New insights into the classical and non-classical actions of estrogen: evidence from estrogen receptor knock-out and knock-in mice

Abstract

Estrogen receptor alpha (ERalpha) mediates estrogen (E2) actions in the brain and is critical for normal reproductive function and behavior. In the classical pathway, ERalpha binds to estrogen response elements (EREs) to regulate gene transcription. ERalpha can also participate in several non-classical pathways, including ERE-independent gene transcription via protein-protein interactions with transcription factors and rapid, non-genotropic pathways. To distinguish between ERE-dependent and ERE-independent mechanisms of E2 action in vivo, we have created ERalpha null mice that possess an ER knock-in mutation (E207A/G208A; "AA"), in which the mutant ERalpha cannot bind to DNA but retains activity in ERE-independent pathways (ERalpha(-/AA) mice). Understanding the molecular mechanisms of ERalpha action will be helpful in developing pharmacological therapies that differentiate between ERE-dependent and ERE-independent processes. This review focuses on how the ERalpha(-/AA) model has contributed to our knowledge of ERalpha signaling mechanisms in estrogen regulation of the reproductive axis and sexual behavior.

Figure 1. ER Signaling

ERα signaling pathways include: (1) genotropic ERE-dependent (“classical”), in which liganded ER dimerizes on an ERE; (2) genotropic ERE-independent (“tethered”), in which liganded ER interacts with other transcription factors bound to their response elements; (3) membrane initiated, in which a membrane associated ER acts through kinases to phosphorylate other transcription factors and eNOS; (4) ligand independent, in which ER is activated by phosphorylation. Pathways 2–4 are collectively referred to as “non-classical”.

Figure 2. The non-classical ERα knock-in mutation

(A) Schematic illustration of the introduced double mutation (E at position 207 to A and G at position 208 to A) in the first zinc finger of the DNA-binding domain of mouse ERα. (B,C) Transfection studies were performed in ER-deficient TSA cells, using either ERE (B) or AP1 (C) reporters. Cells were transfected with WT ERα or the E207A/G208A mutant. Cells were treated with ethanol vehicle, 1 nM E₂, or 100 nM ICI 182,780, and luciferase activity was measured. The E207A/G208A mutant was incapable of activating transcription through EREs but retained the full capacity to activate transcription through AP1. (Jakacka et al., 2002)

Figure 3. Estrogen feedback in the female

ERE-independent ERα signaling is sufficient to convey estrogen negative but not positive feedback. Serum LH from intact, OVX, OVX/estrogen-replaced females killed in the morning for negative feedback and afternoon for positive feedback (n=5–16). Serum LH is significantly elevated in OVX ERα^+/+ females compared to intact ERα^+/+ females (a), and significantly elevated in OVX ERα^−/AA females compared to intact ERα^−/AA females (b). The post-OVX rise in LH was reduced by E₂ treatment (negative feedback) in ERα^+/+ (c) and ERα^−/AA females (d). Estrogen treatment (positive feedback) significantly increased serum LH in ERα^+/+ females (e), but not in ERα^−/− or ERα^−/AA females. (Glidewell-Kenney et al., 2007)

Figure 4. Proposed model for classical and non-classical ERα signaling in estrogen positive and negative feedback

Negative feedback actions of estrogen on GnRH/LH secretion are mediated in part by non-classical (ERE-independent) ERα signaling mechanisms. In contrast, positive feedback actions of estrogen on GnRH/LH surges are mediated by classical (ERE-dependent) ERα signaling mechanisms. Whether these classical and non-classical mechanisms occur in specific cell types that mediate estrogen feedback (e.g. kisspeptin neurons, POMC neurons) remain to be determined.

Figure 5. Non-classical ERα signaling mechanisms are not sufficient to mediate estrogen’s defeminizing effects on the sexually dimorphic AVPV

Immunohistochemical analysis revealed that brains from both ERα^−/− and ERα^−/AA males have significantly more TH-ir neurons in the medial AVPV than wild-type male counterparts. *p<0.05, compared to ERα^+/+ males.

Figure 6. Serum T is rescued by the non-classical knock-in mutation

Serum testosterone is significantly elevated in ERα^−/− males (p<0.05) but not in ERα^−/AA males (n=9–13). (McDevitt et al., 2007)