RESUMO
The activation of cytosolic phospholipase A(2)α (cPLA(2)α) plays an important role in initiating the inflammatory response. The regulation of cPLA(2)α mRNA turnover has been proposed to control cPLA(2)α gene expression under cytokine and growth factor stimulation. However, the detailed mechanism is still unknown. In this report, we have demonstrated that the cPLA(2)α mRNA stability was increased under IL-1ß treatment in A549 cells. By using EMSAs, HuR was identified as binding with the cPLA(2)α mRNA 3'-UTR, and the binding region was located at nucleotides 2716-2807, a fragment containing AUUUA flanked by U-rich sequences. IL-1ß treatment enhanced the association of cPLA(2)α mRNA with cytosolic HuR. The reduction of HuR expression by RNA interference technology inhibited IL-1ß-induced cPLA(2)α mRNA and protein expression. Furthermore, blocking the p38 MAPK signaling pathway with SB203580 abolished the effect of IL-1ß-induced cPLA(2)α gene expression. Phosphorylation at residue Thr-118 of HuR is crucial in regulating the interaction between HuR and its target mRNAs. Mutation of HuR Thr-118 reduced the association between HuR and cPLA(2)α mRNA under IL-1ß treatment. This inhibitory effect was also observed in binding with COX-2 mRNA. This result indicated that p38 MAPK-mediated Thr-118 phosphorylation may play a key role in regulating the interaction of HuR with its target mRNAs in inflammation.
Assuntos
Regiões 3' não Traduzidas , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proteínas ELAV/metabolismo , Fosfolipases A2 do Grupo IV/biossíntese , Interleucina-1beta/farmacologia , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Estabilidade de RNA/efeitos dos fármacos , RNA Neoplásico/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Proteínas ELAV/genética , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Fosfolipases A2 do Grupo IV/genética , Humanos , Imidazóis/farmacologia , Inflamação/genética , Inflamação/mortalidade , Neoplasias Pulmonares/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Mutação , Proteínas de Neoplasias/genética , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Piridinas/farmacologia , RNA Neoplásico/genética , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The expression of cPLA2 is critical for transformed growth of non-small cell lung cancer (NSCLC). It is known that phorbol 12-myristate 13-acetate (PMA)-activated signal transduction pathway is thought to be involved in the oncogene action in NSCLC and enzymatic activation of cPLA2. However, the transcriptional regulation of cPLA2alpha in PMA-activated NSCLC is not clear. In this study, we found that PMA induced the mRNA level and protein expression of cPLA2alpha. In addition, two Sp1-binding sites of cPLA2alpha promoter were required for response to PMA and c-Jun overexpression. Small interfering RNA (siRNA) of c-Jun and nucleolin inhibited PMA induced the promoter activity and protein expression of cPLA2alpha. Furthermore, PMA stimulated the formation of c-Jun/Sp1 and c-Jun/nucleolin complexes as well as the binding of these transcription factor complexes to the cPLA2alpha promoter. Although Sp1-binding sites were required for the bindings of Sp1 and nucleolin to the promoter, the binding of nucleolin or Sp1 to the promoter was independent of each other. Our results revealed that c-Jun/nucleolin and c-Jun/Sp1 complexes play an important role in PMA-regulated cPLA2alpha gene expression. It is likely that nucleolin binding at place of Sp1 on gene promoter could also mediate the regulation of c-Jun/Sp1-activated genes.
