Transcriptional Regulation of human Plasminogen Activator Inhibitor-1 Gene Expression by Insulin-like Growth Factor-1, Insulin and Upstream Stimulatory Factor-2

PAI-1 inhibits conversion of the proenzyme plasminogen to the active protease plasmin and modulates proteolytic processes associated with pathophysiological conditions such as prethrombotic events, hemorrhage and thrombus formations which are positively correlated with increased PAI-1 plasma levels and hypoxic conditions. Insulin-resistant states are characterized by enhanced PAI-1 levels. We investigated the mechanisms that mediate IGF-1- and insulin-dependent induction of PAI-1 transcription in human HepG2 hepatoma cells. Treatment of HepG2 cells with IGF-1 and insulin enhanced PAI-1 mRNA, protein expression and PAI-1 promoter-Luc reporter activity. We found that mutation of the hypoxia-responsive element (HRE), a target for HIF-1, nearly abolished the IGF-1- and insulin-dependent PAI-1 promoter activity. Mutation of E4 and E5 did not affect the IGF-1-dependent induction of PAI-1 promoter constructs under normoxia but abolished the effect of IGF-1 under hypoxia. We found that IGF-1-induced up-regulation of PAI-1 expression was associated with activation of HIF-1 alpha. IGF-1 enhanced HIF-1 alpha protein levels and HIF-1 DNA-binding to each HRE, and E-boxes E4 and E5 as shown by EMSAs. Inhibition of either PI(3)K by LY294002 or ERK1/2 by U0126 reduced HIF-1 alpha protein levels while both inhibitors together completely abolished the IGF-1 effect on HIF-alpha. Expression of a dominant-negative Raf inhibited the IGF-1 effect on HIF-1-alpha while no effect was observed by using dominant-negative PDK1, or TRB3 which inhibits Akt/PKB activation. Thus, IGF-1 activates human PAI-1 expression through activation of the PI(3)K and ERK1/2. The insulin-induced enhancement of HIF-1 alpha protein levels by specific PI(3)K inhibitors was inhibited under normoxia and only slightly reduced under hypoxia. The overexpression of dominant negative PDK1 as well as TRB3 reduced the basal HIF-1 alpha protein levels but did not influence the insulin-induced HIF-1 alpha protein levels. Thus, in the case of insulin, activation of PAI-1 expression occurred mainly via the MAPK pathway. In addition to HIF-1, USF-2 can induce human PAI-1 gene expression as well via HRE, E4 and E5. EMSAs showed that E5 and E4 bound USFs and although the HRE contributed to the USF-dependent regulation of the human PAI-1 gene, it did not bind them. By contrast, USF-2 inhibited PAI-1 promoter activity in primary rat hepatocytes suggesting that PAI-1 expression depends on either the promoter context or the activity of USF which might be cell-type specific. We found that the different effects of USF on PAI-1 expression depend on the cell-type rather than the promoter context and that the USR domain of USF accounts for the observed effects. Together, we have shown that IGF-1, insulin and even hypoxia activate human PAI-1 expression through activation of the PI(3)Kinase and MAPK signaling cascade via HIF-1 binding to HRE. We described a different USF-mediated regulation of the PAI-1 gene in hepatoma cells and primary rat hepatocytes showing a cell-type-dependent regulation.