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1.
J Immunol ; 208(1): 38-48, 2022 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-34862257

RESUMEN

RNA-binding protein HuR (ELAVL1) is a master regulator of gene expression in human pathophysiology. Its dysregulation plays an important role in many diseases. We hypothesized that HuR plays an important role in Th2 inflammation in asthma in both mouse and human. To address this, we used a model of airway inflammation in a T cell-specific knockout mouse model, distal lck-Cre HuRfl/fl, as well as small molecule inhibitors in human peripheral blood-derived CD4+ T cells. Peripheral CD4+ T cells were isolated from 26 healthy control subjects and 45 asthmatics (36 type 2 high and 9 non-type 2 high, determined by blood eosinophil levels and fraction of exhaled NO). Our mouse data showed conditional ablation of HuR in T cell-abrogated Th2 differentiation, cytokine production, and lung inflammation. Studies using human T cells showed that HuR protein levels in CD4+ T cells were significantly higher in asthmatics compared with healthy control subjects. The expression and secretion of Th2 cytokines were significantly higher in asthmatics compared with control subjects. AMP-activated protein kinase activator treatment reduced the expression of several cytokines in both type 2 high and non-type 2 high asthma groups. However, the effects of CMLD-2 (a HuR-specific inhibitor) were more specific to endotype-defining cytokines in type 2 high asthmatics. Taken together, these data suggest that HuR plays a permissive role in both allergen and non-allergen-driven airway inflammation by regulating key genes, and that interfering with its function may be a novel method of asthma treatment.


Asunto(s)
Asma/metabolismo , Proteína 1 Similar a ELAV/metabolismo , Células Th2/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Alérgenos/inmunología , Animales , Antiinflamatorios/farmacología , Asma/genética , Asma/terapia , Benzopiranos/farmacología , Células Cultivadas , Modelos Animales de Enfermedad , Proteína 1 Similar a ELAV/antagonistas & inhibidores , Proteína 1 Similar a ELAV/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Ovalbúmina/inmunología , Pirrolidinas/farmacología , Adulto Joven
2.
J Biol Chem ; 292(35): 14532-14543, 2017 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-28684423

RESUMEN

In both multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), the C-C chemokine receptor 6 (CCR6) is critical for pathogenic T helper 17 (Th17) cell migration to the central nervous system (CNS). Whereas many cytokines and their receptors are potently regulated via post-transcriptional mechanisms in response to various stimuli, how CCR6 expression is post-transcriptionally regulated in Th17 cells is unknown. Here, using RNA-binding protein HuR conditional knock-out (KO) and wild-type (WT) mice, we present evidence that HuR post-transcriptionally regulates CCR6 expression by binding to and stabilizing Ccr6 mRNA and by promoting CCR6 translation. We also found that HuR down-regulates several microRNA expressions, which could target the 3'-UTR of Ccr6 mRNA for decay. Accordingly, knock-out of HuR reduced CCR6 expression on Th17 cells and impaired their migration to CNS compared with the response of WT Th17 cells and thereby ameliorated EAE. Together, these findings highlight how HuR contributes to Th17 cell-mediated autoimmune neuroinflammation and support the notion that targeting HuR might be a potential therapeutic intervention for managing autoimmune disorders of the CNS.


Asunto(s)
Proteína 1 Similar a ELAV/metabolismo , Regulación de la Expresión Génica , ARN Mensajero/metabolismo , Receptores CCR6/agonistas , Linfocitos T Colaboradores-Inductores/metabolismo , Regiones no Traducidas 3' , Animales , Enfermedades Autoinmunes del Sistema Nervioso/inmunología , Enfermedades Autoinmunes del Sistema Nervioso/metabolismo , Enfermedades Autoinmunes del Sistema Nervioso/patología , Línea Celular , Movimiento Celular , Células Cultivadas , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Proteína 1 Similar a ELAV/antagonistas & inhibidores , Proteína 1 Similar a ELAV/genética , Encefalomielitis/inmunología , Encefalomielitis/metabolismo , Encefalomielitis/patología , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , MicroARNs/metabolismo , Biosíntesis de Proteínas , Interferencia de ARN , Estabilidad del ARN , Receptores CCR6/antagonistas & inhibidores , Receptores CCR6/genética , Receptores CCR6/metabolismo , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/patología
3.
Am J Pathol ; 185(3): 679-92, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25572154

RESUMEN

High-risk human papillomavirus (HPV) is a causative agent for an increasing subset of oropharyngeal squamous cell carcinomas (OPSCCs), and current evidence supports these tumors as having identifiable risk factors and improved response to therapy. However, the biochemical and molecular alterations underlying the pathobiology of HPV-associated OPSCC (designated HPV(+) OPSCC) remain unclear. Herein, we profile miRNA expression patterns in HPV(+) OPSCC to provide a more detailed understanding of pathologic molecular events and to identify biomarkers that may have applicability for early diagnosis, improved staging, and prognostic stratification. Differentially expressed miRNAs were identified in RNA isolated from an initial clinical cohort of HPV(+/-) OPSCC tumors by quantitative PCR-based miRNA profiling. This oncogenic miRNA panel was validated using miRNA sequencing and clinical data from The Cancer Genome Atlas and miRNA in situ hybridization. The HPV-associated oncogenic miRNA panel has potential utility in diagnosis and disease stratification and in mechanistic elucidation of molecular factors that contribute to OPSCC development, progression, and differential response to therapy.


Asunto(s)
Carcinoma de Células Escamosas/genética , MicroARNs , Neoplasias Orofaríngeas/genética , Infecciones por Papillomavirus/genética , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/virología , Línea Celular Tumoral , Biología Computacional , ADN Viral , Papillomavirus Humano 16 , Humanos , Persona de Mediana Edad , Neoplasias Orofaríngeas/patología , Neoplasias Orofaríngeas/virología , Infecciones por Papillomavirus/patología , Infecciones por Papillomavirus/virología
4.
J Immunol ; 191(11): 5441-50, 2013 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-24166976

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 Noqueados
5.
Mol Med ; 20: 93-108, 2014 Mar 20.
Artículo en Inglés | MEDLINE | ID: mdl-24477678

RESUMEN

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/citología
6.
Clin Epigenetics ; 16(1): 139, 2024 Oct 08.
Artículo en Inglés | MEDLINE | ID: mdl-39380119

RESUMEN

BACKGROUND: DNA methylation plays a critical role in asthma development, but differences in DNA methylation among adults with varying asthma severity are less well-defined. OBJECTIVE: To examine how DNA methylomic patterns differ among adults with asthma based on asthma severity and airway inflammation. METHODS: Peripheral blood T cells from 35 adults with asthma in Beijing, China, were serially collected over time (130 samples total) and analyzed for global DNA methylation using the Illumina MethylationEPIC Array. Differential methylation was compared among subjects with varying airway inflammation and severity, as measured by fraction of exhaled nitric oxide, forced expiratory volume in one second (FEV1), and Asthma Control Test (ACT) scores. RESULTS: Significant differences in DNA methylation were noted among subjects with different degrees of airway inflammation and asthma severity. These differences in DNA methylation were annotated to genes that were enriched in pathways related to asthma or T cell function and included gene ontology categories related to MHC class II assembly, T cell activation, interleukin (IL)-1, and IL-12. Genes related to P450 drug metabolism, glutathione metabolism, and developmental pathways were also differentially methylated in comparisons between subjects with high vs low FEV1 and ACT. Notable genes that were differentially methylated based on asthma severity included RUNX3, several members of the HLA family, AGT, PTPRC, PTPRJ, and several genes downstream of the JAK2 and TNF signaling pathway. CONCLUSION: These findings demonstrate how adults with asthma of varying severity possess differences in peripheral blood T cell DNA methylation that contribute to differences in clinical indices of asthma.


Asunto(s)
Asma , Metilación de ADN , Índice de Severidad de la Enfermedad , Linfocitos T , Humanos , Asma/genética , Asma/inmunología , Metilación de ADN/genética , Femenino , Masculino , Adulto , Persona de Mediana Edad , Linfocitos T/inmunología , Linfocitos T/metabolismo , Epigenoma/genética , China , Epigénesis Genética
7.
Res Sq ; 2024 Jun 10.
Artículo en Inglés | MEDLINE | ID: mdl-38946998

RESUMEN

Background: DNA methylation plays a critical role in asthma development, but differences in DNA methylation among adults with varying asthma severity or asthma endotypes are less well-defined. Objective: To examine how DNA methylomic patterns differ among adults with asthma based on asthma severity and airway inflammation. Methods: Peripheral blood T cells from 35 adults with asthma in Beijing, China were serially collected over time (130 samples total) and analyzed for global DNA methylation using the Illumina MethylationEPIC Array. Differential methylation was compared among subjects with varying airway inflammation and severity, as measured by fraction of exhaled nitric oxide, forced expiratory volume in one second (FEV1), and Asthma Control Test (ACT) scores. Results: Significant differences in DNA methylation were noted among subjects with different degrees of airway inflammation and asthma severity. These differences in DNA methylation were annotated to genes that were enriched in pathways related to asthma or T cell function and included gene ontology categories related to MHC class II assembly, T cell activation, interleukin (IL)-1, and IL-12. Genes related to P450 drug metabolism, glutathione metabolism, and developmental pathways were also differentially methylated in comparisons between subjects with high vs low FEV1 and ACT. Notable genes that were differentially methylated based on asthma severity included RUNX3, several members of the HLA family, AGT, PTPRC, PTPRJ, and several genes downstream of the JAK2 and TNF signaling pathway. Conclusion: These findings demonstrate how adults with asthma of varying severity possess differences in peripheral blood T cell DNA methylation that contribute to the phenotype and severity of their overall disease.

8.
J Immunol ; 186(4): 2482-94, 2011 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-21220697

RESUMEN

HuR is a regulator of mRNA turnover or translation of inflammatory genes through binding to adenylate-uridylate-rich elements and related motifs present in the 3'untranslated region (UTR) of mRNAs. We postulate that HuR critically regulates the epithelial response by associating with multiple ARE-bearing, functionally related inflammatory transcripts. We aimed to identify HuR targets in the human airway epithelial cell line BEAS-2B challenged with TNF-α plus IFN-γ, a strong stimulus for inflammatory epithelial responses. Ribonucleoprotein complexes from resting and cytokine-treated cells were immunoprecipitated using anti-HuR and isotype-control Ab, and eluted mRNAs were reverse-transcribed and hybridized to an inflammatory-focused gene array. The chemokines CCL2, CCL8, CXCL1, and CXCL2 ranked highest among 27 signaling and inflammatory genes significantly enriched in the HuR RNP-IP from stimulated cells over the control immunoprecipitation. Among these, 20 displayed published HuR binding motifs. Association of HuR with the four endogenous chemokine mRNAs was validated by single-gene ribonucleoprotein-immunoprecipitation and shown to be 3'UTR-dependent by biotin pull-down assay. Cytokine treatment increased mRNA stability only for CCL2 and CCL8, and transient silencing and overexpression of HuR affected only CCL2 and CCL8 expression in primary and transformed epithelial cells. Cytokine-induced CCL2 mRNA was predominantly cytoplasmic. Conversely, CXCL1 mRNA remained mostly nuclear and unaffected, as CXCL2, by changes in HuR levels. Increase in cytoplasmic HuR and HuR target expression partially relied on the inhibition of AMP-dependent kinase, a negative regulator of HuR nucleocytoplasmic shuttling. HuR-mediated regulation in airway epithelium appears broader than previously appreciated, coordinating numerous inflammatory genes through multiple posttranscriptional mechanisms.


Asunto(s)
Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Quimiocinas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Mucosa Respiratoria/inmunología , Proteínas Quinasas Activadas por AMP/fisiología , Biotinilación , Bronquios/inmunología , Bronquios/metabolismo , Bronquios/patología , Línea Celular Transformada , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL8/genética , Quimiocina CCL8/metabolismo , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Quimiocina CXCL2/genética , Quimiocina CXCL2/metabolismo , Quimiocinas/genética , Proteínas ELAV , Proteína 1 Similar a ELAV , Humanos , Mediadores de Inflamación/fisiología , Unión Proteica/genética , Unión Proteica/inmunología , Estabilidad del ARN/genética , Estabilidad del ARN/inmunología , Reproducibilidad de los Resultados , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Transducción de Señal/genética , Transducción de Señal/inmunología , Tráquea/inmunología , Tráquea/metabolismo , Tráquea/patología , Transcripción Genética/inmunología
9.
J Immunol ; 187(1): 441-9, 2011 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-21613615

RESUMEN

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/inmunología
10.
J Immunol Res ; 2021: 9937243, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34395636

RESUMEN

After antigen and/or different cytokine stimulation, CD4+ T cells activated and differentiated into distinct T helper (Th) cells via differential T cell signaling pathways. Transcriptional regulation of the activation and differentiation of naïve CD4+ T cells into distinct lineage Th cells such as Th17 cells has been fully studied. However, the role of RNA-binding protein HuR in the signaling pathways of their activation and differentiation has not been well characterized. Here, we used HuR conditional knockout (HuR KO) CD4+ T cells to study mechanisms underlying HuR regulation of T cell activation and differentiation through distinct signaling pathways. Our work showed that, mechanistically, HuR positively promoted CD3g expression by binding its mRNA and enhanced the expression of downstream adaptor Zap70 and Malt1 in activated CD4+ T cells. Compared to WT Th0 cells, HuR KO Th0 cells with reduced Bcl-2 expression are much more susceptible to apoptosis than WT Th0 cells. We also found that HuR stabilized IL-6Rα mRNA and promoted IL-6Rα protein expression, thereby upregulating its downstream phosphorylation of Jak1 and Stat3 and increased level of phosphorylation of IκBα to facilitate Th17 cell differentiation. However, knockout of HuR increased IL-22 production in Th17 cells, which was due to HuR deficiency in reducing IL-22 transcription repressor c-Maf expression. These results highlight the importance of HuR in TCR signaling and IL-6/IL-6R axis driving naïve CD4+ T cell activation and differentiation into Th17 cells.


Asunto(s)
Proteína 1 Similar a ELAV/metabolismo , Activación de Linfocitos/inmunología , Transducción de Señal , Células Th17/inmunología , Células Th17/metabolismo , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Citocinas/metabolismo , Proteína 1 Similar a ELAV/genética , Inmunofenotipificación , Interleucina-6/metabolismo , Activación de Linfocitos/genética , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Polirribosomas/metabolismo , Receptores de Interleucina-6/metabolismo , Factor de Transcripción STAT3/metabolismo
11.
J Exp Med ; 218(11)2021 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-34477806

RESUMEN

The autoimmune regulator (AIRE) is essential for the establishment of central tolerance and prevention of autoimmunity. Interestingly, different AIRE mutations cause autoimmunity in either recessive or dominant-negative manners. Using engineered mouse models, we establish that some monoallelic mutants, including C311Y and C446G, cause breakdown of central tolerance. By using RNAseq, ATACseq, ChIPseq, and protein analyses, we dissect the underlying mechanisms for their dominancy. Specifically, we show that recessive mutations result in a lack of AIRE protein expression, while the dominant mutations in both PHD domains augment the expression of dysfunctional AIRE with altered capacity to bind chromatin and induce gene expression. Finally, we demonstrate that enhanced AIRE expression is partially due to increased chromatin accessibility of the AIRE proximal enhancer, which serves as a docking site for AIRE binding. Therefore, our data not only elucidate why some AIRE mutations are recessive while others dominant, but also identify an autoregulatory mechanism by which AIRE negatively modulates its own expression.


Asunto(s)
Homeostasis/genética , Mutación/genética , Factores de Transcripción/genética , Animales , Autoinmunidad/genética , Cromatina/genética , Disección/métodos , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Modelos Animales , Proteína AIRE
12.
BMC Cancer ; 10: 126, 2010 Apr 06.
Artículo en Inglés | MEDLINE | ID: mdl-20370918

RESUMEN

BACKGROUND: The discordance between steady-state levels of mRNAs and protein has been attributed to posttranscriptional control mechanisms affecting mRNA stability and translation. Traditional methods of genome wide microarray analysis, profiling steady-state levels of mRNA, may miss important mRNA targets owing to significant posttranscriptional gene regulation by RNA binding proteins (RBPs). METHODS: The ribonomic approach, utilizing RNA immunoprecipitation hybridized to microarray (RIP-Chip), provides global identification of putative endogenous mRNA targets of different RBPs. HuR is an RBP that binds to the AU-rich elements (ARE) of labile mRNAs, such as proto-oncogenes, facilitating their translation into protein. HuR has been shown to play a role in cancer progression and elevated levels of cytoplasmic HuR directly correlate with increased invasiveness and poor prognosis for many cancers, including those of the breast. HuR has been described to control genes in several of the acquired capabilities of cancer and has been hypothesized to be a tumor-maintenance gene, allowing for cancers to proliferate once they are established. RESULTS: We used HuR RIP-Chip as a comprehensive and systematic method to survey breast cancer target genes in both MCF-7 (estrogen receptor positive, ER+) and MDA-MB-231 (estrogen receptor negative, ER-) breast cancer cell lines. We identified unique subsets of HuR-associated mRNAs found individually or in both cell types. Two novel HuR targets, CD9 and CALM2 mRNAs, were identified and validated by quantitative RT-PCR and biotin pull-down analysis. CONCLUSION: This is the first report of a side-by-side genome-wide comparison of HuR-associated targets in wild type ER+ and ER- breast cancer. We found distinct, differentially expressed subsets of cancer related genes in ER+ and ER- breast cancer cell lines, and noted that the differential regulation of two cancer-related genes by HuR was contingent upon the cellular environment.


Asunto(s)
Antígenos de Superficie/genética , Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Receptores de Estrógenos/biosíntesis , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Superficie/metabolismo , Biotina/química , Neoplasias de la Mama/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Línea Celular Tumoral , Proteínas ELAV , Proteína 1 Similar a ELAV , Humanos , Inmunoprecipitación , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/biosíntesis , Proteínas de Unión al ARN/metabolismo , Receptores de Estrógenos/genética , Tetraspanina 29
13.
J Allergy Clin Immunol ; 121(4): 853-9.e4, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18279945

RESUMEN

BACKGROUND: IL-13, a critical cytokine in allergy, is regulated by as-yet-elusive mechanisms. OBJECTIVE: We investigated IL-13 posttranscriptional regulation by HuR, a protein associating with adenylate-uridylate-rich elements in the 3' untranslated regions (UTRs) of mRNA, promoting mRNA stability and translation. METHODS: IL-13 mRNA decay was monitored in human T(H)2-skewed cells by using the transcriptional inhibitor actinomycin D. The IL-13 3'UTR was subcloned into an inducible beta-globin reporter transiently expressed in H2 cells in the absence or presence of overexpressed HuR. Association of HuR with IL-13 mRNA was detected by means of immunoprecipitation of ribonucleoprotein complexes and a biotin pull-down assay. The effects of HuR transient overexpression and silencing on IL-13 expression were investigated. RESULTS: IL-13 mRNA half-life increased significantly in restimulated T(H)2-skewed cells compared with baseline values. Decay of beta-globin mRNA was significantly faster in H2 cells transfected with the IL-13 3'UTR-containing plasmid than in those carrying a control vector. HuR overexpression increased the beta-globin IL-13 3'UTR reporter half-life. Significant enrichment of IL-13 mRNA was produced by means of immunoprecipitation of Jurkat cell ribonucleoprotein complexes with anti-HuR. HuR binding to the IL-13 3'UTR was confirmed by means of pull-down assay of biotin-labeled RNA probes spanning the IL-13 3'UTR. Two-dimensional Western blot analysis showed stimulus-induced posttranslational modification of HuR. In Jurkat cells mitogen-induced IL-13 mRNA was significantly affected by HuR overexpression and silencing. CONCLUSIONS: Mitogen-induced IL-13 expression involves changes in transcript turnover and a change in phosphorylation of HuR and its association with the mRNA 3'UTR.


Asunto(s)
Antígenos de Superficie/fisiología , Interleucina-13/genética , Interleucina-13/metabolismo , Procesamiento Postranscripcional del ARN/inmunología , Proteínas de Unión al ARN/fisiología , Células Th2/inmunología , Células Th2/metabolismo , Regiones no Traducidas 3'/genética , Regiones no Traducidas 3'/inmunología , Secuencia de Bases , Línea Celular Tumoral , Células Cultivadas , Proteínas ELAV , Proteína 1 Similar a ELAV , Humanos , Interleucina-13/biosíntesis , Células Jurkat , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Datos de Secuencia Molecular , Monensina/farmacología , ARN Mensajero/metabolismo , Acetato de Tetradecanoilforbol/farmacología
14.
Sci Signal ; 11(551)2018 10 09.
Artículo en Inglés | MEDLINE | ID: mdl-30301788

RESUMEN

Interleukin-17A (IL-17A) not only stimulates immunity to fungal pathogens but also contributes to autoimmune pathology. IL-17 is only a modest activator of transcription in experimental tissue culture settings. However, IL-17 controls posttranscriptional events that enhance the expression of target mRNAs. Here, we showed that the RNA binding protein (RBP) Arid5a (AT-rich interactive domain-containing protein 5a) integrated multiple IL-17-driven signaling pathways through posttranscriptional control of mRNA. IL-17 induced expression of Arid5a, which was recruited to the adaptor TRAF2. Arid5a stabilized IL-17-induced cytokine transcripts by binding to their 3' untranslated regions and also counteracted mRNA degradation mediated by the endoribonuclease MCPIP1 (Regnase-1). Arid5a inducibly associated with the eukaryotic translation initiation complex and facilitated the translation of the transcription factors (TFs) IκBζ (Nfkbiz ) and C/EBPß (Cebpb). These TFs in turn transactivated IL-17-dependent promoters. Together, these data indicated that Arid5a orchestrates a feed-forward amplification loop, which promoted IL-17 signaling by controlling mRNA stability and translation.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Interleucina-17/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Regiones no Traducidas 3' , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Citocinas/metabolismo , Fibroblastos/metabolismo , Células HEK293 , Humanos , Inflamación , Queratinocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/metabolismo , Unión Proteica , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleasas/metabolismo , Factor 2 Asociado a Receptor de TNF/metabolismo
15.
Mol Cell Biol ; 23(14): 4991-5004, 2003 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-12832484

RESUMEN

In this report, we investigate the role of the RNA-binding protein HuR during skeletal myogenesis. At the onset of myogenesis in differentiating C2C12 myocytes and in vivo in regenerating mouse muscle, HuR cytoplasmic abundance increased dramatically, returning to a predominantly nuclear presence upon completion of myogenesis. mRNAs encoding key regulators of myogenesis-specific transcription (myogenin and MyoD) and cell cycle withdrawal (p21), bearing AU-rich regions, were found to be targets of HuR in a differentiation-dependent manner. Accordingly, mRNA half-lives were highest during differentiation, declining when differentiation was completed. Importantly, HuR-overexpressing C2C12 cells displayed increased target mRNA expression and half-life and underwent precocious differentiation. Our findings underscore a critical function for HuR during skeletal myogenesis linked to HuR's coordinate regulation of muscle differentiation genes.


Asunto(s)
Antígenos de Superficie , Regulación del Desarrollo de la Expresión Génica , Desarrollo de Músculos/fisiología , Músculo Esquelético/fisiología , Proteínas de Unión al ARN/fisiología , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , Citoplasma/efectos de los fármacos , Citoplasma/genética , Citoplasma/metabolismo , Proteínas ELAV , Proteína 1 Similar a ELAV , Semivida , Insulina/farmacología , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Músculo Esquelético/citología , Proteína MioD/genética , Mioblastos/fisiología , Miogenina/genética , ARN Mensajero/metabolismo , Regeneración/genética , Selenio/farmacología , Transferrina/farmacología
16.
Sci Rep ; 7(1): 17233, 2017 12 08.
Artículo en Inglés | MEDLINE | ID: mdl-29222492

RESUMEN

Granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by T helper 17 (Th17) cells plays an essential role in autoimmune diseases. Transcriptional regulation of Th17 cell differentiation has been extensively studied, but post-transcriptional regulation of Th17 cell differentiation has remained less well characterized. The RNA-binding protein HuR functions to promote the stability of target mRNAs via binding the AU-rich elements of the 3' untranslated region (3'UTR) of numerous pro-inflammatory cytokines including IL-4, IL-13, IL-17 and TNF-α. However, whether HuR regulates GM-CSF expression in Th17 cells has not been fully investigated. Here we showed that HuR conditional knockout (KO) Th17 cells have decreased GM-CSF mRNA in comparison with wild-type (WT) Th17 cells, and that HuR binds directly to GM-CSF mRNA 3'UTR. Interestingly, HuR deficiency increased the levels of certain microRNA expression in Th17 cells; for example, miR-466i functioned to mediate GM-CSF and IL-17 mRNA decay, which was confirmed by in vitro luciferase assay. Furthermore, we found that HuR promoted Mxi1 expression to inhibit certain miRNA expression. Taken together, these findings indicate that interaction of HuR and miR-466i orchestrates GM-CSF expression in Th17 cells.


Asunto(s)
Proteína 1 Similar a ELAV/metabolismo , Regulación de la Expresión Génica/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , MicroARNs/genética , MicroARNs/metabolismo , Regiones no Traducidas 3'/genética , Animales , Secuencia de Bases , Línea Celular , Proteína 1 Similar a ELAV/deficiencia , Proteína 1 Similar a ELAV/genética , Técnicas de Inactivación de Genes , Interleucina-17/genética , Macrófagos/metabolismo , Ratones , Unión Proteica , Estabilidad del ARN , Células Th17/metabolismo
17.
Immunohorizons ; 1(6): 109-123, 2017 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-30035254

RESUMEN

Posttranscriptional gene regulation by RNA-binding proteins, such as HuR (elavl1), fine-tune gene expression in T cells, leading to powerful effects on immune responses. HuR can stabilize target mRNAs and/or promote translation by interacting with their 3' untranslated region adenylate and uridylate-rich elements. It was previously demonstrated that HuR facilitates Th2 cytokine expression by mRNA stabilization. However, its effects upon IL-2 homeostasis and CD4+ Th2 differentiation are not as well understood. We found that optimal translation of Il2ra (CD25) required interaction of its mRNA with HuR. Conditional HuR knockout in CD4+ T cells resulted in loss of IL-2 homeostasis and defects in JAK-STAT signaling, Th2 differentiation, and cytokine production. HuR-knockout CD4+ T cells from OVA-immunized mice also failed to proliferate in response to Ag. These results demonstrate that HuR plays a pivotal role in maintaining normal IL-2 homeostasis and initiating CD4+ Th2 differentiation.

18.
PLoS One ; 10(7): e0129321, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26162078

RESUMEN

Due to poor correlation between steady state mRNA levels and protein product, purely transcriptomic profiling methods may miss genes posttranscriptionally regulated by RNA binding proteins (RBPs) and microRNAs (miRNAs). RNA immunoprecipitation (RIP) methods developed to identify in vivo targets of RBPs have greatly elucidated those mRNAs which may be regulated via transcript stability and translation. The RBP HuR (ELAVL1) and family members are major stabilizers of mRNA. Many labs have identified HuR mRNA targets; however, many of these analyses have been performed in cell lines and oftentimes are not independent biological replicates. Little is known about how HuR target mRNAs behave in conditional knock-out models. In the present work, we performed HuR RIP-Seq and RNA-Seq to investigate HuR direct and indirect targets using a novel conditional knock-out model of HuR genetic ablation during CD4+ T activation and Th2 differentiation. Using independent biological replicates, we generated a high coverage RIP-Seq data set (>160 million reads) that was analyzed using bioinformatics methods specifically designed to find direct mRNA targets in RIP-Seq data. Simultaneously, another set of independent biological replicates were sequenced by RNA-Seq (>425 million reads) to identify indirect HuR targets. These direct and indirect targets were combined to determine canonical pathways in CD4+ T cell activation and differentiation for which HuR plays an important role. We show that HuR may regulate genes in multiple canonical pathways involved in T cell activation especially the CD28 family signaling pathway. These data provide insights into potential HuR-regulated genes during T cell activation and immune mechanisms.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Proteína 1 Similar a ELAV/inmunología , Regulación de la Expresión Génica , Activación de Linfocitos , ARN Mensajero/inmunología , Transcriptoma , Animales , Antígenos CD28/genética , Antígenos CD28/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular , Células Cultivadas , Proteína 1 Similar a ELAV/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/genética , Células Th2/citología , Células Th2/inmunología , Células Th2/metabolismo
19.
Gene ; 317(1-2): 79-87, 2003 Oct 23.
Artículo en Inglés | MEDLINE | ID: mdl-14604794

RESUMEN

Coordinated gene expression is influenced by transcriptional and posttranscriptional events and is necessary for efficient cell growth and differentiation. Genomic array technologies have afforded great advances in identifying global changes of gene expression in response to a variety of environmental stimuli. However, it has been a challenge to assess whether a concomitant effect on protein expression reflects the coordinated regulation of distinct subsets of mRNAs detected by cDNA arrays [Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 7018]. We have expanded the utility of cDNA arrays by using them to assist in elucidating combinatorial posttranscriptional eukaryotic operons [Mol. Cell 9 (2002) 1161]. In this study, we have used two mRNA partitioning methods in which: (1) subsets of mRNAs are isolated as endogenous mRNP complexes using autoimmune patient sera, and (2) transcriptional contributions to gene expression are assessed using cDNA array analysis of an en masse nuclear run-on assay (emRUN). The combination of these methods can provide an additional 'systems biology' discovery approach to gene expression analysis based upon the physical partitioning of mRNA subsets, as well as a functional partitioning of transcriptional and posttranscriptional processes. We demonstrate how these approaches can reduce transcriptomic complexity by partitioning mRNAs into biologically relevant subsets in order to derive information about the expression of multiple, but functionally linked, genes.


Asunto(s)
Núcleo Celular/genética , Perfilación de la Expresión Génica/métodos , Genoma Humano , Ribonucleoproteínas/genética , Enfermedades Autoinmunes/sangre , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Western Blotting , Núcleo Celular/metabolismo , Células HeLa , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Pruebas de Precipitina , Procesamiento Postranscripcional del ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/inmunología , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/inmunología , Ribonucleoproteínas/metabolismo , Transcripción Genética
20.
J Vis Exp ; (67)2012 Sep 29.
Artículo en Inglés | MEDLINE | ID: mdl-23051702

RESUMEN

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.


Asunto(s)
Inmunoprecipitación/métodos , MicroARNs/química , Análisis por Micromatrices/métodos , ARN Mensajero/química , Ribonucleoproteínas/química , MicroARNs/aislamiento & purificación , ARN Mensajero/aislamiento & purificación , Ribonucleoproteínas/aislamiento & purificación
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