Thyroid hormone causes mitogen-activated protein kinase-dependent phosphorylation of the nuclear estrogen receptor

Activated by thyroid hormone, the MAPK (ERK1/2) signaling pathway causes serine phosphorylation by MAPK of several nucleoproteins, including the nuclear thyroid hormone receptor beta1. Because estrogen can activate MAPK and cause MAPK-dependent serine phosphorylation of nuclear estrogen receptor (ER)alpha, we studied whether thyroid hormone also promoted MAPK-mediated ERalpha phosphorylation. Human breast cancer (MCF-7) cells were incubated with physiological concentrations of L-T-4 or 17beta-estradiol (E-2) for 15 min to 24 h, and nuclear ERalpha and serine-118-phosphorylated ERalpha were identified by Western blotting. Serine-118-phosphorylated ERalpha was recovered at 15 min in nuclei of MCF-7 cells exposed to either T-4 or E-2. The T-4 effect was apparent at 15 min and peaked at 2 h, whereas the E-2 effect was maximal at 4-6 h. T-4-agarose was as effective as T-4 in causing phosphorylation of ERalpha. T-4 action on ERalpha was inhibited by PD 98059, an inhibitor of ERK1/2 phosphorylation, and by tetraiodothyroacetic acid, a T-4 analog that blocks cell surface-initiated actions of T-4 but is not itself an agonist. Electrophoretic mobility shift assay of nuclear extracts from T-4-treated and E-2-treated cells showed similar specific protein-DNA-binding. Indexed by [H-3]thymidine incorporation and nuclear proliferating cell nuclear antigen, MCF-7 cell proliferation was stimulated byT(4) and T-4-agarose to an extent comparable with the effect of E-2. This T-4 effect was blocked by either PD 98059 or ICI 182,780, an ER antagonist. Thus, T-4, like E-2, causes phosphorylation by MAPK of nuclear ERalpha at serine-118 in MCF-7 cells and promotes cell proliferation through the ER by a MAPK-dependent pathway.