On the role of c-Jun in the induction of PAI-1 gene expression by phorbol ester, serum, and IL-1 alpha in HepG2 cells

We have characterized the regulation of plasminogen activator inhibitor-1 ( PAI-1) gene expression by phorbol 12-myristate 13-acetate (PMA), serum, and interleukin-1 alpha (IL-1 alpha) in the human hepatoma cell Line HepG2. PM A, serum, and IL-1 alpha induced a rapid and transient 28-fold (PMA), 9-fol d (serum), and 23-fold (IL-1 alpha) increase in PAI-1 mRNA, peaking after a pproximate to 4 hours. These inductions of PAI-1 mRNA accumulation were red uced by pretreatment of the HepG2 cells with the protein tyrosine kinase in hibitor genistein. Conversely, stimulation of tyrosine phosphorylation by s odium orthovanadate, an inhibitor of protein tyrosine phosphatases, caused an increase in PAI-1 mRNA levels. The effects of PMA, serum, and IL-1 alpha on PAI-1 mRNA expression have been compared with their ability to modulate the expression of a chloramphenicol acetyltransferase (CAT) reporter plasm id, which was under control of the -489 to +75 region of the PAI-1 promoter , and stably transfected into HepG2 cells. This region of the PAI-1 promote r was previously found to contain a tetradecanoyl phorbol acetate-response element (TRE; between -58 and -50) necessary for PMA responsiveness and wit h a high affinity for c-Jun homodimers. Whereas incubation of these transfe cted HepG2 cells with PMA and serum showed an induction profile of CAT mRNA similar to that of PAT-I mRNA, hardly any induction of CAT mRNA was found with IL-1 alpha. In line with these findings, IL-1 alpha poorly induced c-J un homodimer binding to the PAI-I TRE in gel mobility-shift assays. Pretrea tment of HepG2 cells with the protein kinase C inhibitor Ro 31-8220 or the mitogen-activated protein kinase kinase (MAPKK)(1,2) activity blocker PD980 59 selectively suppressed the induction of PAI-1 (and CAT) expression by PM A, but not that by IL-la. In contrast, the protein tyrosine kinase inhibito r herbimycin A blocked PAI-T mRNA induction by IL-1 alpha only. We propose 2 separate PAI-I inductory pathways for PMA and LL-1 alpha in HepG2, both i nvolving protein tyrosine kinase activation; the serum-induced signaling pa thway may (partially) overlap with the PMA-activated protein kinase C/mitog en-activated protein kinase kinase pathway, leading to c-Jun homodimer bind ing to the PAI-1 TRE.