Transcriptional regulation of plasminogen activator inhibitor type I gene by insulin - Insights into the signaling pathway

Impairment of the fibrinolytic system, caused primarily by increases in the plasma levels of plasminogen activator inhibitor (PAI) type 1, are frequen tly found in diabetes and the insulin-resistance syndrome. Among the factor s responsible for the increases of PAT-1, insulin has recently attracted at tention. In this study, we analyzed the effects of insulin on PAI-1 biosynt hesis in HepG2 cells, paying particular attention to the signaling network evoked by this hormone. Experiments performed in CHO cells overexpressing t he insulin receptor indicate that insulin increases PAI-1 gene transcriptio n through interaction with its receptor. By using inhibitors of the differe nt signaling pathways evoked by insulin-receptor binding, it has been shown that the biosynthesis of PAI-1 is due to phosphatidylinositol (PI) 3-kinas e activation, followed by protein kinase C and ultimately by mitogen-activa ted protein (MAP) kinase activation and extracellular signal-regulated kina se 2 phosphorylation, We also,showed that this pathway is Ras-independent. Transfection of HepG2 cells with several truncations of the PAT-1 promoter coupled to a CAT gene allowed vs to recognize two major response elements l ocated in the regions between -804 and -708 and between -211 and -54, Elect rophoretic mobility shift assay identified three binding sites for insulin- induced factors, all colocalized with putative Spl binding sites. Using sup ershifting antibodies, the binding of Spl could only be confirmed at the bi nding site located just upstream from the transcription start site of the P AI-1 promoter. A construct comprising four tandem repeat copies of the -93/ -62 region of the PAT-1 promoter linked to CAT was transcriptionally activa ted in HepG2 cells by insulin, These results outline the central role of MA P kinase activation in the regulation of PAI-1 induced by insulin.