ESTROGEN INDUCTION OF TGF-ALPHA IS MEDIATED BY AN ESTROGEN RESPONSE ELEMENT COMPOSED OF 2 IMPERFECT PALINDROMES

To investigate the molecular mechanisms underlying the two- to three-f old induction of human transforming growth factor-alpha (hTGF-alpha) m RNA and two- to five-fold induction of hTGF-alpha protein observed fol lowing estrogen treatment of hormone-responsive human breast cancer ce ll lines, the hTGF-alpha promoter was assayed for ERE-like sequences a ble 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-alpha 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 t wo- to five-fold induction of CAT activity by estrogen. Although no co nsensus estrogen response element (ERE) exists in the hTGF-alpha promo ter, 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 heter ologous promoter. Transient co-transfection of this construct with an estrogen receptor expression vector into either COS-7 cells or MCF-7 c ells resulted in an average 30-fold estrogen induction of CAT activity . Gel shift assays with human recombinant estrogen receptor (ER) and P -32-labelled fragments revealed that the ER could specifically bind to this sequence. These results indicate that this 53 bp sequence can fu nction as an ERE, and is likely to be responsible for the observed ind uction of TGF-alpha message and protein in response to estrogen. These data also indicate that the level of estrogen inducibility mediated b y this element may be positively or negatively modulated by interactio n or competition with other transcription factors. Copyright (C) 1996 Elsevier Science Ltd.