DIFFERENTIAL BINDING OF CAMP-RESPONSIVE-ELEMENT(CRE)-BINDING PROTEIN-1 AND ACTIVATING TRANSCRIPTION FACTOR-II TO A CRE-LIKE ELEMENT IN THE HUMAN TISSUE-TYPE PLASMINOGEN-ACTIVATOR (T-PA) GENE PROMOTER CORRELATES WITH OPPOSITE REGULATION OF T-PA BY PHORBOL ESTER IN HT-1080 AND HELA-CELLS

The human tissue-type plasminogen activator gene (t-PA) is induced by the phorbol ester, phorbol 12-myristate 13-acetate (PMA), in HeLa cell s. Previous studies in transfected HeLa cells identified two cis-actin g regulatory elements within the t-PA gene promoter responsible for bo th constitutive and PMA-inducible expression. One element differs from the consensus cAMP response element (CRE) by a single nucleotide subs titution (referred to in this report as t-PACRE) and another which bea rs similarity to the AP-2 recognition sequence. In HT-1080 fibrosarcom a cells, t-PA mRNA levels are expressed at higher constitutive levels and are suppressed by PMA. Nuclear run-on transcription experiments in dicate that PMA-mediated suppression of t-PA in these cells is associa ted with a decrease in t-PA gene template. activity. We designed exper iments to determine whether nuclear t-PACRE or AP-2-like binding prote ins were differentially expressed in HeLa and HT-1080 cells and, accor dingly, if these could be correlated with the opposite effect of PMA o n t-PA expression. Band shift analyses indicated that the migration pr ofiles of HeLa and HT-1080 nuclear proteins interacting with the AP-2- like site were indistinguishable; however, those produced with the t-P ACRE binding site were qualitatively and quantitatively distinct. The distribution of t-PACRE binding proteins in these cells was investigat ed in a supershift assay using specific antibodies against members of the fos/jun and CRE-binding protein (CREB)/activating transcription fa ctor (ATF) families. In HT-1080 cells, CREB-1 was the most prominent t -PACRE-binding activity detected and was greatly increased in cells tr eated with PMA. In contrast, CREB-1 activity was absent in HeLa cells, but antibodies specific for ATF-2 produced a marked supershifted comp lex which was unaffected by PMA treatment. Since CREB-1 can repress tr anscription of other target genes (including c-jun) via association wi th identical cis-acting CRE-like sequences, we suggest that the mechan ism for the transcriptional down-regulation of t-PA by PMA in HT-1080 cells requires CREB-1 binding to the t-PACRE while ATF-2, by associati ng with the same site, plays a role in PMA mediated induction of t-PA in HeLa cells.