John Kuo

Membrane Estrogen Receptors Stimulate Intracellular Calcium Release and Progesterone Synthesis in Hypothalamic Astrocytes

In hypothalamic astrocytes obtained from adult female rats, estradiol rapidly increased free cytoplasmic calcium concentrations ([Ca2+]i) that facilitate progesterone synthesis. The present study demonstrated that estradiol (1 nm) significantly and maximally stimulated progesterone synthesis within 5 min, supporting a rapid, nongenomic mechanism. The group I metabotropic glutamate receptor (mGluR1a) antagonist LY 367385 [(S)-(+)-a-amino-4-carboxy-2-methylbenzeneacetic acid] attenuated both the estradiol-induced [Ca2+]i release and progesterone synthesis. To investigate membrane-associated estrogen receptors (mERs), agonists for ERα, ERβ, STX-activated protein, and GPR30 were compared. The selective ERα agonist propylpyrazole triole (PPT) and STX most closely mimicked the estradiol-induced [Ca2+]i responses, where PPT was more potent but less efficacious than STX. Only high doses (100 nm) of selective ERβ agonist diarylpropionitrile (DPN) and GPR30 agonist G-1 induced estradiol-like [Ca2+]i responses. With the exception of DPN (even at 100 nm), all agonists stimulated progesterone synthesis. The PPT- and STX-induced [Ca2+]i release and progesterone synthesis were blocked by LY 367385. While the G-1-stimulated [Ca2+]i release was blocked by LY 367385, progesterone synthesis was not. Since GPR30 was detected intracellularly but not in the membrane, we interpreted these results to suggest that G-1 could activate mGluR1a on the membrane and GPR30 on the smooth endoplasmic reticulum to release intracellular calcium. Although STX and G-1 maximally stimulated [Ca2+]i release in astrocytes from estrogen receptor-α knock-out (ERKO) mice, estradiol in vivo did not stimulate progesterone synthesis in the ERKO mice. Together, these results indicate that mERα is mainly responsible for the rapid, membrane-initiated estradiol-signaling that leads to progesterone synthesis in hypothalamic astrocytes.

Estradiol-Induced Estrogen Receptor-α Trafficking

Estradiol has rapid actions in the CNS that are mediated by membrane estrogen receptors (ERs) and activate cell signaling pathways through interaction with metabotropic glutamate receptors (mGluRs). Membrane-initiated estradiol signaling increases the free cytoplasmic calcium concentration ([Ca2+]i) that stimulates the synthesis of neuroprogesterone in astrocytes. We used surface biotinylation to demonstrate that ERα has an extracellular portion. In addition to the full-length ERα [apparent molecular weight (MW), 66 kDa], surface biotinylation labeled an ERα-immunoreactive protein (MW, ∼52 kDa) identified by both COOH- and NH2-directed antibodies. Estradiol treatment regulated membrane levels of both proteins in parallel: within 5 min, estradiol significantly increased membrane levels of the 66 and 52 kDa ERα. Internalization, a measure of membrane receptor activation, was also increased by estradiol with a similar time course. Continuous treatment with estradiol for 24–48 h reduced ERα levels, suggesting receptor downregulation. Estradiol also increased mGluR1a trafficking and internalization, consistent with the proposed ERα–mGluR1a interaction. Blocking ER with ICI 182,780 or mGluR1a with LY 367385 prevented ERα trafficking to and from the membrane. Estradiol-induced [Ca2+]i flux was also significantly increased at the time of peak ERα activation/internalization. These results demonstrate that ERα is present in the membrane and has an extracellular portion. Furthermore, membrane levels and internalization of ERα are regulated by estradiol and mGluR1a ligands. The pattern of trafficking into and out of the membrane suggests that the changing concentration of estradiol during the estrous cycle regulates ERα to augment and then terminate membrane-initiated signaling.

Membrane Estrogen Receptor-α Interacts with Metabotropic Glutamate Receptor Type 1a to Mobilize Intracellular Calcium in Hypothalamic Astrocytes

Estradiol, acting on a membrane-associated estrogen receptor-alpha (mERalpha), induces an increase in free cytoplasmic calcium concentration ([Ca(2+)](i)) needed for progesterone synthesis in hypothalamic astrocytes. To determine whether rapid estradiol signaling involves an interaction of mERalpha with metabotropic glutamate receptor type 1a (mGluR1a), changes in [Ca(2+)](i) were monitored with the calcium indicator, Fluo-4 AM, in primary cultures of female postpubertal hypothalamic astrocytes. 17beta-Estradiol over a range of 1 nm to 100 nm induced a maximal increase in [Ca(2+)](i) flux measured as a change in relative fluorescence [DeltaF Ca(2+) = 615 +/- 36 to 641 +/- 47 relative fluorescent units (RFU)], whereas 0.1 nm of estradiol stimulated a moderate [Ca(2+)](i) increase (275 +/- 16 RFU). The rapid estradiol-induced [Ca(2+)](i) flux was blocked with 1 microm of the estrogen receptor antagonist ICI 182,780 (635 +/- 24 vs. 102 +/- 11 RFU, P < 0.001) and 20 nmof the mGluR1a antagonist LY 367385 (617 +/- 35 vs. 133 +/- 20 RFU, P < 0.001). Whereas the mGluR1a receptor agonist (RS)-3,5-dihydroxyphenyl-glycine (50 microm) also stimulated a robust [Ca(2+)](i) flux (626 +/- 23 RFU), combined treatment of estradiol (1 nm) plus (RS)-3,5-dihydroxyphenyl-glycine (50 microm) augmented the [Ca(2+)](i) response (762 +/- 17 RFU) compared with either compound alone (P < 0.001). Coimmunoprecipitation demonstrated a direct physical interaction between mERalpha and mGluR1a in the plasma membrane of hypothalamic astrocytes. These results indicate that mERalpha acts through mGluR1a, and mGluR1a activation facilitates the estradiol response, suggesting that neural activity can modify estradiol-induced membrane signaling in astrocytes.

Physiology of Membrane Oestrogen Receptor Signalling in Reproduction

The best characterised oestrogen receptors (ERs) that are responsible for membrane‐initiated oestradiol signalling are the classic ERs, ERα and ERβ. When in the nucleus, these proteins are oestradiol activated transcription factors but, when trafficked to the cell membrane, ERα and ERβ rapidly activate protein kinase pathways, alter membrane electrical properties, modulate ion flux and can mediate long‐term effects through gene expression. To initiate cell signalling, membrane ERs transactivate metabotropic glutamate receptors (mGluRs) to stimulate Gq signalling through pathways using PKC and calcium. In this review, we discuss the interaction of membrane ERα with metabotropic glutamate receptor 1a (mGluR1a) to initiate rapid oestradiol cell signalling and its critical roles in female reproduction: sexual behaviour and oestrogen positive feedback of the luteinising hormone (LH) surge. Although long considered to be regulated by the long‐term actions of oestradiol on gene transcription, recent results indicate that membrane oestradiol cell signalling is vital for a full display of sexual receptivity. Similarly, the source of pre‐ovulatory progesterone necessary for initiating the LH surge is hypothalamic astrocytes. Oestradiol rapidly amplifies progesterone synthesis through the release of intracellular calcium stores. The ERα‐mGluR1a interaction is necessary for critical calcium flux. These two examples provide support for the hypothesis that membrane ERs are not themselves G‐protein receptors; rather, they use mGluRs to signal.

Estrogen Actions on Neuroendocrine Glia

Astrocytes are the most abundant cells in the central nervous system (CNS). It appears that astrocytes are as diverse as neurons, having different phenotypes in various regions throughout the brain and participating in intercellular communication that involves signaling to neurons. It is not surprising then that astrocytes in the hypothalamus have an active role in the CNS regulation of reproduction. In addition to the traditional mechanism involving ensheathment of neurons and processes, astrocytes may have a critical role in regulating estrogen-positive feedback. Work in our laboratory has focused on the relationship between circulating estradiol and progesterone synthesized de novo in the brain. We have demonstrated that circulating estradiol stimulates the synthesis of progesterone in adult hypothalamic astrocytes, and this neuroprogesterone is critical for initiating the LH surge. Estradiol cell signaling is initiated at the cell membrane and involves the transactivation of metabotropic glutamate receptor type 1a (mGluR1a) leading to the release of intracellular stores of calcium. We used surface biotinylation to demonstrate that estrogen receptor-α (ERα) is present in the cell membrane and has an extracellular portion. Like other membrane receptors, ERα is inserted into the membrane and removed via internalization after agonist stimulation. This trafficking is directly regulated by estradiol, which rapidly and transiently increases the levels of membrane ERα, and upon activation, increases internalization that finally leads to ERα degradation. This autoregulation temporally limits membrane-initiated estradiol cell signaling. Thus, neuroprogesterone, the necessary signal for the LH surge, is released when circulating levels of estradiol peak on proestrus and activate progesterone receptors whose expression has been induced by the gradual rise of estradiol during follicular development.

Sex differences in hypothalamic astrocyte response to estradiol stimulation

BackgroundReproductive functions controlled by the hypothalamus are highly sexually differentiated. One of the most dramatic differences involves estrogen positive feedback, which leads to ovulation. A crucial feature of this positive feedback is the ability of estradiol to facilitate progesterone synthesis in female hypothalamic astrocytes. Conversely, estradiol fails to elevate hypothalamic progesterone levels in male rodents, which lack the estrogen positive feedback-induced luteinizing hormone (LH) surge. To determine whether hypothalamic astrocytes are sexually differentiated, we examined the cellular responses of female and male astrocytes to estradiol stimulation.MethodsPrimary adult hypothalamic astrocyte cultures were established from wild type rats and mice, estrogen receptor-α knockout (ERKO) mice, and four core genotype (FCG) mice, with the sex determining region of the Y chromosome (Sry) deleted and inserted into an autosome. Astrocytes were analyzed for Sry expression with reverse transcription PCR. Responses to estradiol stimulation were tested by measuring free cytoplasmic calcium concentration ([Ca2+]i) with fluo-4 AM, and progesterone synthesis with column chromatography and radioimmunoassay. Membrane estrogen receptor-α (mERα) levels were examined using surface biotinylation and western blotting.ResultsEstradiol stimulated both [Ca2+]i release and progesterone synthesis in hypothalamic astrocytes from adult female mice. Male astrocytes had a significantly elevated [Ca2+]i response but it was significantly lower than in females, and progesterone synthesis was not enhanced. Surface biotinylation demonstrated mERα in both female and male astrocytes, but only in female astrocytes did estradiol treatment increase insertion of the receptor into the membrane, a necessary step for maximal [Ca2+]i release. Regardless of the chromosomal sex, estradiol facilitated progesterone synthesis in astrocytes from mice with ovaries (XX and XY-), but not in mice with testes (XY-Sry and XXSry).ConclusionsAstrocytes are sexually differentiated, and in adulthood reflect the actions of sex steroids during development. The response of hypothalamic astrocytes to estradiol stimulation was determined by the presence or absence of ovaries, regardless of chromosomal sex. The trafficking of mERα in female, but not male, astrocytes further suggests that cell signaling mechanisms are sexually differentiated.

Estradiol Modulates Translocator Protein (TSPO) and Steroid Acute Regulatory Protein (StAR) via Protein Kinase A (PKA) Signaling in Hypothalamic Astrocytes

The ability of the central nervous system to synthesize steroid hormones has wide-ranging implications for physiology and pathology. Among the proposed roles of neurosteroids is the regulation of the LH surge. This involvement in the estrogen-positive feedback demonstrates the integration of peripheral steroids with neurosteroids. Within the female hypothalamus, estradiol from developing follicles stimulates progesterone synthesis in astrocytes, which activate neural circuits regulating gonadotropin (GnRH) neurons. Estradiol acts at membrane estrogen receptor-α to activate cellular signaling that results in the release of inositol trisphosphate-sensitive calcium stores that are sufficient to induce neuroprogesterone synthesis. The purpose of the present studies was to characterize the estradiol-induced signaling leading to activation of steroid acute regulatory protein (StAR) and transporter protein (TSPO), which mediate the rate-limiting step in steroidogenesis, ie, the transport of cholesterol into the mitochondrion. Treatment of primary cultures of adult female rat hypothalamic astrocytes with estradiol induced a cascade of phosphorylation that resulted in the activation of a calcium-dependent adenylyl cyclase, AC1, elevation of cAMP, and activation of both StAR and TSPO. Blocking protein kinase A activation with H-89 abrogated the estradiol-induced neuroprogesterone synthesis. Thus, together with previous results, these experiments completed the characterization of how estradiol action at the membrane leads to the augmentation of neuroprogesterone synthesis through increasing cAMP, activation of protein kinase A, and the phosphorylation of TSPO and StAR in hypothalamic astrocytes.

Membrane-initiated estradiol signaling in immortalized hypothalamic N-38 neurons

Regulation of sexual reproduction by estradiol involves the activation of estrogen receptors (ERs) in the hypothalamus. Of the two classical ERs involved in reproduction, ERα appears to be the critical isoform. The role of ERα in reproduction has been found to involve a nuclear ERα that induces a genomic mechanism of action. More recently, a plasma membrane ERα has been shown to trigger signaling pathways involved in reproduction. Mechanisms underlying membrane-initiated estradiol signaling are emerging, including evidence that activation of plasma membrane ERα involves receptor trafficking. The present study examined the insertion of ERα into the plasma membrane of N-38 neurons, an immortalized murine hypothalamic cell line. We identified, using western blotting and PCR that N-38 neurons express full-length 66kDa ERα and a 52kDa ERα spliced variant missing the fourth exon - ERαΔ4. Using surface biotinylation, we observed that treatment of N-38 neurons with estradiol or with a membrane impermeant estradiol elevated plasma membrane ERα protein levels, indicating that membrane signaling increased receptor insertion into the cell membrane. Insertion of ERα was blocked by the ER antagonist ICI 182,780 or with the protein kinase C (PKC) pathway inhibitor bisindolylmaleimide (BIS). Downstream membrane-initiated signaling was confirmed by estradiol activation of PKC-theta (PKCθ) and the release of intracellular calcium. These results indicate that membrane ERα levels in N-38 neurons are dynamically autoregulated by estradiol.

An interaction of oxytocin receptors with metabotropic glutamate receptors in hypothalamic astrocytes.

Hypothalamic astrocytes play a critical role in the regulation and support of many different neuroendocrine events, and are affected by oestradiol. Both nuclear and membrane oestrogen receptors (ERs) are expressed in astrocytes. Upon oestradiol activation, membrane‐associated ER signals through the type 1a metabotropic glutamate receptor (mGluR1a) to induce an increase of free cytoplasmic calcium concentration ([Ca2+]i). Because the expression of oxytocin receptors (OTRs) is modulated by oestradiol, we tested whether oestradiol also influences oxytocin signalling. Oxytocin at 1, 10, and 100 nm induced a [Ca2+]i flux measured as a change in relative fluorescence [ΔF Ca2+ = 330 ± 17 relative fluorescent units (RFU), ΔF Ca2+ = 331 ± 22 RFU, and ΔF Ca2+ = 347 ± 13 RFU, respectively] in primary cultures of female post‐pubertal hypothalamic astrocytes. Interestingly, OTRs interacted with mGluRs. The mGluR1a antagonist, LY 367385 (20 nm), blocked the oxytocin (1 nm)‐induced [Ca2+]i flux (ΔF Ca2+ = 344 ± 19 versus 127 ± 11 RFU, P < 0.001). Conversely, the mGluR1a receptor agonist, (RS)‐3,5‐dihydroxyphenyl‐glycine (100 nm), increased the oxytocin (1 nm)‐induced [Ca2+]i response (ΔF Ca2+ = 670 ± 31 RFU) compared to either compound alone (P < 0.001). Because both oxytocin and oestradiol rapidly signal through the mGluR1a, we treated hypothalamic astrocytes sequentially with oxytocin and oestradiol to determine whether stimulation with one hormone affected the subsequent [Ca2+]i response to the second hormone. Oestradiol treatment did not change the subsequent [Ca2+]i flux to oxytocin (P > 0.05) and previous oxytocin exposure did not affect the [Ca2+]i response to oestradiol (P > 0.05). Furthermore, simultaneous oestradiol and oxytocin stimulation failed to yield a synergistic [Ca2+]i response. These results suggest that the OTR signals through the mGluR1a to release Ca2+ from intracellular stores and rapid, nongenomic oestradiol stimulation does not influence OTR signalling in astrocytes.