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 putative phorbol 12-myristate-13-acetate-(PMA)-responsive elements (TREs) in the induction of plasminogen-activator inhibitor 1 (PAI-1) gene transcription in the human hepatoma cell line HepG2 by activators of protein kinase C (PKC). Treatment of HepG2 cells with the phorbol ester PMA or serum rapidly and transiently increased c-Jun and c-Fos mRNA and protein levels prior to PAI-1 induction. This induction of PAI-1 gene transcription was found to be dependent on ongoing protein synthesis. An essential role of c-Jun and c-Fos in basal and PMA-stimulated transcription of the PAI-1 gene is demonstrated by our finding that antisense c-jun and c-fos oligodeoxynucleotides both strongly reduced basal and PMA-stimulated PAI-1 synthesis. Since it has already been shown that 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 promoter activity ([16]), we examined binding of nuclear proteins to these elements. The protein-binding activity to the TRE between positions -79 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 -58 and -50 forms two complexes, both containing c-Jun protein. The faster migrating complex primarily contains c-Jun homodimers. The amount of the faster migrating complex is enhanced more than 30-fold in PMA-treated cells, due to a strongly increased binding of c-Jun homodimers and, to a minor extent, to binding of c-Jun/c-Fos heterodimers. Dissociation 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 oligodeoxynucleotides reduced the amount of c-Jun homodimer, we conclude that binding of c-Jun homodimer to the TRE at positions -58 to -50 is important in the basal activity and PMA activation of the PAI-1 promoter in HepG2 cells.
Arts
J
J
Gaubius Laboratory, TNO-PG, Leiden, The Netherlands.
Grimbergen
J
J
Bosma
P J
PJ
Rahmsdorf
H J
HJ
Kooistra
T
T
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D003470:Culture Media; D003513:Cycloheximide / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D016762:Genes, fos / Q000187:drug effects; D016758:Genes, jun / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis / Q000235:genetics; D016755:Proto-Oncogene Proteins c-jun / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D018808:Transcription Factor AP-1 / Q000378:metabolism
D003470:Culture Media; D016376:Oligonucleotides, Antisense; D017395:Plasminogen Activator Inhibitor 1; D011500:Protein Synthesis Inhibitors; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D003513:Cycloheximide; D013755:Tetradecanoylphorbol Acetate