Objective: The effects of estrogen on cells are mediated by the estrogen receptor α (ERα) which localizes at the peri-membrane, cytoplasm, and the nucleus of cells. Therefore, we intended to investigate how cytonuclear ERα plays its roles in different cellular activities.
Methods: We used amino acid substituted ERα that localized at the cytoplasm and nucleus but has no direct DNA-binding activities. ERα-negative endometrial carcinoma cells (ERα-) were stably transfected with plasmid of human ERα carrying a substituted phenylalanine at position 445 with alanine (ERα-F445A). Treated with 17β-estrogen (E2) or bazedoxifene (BDF), cell proliferation, migration, and expression of kinases related to ERα signal transduction pathways were observed.
Results: E2 (40 nM) significantly activated proliferation in ERα-F445A cells, but not in ERα- cells. Similarly, E2 significantly activated cell migration in ERα-F445A cells, rather than that in ERα- cells. While no obvious change in the amount of the non-phosphorylated mammalian target of Rapamycin (mTOR), the expression of mTOR phosphorylated at serine 2448 decreased, which was recovered in presence of 17β-estrogen (E2) in the ERα-F445A cells. On the other hand, the expression of focal adhesion kinase (FAK) phosphorylated at tyrosine at 297 was attenuated in the ERα-F445A cells treated with E2.
Conclusion: It is suggested that the cytonuclear ERα-F445A induces phosphorylation of kinases in downstream pathways, which regulate cell proliferation and migration.