Estradiol (E) and progesterone are ovarian steroid hormones that are important for normal development and function of the mammary gland and uterus. Epidemiologic and experimental data support that one or both of these steroids contribute to the formation and growth of breast cancers. Previous studies made by several laboratories have indicated that the proliferative action of ovarian steroids is mediated by the epidermal growth factor (EGF) receptor pathway. We have found that the transcripts of two known EGF-like ligands, transforming growth factor-alpha (TGF- alpha) and amphiregulin, are stimulated in the mammary glands of ovariectomized nice treated with E. In order to determine the promoter elements of the TGF-alpha gene that confer E stimulation, selected deletions were made of 1.1 kb of the 5'-flanking region of the human TGF- alpha gene linked to a chloramphenicol acetyl transferase (CAT) reporter gene. Constructs were cotransfected with a human estrogen receptor (ER) plasmid into HeLa cells and exposed to E (10-8M). So far, our findings reveal that deletion of two imperfect (putative) E response elements (EREs) failed to prevent E-induced CAT activity. In addition, a 52 mer oligonucleotide containing the putative EREs failed to demonstrate ER binding by gel mobility shift analysis when reacted with an extract of nuclei from mouse uteri or yeast expressing the human ER. Binding was evident when the 52 mer oligo was reacted with purified c-jun; binding did not occur with SP-1. It is unlikely, then, that the hormonal stimulation of the TGF-alpha gene is conferred by these regions of the promoter that have high homology with known EREs. Further mutation deletions of the hTGF-alpha 5' flanking region have been made and are currently being evaluated. These data support that hormonal regulation of TGF-alpha gene expression by the E-ER complex is made through on alternate mechanism probably not requiring direct interaction of the ER with the TGF-alpha promoter. In a related study, we investigated the tyrosine (Tyr) phosphorylation of EGFR in the mouse uterus and mammary gland in vivo as a determinant of ovarian steroid stimulation of EGFR ligands. Increased phosphorylation of EGFR (180kDa) was apparent by 6 hours following treatment of castrated female mice with E Phosphoration of EGFR serine or threonine was not observed. The cumulative findings support the notion that the proliferative action of sex steroids is conferred locally by stimulation of the levels of EGF-like ligands that activate the EGFR through an autocrine/paracrine mechanism.
