RESUMO
Virus-derived double-stranded RNAs (dsRNAs) are sensed in the cytosol by retinoic acid-inducible gene (RIG)-I-like receptors (RLRs). These induce the expression of type I IFN and proinflammatory cytokines through signaling pathways mediated by the mitochondrial antiviral signaling (MAVS) protein. TNF receptor-associated factor (TRAF) family proteins are reported to facilitate the RLR-dependent expression of type I IFN by interacting with MAVS. However, the precise regulatory mechanisms remain unclear. Here, we show the role of FK506-binding protein 51 (FKBP51) in regulating the dsRNA-dependent expression of type I IFN. The binding of FKBP51 to TRAF6 was first identified by "in vitro virus" selection and was subsequently confirmed with a coimmunoprecipitation assay in HEK293T cells. The TRAF-C domain of TRAF6 is required for its interaction, although FKBP51 does not contain the consensus motif for interaction with the TRAF-C domain. Besides TRAF6, we found that FKBP51 also interacts with TRAF3. The depletion of FKBP51 reduced the expression of type I IFN induced by dsRNA transfection or Newcastle disease virus infection in murine fibroblasts. Consistent with this, the FKBP51 depletion attenuated dsRNA-mediated phosphorylations of IRF3 and JNK and nuclear translocation of RelA. Interestingly, dsRNA stimulation promoted the accumulation of FKBP51 in the mitochondria. Moreover, the overexpression of FKBP51 inhibited RLR-dependent transcriptional activation, suggesting a scaffolding function for FKBP51 in the MAVS-mediated signaling pathway. Overall, we have demonstrated that FKBP51 interacts with TRAF proteins and facilitates the expression of type I IFN induced by cytosolic dsRNA. These findings suggest a novel role for FKBP51 in the innate immune response to viral infection.
Assuntos
Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Interferon Tipo I/genética , Mitocôndrias/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Animais , Linhagem Celular , Humanos , Imunidade Inata , Interferon Tipo I/metabolismo , Camundongos , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/imunologia , Vírus da Doença de Newcastle/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Transporte Proteico , RNA de Cadeia Dupla/metabolismo , Transdução de Sinais , Fator 3 Associado a Receptor de TNF/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Proteínas de Ligação a Tacrolimo/genéticaRESUMO
Medullary thymic epithelial cells (mTECs) are essential for thymic negative selection to prevent autoimmunity. Previous studies show that mTEC development is dependent on the signal transducers TRAF6 and NIK. However, the downstream target genes of signals controlled by these molecules remain unknown. We performed a microarray analysis on mRNAs down-regulated by deficiencies in TRAF6 or functional NIK in an in vitro organ culture of fetal thymic stromata (2DG-FTOC). An in silico analysis of transcription factor binding sites in plausible promoter regions of differentially expressed genes suggests that STAT1 is involved in TRAF6- and NIK-dependent gene expression. Indeed, the signal of RANK, a TNF receptor family member that activates TRAF6 and NIK, induces the activation of STAT1 in 2DG-FTOC. Moreover, RANK signaling induces the up-regulation of interferon (IFN)-stimulated gene (ISG) expression, suggesting that the RANKL-dependent activation of STAT1 up-regulates ISG expression. The RANKL-dependent expression levels of ISGs were reduced but not completely abolished in interferon α receptor 1-deficient (Ifnar1(-/-)) 2DG-FTOC. Our data suggest that RANK signaling induces ISG expression in both type I interferon-independent and interferon-dependent mechanisms.
Assuntos
Células Epiteliais/imunologia , Regulação da Expressão Gênica , Interferon Tipo I/imunologia , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Tolerância a Antígenos Próprios/genética , Timo/imunologia , Animais , Feto , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/genética , Receptor de Interferon alfa e beta/genética , Transdução de Sinais , Células Estromais/imunologia , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Quinase Induzida por NF-kappaBRESUMO
Immunoglobulin mu-binding protein 2 (IGHMBP2/Smubp-2) is a helicase motif-containing DNA-binding protein that has been suggested to regulate various nuclear functions. Recent studies indicated that mutations in the IGHMBP2 gene are responsible for spinal muscular atrophy with respiratory distress type I (SMARD1). However, the mechanism of regulation of IGHMBP2 gene expression remains unclear. In the present study, a 2.0-kb fragment of the 5'-flanking (promoter) region of the human IGHMBP2 gene was isolated from the HL-60 genome by PCR and ligated into a luciferase (Luc) expression vector, pGL3, to generate the pSmu-Luc plasmid. Deletion analyses revealed that a 108-bp region is essential for basal promoter activity with a response to TPA in HL-60 cells. TF-SEARCH analysis showed that overlapping ets (GGAA) motifs are located upstream of the transcription start sites. Chromatin immunoprecipitation (ChIP) assay, electropheretic mobility shift assay (EMSA) and competition analyses indicated that PU.1 (Spi-1) recognizes and binds to the duplicated ets motifs in this 108-bp region. Moreover, co-transfection of the PU.1 expression plasmid and pSmu-Luc into HL-60 cells revealed that PU.1 modulates TPA-induced IGHMBP2 promoter activity. Taken together, these observations suggest that the duplicated GGAA motifs are essential for the IGHMBP2 promoter activity and its positive response to TPA in HL-60 cells.
