RESUMO
The rapid induction of interferon-gamma (IFN-gamma) by innate cytokines such as interleukin 12 (IL-12) and IL-18 is critical for immunity against infectious pathogens. We investigated the molecular mechanisms underlying this response. IL-12 and IL-18 rapidly and synergistically induced the secretion of IFN-gamma by freshly purified human peripheral blood lymphocytes. At early time points, IFN-gamma was expressed almost exclusively by natural killer cells and in both CD56bright and CD56dim subpopulations. Mitogen-activated protein kinase p38 was activated strongly by IL-18 and weakly by IL-12 in natural killer cells but was not activated by either cytokine in T cells. The expression of IFN-gamma mRNA and protein was dose-dependently blocked by SB203580, a specific inhibitor of mitogen-activated protein kinase p38, which also caused a dramatic destabilization of IFN-gamma mRNA. The 3' untranslated region (UTR) of IFN-gamma mRNA conferred p38 responsiveness to a heterologous reporter mRNA. Therefore, the synergistic induction of IFN-gamma by IL-12 and IL-18 in natural killer cells is mediated at least in part by p38-dependent and 3' UTR-mediated stabilization of IFN-gamma mRNA.
Assuntos
Interferon gama/genética , Interleucina-12/farmacologia , Interleucina-18/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Estabilidade de RNA/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Células Cultivadas , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Humanos , Interferon gama/biossíntese , Interferon gama/metabolismo , Células Matadoras Naturais/enzimologia , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/efeitos dos fármacos , RNA Mensageiro/genética , Transdução de SinaisRESUMO
The p38 mitogen-activated protein kinase (MAPK) pathway plays an important role in the post-transcriptional regulation of inflammatory genes. p38 has been found to regulate both the translation and the stability of inflammatory mRNAs. The mRNAs regulated by p38 share common AU-rich elements (ARE) present in their 3'-untranslated regions. AREs act as mRNA instability determinants but also confer stabilisation of the mRNA by the p38 pathway. In recent years, AREs have shown to be binding sites for numerous proteins including HuR, TTP, AUF1, AUF2, FBP1, FBP2 (KSRP), TIA-1, and TIAR. However, it is unclear which protein is responsible for mRNA stabilisation by p38. This review gives an overview of the major ARE-binding proteins and discusses reasons for and against their involvement in p38-mediated mRNA stabilisation.
Assuntos
Sistema de Sinalização das MAP Quinases/fisiologia , Estabilidade de RNA/fisiologia , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Humanos , Ligação Proteica , Estabilidade de RNA/genéticaRESUMO
COX-2 (cyclo-oxygenase-2) mRNA is degraded rapidly in resting cells, but is stabilized by the mitogen-activated protein kinase p38 signalling pathway in response to pro-inflammatory stimuli. A conserved ARE (AU-rich element) of the COX-2 3' untranslated region, CR1 (conserved region 1), acts as a potent instability determinant, and mediates stabilization in response to p38 activation. A detailed structural and functional analysis of this element was performed in an attempt to identify RNA-binding proteins involved in the regulation of COX-2 mRNA stability. Destabilization of a beta-globin reporter mRNA was dependent upon two distinct AREs within CR1, each containing three copies of the sequence AUUUA. CR1 was shown to bind AUF-1 [ARE/poly(U)-binding/degradation factor-1] and/or AUF-2, HuR (Hu antigen R), TTP (tristetraprolin) and FBP1 (far-upstream-sequence-element-binding protein 1), yet these factors did not appear to account for the effects of CR1 upon mRNA stability. Mutant sequences were identified that were incapable of destabilizing a reporter mRNA, yet showed unimpaired binding of FBP1 and AUF-1 and/or -2. TTP was absent from the HeLa cell line used in this analysis. Finally, RNA interference experiments argued against a prominent role for HuR in the CR1-mediated regulation of mRNA stability. We conclude that at least one critical regulator of COX-2 mRNA stability is likely to remain unidentified at present.
Assuntos
Antígenos de Superfície , Sequência Conservada/fisiologia , Proteínas de Ligação a DNA/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Proteínas Imediatamente Precoces/genética , Isoenzimas/genética , Prostaglandina-Endoperóxido Sintases/genética , Proteínas de Ligação a RNA/genética , Sequências Reguladoras de Ácido Ribonucleico/fisiologia , Proteínas Repressoras/genética , Regiões 3' não Traduzidas/genética , Regiões 3' não Traduzidas/metabolismo , Regiões 3' não Traduzidas/fisiologia , Composição de Bases , Sequência de Bases , Linhagem Celular Tumoral , Sequência Conservada/genética , Ciclo-Oxigenase 2 , Análise Mutacional de DNA , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Ensaio de Desvio de Mobilidade Eletroforética , Células HeLa , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Humanos , Proteínas Imediatamente Precoces/metabolismo , Isoenzimas/fisiologia , Proteínas de Membrana , Dados de Sequência Molecular , Prostaglandina-Endoperóxido Sintases/fisiologia , Estabilidade de RNA/genética , Estabilidade de RNA/fisiologia , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , RNA Neoplásico/fisiologia , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/fisiologia , Sequências Reguladoras de Ácido Ribonucleico/genética , Relação Estrutura-Atividade , TristetraprolinaRESUMO
The AU-rich element (ARE) is an important instability determinant for a large number of early-response-gene mRNAs. AREs also mediate the stabilization of certain pro-inflammatory mRNAs, such as tumour necrosis factor (TNF)-alpha and cyclo-oxygenase-2 (COX-2), in response to inflammatory stimuli. To understand how AREs control mRNA stability, it is necessary to identify trans-acting factors. We have purified a new ARE-binding protein and identified it as CArG box-binding factor-A (CBF-A). The amino acid sequence of CBF-A is highly similar to that of the ARE-binding protein AUF1. Recombinant CBF-A bound the COX-2 and TNF-alpha AREs, but not a non-specific control RNA. In contrast, in an electrophoretic-mobility-shift assay (EMSA) of crude RAW 264.7 macrophage-like cell extracts, an antiserum that recognizes both AUF1 and CBF-A failed to supershift complexes formed on the TNF-alpha ARE, but did supershift a complex specific for the COX-2 ARE. CBF-A exists as two isoforms, p37 and p42, that differ by a 47-amino-acid insertion close to the C-terminus. By expressing epitope-tagged isoforms of CBF-A it was shown that the p42 isoform binds the COX-2 ARE in EMSA of crude cell extracts. In a HeLa-cell tetracycline-regulated reporter system, overexpression of the p42 CBF-A isoform resulted in stabilization of a COX-2 ARE reporter mRNA. Epitope-tagged p42 CBF-A expressed in HeLa cells co-immunoprecipitated with endogenous COX-2 mRNA, but not glyceraldehyde-3-phosphate dehydrogenase mRNA, as shown by reverse-transcription PCR. The similarity between CBF-A and AUF1 suggests that CBF-A could be re-named AUF2.