Retinoic Acid-induced Gene Expression Research Articles

Amanita muscaria extract potentiates production of proinflammatory cytokines by dsRNA-activated human microglia.

Recent interest in mushrooms and their components as potential therapies for mental health, along with recent government and health authority approvals, has necessitated a more comprehensive understanding of their effects on the cellular microenvironment of the brain. Amanita muscaria has been ingested as a treatment for a variety of ailments for centuries, most notably those affecting the central nervous system and conditions associated with neuroinflammation. However, the effects of these extracts on neuroinflammatory cells, such as microglia, are unknown. The effect of commercially-sourced A. muscaria extract (AME-1) on human microglial cell line (HMC3) expression of surface receptors such as CD86, CXCR4, CD45, CD125 and TLR4 was determined by flow cytometry. AME-1 upregulated expression of all of these receptors. The effect of AME-1 on HMC3 production of IL-8 and IL-6 was determined and compared to tumor necrosis factor (TNF), polyinosinic-polycytidylic acid [poly(I:C)], substance P and lipopolysaccharide (LPS), all known activators of HMC-3 and primary microglia. HMC3 produced both IL-8 and IL-6 when activated with LPS, TNF and poly(I:C) but not when they were activated with substance P. Although AME-1 at higher concentrations increased IL-8 production of HMC3 on its own, AME-1 notably potentiated HMC3 production of IL-8 in response to poly(I:C). AME-1 altered expression of toll-like receptor 3 (TLR3) mRNA but not surface protein by HMC3. AME-1 also did not significantly alter expression of retinoic acid-inducible gene I (RIG-I) or melanoma differentiation-associated protein 5 (MDA5), both cytosolic sensors of dsRNA. Metabolomics analysis showed that AME-1 contained several metabolites, including the autophagy inducer, trehalose. Like AME-1, trehalose also potentiated HMC3 poly(I:C) mediated production of IL-8. This study suggests that A. muscaria extracts can modify HMC3 inflammatory responses, possibly due to their trehalose content.

Vitamin C Enhances Antiviral Functions of Lung Epithelial Cells.

Vitamin C is well documented to have antiviral functions; however, there is limited information about its effect on airway epithelial cells—the first cells to encounter infections. Here, we examined the effect of vitamin C on human bronchial epithelium transformed with Ad12-SV40 2B (BEAS-2B) cells, and observed that sodium-dependent vitamin C transporter 2 (SVCT2) was the primary vitamin C transporter. Transcriptomic analysis revealed that treating BEAS-2B cells with vitamin C led to a significant upregulation of several metabolic pathways and interferon-stimulated genes (ISGs) along with a downregulation of pathways involved in lung injury and inflammation. Remarkably, vitamin C also enhanced the expression of the viral-sensing receptors retinoic acid-inducible gene 1 (RIG-1) and melanoma differentiation-associated protein 5 (MDA-5), which was confirmed at the protein and functional levels. In addition, the lungs of l-gulono-γ-lactone oxidase knockout (GULO-KO) mice also displayed a marked decrease in these genes compared to wild-type controls. Collectively, our findings indicate that vitamin C acts at multiple levels to exert its antiviral and protective functions in the lungs.

Knock-in of the duck retinoic acid-inducible gene I (RIG-I) into the Mx gene in DF-1 cells enables both stable and immune response-dependent RIG-I expression

Waterfowls, such as ducks, are natural hosts of avian influenza virus (AIV) and can genetically limit the pathogenicity. On the other hand, some AIV strains cause severe pathogenicity in chickens. It is suggested that differences in the pathogenicity of AIV infection between waterfowls and chickens are related to the expression of retinoic acid-inducible gene I (RIG-I), a pattern recognition receptor that chickens evolutionally lack. Here, we knocked-in the duck RIG-I bearing the T2A peptide sequence at the 3′ region of the Mx, an interferon-stimulated gene (ISG), in chicken embryo fibroblast cells (DF-1) using the precise integration into target chromosome (PITCh) system to control the duck RIG-I expression in chickens. The expression patterns of the duck RIG-I were then analyzed using qPCR. The knocked-in DF-1 cells expressed RIG-I via the stimulation of IFN-β and poly(I:C) in a dose-dependent manner. Moreover, poly(I:C) stimulation in the knocked-in DF-1 cells upregulated RIG-I-like receptor (RLR) family signaling pathway-related genes IFN-β, OASL, and IRF7. The IFN-β-dependent expression of RIG-I and upregulation of IFN-β in the poly(I:C) stimulation demonstrated a positive-feedback loop via RIG-I, usually evident in ducks. Overall, this novel strategy established RIG-I-dependent immune response in chickens without overexpression of RIG-I and disruption of the host genes.

Pontin Functions as A Transcriptional Co-activator for Retinoic Acid-induced HOX Gene Expression

Pontin is a AAA+ ATPase protein that has functions in various biological contexts including gene transcription regulation, chromatin remodeling, DNA damage sensing and repair, as well as assembly of protein and ribonucleoprotein complexes. Pontin is known to regulate the transcription of several important signaling pathways, including Wnt signaling. However, its role in early embryonic signaling regulation remains unclear. Retinoic acid (RA) signaling plays a central role in vertebrate development. Using an in vivo biotin tagging technology, we mapped the genome-wide binding pattern of Pontin before and after RA-induced differentiation in the pluripotent embryo carcinoma cell line NTERA-2. Biotin ChIP-seq revealed significant changes in genome-wide Pontin binding sites upon RA stimulation. We also identified a substantial amount of overlapping binding peaks between Pontin and RARα, especially on all of the HOX gene loci (A-D clusters). Pontin knockdown experiments showed that its chromatin binding at the HOX gene clusters is required for RA-induced HOX gene expression. Furthermore, we performed Global Run-On sequencing (GRO-seq) to map de novo transcripts genome-wide and found that Pontin knockdown significantly diminished nascent HOX gene transcripts, indicating that Pontin regulates HOX gene expression at the transcriptional level. Finally, proteomic analysis demonstrated that Pontin associates with chromatin organization/remodeling complexes and various other functional complexes. Altogether, we have demonstrated that Pontin is a critical transcriptional co-activator for RA-induced HOX gene activation.

MicroRNA-30a Modulates Type I Interferon Responses to Facilitate Coxsackievirus B3 Replication Via Targeting Tripartite Motif Protein 25.

Viral myocarditis is caused by a viral infection and characterized by the inflammation of the myocardium. Coxsackievirus B3 (CVB3) infection is one of the most common among the infections caused by this virus. The host’s early innate immune response to CVB3 infection particularly depends on the functions of type I interferons (IFNs). In this study, we report that a host microRNA, miR-30a, was upregulated by CVB3 to facilitate its replication. We demonstrated that miR-30a was a potent negative regulator of IFN-I signaling by targeting tripartite motif protein 25 (TRIM25). In addition, we found that TRIM25 overexpression significantly suppressed CVB3 replication, whereas TRIM25 knockdown increased viral titer and VP1 protein expression. MiR-30a inhibits the expression of TRIM25 and TRIM25-mediated retinoic acid-inducible gene (RIG)-I ubiquitination to suppress IFN-β activation and production, thereby resulting in the enhancement of CVB3 replication. These results indicate the proviral role of miR-30a in modulating CVB3 infection for the first time. This not only provides a new strategy followed by CVB3 in order to modulate IFN-I–mediated antiviral immune responses by engaging host miR-30a but also improves our understanding of its pathogenesis.

SRC family kinase inihibitors enhance all-trans-retinoic acid or arsenic trioxide induced differentiation of NB4 Leukemia Cells

Background & Aim Background: The Src family kinases (SFK) are non-receptor tyrosine kinases that have been implicated in a variety of process, including proliferation, survival, differentiation and migration. Recent studies showed that inhibition of SFKs resulted in enhancement of retinoic acid-induced myeloid differentiation. In this study, we investigated whether SFK inhibitor PP2 and FDA approved dual Src/ABL inhibitor (bosutinib or dasatinib) could enhance the differentiation of NB4 leukemia cells when combined with all-trans-retinoic acid (ATRA) or arsenic trioxide (ATO). Methods, Results & Conclusion Methods: We demonstrated the SFK inhibitor PP2 enhanced the differentiation of NB4 cells when combined with ATRA or ATO. Subsequently, we investigated whether bosutinib and dasatinib could enhance the differentiation of NB4 cells when combined with ATRA or ATO. The cells were analyzed for myeloid differentiation marker CD11b expression using flow cytometry. These results were confirmed in morphologic analysis by Wright stain and NBT reduction assay. In addition, we attempted to determine the difference in retinoic acid-induced gene expression. Results Our data showed that SFK inhibitor PP2 enhanced myeloid differentiation when combined with ATRA or ATO. The synergistic effect was significantly stronger when PP2 was combined with ATRA and ATO (triple treatment) than when PP2 was combined with ATRA or ATO (dual treatment). The similar results were obtained when bosutinib and dasatinib were applied instead of PP2. Co-treatment with bosutinib plus ATRA or ATO, and dasatinib plus ATRA or ATO resulted in significant enhancement of CD11b-positive cells when compared to the each treatment group in bosutinib, dasatinib, ATRA, or ATO. Also, the synergistic effects of bosutinib and dasatinib in combination with ATRA were greater than in combination with ATO. Conclusions SFK inhibitor PP2 and dual Src/ABL inhibitor (bosutinib or dasatinib) can enhances the differentiation of NB4 leukemia cells when combined ATRA and/or ATO. These findings suggest that ATRA or ATO combinated with dual Src/ABL inhibitors may have therapeutic beneficial for the treatment of APL patients.

Japanese Encephalitis Virus infection induces inflammation of swine testis through RIG-I—NF-ĸB signaling pathway

Japanese Encephalitis Virus (JEV) is an important zoonotic flavivirus transmitted by mosquitos. JEV infection in sows primarily manifests as a reproductive disease such as abortion and transient infertility while in infected boars, it can cause orchitis. Previous studies mainly focused on the pathogenesis of human encephalitis caused by JEV infection, while few concentrations have been made to unveil the potential mechanism of reproductive dysfunction in JEV-infected pigs. In this study, histopathological analysis and immunohistochemistry staining was performed on testis of JEV-infected boars, indicating that JEV could infect testicular cells and cause inflammatory changes in testis. In vitro assays reveal that primary swine testicular cells and swine testis (ST) cells are highly permissive to JEV and significant inflammatory response was shown during JEV infection. Mechanically, we found that JEV infection increases the expression of retinoic acid-inducible gene I (RIG-I) and activates transcription factor NF-κB. Production of pro-inflammatory cytokines was greatly reduced in JEV infected testicular cells after knockout of RIG-I or treatment with the NF-κB specific inhibitor. In addition, activation of NF-κB was also significantly suppressed upon RIG-I knockout. Taken together, our results reveal that JEV could infect boar testicles, and RIG-I—NF-κB signaling pathway is involved in JEV-induced inflammation in swine testicular cells.

Alfalfa polysaccharide prevents H2O2-induced oxidative damage in MEFs by activating MAPK/Nrf2 signaling pathways and suppressing NF-\u03baB signaling pathways

Alfalfa polysaccharide (APS) is a bioactive component extracted from alfalfa that exhibits potent antioxidant properties. However, the cellular and molecular mechanisms underlying these properties remain unclear. To explore the molecular mechanism by which APS exerts antioxidant effects, an H2O2-induced oxidative stress mouse embryonic fibroblast (MEF) model was established. Cell proliferation, antioxidant enzyme activity, immune cytokine expression, and related protein expression were examined in APS-supplemented or non-supplemented conditions. The results suggested that APS strengthened the antioxidative capacity of MEFs, increasing cell proliferation, superoxide dismutase activity (SOD), and the total antioxidant capacity (T-AOC). In addition, APS reduced the secretion of interleukin (IL)-6 and IL-8 as well as expression of the proinflammatory gene retinoic acid-inducible gene I (RIG-I). APS was also able to activate the mitogen-activated protein kinase (MAPK) pathway, which promoted the translocation of nuclear factor erythroid 2–related factor 2 (Nrf2) to the nucleus. However, expression of nuclear factor-κB (NF-κB) was decreased after APS treatment. Overall, these results suggest that APS relieves H2O2-induced oxidative stress in MEFs by activating MAPK/Nrf2 signaling and suppressing NF-κB signaling. To the best of our knowledge, this is the first study to link APS with MAPK/Nrf2, NF-κB and RIG-I, thus providing new perspectives regarding the mechanisms of the antioxidant activity of APS.

Expression of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) in third-trimester placentas following cytomegalovirus infection ex vivo

Expression of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) in third-trimester placentas following cytomegalovirus infection ex vivo

Comparison of transcriptional profiles of interferons, CXCL10 and RIG-1 in influenza infected A549 cells stimulated with exogenous interferons.

Type I and type III interferons (IFNs) are induced by viral infection. It was concluded that these IFN species are identical in regulation and biological functions. However, these two systems differ in the tissue expression of their receptors and their transcriptional regulation is fundamentally different as well as cellular signaling pathways that drive expression of each IFN. Here, we have investigated the transcriptional profile of endogenous IFNs after stimulation of cells with exogenous IFNs and subsequent infection of A549 cells with A/chicken/Germany/27 [H7N7] influenza virus. Both type I and type III IFNs exhibit high degree of the cross-induction. Our results show that type III IFNs (IFN-λ1, IFN-λ2 and IFN-λ3) are better inducers of CXCL10 than type I IFNs. The IFN-β1a and IFN-λ2 were the most potent IFNs and they highly increased the level of IFN-α, IFN-β, IFN-λ, and CXCL10 mRNAs. Since type I IFNs up regulated expression of retinoic acid-inducible gene 1 (RIG-1) mRNA, type III IFNs-λ down regulated expression of RIG-1 mRNA in influenza infected cells. IFN-α and IFN-ω induced similar amount of IFN-α, IFN-β and IFN-λ mRNA but differ in induction of CXCL10 and RIG-1 mRNA.

RIG-I contributes to the innate immune response after cerebral ischemia.

BackgroundFocal cerebral ischemia induces an inflammatory response that when exacerbated contributes to deleterious outcomes. The molecular basis regarding the regulation of the innate immune response after focal cerebral ischemia remains poorly understood.MethodsIn this study we examined the expression of retinoic acid-inducible gene (RIG)-like receptor-I (RIG-I) and its involvement in regulating inflammation after ischemia in the brain of rats subjected to middle cerebral artery occlusion (MCAO). In addition, we studied the regulation of RIG-I after oxygen glucose deprivation (OGD) in astrocytes in culture.ResultsIn this study we show that in the hippocampus of rats, RIG-I and IFN-α are elevated after MCAO. Consistent with these results was an increased in RIG-I and IFN-α after OGD in astrocytes in culture. These data are consistent with immunohistochemical analysis of hippocampal sections, indicating that in GFAP-positive cells there was an increase in RIG-I after MCAO. In addition, in this study we have identified n-propyl gallate as an inhibitor of IFN-α signaling in astrocytes.ConclusionOur findings suggest a role for RIG-I in contributing to the innate immune response after focal cerebral ischemia.

Abstract 5400: A novel combinatorial therapy for hepatocellular carcinoma (HCC)

Abstract The incidence of hepatocellular carcinoma (HCC) is rising in the US with parallel increase in mortality rate. Lack of effective therapy for advanced HCC mandates development of novel targeted therapies to counteract this fatal malady. Astrocyte elevated gene-1 (AEG-1), also known as metadherin (MTDH) and LYRIC, plays a pivotal role in hepatocarcinogenesis and serves as an ideal target for anti-HCC therapy. AEG-1 interacts with retinoid X receptor (RXR) and inhibits retinoic acid-induced gene expression and cell death. Retinoic acid has been evaluated for HCC treatment without promising result. Overexpression of AEG-1 might underlie the poor performance of retinoic acid in HCC clinical trials. We documented that combination of a lentivirus expressing AEG-1 shRNA (lenti.shAEG-1) and all-trans retinoic acid (ATRA) profoundly and synergistically inhibited subcutaneous human HCC xenografts in nude mice. We now have developed liver-targeted nanoplexes by conjugating poly(amidoamine) (PAMAM) dendrimers with polyethylene glycol (PEG) and lactobionic acid (PAMAM-PEG-Gal) which were complexed with AEG-1 siRNA (PAMAM-AEG-1si). PAMAM has a net positive charge that complexes with the net negative charge of the siRNA backbone, PEG increases stability of the nanoplexes and increases circulation time and the galactose binds to asialoglycoprotein receptors that are upregulated on liver cells. The polymer conjugate was characterized by 1H-NMR and optimal nanoplex formulations were characterized for surface charge and size using a zetasizer Nano ZS. We established orthotopic xenografts of human HCC cell QGY-7703 expressing luciferase (QGY-luc) in the livers of athymic nude mice and monitored tumor development by bioluminescence imaging (BLI). One week after tumor establishment mice were treated with PAMAM-siCon, PAMAM-siCon+ATRA, PAMAM-AEG-1si and PAMAM-AEG-1si+ATRA by 8 i.v. injections over 4 weeks, and sacrificed 2 weeks after the last injection. In the control group the tumor developed aggressively. ATRA showed little effect due to high AEG-1 levels in QGY-luc cells. PAMAM-AEG-1si showed significant reduction in tumor growth and the combination of PAMAM-AEG-1si+ATRA showed profound and synergistic inhibition so that the tumors were almost undetectable by BLI. Measurement of liver weight at the end of the experiment corroborated these findings. Analysis of AEG-1 mRNA in tumor samples showed a marked decrease in AEG-1 level by PAMAM-AEG-1si indicating efficacy of in vivo knockdown. The group treated with PAMAM-AEG-1si+ATRA nanoplexes showed increased necrosis, inhibition of proliferation and increased apoptosis when compared to other groups. Liver is an ideal organ for RNAi therapy and ATRA is an approved anti-cancer agent. Our exciting observations suggest that the combinatorial approach might be an effective way to combat HCC and need to be evaluated stringently in endogenous mouse models of HCC for a potential Phase I/II clinical trial. Citation Format: Jyoti Srivastava, Devaraja Rajasekaran, Ayesha Siddiq, Rachel Gredler, Chadia L. Robertson, Maaged A. Akiel, Xue-Ning Shen, Kareem A.N. Ebeid, Aliasger K. Salem, Paul B. Fisher, Devanand Sarkar. A novel combinatorial therapy for hepatocellular carcinoma (HCC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5400. doi:10.1158/1538-7445.AM2015-5400

Combination of Nanoparticle-Delivered siRNA for Astrocyte Elevated Gene-1 (AEG-1) and All-trans Retinoic Acid (ATRA): An Effective Therapeutic Strategy for Hepatocellular Carcinoma (HCC).

Hepatocellular carcinoma (HCC) is a fatal cancer with no effective therapy. Astrocyte elevated gene-1 (AEG-1) plays a pivotal role in hepatocarcinogenesis and inhibits retinoic acid-induced gene expression and cell death. The combination of a lentivirus expressing AEG-1 shRNA and all-trans retinoic acid (ATRA) profoundly and synergistically inhibited subcutaneous human HCC xenografts in nude mice. We have now developed liver-targeted nanoplexes by conjugating poly(amidoamine) (PAMAM) dendrimers with polyethylene glycol (PEG) and lactobionic acid (Gal) (PAMAM-PEG-Gal) which were complexed with AEG-1 siRNA (PAMAM-AEG-1si). The polymer conjugate was characterized by (1)H-NMR, MALDI, and mass spectrometry; and optimal nanoplex formulations were characterized for surface charge, size, and morphology. Orthotopic xenografts of human HCC cell QGY-7703 expressing luciferase (QGY-luc) were established in the livers of athymic nude mice and tumor development was monitored by bioluminescence imaging (BLI). Tumor-bearing mice were treated with PAMAM-siCon, PAMAM-siCon+ATRA, PAMAM-AEG-1si, and PAMAM-AEG-1si+ATRA. In the control group the tumor developed aggressively. ATRA showed little effect due to high AEG-1 levels in QGY-luc cells. PAMAM-AEG-1si showed significant reduction in tumor growth, and the combination of PAMAM-AEG-1si+ATRA showed profound and synergistic inhibition so that the tumors were almost undetectable by BLI. A marked decrease in AEG-1 level was observed in tumor samples treated with PAMAM-AEG-1si. The group treated with PAMAM-AEG-1si+ATRA nanoplexes showed increased necrosis, inhibition of proliferation, and increased apoptosis when compared to other groups. Liver is an ideal organ for RNAi therapy and ATRA is an approved anticancer agent. Our exciting observations suggest that the combinatorial approach might be an effective way to combat HCC.

Involvement of NLRP3 inflammasome in CVB3-induced viral myocarditis.

Viral myocarditis, which is most prevalently caused by coxsackievirus B3 (CVB3) infection, is a serious clinical condition characterized by cardiac inflammation. Inflammasome plays an essential role in the regulation of diverse inflammatory responses by serving as a platform for caspase-1 activation and caspase-1-dependent proteolytic maturation and secretion of IL-1β. Although inflammasome has been reported to be crucial for the development of many inflammatory diseases, its role in the pathogenesis of viral myocarditis is still elusive. The present study aims to investigate whether CVB3 infection activates inflammasome and whether the activation of inflammasome contributes to CVB3-induced myocarditis. Our results showed that CVB3 infection induced inflammasome activation both in vitro and in vivo. With the inhibition of inflammasome activation, the severity of CVB3-induced myocarditis was significantly alleviated as evidenced by less weight loss, decreased serological indexes of creatine kinase and creatinekinase-MB activities, as well as less severe myocardial injury. Of importance, echocardiography results showed that inhibition of inflammasome activation also efficiently improved cardiac function as revealed by enhanced left ventricular ejection fraction and left ventricular fractional shortening. Despite that CVB3 infection significantly increased the expression of both retinoic acid-inducible gene 1 and NOD-like receptor family, pyrin domain containing 3 (NLRP3) in cardiac myocytes, CVB3-induced inflammasome activation was NLRP3-, but not retinoic acid-inducible gene 1, dependent. Further study showed that reactive oxygen species production and K(+) efflux were critical for the activation of NLRP3 inflammasome upon CVB3 infection. Collectively, our study demonstrated a crucial role of the NLRP3 inflammasome in the pathogenesis of CVB3-induced myocarditis, and modulation of inflammasome activation might represent a promising therapeutic strategy for viral myocarditis.

Duck MDA5 functions in innate immunity against H5N1 highly pathogenic avian influenza virus infections.

Melanoma differentiation-associated gene 5 (MDA5) is an important intracellular receptor that recognizes long molecules of viral double-stranded RNA in innate immunity. To understand the mechanism of duck MDA5-mediated innate immunity, we cloned the MDA5 cDNA from the Muscovy duck (Cairina moschata). Quantitative real-time PCR analysis indicates that duck MDA5 mRNA was constitutively expressed in all sampled tissues. A significant increase of MDA5 mRNA was detected in the brain, spleen and lungs of ducks after infection with an H5N1 highly pathogenic avian influenza virus (HPAIV). We investigated the role of the predicted functional domains of MDA5. The results indicate the caspase activation and recruitment domain (CARD) of duck MDA5 had a signal transmission function through IRF-7-dependent signaling pathway. Overexpression of the CARD strongly activated the chicken IFN-β promoter and upregulated the mRNA expression of antiviral molecules (such as OAS, PKR and Mx), proinflammatory cytokines (such as IL-2, IL-6, IFN-α and IFN-γ, but not IL-1β and IL-8) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) (RIG-I and LGP2) without exogenous stimulation. We also demonstrate the NS1 of the H5N1 HPAIV inhibited the duck MDA5-mediated signaling pathway in vitro. These results suggest that duck MDA5 is an important receptor for inducing antiviral activity in the host immune response of ducks.

Src Family Kinase Inhibitor PP2 Has Different Effects on All-Trans-Retinoic Acid or Arsenic Trioxide-Induced Differentiation of an Acute Promyelocytic Leukemia Cell Line

PurposeLeukemic promyelocytes have the unique ability to undergo differentiation after exposure to retinoic acid and both differentiation and apoptosis after exposure to arsenic trioxide (ATO). Recent studies have shown that inhibition of Src family kinases (SFKs) resulted in enhancement of retinoic acid-induced myeloid differentiation.Materials and MethodsIn this study, we investigated the question of whether the SFK inhibitor PP2 enhanced the differentiation of NB4 cells when combined with ATO as well as when combined with all-trans-retinoic acid (ATRA). In addition, we attempted to determine the difference in retinoic acid-induced gene expression between cells treated with PP2 in combination with ATRA and in combination with ATO.ResultsSFK inhibitor PP2 induced significant enhancement of ATRA- or ATO-induced differentiation of NB4 cells. A significantly stronger synergistic effect was observed when PP2 was combined with ATRA than when combined with ATO. Flow cytometric analysis demonstrated a significant increase in CD11b-positive granulocytes up to 60.73% and 31.58%, respectively. These results were confirmed by nitroblue tetrazolium staining. These effects were not related to apoptosis. Results of Annexin-V-fluorescein staining revealed that PP2 combined with ATRA or PP2 combined with ATO did not induce apoptosis in NB4 cells. Retinoic acid-induced gene expression was different in both groups. Intercellular adhesion molecule-1 expression showed a significant increase in cells treated with PP2 in combination with ATRA, whereas cathepsin D expression showed a significant increase in cells treated with PP2 in combination with ATO.ConclusionOur data showed that SFK inhibitor PP2 enhanced acute promyelocytic leukemia cell differentiation when combined with either ATRA or ATO with difference in activation of retinoic acid-induced genes.

Expression of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) in the human term placenta

Event Abstract Back to Event Expression of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) in the human term placenta Agnieszka Jabłońska1, Patrycja Suski1, Mirosława Studzińska1, Jarosław Kalinka2 and Edyta Paradowska1* 1 Institute of Medical Biology Polish Academy of Sciences, Laboratory of Molecular Virology and Biological Chemistry, Poland 2 Medical University of Lodz, Department of Perinatology, First Chair of Gynecology and Obstetrics, Poland Innate recognition of viruses is mediated by germline-encoded pattern recognition receptors (PRRs) that initiate signaling events, resulting in the production of type I interferon and proinflammatory cytokines. Double-stranded RNA and 5’triphosphate group of ssRNA generated during viral replication are detected by sensory molecules, named RIG-I-like receptors (RLRs). Three members of the RLRs family have been characterized so far, including RIG-I (retinoic acid-inducible gene-I), MDA5 (melanoma differentation-associated gene 5) and LGP2 (laboratory of genetics and physiology 2). RIG-I participates in the recognition of Paramyxoviruses, Rhabdoviruses, Flaviviruses and Orthomyxoviruses, whereas MDA-5 is essential for the recognition of Picornaviruses. In this study, we investigated the RLRs gene expression in fetal and maternal parts of the human placentas during viral infection. The explants of third-trimester chorionic villi and deciduas were exposed to infection with dsDNA viruses (human cytomegalovirus, HCMV; herpes simplex virus type 1, HSV1) or ssRNA virus (vesicular stomatitis virus, VSV). RIG-I, MDA5 and LGP2 mRNA levels were assessed by real-time PCR. Our results demonstrated that all RLRs were expressed in term human placentas with the highest level of LGP2. RIG-I and MDA5 (in fetal part) have been identified as the main mediators of VSV recognition, whereas RLRs were not involved in the identification of dsDNA viruses. The expression of LGP2 in HCMV-infected explants was decreased compared to mock-infected. The results clearly indicated that RLRs play essential role in RNA viruses’ recognition in term placenta. Acknowledgements Project financed by the European Regional Development Found under the Operational Programme Innovative Economy, Grant No. POIG.01.01.02-10-107/09. Keywords: human placenta, Virus infection, RLRs, RIG-I-like receptors, LGP2 Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Host-pathogen interactions Citation: Jabłońska A, Suski P, Studzińska M, Kalinka J and Paradowska E (2013). Expression of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) in the human term placenta. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00293 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 13 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Edyta Paradowska, Institute of Medical Biology Polish Academy of Sciences, Laboratory of Molecular Virology and Biological Chemistry, Lodz, 93-232, Poland, eparadowska@cbm.pan.pl Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Agnieszka Jabłońska Patrycja Suski Mirosława Studzińska Jarosław Kalinka Edyta Paradowska Google Agnieszka Jabłońska Patrycja Suski Mirosława Studzińska Jarosław Kalinka Edyta Paradowska Google Scholar Agnieszka Jabłońska Patrycja Suski Mirosława Studzińska Jarosław Kalinka Edyta Paradowska PubMed Agnieszka Jabłońska Patrycja Suski Mirosława Studzińska Jarosław Kalinka Edyta Paradowska Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Src Family Kinase Inhibitor PP2 Has Different Effects On ATRA or Arsenic Trioxide-Induced Differentiation of Acute Promyelocytic Leukemia Cell Line

AbstractAbstract 4800Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML) with distinctive biologic and clinical features that is now highly curable. Leukemic promyelocytes have the unique ability to undergo differentiation after exposure to retinoic acid and both differentiation and apoptosis after exposure to arsenic trioxide (As2O3, ATO). Emerging evidence has implicated Src family kinases (SFKs) as regulators of proliferation and survival of myeloid lineage cells. Recent studies showed that inhibition of SFKs resulted in enhancement of retinoic acid-induced myeloid differentiation. In this study, we demonstrated that the SFK inhibitor PP2 enhanced the differentiation of NB4 cells when combined with ATO as well when combined with as all-trans-retinoic acid (ATRA) and the difference in the retinoic acid-induced gene expression between the cells treated with PP2 in combination with ATRA and the cells treated with PP2 in combination with ATO.SFK inhibitor PP2 significantly enhanced ATRA- or ATO-induced differentiation of NB4 cells. The synergistic effect was significantly stronger when PP2 was combined with ATRA than when PP2 was combined with ATO. Flow cytometric analysis demonstrated a significant increase in CD11b-positive granulocytes up to 60.73% and 31.58%, respectively. These results were confirmed by morphologic analysis using Wright stain and NBT staining. These effects were not related to apoptosis. Annexin-V-fluorescein staining revealed that PP2 combined with ATRA or PP2 combined with ATO did not induce apoptosis in NB4 cells. Retinoic acid-induced gene expression was different in both groups. ICAM-1 expression was significantly increased in the cells treated with PP2 in combination with ATRA whereas cathepsin D expression was significantly increased in the cells treated with PP2 in combination with ATO. These findings suggested that the synergistic effect of SFK inhibitor PP2 in combination with ATRA was significantly stronger than that of PP2 in combination with ATO on NB4 myeloid leukemia cell differentiation and this difference was related to the disparate activation of retinoic acid-induced genes. Disclosures:No relevant conflicts of interest to declare.

Transcriptional Elongation Factor Elongin A Regulates Retinoic Acid-Induced Gene Expression during Neuronal Differentiation

Elongin A increases the rate of RNA polymerase II (pol II) transcript elongation by suppressing transient pausing by the enzyme. Elongin A also acts as a component of a cullin-RING ligase that can target stalled pol II for ubiquitylation and proteasome-dependent degradation. It is not known whether these activities of Elongin A are functionally interdependent in vivo. Here, we demonstrate that Elongin A-deficient (Elongin A(-/-)) embryos exhibit abnormalities in the formation of both cranial and spinal nerves and that Elongin A(-/-) embryonic stem cells (ESCs) show a markedly decreased capacity to differentiate into neurons. Moreover, we identify Elongin A mutations that selectively inactivate one or the other of the aforementioned activities and show that mutants that retain the elongation stimulatory, but not pol II ubiquitylation, activity of Elongin A rescue neuronal differentiation and support retinoic acid-induced upregulation of a subset of neurogenesis-related genes in Elongin A(-/-) ESCs.

Pathogen recognition receptors in channel catfish: II. Identification, phylogeny and expression of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs)

Vertebrates including teleost fish have evolved an array of pathogen recognition receptors (PRRs) for detecting and responding to various pathogen-associated molecular patterns (PAMPs), including Toll-like receptors (TLRs), nucleotide-binding domain, leucine-rich repeat containing receptors (NLRs), and the retinoic acid inducible gene I (RIG-I) like receptors (RLRs). As a part of the series of studies targeted to characterize catfish PRRs, we described 22 NLR receptors in the sister contribution. Here in this study, we focused on cytosolic PRRs recognizing nucleotide pathogen-associated molecular patterns (PAMPs) of invading viruses, the retinoic acid-inducible gene I (RIG-I)-like receptors (RLR receptors). Three RLRs with DExD/H domain containing RNA helicases, retinoic acid inducible gene-I (RIG-I), melanoma differentiation-associated gene 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2), were identified from channel catfish, Ictalurus punctatus. The catfish RIG-I encodes 937 amino acids that contains two CARDs, a DExDc, a HELICc and a RD domains. MDA5 encodes 1005 amino acids with all the domains identified for RIG-I. LGP2 encodes 677 amino acids that contain other domains but not the CARD domain at the N-terminus. Phylogenetic analyses of the three genes of catfish showed close clustering with their counterparts from other teleost fish. All the genes were found to be constitutively expressed in various tissues of catfish with minor variations. Channel catfish ovarian cells when infected with channel catfish virus showed significant increase in the transcript abundance of all the three genes. Further, RLR genes showed significant increases in expression in the liver tissue collected at different time-points after bacterial infection as well. The results indicate that the catfish RLRs may play important roles in antiviral and anti-bacterial immune responses.