsK-1, a highly selective K(v)1.3 inhibitor, blocked the expression of K(v)1.3 induced by C5b-9. rOsK-1 inhibited Akt phosphorylation and activation by C5b-9 but had no effect on ERK1 activation. These data strongly suggest a role for K(v)1.3 in controlling the Akt activation induced by C5b-9. Since Akt plays a major role in C5b-9-induced cell cycle activation, we also investigated the effect of inhibiting K(v)1.3 channels on DNA synthesis. rOsK-1 significantly inhibited the DNA synthesis induced by C5b-9 in OLG, indicating that K(v)1.3 plays an important role in the C5b-9-induced cell cycle. In addition, C5b-9-mediated myelin basic protein and proteolipid protein mRNA decay was completely abrogated by inhibition of K(v)1.3 expression. In the brains of multiple sclerosis patients, C5b-9 co-localized with NG2(+) OLG progenitor cells that expressed K(v)1.3 channels. Taken together, these data suggest that K(v)1.3 channels play an important role in controlling C5b-9-induced cell cycle activation and OLG dedifferentiation, both in vitro and in vivo.

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.