ROLE OF C-JUN AND PROXIMAL PHORBOL 12-MYRISTATE-13-ACETATE-(PMA)-RESPONSIVE ELEMENTS IN THE REGULATION OF BASAL AND PMA-STIMULATED PLASMINOGEN-ACTIVATOR INHIBITOR-1 GENE-EXPRESSION IN HEPG2

Experiments were designed to clarify the role of c-Jun/c-Fos and of pu tative phorbol 12-myristate-13-acetate-(PMA)-responsive elements (TREs ) in the induction of plasminogen-activator inhibitor 1 (PAI-1) gene t ranscription in the human hepatoma cell line HepG2 by activators of pr otein kinase C (PKC). Treatment of HepG2 cells with the phorbol ester PMA or serum rapidly and transiently increased c-Jun and c-Fos mRNA an d protein levels prior to PAI-1 induction. This induction of PAI-1 gen e transcription was found to be dependent on ongoing protein synthesis . An essential role of c-Jun and c-Fos in basal and PMA-stimulated tra nscription of the PAI-1 gene is demonstrated by our finding that antis ense c-jun and c-fos oligodeoxynucleotides both strongly reduced basal and PMA-stimulated PAI-1 synthesis. Since it has already been shown t hat two TREs between positions -58 and -50 and between -79 and -72 of the PAI-1 promoter are essential for basal and PMA-induced PAI-1 promo ter activity ([16]), we examined binding of nuclear proteins to these elements. The protein-binding activity to the TRE between positions -7 9 and -72 shows very strong PMA induction of an unknown factor, which is not related to c-Jun or c-Fos. The TRE binding between positions -5 8 and -50 forms two complexes, both containing c-Jun protein. The fast er migrating complex primarily contains c-Jun homodimers. The amount o f the faster migrating complex is enhanced more than 30-fold in PMA-tr eated cells, due to a strongly increased binding of c-Jun homodimers a nd, to a minor extent, to binding of c-Jun/c-Fos heterodimers. Dissoci ation experiments suggest that the c-Jun/c-Fos heterodimers bind with much lower affinity compared to binding of c-Jun homodimers. Together with the finding that both antisense c-Jun and antisense c-fos oligode oxy-nucleotides reduced the amount of c-Jun homodimer, we conclude tha t binding of c-Jun homodimer to the TRE at positions -58 to -50 is imp ortant in the basal activity and PMA activation of the PAI-1 promoter in HepG2 cells.