Membrane-Localized Estrogen Receptor α Is Required for Normal Organ Development and Function

SummarySteroid receptors are found in discrete cellular locations, but it is unknown whether extranuclear pools are necessary for normal organ development. To assess this, we developed a point mutant estrogen receptor α (ERα) knockin mouse (C451A) that precludes palmitoylation and membrane trafficking of the steroid receptor in all organs. Homozygous knockin female mice (nuclear-only ERα [NOER]) show loss of rapid signaling that occurs from membrane ERα in wild-type mice. Multiple developmental abnormalities were found, including infertility, relatively hypoplastic uteri, abnormal ovaries, stunted mammary gland ductal development, and abnormal pituitary hormone regulation in NOER mice. These abnormalities were rescued in heterozygous NOER mice that were comparable to wild-type mice. mRNAs implicated in organ development were often poorly stimulated by estrogen only in homozygous NOER mice. We conclude that many organs require membrane ERα and resulting signal transduction to collaborate with nuclear ERα for normal development and function

Estrogen receptor-beta prevents cardiac fibrosis.

Development of cardiac fibrosis portends the transition and deterioration from hypertrophy to dilation and heart failure. Here we examined how estrogen blocks this important development. Angiotensin II (AngII) and endothelin-1 induce cardiac hypertrophy and fibrosis in humans. and we find that these agents directly stimulate the transition of the cardiac fibroblast to a myofibroblast. AngII and endothelin-1 stimulated TGFβ1 synthesis in the fibroblast, an inducer of fibrosis that signaled via c-jun kinase to Sma- and Mad-related protein 3 phosphorylation and nuclear translocation in myofibroblasts. As a result, mesenchymal proteins fibronectin and vimentin were produced, as were collagens I and III, the major forms found in fibrotic hearts. 17β-Estradiol (E2) or dipropylnitrile, an estrogen receptor (ER)β agonist, comparably blocked all these events, reversed by estrogen receptor (ER)β small interfering RNA. E2 and dipropylnitrile signaling through cAMP and protein kinase A prevented myofibroblast formation and blocked activation of c-jun kinase and important events of fibrosis. In the hearts of ovariectomized female mice, cardiac hypertrophy and fibrosis were induced by AngII infusion and prevented by E2 administration to wild type but not ERβ knockout rodents. Our results establish the cardiac fibroblast as an important target for hypertrophic/fibrosis-inducing peptides the actions of which were mitigated by E2/ERβ acting in these stromal cells

Heat Shock Protein 27 Is Required for Sex Steroid Receptor Trafficking to and Functioning at the Plasma Membrane ▿

Classical sex steroid receptors (SRs) localize at the plasma membranes (PMs) of cells, initiating signal transduction through kinase cascades that contribute to steroid hormone action. Palmitoylation of the SRs is required for membrane localization and function, but the proteins that facilitate this modification and subsequent receptor trafficking are unknown. Initially using a proteomic approach, we identified that heat shock protein 27 (Hsp27) binds to a motif in estrogen receptor alpha (ERα) and promotes palmitoylation of the SR. Hsp27-induced acylation occurred on the ERα monomer and augmented caveolin-1 interactions with ERα, resulting in membrane localization, kinase activation, and DNA synthesis in breast cancer cells. Oligomerization of Hsp27 was required, and similar results were found for the trafficking of endogenous progesterone and androgen receptors to the PMs of breast and prostate cancer cells, respectively. Small interfering RNA (siRNA) knockdown of Hsp27 prevented sex SR trafficking to and signaling from the membrane. These results identify a conserved and novel function for Hsp27 with potential as a target for interrupting signaling from membrane sex SRs to tumor biology in hormone-responsive cancers

Membrane and nuclear estrogen receptor α collaborate to suppress adipogenesis but not triglyceride content.

Estrogen and estrogen receptor (ER)-α suppress visceral fat development through actions in several organs via unclear mechanisms that we sought to identify. Using mice that express only nuclear ER-α [nuclear-only ER-α (NOER) mice] or plasma membrane ER-α [membrane-only ER-α (MOER) mice], we found that 10-wk-old mice that lacked either receptor pool showed extensive abdominal visceral fat deposition and weight gain compared with wild-type (WT) mice. Differentiation of cultured bone marrow stem cells (BMSCs) into the adipocyte lineage was suppressed by 17-β-estradiol (E2) in WT female mice but not in NOER or MOER mice. This finding correlated with E2 inhibition of prominent differentiation genes in WT BMSCs. In contrast, triglyceride content in differentiated BMSCs or 3T3-L1 cells was suppressed as a result of membrane ER-α signaling through several kinases to inhibit carbohydrate response element-binding protein-α and -β. We concluded that extranuclear and nuclear ER-α collaborate to suppress adipocyte development, but inhibition of lipid synthesis in mature cells does not involve nuclear ER-α

Functional Estrogen Receptors in the Mitochondria of Breast Cancer Cells

Steroid hormones have been reported to indirectly impact mitochondrial functions, attributed to nuclear receptor-induced production of proteins that localize in this cytoplasmic organelle. Here we show high-affinity estrogen receptors in the mitochondria of MCF-7 breast cancer cells and endothelial cells, compatible with classical estrogen receptors ERα and ERβ. We report that in MCF-7, estrogen inhibits UV radiation-induced cytochrome C release, the decrease of the mitochondrial membrane potential, and apoptotic cell death. UV stimulated the formation of mitochondrial reactive oxygen species (mROS), and mROS were essential to inducing mitochondrial events of cell death. mROS mediated the UV activation of c-jun N-terminal kinase (JNK), and protein kinase C (PKC) δ, underlying the subsequent translocation of Bax to the mitochondria where oligomerization was promoted. E2 (estradiol) inhibited all these events, directly acting in mitochondria to inhibit mROS by rapidly up-regulating manganese superoxide dismutase activity. We implicate novel functions of ER in the mitochondria of breast cancer that lead to the survival of the tumor cells

Identification of a Structural Determinant Necessary for the Localization and Function of Estrogen Receptor α at the Plasma Membrane

Estrogen receptors (ER) have been localized to the cell plasma membrane (PM), where signal transduction mediates some estradiol (E2) actions. However, the precise structural features of ER that result in membrane localization have not been determined. We obtained a partial tryptic peptide/mass spectrometry analysis of membrane mouse ERα protein. Based on this, we substituted alanine for the determined serine at amino acid 522 within the E domain of wild-type (wt) ERα. Upon transfection in CHO cells, the S522A mutant ERα resulted in a 62% decrease in membrane receptor number and reduced colocalization with caveolin 1 relative to those with expression of wt ERα. E2 was significantly less effective in stimulating multiple rapid signals from the membranes of CHO cells expressing ERα S522A than from those of CHO cells expressing wt ERα. In contrast, nuclear receptor expression and transcriptional function were very similar. The S522A mutant was also 60% less effective than wt ERα in binding caveolin 1, which facilitates ER transport to the PM. All functions of ERα mutants with other S-to-A substitutions were comparable to those of wt ER, and deletion of the A/B or C domain had little consequence for membrane localization or function. Transfection of ERα S522A into breast cancer cells that express native ER downregulated E2 binding at the membrane, signaling to ERK, and G(1)/S cell cycle events and progression. However, there was no effect on the E2 transactivation of an ERE-luciferase reporter. In summary, serine 522 is necessary for the efficient translocation and function of ERα at the PM. The S522A mutant also serves as a dominant-negative construct, identifying important functions of E2 that originate from activating PM ER

Membrane and nuclear estrogen receptor a collaborate to suppress adipogenesis but not triglyceride content

Estrogen and estrogen receptor (ER)-α suppress visceral fat development through actions in several organs via unclear mechanisms that we sought to identify. Using mice that express only nuclear ER-α [nuclear-only ER-α (NOER) mice] or plasma membrane ER-α [membrane-only ER-α (MOER) mice], we found that 10-wk-old mice that lacked either receptor pool showed extensive abdominal visceral fat deposition and weight gain compared with wild-type (WT) mice. Differentiation of cultured bone marrow stem cells (BMSCs) into the adipocyte lineage was suppressed by 17-β-estradiol (E2) in WT female mice but not in NOER or MOER mice. This finding correlated with E2 inhibition of prominent differentiation genes in WT BMSCs. In contrast, triglyceride content in differentiated BMSCs or 3T3-L1 cells was suppressed as a result of membrane ER-α signaling through several kinases to inhibit carbohydrate response element-binding protein-α and -β. We concluded that extranuclear and nuclear ER-α collaborate to suppress adipocyte development, but inhibition of lipid synthesis in mature cells does not involve nuclear ER-α