Identification and characterization of a cAMP-responsive element in the region upstream from promoter 1.3 of the human aromatase gene

Aromatase converts androgens to estrogens. The expression of this enzyme is driven by multiple tissue-specific promoters which are differentially regu lated. Aromatase expression in breast cancer and the surrounding adipose ce lls is directed mainly by promoters I.3 and II, while its expression in the normal breast adipose tissue is driven by promoter I.4. Like promoter II, promoters I.3 is thought to be a cAMP-driven promoter, demonstrated previou sly by cell culture experiments. In the present study, we have identified a nd characterized a cAMP-responsive element (CREaro) upstream from promoter 1.3. This positive element, TGAAGTCA, between -66 and -59 bp relative to th e transcriptional start site of promoter 1.3 was identified by DNA deletion and mutation analyses. The sequence of CREaro is one base different from t he consensus CRE sequence (CREpal; TGACGTCA), and the mutational analysis r evealed that CREaro had a higher enhancer activity to promoter 1.3 than CRE pal. Nuclear proteins from both WS3TF breast tumor fibroblasts and SK-BR-3 breast cancer cells bound to this CREaro, as demonstrated by DNA mobility s hift assay. The molecular weight of the major binding protein in fibroblast s was determined to be approximately 60 kDa, as shown by UV crosslinking, w hich is different from those of known CRE-binding proteins. It is thought t hat CREB1 is not expressed in tumor fibroblasts because the Western blot an alysis using anti-CREB1 antibody was not able to detect CREB1 in the nuclea r protein extract from these cells. DNA mobility shift analysis using a nuc lear protein extract from SK-BR-3 cells revealed that at least two proteins bound to the CREaro and that one of these proteins was identified to be CR EB1. These studies provide direct evidence that promoter I.3 is a cAMP-resp onsive promoter, (C) 1999 Academic Press.