Detection and localization of an estrogen receptor beta splice variant protein (ERβ2) in the adult female rat forebrain and midbrain regions

Abstract

Estrogens regulate neural processes such as neuronal development, reproductive behavior, and hormone secretion, and signal through estrogen receptor (ER) alpha and ERbeta (here called ERbeta1). Recent studies have found variations in ERalpha and ERbeta1 mRNA splicing in rodents and humans. Functional reporter gene assays suggest that these splicing variations alter ER-mediated transcriptional regulation. Estrogen receptor beta 2 (ERbeta2), an ERbeta1 splice variant containing an 18 amino acid (AA) insert in the ligand binding domain, binds estradiol with approximately 10-fold lower affinity than ERbeta1, suggesting that it may serve as a low-affinity ER. Moreover, ERbeta2 reportedly acts in a dominant-negative fashion when heterodimerized with ERbeta1 or ERalpha. To explore the function of ERbeta2 in brain, an antiserum (TwobetaER.1) targeting the 18 AA insert was developed and characterized. Western blot analysis and transient expression of ERbeta2 in cell lines demonstrated that TwobetaER.1 recognizes ERbeta2. In the adult female rat brain, ERbeta2 immunoreactivity is localized in the cell nucleus and is expressed with a distribution similar to that of ERbeta1 mRNA. ERbeta2 immunoreactive cell numbers were high in, for example, piriform cortex, paraventricular nucleus, supraoptic nucleus, arcuate nucleus, and hippocampal CA regions, whereas it was low in the dentate gyrus. Moreover, ERbeta2 is coexpressed in gonadotropin-releasing hormone and oxytocin neurons. These studies demonstrate ERbeta splice variant proteins in brain and support the hypothesis that ER signaling diversity depends not only on ligand or coregulatory proteins, but also on regional and phenotypic selectivity of ER splice variant proteins.

Spatiotemporal dynamics of the expression of estrogen receptors in the postnatal mouse brain

“…These are brain areas that are highly sensitive to circulating E2. If Handa et al 22 are correct, our result that ERb ins mRNA decreases just after birth, suggests that this splice variant of ERb may be playing some modulatory role during embryonic life. One such function could be protection of the fetus from circulating high-level E2 by being a dominant repressor of both ERa and ERb1.…”

“…Handa et al 22 have hypothesized that ERb ins may act as a dominant negative, low-affinity ER in the brain. Their hypothesis is based on their IHC observation of colocalization of ERb1 and ERb ins in the preoptic area, the bed nucleus of stria terminalis, and the Dynamics of estrogen receptors in the brain N Sugiyama et al amygdala of the adult rat brain.…”

“…[19][20][21] It has been proposed that this receptor isoform might act as a dominant negative regulator of estrogen signaling. 19,22 Interestingly, the affinity of ERb ins for the antiestrogen, Tamoxifen, is similar to that of ERb1. 23 The fetal brain is very well protected from circulating E2 (either coming from maternal circulation or from siblings developing in close proximity), 24 because of the presence of the high-affinity estrogenbinding protein a-fetoprotein (AFP), which circulates at high concentrations in the fetal blood.…”

Insights into Rapid Modulation of Neuroplasticity by Brain Estrogens

“…There is also increasing evidence that a subpopulation of ERs are found at extranuclear sites and specifically at synapses, a subcellular localization consistent with the ability of these receptors to couple to second messenger signaling pathways. Others have suggested that rapid actions of estrogens are mediated by splice variants of ERs (Toran-Allerand, 2004;Zhao et al, 2005;Chung et al, 2007;Ishunina and Swaab, 2008;Ishii et al, 2011;Kobayashi et al, 2011;Wu et al, 2012).…”

“…Another potential mechanism that has been proposed is the identification of splice variants of both ERa and ERb in neuronal tissue with extranuclear expression (Price et al, 2001;Ishii et al, 2011). Splice variants for both receptors can generate receptors lacking specific motifs, such as the nuclear localizing signal, or even the N or C termini (Price et al, 2001;Chung et al, 2007;Ishunina and Swaab, 2008;Ishii et al, 2011;Kobayashi et al, 2011). Therefore, it is plausible to suggest that these splice variants could function solely as a membrane ER, capable of coupling to second messenger signaling pathways.…”