RESUMO
BACKGROUND: Plasminogen activator inhibitor (PAI)-1 is a key regulator of the fibrinolytic system. PAI-1 levels are markedly elevated in the asthmatic airways. The 4G/5G polymorphism of the PAI-1 gene is associated with allergic asthma. OBJECTIVE: To characterize the mechanisms of the 4G/5G-dependent PAI-1 expression in mast cells (MCs), a major source of PAI-1 and key effector cells in asthma. METHODS: Transcription of PAI-1 was assessed by transiently transfecting human MC line (HMC-1) cells with the luciferase-tagged PAI-1 promoters containing the 4G or 5G allele (4G-PAI-1 or 5G-PAI-1 promoter). Upstream stimulatory factor (USF)-1 and the E-box interactions were studied by electrophoretic mobility shift assays and supershift assays. Expression of USF-1 was determined by Western blot analysis. RESULTS: The 4G-PAI-1 promoter has higher promoter activity than the 5G-PAI-1 promoter in stimulated HMC-1 cells, and the E-box adjacent to the 4G/5G site (E-4G/5G) regulates the genotype-specific PAI-1 transcription. USF-1 binds to the E-4G with greater affinity than to the E-5G. USF-1 level is increased in HMC-1 cells after stimulation, and elevated USF-1 enhances PAI-1 transcription. Overexpression of wild-type USF-1 or dominant-negative USF remedies the 4G/5G-dependent PAI-1 transcription. CONCLUSION: Binding of USF-1 to the E-4G/5G regulates the 4G/5G polymorphism-dependent PAI-1 expression in MCs.
Assuntos
Alelos , Elementos E-Box/fisiologia , Nucleotídeos de Guanina/genética , Mastócitos/metabolismo , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Inibidor 1 de Ativador de Plasminogênio/genética , Polimorfismo Genético/fisiologia , Fatores Estimuladores Upstream/metabolismo , Asma/genética , Asma/metabolismo , Asma/patologia , Linhagem Celular , Elementos E-Box/genética , Nucleotídeos de Guanina/metabolismo , Humanos , Mastócitos/imunologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ligação Proteica/genética , Regulação para Cima/genética , Fatores Estimuladores Upstream/biossíntese , Fatores Estimuladores Upstream/genéticaRESUMO
Plasminogen activator inhibitor (PAI)-1 promotes development of asthma. PAI-1 mRNA and protein are markedly induced in activated mast cells (MCs), a major effector cell type in asthma. However, regulatory mechanisms of PAI-1 transcription in MCs are unknown. We present first evidence that PAI-1 is transcriptionally regulated in human MCs (hMCs). In addition to three enhancer regions, we demonstrated that the E-box at -566 bp to -561 bp is the negative regulatory element, and the specific and constitutive binding of the upstream stimulating factor-1 to this E-box is the key mechanism of the negative regulation of PAI-1 expression in hMCs.