FRA-1 EXPRESSION LEVEL MODULATES REGULATION OF ACTIVATOR PROTEIN-1 ACTIVITY BY ESTRADIOL IN BREAST-CANCER CELLS

We compared the effect of estradiol on activator protein-1 (AP-1) acti vity in estrogen receptor positive (ER alpha+) and estrogen receptor n egative (ER alpha-) human breast cancer cell lines transiently transfe cted with the AP-l-responsive reporter plasmid AP-1-TK-CAT and an ER a lpha expression vector. While estradiol increased AP-I activity in the ER alpha+ cell lines MCF7, ZR75.1, and T47D, it decreased (MDA-MB231 and BT20 cells) or had no significant effect (MDA-MB435 cells) on AP-l -mediated transcription in ER alpha- cells. Estradiol also inhibited A P-1 activity in ER alpha-MDA-MB231 cells stably transfected with ERa a nd in which ERa levels are close to those found in MCF7. Use of ER alp ha mutant expression vectors demonstrated that the DNA-binding domain of ER alpha was needed for stimulation or inhibition of AP-1 activity by estradiol but suggested that ER alpha binding to estrogen-responsiv e elements was not required for these effects. Changes in regulation p aralleled quantitative and qualitative changes in protein binding to A P-1 sites, as demonstrated by gel shift assay: protein binding was gre ater and DNA/protein complexes migrated faster for ER alpha- than for ER alpha+ cells. In fact, by Northern blot, a high level of Fra-1 mRNA was found in BT20 and MDA-MB231 cells as compared with ER alpha+ cell s, and MDA-MB435 cells showed an intermediary level of expression. The differential expression of Fra-1 in MCF7 and MDA-MB231 cells was conf irmed at the protein level by supershift experiments. In addition, ove rexpression of Fra-1 in MCF7 cells decreased the positive effect of es tradiol while inhibition of Fra-1 expression in MDA-MB231 cells, by tr ansient transfection of the Fra-1 antisense expression vector, abolish ed the negative effect of the hormone. In conclusion, we demonstrated that ER alpha- breast cancer cell lines differ from ER+ cells by a hig h level of AP-1 DNA-binding activity doe, at least in part, to high Fr a-1 constitutive expression. High Fra-1 concentration is crucial for t he negative regulation of AP-1 activity by estradiol and thus may take part in estradiol-induced inhibition of cell proliferation in ER alph a- breast cancer cells transfected with ER alpha expression construct.