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At least 19 recordsLinked to original sources

PubMed1993

Estrogen inhibits the growth of estrogen receptor-negative, but not estrogen receptor-positive, human mammary epithelial cells expressing a recombinant estrogen receptor.

Estrogen is essential for the growth of the normal mammary gland and most estrogen receptor (ER)-positive mammary carcinomas. To better understand the differences between the estrogen response pathways in normal and tumor cells, we have stably transfected ER-negative immortal, nontumorigenic human mammary epithelial cells and ER-negative breast cancer cells with an ER-encoding expression vector. Unexpectedly, estrogen treatment (1.0 nM) inhibited the proliferation of ER-transfected nontumorigenic and tumor-derived cells. The control transfectants and parental cells exhibited no response to estrogen concentrations as high as 1.0 microM. This inhibitory effect was attributed to a decreased growth rate and a perturbation of the cell cycle distribution by estrogen treatment of the ER transfectants. The inhibitory response was blocked by cotreatment with the antiestrogen ICI 164,384 as predicted for a pure antagonist of estrogen action. However, treatment with the antiestrogen hydroxytamoxifen caused growth inhibition, implying that hydroxytamoxifen acts as an agonist of estrogen action in ER-transfected cells. Since estrogen is a mitogenic and not a growth-inhibitory stimulus for ER-positive breast cancers and cell lines, we tested the effect of constitutive, high level expression of the ER in ER-positive tumor cells. Stable transfection of ER-positive MCF-7 and T47D cells with the ER expression vector yielded cells with varying amounts of ER. At ER levels comparable to those found in the ER-negative transfected cells, the MCF-7 and T47D ER transfectants were not inhibited by estrogen. These data suggest that ER-positive breast cancer cells can tolerate higher constitutive levels of ER expression than ER-negative cells. The mechanism by which this is accomplished may be an essential step in the process which yields ER-positive tumors.

Animals↗

PubMed2001

Natural killer cells express estrogen receptor-alpha and estrogen receptor-beta and can respond to estrogen via a non-estrogen receptor-alpha-mediated pathway.

Natural killer (NK) cells play a crucial role in host defense against pathogens and immune surveillance against cancer. Given that estrogens have been reported to suppress NK cell activity, we sought to elucidate the mechanisms by which estrogen mediates this effect. We demonstrate by immunocytochemical staining with estrogen receptor-alpha (ERalpha)- and estrogen receptor-beta (ERbeta)-specific antibodies that both ERalpha and ERbeta are expressed in murine NK cells. We also compared the ability of high doses of 17beta-estradiol ( approximately 800 pg/ml) to regulate NK cell activity in wild-type and estrogen receptor-alpha-deficient (ERalphaKO) mice. 17beta-estradiol elicited a significant decrease in NK cell activity in both wild-type and ERalphaKO mice (P < 0.001). These data suggest that ERbeta or possibly a novel receptor is involved in mediating estrogen action on NK cell activity and raise the potential for therapeutic modulation of NK cell activity with selective estrogen receptor modulators (SERMS).

Animals↗

PubMed2000

Estrogen receptor transcription and transactivation: Estrogen receptor alpha and estrogen receptor beta: regulation by selective estrogen receptor modulators and importance in breast cancer.

Estrogens display intriguing tissue-selective action that is of great biomedical importance in the development of optimal therapeutics for the prevention and treatment of breast cancer, for menopausal hormone replacement, and for fertility regulation. Certain compounds that act through the estrogen receptor (ER), now referred to as selective estrogen receptor modulators (SERMs), can demonstrate remarkable differences in activity in the various estrogen target tissues, functioning as agonists in some tissues but as antagonists in others. Recent advances elucidating the tripartite nature of the biochemical and molecular actions of estrogens provide a good basis for understanding these tissue-selective actions. As discussed in this thematic review, the development of optimal SERMs should now be viewed in the context of two estrogen receptor subtypes, ERalpha and ERbeta, that have differing affinities and responsiveness to various SERMs, and differing tissue distribution and effectiveness at various gene regulatory sites. Cellular, biochemical, and structural approaches have also shown that the nature of the ligand affects the conformation assumed by the ER-ligand complex, thereby regulating its state of phosphorylation and the recruitment of different coregulator proteins. Growth factors and protein kinases that control the phosphorylation state of the complex also regulate the bioactivity of the ER. These interactions and changes determine the magnitude of the transcriptional response and the potency of different SERMs. As these critical components are becoming increasingly well defined, they provide a sound basis for the development of novel SERMs with optimal profiles of tissue selectivity as medical therapeutic agents.

Animals↗

PubMed2000

Interactions of dietary estrogens with human estrogen receptors and the effect on estrogen receptor-estrogen response element complex formation.

Epidemiologic and experimental studies support the hypothesis that dietary estrogens from plant sources (phytoestrogens) may play a role in the prevention of breast and prostate cancer. The molecular mechanisms for such chemopreventive effect are still unclear. We investigated the possibility that phytoestrogens may bind differentially to estrogen receptor proteins (ER[alpha] and ERss) and affect the interactions of the ligand-ER complexes with different estrogen response element (ERE) sequences. We used fluorescence polarization to measure the binding affinities of genistein, coumestrol, daidzein, glyceollin, and zearalenone for human ER[alpha] and ERss. Competition binding experiments revealed higher affinity of the phytoestrogens for ERss than for ER[alpha]. Genistein [median inhibitory concentration 12nM] is the most potent and has the same relative binding affinity for ERss as 17ss-estradiol. We also studied the effect of these phytoestrogens on the ability of ER[alpha] and ERss to associate with specific DNA sequences (EREs). The direct binding of human recombinant estrogen receptors to fluorescein-labeled EREs indicates that phytoestrogens can cause conformational changes in both human ERs, which results in altered affinities of the complexes for the ERE from the Xenopus vitellogenin A2 gene and an ERE from the human pS2 gene.

Animals↗

PubMed2003

Placental expression of estrogen receptor beta and its hormone binding variant--comparison with estrogen receptor alpha and a role for estrogen receptors in asymmetric division and differentiation of estrogen-dependent cells.

During human pregnancy, the production of 17-beta-estradiol (E2) rises steadily to eighty fold at term, and placenta has been found to specifically bind estrogens. We have recently demonstrated the expression of estrogen receptor alpha (ER-alpha) protein in human placenta and its localization in villous cytotrophoblast (CT), vascular pericytes, and amniotic fibroblasts. In vitro, E2 stimulated development of large syncytiotrophoblast (ST) aggregates. In the present study we utilized ER-beta affinity purified polyclonal (N19:sc6820) and ER-alpha monoclonal (clone h-151) antibodies. Western blot analysis revealed a single approximately 52 kDa ER-beta band in chorionic villi (CV) protein extracts. In CV, strong cytoplasmic ER-beta immunoreactivity was confined to ST. Dual color immunohistochemistry revealed asymmetric segregation of ER-alpha in dividing villous CT cells. Prior to separation, the cell nuclei more distant from ST exhibited high ER-alpha, while cell nuclei associated with ST showed diminution of ER-alpha and appearance of ER-beta. In trophoblast cultures, development of ST aggregates was associated with diminution of ER-alpha and appearance of ER-beta immunoreactivity. ER-beta was also detected in endothelial cells, amniotic epithelial cells and fibroblasts, extravillous trophoblast (nuclear and cytoplasmic) and decidual cells (cytoplasmic only). In addition, CFK-E12 (E12) and CWK-F12 (F12) monoclonal antibodies, which recognize approximately 64 kDa ER-beta with hormone binding domain, showed nuclear-specific reactivity with villous ST, extravillous trophoblast, and amniotic epithelium and fibroblasts. Western blot analysis indicated abundant expression of a approximately 64 kDa ER-beta variant in trophoblast cultures, significantly higher when compared to the chorionic villi and freshly isolated trophoblast cell protein extracts. This is the first report on ER-beta expression in human placenta and cultured trophoblast. Our data indicate that during trophoblast differentiation, the ER-alpha is associated with a less, and ER-beta with the more differentiated state. Enhanced expression of approximately 64 kDa ER-beta variant in trophoblast cultures suggests a unique role of ER-beta hormone binding domain in the regulation of trophoblast differentiation. Our data also indicate that asymmetric segregation of ER-alpha may play a role in asymmetric division of estrogen-dependent cells.

Amnion↗

PubMed2006

Select estrogens within the complex formulation of conjugated equine estrogens (Premarin) are protective against neurodegenerative insults: implications for a composition of estrogen therapy to promote neuronal function and prevent Alzheimer's disease.

BACKGROUND: Results of the Women's Health Initiative Memory Study (WHIMS) raised concerns regarding the timing and formulation of hormone interventions. Conjugated equine estrogens (CEE), used as the estrogen therapy in the WHIMS trial, is a complex formulation containing multiple estrogens, including several not secreted by human ovaries, as well as other biologically active steroids. Although the full spectrum of estrogenic components present in CEE has not yet been resolved, 10 estrogens have been identified. In the present study, we sought to determine which estrogenic components, at concentrations commensurate with their plasma levels achieved following a single oral dose of 0.625 mg CEE (the dose used in the WHIMS trial) in women, are neuroprotective and whether combinations of those neuroprotective estrogens provide added benefit. Further, we sought, through computer-aided modeling analyses, to investigate the potential correlation of the molecular mechanisms that conferred estrogen neuroprotection with estrogen interactions with the estrogen receptor (ER). RESULTS: Cultured basal forebrain neurons were exposed to either beta-amyloid(25-35) or excitotoxic glutamate with or without pretreatment with estrogens followed by neuroprotection analyses. Three indicators of neuroprotection that rely on different aspects of neuronal damage and viability, LDH release, intracellular ATP level and MTT formazan formation, were used to assess neuroprotective efficacy. Results of these analyses indicate that the estrogens, 17alpha-estradiol, 17beta-estradiol, equilin, 17alpha-dihydroequilin, equilinen, 17alpha-dihydroequilenin, 17beta-dihydroequilenin, and Delta8,9-dehydroestrone were each significantly neuroprotective in reducing neuronal plasma membrane damage induced by glutamate excitotoxicity. Of these estrogens, 17beta-estradiol and Delta8,9-dehydroestrone were effective in protecting neurons against beta-amyloid25-35-induced intracellular ATP decline. Coadministration of two out of three neuroprotective estrogens, 17beta-estradiol, equilin and Delta8,9-dehydroestrone, exerted greater neuroprotective efficacy than individual estrogens. Computer-aided analyses to determine structure/function relationships between the estrogenic structures and their neuroprotective activity revealed that the predicted intermolecular interactions of estrogen analogues with ER correlate to their overall neuroprotective efficacy. CONCLUSION: The present study provides the first documentation of the neuroprotective profile of individual estrogens contained within the complex formulation of CEE at concentrations commensurate with their plasma levels achieved after an oral administration of 0.625 mg CEE in women. Our analyses demonstrate that select estrogens within the complex formulation of CEE contribute to its neuroprotective efficacy. Moreover, our data predict that the magnitude of neuroprotection induced by individual estrogens at relatively low concentrations may be clinically undetectable and ineffective, whereas, a combination of select neuroprotective estrogens could provide an increased and clinically meaningful efficacy. More importantly, these data suggest a strategy for determining neurological efficacy and rational design and development of a composition of estrogen therapy to alleviate climacteric symptoms, promote neurological health, and prevent age-related neurodegeneration, such as AD, in postmenopausal women.

Adenosine Triphosphate↗

PubMed1998

Estrogen and estrogen-androgen replacement in postmenopausal women dissatisfied with estrogen-only therapy. Sexual behavior and neuroendocrine responses.

OBJECTIVE: To investigate the efficacy of esterified estrogens alone and combined with oral androgen on sexual function and menopausal symptoms in postmenopausal women. STUDY DESIGN: Twenty postmenopausal women dissatisfied with their estrogen or estrogen-progestin therapy volunteered to enter a double-blind, randomized trial in which they received either oral esterified estrogens or esterified estrogens + androgen for eight weeks after a single-blind, placebo, lead-in period. Sexual function was assessed with a questionnaire used in the Yale midlife survey, and plasma levels of estradiol, estrone, sex hormone binding globulin (SHBG) and beta-endorphin were measured at two- to four-week intervals. RESULTS: Estrogen-androgen therapy significantly improved sexual sensation and desire after four and eight weeks of double-blind treatment in comparison to previous estrogen therapy and postplacebo baseline assessments. Plasma levels of estradiol and estrone increased significantly in all patients as compared to the postplacebo baseline and decreased in comparison to circulating estrogen concentrations on previous therapy. Relative proportions of free and bound steroid hormone exhibited contrasting shifts during estrogen and estrogen-androgen therapy. SHBG increased in the estrogen group and decreased in the estrogen-androgen group, leading to lower amounts of free androgens during estrogen therapy and increased free androgen levels during estrogen-androgen therapy. Since proportions of free (bioavailable) ovarian steroids would correlate inversely with plasma protein binding capacity, the beneficial effects of oral estrogen-androgen therapy on sexual sensation and desire may be due either to the administered androgen or to the increased availability of endogenous and exogenous androgens, particularly in the central nervous system. CONCLUSION: Sexual desire, satisfaction and frequency in postmenopausal women taking hormonal therapy were improved significantly by combined estrogen-androgen therapy but not by estrogen or estrogen-progestin therapy. Sexual function improved with estrogen-androgen therapy even though circulating estrogen levels were lower than those measured during previous estrogen therapy. This leads to the conclusion that androgens play a pivotal role in sexual function but that estrogens are not a significant factor determining levels of sexual drive and enjoyment.

Double-Blind Method↗

PubMed2004

Estrogen receptor-independent catechol estrogen binding activity: protein binding studies in wild-type, Estrogen receptor-alpha KO, and aromatase KO mice tissues.

Primary evidence for novel estrogen signaling pathways is based upon well-documented estrogenic responses not inhibited by estrogen receptor antagonists. In addition to 17beta-E2, the catechol estrogen 4-hydroxyestradiol (4OHE2) has been shown to elicit biological responses independent of classical estrogen receptors in estrogen receptor-alpha knockout (ERalphaKO) mice. Consequently, our research was designed to biochemically characterize the protein(s) that could be mediating the biological effects of catechol estrogens using enzymatically synthesized, radiolabeled 4-hydroxyestrone (4OHE1) and 4OHE2. Scatchard analyses identified a single class of high-affinity (K(d) approximately 1.6 nM), saturable cytosolic binding sites in several ERalphaKO estrogen-responsive tissues. Specific catechol estrogen binding was competitively inhibited by unlabeled catechol estrogens, but not by 17beta-E2 or the estrogen receptor antagonist ICI 182,780. Tissue distribution studies indicated significant binding differences both within and among various tissues in wild-type, ERalphaKO, and aromatase knockout female mice. Ligand metabolism experiments revealed extensive metabolism of labeled catechol estrogen, suggesting that catechol estrogen metabolites were responsible for the specific binding. Collectively, our data provide compelling evidence for the interaction of catechol estrogen metabolites with a novel binding protein that exhibits high affinity, specificity, and selective tissue distribution. The extensive biochemical characterization of this binding protein indicates that this protein may be a receptor, and thus may mediate ERalpha/beta-independent effects of catechol estrogens and their metabolites.

Animals↗

PubMed2001

Re-expression of estrogen receptor alpha in estrogen receptor alpha-negative MCF-7 cells restores both estrogen and insulin-like growth factor-mediated signaling and growth.

Estrogen can increase insulin-like growth factor-I receptor (IGF-IR) and insulin receptor substrate-1 (IRS-1) expression, two key components of IGF-I-mediated signaling. The result is sensitization of breast cancer cells to IGF-I and synergistic growth in the presence of estrogen and IGF-I. We hypothesized that loss of estrogen receptor alpha (ERalpha) would result in reduced IGF-mediated signaling and growth. To test this hypothesis, we examined IGF-I effects in MCF-7 breast cancer cell sublines that have been selected for loss of ERalpha (C4 and C4-12 cells are ERalpha-negative) by long-term estrogen withdrawal. C4 and C4-12 cells had reduced IGF-IR and IRS-1 mRNA and protein expression (compared with MCF-7 cells) that was not inducible by estrogen. Furthermore, C4 and C4-12 cells showed reduced IGF-I signaling and failed to show any growth response to either estrogen or IGF-I. To prove that loss of IGF and estrogen-mediated signaling and growth was a consequence of loss of ERalpha, we re-expressed ERalpha in C4-12 cells by stable transfection with HA-tagged ERalpha. Three independent C4-12 ERalpha-HA clones expressed a functional ERalpha that (a) was down-regulated by estrogen, (b) conferred estrogen-induction of cyclin D1 expression, and (c) caused estrogen-mediated increase in the number of cells in S phase. All of the effects were completely blocked by antiestrogens. Interestingly, ERalpha-HA expression in C4-12 cells did not restore estrogen induction of progesterone receptor expression. However, ERalpha-positive C4-12 cells now exhibited estrogen-induction of IGF-IR and IRS-1 levels and responded mitogenically to both estrogen and IGF-I. These data show that ERalpha is a critical requirement for IGF signaling, and to our knowledge this is the first report of functional ERalpha expression that confers estrogen-mediated growth of an ER-negative breast cancer cell line.

Breast Neoplasms↗

PubMed1997

Evaluation of clinical and environmental anti-estrogens with human estrogen receptor expressed in Saccharomyces cerevisiae: a novel role for ABC-cassette transporters in mediating anti-estrogenic activity.

The effectiveness of anti-estrogens in treating estrogen-dependent diseases is limited by the acquired resistance of some diseases to anti-estrogens. This effect could occur by the export of anti-estrogens by cell membrane transport proteins. To study this phenomenon we have expressed human estrogen receptor (hER) and an estrogen-sensitive reporter in wild-type yeast and two transport-defective strains. In the wild-type strain, the most effective anti-estrogen was nafoxidine. 4-Hydroxy tamoxifen and clomiphene were inactive whereas tamoxifen had significant inhibitory activity in the wild-type strain. Using a strain missing the ABC-cassette transporter Snq2, clomiphene had anti-estrogenic activity. 4-Hydroxy tamoxifen had anti-estrogenic activity only in yeast lacking the transporter Pdr5. Whole cell binding assays indicated that 4-hydroxy tamoxifen is exported by Pdr5. Environmental chemicals such as polychlorinated biphenyls function as partial estrogens and anti-estrogens in yeast. In the absence of Pdr5 or Snq2, the estrogenic activity of 4-hydroxy, 2',4',6'-trichloro biphenyl (3-PCB) was substantially reduced in comparison to its activity in the wild-type strain. Interestingly, the antiestrogenic activity of 3-PCB was equivalent in the wild-type and transporter-defective strains. Our results suggest a novel role for ABC-cassette transporters in regulating the activity of clinical and environmental anti-estrogens.

ATP-Binding Cassette Transporters↗

PubMed1999

Estrogen receptor beta activates the human retinoic acid receptor alpha-1 promoter in response to tamoxifen and other estrogen receptor antagonists, but not in response to estrogen.

Human estrogen receptor-alpha (hERalpha) or -beta (hERbeta) transfected into Hep G2 or COS1 cells each responded to estrogen to increase transcription from an estrogen-responsive element (ERE)-driven reporter vector with similar fold induction through a classical mechanism involving direct receptor binding to DNA. ER antagonists inhibited this estrogen induction through both hERalpha and hERbeta, although raloxifene was more potent through ERalpha than ERbeta, and tamoxifen was more potent via ERbeta than ERalpha. We have shown previously that estrogen stimulated the human retinoic acid receptor-alpha-1 (hRARalpha-1) promoter through nonclassical EREs by a mechanism that was ERalpha dependent, but that did not involve direct receptor binding to DNA. We show here that in contrast to hERalpha, hERbeta did not induce reporter activity driven by the hRARalpha-1 promoter in the presence of estrogen. While hERbeta did not confer estrogen responsiveness on this promoter, it did elicit transcriptional activation in the presence of 4-hydroxytamoxifen (4-OH-Tam). Additionally, this 4-OH-Tam agonist activity via ERbeta was completely blocked by estrogen. Like ERalpha, transcriptional activation of this promoter by ERbeta was not mediated by direct receptor binding to DNA. While hERalpha was shown to act through two estrogen-responsive sequences within the promoter, hERbeta acted only at the 3'-region, through two Sp1 sites, in response to 4-OH-Tam. Other ER antagonists including raloxifene, ICI-164,384 and ICI-182,780 also acted as agonists through ERbeta via the hRARalpha-1 promoter. Through the use of mutant and chimeric receptors, it was shown that the 4-OH-Tam activity via ERbeta from the hRARalpha-1 promoter in Hep G2 cells required the amino-terminal region of ERbeta, a region that was not necessary for estrogen-induced ERbeta activity from an ERE in Hep G2 cells. Additionally, the progesterone receptor (PR) antagonist RU486 acted as a weak (IC50 >1 microM) antagonist via hERalpha and as a fairly potent (IC50 approximately 200 nM) antagonist via hERbeta from an ERE-driven reporter in cells that do not express PR. Although RU486 bound only weakly to ERalpha or ERbeta in vitro, it did bind to ERbeta in whole-cell binding assays, and therefore, it is likely metabolized to an ERbeta-interacting compound in the cell. Interestingly, RU486 acted as an agonist through ERbeta to stimulate the hRARalpha-1 promoter in Hep G2 cells. These findings may have ramifications in breast cancer treatment regimens utilizing tamoxifen or other ER antagonists and may explain some of the known estrogenic or antiestrogenic biological actions of RU486.

Animals↗

PubMed1995

Estrogen synthesis, estrogen metabolism, and functional estrogen receptors in rat arterial smooth muscle cells in culture.

To investigate the mechanisms by which estrogen hormones influence the vascular system, the metabolism of these hormones and the functionality of estrogen receptors were characterized in rat aortic smooth muscle cells from secondary cultures, a widely studied model of vascular biology. Aromatase, estradiol-17 beta-hydroxysteroid dehydrogenase and 17-ketoreductase enzyme activities were demonstrated in these cells. The presence of functional estrogen receptor could also be demonstrated by estrogen-induced transactivating ability in transfection experiments using the luciferase gene reporter and an estrogen responsive element as transcriptional enhancer although the amplitude of the response was only in the range of 140 to 150%. Immunocytochemical analyses, using monoclonal antibodies that recognize epitopes in the A/B domain of the molecule, showed a predominant cytoplasmic localization of these estrogen receptors, even after estrogen addition to the culture medium. Western blot analysis using antibodies that recognize epitopes in the A/B or F domain gave a mol wt of 67,000. Analysis of the estrogen receptor messenger RNA showed that there was no deletion of the proto-signals for nuclear accumulation. The aromatase and dehydrogenase activity results, coupled with the estrogen receptor immunological, RNA analysis, and transfection data strongly support the contention that rat aortic smooth muscle cells are estrogen target cells. This in vitro model is convenient for studying the mechanisms of action of estrogen hormones that seem very peculiar in this cell population.

17-Hydroxysteroid Dehydrogenases↗

PubMed2005

Estrogen provides neuroprotection against activated microglia-induced dopaminergic neuronal injury through both estrogen receptor-alpha and estrogen receptor-beta in microglia.

Estrogen provides neuroprotection against neurodegenerative diseases, including Parkinson's disease. Its effects may stem from interactions with neurons, astrocytes, and microglia. We demonstrate here in primary cultures of rat mesencephalic neurons that estrogen protects them from injury induced by conditioned medium obtained from lipopolysaccharide (LPS)-activated microglia. LPS-induced nitrite production and tumor necrosis factor-alpha up-regulation in microglia were blocked by estrogen pretreatment. Estrogen neuroprotection was related to microglial activation of estrogen receptors (ERs), insofar as the protective effect of the microglia-conditioned medium was overridden by pretreatment of microglia with the ER antagonist ICI 182,780. On the other hand, the specific ERalpha antagonist, MPP dihydrochloride, only partially blocked the effects of estrogen, suggesting that estrogen protection was mediated via both ERalpha and ERbeta. LPS treatment did not change ERalpha mRNA levels in microglia, astrocytes, and neurons, but it up-regulated ERbeta mRNA levels in microglia and astrocytes. Similarly, increased ERbeta protein levels were detected in LPS-activated microglia. More interesting was that immunocytochemical analysis revealed that ERbeta was localized in the cytoplasm of microglia and in the cell nucleus of astrocytes and neurons. In summary, our results support the notion that estrogen inhibits microglial activation and thus exhibits neuroprotective effects through both ERalpha and ERbeta activation. The cytoplasm location of microglial ERbeta suggests the possible involvement of nonclassical effects of estrogen on microglia. Changes in microglial ERbeta expression levels may modulate such effects of estrogen.

Animals↗

PubMed1997

Estrogenic responses in estrogen receptor-alpha deficient mice reveal a distinct estrogen signaling pathway.

Estrogens are thought to regulate female reproductive functions by altering gene transcription in target organs primarily via the nuclear estrogen receptor-alpha (ER-alpha). By using ER-alpha "knock-out" (ERKO) mice, we demonstrate herein that a catecholestrogen, 4-hydroxyestradiol-17beta (4-OH-E2), and an environmental estrogen, chlordecone (kepone), up-regulate the uterine expression of an estrogen-responsive gene, lactoferrin (LF), independent of ER-alpha. A primary estrogen, estradiol-17beta (E2), did not induce this LF response. An estrogen receptor antagonist, ICI-182,780, or E2 failed to inhibit uterine LF gene expression induced by 4-OH-E2 or kepone in ERKO mice, which suggests that this estrogen signaling pathway is independent of both ER-alpha and the recently cloned ER-beta. 4-OH-E2, but not E2, also stimulated increases in uterine water imbibition and macromolecule uptake in ovariectomized ERKO mice. The results strongly imply the presence of a distinct estrogen-signaling pathway in the mouse uterus that mediates the effects of both physiological and environmental estrogens. This estrogen response pathway will have profound implications for our understanding of the physiology and pathophysiology of female sex steroid hormone actions in target organs.

Animals↗

PubMed1997

Resistance of human breast-cancer cells to the pure steroidal anti-estrogen ICI 182,780 is not associated with a general loss of estrogen-receptor expression or lack of estrogen responsiveness.

To elucidate the mechanisms responsible for the development of anti-estrogen resistance, we have cloned and established 3 stable ICI-182,780-resistant sub-lines, MCF-7/182R-1, MCF-7/182R-6 and MCF-7/182R-7 from the estrogen-receptor(ER)-positive and estrogen-responsive human breast-cancer MCF-7 cell line by long-term treatment with 10(-7) M ICI 182,780. The ICI-182,780-resistant MCF-7 sub-lines express ER, but compared with MCF-7 cells the level is significantly lower in all 3 sub-lines. In the MCF-7 cell line we find that ER expression is regulated by estrogen and anti-estrogens at the transcriptional and post-transcriptional level. This is in contrast to the ICI-182,780-resistant sub-lines, in which we find very little hormonal effects on the ER mRNA expression level. The resistant sub-lines also deviate from parent characteristics by the complete lack of expression of progesterone receptor even when grown in the presence of estradiol. All 3 resistant sub-lines have a lower basal expression of cathepsin-D mRNA comparable with the lower ER expression, but, in contrast, they have higher basal expression of the pS2 mRNA than the parent MCF-7 cell line. Although there are different basal expression levels of the pS2 and cathepsin-D genes, the resistant sub-lines behave like the parent MCF-7 cell line with respect to the hormonal regulation of both genes. The estrogen receptors in the resistant sub-lines have also maintained wild-type characteristics with respect to estrogen and anti-estrogen regulation of the estrogen-regulated proteins procathepsin D, alpha1-antitrypsin and a 42-kDa protein. The resistant cells require estrogen for growth in athymic nude mice. Our results clearly demonstrate that the ER in the resistant sub-lines have a normal function for most parameters investigated, supporting our earlier observation that only wild-type ER protein is expressed in these cells. The few observed differences in ER function between the parent MCF-7 cell line and the resistant sub-lines are not likely to be responsible for the ICI-182,780-resistant phenotype.

Animals↗

PubMed1985

Specific binding of estrogen and estrogen-receptor complex by microsomes from estrogen-responsive tissues of the rat.

Rat uterine and anterior pituitary microsomes each contain a population of specific estrogen-binding sites. Saturation binding of estradiol is demonstrable, with an affinity similar to that of the cytosol estrogen receptor (Ka = 1-2 X 10(10) M-1). Dissociation rate kinetic determinations, however, revealed that estrogen-microsomal complexes are 4 times as stable as cytosol estrogen-receptor complexes. Sedimentation properties in sucrose gradients were salt-dependent, yielding values of 10S in KCl-free buffer and 5.5S in the presence of 0.4 M KCl. The concentration of microsomal sites varies in proportion to the level of cytosol estrogen receptor, such that microsomal binding constitutes a consistent 20% of the total extranuclear binding capacity. Binding is sensitive to pronase, but not to ribonuclease or deoxyribonuclease; steroidal specificity differs from cytosol receptor only with respect to a greater extent of competition by progesterone. Microsomal binding sites are readily extractable with KCl-free hypotonic buffer or with 0.4 M KCl, but are resistant to extraction by 0.15 M KCl. The presence of estradiol lends stability to the microsomal binding sites, while high salt has a deleterious effect on their longevity. After exhaustive extraction of binding sites, microsomes are capable of accepting cytosol estradiol-receptor complexes to a level corresponding to the concentration of depleted binding sites; microsomes from nontarget tissue do not manifest such capability. However, the original microsomal estrogen-binding sites are not simply cytosol receptor contaminants, as evidenced by the observations that the microsomal binding site concentration is independent of the volume of tissue homogenate (indicating that a trapping phenomenon is not operative) and that nonextracted microsomes are not potential acceptor sites for cytosol estradiol-receptor complexes. In considering total cellular dynamics of estrogen and estrogen receptor turnover, it thus becomes important to explore the role of the microsomal compartment, since it functions as a repository of specific estrogen-binding sites and may have significant acceptor capability for the cytosol estrogen-receptor complex.

Animals↗

PubMed1985

Estrogen 2- and 4-hydroxylase activity, catechol estrogen formation, and implications for estrogen carcinogenesis in the hamster kidney.

Estrogen 2- and 4-hydroxylase (ESH), a microsomal enzyme which mediates the formation of catechol estrogens, has been studied in the kidneys of castrated male Syrian hamsters, a species uniquely susceptible to induction of renal carcinomas by both steroidal and stilbene estrogens. The apparent Km for estrone was 17.0 microM, and Vmax was 0.5 pmol per mg protein per min for ESH in renal microsomes derived from castrated hamsters. Different steroidal estrogen substrates exhibited decreasing catechol formation with hamster kidney microsomal preparations in the following order: estrone greater than d-equilenin greater than 17 beta-estradiol greater than equilin greater than ethynyl estradiol greater than estriol. Except for beta-dienestrol, the stilbene estrogens revealed levels of catechol formation that were similar to 17 beta-estradiol. These findings provide a rationale for the weak carcinogenic activity of ethynyl estradiol, estriol, and beta-dienestrol, since they were poor substrates for hamster renal ESH and for the relatively potent carcinogenic activity of the distal metabolite of diethylstilbestrol, indenestrol B/A, which exhibited substantial levels of o-hydroxylation when used as a substrate. Interestingly, ESH activity was significantly greater in the hamster kidney compared to corresponding rat tissue, and catechol estrogen formation was found to be 2.5- to 19-fold higher in the hamster kidney compared to the rat, using various steroidal and stilbene estrogen substrates. Moreover, the finding that a 3.5- to nearly 6-fold decrease, compared to untreated levels, in catechol formation in kidneys but not in livers of alpha-naphthoflavone-exposed hamsters, depending on the steroidal or stilbene estrogen substrate used, is consistent with the belief that the catechol estrogen pathway is pertinent to events leading to estrogen-induced renal tumorigenesis in the hamster.

Animals↗

PubMed2001

Differential neonatal imprinting and regulation by estrogen of estrogen receptor subtypes alpha and beta and of the truncated estrogen receptor product (TERP-1) mRNA expression in the male rat pituitary.

Two distinct nuclear estrogen receptors (ERs) have been identified, the classical one, renamed ERalpha, and the more recently cloned ERbeta. In a variety of tissues, gene expression of both receptor subtypes results in the generation of multiple transcripts encoding the full-length as well as several alternately spliced isoforms. In the rat pituitary, a truncated, tissue-specific variant of ERalpha, called TERP-1, has been identified and found able to modulate ERalpha and ERbeta activity. So far, its pattern of expression and hormonal regulation have been mostly studied in females. The present study was designed to analyze the pattern of expression of TERP-1 mRNA in the male rat pituitary at different stages of postnatal development, and to evaluate the impact of neonatal imprinting and estrogen treatment upon TERP-1 expression in the male pituitary. Assessment of TERP-1 mRNA levels by semi-quantitative RT-PCR, using a variant-specific primer pair, revealed that TERP-1 is also expressed in the male rat pituitary. Relative mRNA expression levels changed markedly during postnatal development, with moderate expression of the TERP-1 transcript at birth, barely detectable levels during the infantile-prepubertal period, and maximal values in adulthood. Expression of TERP-1 was sensitive to neonatal estrogen exposure, which resulted in a significant, persistent increase in mRNA levels from the infantile period until puberty. This phenomenon was not mimicked by neonatal blockade of endogenous GnRH. In addition, estrogen was able to acutely up-regulate pituitary TERP-1 mRNA expression levels in prepubertal (30-day-old) and adult (75-day-old) males. Interestingly, neonatal imprinting as well as acute estrogen treatment resulted in opposite effects on TERP-1 and full-length ERalpha and ERbeta transcripts, the latter being decreased under both conditions. In conclusion, our data indicate that TERP-1 mRNA is expressed in a developmentally regulated manner in the male rat pituitary, and is affected by neonatal estrogen imprinting and acute estrogen treatment. Regulation of TERP-1 expression by neonatal or acute estrogen treatment may thus represent an additional tuning mechanism for estrogen actions in the male rat pituitary.

Aging↗