During the past decade there has been a substantial advance in our understanding of estrogen signaling both from a clinical as well as a preclinical perspective. Estrogen signaling is a balance bet...

Estrogen Receptors: How Do They Signal and What Are Their Targets

I. Introduction II. Mechanisms of Estrogen Action A. “Genomic” and “nongenomic” mechanisms B. Steroid hormone actions on gene expression C. Subtypes of estrogen receptors D. Steroid hormone actions on putative receptors on membranes E. Rapid actions of steroids on neuronal excitability F. Steroid hormone actions via second messengers G. Neuroprotective effects of estrogens H. Summary III. Areas of the Brain Affected Outside of the Hypothalamus A. Studies of hypothalamic and extrahypothalamic actions of estrogens B. Estrogens and the cholinergic system C. Estrogens and the serotonergic system D. Catecholaminergic neurons E. Spinal cord F. Hippocampus G. Glial cells, endothelial cells, and the blood-brain barrier H. Summary IV. Effects of Estrogens on Learning and Memory V. Estrogens, Neuroprotection, and Alzheimer’s Disease VI. Conclusions

Estrogen actions in the central nervous system.

Estrogen receptors (ERs) are localized to many sites within the cell, potentially contributing to overall estrogen action. In the nucleus, estrogen mainly modulates gene transcription, and the resulting protein products determine the cell biological actions of the sex steroid. In addition, a small pool of ERs localize to the plasma membrane and signal mainly though coupling, directly or indirectly, to G proteins. In response to steroid, signal transduction modulates both nontranscriptional and transcriptional events and impacts both the rapid and more prolonged actions of estrogen. Cross-talk from membrane-localized ERs to nuclear ERs can be mediated through growth factor receptor tyrosine kinases, such as epidermal growth factor receptor and IGF-I receptor. Growth factor receptors enact signal transduction to kinases such as ERK and phosphatidylinositol 3-kinase that phosphorylate and activate nuclear ERs, and this can also occur in the absence of sex steroid. A complex relationship between the membrane and nuclear effects of estrogen also involves membrane-initiated phosphorylation of coactivators, recruiting these proteins to the nuclear transcriptosome. Finally, large pools of cytoplasmic ERs exist, and some are localized to mitochondria. The integration of sex steroid effects at distinct cellular locations of its receptor leads to important cellular physiological outcomes and are manifest in both reproductive and nonreproductive organs.

/pdf/integration-of-the-extranuclear-and-nuclear-actions-of-1jsyitbsya.pdf

Integration of the Extranuclear and Nuclear Actions of Estrogen

The corpus luteum (CL) is one of the few endocrine glands that forms from the remains of another organ and whose function and survival are limited in scope and time. The CL is the site of rapid remodeling, growth, differentiation, and death of cells originating from granulosa, theca, capillaries, and fibroblasts. The apparent raison d'etre of the CL is the production of progesterone, and all the structural and functional features of this gland are geared toward this end. Because of its unique importance for successful pregnancies, the mammals have evolved a complex series of checks and balances that maintains progesterone at appropriate levels throughout gestation. The formation, maintenance, regression, and steroidogenesis of the CL are among the most significant and closely regulated events in mammalian reproduction. During pregnancy, the fate of the CL depends on the interplay of ovarian, pituitary, and placental regulators. At the end of its life span, the CL undergoes a process of regression leading to its disappearance from the ovary and allowing the initiation of a new cycle. The generation of transgenic, knockout and knockin mice and the development of innovative technologies have revealed a novel role of several molecules in the reprogramming of granulosa cells into luteal cells and in the hormonal and molecular control of the function and demise of the CL. The current review highlights our knowledge on these key molecular events in rodents.

The Molecular Control of Corpus Luteum Formation, Function, and Regression

Mice lacking estrogen receptors α and β were generated to clarify the roles of each receptor in the physiology of estrogen target tissues. Both sexes of αβ estrogen receptor knockout (αβERKO) mutants exhibit normal reproductive tract development but are infertile. Ovaries of adult αβERKO females exhibit follicle transdifferentiation to structures resembling seminiferous tubules of the testis, including Sertoli-like cells and expression of Mullerian inhibiting substance, sulfated glycoprotein-2, and Sox9. Therefore, loss of both receptors leads to an ovarian phenotype that is distinct from that of the individual ERKO mutants, which indicates that both receptors are required for the maintenance of germ and somatic cells in the postnatal ovary.

/pdf/postnatal-sex-reversal-of-the-ovaries-in-mice-lacking-23olt1xakt.pdf

Postnatal Sex Reversal of the Ovaries in Mice Lacking Estrogen Receptors α and β

The steroid 17beta-estradiol (E2) acts to modulate transcription through classical nuclear estrogen receptors (ER-alpha and ER-beta). However, E2 also induces a number of rapid responses (<10 min) within cells, including cells devoid of classical ERs, consistent with the presence of a membrane receptor for E2. Membrane impermeable steroids, typically bovine serum albumin (BSA) conjugates, are commonly used to characterize these non-genomic actions of E2 to exclude the involvement of nuclear ERs. Here we report that E2-BSA conjugate preparations, but not unconjugated E2, activate extracellular signal-regulated protein kinases (ERK1 and ERK2) in the SK-N-SH neuroblastoma cell line, raising concerns regarding the use of these reagents as E2 mimics. Freshly prepared solutions of E2-BSA were found to contain free immunoassayable E2 (iE2), which could be removed by filtration. E2-BSA solutions devoid of free iE2 failed to compete for binding of 125I16alpha-iodo-E2 to ER-alpha or ER-beta. Furthermore, in contrast to E2, E2-BSA conjugates did not bind to ER-alpha or ER-beta as assessed by electrophoretic mobility shift analyses. Protein analysis demonstrated that certain E2-BSA preparations were of very high molecular weight, suggesting extreme protein cross-linking. These findings suggest that E2-BSA does not mimic E2 and is not an appropriate ligand for investigating estrogen receptors. This underscores the need to design stable, cell impermeable analogs of estrogen for the characterization of membrane estrogen receptors.

Differential effects of estradiol and estradiol-BSA conjugates.

Estrogen is an essential hormone for the LH surge and ovulation. The primary source of estrogen is from ovarian granulosa cells and in rats, estrogen, in turn, increases granulosa cell number and enhances FSH-stimulated gene expression in these cells. Thus, rat granulosa cells both respond to and synthesize estrogen. To further elucidate the mechanisms mediating the actions of estrogen in granulosa cells, we have identified and characterized the estrogen receptor-β (ER-β) subtype in rodent granulosa cells. ER-β protein was localized to the nuclei of rat granulosa cells in preantral and antral follicles by immunocytochemistry, coincident with the location of ER-β messenger RNA (mRNA). Immunoprecipitation and Western blot analysis using ER-β specific antisera demonstrated a protein of approximately 60 kDa in granulosa cells prepared from PMSG-primed immature mice and estrogen-treated immature rats. Extracts from granulosa cells specifically bound an estrogen response element and the complex was recognized b...

/pdf/expression-of-estrogen-receptor-beta-protein-in-rodent-ovary-4ksa7w7y0l.pdf

Expression of estrogen receptor-beta protein in rodent ovary.

Under physiological conditions, FSH is secreted into the circulation as a complex mixture of several isoforms that vary in the degree of glycosylation. Although it is well established that the glycosylation of FSH is important for the serum half-life of the hormone and coupling of the receptor to adenylate cyclase, little is known concerning how physiologically occurring glycosylation patterns of this hormone affect receptor signaling. In this study, we have examined the biological activity of deglycosylated human FSH (DeGly-phFSH), recombinant mammalian-expressed hFSH (CHO-hFSH), and insect cell-expressed hFSH (BV-hFSH, alternatively glycosylated) as compared with that of purified human pituitary FSH (phFSH) using a Chinese hamster ovarian cell line stably expressing the hFSH receptor (3D2 cells). Differentially glycosylated forms of FSH did not bind to the FSH receptor in the same manner as phFSH. Although all hormones showed similar potency in competing for [125I]phFSH binding to the hFSH receptor, competition curves for deglycosylated and insect cell-produced FSH were steeper. Similarly, glycosylation of FSH had a profound effect on bioactivity of the hormone. Purified hFSH produced a sigmoidal dose-dependent stimulation in cAMP production, whereas DeGly-phFSH and BV-hFSH induced biphasic (bell-shaped) dose-response curves. BV-hFSH also elicited biphasic effects on steroidogenesis in primary cultures of rat granulosa cells. The cellular response to BV-hFSH was dependent on the degree of receptor-transducer activation. BV-hFSH bioactivity was strictly inhibitory when combined with the ED80 of phFSH. Lower concentrations of phFSH resulted in a gradual shift from inhibition to a biphasic activity in the presence of the ED20 of phFSH. Biphasic responses to BV-hFSH were attributed to activation of different G protein subtypes, since treatment of 3D2 cells with cholera toxin or pertussis toxin differentially blocked the two phases of BV-hFSH bioactivity. These data suggest that alternative glycosylation of FSH leads to a functionally altered form of the hormone. Functionally different hormones appear to convey distinct signals that are transduced by the receptor-transduction system as either stimulatory or inhibitory intracellular events via promiscuous, glycosylation-dependent G protein coupling. Promiscuity in signaling of the FSH receptor, in turn, may represent a potentially novel mechanism for FSH action, whereby the gonad may respond in diverse ways to complex hormonal signals such as those presented by circulating FSH isoforms.

/pdf/induction-of-promiscuous-g-protein-coupling-of-the-follicle-47pak180y1.pdf

Induction of promiscuous G protein coupling of the follicle-stimulating hormone (FSH) receptor: a novel mechanism for transducing pleiotropic actions of FSH isoforms.

The activity of estradiol on the LHRH neuronal network is crucial in the regulation of reproduction. In vivo, estradiol induces galanin (GAL) gene expression in LHRH neurons and GAL/LHRH colocalization is sexually dimorphic and neonatally determined by steroid exposure. The effects of estradiol on LHRH neurons, however, are considered to be indirect because estrogen receptors (ER) have not been detected in LHRH neurons in vivo. Using immortalized mouse LHRH neurons (GT1-7 cells), we demonstrated by RT-PCR and Southern blotting that GT1-7 cells express ER messenger RNA (mRNA). Sequencing of the amplification products indicated that GT1-7 ER is of the alpha-subtype (ER alpha). Additionally, estrogen receptors in GT1-7 cells were characterized by competitive radioligand receptor binding and IC50 values for 17beta-estradiol and ICI-182,780 were found to be 0.24 and 4.1 nM, respectively. The ability of endogenous GT1-7 cell ER to regulate transcription was determined in transient transfection studies using a construct that consisted of a luciferase reporter gene that is driven by tandem estrogen response elements (ERE) and a minimal herpes simplex virus thymidine kinase promoter. 17Beta-estradiol was found to enhance luciferase activity by 2.5-fold at physiological concentrations with an ED50 value of 47 pM. This induction was completely inhibited by ICI-182,780 which had an IC50 value of 4.8 nM. Raloxifene, tamoxifen, 4-hydroxytamoxifen, and droloxifene also fully blocked estrogen-mediated luciferase induction with IC50 values of 58.4, 89.2, 33.2, and 49.8 nM, respectively. In addition, GAL mRNA was detected and identified by RT-PCR followed by Southern blotting using a rat GAL complementary DNA (cDNA) probe. The ability of 17beta-estradiol to modulate expression of the endogenous GAL gene in immortalized LHRH neurons was also determined. Quantitative RT-PCR demonstrated that physiological concentrations of estrogen increase GAL gene expression by 2-fold with an ED50 value of 23 pM. ICI-182,780, raloxifene, and droloxifene completely blocked this induction. In summary, our data demonstrate the presence of ER alpha and GAL mRNA in GT1-7 cells. The ER in GT1-7 cells is biologically active because 17beta-estradiol enhances both endogenous GAL gene expression and an ERE-driven reporter gene. These results suggest that estrogenic control of GAL gene expression in immortalized LHRH neurons may be transduced by ER. Thus, hypothalamic-derived LHRH neurons appear to have the capacity to be directly regulated by estrogen.

/pdf/expression-of-functional-estrogen-receptors-and-galanin-4l1wi2tld5.pdf

Expression of Functional Estrogen Receptors and Galanin Messenger Ribonucleic Acid in Immortalized Luteinizing Hormone-Releasing Hormone Neurons: Estrogenic Control of Galanin Gene Expression

The glycoprotein hormones (LH, FSH, and TSH) are critical to the maintenance of physiological homeostasis and control of reproduction. However, despite an obvious utility for synthetic pharmacological agents, there are few reports of selective, nonpeptide agonists or antagonists to receptors for these hormones. We have identified and characterized a novel synthetic molecule capable of inhibiting the action of FSH. This compound, 7-{4-[Bis-(2-carbamoyl-ethyl)-amino]-6-chloro(1,3,5)-triazin-2-ylamino)-4-hydroxy-3-(4-methoxy-phenylazo)naphthalene}-2-sulfonic acid, sodium salt (compound 1), is a selective, noncompetitive inhibitor of the human (h) and rat (r) FSH receptors (FSHRs). Compound 1 selectively inhibited binding of [ 125 I]hFSH with an IC50 value of 5.4 2.3 M. Radioligand-binding assays were performed using the baculovirus expressed extracellular domain of hFSHR (BV-tFSHR) to demonstrate site-specific interaction. Compound 1 competed for [ 125 I]hFSH binding to BV-tFSHR with an IC50 value of 10 2.8 M. Functionally, compound 1 inhibited hFSH-induced cAMP accumulation and steroidogenesis in vitro with an IC50 value of 3 0.6 M. Competition of compound 1 for binding to other glycoprotein hormone receptors and other G protein-coupled receptors demonstrated select activity for FHSRs. Compound 1 inhibited ovulation in immature and cycling adult rats. These data provide proof of concept that selective, small molecule antagonists can be designed for glycoprotein hormone receptors. (Endocrinology 143: 3822–3829, 2002)

Darlene C. Deecher

Papers

Differential effects of estradiol and estradiol-BSA conjugates.

Expression of estrogen receptor-beta protein in rodent ovary.

Induction of promiscuous G protein coupling of the follicle-stimulating hormone (FSH) receptor: a novel mechanism for transducing pleiotropic actions of FSH isoforms.

Expression of Functional Estrogen Receptors and Galanin Messenger Ribonucleic Acid in Immortalized Luteinizing Hormone-Releasing Hormone Neurons: Estrogenic Control of Galanin Gene Expression

Identification and characterization of a selective, nonpeptide follicle-stimulating hormone receptor antagonist