Mutation of the palmitoylation site of estrogen receptor α in vivo reveals tissue-specific roles for membrane versus nuclear actions

Abstract

Estrogen receptor alpha (ERα) activation functions AF-1 and AF-2 classically mediate gene transcription in response to estradiol (E2). A fraction of ERα is targeted to plasma membrane and elicits membrane-initiated steroid signaling (MISS), but the physiological roles of MISS in vivo are poorly understood. We therefore generated a mouse with a point mutation of the palmitoylation site of ERα (C451A-ERα) to obtain membrane-specific loss of function of ERα. The abrogation of membrane localization of ERα in vivo was confirmed in primary hepatocytes, and it resulted in female infertility with abnormal ovaries lacking corpora lutea and increase in luteinizing hormone levels. In contrast, E2 action in the uterus was preserved in C451A-ERα mice and endometrial epithelial proliferation was similar to wild type. However, E2 vascular actions such as rapid dilatation, acceleration of endothelial repair, and endothelial NO synthase phosphorylation were abrogated in C451A-ERα mice. A complementary mutant mouse lacking the transactivation function AF-2 of ERα (ERα-AF2 0 ) provided selective loss of function of nuclear ERα actions. In ERα-AF2 0 , the acceleration of endothelial repair in response to estrogen-dendrimer conjugate, which is a membrane-selective ER ligand, was unaltered, demonstrating integrity of MISS actions. In genome-wide analysis of uterine gene expression, the vast majority of E2-dependent gene regulation was abrogated in ERα-AF2 0 , whereas in C451A-ERα it was nearly fully preserved, indicating that membrane-to-nuclear receptor cross-talk in vivo is modest in the uterus. Thus, this work genetically segregated membrane versus nuclear actions of a steroid hormone receptor and demonstrated their in vivo tissuespecific roles.fertility | vascular effects | nongenomic effects | genomic actions A lthough estrogens classically serve as reproductive hormones, they induce cellular responses in almost all tissues in mammalian species. The biological effects of estrogens, and particularly of 17β-estradiol (E2), are initiated by their binding to intracellular estrogen receptors (ERs), ERα and ERβ, which classically serve as nuclear transcription factors (1, 2). The ERs regulate the transcription of hundreds of genes in a cell-and tissue-specific manner through their two activation functions (AFs), AF-1 and AF-2. The roles of the activation functions of ERα have been studied in vivo using mice deleted for ERαAF-1 or ERαAF-2 (3-5). These results, in particular the two models of ERαAF-2 inactivation (4, 6), suggested that many physiological functions strongly rely on nuclear ERα and gene transcription regulation. However, in addition to the nuclear, termed "genomic actions of ER," the receptors stimulate rapid (from seconds to minutes), nonnuclear signal transduction, usually termed "nongenomic" or "extranuclear" effects. The rapid mobilization of intracellular calcium and the generation of cAMP by E2 were demonstrated several decades ago (7,8). More recently, the modulation of potassium currents, phospholipase C activatio...

Predominant Role of Nuclear Versus Membrane Estrogen Receptor α in Arterial Protection: Implications for Estrogen Receptor α Modulation in Cardiovascular Prevention/Safety

“…To this aim, on the basis of in vitro work demonstrating that the palmitoylation site at Cys447 of human ERα (the mouse counterpart of ERα Cys451) is crucial for the membrane localization of ERα,39 we generated a mouse model with a point mutation of this amino acid (ERα‐C451A) 22. The decrease of membrane localization of ERα was confirmed in primary hepatocytes and resulted in female infertility, an increase in luteinizing hormone levels, and hemorrhagic ovaries lacking corpora lutea 22. By contrast, the transcriptional action of 17β‐estradiol on the uterus was preserved: gene expression was similar in ERα‐C451A and WT mice 22.…”

“…The investigation conforms to the directive 2010/63/EU of the European parliament. To generate the double‐deficient mice, LDLr −/− female mice, purchased from Charles River (L'Arbresle, France), were crossed with ERα‐C451A +/− mice 22. ERα‐AF20 mice were generated, as previously described,32 and were kindly provided by Professor P. Chambon (Strasbourg, France).…”

“…Thus, the endothelium, a guardian of arterial integrity, represents a promising cellular target for 17β‐estradiol via the activation of ERα 18. Beside the well‐recognized role of nuclear ERα, which regulates target gene transcription (genomic action) through activation functions (AFs) 1 and 2,16, 20 a subpopulation of ERα is present at or near the plasma membrane, where it can elicit rapid, nongenomic, membrane‐initiated steroid signaling (MISS) effects 21, 22, 23, 24, 25…”

“…Recently, we generated a mouse model in which membrane ERα localization was abrogated by a point mutation of the palmitoylation site of ERα (mice with ER α mutated at cysteine 451 [ERα‐C451A]), leading to an abrogation of endothelial NO synthase (eNOS) phosphorylation and acceleration of reendothelialization in response to 17β‐estradiol 22. The estrogen‐dendrimer conjugate (EDC) and “pathway preferential estrogens” (PaPEs) are highly effective in stimulating nonnuclear signaling, but they are inefficient in stimulating nuclear ER target gene expression 23, 26.…”

“…More important, in vivo administration of EDC or PaPE‐1 accelerates repair of endothelial damage 23, 26. In addition, EDC and PaPE‐1 both favor the production of endothelial NO,22 a vasculoprotective agent of the arteries, initially viewed as an important mechanism of the protective action of 17β‐estradiol against the development of atheroma and hypertension 27. Our arsenal of pharmacological tools has recently been enriched with estetrol, a fetal estrogen produced exclusively by the liver of humans (and to a smaller extent in large apes).…”