Richard B. Hochberg

International Union of Pharmacology. LXV. The pharmacology and classification of the nuclear receptor superfamily: glucocorticoid, mineralocorticoid, progesterone, and androgen receptors.

The glucocorticoid receptor (GR[1][1]), mineralocorticoid receptor (MR), progesterone receptor (PR), and androgen receptor (AR) are classic members of the nuclear receptor superfamily, composing subfamily 3C. Members of this subfamily are among those receptors that were cloned the earliest, with the

NFkappaB selectivity of estrogen receptor ligands revealed by comparative crystallographic analyses

Our understanding of how steroid hormones regulate physiological functions has been significantly advanced by structural biology approaches. However, progress has been hampered by misfolding of the ligand binding domains in heterologous expression systems and by conformational flexibility that interferes with crystallization. Here, we show that protein folding problems that are common to steroid hormone receptors are circumvented by mutations that stabilize well-characterized conformations of the receptor. We use this approach to present the structure of an apo steroid receptor that reveals a ligand-accessible channel allowing soaking of preformed crystals. Furthermore, crystallization of different pharmacological classes of compounds allowed us to define the structural basis of NFkappaB-selective signaling through the estrogen receptor, thus revealing a unique conformation of the receptor that allows selective suppression of inflammatory gene expression. The ability to crystallize many receptor-ligand complexes with distinct pharmacophores allows one to define structural features of signaling specificity that would not be apparent in a single structure.

Cellular variations in estrogen receptor mRNA translation in the developing brain: evidence from combined [125I]estrogen autoradiography and non-isotopic in situ hybridization histochemistry.

The spatial distribution of cells in the adult rodent forebrain which express estrogen receptor mRNA, as shown by in situ hybridization histochemistry with isotopically-labeled probes, has been reported to overlap with regions that are known targets of estrogen and which bind estrogen. The extent to which detection of estrogen receptor mRNA within developing forebrain neurons of the postnatal day 10-12 female rat is accompanied by translation into estrogen binding sites was investigated by combining [125I]estrogen autoradiography with non-isotopic (digoxigenin) in situ hybridization, using a 48-base oligodeoxyribonucleotide probe encoding a sequence of the estrogen-binding domain of rat uterine estrogen receptor cDNA. Estrogen receptor mRNA and estrogen binding sites appeared to be restricted to neurons. No mRNA or binding was seen in ependymal cells. Cells expressing estrogen receptor mRNA were widely distributed in the developing rat forebrain and were found in brain regions generally corresponding to those previously shown in the adult, with the addition of some regions not previously described, such as the medial habenula and dorsal endopiriform nucleus. Although there was widespread overlapping of estrogen receptor mRNA expression with known estrogen binding sites, there were regional and cellular variations in the extent of receptor mRNA translation. This pattern was true for developing forebrain regions previously defined as estrogen receptor-containing (hypothalamus, preoptic area, medial and lateral septum, vertical and horizontal nuclei of the diagonal band, cerebral cortex, hippocampus and amygdala) as well as for regions heretofore not considered estrogen targets (the thalamus, dorsal endopiriform nucleus, claustrum, ventral pallidum/substantia innominata and the basal nucleus of Meynert) or characterized as estrogen-responsive in the adult without previously documented estrogen binding [caudate-putamen (striatum)]. While estrogen binding and receptor mRNA expression always co-localized, neurons expressing estrogen receptor mRNA did not always exhibit ligand binding and there was no clear-cut relationship between the intensity of the hybridization signal and estrogen binding. Little, however, is known about translational control of estrogen receptor expression in the brain. Localization of estrogen binding sites to regions not generally considered targets of estrogen would appear to reflect the greater sensitivity of the iodinated ligand than the tritiated estrogens more commonly used for autoradiography. Non-isotopic in situ hybridization histochemistry combined with [125I]estrogen autoradiography represents a very powerful tool with which to study regulation of estrogen receptor gene expression at the single cell level with an exceptional degree of cellular and anatomical resolution.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of Estrogen Receptor Concentrations in the Rat Brain: Effects of Sustained Androgen and Estrogen Exposure

To determine whether estrogen and androgens either alone or in combination downregulate estrogen receptors in the brain, ovariectomized/adrenalectomized female rats received one of the following four treatments: (1) one subcutaneously placed Silastic capsule containing 10% estradiol in cholesterol, (2) one capsule containing 10% estradiol and two capsules containing 100% 5 alpha-dihydrotestosterone (DHT), (3) two capsules containing DHT, or (4) empty Silastic capsules (control animals). Animals were killed 4 or 8 days after capsule insertion and the occupied, unoccupied and total estrogen receptor content in specific brain nuclei was determined by quantitative in vitro autoradiography. To determine if the effects of the androgen were reversible, DHT capsules were removed after 4 days from half of the estradiol+DHT-treated rats, and the animals were killed 4 days later. Estradiol downregulated estrogen receptor expression in the periventricular preoptic area, medial preoptic area, bed nucleus of the stria terminalis (BNST), arcuate nucleus (ARC), ventromedial nucleus (VMN), and medial and cortical amygdala, decreasing receptor content by 30-41% in animals treated for 4 days, and by 44-60% in animals treated for 8 days with estradiol alone. DHT treatment in combination with estradiol further decreased estrogen receptor content in the BNST, ARC and VMN, relative to the estradiol-only animals. DHT in the absence of estrogen was without effect. In animals in which the DHT capsules were removed after 4 days of exposure, allowing the estradiol to remain for a further 4 days, estrogen receptor levels were indistinguishable from those measured in control animals treated for 8 days with estradiol alone. These results demonstrate that sustained estrogen exposure downregulates levels of estrogen receptor in the brain and confirm that DHT synergizes with estrogen in inducing this response in some, but not all, target neuronal groups.

The naturally occurring C-17 fatty acid esters of estradiol are long-acting estrogens.

C-17 fatty acid esters of estradiol are naturally occurring biosynthetic metabolites of estradiol. A representative component of this family of esters, estradiol-17-stearate, was studied in order to determine the estrogenic properties of these unusual hydrophobic steroids. Following the classical estrogen bioassay, a solution of this ester in oil was injected subcutaneously into immature rats once a day for 3 days. There was little effect on the uterus on the first day after the third injection. However, on subsequent days a large stimulation of uterine growth occurred. The course of this estrogenic effect was exactly opposite to that obtained with estradiol. In order to eliminate the possibility that this effect on the time course of estrogenic stimulation was caused by increased solubility of the hydrophobic esters in the carrier oil, the steroids were administered to adult ovariectomized animals in aqueous medium via a single intravenous injection. The uterotrophic response to estradiol was maximal at 12 h and was completely dissipated in 48-60 h. Estradiol-17-stearate produced a uterotrophic effect of twice the duration of estradiol. In the immature rat, aqueous intravenous injections of estradiol-17-stearate produced a greater uterotrophic effect than estradiol and this effect was still maximal 96 h later. In addition, this single injection of estradiol-17-stearate advanced the time of vaginal opening, a marker for puberty in the female rat. The mechanism of the prolonged estrogenic stimulation was investigated by studying the steroidal content of the uterus after injecting [3H]estradiol and [3H]estradiol-17 -stearate i.v. into immature rats. At 1 and 4 h there was significantly more radioactivity in the uteri of the [3H]estradiol treated animals. At later times (8 h and onwards) the total radioactivity in the uterus did not differ appreciably between the two groups. However at these later times, the amount of [3H]estradiol was far greater in the uteri of animals receiving [3H]estradiol-17-stearate. Consequently, the prolonged estrogenic effects of the endogenous C-17 fatty acid esters of estradiol are caused by the increased duration of the estrogenic signal. It is hypothesized that one of the roles of the fatty acid is to protect the steroid nucleus from metabolism and thereby prolong the life of the parent C18 steroid. Thus, the results of these experiments are consistent with the family of endogenous alkyl esters of estradiol having a physiological role as long-acting estrogens.

Intrinsic estrogenicity of some progestagenic drugs

The intrinsic estrogenic activity of some progestins cannot be properly evaluated by using hormone responsive systems when the chosen end-points are also sensitive to progestagenic activity, usually antagonistic of estrogenic actions. We have therefore applied to the evaluation of some drugs commonly used in contraceptive and hormone replacement formulations a recently developed in vitro method to estimate estrogenic activities, which is based on measurements of the estrogen-stimulated alkaline phosphatase activity in cells of the Ishikawa-Var I human endometrial adenocarcinoma line, a response not influenced by progestins. Whereas progesterone, medroxyprogesterone acetate and danazol were found to be devoid of estrogenic activity in this assay, Org OD-14, norethynodrel, gestrinone (R 2323), norethindrone and dl-norgestrel provoked half maximal increases in alkaline phosphatase activity at concentrations (EC-50) of 7, 14, 140, 200 and 2900 nM, respectively, under conditions in which the corresponding value for estradiol was 8 pM. This intrinsic estrogenic activity can be inhibited by antiestrogens, as verified by reversing the effect of R 2323 with 4-hydroxytamoxifen. Since prostaglandin F2 alpha output by secretory endometrium is increased by estrogens and diminished by progestins, this end-point can serve to evaluate the net effect of drugs with intrinsic estrogenic and progestagenic activities. For instance, R 2323 showed estrogenic activity in this assay whereas Org OD-14 did not. The same in vitro system can be used to evaluate estrogen antagonistic activities of test compounds, using estradiol as the agonist. These in vitro systems are useful in establishing a profile of activities of a drug on a relevant human target tissue, in the screening of synthetic or natural compounds under investigation, and in studies on structure/action relationships.

Sex and the developing brain : Suppression of neuronal estrogen sensitivity by developmental androgen exposure

The developmental effects of androgen play a central role in sexual differentiation of the mammalian central nervous system. The cellular mechanisms responsible for mediating these effects remain incompletely understood. A considerable amount of evidence has accumulated indicating that one of the earliest detectable events in the mechanism of sexual differentiation is a selective and permanent reduction in estrogen receptor concentrations in specific regions of the brain. Using quantitative autoradiographic methods, it has been possible to precisely map the regional distribution of estrogen receptors in the brains of male and female rats, as well as to study the development of sexual dimorphisms in receptor distribution. Despite previous data suggesting that the left and right sides of the brain may be differentially responsive to early androgen exposure, there is no significant right-left asymmetry in estrogen receptor distribution, in either sex. Significant sex differences in receptor density are, however, observed in several regions of the preoptic area, the bed nucleus of the stria terminalis and the ventromedial nucleus of the hypothalamus, particularly in its most rostral and caudal aspects. In the periventricular preoptic area of the female, highest estrogen receptor density occurs in the anteroventral periventricular region: binding in this region is reduced by approximately 50% in the male, as compared to the female. These data are consistent with the hypothesis that androgen-induced defeminization of feminine behavioral and neuroendocrine responses to estrogen may involve selective reductions in the estrogen sensitivity of critical components of the neural circuitry regulating these responses, mediated in part through a reduction in estrogen receptor biosynthesis.

Distribution of estrogen target sites in the 2-day-old mouse forebrain and pituitary gland during the ‘critical period’ of sexual differentiation

The present study provides a detailed anatomical description of estrogen target cells in the mouse forebrain and pituitary gland during the sexual imprinting stage of the brain. Six 2-day-old mice (3 males and 3 females) were s.c. injected with 16 alpha-[125I]iodo-11 beta-methoxy-17 beta-estradiol ([125I]MIE2) and two additional mice (one male and one female) were s.c. injected with 1000x unlabeled 17 beta-estradiol 1 h before [125I]MIE2 to check the specificity of estradiol binding. Two hours after injection the mice were decapitated, the brains dissected, frozen sectioned, and processed for thaw mount autoradiography. The highest intensity of nuclear labeling was observed in the preoptic-anterior hypothalamic area, amygdala and cortex entorhinalis. Strong labeling was present in the cerebral cortex and moderate to strong labeling in the lateral septum, bed nucleus of stria terminalis and pituitary gland. Weak to moderate labeling was observed in the bulbus olfactorius, circumventricular organs, basal ganglia, ventral striatum, thalamus, hippocampus and pineal gland. No sex differences were observed in the intensity of labeling and distribution of the estrogen target sites. The topographic distributions of estrogen-concentrating cells in the hypothalamus of the 2-day-old mouse forebrain was similar to the adult pattern but differed prominently in the cerebral cortex, entorhinal cortex and thalamus: the cerebral cortex showed an extensive and intensive labeling, the intensity of labeling in the entorhinal cortex greatly exceeded that observed in the adult and the nucleus anterior medialis thalami was distinctly labeled.

Sexual differentiation of estrogen receptor concentrations in the rat brain: effects of neonatal testosterone exposure

This study tests the hypothesis that sex differences in estrogen receptor (ER) expression in the rat hypothalamus and preoptic area may at least partly result from androgen exposure during the immediate postnatal period. Male rats were castrated and female rats were injected with androgen, at either 15-30 min, 24 h or 10 days after birth. ER distribution in the brain was evaluated by in vitro autoradiography at 28 days of age. Males castrated immediately after birth exhibited higher ER levels in the preoptic area and the ventromedial and arcuate nuclei of the hypothalamus than either control males or males castrated on day 10. Females injected at birth with testosterone propionate exhibited reduced ER binding in the same brain regions. These data suggest that postnatal androgen secretion prior to postnatal day 10 permanently alters patterns of ER expression in the brain.

Steroidal fatty acid esters

Several years ago we discovered an unexpected family of steroidal metabolites, steroidal fatty acid esters. We found that fatty acid esters of 5-ene-3 beta-hydroxysteroids, pregnenolone and dehydroisoandrosterone are present in the adrenal. Subsequently, others have shown the existence of these non-polar 5-ene-3 beta-hydroxysteroidal esters in blood, brain and ovaries. Currently, almost every family of steroid hormone is known to occur in esterified form. We have studied the esters of the estrogens and glucocorticoids in some detail, and have found that these two steroidal families are esterified by separate enzymes. In a biosynthetic experiment performed simultaneously with estradiol and corticosterone, we established that the fatty acid composition of the steroidal esters is quite different. The corticoid is composed predominantly of one fatty acid, oleate, while the estradiol esters are extremely heterogeneous. Our studies have demonstrated that the estrogens are extremely long-lived hormones, that they are protected by the fatty acid from metabolism. They are extremely potent estrogens, with prolonged activity. Esterification appears to be the only form of metabolism that does not deactivate the biological effects of estradiol. We have demonstrated the biosynthesis of fatty acid esters of estriol, monoesters at both C-16 alpha and C-17 beta. They too are very potent estrogens. These fatty acid esters of the estrogens are the endogenous analogs of estrogen esters, like benzoate, cypionate, etc., which have been used for decades, pharmacologically because of their prolonged therapeutic potency. We have found that the estradiol esters are located predominantly in hydrophobic tissues, such as fat. Sequestered in these tissues, they are an obvious reservoir of estrogenic reserve, requiring only an esterase for activation. To the contrary the biological activity of the fatty acid esters of the glucocorticoid, corticosterone, is not different from that of its free parent steroid. We have shown that the rapid kinetics of its induction of gluconeogenic responses is caused by its labile C-21 ester which is rapidly hydrolyzed by esterase enzymes. While it appears that the physiological role of the estrogen esters may be related to their long-lived hormonal activity, the role of the other families of steroidal esters is not yet apparent. They, and perhaps the estrogen esters as well, must serve other purposes. Indeed they may serve important biological functions beyond those which we ordinarily associate with steroid hormones.