Endocrine disruption through membrane estrogen receptors and novel pathways leading to rapid toxicological and epigenetic effects

Abstract

Estrogen binding to estrogen receptors (ESR) triggers signaling cascades within cells. Historically, a major emphasis has been characterizing estrogen-induced genomic actions resulting from binding to nuclear estrogen receptor 1 (nESR1). However, recent evidence indicates the first receptors estrogens encounter as they enter a cell, membrane ESR1 (mESR1), also play crucial roles. Membrane and nuclear ESR are derived from the same transcripts but the former are directed to the membrane via palmitoylation. Binding and activation of mESR1 leads to rapid fluctuations in cAMP and Ca⁺² and stimulation of protein kinase pathways. Endocrine disrupting chemicals (EDC) that mimic 17β-estradiol can signal through mESR1 and elicit non-genomic effects. Most current EDC studies have focused on genomic actions via nESR1. However, increasing number of studies have begun to examine potential EDC effects mediated through mESR1, and some EDC might have higher potency for signaling through mESR1 than nESR1. The notion that such chemicals might also affect mESR1 signaling via palmitoylation and depalmitoylation pathways has also begun to gain currency. Recent development of transgenic mice that lack either mESR1 or nESR1, while retaining functional ESR1 in the other compartment, will allow more precise in vivo approaches to determine EDC effects through nESR1 and/or mESR1. It is increasingly becoming apparent in this quickly evolving field that EDC directly affect mESR and estrogen signaling, but such chemicals can also affect proportion of ESR reaching the membrane. Future EDC studies should be designed to consider the full range of effects through mESR alone and in combination with nESR.

Keywords: Calcium signaling; ERK; MAPK; NOER; Non-genomic actions; Xenoestrogen.

Fig. 1.. Pathways for the palmitoylation or depalmitoylation of ESR1.

Palmitoylation of estrogen receptor 1 (ESR1) is stimulated by palmitoyl-acyltransferase(s) (DHHC-7 and -21), and this results in palmitoylated ESR1 becoming associated with the plasma membrane; this reaction can be inhibited with 2-bromohexadecanoic acid (2-Br). Conversely, acyl protein thioesterase(s) (APT-1 and -2) induce depalmitoylation of ESR1, and this reaction can be inhibited by with palmostatin.

Fig. 2.. Estrogen receptor 1 signaling in wild-type mice and in various transgenic mouse models used to study actions of ESR1.

A) In wild-type (WT) mice, genomic estrogen receptor 1 (ESR1,ER) signaling occurs when estrogens bind to cytoplasmic/nuclear receptors, dimerize and then bind to estrogen response element (ERE) within target gene promoter regions and generally induce gene transcription that ultimately leads to translation and increase expression of specific proteins. In the second (non-genomic) pathway for estrogen receptor 1 responses, estrogens bind to membrane estrogen receptor (mESR1,ER), where they can stimulate several intracellular protein kinase cascades and/or G-protein coupled receptors (GPCR), which can result in enhanced cAMP response element-binding protein (CREB)-induced protein transcription, as well as activation of the phosphoinositol 3-kinase/ protein kinase B (PI3K/AKT) and mitogen-activated protein kinase (MAPK) pathways. In addition, 17β-estradiol (E2) or other estrogens could also bind and signal through membrane G protein-coupled estrogen receptor (GPER), which also results in increases in cAMP as well as increased release of epidermal growth factor (EGF) family ligands. In panel A, as well as all the other panels, nuclear and membrane ESR2, as well as GPER, would also be present, but these are not shown in the interest of simplicity. B) Nuclear-only ESR1 (NOER) mice lack membrane ESR1 due to a point mutation in the palmitolyation site (C451A, equivalent site in hESR1 would be Cys447) on this molecule, but the nESR1 pathway remains functional. C) Membrane-only ESR1 (MOER) mice lack both the nESR1 and mESR1 pathways, but a transgene consisting of the E-domain of human ESR1 (hER-E) and another protein fragment that directs all of the protein encoded by this transgene to the membrane has been knocked into these mice, and this mouse has the E-domain of human ESR1 expressed in the plasma membrane. D) Transgenic mice that retain non-genomic ESR1 signaling but lack genomic ESR1 signaling due to mutation in the hinge region of ESR1 (H2NES) possess mutations in the estrogen receptor 1 D-domain, which prevents retention of estrogen-bound ESR1 in the nucleus and thus blocks the classic nuclear pathway involving ligand-bound ESR1 associating with ERE in target genes. Signaling through mESR1 should be unaffected in this transgenic mouse model.

Fig. 3.. Mechanisms by which endocrine disrupting chemicals (EDC) can disrupt membrane estrogen receptor signaling.

A) In this model, EDC bind with equal affinity to estrogen receptor 1 (ESR1, ER) located in the plasma membrane and cytoplasm/nucleus, and can also act through the G protein-coupled estrogen receptor (GPER) pathway. The EDC can also bind and signal through all of these receptors and disrupt estrogen signaling in this manner. B) Conversely, EDC may preferentially bind and activate membrane ESR1 (mESR1) relative to those located in the cytoplasm/nucleus, resulting in the EDC have more potent non-genomic effects in terms of disrupting mESR1 rather than nuclear ESR1 (nESR1) signaling. C) By inhibiting the DHHC-7 and -21 palmitoylacyltransferases, some EDC might reduce palmitoylation and shuttling of ESRs to the plasma membrane, which would result in decreased mESR1 signaling compared to nESR1 signaling. EDCs might also disrupt membrane ESR1 action by reducing expression of caveolin 1 3, which are essential for mESR1 action. D) EDC can potentially increase the transcription and translation of the depalmitoylating enzymes, acyl protein thioesterase(s) (APT1 and APT2), leading to depalmitoylation of membrane ESR1, decreased caveolin binding, and reduced signaling through the mESR1 pathway.

Fig. 4.. Membrane estrogen receptor activation can have effects on enhancer of zeste homolog 2 (EZH2) and epigenetic imprinting.

Left panel: Binding of E2, other estrogens or EDC can activate the phosphoinositol 3-kinase/ protein kinase B (PI3K/AKT) pathway, resulting in phosphorylation EZH2, which inactivates it and blocks it from methylating histone 3 (H3) proteins at certain sites (e.g., H3K27me3), resulting in a more open chromatin (euchromatin) arrangement that favors gene transcription. Right panel: In contrast, lack of mESR1 activation results in decreased phosphorylation of EZH2, which causes more extensive methylation of histones by EZH2 and a more compacted chromatin (heterochromatin) that is less transcriptionally active.