Estrogen and its receptors influence growth and differentiation by stimulating the production and secretion of growth factors. Our previous studies indicate an increased expression of estrogen receptor (ER)-alpha and decreased growth factor synthesis in the olfactory bulb of reproductive senescent female rats as compared with young animals. The present study tests the hypothesis that abnormal overexpression of ERalpha contributes to decreased growth factor synthesis. We developed the HeLa-Tet-On cell line stably transfected with ERalpha (HTERalpha) that expresses increasing amounts of ERalpha with increasing doses of doxycycline (Dox). Increasing doses of Dox had no effect on vascular endothelial growth factor (VEGF) secretion in HTERalpha cells. However, in the presence of 40 nm 17beta-estradiol, VEGF secretion increased in low-dose Dox-exposed HTERalpha cultures, which was attenuated by the ERalpha antagonist, 1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]1H-pyrazole dihydrochloride. However, at high-dose Dox and, consequently, high ERalpha levels, estradiol failed to increase VEGF. In the HeLa X6 cell line in which the Tet-On construct is upstream of an unrelated gene (Pitx2A), estradiol failed to induce VEGF at any Dox dose. Furthermore, in the HTERalpha cell line, estradiol selectively down-regulates phospho-ERK2 and phospho-Akt at high ERalpha expression. This study clearly demonstrates that the dose of receptor critically mediates estradiol's ability to regulate growth factors and survival kinases. The present data also support the hypothesis that 17beta-estradiol treatment to an ERalpha overexpressing system, such as the senescent brain, could reverse the normally observed beneficial effect of estrogen.