Differential binding of creb-1 and atf-2 to a cre-like element in the t-pa gene promoter correlates with the opposite regulation of t-Pa in ht-1080 and hela cells

M. Costa, R. L. Medcalf

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The human tissue-type plasminogcn activator gene (t-PA) is induced by the tumour promoter phorbol 13-myristate 12-acetate (PMA) in HeLa cells. Previous studies in transfected HeLa cells identified two cisacting regulatory elements within the t-PA gene promoter responsible for constitutive and PMA-inducible expression. One element differs from the consensus cAMP response element (CRE) by a single nucleotide substitution (referred to as t-PACRE) and another which bears homology 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 HT1080 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 CREB/ATF families. In HT-1080 cells, CREB-1 was the most prominent tPACRE-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 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.

Original languageEnglish
Number of pages1
Issue numberSUPPL. 3
Publication statusPublished - 1 Dec 1996

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