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The higher vocal center (HVC) of the songbird forebrain exhibits persistent neurogenesis in adulthood, particularly in a region of the mediocaudal neostriatum that is associated with a subventricular layer of estrogen receptive cells. We asked whether estrogens might influence adult neurogenesis, by assessing the effect of ovariectomy on HVC neuronal production in the adult female canary. Fifteen 1-year-old females were separated into groups of ovariectomized, estradiol-replaced ovariectomized, and gonadally intact birds. To label dividing cells and their progeny, the birds were given [3H]thymidine for 8 days, killed 32 days later, and their brains autoradiographed. A significant rise was noted in the number of HVC neurons per section in estradiol-treated birds relative to the untreated control birds. The number of [3H]thymidine-labeled HVC neurons was also higher in the estrogen-treated birds; however, the neuronal labeling index (LI) did not vary as a function of estradiol replacement, as the total number of HVC neurons rose in parallel with the added new neurons. In contrast, the neuronal LI did rise as a result of ovariectomy, and this ovariectomy-associated increase in the LI was not reversed by estradiol. Among non-neuronal cell types, the endothelial LI was higher in estrogen-treated birds than in their untreated counterparts, suggesting estrogen-associated angiogenesis. Radioimmunoassay confirmed that serum estradiol was reduced in the castrated birds. Since estrogen appeared to promote the survival of [3H]thymidine+ neurons, we next sought to determine whether estrogen acted directly on the newly generated neurons, or rather indirectly through an intermediary cell population. To this end, we asked whether the new neurons or their precursors expressed estrogen receptor immunoreactivity (ER-IR). Five adult male canaries were given [3H]thymidine for periods ranging from 2 to 28 days, killed at varying times up to 3 weeks thereafter, then probed for ER-IR and autoradiographed. [3H]thymidine+ cells displayed no detectable ER-IR within their first 4 weeks of postmitotic life. Rather, during migration from the ventricular zone (VZ), the new neurons traversed a layer of mitotically quiescent, ER+ subventricular cells. Double labeling for ER-IR and cell-type selective antigens confirmed that these ER+ cells were neurons. These results indicate that the early survival of new neurons in the adult songbird HVC is promoted by estrogen, and may be mediated by the estrogen-stimulated paracrine release of neurotrophic agents by ER-IR subventricular neurons. Our data suggest that estrogen's promotion of neuronal survival may operate concurrently with an estrogen-independent ovarian suppression of neuronal mitogenesis.
We have previously demonstrated the existence of nuclear estrogen receptors in isolated adipocytes (Pedersen et al. (1991) Biochim. Biophys. Acta 1093, 80-86). In the present study we have investigated the regulatory properties of these nuclear estrogen receptors, in addition to the metabolic effects of estrogen on adipose tissue metabolism. Estrogen treatment (20 micrograms 17 beta-estradiol in NaCl for 7 days) decreased lipoprotein lipase activity (LPL) in the adipose tissue by 62% (p less than 0.05), decreased adipocyte size by 27% (p less than 0.01) and diminished the normal postovariectomy weight gain. Furthermore, estrogen treatment increased the nuclear estrogen receptor binding in adipocytes; in addition, there was a tendency for increased cytosolic estrogen receptor content as well. Time course studies revealed that already 6 h after a single estrogen injection the Bmax increased from 3.82 +/- 0.3 fmol/10(6) cells to 9.8 +/- 3.6 fmol/10(6) cells (p less than 0.1) and 24 h after a single injection the Bmax was maximally increased to 12.7 +/- 5.5 fmol/10(6) cells (p less than 0.05). The Kd was similar at all time points (about 3-5 nM). Furthermore, the specific insulin receptor binding was increased in adipocytes from estrogen treated rats. The specific insulin binding was maximally increased by 149 +/- 6% (p less than 0.001) after 4 days of daily estrogen injections. The increased binding seemed to be due to an increased number of insulin receptors on adipocytes from estrogen treated rats with no alteration of the ED50 value. In conclusion it was found that estrogen treatment has a positive feedback effect on its own nuclear receptor.(ABSTRACT TRUNCATED AT 250 WORDS)
Catechol estrogens, the cytochromes P450 mediated metabolites of 17 beta-estradiol, undergo further metabolism either via catechol O-methyltransferase (COMT) catalyzed methylation, or by oxidation and subsequent thioether formation with glutathione (GSH). Secondary metabolites of 17 beta-estradiol arising from both these metabolic pathways have been identified in vivo. However, the relative contribution of catechol O-methylation, and catechol oxidation followed by GSH conjugation, to the disposition of the catechol estrogens is unclear. We have therefore quantified both pathways of catechol estrogen disposition, generated in situ from 17 beta-estradiol, in hamster hepatic microsomes. 17 beta-Estradiol was readily converted to 2- and 4-hydroxy-17 beta-estradiol, both of which were effectively methylated in the presence of COMT (300 units/mL). Addition of GSH (50 microM-1 mM) to microsomal incubations resulted in the formation of four catechol estrogen-derived GSH conjugates. Three conjugates of 2-hydroxy-17 beta-estradiol were identified: 2-hydroxy-1,4-bis(glutathione-S-yl)-17 beta-estradiol, 2-hydroxy-1-glutathione-S-yl-17 beta-estradiol, and 2-hydroxy-4-glutathione-S-yl-27 beta-estradiol. In contrast, just one GSH conjugate of 4-hydroxy-17 beta-estradiol was identified: 4-hydroxy-1-glutathione-S-yl-17 beta-estradiol. When a combination of COMT and GSH were simultaneously added to microsomal incubations, both metabolic pathways competed for the same pool of catechol estrogens, and ascorbate dramatically influenced which of these two pathways predominate. In the presence of ascorbate, catechol estrogen methylation predominated over catechol estrogen oxidation and GSH conjugation. In the absence of ascorbic acid, catechol estrogen methylation, and catechol estrogen oxidation inked to GSH conjugation, contributed equally to the disposition of the catechol estrogens. 17 beta-Estradiol 2- and 4-hydroxylase activity was always higher in the absence of ascorbate, irrespective of whether GSH or COMT was used as the trapping agent. Thus, the usual method (COMT plus ascorbate) of determining 17 beta-estradiol 2- and 4-hydroxylase activity underestimates enzyme activity by approximately 50% when compared to the value obtained when GSH is used to trap the o-quinones in the absence of ascorbate. A reassessment of 17 beta-estradiol 2- and 4-hydroxylase activity in different species and tissues is required to permit a more informed evaluation of the role of catechol estrogens in estrogen-induced carcinogenesis.
Neonatal exposure to estrogens results in permanent imprints of the rat prostate gland. To delineate the direct target of estrogen action within that tissue, the present study examined estrogen receptor (ER) expression by immunocytochemistry and in situ hybridization. ER were confined to mesenchymal cells in the urogenital sinus and proximal regions of the budding prostate lobes of newborn control rat prostates, and this expression declined after morphogenesis. Exposure to estradiol benzoate on days 1, 3, and 5 resulted in induction of ER expression in periductal smooth muscle cells from the proximal regions out to the distal tips of the developing prostate lobes. This ER expression was associated with the appearance of ER messenger RNA in those cells; thus, it was concluded that the up-regulation of ER by estrogens is mediated at the message level. Autoregulation of ER expression was next examined in adult prostates that had been exposed to oil or estrogens neonatally. Day 70 rats were castrated and given testosterone with or without estradiol for 7 days before death. Estrogen exposure in adulthood induced low levels of epithelial cell ER in the lateral lobe. Neonatal estrogenization increased the sensitivity of lateral lobe epithelial cells to this autoregulation, as the incidence and intensity of ER immunostaining were markedly increased. No autoinduction of ER was observed in adult ventral or dorsal prostatic lobes. From the present study we conclude that smooth muscle cells are the targets of estrogen action in the developmentally estrogenized prostate and that estrogen amplifies its own effects through auto-up-regulation of ER. In addition, lateral lobe epithelial cells are sensitive to estrogen up-regulation of ER, which may in part account for the lobe-specific effects observed after neonatal estrogenization of the prostate gland.
The effects of estrogen and estrogen agonists can be mediated by estrogen receptor alpha (ER alpha) and estrogen receptor beta (ER beta). We now report the identification and initial characterization of several novel isoforms of rat ER beta messenger RNA (mRNA). The most abundant of these mRNA variants we have called ER beta2. ER beta2 had an in-frame insertion of 54 nucleotides that resulted in the predicted insertion of 18 amino acids within the ligand binding domain. We demonstrated by semiquantitative RT-PCR and RNase protection that ER beta2 mRNA was expressed at levels equal to those of the previously published ER beta (ER beta1) in ovary, prostate, pituitary, and muscle. In tissues of the nervous system, including frontal cortex, hippocampus, and hypothalamus, ER beta1 was present in a 2- to 6-fold greater abundance than ER beta2. We have also detected variants of both ER beta1 and ER beta2 mRNAs that contained deletions of 117 bp encompassing the region encoding the second zinc finger of the DNA binding domain. All four mRNA species were efficiently translated into functional protein in a heterologous system. ER beta2 bound estradiol with a lower affinity (Kd 5.1 nM) than either ER alpha (0.19 nM) or ER beta1 (0.14 nM). The binding of ER beta2 was selective in that cortisol, testosterone, aldosterone, and progesterone among other agents did not compete for estradiol binding. However, a variety of known estrogenic agents, including physiological estrogens (estrone and estriol), plant and environmental estrogens (genistein, coumestrol, bisphenol A, methoxychlor), and pharmocological agents (tamoxifen, 4-hydroxytamoxifen) did effectively compete for estradiol binding to both ER beta1 and ER beta2. Interestingly, the binding pharmacology differed among the agents tested. For example, genistein competed effectively for estradiol binding to ER beta1 but was > 150-fold weaker at competing from ER beta2. In contrast, 4-hydroxytamoxifen competed equally well at both receptors. We have also demonstrated by a gel shift assay that both ER beta1 and ER beta2 bound specifically to DNA containing a consensus estrogen response element. ER beta1 and ER beta2 could heterodimerize with each other and with ER alpha. Both ER beta1 and ER beta2 activated transcription in response to estradiol, however, ER beta2 required a 1000-fold greater estradiol concentration for activity than did ER beta1. Cotransfection of ER beta2 had no effect on ER beta1 activation when used in a equal ratio. A 10-fold excess of ER beta2 did raise the half-maximal dose of estradiol required for transcriptional activation, whereas the maximal level of induction did not change. The ER beta complementary DNAs deleted within the DNA binding domain could not bind to DNA or activate transcription from this reporter in the cell backgrounds tested. In conclusion, although the physiological significance of these ER beta variants warrants further investigation, ER beta2 mRNA encodes a specific, functional receptor for estradiol and estrogenic agents. We propose that ER beta2 should also be considered in addition to ER beta1 and ER alpha when describing the effects of estrogen, estrogen agonists/antagonists, or environmental estrogens.
Certain metabolic effects were investigated in post-menopausal women undergoing oral estrogen replacement therapy for 6 months using various substances. The increases in serum concentration of the estrogen-sensitive proteins, pregnancy zone protein (PZP), and sex hormone binding globulin (SHBG) had very similar and dose-dependent patterns. Ethinyl-estradiol was found to be much more potent than the "natural" estrogens. Estriol in various doses did not increase the protein level. Gonadotropin inhibition occurred in a dose-dependent manner. In terms of FSH suppression ethinyl-estradiol was approximately 120 times as potent as the "natural" estrogens. There was a striking resemblance between the "estrogenicity" of four different estrogens when expressed both in inhibition of gonadotropins and in induction of the two serum proteins SHBG and ceruloplasmin. Estriol caused a significant depression of FSH when given orally in a dose of 2 mg three times daily. Prolactin was found to decrease during treatment with low doses of estrogens. Estrogen therapy was found to have only moderate effects on adrenal androgens. Tamoxifen, and anti-estrogen, was found to exert distinctly estrogenic effects during treatment of post-menopausal women. In post-menopausal women with low amounts of circulating estrogens the tamoxifen-receptor complex itself may produce a net estrogenic response. Serum samples from post-menopausal women treated with ethinyl estradiol 0.05 mg and estrone sulphate 2.5 mg daily were found to reduce the lymphocyte reactivity in mixed lymphocyte cultures.
Anti-estrogenic effects of yuehchukene were observed in rat uterotrophic, mice vaginal smear and MCF-7 cell growth assays. Whereas yuehchukene per se was estrogenic in these bioassay models, the co-administration of yuehchukene and an optimal dose of 3,17 beta-estradiol (estradiol) could attenuate the maximum estrogenic response due to estradiol alone. The anti-estrogenic effect of yuehchukene in rat uterine hypertrophy was corroborated by a parallel attenuation of ornithine decarboxylase activity in these tissues. Yuehchukene binds to rat, mice and MCF-7 cell estrogen receptors with a relative binding affinity of 1/150 to 1/300. This binding affinity was positively related to estrogenicity as determined by uterotrophic assay and MCF-7 cell growth. However, this estrogenic effect did not correlate with the degree of competitive receptor binding by a weaker agonist. Indole-3-carbinol and methylbutadienylindole could induce ethoxyresorufin O-deethylase and estradiol-2-hydroxylase in rat liver and MCF-7 cells. It is postulated that the 'free' indole moiety of yuehchukene could possess similar induction activity. Thus yuehchukene may have a dual pharmacological function. While the intact molecule is a weak estrogen, the 'free' indole moiety in yuehchukene may induce an enhancement of estradiol-2-hydroxylase, thus terminating the biological activity of the endogenous estrogen pool. There is obvious benefit in attenuating the estrogen level in post-menopausal breast cancer patients without going directly to the use of tamoxifen or aromatase inhibitor. Yuehchukene may serve this purpose. In this context, the pharmacological evaluation of a hydroxylated yuehchukene analogue and the anti-estrogenic effect of methylbutadienylindole acid-condensation products are now being studied in earnest.
A wide variety of environmental contaminants have been shown to exert estrogenic actions in wildlife and laboratory animals through binding to nuclear estrogen receptors (ERs) and subsequent transcription of estrogen responsive genes. We show here that several of these environmental estrogens also bind to the novel seven-transmembrane estrogen receptor, GPR30, to activate alternative estrogen signaling pathways in an ER-negative cell line (HEK293) stably transfected with the receptor. Genestein was the most effective competitor for the receptor (IC(50) 133 nM), with a relative binding affinity (RBA) 13% that of estradiol-17beta (E2). Bisphenol A, zearalonone, and nonylphenol also had relatively high binding affinities for GPR30 with RBAs of 2-3%. Kepone, p,p'-DDT, 2,2',5',-PCB-4-OH and o,p'-DDE had lower affinities with RBAs of 0.25-1.3%, whereas o,p'-DDT, p,p'-DDE, methoxychlor and atrazine caused less than 50% displacement of [(3)H]-E2 at concentrations up to 10 microM. Overall, the binding affinities of these compounds for GPR30 are broadly similar to their affinities to the ERs. Environmental estrogens with relatively high binding affinities for GPR30 (genestein, bisphenol A, nonylphenol and Kepone) also displayed estrogen agonist activities in an in vitro assay of membrane-bound adenylyl cyclase activity, a GPR30-dependent signaling pathway activated by estrogens. The results indicate that nontraditional estrogen actions mediated through GPR30 are potentially susceptible to disruption by a variety of environmental estrogens.
Excessive exposure to synthetic and endogenous estrogens has been associated with the development of cancer in several tissues. 4-Hydroxyequilenin (4-OHEN), a major metabolite of equine estrogens present in estrogen replacement formulations, has been shown to induce cytotoxic/carcinogenic effects. In the present study, we have found that 4-OHEN caused DNA damage in breast cancer cells, and cells that contain estrogen receptor alpha (S30) are more sensitive to 4-OHEN-mediated DNA damage as compared to estrogen receptor negative cells (MDA-MB-231). For example, concentration-dependent increases in 8-oxo-deoxyguanosine (8-oxo-dG), as measured by LC-MS-MS or by the Fpg comet assay, were only detected in the S30 cells, and the amount of this lesion could be enhanced by agents, which catalyze redox cycling (NADH) or deplete GSH (diethyl maleate). The role of the estrogen receptor in modulating DNA damage was further established in incubations with the ER antagonist tamoxifen, where decreases in 8-oxo-deoxyguanosine were observed. Another equine estrogen metabolite, 4,17 beta-hydroxyequilenin (4,17 beta-OHEN), was found to have the same cytotoxicity and a similar ability to induce reactive oxygen species (ROS), and caused the same oxidative DNA damage in S30 cells as compared to 4-OHEN. However, 4,17 beta-OHEN induced twice as much single strand DNA breaks in S30 cells compared to 4-OHEN. Also 4,17 beta-OHEN was more estrogenic than 4-OHEN as demonstrated by a higher binding affinity for ER alpha and an enhanced induction in activity of estrogen-dependent alkaline phosphatase in Ishikawa cells. These data suggest that the mechanism of DNA damage induced by equine estrogen metabolites could involve oxidative stress and that the estrogen receptor may play a role in this process.
Estrogen is of importance for the regulation of hair growth and epidermal thickness. The effects of estrogen have predominantly been studied in females; however, recent studies demonstrate that estrogen also is critical for males. The aim of this study was to investigate the relative functional importance of estrogen receptor alpha and estrogen receptor beta in the regulation of the hair follicle cycling and epidermal thickness in male mice. Seven month old transgenic male mice, lacking estrogen receptor alpha (ERKO), estrogen receptor beta (BERKO), or both receptors (DERKO), were orchidectomized and treated for 3 week with 17beta-estradiol or vehicle. Orchidectomy induced a synchronized anagen phase of the hair follicles, which was inhibited by 17beta-estradiol treatment in wild-type and BERKO mice, but not in ERKO and DERKO mice. Furthermore, 17beta-estradiol treatment increased the thickness of epidermis in wild-type and BERKO mice, but not in ERKO and DERKO. This study demonstrates that estrogen is of importance for the regulation of hair follicle cycling and epidermal thickness in male mice. The effect on hair follicle cycling is caused by an estrogen receptor alpha mediated inhibition of telogen-anagen transition and the effect of estrogen to increase epidermal thickness is associated with an estrogen receptor alpha mediated increase in the proliferative rate of the keratinocytes in the basal cell layer of the epidermis.
The status of the 66-kDa human estrogen receptor-alpha (hER-alpha66) is a critical determinant in the assessment of the prognosis and in the design of treatment strategies of human breast cancer. Recently, we cloned the cDNA of an alternatively spliced variant of hER-alpha66, termed hER-alpha36; the predicted protein lacks both transcriptional activation domains of hER-alpha66 but retains its DNA-binding domain, partial dimerization, and ligand-binding domains and three potential myristoylation sites located near the N terminus. These findings thus predict that hER-alpha36 functions very differently from hER-alpha66 in response to estrogen signaling. We now demonstrate that hER-alpha36 inhibits the estrogen-dependent and estrogen-independent transactivation activities of hER-alpha66 and hER-beta. We further demonstrate that hER-alpha36 is predominantly associated with the plasma membrane where it transduces both estrogen- and antiestrogen-dependent activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and stimulates cell growth. We conclude that hER-alpha36 is a predominantly membrane-based, unique alternatively spliced variant of hER-alpha66 that acts as a dominant-negative effector of both estrogen-dependent and estrogen-independent transactivation functions signaled through hER-alpha66 and ER-beta; it also transduces membrane-initiated estrogen-dependent activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase mitogenic signaling pathway. The estrogen and antiestrogen signaling pathways mediated by hER-alpha36 provide an alternative explanation for why some human breast cancers are resistant to and others are worsened by antiestrogen therapy; the data suggest that hER-alpha36 also may be an important marker to direct therapy in human breast cancers, and perhaps hER-alpha36 also may transduce estrogen-dependent signaling in other estrogen target tissues.
The action of diethylpyrocarbonate on lamb uterine estrogen receptor produced an homogeneous population of the receptor (approximately 55%) which still bound triarylethylene antiestrogens such as 4-hydroxytamoxifen with a high affinity but bound classical potent estrogens such as estradiol or diethylstilbestrol with a very low affinity. To specify the structural features of the ligands involved in the decrease of ligand affinity upon modification of the estrogen receptor, we determined the relative affinity constants of 17 steroidal estrogens or antiestrogens (deriving from estradiol by a 7 alpha- or 11 beta-substitution) and 14 nonsteroidal estrogens or antiestrogens (all including the 1,2-trans-diphenylethylene structure of diethylstilbestrol) for native and diethylpyrocarbonate-modified estrogen receptors. Then the ratio of the relative affinity constant for the native receptor to that for the modified receptor (rho) was calculated for each ligand, to compare the variation in the affinity of the ligand upon modification of the receptor to that of 4-hydroxytamoxifen (rho = 1). The results showed that the strong decrease of ligand affinity upon modification of the receptor displayed by classical estrogens (rho greater than or equal to 200) is strictly dependent on the presence of the 17 beta-hydroxyl group in steroidal compounds or its alpha-4- and beta-4-counterparts in diethylstilbestrol-related compounds. However, for the 7 alpha- or 11 beta-derivatives of estradiol displaying potent antiestrogenic properties, the relative decrease in affinity was much more limited (rho less than or equal to 19). For 11 beta-derivatives displaying a relative estrogenic activity weaker than that of estradiol itself, an average decrease in affinity was observed (23 less than or equal to rho less than or equal to 62). With the diethylstilbestrol-related compounds, bearing or not the alpha-4-hydroxyl and/or the beta-4-hydroxy functions and showing either weak relative estrogenic or antiestrogenic properties, the relative variation in affinity was weak (0.6 less than or equal to rho less than or equal to 24). These results indicate that the interaction of 7 alpha- or 11 beta-substituted steroidal antiestrogens and of 1,2-trans-diphenylethylene or triphenylethylene derivatives, displaying either weak relative estrogenic or antiestrogenic properties with the receptor, differs at the 17 beta-hydroxy or at the alpha-4-/beta-4-hydroxy functions from that of potent estrogens. They suggest that the strong decrease in the relative affinity of ligands upon receptor modification may reflect the high efficiency of the ligands to activate the receptor properly.(ABSTRACT TRUNCATED AT 400 WORDS)
The rat, mouse and human estrogen receptor (ER) exists as two subtypes, ER alpha and ER beta, which differ in the C-terminal ligand-binding domain and in the N-terminal transactivation domain. In this study, we investigated the estrogenic activity of environmental chemicals and phytoestrogens in competition binding assays with ER alpha or ER beta protein, and in a transient gene expression assay using cells in which an acute estrogenic response is created by cotransfecting cultures with recombinant human ER alpha or ER beta complementary DNA (cDNA) in the presence of an estrogen-dependent reporter plasmid. Saturation ligand-binding analysis of human ER alpha and ER beta protein revealed a single binding component for [3H]-17beta-estradiol (E2) with high affinity [dissociation constant (Kd) = 0.05 - 0.1 nM]. All environmental estrogenic chemicals [polychlorinated hydroxybiphenyls, dichlorodiphenyltrichloroethane (DDT) and derivatives, alkylphenols, bisphenol A, methoxychlor and chlordecone] compete with E2 for binding to both ER subtypes with a similar preference and degree. In most instances the relative binding affinities (RBA) are at least 1000-fold lower than that of E2. Some phytoestrogens such as coumestrol, genistein, apigenin, naringenin, and kaempferol compete stronger with E2 for binding to ER beta than to ER alpha. Estrogenic chemicals, as for instance nonylphenol, bisphenol A, o, p'-DDT and 2',4',6'-trichloro-4-biphenylol stimulate the transcriptional activity of ER alpha and ER beta at concentrations of 100-1000 nM. Phytoestrogens, including genistein, coumestrol and zearalenone stimulate the transcriptional activity of both ER subtypes at concentrations of 1-10 nM. The ranking of the estrogenic potency of phytoestrogens for both ER subtypes in the transactivation assay is different; that is, E2 >> zearalenone = coumestrol > genistein > daidzein > apigenin = phloretin > biochanin A = kaempferol = naringenin > formononetin = ipriflavone = quercetin = chrysin for ER alpha and E2 >> genistein = coumestrol > zearalenone > daidzein > biochanin A = apigenin = kaempferol = naringenin > phloretin = quercetin = ipriflavone = formononetin = chrysin for ER beta. Antiestrogenic activity of the phytoestrogens could not be detected, except for zearalenone which is a full agonist for ER alpha and a mixed agonist-antagonist for ER beta. In summary, while the estrogenic potency of industrial-derived estrogenic chemicals is very limited, the estrogenic potency of phytoestrogens is significant, especially for ER beta, and they may trigger many of the biological responses that are evoked by the physiological estrogens.
Estrogen seems to promote and maintain the typical female type of fat distribution that is characterized by accumulation of adipose tissue, especially in the sc fat depot, with only modest accumulation of adipose tissue intraabdominally. However, it is completely unknown how estrogen controls the fat accumulation. We studied the effects of estradiol in vivo and in vitro on human adipose tissue metabolism and found that estradiol directly increases the number of antilipolytic alpha2A-adrenergic receptors in sc adipocytes. The increased number of alpha2A-adrenergic receptors caused an attenuated lipolytic response of epinephrine in sc adipocytes; in contrast, no effect of estrogen on alpha2A-adrenergic receptor mRNA expression was observed in adipocytes from the intraabdominal fat depot. These findings show that estrogen lowers the lipolytic response in sc fat depot by increasing the number of antilipolytic alpha2A-adrenergic receptors, whereas estrogen seems not to affect lipolysis in adipocytes from the intraabdominal fat depot. Using estrogen receptor subtype-specific ligands, we found that this effect of estrogen was caused through the estrogen receptor subtype alpha. These findings demonstrate that estrogen attenuates the lipolytic response through up-regulation of the number of antilipolytic alpha2A-adrenergic receptors only in sc and not in visceral fat depots. Thus, our findings offer an explanation how estrogen maintains the typical female sc fat distribution because estrogen seems to inhibit lipolysis only in sc depots and thereby shifts the assimilation of fat from intraabdominal depots to sc depots.
We investigated the estrogenic activity of various environmental pollutants (xenobiotics), in particular the xenoestrogen o,p-DDT, and compared their effects with those of endogenous estrogens, phytoestrogens, and mycoestrogens on estrogen receptor binding capacity, induction of estrogen end products, and activation of cell proliferation in estrogen-sensitive human breast cancer cells in monolayer culture. We also quantified the levels of phytoestrogens in extracts of some common foods, herbs, and spices and in human saliva following consumption of a high phytoestrogen food source (soy milk) to compare phytoestrogen abundance and bioavailability relative to the reported xenoestrogen burden in humans. Results show that natural endogenous estrogens, phytoestrogens, mycoestrogens, and xenoestrogens bind estrogen receptor (ER) in intact cells, but demonstrate marked differences in their ability to induce end products of estrogen action and to regulate cell proliferation. All of the different classes of estrogens stimulated cell proliferation at concentrations that half-saturated ER, but only some classes were able to induce estrogen-regulated end products. Genistein, a common phytoestrogen found in soy foods, differed from the xenoestrogen DDT in its effects on cell proliferation and ability to induce estrogen-regulated end products. Moreover, we found that many of the foods, herbs, and spices commonly consumed by humans contain significant amounts of phytoestrogens, and consumption of soy milk, a phytoestrogen-rich food, markedly increases the levels of phytoestrogens in saliva. In conclusion, our in vitro results predict that a diet high in phytoestrogens would significantly reduce the binding of weak xenoestrogens to ER in target tissues in vivo.
Recent studies have revealed that hundreds of genes in the uterus are activated by estrogen. Their expression profiles differ over time and doses and it is not clear whether all these genes are directly regulated by estrogen via the estrogen receptor. To select the genes that may be regulated by estrogen, we treated mice with several doses of estrogen and searched for those genes whose dose-response expression pattern mirrored the uterine growth pattern. Among those genes, we found that the dose-dependent expression of the adrenomedullin (ADM) gene correlated well with the uterotrophic effect of estrogen. ADM expression is induced early after estrogen administration and is restricted to the endometrial stroma. The spatiotemporal gene expression pattern of ADM was similar to that of receptor-modifying protein 3 (RAMP3). RAMP3 is known to modify calcitonin gene-related receptor (CRLR) so that it can then serve as an ADM receptor. Chromatin immunoprecipitation assays indicated that the estrogen receptor binds directly to the ADM promoter region and RAMP3 intron after estrogen administration. It was also shown that neither the ADM nor RAMP3 gene could be activated in estrogen receptor-alpha-null mouse. Although uterine ADM expression has been reported to occur in the myometrium, our observations indicate that estrogen-induced ADM is also expressed in the uterine stroma and that such variable, spatiotemporally regulated ADM expression contributes to a wider range of biological effects than previously expected.