Estrogen Receptor In Rat Uterus Research Articles

Cadmium modulates expression of aryl hydrocarbon receptor-associated genes in rat uterus by interaction with the estrogen receptor

Estrogen-like effects of the heavy metal cadmium have been reported in both in vitro and in vivo studies. Yet, the molecular mechanisms involved in the hormonal activity of cadmium ions have not been fully elucidated. There are extensive data on cross-talk between aryl hydrocarbon receptor (AhR) and estrogen receptor (ER). Recently, 17β-estradiol (E(2)) was found to modulate the expression of AhR and AhR-regulated genes in rat uterus (Kretzschmar et al. in Mol Cell Endocrinol 321:253-257, 2010). Thus, we hypothesized that cadmium may also affect AhR signaling and examined whether cadmium or E(2) modulate AhR-associated genes via the ER in rat uterus. Ovariectomized Wistar rats received E(2) (0.5 mg/kg bw) or cadmium chloride (0.05 and 2 mg/kg bw i.p.) alone and in combination with the pure anti-estrogen ZK191703. We also co-treated a group with E(2) and cadmium 2 mg/kg bw to assess how they act in concert. Uterus wet weight, uterus epithelial height, complement C3 mRNA, and progesterone receptor (PR) protein expression served as estrogen response parameters, and expression of Mt1a mRNA was analyzed as a cadmium responsive gene. The expression of AhR protein and AhR-associated gene expression, i.e., Ahr, Arnt1, Arnt2, Cyp1a1, and Gsta2, were analyzed to examine effects on AhR-mediated signaling pathways in the uterus of all groups. Both, E(2) and cadmium induced C3 and PR expression, and this was antagonized by ZK191703. Mt1a expression was clearly induced by cadmium but slightly reduced by E(2) compared to controls. Uterine Ahr, Arnt1, Arnt2, and Cyp1a1 expression was modulated by E(2) via the ER since down-regulation by E(2) was reversed by anti-estrogen. Cadmium apparently also modulated Cyp1a1 expression via the ER. Furthermore, cadmium-induced AhR was antagonized by E(2,) and anti-estrogen-induced Gsta2 expression was antagonized by cadmium. Together our findings provide evidence for cross-talk of ER and AhR in the rat uterus.

Expression of estrogen receptor co-regulators SRC-1, RIP140 and NCoR and their interaction with estrogen receptor in rat uterus, under the influence of ormeloxifene

Ormeloxifene binds competitively to ERs and antagonizes estrogen-induced gene expression in the uterus. However its detailed molecular mechanisms are not well understood. Present study was aimed to examine the changes in expression pattern of co-regulatory proteins SRC-1 (co-activator), RIP140 and NCoR (co-repressors) and their interaction with ERα in rat uterus under the influence of ormeloxifene (Orm) and tamoxifen (Tam). Adult ovariectomized rats were treated with estradiol (E 2) (5 μg/100 g), or Orm or Tam (200 μg/100 g, s.c.) alone or along with E 2, for 3 days. RT-PCR analysis of uterine RNA and immunoblotting of uterine extracts revealed that expression of SRC-1, RIP140 and NCoR was insensitive to E 2 or Orm or Tam treatment. Direct protein–protein interaction experiments using co-immunoprecipitation revealed that E 2-induced the interaction of ERα with co-activator SRC-1. In rats given Orm alone or along with E 2, there was a significant reduction in E 2-induced effect on ERα–SRC-1 interaction. In case of ERβ and SRC-1, Orm reduced interaction only in the absence of E 2. Interaction of RIP140 or NCoR with ERα was found to be more in rats treated with Orm along with E 2 as compared to that in E 2-treated rats whereas no such recruitment was found in Tam treated rats. Interaction of RIP140 with ERβ was insensitive to Orm or Tam treatment whereas the interaction of NCoR with ERα and ERβ was increased in Orm treated rats. Ormeloxifene also showed inhibitory effects on uterine ER–ERE binding and estrogen-induced expression of progesterone receptor. Taken together, these findings demonstrate that ormeloxifene antagonizes ERα-mediated transcription by inhibiting the recruitment of SRC-1 and inducing the recruitment of RIP140 and NCoR.

Estrogenic Activity of Stilbene Derivatives

We previously reported that trans-stilbene is metabolically activated to estrogenic compounds by a liver microsomal enzyme system. In this study, we demonstrated the structural requirement for estrogenic activity of various stilbene derivatives including proestrogens. High estrogenic activity in 4,4′-dihydroxystilbene, 4-amino-4′-hydroxystilbene, 4,4′-dihydroxy-α-methylstilbene, hexestrol, and diethylstilbestrol (DES), moderate activity in 4-hydroxystilbene, 4-aminostilbene, 4-hydroxyazobenzene, and 4-hydroxy-4′-nitrostilbene, low activity in 4-nitrostilbene, 4,4′-dihydroxydibenzyl, resveratrol, and 4-hydroxy-α-methylstyrene, and marginal activity in 4,4′-dimethoxystilbene and 4-hydroxymethylstilbene were observed in an estrogen reporter assay using the estrogen-responsive human breast cancer cell line MCF-7 and a binding assay with rat uterus estrogen receptor. In contrast, α-methylstilbene, 4,4′-dimethoxystilbene, 4-hydroxymethylstilbene, dibenzyl, tolan and azobenzene also exhibited estrogenic activities after incubation with liver microsomes of 3-methylcholanthrene- or phenobarbital-treated rats in the presence of NADPH. These results suggest that the structural requirements for the estrogenic activities of stilbene derivatives are a p-hydroxyl group in the A-phenyl ring, vinyl linkage, and a B-phenyl ring for the maximal activity, and hydrophobicity of the linkage for higher activity as observed in DES. p-Nitro and amino groups in the A-phenyl ring are also effective for the estrogenic activity.

Metabolic Activation of Proestrogenic Diphenyl and Related Compounds by Rat Liver Microsomes

In this study, liver microsome-mediated activation of diphenyl (DP), diphenylmethane (DPM) and 2,2-diphenylpropane (DPP) to estrogens was demonstrated. These three compounds were negative in estrogen reporter assay using estrogen-responsive human breast cancer cell line MCF-7. However, they exhibited estrogenic activity after incubation with liver microsomes of 3-methylcholanthrene-treated rats in the cases of DP and DPM, or of phenobarbital-treated rats in the cases of DP and DPP, in the presence of NADPH. When these compounds were incubated with liver microsomes in the presence of NADPH, monohydroxyl and dihydroxyl derivatives were formed. These hydroxylated metabolites, 4-hydroxydiphenyl, 3-hydroxydiphenyl, 2-hydroxydiphenyl, 4-hydroxydiphenylmethane, 2-(4-hydroxyphenyl)-2-phenylpropane (4-OH-DPP), 4,4′-dihydroxydiphenyl, 4,4′-dihydroxydiphenylmethane and 2,2-bis(4-hydroxyphenyl)propane (bisphenol A), all exhibited estrogenic activity in MCF-7 cells. Binding assay of these hydroxylated compounds with rat uterus estrogen receptor was also positive. These results suggest that the estrogenic activities of DP, DPM and DPP were due to the formation of hydroxylated metabolites by the liver cytochrome P450 system.

Effect of Estrogen on Ontogenic Expression of Progesterone and Estrogen Receptors in Rat Uterus

The ontogenic expression of progesterone and estrogen receptors (PR and ER) and effect of estrogen on these receptors were investigated immunohistochemically in rat uterus from the day of birth ( = 0 day) to 30 days of age. Uterine epithelial and stromal cells showed a negative PR immunoreaction at 0 day. The PR in the epithelial cell nuclei appeared by 5 days, while the stromal cells showed a negative PR reaction until 12 days. The staining of the stromal cells appeared from 12 to 15 days. In both the epithelial and stromal cells, the initiation of the PR appearance was not affected by ovariectomy performed at 0 day or 5 days prior to the appearance of PR in the epithelial and stromal cells. Estrogen injections from 0 day failed to initiate the appearance of PR in the epithelial cells, regardless of doses of estradiol-17 beta (0.1, 1 and 10 micrograms daily), but induced PR in the stromal cells. The staining of ER appeared at 5 days in the epithelial cells and at 1 day in the stromal cells, respectively. ER appeared after 2-3 daily injections of estrogen from 0 day depending upon the doses. These results suggest that steroid hormones secreted from neonatal ovary do not play any important role in ontogenic expression of PR during the postnatal uterine maturation.

Androgen regulation of the insulin-like growth factor-I and the estrogen receptor in rat uterus and liver

For the first time testosterone is shown to be an important regulator of the insulin-like growth factor-I (IGF-I) in the rat uterus under in vivo conditions. In this study the regulation of IGF-I and the estrogen receptor (ER) by gonadal steroids in the uterus and liver of female rats was monitored. The ER level was assayed by hormone binding after treatment with testosterone, 5α-dihydrotestosterone or estradiol and specific mRNA species were analyzed by a solution hybridization/RNase protection assay using 35S-labeled RNA probes. Ovariectomized rats restored uterine weight after treatment with testosterone. Uterine IGF-I mRNA was more than 20-fold higher in testosterone treated rats compared to untreated ovariectomized controls after 48 h treatment. The effects of testosterone on ovariectomized animals was followed in a timecourse study. Testosterone administration increased uterine IGF-I mRNA expression during the first 48 h and the maximally induced level was maintained throughout the duration of the experiment (168 h). Since induction of IGF-I mRNA by estrogen is transient, these data indicate that androgen and estrogen increase IGF-I mRNA by different mechanisms. Regulation of IGF-I mRNA by gonadal steroids was also studied in hypophysectomized animals. The rats were given either testosterone, 5α-dihydrotestosterone or estradiol, and uterine IGF-I mRNA was measured after 1 week of treatment. At this timepoint estrogen treated rats showed levels of IGF-I mRNA not significantly different from those of hypophysectomized controls. In contrast testosterone and 5α-dihydrotestosterone increased the IGF-I mRNA level 30 and 40 times, respectively, relative to hypophysectomized control animals. Since 5α-dihydrotestosterone is not convertable to estrogen, the induction by testosterone was considered to be a true androgenic phenomenon.

Evidence for involvement of tyrosine in estradiol binding by rat uterus estrogen receptor

The possibility of tyrosine involvement in steroid binding by rat uterus estrogen receptor (rER) was investigated. Chemical modification of rER with reagents such as tetranitromethane (TNM) and N-acetylimidazole (NAcI) inhibited estradiol binding. Steroid binding was inhibited to a greater extent at pH 8 than at pH 6, indicating the participation of tyrosine (TNM has increasing affinity for cysteine over tyrosine at pH 6). Inhibition patterns remained similar for incubations at 0–4°C or 37°C. NAcI inhibited rER steroid binding at 37°C, but not at 0–4°C. Hydroxylamine incubated in the presence of rER and NAcI appeared to reverse this inhibition. Thus, these findings indicate that the phenyl ring and possibly the phenolic hydroxyl of tyrosine are involved in steroid binding of the receptor.

Binding of aromatic amines to the rat hepatic Ah receptor in vitro and in vivo and to the 8S and 4S estrogen receptor of rat uterus and rat liver.

Studies on structurally related aromatic amines with different carcinogenic properties have shown that 2-acetylaminofluorene (2-AAF) and 2-acetylaminophenanthrene (AAP) inhibit the binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin to the Ah receptor in vitro. The apparent inhibitor constants (Ki) are 2.3 microM for 2-AAF and 2.7 microM for AAP. In contrast, 4-acetylaminofluorene, an isomer of 2-AAF, and trans-4-acetylaminostilbene do not bind to the rat hepatic cytosolic Ah receptor. Pretreating female Wistar rats with 2-AAF or AAP leads to the induction of the P-450 isoenzymes that are under the control of the Ah receptor. Ornithine decarboxylase activity is induced by all aromatic amines tested irrespective of their Ah receptor affinity. The aromatic amines used as model compounds do not inhibit the binding of 17-beta-estradiol to the 8S and 4S estrogen receptor of rat uterus or rat liver in a competition assay analyzed using sucrose density gradient centrifugation. On the other hand, the aromatic amines bind to varying extents to another estrogen-binding protein of rat liver whose function and identity is still unknown. Our study demonstrates that structurally related aromatic amines in their unmetabolized form interact differentially with a cellular target protein, the Ah receptor, in vitro as well as in vivo. However, a relationship between these effects and the postulated promoting properties of 2-AAF remains to be established.

Binding of Aromatic Amines to the Rat Hepatic Ah Receptor In Vitro and In Vivo and to the 8S and 4S Estrogen Receptor of Rat Uterus and Rat Liver

Binding of Aromatic Amines to the Rat Hepatic Ah Receptor In Vitro and In Vivo and to the 8S and 4S Estrogen Receptor of Rat Uterus and Rat Liver

Tissue-specific regulation of rat estrogen receptor mRNAs.

The estrogen receptor (ER) is present in a wide variety of mammalian tissues and is required for physiological estrogen responses, including estrogen-induced tissue-specific changes in gene expression. We studied the estrogen regulation of the mRNAs encoding the ER in rat uterus, liver, and pituitary. Ovariectomized (21-28 day post surgery) female CD-1 rats were injected daily with 17 beta-estradiol (E2, 10 micrograms/100 g BW) for 0, 1, or 4 h, 1, 3, or 7 days and compared with intact controls. Steady-state levels of ER mRNA were quantified using a human ER cDNA probe. Only one hybridizing species of approximately 6.2 kilobase (kb) was detected in uterine and liver RNA, similar to that observed in MCF7 human breast cancer cells. However, the ER mRNA regulation by E2 differed in direction depending on the tissue examined. In uterus, ER mRNA increased 3- to 6-fold after ovariectomy, and returned to intact levels within 24 h of E2 replacement. In contrast, liver ER mRNA declined 1.5- to 3-fold after ovariectomy and returned to intact levels after 1-3 days of E2. In pituitary tissue two hybridizing forms of ER mRNA were observed, with one species migrating at 6.2 kb, equivalent to the form in other tissues, and a second smaller species at approximately 5.5 kb. The lower molecular weight species varied somewhat in abundance from animal to animal, averaging about 20% of the intensity of the 6.2 kb band. The ER mRNA forms were regulated positively with E2.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of estrogen-nitrosourea conjugates with the estrogen receptor in rat uterus

Several estrogen-nitrosourea conjugates have been synthesized with the aim of producing more selective cytotoxic agents. The conjugates were shown to compete with estradiol for binding to cytosolic estrogen receptor in rat uterus; the relative binding affinities for N-(2-chloroethyl)- N′-(3,17 β-dihydroxyestra-1,3,5 (10)-trien-17 α-yl)methyl- N-nitrosourea (17α-CNU), N-(2-chloroethyl)- N′-(3-hydroxyestra-1, 3,5(10)-trien-17 β-yl)- N-nitrosourea (17β-CNU), and N-(2-chloroethyl)- N′-2,3-di( p-hydroxyphenyl)-pentanyl- N-nitrosourea (HEX-CNU) were 2,0.4, and 0.2, respectively, using a binding affinity of 100 for estradiol. In the ligand exchange assay, cytosolic receptors preloaded with 17α-CNU and HEX-CNU were found to lose some of their estradiol (E 2) binding sites, suggesting that binding to estrogen receptor (ER) may be irreversible. An increase of nuclear accumulation of ER was observed in the presence of 17α-CNU and HEX-CNU. In the rat system, even at a 10,000-fold excess, these two agents failed to show any antagonism of the uterotrophic effect of E 2 in vivo. The low binding affinity and instability of these conjugates may account for their lack of antiestrogen activity. On the other hand, 17α-CNU at 100 or 1000 μg/day and HEX-CNU at 1000 μg/day demonstrated significant uterotrophic activity. This study did not resolve whether the stimulation of uterine growth was due to the parent estrogen-nitrosourea conjugate or to decomposition and/or metabolic products.

Estrogen receptor binding material in blood of patients after clomiphene citrate administration: determination by a radioreceptor assay

The aim of the current study was to assess whether clomiphene citrate (CC) and/or active metabolites are present at presumed time of ovulation, nidation, or steroid-sensitive organogenesis, in serum of patients receiving CC for induction of ovulation. A radioreceptor assay, based on competitive replacement of 3 H-estradiol on the rat uterus estrogen receptor, by ligands present in serum of patients after CC administration, was developed. Ligands reached maximal concentration 4 to 5 hours after a single dose of CC was administered, and declined with a half-life of 4.5 to 10 hours. In patients receiving CC on day 5 to day 9 in the cycle, ligands are still present on day 14 in the cycle and in some patients on day 22 of the cycle, but no ligands were detected 60 days after CC treatment.

Different interaction of estradiol and antiestrogens with the estrogen receptor of rat uterus

Cytosoluble estradiol-receptor (ER) complexes obtained from uteri of castrated rats show a 4.3 S sedimentation coefficient when analysed by sucrose gradient in high salt buffer both in the absence and presence of 0.1 M ammonium thiocyanate. After incubation with nuclei, the complexes sediment at 4.3 S and at 3.2 S, in the absence and presence of 0.1 M ammonium thiocyanate respectively. This structural modification of ER is also confirmed by gel filtration analysis. In similar conditions hydroxytamoxifen-ER complexes apparently do not undergo such a modification. However, increasing the molarity of the chaotropic ion up to 0.5 M shows that, in fact, the modification occurs. Sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis of covalently linked tamoxifen aziridine-ER ‘complexes’ confirms that the nuclear treatment reduces the apparent molecular weight of ER from 62 000 to 47 000. These data demonstrate that, in vitro, nuclear ER is cleaved into a smaller molecular form and that the receptor fragments can be held together by the triphenylethylene antiestrogen hydroxytamoxifen.

Characterization of an estrogen-binding protein in the yeast Saccharomyces cerevisiae.

This paper further characterizes the estrogen-binding protein we have described in the cytosol of the yeast Saccharomyces cerevisiae. [3H]Estradiol was used as the radioprobe, and specific binding of cytosol fractions was measured by chromatography on Sephadex minicolumns. Other 3H-steroids did not exhibit specific binding. [3H]Estradiol binding was destroyed by treatment with trypsin, but not RNase, DNase, or phospholipase; N-ethylmaleimide substantially decreased the binding. The yeast did not metabolize estradiol added to the medium, and extraction and chromatography of the bound moiety showed it to be unmetabolized estradiol. Scatchard analysis of cytosol from both a and alpha mating types as well as the a/alpha diploid cell revealed similar binding properties: an apparent dissociation constant or Kd(25 degrees) for [3H]estradiol of 1.6-1.8 nM and a maximal binding capacity or Nmax of approximately 2000-2800 fmol/mg of cytosol protein. Gel exclusion chromatography on Sephacryl S-200 and high performance liquid chromatography suggested a Stokes radius of approximately 30 A. Sucrose gradient centrifugation showed a sedimentation coefficient of approximately 5 S, and the complex did not exhibit ionic dependent aggregation. The estrogen binder in S. cerevisiae differed in its steroidal specificities from classical mammalian estrogen receptors in rat uterus. 17 beta-Estradiol was the best competitor, 17 alpha-estradiol had about 5% the activity, and diethylstilbestrol exhibited negligible binding affinity as did tamoxifen, nafoxidine, and the zearalenones. In summary, a high affinity, stereospecific, steroid-selective binding protein has been demonstrated in the cytosol of the simple yeast S. cerevisiae. We speculate that this molecule may represent a primitive hormone receptor system, possibly for an estrogen-like message molecule.

Effect of pregnancy and local effect of implantation on thymidine kinase and estrogen receptor in rat uterus (author's transl)

We have previously reported that estradiol (E2) administration induces a particular isozyme of thymidine kinase (TK) in the uterus of immature rats but not of adult rats. This peculiar TK isozyme, which seems to be related to DNA synthesis in myometrial cells induced by E2 diminishes with the maturation of the rat. Growth with age and hypertrophy during pregnancy are similar phenomena from the standpoint of physiological uterine development. We are interested in the effect of pregnancy on TK activity and the cytoplasmic and nuclear estrogen receptor (ER) in the uterus. In the present study, an oviduct on one side was sectioned previous to mating, in order to compare TK and ER between the non-implanted and implanted uterine horns of the same rat during one to three weeks of pregnancy. No implantation was found in the uterine horn with the sectioned oviduct. In both the nonimplanted and implanted uterine horns, no increment of TK activity and no appearance of the specific TK isozyme were found during pregnancy. It seems that uterine growth with maturation is not similar to uterine hypertrophy during pregnancy. A local effect of E2 supplied by trophoblast is not evident in the level of TK. The cytoplasmic ER level of the nonimplanted horn was approximately twice that of the implanted horn. Unchanged levels of nuclear ER were shown in both uterine horns during pregnancy. The lower concentration of cytoplasmic ER in the implanted horn than in the nonimpalnted horn during pregnancy might be caused by the local effect of E2 supplied by trophoblast. These results indicated that uterine development during pregnancy might be caused by an enlargement of individual myometrial cells, and the local effect of steroids supplied by trophoblast might support ovum-implantation to the adjacent endometrium.

Studies on Estrogen Receptors in Rat Uterus during Protein Malnutrition and Steroid-Maintained Pregnancy

The present studies were performed to determine the nature and subcellular distribution of estrogen receptors (ER) in rat uterus, exposed to several cellular and hormonal responses as a result of dietary protein restriction. In protein-deficient rats, the concentration of estrogen receptors in uterine nuclei was lowered as compared to animals fed an 18% casein diet. No remarkable changes were observed in the cytosol estrogen receptor concentration in protein-deficient groups when values were expressed per milligram DNA. On day 15 of normal and steroid-maintained pregnancy, most of the receptor population was localized in the nuclear compartment with a significant decrease in cytosol receptor levels, as compared to virgin controls. However, on day 20 of steroid-maintained pregnancy, a marked reduction was recorded both in nuclear and cytosol estrogen receptors, as compared to that of day 15. Translocation study with protein-deficient rats revealed that the pattern of nuclear accumulation of estrogen receptors as a function of time was identical to 18% casein-fed group. However, the magnitude of translocated receptors was significantly higher at 2 hours after hormone treatment in protein-deficient animals. Additionally, the antagonistic, antiestrogenic effect of progesterone on estrogen receptor distribution was more pronounced in the protein-deficient group. The changes in estrogen receptor concentrations and their correlation with hormonal variations during protein malnutrition have been discussed.

ラット子宮エストロジェン受容体とッェラレノンの相互作用

ラット子宮エストロジェン受容体とッェラレノンの相互作用

Mechanism of interaction of digitalis with estradiol binding sites in rat uteri.

Digitalis preparations have a weak estrogenic effect in man. The data in animals are equivocal. We have studied.the biologic effect of both digitoxin and digoxin on the rat uterus in vivo and the interaction of these drugs with the rat uterus estrogen receptor in vitro. Digitoxin and estradiol significantly increased the uterine weight of immature rats, while digoxin did not. The interaction of digitoxin and digoxin with the rat uterus estrogen cytosol receptor was studied using protamine sulfate precipitation and dextran-coated charcoal (DCC) assays. Both methods gave a Kd for the estradiol-receptor interaction between 0.8-3.1 X 10(-9) M (n = 20). Digitoxin at concentrations of 0.5-2.0 X 10(-6) M significantly inhibited the binding of estradiol to the specific or saturable binding sites with minimal inhibition of hormonal binding to nonspecific sites. The binding was competitive with a calculated Ki for digitoxin of 5.2-7.8 X 10(-7) M (n = 18). Digoxin failed to inhibit estradiol binding to the receptor protein in vitro. We conclude that digitoxin probably acts directly as a weak estrogen and that this effect probably explains the estrogen-like side effects seen with digitoxin therapy in man.