A common response element mediates differential effects of phorbol esters and forskolin on type-1 plasminogen activator inhibitor gene expression in human breast carcinoma cells.

We have characterized regulation of type-1 plasminogen activator inhibitor (PAI-1) gene expression by phorbol 12-myristate 13-acetate (PMA) and the cAMP-inducing agent forskolin in the human breast carcinoma cell line MCF-7. PMA caused a strong induction of PAI-1, while forskolin suppressed the PMA response. Transfection experiments with fusion genes showed that sequences mediating PMA induction as well as forskolin suppression were present between base pairs -100 and -30 of the 5'-flanking region of the PAI-1 gene. The region was found to contain two Sp1 binding sites. A proximal sequence in the region, TGAGTTCA (P box), with sequence similarity to phorbol ester response elements (TRE) as well as to cAMP response elements (CRE), bound a low-abundance, as yet unidentified nuclear protein in MCF-7 cells. This sequence had a higher affinity to purified c-jun homodimer than to c-jun/c-fos heterodimer in MCF-7 nuclear extracts; it had no affinity to the proteins binding to CRE consensus sequences in these extracts. A distal TRE-like sequence, TGAGTGG (D box), had a weak affinity to c-jun/c-fos heterodimer and c-jun homodimer; binding of proteins to this sequence was facilitated by binding of proteins to the P box. Both the P box and the D box were necessary for PMA responsiveness, suggesting a cooperativity between the two binding sites. A mutation of the P box removing the CRE similarity abolished the forskolin suppression of the PMA response. We propose that the protein kinase C and the protein kinase A signal-transduction pathways, with opposite effects on PAI-1 gene expression converge by modulating differently P-box-binding proteins.