REGULATION BY ESTROGEN THROUGH THE 5'-FLANKING REGION OF THE TRANSFORMING GROWTH-FACTOR ALPHA GENE

Expression of transforming growth factor-alpha (TGF-alpha) mRNA and protein can be stimulated by estrogens such as 17-beta-estradiol (E2) in estrogen-responsive rodent and human breast cancer cells. To ascertain if E2 can directly regulate TGF-alpha expression through the 5'-flanking region of the human TGF-alpha gene, E2-responsive MCF-7 or ZR-75-1 human breast cancer cells or E2-nonresponsive MDA-MB-231 breast cancer cells were transiently transfected with a plasmid containing an 1140-base pair (bp) Sac-I fragment of the TGF-alpha 5'-flanking region ligated to the chloramphenicol acetyltransferase (CAT) gene. Cells that were transfected and subsequently treated with physiological concentrations of E2 (10(-11)-10(-8) M) for 24 h exhibited a 2- to 10-fold increase in CAT activity. The E2 stimulation of CAT activity was dose-dependent with an increase first found at 10(-10) M E2. The increase in CAT activity could be detected within 24-36 h after the addition of E2. There was no significant change in CAT activity in transiently transfected MDA-MB-231 cells as mediated through the TGF-alpha 5'-flanking region after E2 treatment. MCF-7 cells were also transiently transfected with different fragments of the TGF-alpha 5'-flanking region ligated to the luciferase gene. In the absence of E2 treatment, no detectable luciferase activity was found. E2 was able to stimulate, in a dose-dependent manner, a 30- to 300-fold increase in luciferase activity in MCF-7 cells, which have been transfected with either a 2813-bp Pst-I, a 1565-bp Spe-I, a 1140-bp Sac-I, or a 370-bp Bam-HI TGF-alpha-luciferase fragment. However, a loss in E2 responsiveness occurred when MCF-7 cells were transfected with a 77-bp Sac-II TGF-alpha-luciferase plasmid. The induction of luciferase activity by E2 could be effectively blocked by simultaneous treatment of the cells with the antiestrogens tamoxifen or droloxifene. In addition, the increase in luciferase activity was specific for E2 since progesterone or dexamethasone were ineffective in modifying luciferase activity through the TGF-alpha 5'-flanking sequence. The results show that the TGF-alpha 5'-flanking region contains a potential estrogen-responsive element(s) and that this region is upstream of the Sac-II restriction site.