Membrane-initiated actions of estradiol that regulate reproduction, energy balance and body temperature

Abstract

It is well known that many of the actions of estrogens in the central nervous system are mediated via intracellular receptor/transcription factors that interact with steroid response elements on target genes. However, there now exists compelling evidence for membrane estrogen receptors in hypothalamic and other brain neurons. But, it is not well understood how estrogens signal via membrane receptors, and how these signals impact not only membrane excitability but also gene transcription in neurons. Indeed, it has been known for sometime that estrogens can rapidly alter neuronal activity within seconds, indicating that some cellular effects can occur via membrane delimited events. In addition, estrogens can affect second messenger systems including calcium mobilization and a plethora of kinases to alter cell signaling. Therefore, this review will consider our current knowledge of rapid membrane-initiated and intracellular signaling by estrogens in the hypothalamus, the nature of receptors involved and how they contribute to homeostatic functions.

Figure 1. Cellular actions of 17β-estradiol (E2) and leptin in POMC neurons

Schematic overview of the E2-mediated modulation of Gαi,o-coupled (μ-opioid and GABA_B) receptors via a membrane-associated receptor (mER) in hypothalamic POMC neurons. E2 binds to a mER that is Gαq –coupled to activate phospholipase C and catalyzes the hydrolysis of membrane-bound phosphatidylinositol 4,5-biphosphate (PIP₂) to inositol 1,4,5 triphosphate (IP₃) and diacylglycerol (DAG). Calcium is released from intracellular stores (endoplasmic reticulum) by IP₃, and DAG activates protein kinase C (PKC). Through phosphorylation, adenylyl cyclase (AC) activity is upregulated by PKC. The generation of cAMP activates PKA, which can uncouple μ-opioid (μ) and GABA_B receptors from their signaling pathway through phosphorylation of a downstream effector molecule (e.g., G protein-coupled, inwardly rectifying K⁺, GIRK, channels). PKA can also potentially phosphorylate other channels (e.g., TRPC channels) to alter their function (not shown). In addition, the membrane-initiated E2 signaling through PKA can phosphorylate cAMP-responsive element binding protein (pCREB), which can alter gene transcription through its interaction with the cAMP responsive element (CRE). Therefore, the rapid membrane-initiated signaling can alter gene expression in an estrogen-response element-independent fashion. Leptin binds to its receptor (LRb) to activate Jak2, which phosphorylates IRS proteins and in turn activates PI3 kinase (PI3-K). PI3 kinase subsequently activates PLCγ1 to augment TRPC channel activity. All of these signaling events initiated by E2 and leptin enhance POMC neuronal excitability.

Figure 2. 17β-Estradiol and STX attenuate the body weight gain in female guinea pigs after ovariectomy

Female guinea pigs were ovariectomized and after a one-week recovery period were given bi-daily subcutaneous injections of vehicle, 17β-estradiol benzoate (EB), or STX starting on day 0. A two-way ANOVA (repeated measures) revealed an overall significant effect of both EB and STX (p<0.001), and post hoc Newman-Keuls analysis revealed daily significant differences between EB and vehicle-treated, and STX and oil-treated groups (** p< 0.01). (Reproduced from Roepke et al. Endocrinology 149: 6113–24, 2008, with permission from the Endocrine Society)

Figure 3. Genes regulated by E2 and STX in guinea pig arcuate nucleus

Quantitative real-time PCR confirmed regulation of multiple genes by STX in the arcuate nucleus [172]. The relative mRNA expression for selected control genes (A, B) and selected genes of interest (C, D) from microdissected arcuate tissue from vehicle-treated (PPG (n-5) or oil (n=7)) females and STX- (n=7) or EB-treated (n=6) females. The expression values were calculated using the CT method where the calibrator was the average CT of the vehicle-treated samples. Bar graphs represent the mean ± SEM. Statistics: two-tailed Student’s t-test (p<0.05*; p<0.01**; p<0.001*** compared to vehicle). AKAP11, A-kinase anchoring protein 11; CaM-1, calmodulin 1; CaMKIIα, calcium/calmodulin-dependent kinase II αsubunit; Ca_v3.1, T-type calcium channel 3.1 subunit; ERα, estrogen receptor α; ERβ, estrogen receptor β; GEC-1, GABA-A receptor associated protein-like 1; GLR, Glycine Receptor β; KCNQ5, M-current potassium channel subunit 5; NPY, neuropeptide Y; PI3K p85, phosphatidylinositol 3-kinase, p85α subunit; PKAα₁, protein kinase A α₁ subunit; PITP, phosphatidylinositol transfer protein β; POMC, pro-opiomelanocortin; SUR1, sulfonylurea receptor 1; TH, tyrosine hydroxylase. (Reproduced from Roepke et al. Endocrinology 149: 6113–24, 2008, with permission from the Endocrine Society)

Figure 4. E2 and STX reduce core body temperature (Tc) of ovariectomized female guinea pigs

Similar to 17β-estradiol benzoate (EB)-treated females (8 g/kg), the mER selective ligand STX (6 mg/kg) significantly decreased the Tc compared to the propylene glycol vehicle (PPG). Females were given daily injections of drug at 1000. The data are presented as the mean ± SEM for each hour of the day averaged over three weeks of temperature probe recordings. The data were analyzed using a two-way ANOVA (p < 0.05, F = 4.787, df = 2) with post-hoc Newman-Keuls Multiple Comparison Test. All data points for both STX and EB were significantly different from vehicle (p<0.01) except for the STX 1400–1600 hr time points (p<0.05). The solid bars above the x-axis represent lights off or nighttime. (Reproduced from Roepke et al. Endocrinology 151: 4926–37, 2010, with permission from the Endocrine Society)

Figure 5. E2 and STX increase cancellous bone density of the proximal tibia in ovariectomized female guinea pigs

Ovariectomized females treated with EB (20 µg/kg) or STX (12 mg/kg) for 52 days had significantly higher bone density in the proximal tibia compared to the vehicle-treated females. On day 52 the animals were killed, the tibia was harvested and the trabecular bone region was scanned using quantitative computer tomography [165]. A one-way ANOVA (p<0.05, F = 3.616, df = 2) followed by a post-hoc Newman-Keuls Multiple Comparison Test (* = p<0.05) was used to compare significance between each treatment. (Reproduced from Roepke et al. Endocrinology 151: 4926–37, 2010, with permission from the Endocrine Society)