RESUMEN
IL-17 is a proinflammatory cytokine produced by activated Th17 cells and other immune cells. IL-17-producing Th17 cells are major contributors to chronic inflammatory and autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. Although the transcriptional regulation of Th17 cells is well understood, the posttranscriptional regulation of IL-17 gene expression remains unknown. The RNA-binding protein HuR positively regulates the stability of many target mRNAs via binding the AU-rich elements present in the 3' untranslated region of many inflammatory cytokines including IL-4, IL-13, and TNF-α. However, the regulation of IL-17 expression by HuR has not been established. CD4(+) Th17 cells from HuR knockout mice had decreased IL-17 steady-state mRNA and protein levels compared with wild-type Th17 cells, as well as decreases in frequency of IL-17(+) cells. Moreover, we demonstrated that HuR directly binds to the IL-17 mRNA 3' untranslated region by using RNA immunoprecipitation and biotin pulldown assays. In addition, the knockout of HuR decreased cellular proliferation of CD4(+) T cells. Mice with adoptively transferred HuR KO Th17 cells had delayed initiation and reduced disease severity in the onset of experimental autoimmune encephalomyelitis compared with wild-type Th17 cells. Our results reveal a HuR-induced posttranscriptional regulatory mechanism of Th17 differentiation that influences IL-17 expression. These findings may provide novel therapeutic targets for the treatment of Th17-mediated autoimmune neuroinflammation.
Asunto(s)
Proteínas ELAV/metabolismo , Encefalomielitis Autoinmune Experimental/inmunología , Interleucina-17/inmunología , Interferencia de ARN , ARN Mensajero/metabolismo , Células Th17/inmunología , Traslado Adoptivo , Animales , Procesos de Crecimiento Celular/genética , Células Cultivadas , Proteínas ELAV/genética , Proteínas ELAV/inmunología , Femenino , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
The posttranscriptional mechanisms by which RNA binding proteins (RBPs) regulate T-cell differentiation and cytokine production in vivo remain unclear. The RBP HuR binds to labile mRNAs, usually leading to increases in mRNA stability and/or translation. Previous work demonstrated that HuR binds to the mRNAs encoding the Th2 transcription factor trans-acting T-cell-specific transcription factor (GATA-3) and Th2 cytokines interleukin (IL)-4 and IL-13, thereby regulating their expression. By using a novel conditional HuR knockout (KO) mouse in which HuR is deleted in activated T cells, we show that Th2-polarized cells from heterozygous HuR conditional (OX40-Cre HuR(fl/+)) KO mice had decreased steady-state levels of Gata3, Il4 and Il13 mRNAs with little changes at the protein level. Surprisingly, Th2-polarized cells from homozygous HuR conditional (OX40-Cre HuR(fl/fl)) KO mice showed increased Il2, Il4 and Il13 mRNA and protein via different mechanisms. Specifically, Il4 was transcriptionally upregulated in HuR KO T cells, whereas Il2 and Il13 mRNA stabilities increased. Additionally, when using the standard ovalbumin model of allergic airway inflammation, HuR conditional KO mice mounted a robust inflammatory response similar to mice with wild-type HuR levels. These results reveal a complex differential posttranscriptional regulation of cytokines by HuR in which gene dosage plays an important role. These findings may have significant implications in allergies and asthma, as well as autoimmune diseases and infection.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Citocinas/genética , Proteínas ELAV/genética , Alérgenos , Animales , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Linfocitos T CD4-Positivos/metabolismo , Recuento de Células , Células Cultivadas , Citocinas/metabolismo , Proteínas ELAV/metabolismo , Factor de Transcripción GATA3/genética , Factor de Transcripción GATA3/metabolismo , Dosificación de Gen , Ratones Noqueados , Ovalbúmina , Neumonía/genética , Neumonía/inmunología , Neumonía/metabolismo , ARN Mensajero/metabolismo , Hipersensibilidad Respiratoria/genética , Hipersensibilidad Respiratoria/inmunología , Hipersensibilidad Respiratoria/metabolismo , Bazo/citologíaRESUMEN
The posttranscriptional mechanisms whereby RNA-binding proteins (RBPs) regulate T cell differentiation remain unclear. RBPs can coordinately regulate the expression of functionally related genes via binding to shared regulatory sequences, such as the adenylate-uridylate-rich elements (AREs) present in the 3' untranslated region (UTR) of mRNA. The RBP HuR posttranscriptionally regulates IL-4, IL-13, and other Th2 cell-restricted transcripts. We hypothesized that the ARE-bearing GATA-3 gene, a critical regulator of Th2 polarization, is under HuR control as part of its coordinate posttranscriptional regulation of the Th2 program. We report that in parallel with stimulus-induced increase in GATA-3 mRNA and protein levels, GATA-3 mRNA half-life is increased after restimulation in the human T cell line Jurkat, in human memory and Th2 cells, and in murine Th2-skewed cells. We demonstrate by immunoprecipitation of ribonucleoprotein complexes that HuR associates with the GATA-3 endogenous transcript in human T cells and found, using biotin pulldown assay, that HuR specifically interacts with its 3'UTR. Using both loss-of-function and gain-of-function approaches in vitro and in animal models, we show that HuR is a critical mediator of stimulus-induced increase in GATA-3 mRNA and protein expression and that it positively influences GATA-3 mRNA turnover, in parallel with selective promotion of Th2 cytokine overexpression. These results suggest that HuR-driven posttranscriptional control plays a significant role in T cell development and effector function in both murine and human systems. A better understanding of HuR-mediated control of Th2 polarization may have utility in altering allergic airway inflammation in human asthmatic patients.
Asunto(s)
Antígenos de Superficie/fisiología , Citocinas/biosíntesis , Citocinas/genética , Factor de Transcripción GATA3/biosíntesis , Factor de Transcripción GATA3/genética , Regulación de la Expresión Génica/inmunología , Proteínas de Unión al ARN/fisiología , Células Th2/inmunología , Células Th2/metabolismo , Animales , Secuencia de Bases , Línea Celular Tumoral , Proteínas ELAV , Proteína 1 Similar a ELAV , Femenino , Humanos , Células Jurkat , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Células 3T3 NIH , Estabilidad del ARN/inmunología , Transcripción Genética/inmunologíaRESUMEN
As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.