Estrogen receptors alpha and beta are differentially expressed in developing human bone

Estrogen plays an essential role in the development and maintenance of the skeleton; its effects are mediated via interactions with two estrogen receptor (ER) subtypes, alpha and beta. The aim of this study was to establish the cellular distribution of ERalpha and ERbeta in neonatal human rib bone. ERalpha and ERbeta immunoreactivity was seen in proliferative and prehypertrophic chondrocytes in the growth plate, with lower levels of expression in the late hypertrophic zone. Different patterns of expression of the two ERs were seen in bone. In cortical bone, intense staining for ERalpha was observed in osteoblasts and osteocytes adjacent to the periosteal-forming surface and in osteoclasts on the opposing resorbing surface. In cancellous bone, ERbeta was strongly expressed in both osteoblasts and osteocytes, whereas only low expression of ERalpha was seen in these areas. Nuclear and cytoplasmic staining for ERbeta was apparent in osteoclasts. These observations demonstrate distinct patterns of expression for the two ER subtypes in developing human bone and indicate functions in both the growth plate and mineralized bone. In the latter, ERalpha is predominantly expressed in cortical bone, whereas ERbeta shows higher levels of expression in cancellous bone.     

Estrogen receptors alpha and beta in the rodent mammary gland

An obligatory role for estrogen in growth, development, and functions of the mammary gland is well established, but the roles of the two estrogen receptors remain unclear. With the use of specific antibodies, it was found that both estrogen receptors, ERalpha and ERbeta, are expressed in the rat mammary gland but the presence and cellular distribution of the two receptors are distinct. In prepubertal rats, ERalpha was detected in 40% of the epithelial cell nuclei. This decreased to 30% at puberty and continued to decrease throughout pregnancy to a low of 5% at day 14. During lactation there was a large induction of ERalpha with up to 70% of the nuclei positive at day 21. Approximately 60-70% of epithelial cells expressed ERbeta at all stages of breast development. Cells coexpressing ERalpha and ERbeta were rare during pregnancy, a proliferative phase, but they represented up to 60% of the epithelial cells during lactation, a postproliferative phase. Western blot analysis and sucrose gradient centrifugation confirmed this pattern of expression. During pregnancy, the proliferating cell nuclear antigen was not expressed in ERalpha-positive cells but was observed in 3-7% of ERbeta-containing cells. Because more than 90% of ERbeta-bearing cells do not proliferate, and 55-70% of the dividing cells have neither ERalpha nor ERbeta, it is clear that the presence of these receptors in epithelial cells is not a prerequisite for estrogen-mediated proliferation.     

Estrogen receptor beta expression in human prostate tissue

Estrogen receptor subtype beta (ERbeta) is highly expressed in rat prostate epithelium, but its presence in human prostate needs to be confirmed. Here we investigated the expression of ERbeta in five benign (normal and/or hyperplastic) and 10 malignant (Gleasons' score 2-7) prostate tissue specimens using immunohistochemistry. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue sections, using a commercially available ERbeta polyclonal antibody developed against the C-terminal amino acid residue. Nuclear ERbeta expression was found in the nuclei of glandular epithelium of benign prostate tissue specimens; faint nuclear ERbeta positivity was also present in a few stromal cells around normal epithelium. Nuclear ERbeta specific immunostaining was undetectable in all prostate cancer sections.     

Kinetic parameters of 5 alpha-reductase activity in stroma and epithelium of normal, hyperplastic, and carcinomatous human prostates

To study the influence of 5 alpha-reductase on the concentration of dihydrotestosterone in prostatic tissue, we measured the activity of this enzyme in stroma and epithelium from 15 normal, 50 hyperplastic, and 20 carcinomatous prostates. Maximum velocity (Vmax) and Km parameters based on the Lineweaver-Burk and Eadie-Hofstee transformations of the Michaelis-Menten equation were related to the stromal and epithelial concentrations of dihydrotestosterone. On the basis of relative Vmax values, there was 6-15 times more 5 alpha-reductase activity in stroma than in epithelium regardless of the histology of the prostate. Stromal enzyme activity also was unique in having a 2- to 5-fold larger mean Km value and greater resistance to competitive and noncompetitive inhibition. Despite the enrichment of 5 alpha-reductase activity in stroma, the dihydrotestosterone concentrations in the stromal and epithelial fractions were very similar. In addition, similar concentrations were found in the stromal fractions of hyperplastic and carcinomatous tissues, notwithstanding a 4-fold difference in the mean Vmax values. This anomaly occurred in association with a large disparity in mean Km values, i.e. 68.3 +/- 1.6 (+/- SE) nmol/L in hyperplasia vs. 23.0 +/- 2.9 nmol/L in carcinoma. The dissociation between parameters of 5 alpha-reductase activity and tissue dihydrotestosterone concentrations was apparent to some extent in benign prostatic hyperplasia, in which the lowest stromal androgen concentrations were found in prostates with the largest Vmax and Km values; also, a rise in stromal Km was almost invariably associated with a proportional increase in Vmax (correlation coefficient = 0.95). These data strongly suggest that the stromal and epithelial forms of 5 alpha-reductase are separate isoenzymes, and that the excess of 5 alpha-reductase in stroma does not promote accumulation of an abnormal amount of dihydrotestosterone. They also imply that both the augmentation of 5 alpha-reductase activity in hyperplastic stroma and the condition of benign hyperplasia of the prostate are mutual consequences of a primary increase in Km.     

Estrogen receptors ER alpha and ER beta in proliferation in the rodent mammary gland

Most evidence supports the view that ER alpha is responsible for estrogen (ovarian estradiol, E(2))-induced proliferation in the epithelial cells of the mammary gland, but despite this, proliferating epithelial cells do not express ER alpha. We have examined this apparent paradox by studying the role of ER alpha and ER beta in E(2)-induced proliferation in mammary glands (measured by BrdUrd incorporation into DNA) in mice with intact ER beta (WT mice) and those in which the ER beta gene has been inactivated (ER beta(-/-) mice). On treatment of ER beta(-/-) mice with E(2) or ovariectomized WT mice with E(2), tamoxifen, or a specific ER beta agonist (BAG), the number of BrdUrd-labeled cells in mammary glands increased from 3.4% in controls to 28-38% in the treated mice. This indicates that both ER alpha and ER beta can mediate E(2)-induced proliferation independently of each other. With specific antibodies, ER beta was found in both epithelial and stromal cells, whereas ER alpha was strictly epithelial. Within 4 h of a single dose of E(2), ER alpha was lost from the nuclei of epithelial cells. In WT mice, ER alpha reappeared by 24 h, but in ER beta(-/-) mice, return to the nucleus was delayed by 24 h. At 4 h after E(2), neither ER alpha nor progesterone receptor was detectable in BrdUrd-labeled nuclei but by 48 h after E(2), 29% of the BrdUrd-labeled cells expressed ER alpha, and 21-38% expressed progesterone receptor. During 3 weeks of continuous E(2) treatment, ER beta remained in the nucleus, but there was no detectable ER alpha. With tamoxifen treatment, ER alpha remained in the nucleus, but ER beta was lost. From these results, we conclude that ER alpha receives the proliferation signal from E(2), initiates DNA synthesis, and is then lost from cells. The subsequent steps in proliferation can proceed in the absence of either ER alpha or ER beta. ER beta facilitates the return of ER alpha to the nucleus and restores responsiveness to E(2). By down-regulating ER beta, tamoxifen may prolong refractoriness to E(2) in mammary epithelium.     

Effect of aging on kinetic parameters of 3 alpha(beta)-hydroxysteroid oxidoreductases in epithelium and stroma of human normal and hyperplastic prostate.

The accumulation of 5 alpha-dihydrotestosterone (DHT), particularly in stroma, is a possible etiological factor in regard of the age-dependent development of benign prostatic hyperplasia (BPH). In this context, we have recently demonstrated age-dependent alterations of 5 alpha-reductase, which is responsible for the irreversible conversion of testosterone to DHT. Therefore, it was also of interest to study possible age-dependent alterations of those enzymes mainly involved in the reversible metabolism of DHT to 5 alpha-androstanediols. Thus, we determined, in the presence of NADPH/NADP+, kinetic parameters [Km and maximum velocity (Vmax)] of 3-hydroxysteroid oxidoreductases (3 alpha-HSORred, 3 beta-HSORred, and 3 alpha-HSORox) in separated epithelium and stroma of 10 normal (NPR) and 20 hyperplastic prostates (BPH) and correlated the data with the age of the donors (15-86 yr). The mean Km (nanomolar concentrations +/- SEM) of 3 alpha-HSORred was significantly (P less than 0.01) higher in epithelium (NPR, 1391 +/- 181; BPH, 2150 +/- 157) than in stroma (NPR, 778 +/- 22; BPH, 749 +/- 62), indicating the presence of epithelial and stromal enzymes. The mean Km values of 3 beta-HSORred and 3 alpha-HSORox were similar. Concerning 3 alpha-HSORred, the mean potential capacity, i.e. the quotient of Vmax/Km (+/- SEM), was significantly (P less than 0.01) higher in epithelium (0.56 +/- 0.08) than in stroma (0.19 +/- 0.02) of NPR, while in BPH nearly identical mean potential capacities were found in epithelium (0.33 +/- 0.04) and stroma (0.26 +/- 0.02). The respective Vmax/Km of 3 beta-HSORred and 3 alpha-HSORox were significantly (P less than 0.05) lower. In addition, the potential capacity of all three enzymes was distinctly lower than the potential DHT-forming capacity of 5 alpha-reductase. With advancing age, the Vmax/Km decreased significantly (P less than 0.001; 3 alpha-HSORred and 3 beta-HSORred) or tendentiously (3 alpha-HSORox) in epithelium, while in stroma a significant (P less than 0.001; 3 alpha-HSORred and 3 alpha-HSORox) or tendentious (3 beta-HSORred) increase with age was found. Our results indicate that aging has a significant impact on DHT-removing enzymes. However, these enzymes counterbalance only in part the strong potential capacity of 5 alpha-reductase.     

Estrogen receptors alpha and beta have similar activities in multiple endothelial cell pathways

The presence of both estrogen receptor alpha (ERalpha) and ERbeta in vascular cells has greatly increased the complexity of potential estrogen regulatory pathways in the cardiovascular system. Here, human umbilical vein endothelial cells were engineered using adenovirus vectors to express either ERalpha or ERbeta. The activities of ERalpha and ERbeta were compared in three distinct gene regulatory pathways, including inhibition of IL-1beta induction of E-selectin expression, inhibition of basal endothelin-1 production, and the ability to induce two matrix-stabilizing enzymes: tissue transglutaminase and a novel member of the lysyl oxidase family. Both ERs were active on these end points, although ERbeta was typically less efficacious than ERalpha. As no class of gene regulation could differentiate ERalpha from ERbeta activity, we characterized a novel steroid (7alpha-thiophenyl-E2) that bound with similar affinities to ERalpha and ERbeta, but functioned as an ERalpha agonist and ERbeta antagonist for all of these endothelial responses. This pattern of receptor subtype-selective activity was not unique to endothelial cells, but was also seen in metabolically active HepG2 cells, suggesting potential in vivo utility. The panel of endothelial responses coupled with a selective modulator should provide a means to characterize the roles of ERalpha and ERbeta in endothelial cells in vivo.     

Increased ratio of 5 alpha-reductase: 3 alpha (beta)-hydroxysteroid dehydrogenase activities in the hyperplastic human prostate.

The activities of 5 alpha-reductase and 3 alpha (beta)-hydroxysteroid dehydrogenase were assayed in homogenates of eight normal, 21 hyperplastic and four carcinomatous human prostates. Samples consisting of 300--500 microgram tissue protein in Tris buffer, pH 7.0, were incubated at 37 degrees C for 30 min in the presence of 50 nM-[3H]androgen and an NADPH-generating system started with 5 X 10(-4)M-NADP. The yield of 5 alpha- and 3 alpha-reduced metabolites, as established by using t.l.c. and g.l.c., gave an estimate of enzyme activity. The formation of metabolites denoting 5 alpha-reductase activity in normal, hyperplastic and carcinomatous tissue respectively was 28.8 +/- 47 (S.E.M.), 76.8 +/- 8.9 and 3.5 +/- 0.7 pmol 30 min-1 mg protein-1; similarly, that denoting 3 alpha (beta)-hydroxysteroid dehydrogenase activity was 69.3 +/- 6.7, 46.6 +/- 5.7 and 38.8 +/- 22.1 pmol 30 min-1 mg protein-1. In all normal prostates 5 alpha-reductase activity was lower than 3 alpha (beta)-hydroxysteroid dehydrogenase activity. Conversely, in 18 out of 21 hyperplastic prostates, 5 alpha-reductase activity was higher than 3 alpha (beta)-hydroxysteroid dehydrogenase activity. The effect of the increase in 5 alpha-reductase activity without a compensatory change in 3 alpha (beta)-hydroxysteroid dehydrogenase activity was to alter the mean ratio between 5 alpha-reductase and 3 alpha (beta)-hydroxysteriod dehydrogenase activities from 0.47 +/- 0.11 in the normal prostate to 1.84 +/- 0,19 in hyperplastic tissue. It is inferred that this change may predispose the hyperplastic prostate to asymmetrical rates of androgen metabolism and thereby contribute to the abnormal accumulation of dihydrotestosterone.     

In vitro studies of the regulation of androgen-tissue relationship in normal canine and hyperplastic human prostate

Androgen-tissue relationship was investigated in vitro in human hyperplastic and normal canine prostate. A modified procedure permitted the use of steroid concentrations similar to those found in vivo. Entry, uptake or retention, metabolism, release and tissue clearance of testosterone and 5α-dihydrotestosterone were measured. Oestradiol-17β or cyproterone acetate, added to the superfusing medium, increased entry and modified uptake and metabolism of the androgens. Changes observed with human hyperplastic tissue differed from those with canine normal tissue. The former tissue appeared to have lost the ability to regulate uptake and the removal of the androgens observed with canine normal tissue.When stilboestrol was administered to men before operation, tissue from the periurethral region of the prostate gave results differing from those obtained with tissue from the outer part of the gland.     

Immunocytochemical localization of estrogen receptors alpha and beta in the human reproductive organs

To better define the role of estrogens in reproductive functions, we have proceeded to the immunocytochemical localization of the estrogen receptor (ER) subtypes, ERalpha and the recently discovered ERbeta+, in human reproductive tissues. In the ovary, ERbeta+ immunoreactivity was found in nuclei of granulosa cells of growing follicles at all stages from primary to mature follicles, interstitial gland, and germinal epithelium cells. Nuclear staining for ERalpha occurred in thecal, interstitial gland, and germinal epithelium cells. In the uterus, strong ERalpha immunoreactivity was detected in nuclei of epithelial, stromal, and muscle cells. Similar localization was obtained for ERbeta+, although the staining was much weaker. In the vagina, only ERalpha could be detected; a nuclear reaction was observed in deep layers of the stratified epithelium as well as in stromal and muscle cells. In the mammary gland, both ER subtypes were observed in epithelial and stromal cells. In the testis, ERbeta+ was detected in nuclei of Sertoli and Leydig cells, whereas ERalpha immunoreactivity was only observed in Leydig cells, with no tubular labeling. In the efferent ducts, only ERbeta+ could be detected, whereas neither ERbeta+ nor ERalpha could be found in the epididymis. In the prostate, ERbeta+ nuclear immunolabeling was observed in both basal and secretory cells in alveoli as well as in stromal cells, whereas ERalpha could not be detected. The present results demonstrate that there is a cell-specific localization for each of the ER subtypes in the majority of the reproductive organs studied. Moreover, they contribute to establish the exact sites of action of estrogens in male and female human reproductive systems.     

Demonstration of a specific cytosol receptor in the normal and hyperplastic canine prostate for 5alpha-androstane-3alpha, 17alpha-diol.

A specific receptor protein has been demonstrated for 5alpha-androstane-3alpha, 17alpha-diol in cytoplasmic extracts of normal and hyperplastic canine prostates. The receptor molecule, with a sedimentation coefficient of 4-5S, has been identified by the use of sucrose gradient centrifugation of tissue fractions which had been labelled in vitro with tritiated 5alpha-androstane-3alpha, 17alpha-diol. The receptor showed a relatively high affinity for this compound whereas binding could not be demonstrated with other labelled C19 steroids. In addition binding of tritiated 5alpha-androstane-3alpha, 17alpha-diol was not affected by the presence of 50-fold excesses of other C19 steroids.     

Estrogen and progesterone receptors in the human vagina

Estrogen (E) and progesterone (Pg) receptor (R) levels were determined in the human vagina in relation to menopausal status, day of ovarian cycle and pregnancy. The results obtained confirmed that the human vagina contains ER and, in addition, demonstrated for the first time the presence of PgR in this organ in humans. In cycling women, ER and PgR did not vary significantly during the ovarian cycle; however low (less than or equal to 10 fmoles/mg cytosol protein) concentrations of PgR were more frequently (6 out of 8 cases) detected during the secretory phase. No substantial difference was seen in ER and PgR values between anterior and posterior wall of the vagina. In postmenopausal patients the levels of ER (range: 10-83 fmoles/mg) were similar to those found in premenopause (range: 12-78 fmoles/mg). As regards PgR, the majority (14 out of 20) of vaginae were devoid of PgR, 4 had a very low (less than or equal to 6 fmoles/mg) PgR content and only 2 cases had a PgR level higher than 10 fmol/mg cytosol protein. In pregnant patients (6th to 8th week) ER were found in all vaginae, while PgR were present only in some cases (3 out of 8). It was concluded that the behavior of ER in the human vagina seems different from that in the human endometrium, since ER levels do not vary in relation to changes in the concentrations of sexual hormones in the circulation. On the contrary, PgR levels appear to depend on blood estradiol and progesterone concentration, as in other target tissues.     

Expression and cellular localization of estrogen receptors alpha and beta in the human fetus

Estrogens exert various biological effects by acting through their native receptors, two of which have been identified to date: estrogen receptors alpha (ERalpha) and beta (ERbeta). In this study we examined the expression and cellular localization of ERalpha and ERbeta in various human fetal tissues by semiquantitative RT-PCR (13 and 20 gestational weeks) and immunohistochemistry (13, 20, and 38 gestational weeks), respectively, to study the possible effects of estrogens on human fetal tissues during development. Relatively high levels of ERbeta expression were detected in various human fetal tissues, whereas those tissues expressing ERbeta had markedly lower levels of ERalpha expression. ERbeta messenger ribonucleic acid expression was especially high in the adrenal gland. ERbeta-immunoreactive protein was localized to the definitive zone, but not in the fetal zone, of the adrenal cortex. Although low levels of ERbeta messenger ribonucleic acid were present in the brain, heart, lung, and kidney, ERbeta immunoreactivity was not detected in these tissues. These results suggest that the effects of estrogens in these tissues are predominantly mediated through ERbeta. ERbeta immunoreactivity was detected in Sertoli cells and spermatogonia in the male reproductive tract and in germ cells in the fetal testis and epididymis. In the female reproductive tract, both ERalpha and ERbeta were immunopositive in epithelium of the oviduct. The results of the present study have demonstrated the possible sites for estrogenic action in the human fetus and suggest that the effects of estrogen via ERbeta may play important roles in human fetal development, especially in the definitive zone of the adrenal cortex, and in the reproductive tissues of the developing fetus.     

Estrogen and progesterone receptors in human gallbladder

Gallbladder disease is more prevalent in women than men. Estrogen therapy has been associated with an increased incidence of gallbladder disease in both sexes. Further, increased progesterone levels have been implicated in impairment of gallbladder motility in pregnancy. Because sex hormones often exert their action through specific receptors, we investigated whether human gallbladder contains receptors for estrogen and progesterone. Binding of radiolabeled hormones in cytosol and nuclei prepared from human gallbladder of both sexes is indicative of the presence of receptors for both estrogen and progesterone. The binding is saturable, of high affinity and highly specific for its particular type of hormone but not other steroids. Fractionation of sodium molybdate-stabilized gallbladder cytosol on Sephadex G-100 demonstrates that the labeled hormones are not bound to defined proteins such as sex steroid binding globulin or albumin. These studies indicate that human gallbladder contains both estrogen and progesterone receptors, that the presence of these receptors may explain the sensitivity of gallbladder tissue to these hormones and that certain aspects of gallbladder function may be mediated by the interaction of steroid hormones with these receptors.     

Antibodies specific to the retinoic acid human nuclear receptors alpha and beta.

Two cDNAs encoding two human receptors for retinoic acid (RA), RAR-alpha and RAR-beta, have been characterized recently. Synthetic peptides corresponding to the cDNA-deduced amino acid sequences unique to RAR-alpha and RAR-beta were used to generate anti-RAR-alpha antiserum (SP171) and anti-RAR-beta antisera (SP172 and SP248). The specificity of these antisera was confirmed both by immunocytochemical detection of these receptors in COS-1 cells transfected with RAR-alpha and RAR-beta expression vectors and by immunoblot analyses performed with whole extracts of these cells. We also demonstrate that these antisera recognize RAR-alpha and RAR-beta endogenously expressed in the RA-responsive human promyelocytic leukemia cell line HL-60.     

Adenomatous and carcinomatous changes within hyperplastic colonic epithelium

Summary Hyperplastic colonic polyps are benign, nonneoplastic proliferations; unlike tubular and villous adenomas, they do not predispose the patient to colonic cancer. Theoretically, these hyperplastic polyps, like normal colonic epithelium, should be able to undergo adenomatous transformation and possibly develop into cancer. In this report, we discuss an unusual case of a patient with numerous hyperplastic polyps, in which adenomatous changes occurred and cancer developed. We also discuss the significance of these changes as they relate to the polyp-cancer sequence.