Sonia I. Vlaicu

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.

Role of response gene to complement 32 in diseases

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 β 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.

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.

The role of complement system in adipose tissue-related inflammation

Abstract As the common factor linking adipose tissue to the metabolic context of obesity, insulin resistance and atherosclerosis are associated with a low-grade chronic inflammatory status, to which the complement system is an important contributor. Adipose tissue synthesizes complement proteins and is a target of complement activation. C3a-desArg/acylation-stimulating protein stimulates lipogenesis and affects lipid metabolism. The C3a receptor and C5aR are involved in the development of adipocytes’ insulin resistance through macrophage infiltration and the activation of adipose tissue. The terminal complement pathway has been found to be instrumental in promoting hyperglycemia-associated tissue damage, which is characteristic of the major vascular complications of diabetes mellitus and diabetic ketoacidosis. As a mediator of the effects of the terminal complement complex C5b-9, RGC-32 has an impact on energy expenditure as well as lipid and glucose metabolic homeostasis. All of this evidence, taken together, indicates an important role for complement activation in metabolic diseases.

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.

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.

Neuroprotective effects of the complement terminal pathway during demyelination: Implications for oligodendrocyte survival

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system that is mediated by activated lymphocytes, macrophages/microglia, and complement. In MS, the myelin-forming oligodendrocytes (OLGs) are the targets of the immune attack. Experimental evidence indicates that C5b-9 plays a role in demyelination during the acute phase of experimental allergic encephalomyelitis (EAE). Terminal complement C5b-9 complexes are capable of protecting OLGs from apoptosis. During chronic EAE complement C5 promotes axonal preservation, remyelination and provides protection from gliosis. These findings indicate that the activation of complement and C5b-9 assembly can also have protective roles during demyelination.

The role of complement activation in atherogenesis: the first 40 years.

The pathogenesis of atherosclerotic inflammation is a multi-step process defined by the interweaving of excess modified lipid particles, monocyte-macrophages populations, and innate immune and adaptive immunity effectors. A part of innate immunity, the complement system, is an important player in the induction and progression of atherosclerosis. The accumulation of either oxidized or enzymatically modified LDL—bound to C-reactive protein or not—prompts complement activation leading to the assembly of the terminal complement C5b-9 complex in the atherosclerotic lesion. The sublytic C5b-9 assembly leads to the activation and proliferation of smooth muscle and endothelial cells, accompanied by the release of various chemotactic, pro-adhesion, and procoagulant cytokines from these cells. Response gene to complement (RGC)-32, an essential effector of the terminal complement complex C5b-9, also affects atherogenesis, propelling vascular smooth muscle cell proliferation and migration, stimulating endothelial proliferation, and promoting vascular lesion formation. A substantial amount of experimental work has suggested a role for the complement system activation during atherosclerotic plaque formation, with the proximal classical complement pathway seemingly having a protective effect and terminal complement contributing to accelerated atherogenesis. All these data suggest that complement plays an important role in atherogenesis.

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.

RGC-32 is expressed in the human atherosclerotic arterial wall: Role in C5b-9-induced cell proliferation and migration.

The complement system is an important player in the development of atherosclerosis. Previously reported as a cell cycle regulator, RGC-32 is an essential effector of the terminal complement complex, C5b-9. In this study, our aims were to determine the expression of RGC-32 in the human atherosclerotic arterial wall and to delineate the mechanisms through which RGC-32 affects C5b-9-induced endothelial cell proliferation and migration. We now demonstrate that RGC-32 is expressed in human aortic atherosclerotic wall and that RGC-32 expression increases with the progression of atherosclerosis. Furthermore, silencing of RGC-32 expression abolished C5b-9-induced human aortic endothelial cell (HAEC) proliferation and migration. Of the 279 genes differentially expressed in HAECs after RGC-32 silencing, the genes involved in cell adhesion and cell cycle activation were significantly regulated by RGC-32. RGC-32 silencing caused a significant reduction in the expression of cyclin D1, cyclin D3, Akt, ROCK1, Rho GDP dissociation inhibitor alpha and profilin. These data suggest that RGC-32 mediates HAEC migration through the regulation of RhoA and ROCK1 expression and is involved in actin cytoskeletal organization. Thus, RGC-32 has promising therapeutic potential with regard to angiogenesis and atherosclerosis.