To investigate the molecular mechanisms underlying the two- to three-fold induction of human transforming growth factor-alpha (hTGF-α) mNA and two- to five-fold induction of hTGF-α protein observed following estrogen treatment of hormone-responsive human breast cancer cell lines, the hTGF-α promoter was assayed for ERE-like sequences able to mediate estrogen induction of a heterologous gene. Transient co-transfection of a chloramphenicol acetyl transferase (CAT) construct consisting of either 1100 bp or 330 bp of hTGF-α promoter sequence and an estrogen receptor expression vector into either COS-7 cells or hormonally responsive MCF-7 human breast cancer cells resulted in a two- to five-fold induction of CAT activity by estrogen. Although no consensus estrogen response element (ERE) exists in the hTGF-α promoter, a sequence consisting of two imperfect ERE palindromes separated by 20 bp is located at -200 to -252. This sequence was inserted into a mouse mammary tumor virus (MMTV) based CAT construct and assayed for its ability to confer estrogen regulation of CAT expression to a heterologous promoter. Transient co-transfection of this construct with an estrogen receptor expression vector into either COS-7 cells or MCF-7 cells resulted in an average 30-fold estrogen induction of CAT activity. Gel shift assays with human recombinant estrogen receptor (ER) and 32P-labelled fragments revealed that the ER could specifically bind to this sequence. These results indicate that this 53 bp sequence can function as an ERE, and is likely to be responsible for the observed induction of TGF-α message and protein in response to estrogen. These data also indicate that the level of estrogen inducibility mediated by this element may be positively or negatively modulated by interaction or competition with other transcription factors.
|Original language||English (US)|
|Number of pages||9|
|Journal||Journal of Steroid Biochemistry and Molecular Biology|
|State||Published - Nov 1 1996|