Expression Of Retinoic Acid-inducible gene-I Research Articles

Synthetic double-stranded RNA induces retinoic acid-inducible gene-I in mouse osteoblastic cells

Retinoic acid inducible gene-I (RIG-I) is a member of the DExH box family of proteins. RIG-I acts as a sensor of viral infections through the recognition of viral double-stranded RNA (dsRNA). Recently, it was demonstrated that polyinosinic acid:polycytidylic acid [poly(I):poly(C)], a synthetic dsRNA analogue, induced the expression of RIG-I in various cell types, such as vascular endothelial cells and gingival fibroblasts. However, it remains unclear whether RIG-I is induced in osteoblasts in response to poly(I):poly(C). In the present study, we investigated the effects of poly(I):poly(C) on the expression of RIG-I in mouse osteoblastic MC3T3-E1(E1) cells. We found that poly(I):poly(C) increased the expression level of RIG-I in E1 cells, and that recombinant interferon-β (IFN-β) induced the expression of RIG-I mRNA in E1 cells. An anti-IFN-β neu-tralizing antibody partially inhibited poly(I):poly(C)-induced RIG-I expression. These results indicate that RIG-I production is induced by poly(I):poly(C)-provoked IFN-β in mouse osteoblastic E1 cells. We suggest that osteoblasts are involved in antiviral defense as well as in bone metabolism. Results of further studies will provide more clues regarding the molecular function of osteoblasts in viral infection.

Tumor necrosis factor-α and Interferon-γ synergistically induce expression of Retinoic acid-inducible gene-I in cultured human keratinocytes

Tumor necrosis factor-α and Interferon-γ synergistically induce expression of Retinoic acid-inducible gene-I in cultured human keratinocytes

Expression of mRNA for functional molecules in urinary sediment in glomerulonephritis

Recent studies have suggested that gene expression studies using urinary sediment might be a non-invasive approach to assessing activity and pathogenesis in glomerulonephritis. However, little information is available regarding the mRNA expression patterns of functional molecules, such as T-bet, GATA-3, FOXP3, and retinoic acid-inducible gene-I (RIG-I), in urinary sediment, from patients with immunocomplex-mediated glomerulonephritis. Fourteen lupus nephritis (LN) patients, 13 IgA nephropathy (IgAN) patients, and 12 healthy controls were enrolled in the study. The mRNA expressions of T-bet, GATA-3, FOXP3 and RIG-I in urinary sediment were measured using real time quantitative polymerase chain reaction. We also studied the expression of RIG-I in kidney tissue specimens obtained from LN and IgAN patients. Significant differences in the expression patterns of GATA-3, FOXP3 and RIG-I, and marginal differences in T-bet expression, were observed between the three study groups. Immunofluorescent staining for RIG-I was observed in the tissue specimens from the LN patients, but not in those from the IgAN patients. The mRNA expression patterns of T-bet, GATA-3, FOXP3 and RIG-I in urinary sediment differ according to diagnostic category. These results suggest that the measurement of these target gene expressions might be a useful, non-invasive method for clinical monitoring and studying of pathogenesis in glomerulonephritis.

Interferon-γ upregulates retinoic acid-inducible gene-I in human pericardial mesothelial cells

Objectives — Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH box family proteins and serves as an intracellular signalling molecule involved in the regulation of various genes. Mesothe-lial cells are active in generating a variety of bioactive factors, and RIG-I may serve as a potential intracellular molecule regulating part of these factors. We have addressed the expression of RIG-I in pericardial mesothelial cells.Methods and results — Cultures of human mesothelial cells separated during cardiac surgery were established. Interferon (IFN)-g was found to stimulate the expression, in cultured mesothelial cells, of RIG-I mRNA and protein in time- and concentration-dependent manners. We also performed immunohistochemical analysis of heart tissues to examine the expression of RIG-I in mesothelial cells. Intense immunoreactivity was detected in reactive pericardial mesothelial cells in non-specific pericarditis and in dilated cardiomyopathy.Conclusion — RIG-I may be involved in the inflammatory reaction in pericardial mesothelial cells.

Innate immunity in hepatitis C virus infection: Interplay among dendritic cells, natural killer cells and natural killer T cells

Sequential activation of innate and adaptive immune response is crucial for virus elimination. We thus sought to clarify the role of innate immune system in the pathogenesis of hepatitis C virus (HCV) infection. Dendritic cells (DC) sense virus infection via toll-like receptors (TLR) or retinoic acid inducible gene-I (RIG-I), resulting in the secretion of type-I interferons (IFN) and inflammatory cytokines. Blood DC consist of two subsets; myeloid DC (MDC) and plasmacytoid DC (PDC). In MDC from HCV-infected patients, regardless of higher expression of TLR2, TLR4 and RIG-I compared to the controls, the levels of TLR/RIG-I-mediated IFN-beta or TNF-alpha induction are lower than those in uninfected donors. These results suggest that the signal transduction in the downstream of TLR/RIG-I in MDC is profoundly impaired in HCV infection. In response to IFN-alpha, DC are able to express MHC class-I related chain A/B (MICA/B) and activate natural killer (NK) cells following ligation of NKG2D. Interestingly, DC from HCV-infected patients are unresponsive to exogenous IFN-alpha to enhance MICA/B expression and fail to activate NK cells. Alternatively, NK cells from HCV-infected patients downregulate DC functions in the presence of human leukocyte antigen E-expressing hepatocytes by secreting interleukin (IL)-10 and transforming growth factor-beta1. Such functional alteration of NK cells in HCV infection is ascribed to the enhanced expression of inhibitory receptor NKG2A/CD94 compared to the healthy counterparts. Invariant NKT cells activated by CD1d-positive DC secrete both T-helper (Th)1 and Th2 cytokines, serving as immune regulators. The frequency of NKT cells in chronic HCV infection does not differ from those in healthy donors. Activated NKT cells produce higher levels of IL-13 but comparable levels of IFN-gamma with those from healthy subjects, showing that NKT cells are biased to Th2-type in chronic HCV infection. In conclusion, cross-talks among DC, NK cells and NKT cells are critical in shaping subsequent adaptive immune response against HCV.

Hepatitis C virus escape from the interferon regulatory factor 3 pathway by a passive and active evasion strategy

Hepatitis C virus (HCV) has been known to replicate with extremely varying efficiencies in different host cells, even within different populations of a single human hepatoma cell line, termed Huh-7. Several reports have implicated the retinoic-acid inducible gene I (RIG-I)/ interferon regulatory factor 3 (IRF-3) pathway of the innate antiviral response with differences in host cell permissiveness to HCV. To investigate the general impact of the IRF-3 response onto HCV replication in cell culture, we generated an ample array of stable Huh-7 cell lines with altered IRF-3 responsiveness. Neither blocking IRF-3 activation in various host cells by expression of dominant negative RIG-I or HCV NS3/4A protease nor reconstitution of RIG-I signaling in Huh7.5, a cell clone known to be defective in this pathway, had any impact on HCV replication. Only by overexpressing constitutively active RIG-I or the signaling adaptor Cardif (also known as interferon-beta promoter stimulator 1, mitochondrial anti-viral signaling protein, or virus-induced signaling adaptor), both leading to a stimulation of the IRF-3 pathway in the absence of inducers, was HCV replication significantly inhibited. We therefore assessed the extent of RIG-I- dependent IRF-3 activation by different species of RNA, including full-length HCV genomes and HCV RNA duplexes, and observed strong induction only in response to double-stranded RNAs. Based on these findings, we propose a refined model of innate immune escape by HCV involving limited initial induction and stringent subsequent control of the IRF-3 response.

Central role of interferon regulatory factor‐1 (IRF‐1) in controlling retinoic acid inducible gene‐I (RIG‐I) expression

Retinoic acid inducible gene-I (RIG-I) functions as the first line of defense against viral infection by sensing dsRNA and inducing type I interferon (IFN) production. The expression of RIG-I itself is induced by IFN-alpha/beta and dsRNA. To comprehend the molecular mechanism of expression regulation, we cloned the RIG-I promoter and analyzed its activity upon IFN-beta and dsRNA treatment. Under basal condition, RIG-I mRNA level and promoter activity were significantly higher in normal cells versus their tumor counterparts. In both normal and cancer cells, RIG-I expression was induced by IFN-beta and dsRNA. A single IRF-1 binding site in the proximal promoter functioned as a crucial regulator of basal, IFN-beta- and dsRNA-mediated induction of the RIG-I promoter. IFN-beta and dsRNA treatment increased IRF-1 binding to the RIG-I promoter. IRF-1 expression was also higher in normal cells than in cancer cells and it was induced by IFN-beta with similar kinetics as RIG-I. These results confirm that by controlling RIG-I expression, IRF-1 plays an essential role in anti-viral immunity. IRF-1 is a tumor suppressor and the expression profile of RIG-I together with its regulation by IRF-1 and the presence of a caspase-recruitment domain in RIG-I suggest that RIG-I might also possess tumor suppressor properties.

Expression of retinoic acid-inducible gene-I in lupus nephritis

Expression of retinoic acid-inducible gene-I in lupus nephritis

Retinoic acid-inducible gene-I mediates RANTES/CCL5 expression in U373MG human astrocytoma cells stimulated with double-stranded RNA

Retinoic acid-inducible gene-I (RIG-I) mediates part of the cell signaling in response to viral infection. Polyinosinic-polycytidilic acid (poly IC) is a synthetic double-stranded RNA (dsRNA) and mimics viral infection when applied to cell cultures. The CC chemokine, RANTES (regulated on activation, normal T-cell expressed and secreted), is a potent attractant for inflammatory cells such as memory T-lymphocytes, monocytes and eosinophils. In the present study, we demonstrated that poly IC enhances the expression of RIG-I in U373MG human astrocytoma cells. The RNA interference of RIG-I resulted in the suppression of the poly IC-induced RANTES expression. Pretreatment of the cells with SB203580, an inhibitor of p38 mitogen-activated protein kinase, and dexamethasone inhibited the poly IC-induced expression of RIG-I. Furthermore, poly IC upregulated RIG-I in normal human astrocytes in culture and the in vivo injection of poly IC into the striatum of the mouse brain induced the expression of RIG-I in astrocytes. We conclude that RIG-I may be involved in immune reactions against viral infection, at least in part, through the regulation of RANTES expression in astrocytes.

Expression of Retinoic Acid-inducible gene-I (RIG-I) in Macrophages: Possible Involvement of RIG-I in Atherosclerosis

Retinoic acid-inducible gene-I (RIG-I) is one of the genes induced by interferon (IFN)-gamma which plays an important role in atherosclerosis. The aim of this study is to examine if RIG-I is involved in atherosclerosis. The expression of RIG-I in atherosclerotic lesions in human aorta was examined by immunohistochemical analysis. The expression of RIG-I in THP-1 monocytic cell line or human monocyte-derived macrophages was studied by western blot and RT-PCR analyses. Intense immunoreactivity for RIG-I was detected in intimal macrophages in atherosclerotic lesions. IFN-gamma slightly enhanced the RIG-I expression in THP-1 cells. Treatment of the cells with phorbol 12-myristate 13-acetate, which induces the differentiation of the cells into macrophage-like cells, significantly enhanced the IFN-gamma -induced RIG-I expression. IFN-gamma also stimulated the expression of RIG-I in monocyte-derived macrophages. These results suggest that RIG-I may be involved in differentiation and activation of macrophages, playing a role in atherosclerosis.

Cytokine modulation of retinoic acid-inducible gene-I (RIG-I) expression in human epidermal keratinocytes

Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH/D box family proteins and designated as a putative RNA helicase, which plays various roles in gene expression and cellular functions in response to a variety of RNA viruses. The present study was designed to investigate the effects of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha on RIG-I expression in human keratinocytes, and the expression of RIG-I in skin lesions of psoriasis vulgaris, in which IFN-gamma and TNF-alpha are considered to be involved in its pathogenesis. The mRNA and protein expression of RIG-I was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Immunohistochemical staining of RIG-I was examined on psoriatic skin section. The levels of RIG-I mRNA and protein were upregulated in HaCaT keratinocytes in the presence of IFN-gamma or TNF-alpha. Immunohistochemically, RIG-I was detected in the cytoplasm of the spinous and basal layers of psoriatic skin. Our results suggest that RIG-I might operate not only as a RNA helicase but also as a mediator of the cytokine network in the inflammatory skin diseases, such as psoriasis vulgaris.

Retinoic acid‐inducible gene‐I is induced in gingival fibroblasts by lipopolysaccharide or poly IC: possible roles in interleukin‐1β, ‐6 and ‐8 expression

Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH family of proteins, and little is known of its biological function in the oral region. We previously reported that interleukin 1beta (IL-1beta) induced RIG-I expression in gingival fibroblasts. In this study, we studied the mechanism of RIG-I expression induced by lipopolysaccharide (LPS) or double-stranded RNA (dsRNA) in gingival fibroblasts. We also addressed the role of RIG-I in the expression of IL-1beta, IL-6 and IL-8 in gingival fibroblasts stimulated with LPS or dsRNA. We stimulated cultured human gingival fibroblasts with LPS or dsRNA, and examined the expression of RIG-I mRNA and protein. The effect of cycloheximide, a protein synthesis inhibitor, on RIG-I induction by these stimuli was examined. The expression of IL-1beta, IL-6 and IL-8 in gingival fibroblasts transfected with RIG-I cDNA stimulated with LPS or dsRNA was examined. LPS or dsRNA induced the expression of mRNA and protein for RIG-I in concentration- and time-dependent manners. We also examined the localization of RIG-I, and found that it was expressed in cytoplasm. Cycloheximide did not suppress the LPS or dsRNA-induced RIG-I expression. Introduction of RIG-I cDNA into gingival fibroblasts resulted in enhanced expression of IL-1beta, IL-6 and IL-8; moreover, overexpression of RIG-I stimulated with LPS or dsRNA synergistically increased expression of IL-1beta, IL-6 and IL-8. RIG-I may have important roles in the innate immune response in the regulation of IL-1beta, IL-6 and IL-8 expression in gingival fibroblasts in response to LPS and dsRNA.

NS1 Protein of Influenza A Virus Inhibits the Function of Intracytoplasmic Pathogen Sensor, RIG-I

Retinoic acid-inducible gene I (RIG-I) has recently been identified as one of the key intracellular sensors of virus infection. RIG-I binds to cytosolic double-stranded RNA and initiates a signaling cascade that leads to the activation of transcription factors required for expression of type I interferon (IFN-I). Previous evidence suggests that nonstructural protein 1 (NS1) encoded by influenza A virus (IAV) suppresses IFN-I secretion in virus-infected cells by an unknown mechanism. In the present study, we demonstrate that RIG-I is required for induction of IFN-I in an IAV-infected human lung epithelial cell line. Knockdown of RIG-I expression by RNA interference greatly impairs production of IFN-beta in cells infected with different strains of wild-type IAV. Furthermore, co-expression of IAV NS1 down-regulates production of IFN-beta induced by RIG-I agonists, and ectopic expression of RIG-I inhibits the replication of IAV. These results provide further information on the mechanism by which IAV NS1 antagonizes the host antiviral response.

Listeria monocytogenes Induces the Expression of Retinoic Acid‐Inducible Gene‐I

Retinoic acid-inducible gene-I (RIG-I) is considered to play a role in innate immunity against virus infections. We showed by immunohistochemical study that RIG-I expression is upregulated in vivo in hepatic Kupffer cells and in splenic reticular cells of mice infected with Listeria monocytogenes. Both heat-killed L. monocytogenes and live L. monocytogenes induced the expression of RIG-I in cultured RAW264.7 murine macrophage-like cells in vitro. RIG-I may also be involved in innate immunity against Listeria infection.

The interferon-inducible RNA helicase, mda-5, is involved in measles virus-induced expression of antiviral cytokines

Activation of host cell antiviral responses is mediated by receptors detecting the presence of viruses. Here we have studied the role of double-stranded RNA (dsRNA) binding molecules melanoma differentiation-associated gene 5 (mda-5), retinoic acid inducible gene I (RIG-I), and Toll-like receptor 3 (TLR3) in measles virus (MV)-induced expression of antiviral cytokines and chemokines in human A549 lung epithelial cells and human umbilical vein endothelial cells (HUVECs). We show that MV infection results in the activation of mda-5, RIG-I, and TLR3 gene expression that is followed by high expression of interferon (IFN)-β, interleukin (IL)-28 and IL-29, CCL5, and CXCL10 genes. We also demonstrate that IFN-α and IFN-β upregulate mda-5, RIG-I, and TLR3 gene expression in epithelial and endothelial cell lines. Forced expression of mda-5, but not that of RIG-I or TLR3, leads to enhanced IFN-β promoter activity in MV-infected A549 cells. Our results suggest that IFN-inducible mda-5 is involved in MV-induced expression of antiviral cytokines.

Retinoic acid inducible gene-I and mda-5 are involved in influenza A virus-induced expression of antiviral cytokines

Activation of host cell antiviral responses is mediated by pattern recognition receptors. Cytoplasmic RNA helicases, retinoic acid inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (mda-5) have been identified to function as receptors for double-stranded RNA. Here we show that interferon (IFN)-α pretreatment enhances influenza A virus-induced expression of IFN-α, IFN-β, interleukin (IL)-28 and IL-29 genes in human dendritic cells and epithelial cell lines. Both IFN-α and IFN-β strongly enhanced RIG-I and mda-5 mRNA and protein expression in these cell types. Expression of RIG-I and mda-5 gene constructs, but not that of TLR3, lead to a dramatic enhancement of IFN-β promoter driven transcription in influenza A virus-infected epithelial cells. Furthermore, dominant negative RIG-I gene construct inhibited influenza A virus-induced IFN-β promoter activity. In conclusion, our results show that in epithelial cells influenza A virus-induced antiviral cytokine gene expression is triggered by RIG-I and mda-5, whose expression is positively regulated by IFN-α.

Responsiveness to double stranded RNA in human biliary epithelial cells: Involvement of innate immunity in the pathogenesis of cholangiopathy

Viral infections including reovirus and rotavirus have been suspected as a possible etiology of biliary atresia and choledochal cysts, but a role of these double stranded RNA (dsRNA) viruses is still unknown in the pathogenesis of cholangiopathies. Toll-like receptor (TLR) 3 and the recently identified cytosolic RNA helicases RIG-I (retinoic acid inducible gene I) and MDA5 (melanoma differentiation-associated gene 5) recognize dsRNA and activate NF-kappaB and interferon regulatory factor 3 (IRF3) leading to the induction of IFN-beta production. In order to clarify the innate immunity to dsRNA virus in cholangiopathies with reference to extrahepatic biliary atresia and choledochal cysts, we examined the expression of TLR3, RIG-I, and MDA5 and the effects of dsRNA using 3 cultured human biliary epithelial cells (HIBECs). The expression of TLR3, RIG-I, and MDA5 were demonstrated in all of three HIBEC cells by RT-PCR. In addition, HIBEC cells produced IFN-beta after the treatment with poly(I:C) (synthetic analog of dsRNA), but not with bacterial components such as peptidoglycan and lipopolysaccharide. Furthermore, the poly(I:C) treatment upregulated the activation of NF-kappaB. In contrast, IRF3 had already activated under basal conditions and its synergistic activation by poly(I:C) was unclear in HIBEC cells. In conclusion, human biliary epithelial cells can cause an innate immunity response to dsRNA virus, supporting the immune-mediated mechanisms in the pathogenesis of cholangiopathies in biliary atresia and choledochal cysts.

Hepatitis C virus NS2 and NS3/4A proteins are potent inhibitors of host cell cytokine/chemokine gene expression

BackgroundHepatitis C virus (HCV) encodes several proteins that interfere with the host cell antiviral response. Previously, the serine protease NS3/4A was shown to inhibit IFN-β gene expression by blocking dsRNA-activated retinoic acid-inducible gene I (RIG-I) and Toll-like receptor 3 (TLR3)-mediated signaling pathways.ResultsIn the present work, we systematically studied the effect of all HCV proteins on IFN gene expression. NS2 and NS3/4A inhibited IFN gene activation. NS3/4A inhibited the Sendai virus-induced expression of multiple IFN (IFN-α, IFN-β and IFN-λ1/IL-29) and chemokine (CCL5, CXCL8 and CXCL10) gene promoters. NS2 and NS3/4A, but not its proteolytically inactive form NS3/4A-S139A, were found to inhibit promoter activity induced by RIG-I or its adaptor protein Cardif (or IPS-1/MAVS/VISA). Both endogenous and transfected Cardif were proteolytically cleaved by NS3/4A but not by NS2 indicating different mechanisms of inhibition of host cell cytokine production by these HCV encoded proteases. Cardif also strongly colocalized with NS3/4A at the mitochondrial membrane, implicating the mitochondrial membrane as the site for proteolytic cleavage. In many experimental systems, IFN priming dramatically enhances RNA virus-induced IFN gene expression; pretreatment of HEK293 cells with IFN-α strongly enhanced RIG-I expression, but failed to protect Cardif from NS3/4A-mediated cleavage and failed to restore Sendai virus-induced IFN-β gene expression.ConclusionHCV NS2 and NS3/4A proteins were identified as potent inhibitors of cytokine gene expression suggesting an important role for HCV proteases in counteracting host cell antiviral response.

Effect of peroxisome proliferator-activated receptor-γ ligands on the expression of retinoic acid-inducible gene-I in endothelial cells stimulated with lipopolysaccharide

Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH box protein family and designated as a putative RNA helicase. RIG-I is implicated in host defense and inflammatory reactions by regulating the expression of various genes. RIG-I is expressed in endothelial cells and upregulated with lipopolysaccharide (LPS). Peroxisome proliferator-activated receptor-γ (PPAR-γ) is a nuclear hormone receptor and regulates gene expressions in response to its specific ligands. In the present study, we examined the effect of PPAR-γ ligands on the LPS-induced RIG-I expression in cultured human umbilical vein endothelial cells (HUVEC). 15-Deoxy-Δ 12,14-prostaglandin J 2 (15d-PGJ 2), a metabolite of PGD 2, is a natural ligand for PPAR-γ and known to modulate inflammatory reactions by regulating the expression of various genes in PPAR-γ-dependent and -independent manners. LPS-induced RIG-I expression in HUVEC was inhibited by pretreatment of the cells with 15d-PGJ 2 in time-and concentration-dependent manners. However, ciglitazone and bisphenol A diglycide ether, authentic and specific ligands for PPAR-γ, did not affect the RIG-I expression. These results suggest that 15d-PGJ 2 inhibits LPS-induced RIG-I expression through a mechanism independent on PPAR-γ. 15d-PGJ 2 may regulate inflammatory reactions, at least in part, by inhibiting the expression of RIG-I.

Involvement of retinoic acid-inducible gene-I in the IFN- /STAT1 signalling pathway in BEAS-2B cells

Bronchial epithelial cells play an important role in airway host defence, and interferon (IFN)-gamma controls immune reactions by regulating the expression of various genes in bronchial epithelial cells. Signal transducer and activator of transcription 1 (STAT1) is the key transcriptional factor in IFN-gamma signalling. Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH box family of proteins and designated a putative RNA helicase. RNA helicases play diverse roles in regulation of gene expression and cellular functions, and RIG-I is implicated in antiviral responses. The aim of the present study was to investigate the effect of IFN-gamma on RIG-I expression in a cell line derived from human bronchial epithelial cells, BEAS-2B. Induction of RIG-I in response to IFN-gamma was found in BEAS-2B cells. Induction of RIG-I by IFN-gamma was also demonstrated in another pulmonary epithelial cell line, NCI-H292. Transfection of BEAS-2B cells with RIG-I complementary DNA resulted in the upregulation of STAT1. Induction of IFN-gamma-inducible protein 10 by IFN-gamma was enhanced in the cells overexpressing RIG-I. It is concluded that retinoic acid-inducible gene-I may play an important role in the regulation of immunological reactions in bronchial epithelial cells elicited by interferon-gamma.