Differential Binding of cAMP‐responsive‐element(CRE)‐binding Protein‐1 and Activating Transcription Factor‐2 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 cells. Previous studies in transfected HeLa cells identified two cis ‐acting regulatory elements within the t‐PA gene promoter responsible for both constitutive and PMA‐inducible expression. One element differs from the consensus cAMP response element (CRE) by a single nucleotide substitution (referred to in this report as t‐PACRE) and another which bears similarity to the AP‐2 recognition sequence. In HT‐1080 fibrosarcoma cells, t‐PA mRNA levels are expressed at higher constitutive levels and are suppressed by PMA. Nuclear run‐on transcription experiments indicate that PMA‐mediated suppression of t‐PA in these cells is associated with a decrease in t‐PA gene template activity. We designed experiments to determine whether nuclear t‐PACRE or AP‐2‐like binding proteins were differentially expressed in HeLa and HT‐1080 cells and, accordingly, if these could be correlated with the opposite effect of PMA on t‐PA expression. Band shift analyses indicated that the migration profiles of HeLa and HT‐1080 nuclear proteins interacting with the AP‐2‐like site were indistinguishable; however, those produced with the t‐PACRE binding site were qualitatively and quantitatively distinct. The distribution of t‐PACRE binding proteins in these cells was investigated in a supershift assay using specific antibodies against members of the fos/jun and CRE‐binding protein (CREB)/activating transcription factor (ATF) families. In HT‐1080 cells, CREB‐1 was the most prominent t‐PACRE‐binding activity detected and was greatly increased in cells treated with PMA. In contrast, CREB‐1 activity was absent in HeLa cells, but antibodies specific for ATF‐2 produced a marked supershifted complex which was unaffected by PMA treatment. Since CREB‐1 can repress transcription of other target genes (including c‐jun ) via association with identical cis ‐acting CRE‐like sequences, we suggest that the mechanism 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 associating with the same site, plays a role in PMA‐mediated induction of t‐PA in HeLa cells.