Fig. 1 Effect of l-T₄ and E₂ on MAPK activation and serine-118 phosphorylation of ERα in MCF-7 cells. A, MCF-7 cells were treated with 10⁻⁷m T₄ (10⁻⁷m, total concentration; 10⁻¹⁰m, free concentration) for 15 min to 24 h. Nuclear proteins were separated by SDS-PAGE and immunoblotted with anti-phospho-MAPK (pERK1/2) or antiserine-118-phosphorylated ERα (pSer-118-ERα) antibody. Early activation of MAPK induced by T₄, shown by nuclear accumulation of phosphorylated ERK1/2, was observed in 15 min, and maximal activation occurred in 2 h, as shown in the upper blot. The appearance of serine-118-phosphorylated ERα is seen at 15 min (middle blot), and was maximal by 2 h. An actin immunoblot demonstrates comparable sample loading. B, Similar studies were carried out with T₄-agarose (T₄-A, 10⁻⁷m), and results are comparable to those with T₄. C, MCF-7 cells were treated with 10⁻¹⁰m E₂ for 15 min to 24 h. Nuclear accumulation of activated MAPK was seen in 15 min and persisted for 24 h. Serine-118 phosphorylation of ERα was apparent by 15 min and reached a peak at 4–6 h. I.O.D., Integrated OD; pMAPK, phospho-MAPK. From: Thyroid Hormone Causes Mitogen-Activated Protein Kinase-Dependent Phosphorylation of the Nuclear Estrogen Receptor Endocrinology. 2004;145(7):3265-3272. doi:10.1210/en.2004-0308 Endocrinology | Copyright © 2004 by The Endocrine Society

Fig. 2 Effect of PD on T₄- and E₂-induced MAPK activation and serine-118 phosphorylation of ERα in MCF-7 cells. A, Cells were treated with T₄ (10⁻⁷m, 15 min) or E₂ (10⁻¹⁰m, 15 min) in the presence or absence of PD (30 μm, cells pretreated for 90 min and treatment continued for 15 min during hormone exposure). T₄ and E₂ induced activation (phosphorylation) of MAPK (pERK1/2) and serine-118 phosphorylation of ERα; these effects were inhibited by PD. B, Nuclear fractions were either: 1) immunoprecipitated (IP) with monoclonal anti-ERα and the precipitated proteins separated by PAGE and immunoblotted with antiphosphoserine (upper blot); or 2) immunoprecipitated with antiphosphoserine and resulting proteins immunoblotted with anti-ERα (lower blot). The blots show that T₄ and E₂ both caused serine phosphorylation of ERα, and that PD inhibited the effect of both hormones. From: Thyroid Hormone Causes Mitogen-Activated Protein Kinase-Dependent Phosphorylation of the Nuclear Estrogen Receptor Endocrinology. 2004;145(7):3265-3272. doi:10.1210/en.2004-0308 Endocrinology | Copyright © 2004 by The Endocrine Society

Fig. 3 Association of phosphorylated ERα with nuclear-activated MAPK (ERK2) in MCF-7 cells treated with T₄ or E₂, and effect of PD on this association. Cells were treated with T₄, (10⁻⁷m) or E₂ (10⁻¹⁰m) for 15 min, in the presence or absence of PD (30 μm, 75 min pretreatment). Nuclear fractions were immunoprecipitated with antibody to phospho-MAPK (pMAPK), and resulting proteins separated by PAGE and immunoblotted with antibody to pSer118-ERα. The nuclear accumulation of pMAPK indicated its activation. Both T₄ and E₂ caused formation of an immunoprecipitable complex of nuclear ERK2 and serine-118-phosphorylated ERα (lanes 3 and 5, respectively). PD inhibited this complex formation, whether induced by T₄ or E₂. From: Thyroid Hormone Causes Mitogen-Activated Protein Kinase-Dependent Phosphorylation of the Nuclear Estrogen Receptor Endocrinology. 2004;145(7):3265-3272. doi:10.1210/en.2004-0308 Endocrinology | Copyright © 2004 by The Endocrine Society

Fig. 4 Effect of PD and tetrac on T₄- and T₄-A-induced MAPK activation and serine-118 phosphorylation of ERα. A, MCF-7 cells were treated with T₄-A (10⁻⁷m, 15 min) or T₄ (10⁻⁷m, 15 min). PD (30 μm) was added to selected cell samples for 90 min pretreatment and continued during hormone treatment. Both T₄ and T₄-A caused activation of MAPK and serine-118 phosphorylation of ERα. These effects were inhibited by PD. As a control, protein A-agarose, containing no T₄, had no effect on MAPK activation or serine phosphorylation (not shown). B, MCF-7 cells were pretreated with tetrac (10⁻⁷m, 90 min); T₄ was added for 15 min (10⁻⁷m), with or without tetrac pretreatment. Although tetrac alone had no effect on serine-118 phosphorylation of ERα, this analog did inhibit the effect of T₄ on ERα phosphorylation. From: Thyroid Hormone Causes Mitogen-Activated Protein Kinase-Dependent Phosphorylation of the Nuclear Estrogen Receptor Endocrinology. 2004;145(7):3265-3272. doi:10.1210/en.2004-0308 Endocrinology | Copyright © 2004 by The Endocrine Society

Fig. 5 EMSA of nuclear extracts of MCF-7 cells treated with T₄ (10⁻⁷m) or E₂ (10⁻¹⁰m). Radiolabeled ERE oligonucleotide was added to extracts before electrophoresis. Specific protein-DNA (oligonucleotide)-binding is shown at band b with both T₄ (lane 2) and E₂ (lane 4). MAPK-dependence of the interaction is shown by reduction in band b-binding with PD treatment of cells that is concurrent with hormone exposure (lanes 3 and 5 for T₄ and E₂, respectively). Specificity of oligonucleotide-binding in band b is shown by addition of excess unlabeled ERE oligonucleotide in E₂-treated sample (lane 7). Addition of excess unlabeled SP-1 oligonucleotide in the E₂-treated sample caused no displacement of labeled oligonucleotide from band b (lane 6). a, protein band that demonstrates nonspecific protein-DNA interaction; b, band exhibiting specific protein-oligonucleotide interaction; F, free radiolabeled oligonucleotide. From: Thyroid Hormone Causes Mitogen-Activated Protein Kinase-Dependent Phosphorylation of the Nuclear Estrogen Receptor Endocrinology. 2004;145(7):3265-3272. doi:10.1210/en.2004-0308 Endocrinology | Copyright © 2004 by The Endocrine Society

Fig. 6 [³H]Thymidine uptake by MCF-7 cells treated with T₄ or E₂. Cells were incubated for the times indicated with T₄ (10⁻⁷m, total concentration; 10⁻¹⁰m, free concentration) or E₂ (10⁻¹⁰m). Significant increases in thymidine incorporation were noted at 6 and 24 h with both hormones (P < 0.05). The actions of both T₄ and E₂ at 24 h were markedly decreased by concurrent treatment of cells with ICI. From: Thyroid Hormone Causes Mitogen-Activated Protein Kinase-Dependent Phosphorylation of the Nuclear Estrogen Receptor Endocrinology. 2004;145(7):3265-3272. doi:10.1210/en.2004-0308 Endocrinology | Copyright © 2004 by The Endocrine Society

Fig. 7 Cell proliferation is induced in MCF-7 cells by both T₄ and E₂. A, Cells were treated with T₄ (10⁻⁷m, 24 h) or E₂ (10⁻¹⁰m, 24 h) in the presence or absence of PD (30 μm). T₄ (lane 3) and E₂ (lane 5) induced cell proliferation, and this effect was blocked by PD (lanes 4 and 6). B, The effects of both T₄ (lane 5) and T₄-A (lane 3) on cell proliferation were blocked by cotreatment of cells with PD (lanes 6 and 4, respectively). C, ICI, a high affinity ER antagonist, inhibited the proliferative effects of both T₄ and E₂ on MCF-7 cells (lanes 4 and 6 compared with lanes 3 and 5, respectively). Together, T₄ and E₂ appeared to have an additive effect on cell proliferation (lane 7), which was also blocked by ICI (lane 8). From: Thyroid Hormone Causes Mitogen-Activated Protein Kinase-Dependent Phosphorylation of the Nuclear Estrogen Receptor Endocrinology. 2004;145(7):3265-3272. doi:10.1210/en.2004-0308 Endocrinology | Copyright © 2004 by The Endocrine Society