Expression Of RIG-1 Research Articles

Abstract Tu013: ETV2 Transcriptionally Activates Rig1 Gene Expression and Promotes Reprogramming of the Endothelial Lineage

ETV2 is a master regulator and pioneer factor for the endothelial lineage. Previous studies have established that this transcription factor was essential for embryogenesis and direct reprogramming of fibroblasts to endothelial cells. ETV2-mediated direct reprogramming holds therapeutic potential in treating ischemic tissues including ischemic heart disease, yet the underlying molecular mechanisms remain to be completely defined. In the current studies, we focused on deciphering the mechanisms that governed ETV2-mediated reprogramming, including the role of RIG1 inflammatory responses. We hypothesized that ETV2 transcriptionally activated Rig1 gene expression, increased expression of its downstream network [RIG1-like receptor (RLR) pathway] and modulated the reprogramming of fibroblasts to endothelial cells. Mouse embryonic fibroblasts (MEFs) with inducible ETV2 expression system were used to overexpress ETV2, and single-cell RNA-seq defined transcript expression involved in RLR signaling pathways. ChIP-seq, electromobility gel shift assays (EMSA), and transcriptional assays were used to verify that ETV2 was a direct upstream activator of Rig1 gene expression. Both RIG1 and RLR pathway proteins were significantly upregulated in ETV2-overexpressing MEFs. ETV2 mediated the Rig1 upregulation through direct binding to the Rig1 promoter and transcriptional activation of Rig1 expression. Knockdown of Rig1 using shRNA significantly (p < 0.05) reduced the efficiency of fibroblast to endothelial cell reprogramming more than 70%. These results highlight that 1) ETV2 activates inflammatory pathways and 2) RIG1 is direct downstream target of ETV2 and together they have an essential role in the reprogramming process. These findings extend our current understanding of the molecular mechanisms underlying ETV2-mediated reprogramming and will be important in the design of a revascularization strategy for the treatment of ischemic heart disease.

Castanea crenata honey reduces influenza infection by activating the innate immune response.

Influenza is an acute respiratory disorder caused by the influenza virus and is associated with prolonged hospitalization and high mortality rates in older individuals and chronically ill patients. Vaccination is the most effective preventive strategy for ameliorating seasonal influenza. However, the vaccine is not fully effective in cases of antigenic mismatch with the viral strains circulating in the community. The emergence of resistance to antiviral drugs aggravates the situation. Therefore, developing new vaccines and antiviral drugs is essential. Castanea crenata honey (CH) is an extensively cultivated food worldwide and has been used as a nutritional supplement or herbal medicine. However, the potential anti-influenza properties of CH remain unexplored. In this study, the in vitro and in vivo antiviral effects of CH were assessed. CH significantly prevented influenza virus infection in mouse Raw264.7 macrophages. CH pretreatment inhibited the expression of the viral proteins M2, PA, and PB1 and enhanced the secretion of proinflammatory cytokines and type-I interferon (IFN)-related proteins in vitro. CH increased the expression of RIG-1, mitochondrial antiviral signaling (MAVS) protein, and IFN-inducible transmembrane protein, which interferes with virus replication. CH reduced body weight loss by 20.9%, increased survival by 60%, and decreased viral replication and inflammatory response in the lungs of influenza A virus-infected mice. Therefore, CH stimulates an antiviral response in murine macrophages and mice by preventing viral infection through the RIG-1-mediated MAVS pathway. Further investigation is warranted to understand the molecular mechanisms involved in the protective effects of CH on influenza virus infection.

Japanese encephalitis virus induces apoptosis by activating the RIG-1 signaling pathway.

Japanese encephalitis virus (JEV) infection can cause brain tissue lesions characterized by neuronal death, and apoptosis is involved in JEV-induced neuronopathy. In the present study, mouse microglia were infected with JEV, and pyknosis with dark-staining nuclei of infected cells was detected using Hoechst 33342 staining. TUNEL staining showed that JEV infection promoted the apoptosis of BV2 cells, and the apoptosis rate was significantly increased at 24-60 hours postinfection (hpi) (P < 0.01) and was the highest at 36 h (P < 0.0001). Western blot results showed that the expression of the Bcl-2 protein in JEV-infected cells was downregulated significantly at 60 hpi (P < 0.001), whereas that of the Bax protein was observably upregulated at 60 hpi (P < 0.001). At the same time, the level of cytochrome c (Cyt c) was significantly increased (P < 0.001), and the expression levels of two apoptosis-related proteins, namely, cleaved caspase-3 (P < 0.01) and caspase-9 (P < 0.001), were elevated significantly. Immunofluorescence staining showed that the amount of Cyt c increased with time after infection. After BV2 cells were infected with JEV, the expression of RIG-1 increased significantly from 24 hpi to 60 h (P < 0.001). The expression of MAVS increased significantly at 24 h (P < 0.001) and decreased gradually from 24 h to 60 hpi. The expression of TBK1 and NF-κB (p65) was not significantly changed. The expression of p-TBK1 and p-NF-κB (p-p65) increased significantly within 24 h (P < 0.001) and decreased from 24 to 60 hpi. The expression levels of IRF3 and p-IRF3 peaked at 24 hpi (P < 0.001) and decreased gradually from 24 to 60 hpi. However, the expression levels of JEV proteins showed no significant change at 24 and 36 hpi but were markedly elevated at 48 and 60 hpi. Interference with the expression of the RIG-1 protein in BV2 cells resulted in a dramatic increase in the expression of the anti-apoptotic protein Bcl-2 (P < 0.05), whereas the pro-apoptotic protein Bax, cleaved caspase-9, and especially cleaved caspase-3 were downregulated (P < 0.05), and viral protein expression was notably reduced (P < 0.05). These results indicate that JEV induces apoptosis through mitochondrial-dependent apoptosis pathways, interfering with the expression of RIG-1 in BV2 cells can inhibit viral replication and inhibit apoptosis.

Cytokine profile of human limbal myofibroblasts: Key players in corneal antiviral response.

Corneal transparency may be compromised by viral infections causing corneal scarring, edema, and neovascularization. Ocular injury results from collateral damage induced by exacerbated immune response in corneal stroma. Myofibroblasts play a key role in this process by producing a disorganized extracellular matrix and inflammatory mediators. However, the immune response profile of myofibroblasts during viral infections is still under study. The aim of this work was to analyze the cytokine profile of human limbal myfibroblasts (HLMs) stimulated with the double-stranded RNA analog polyinosinic:polycytidylic acid (poly I:C) and to identify their signaling pathways. HLMs were isolated from cadaveric sclera-corneal rims and stimulated with poly I:C (10μg/ml) for 12h. The secretion of 36 cytokines was measured using the Human Cytokine Array Panel A. The secretion of IFN-β was quantified by ELISA. The expression of pattern recognition receptors (PRRs) such as TLR3, RIG-1 and MDA5 were analyzed by western blot assays. Furthermore, translocation of the nuclear factors NF-κB, IRF3, and IRF7 was assessed by fluorescence staining. In addition, the differentially expressed cytokines were analyzed using the Core Analysis Tool of the Ingenuity Pathway Analysis IPA software. HLMs stimulated with poly I:C increased (fold change&gt;2) the secretion of G-CSF, sTREM-1, CXCL1, CCL1, CXCL8, CXCL10, CXCL11, CCL2, CCL5, IL-13, IL-6, IL-1ra, and IFN-β compared with HLMs under basal conditions. Poly I:C stimulation also induced the expression of RIG-1 (p&lt;0.001), but the expression of TLR3 and MDA5 was unmodified. Finally, HLMs increased nuclear translocation of NF-κB, IRF3, and IRF7 after poly I:C stimulation. Bioinformatic analysis identified canonical signaling pathways associated with cell adhesion and diapedesis, chemokine signaling, and activation of IRFs by cytosolic pattern recognition receptors. These results demonstrate that HLMs secrete cytokines involved in immune cell activation and chemotaxis. The data suggest a key role for HLMs during viral infections in cornea and extend our knowledge about the signaling pathways they trigger.

Simultaneous Detection of RIG-1, MDA5, and IFIT-1 Expression Is a Convenient Tool for Evaluation of the Interferon-Mediated Response.

In this study, we developed a novel, multiplex qPCR assay for simultaneous detection of RIG-1, MDA5, and IFIT-1 at the mRNA level. The assay was validated in A549 cells transfected with in vitro transcribed RNAs. Both exogenous RNA-GFP and self-amplifying (saRNA-GFP) induced significant expression of RIG-1, MDA5, IFIT-1, as well as type I and III interferons. In contrast, native RNA from intact A549 cells did not upregulate expression of these genes. Next, we evaluated RIG-1, MDA5, and IFIT-1 mRNA levels in the white blood cells of patients with influenza A virus (H3N2) or SARS-CoV-2. In acute phase (about 4 days after disease onset) both viruses induced these genes expression. Clinical observations of SARS-CoV-2 typically describe a two-step disease progression, starting with a mild-to-moderate presentation followed by a secondary respiratory worsening 9 to 12 days after the first onset of symptoms. It revealed that the expression of RIG-1, MDA5, and MxA was not increased after 2 and 3 weeks from the onset the disease, while for IFIT-1 it was observed the second peak at 21 day post infection. It is well known that RIG-1, MDA5, and IFIT-1 expression is induced by the action of interferons. Due to the ability of SOCS-1 to inhibit interferon-dependent signaling, and the distinct antagonism of SARS-CoV-2 in relation to interferon-stimulated genes expression, we assessed SOCS-1 mRNA levels in white blood cells. SARS-CoV-2 patients had increased SOCS-1 expression, while the influenza-infected group did not differ from heathy donors. Moreover, SOCS-1 mRNA expression remained stably elevated during the course of the disease. It can be assumed that augmented SOCS-1 expression is one of multiple mechanisms that allow SARS-CoV-2 to escape from the interferon-mediated immune response. Our results implicate SOCS-1 involvement in the pathogenesis of SARS-CoV-2.

Endothelial Cells as a Key Cell Type for Innate Immunity: A Focused Review on RIG-I Signaling Pathway.

The vascular endothelium consists of a highly heterogeneous monolayer of endothelial cells (ECs) which are the primary target for bacterial and viral infections due to EC’s constant and close contact with the bloodstream. Emerging evidence has shown that ECs are a key cell type for innate immunity. Like macrophages, ECs serve as sentinels when sensing invading pathogens or microbial infection caused by viruses and bacteria. It remains elusive how ECs senses danger signals, transduce the signal and fulfil immune functions. Retinoic acid-inducible gene-I (RIG-I, gene name also known as DDX58) is an important member of RIG-I-like receptor (RLR) family that functions as an important pathogen recognition receptor (PRR) to execute immune surveillance and confer host antiviral response. Recent studies have demonstrated that virus infection, dsRNA, dsDNA, interferons, LPS, and 25-hydroxycholesterol (25-HC) can increase RIG-1 expression in ECs and propagate anti-viral response. Of translational significance, RIG-I activation can be inhibited by Panax notoginseng saponins, endogenous PPARγ ligand 15-PGJ2, tryptanthrin and 2-animopurine. Considering the pivotal role of inflammation and innate immunity in regulating endothelial dysfunction and atherosclerosis, here we provided a concise review of the role of RIG-I in endothelial cell function and highlight future direction to elucidate the potential role of RIG-I in regulating cardiovascular diseases as well as virus infectious disease, including COVID-19. Furthered understanding of RIG-I-mediated signaling pathways is important to control disorders associated with altered immunity and inflammation in ECs.

Innate Immune Responses to Wildtype and Attenuated Sheeppox Virus Mediated Through RIG-1 Sensing in PBMC In-Vitro.

Sheeppox (SPP) is a highly contagious disease of small ruminants caused by sheeppox virus (SPPV) and predominantly occurs in Asia and Africa with significant economic losses. SPPV is genetically and immunologically closely related to goatpox virus (GTPV) and lumpy skin disease virus (LSDV), which infect goats and cattle respectively. SPPV live attenuated vaccines (LAVs) are used for vaccination against SPP and goatpox (GTP). Mechanisms related to innate immunity elicited by SPPV are unknown. Although adaptive immunity is responsible for long-term immunity, it is the innate responses that prevent viral invasion and replication before LAVs generate specific long-term protection. We analyzed the relative expression of thirteen selected genes that included pattern recognition receptors (PRRs), Nuclear factor-κβ p65 (NF-κβ), and cytokines to understand better the interaction between SPPV and its host. The transcripts of targeted genes in sheep PBMC incubated with either wild type (WT) or LAV SPPV were analyzed using quantitative PCR. Among PRRs, we observed a significantly higher expression of RIG-1 in PBMC incubated with both WT and LAV, with the former producing the highest expression level. However, there was high inter-individual variability in cytokine transcripts levels among different donors, with the expression of TNFα, IL-15, and IL-10 all significantly higher in both PBMC infected with either WT or LAV compared to control PBMC. Correlation studies revealed a strong significant correlation between RIG-1 and IL-10, between TLR4, TNFα, and NF-κβ, between IL-18 and IL-15, and between NF-κβ and IL-10. There was also a significant negative correlation between RIG-1 and IFNγ, between TLR3 and IL-1 β, and between TLR4 and IL-15 (P< 0.05). This study identified RIG-1 as an important PRR in the signaling pathway of innate immune activation during SPPV infection, possibly through intermediate viral dsRNA. The role of immunomodulatory molecules produced by SPPV capable of inhibiting downstream signaling activation following RIG-1 upregulation is discussed. These findings advance our knowledge of the induction of immune responses by SPPV and will help develop safer and more potent vaccines against SPP and GTP.

MiR-139 Induces an Interferon-β Response in Prostate Cancer Cells by Binding to RIG-1.

We previously identified a panel of five miRNAs associated with prostate cancer recurrence and metastasis. Expression of one of the down-regulated miRNAs, miR-139-5p, was significantly associated with a lower incidence of biochemical recurrence and metastasis. Transcriptome profiling of miR-139-expressing prostate cancer cells revealed up-regulation of genes involved in interferon (IFN) stimulation. The association between miR-139 and IFN-β was further explored in this study. We examined miR-139 transfected PC3, Du145 and LNCaP cells and the associated IFN response by transcriptome sequencing, immunoblotting and pulldown assays. Treatment of prostate cancer cells by miR-139 resulted in the up-regulation of IFN-related genes. Specifically, miR-139 induced expression of the IFN-β protein. The ability of miR-139 to induce IFN-β was due to its binding to RIG-1 and the induction of IFN-related genes was found to be dependent on RIG-1 expression. miR-139 acts as an immune agonist of RIG-1 to enhance IFN-β response in prostate cancer cells.

Expression Pattern Analysis of Antiviral Genes and Inflammatory Cytokines in PEDV-Infected Porcine Intestinal Epithelial Cells.

Porcine diarrhea disease in newborn and suckling piglets due to infection with porcine epidemic diarrhea virus (PEDV) is a leading cause of economic loss in the pig industry globally. In this study, we investigated the molecular mechanism of the host innate immune response to PEDV infection. The expression dynamics of antiviral genes (e.g., RIG-1, PKR, OAS1, Mx1, and Mx2) and inflammatory cytokines (e.g., IFN-α, IFN-β, TNF-α, IL-6, IL-8, and IL-12) in porcine small intestinal epithelial (IPEC-J2) cells were analyzed following PEDV stimulation. The results showed that the expression of antiviral genes (e.g., PKR, OAS1, and Mx2) and inflammatory cytokines (e.g., IFN-α and TNF-α) were significantly reduced within 0–4 h post-infection (P < 0.05). However, all antiviral genes and inflammatory cytokines were up-regulated from 12 to 24 h (P < 0.05), and cytopathic changes were observed during this time. The expression of RIG-1, PKR, OAS1, Mx1, and Mx2 were significantly and positively correlated to each other during the entire infection (P < 0.01). The results suggested that the RIG-1, PKR, OAS1, Mx1, and Mx2 genes may play an important role in PEDV infection in piglets. Initially, PEDV displayed cellular invasion by inhibiting IFN-α transcription and interfering with the antiviral function of PKR, OAS1, and Mx2, ultimately induced an intense inflammatory response. The relationship between antiviral genes and inflammatory cytokines with PEDV infection at the cellular level provides a reference for studying the mechanism of resistance to PEDV infection in piglets.

Peculiarities of RIG-1 Expression in Placental Villi in Preeclampsia.

The expression of RIG-1 in placenta samples was assessed in women of reproductive age with early- and late-onset preeclampsia and cesarean delivery at 27-39 weeks of gestation. The highest expression of RIG-1 was found in the syncytiotrophoblast of placental villi in the group with uncomplicated full-term pregnancy (normal); RIG-1 expression in groups with early- and late-onset preeclampsia was significantly (p<0.01) lower. In decidual cells, RIG-1 expression was also maximum in normal pregnancy and significantly (p<0.01) lower in lateonset preeclampsia. In the endothelium of villous capillaries, the maximum expression was observed in normal full-term pregnancy and in late-onset preeclampsia, while in early-onset preeclampsia this parameter was significantly (p<0.01) lower. It can be assumed that different variants of preeclampsia are mediated by similar pathogenetic mechanisms, including those related to immature molecular profile of the trophoblast and decidual cells, probably due to impaired stem cell activity in the placenta determining higher vulnerability and reduced regeneration capacity of the placental tissue. This is due to the fact that RIG-1 is one of the important signaling molecules that promote activation of stem cell and tissue regeneration.

Innate immune response in patients with acute Zika virus infection.

Innate immunity receptors (Toll-like receptors/TLRs and RIG-like receptors/RLRs) are important for the initial recognition of Zika virus (ZIKV), modulation of protective immune response, and IFN-α and IFN-β production. Immunological mechanisms involved in protection or pathology during ZIKV infection have not yet been determined. In this study, we evaluated the mRNA expression of innate immune receptors (TLR3, TLR7, TLR8, TLR9, melanoma differentiation-associated protein 5/MDA-5, and retinoic acid inducible gene/RIG-1), its adapter molecules (Myeloid Differentiation Primary Response Gene 88/Myd88, Toll/IL-1 Receptor Domain-Containing Adaptor-Inducing IFN-β/TRIF), and cytokines (IL-6, IL-12, TNF-α, IFN-α, IFN-β, and IFN-γ) in the acute phase of patients infected by ZIKV using real-time PCR in peripheral blood. Patients with acute ZIKV infection had high expression of TLR3, IFN-α, IFN-β, and IFN-γ when compared to healthy controls. In addition, there was a positive correlation between TLR3 expression compared to IFN-α and IFN-β. Moreover, viral load is positively correlated with TLR8, RIG-1, MDA-5, IFN-α, and IFN-β. On the other hand, patients infected by ZIKV showed reduced expression of RIG-1, TLR8, Myd88, and TNF-α molecules, which are also involved in antiviral immunity. Similar expressions of TLR7, TLR9, MDA-5, TRIF, IL-6, and IL-12 were observed between the group of patients infected with ZIKV and control subjects. Our results indicate that acute infection (up to 5days after the onset of symptoms) by ZIKV in patients induces the high mRNA expression of TLR3 correlated to high expression of IFN-γ, IFN-α, and IFN-β, even though the high viral load is correlated to high expression of TLR8, RIG-1, MDA-5, IFN-α, and IFN-β in ZIKV patients.

RIG-1 and MDA5 are the important intracellular sensors against bacteria in septicemia suffering patients

BackgroundMDA5 and RIG-1 are the important intracellular receptors which detect microbial associated molecular patterns. Septicemia is a condition in which infection enters the bloodstream of the patients. The main intracellular mechanisms against septicemia are yet to be clarified. Therefore, this research study was aimed to evaluate expression of MDA5 and RIG-1 in the patients suffering from septicemia in comparison to healthy controls. MethodsMDA5 and RIG-1 expression levels in 40 patients suffering from septicemia and 40 healthy controls were evaluated using Real-Time PCR technique. The sources of bacteria in the bloodstream of the patients suffering from septicemia were determined using microbial cultures. ResultsThe results showed that mRNA levels of MDA5 and RIG-1 were significantly increased in the patients when compared to healthy controls. The results also revealed that the patients were infected with four bacteria including Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii and Pseudomonas aeruginosa. mRNA levels of MDA5 and RIG-1 did not differ among patients with various bacterial infections. ConclusionBased on the results it seems that MDA5 and RIG-1 are the main intracellular immunity against the bacteria during septicemia and could be considered for their roles in induction of immunity.

RIG-1 expression is associated with sexual malfunctions of female type 2 diabetic patients

Introduction: The patients with type 2 diabetes (T2D) suffer from the malfunctions of the sexual behaviors, and several mechanisms have been proposed to describe these disorders. The innate immunity may be involved in the malfunctions of T2D patients. Melanoma differentiation-associated protein 5 (MDA5) and retinoic acid (RA)-inducible gene 1 (RIG-1), as the innate immunity receptors, are the responsible molecules for the activation of some intracellular signaling pathways and the induction of inflammation. Thus, this study aimed to examine the molecules which may participate in the induction/stimulation of sexual malfunctions in the female T2D patients. Methods: Sexual functions were evaluated in 41 female T2D patients using the Female Sexual Function Index (FSFI) questionnaire. Real-time polymerase chain reaction (PCR) technique was used to quantify MDA5 and RIG-1 mRNA levels. Results: Results showed that increased RIG-1 mRNA levels were significantly associated with the bad orgasm in the female T2D patients compared to the female patients with good orgasm. Expression of RIG-1 and MDA5 levels were not associated with other sexual functions’ criteria. Conclusion: The findings of this study demonstrated that bad orgasm is associated with the increased RIG-1 expression. Consequently, the correlation between inflammation and bad orgasm in a RIG-1 dependent manner is suggested.

S129 Inflammatory Cytokines Influence Respiratory Epithelial Anti-viral Immune Responses Via Inducible Epigenetic Control Of Rig1 Expression: A Model Of Early Life Origins Of Asthma?

The development of asthma is linked to early life environmental exposures and the occurrence of severe viral infections. Rapid maturation of adaptive immunity from a tolerant (Th2) to an anti-infective (Th1) state occurs in the neonatal period. We hypothesised that the airway inflammatory milieu, driven by the maturing immune response to environmental exposures may have important effects on the development of anti-viral innate immunity at the level of the epithelium. We studied whether the inflammatory environment of the airway epithelium modulates gene expression via epigenetic regulation of anti-viral genes as a model of the development of a long term abnormality which is a hallmark of asthma. We optimised an in-vitro model using AALEB, human immortalised bronchial epithelial-derived cells which were pre-treated for 24 h with cytokines that mimic Th1 environment (IFNy, 10 ng/ml) and Th2 (IL-13, 10 ng/ml) before being infected with RSV A, MOI=2 for 48 h. Quantitative real-time PCR with Taqman primers was used to assess expression of innate genes. Cells were collected after 48 h and stored in Trizol. Chromatin Immuno Precipitation (ChIP) with antibodies against histone modifications was used to assess epigenetic controls. In order to confirm epigenetic regulation of innate genes we used a panel of HAT, HDAC and histone demethylase inhibitors. We initially studied the impact of cytokines on a range of innate anti-viral genes. RIG1 was differentially expressed and reductions in expression associated with higher viral titres. IFNy priming induced increases in RIG1 mRNA at 48 h that correlated at the promoter with enrichment of H3K9ac and RNApolII (active-promoters) and reduction of H3K9me3 (repressive-promoters). We observed a statistically significant increase of RIG1 expression by IFNγ when co-incubated with SAHA (HDAC I and II inhibitors) and JIB-04 (Pan-Jumanji histone demethylase inhibitor). No effects of Th2 priming were seen at the level of antiviral responses. This in-vitro study suggests the inflammatory environment of naive epithelial cells can induce epigenetic modulation of innate immune responses at the level of histone methylation and acetylation and hence potentially lead to long term impacts on anti-viral immunity. The presence of a Th1 milieu appears key to the development of effective anti-viral responses.

Role of RIG-1 signaling in the Pathogenesis of Alzheimer’s Disease (172.1)

Abstract Recent evidence has accumulated that Alzheimer’s disease (AD) is associated with a central nervous system (CNS) inflammatory response. Neuroinflammation is a complex response against harmful effects of diverse stimuli and plays a critical role in the pathogenesis of AD. An important arm of the inflammatory response is the innate immune response. The innate immune response is regulated by pattern recognition receptors (PRRs) and their signaling pathways. Here we report that the expression of the innate immune response proteins, retinoic acid-inducible gene-I (RIG-1), virus-induced signaling adaptor (VISA), and the NACHT leucine-rich domain and pyrin-containing protein, NLRP12 are significantly elevated in neurons of the temporal cortex, the cerebrospinal fluid and plasma in presymptomatic AD patients, post-mortem diagnosis. Increased expression of RIG-1 and VISA were not found in pathologically confirmed cases of amyotrophic lateral sclerosis, Diffuse Lewy Body disease or Huntington’s disease, suggesting that activation of RIG-1 signaling may be a specific marker for early AD pathogenesis. Moreover, in vitro results show that stimulation of RIG-1 signaling induces expression of amyloid precursor protein and β-amyloid, supporting the idea that activation of the RIG-1 signaling system occurs in presymptomatic AD.

Abstract 4003: Involving of PGD2 signal pathway in the RIG1-mediated suppression of cell invasion in testis cells

Abstract Retinoid-inducible gene 1 (RIG1), also called tazarotene induced gene 3 (TIG3), belongs to the HREV107 gene family that contains five members in humans. Proteins of the HREV107 family share four conserved domains, a proline-rich motif located at the N-terminus, followed by a conserved H-box, the NC domain and a C-terminal transmembrane domain. RIG1 is expressed in high levels in well-differentiated tissues, but the expression of RIG1 decreases in cancer tissues and cancer cell lines. RIG1 stimulates cellular differentiation of keratinocytes, which is mediated by the activation of type I tissue transglutaminase. In addition, RIG1 was shown to inhibit the RAS signal pathways in cervical and gastric cancer cells. However, the direct interactions of RIG1/Type I tissue transglutaminase or RIG1/RAS were observed in cervical cancer cells. Until now, the mechanisms of anti-cancer /induced-cellular differentiation effects caused by RIG1 induced proteins have not been reported. We have found that Prostaglandin D2 synthase (PTGDS) interacted with RIG1 by yeast two hybrid screens. Further, we found PTGDS immunoprecipitated with RIG1 in NT2/D1 testis cell lysate. We also found PTGDS co-localized with RIG1 at the endomembrane. In RIG1-transfected cell, increasing expression of PGD2 production can be observed after stimulating by PTGDS. Thus, this indicated RIG1 can enhance PTGDS activity. Otherwise, cAMP and SOX9 are targets for PGD2 signaling. Our results revealed that cAMP but not SOX9 were upregulated in RIG1-expressing NT2/D1 testis cells. Pervious study indicated that RIG1 is a phospholipid-metabolizing enzyme (PLA) which involved in the phospholipid metabolism and the PLA activity is essential for the PGD2 production. RIG1 upregulated the PGD2 and cAMP production will not be affected in PLA inhibitor AACOCF3 treated-RIG1-expressing NT2/D1 cells. Silencing of PTGDS expression significantly decreased RIG1-mediated cAMP and PGD2 production. These results suggested that RIG1-induced PGD2 production is mediated by increasing the PTGDS activity but not PLA activity of RIG1. Either cell death or cell viability was not affected in RIG1-expressing cells. However, RIG1 significantly inhibited the cell migration and invasion in NT2/D1 cells. Knockdown of PTGDS expression but not treating cells with AACOCF3 can alleviated RIG1-induced invasion and migration suppression of NT2/D1 cells. This result indicated that RIG1 will cause cell invasion suppression though the PGD2 signaling pathway. In conclusion, RIG1 can interact with PTGDS to enhance its function and further suppress the NT2/D1 cell migration and invasion. Our study suggested that RIG1-PGD2 signaling might play an important role in cell differentiation of testis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4003. doi:1538-7445.AM2012-4003

Expression of major guidance receptors is differentially regulated in spinal commissural neurons transfated by mammalian Barh genes

During development, commissural neurons in the spinal cord project their axons across the ventral midline, floor plate, via multiple interactions among temporally controlled molecular guidance cues and receptors. The transcriptional regulation of commissural axon-associated receptors, however, is not well characterized. Spinal dorsal cells are transfated into commissural neurons by misexpression of Mbh1, a Bar-class homeobox gene. We examined the function of another Bar-class homeobox gene, Mbh2, and how Mbh1 and Mbh2 modulate expression of the receptors, leading to midline crossing of axons. Misexpression of Mbh1 and Mbh2 showed the same effects in the spinal cord. The competence of spinal dorsal cells to become commissural neurons was dependent on the embryonic stage, during which misexpression of the Mbh genes was able to activate guidance receptor genes such as Rig1 and Nrp2. Misexpression of Lhx2, which has been recently shown to be involved in Rig1 expression, activated Rig1 but not Nrp2, and was less effective in generating commissural neurons. Moreover, expression of Lhx2 was activated by and required the Mbh genes. These findings have revealed a transcriptional cascade, in which Lhx2-dependent and -independent pathways leading to expression of guidance receptors branch downstream of the Mbh genes.

A Molecular Program for Contralateral Trajectory: Rig-1 Control by LIM Homeodomain Transcription Factors

Despite increasing evidence for transcriptional control of neural connectivity, how transcription factors regulate discrete steps in axon guidance remains obscure. Projection neurons in the dorsal spinal cord relay sensory signals to higher brain centers. Some projection neurons send their axons ipsilaterally, whereas others, commissural neurons, send axons contralaterally. We show that two closely related LIM homeodomain proteins, Lhx2 and Lhx9, are expressed by a set of commissural relay neurons (dI1c neurons) and are required for the dI1c axon projection. Midline crossing by dI1c axons is lost in Lhx2/9 double mutants, a defect that results from loss of expression of Rig-1 from dI1c axons. Lhx2 binds to a conserved motif in the Rig-1 gene, suggesting that Lhx2/9 regulate directly the expression of Rig-1. Our findings reveal a link between the transcriptional programs that define neuronal subtype identity and the expression of receptors that guide distinctive aspects of their trajectory.

Sim1andSim2are required for the correct targeting of mammillary body axons

The mammillary body (MB), and its axonal projections to the thalamus (mammillothalamic tract, MTT) and the tegmentum (mammillotegmental tract, MTEG), are components of a circuit involved in spatial learning. The bHLH-PAS transcription factors SIM1 and SIM2 are co-expressed in the developing MB. We have found that MB neurons are generated and that they survive at least until E18.5 in embryos lacking both Sim1 and Sim2 (Sim1(-/-);Sim2(-/-)). However, the MTT and MTEG are histologically absent in Sim1(-/-);Sim2(-/-) embryos, and are reduced in embryos lacking Sim1 but bearing one or two copies of Sim2, indicating a contribution of the latter to the development of MB axons. We have generated, by homologous recombination, a null allele of Sim1 (Sim1(tlz)) in which the tau-lacZ fusion gene was introduced, allowing the staining of MB axons. Consistent with the histological studies, lacZ staining showed that the MTT/MTEG is barely detectable in Sim1(tlz/tlz);Sim2(+/-) and Sim1(tlz/tlz);Sim2(-/-) brains. Instead, MB axons are splayed and grow towards the midline. Slit1 and Slit2, which code for secreted molecules that induce the repulsion of ROBO1-producing axons, are expressed in the midline at the level of the MB, whereas Robo1 is expressed in the developing MB. The expression of Rig-1/Robo3, a negative regulator of Slit signalling, is upregulated in the prospective MB of Sim1/Sim2 double mutants, raising the possibility that the growth of mutant MB axons towards the midline is caused by a decreased sensitivity to SLIT. Finally, we found that Sim1 and Sim2 act along compensatory, but not hierarchical, pathways, suggesting that they play similar roles in vivo.

The Slit Receptor Rig-1/Robo3 Controls Midline Crossing by Hindbrain Precerebellar Neurons and Axons

During development, precerebellar neurons migrate dorsoventrally from the rhombic lip to the floor plate. Some of these neurons cross the midline while others stop. We have identified a role for the slit receptor Rig-1/Robo3 in directing this process. During their tangential migration, neurons of all major hindbrain precerebellar nuclei express high levels of Rig-1 mRNA. Rig-1 expression is rapidly downregulated as their leading process crosses the floor plate. Interestingly, most precerebellar nuclei do not develop normally in Rig-1-deficient mice, as they fail to cross the midline. In addition, inferior olivary neurons, which normally send axons into the contralateral cerebellum, project ipsilaterally in Rig-1 mutant mice. Similarly, neurons of the lateral reticular nucleus and basilar pons are unable to migrate across the floor plate and instead remain ipsilateral. These results demonstrate that Rig-1 controls the ability of both precerebellar neuron cell bodies and their axons to cross the midline.