Nuclear Estrogen Receptor Complexes Research Articles

Analysis of the antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in human ovarian carcinoma BG-1 cells.

We have used human ovarian carcinoma BG-1 cells to determine which steps in the pathway of estrogen signaling are disrupted by the aryl hydrocarbon receptor (AhR) ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We report that inhibition of estrogen signaling occurs between 7 and 18 h after TCDD treatment and that this effect is not caused by a decrease in estradiol concentration. TCDD decreased estrogen receptor (ER) levels in cells grown in standard medium; however, in estrogen-stripped medium, ER (but not AhR) levels were dramatically reduced (approximately 7-fold) but were not decreased further by TCDD. Because the absolute level of estradiol inducibility and inhibition by TCDD was similar in either medium, decreases in ER are not responsible for the antiestrogenic effect. The AhR also did not bind to the estrogen-responsive element (ERE) in vitro, and ERE binding by nuclear ER complexes was not decreased by TCDD, indicating that the effect of TCDD does not involve direct competition between the AhR and ER for DNA binding. However, inhibition of protein synthesis by cycloheximide blocked the TCDD-induced inhibition of ER-dependent gene expression. Overall, our results are consistent with the action of a TCDD-induced protein at a step(s) after ER-DNA binding, most likely at the level of gene transcription.

6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) is an antiestrogen in human and rodent cancer cell lines: Evidence for the role of the Ah receptor

6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) is a relatively nontoxic analog of 2,3,7,8-tetrachlorodibenzo- p-dioxin. Treatment of aryl hydrocarbon (Ah)-responsive MCF-7 human breast cancer cells with 100 n m MCDF resulted in the inhibition of 17β-estradiol-induced proliferation and the secretion of the 34-, 52-, and 160-kDa proteins. After treatment of the cells with 17β-[ 3H]estradiol, 100 n m of MCDF caused a decrease in the accumulation of the radiolabeled nuclear estrogen receptor (ER) complex in these cells. In parallel experiments, the antiestrogenic effects of MCDF were also determined in Ah-responsive wild-type Hepa 1c1c7 cells and Ah-nonresponsive class 1 and class 2 mutant cells. Treatment of the wild-type cells with 17β-[ 3H]estradiol and 100 n m MCDF caused a decrease in the accumulation of radiolabeled nuclear ER complex in these cells whereas no significant effects were observed in the mutant cells as determined by velocity sedimentation analysis. Comparable results were obtained using ER antibodies to measure the decrease in immunoreactive nuclear ER. In addition, both actinomycin D and cycloheximide inhibited the MCDF-mediated decrease of nuclear ER levels in the Hepa 1c1c7 wild-type cells. Although 100 n m MCDF did not induce cytochrome P-450-dependent monooxygenases in the MCF-7 or Hepa 1c1c7 cell lines, incubation of nuclear extracts from the MCF-7 cells treated with 100 n m MCDF with a synthetic consensus dioxin responsive element (an oligonucleotide duplex of 26 bases) gave a retarded band in a gel-retardation assay. The data suggest that the antiestrogenic effects of MCDF does not require the induction of the CYP1A1 gene expression but may involve the induction of other genes.

Uterine Estrogen Receptor-DNA Complexes: Effects of Different ERE Sequences, Ligands, and Receptor Forms

Previous studies used the gel retardation assay to examine the binding of the mouse estrogen receptor (ER) to the estrogen-responsive element (ERE) from the vitellogenin A2 gene (VitA2ERE). Multiple specific complexes were formed when the ER was bound to various estrogen agonists or antagonists, or in the absence of bound hormone. The ERE from the human PS2 gene, which varies from the consensus ERE by one base change in the right arm, was used in this study to determine the effect of DNA sequence on ER-ERE interaction with various ligand-receptor complexes. Partially purified ligand-free soluble ER showed a 3-fold lower affinity for the PS2ERE than for the VitA2ERE, suggesting a possible influence of the imperfect DNA sequence on certain binding interactions. However, multiple complexes of similar affinity were formed with the PS2 sequence by nuclear ER regardless of the agonist or antagonist bound. In gel retardation experiments, antagonist (LY117018) nuclear ER complexes bound to either PS2 or VitA2ERE migrated more slowly than agonist complexes, indicating that the slower migrating form of the complex was not due to the DNA sequence. Interestingly, soluble ER bound by LY 117018 did not produce this decreased mobility complex, suggesting that it was specific to the nuclear form of the ER antagonist complex. Receptor activation has been linked with exposure to increased temperature, resulting in an ER form that has an increased affinity for DNA. The binding of molybdate-stabilized nonactivated 8S ER to VitA2ERE was studied to determine the effect of temperature on ER binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for the mechanism of action of the 2,3,7,8-tetrachlorodibenzo- p-dioxin-mediated decrease of nuclear estrogen receptor levels in wild-type and mutant mouse hepa 1c1c7 cells

Treatment of wild-type Hepa 1c1c7 cells with 1 nM [ 3H]-17β-estradiol resulted in the rapid accumulation of the nuclear estrogen receptor complex whose levels were maximized within 1 hr. Cotreatment of the cells with 10 nM 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and [ 3H]-17β-estradiol did not affect the nuclear estrogen receptor levels 1 hr after addition of the radioligand; however, pretreatment of the cells for 1, 6, 24 or 42 hr with 10 nM TCDD prior to the addition of the radiolabeled hormone caused a greater than 50% decrease in nuclear estrogen receptor levels (determined by velocity sedimentation analysis) 1 hr after the addition of [ 3H]-17β-estradiol. In parallel experiments in which 10 nM TCDD was added 6 hr prior to the radiolabeled hormone, TCDD caused a 63 and 74% decrease in immunodetectable cytosolic and nuclear estrogen receptor protein levels, respectively, in the wildtype Hepa 1c1c7 cells. The nuclear estrogen receptor was also detected in two Hepa 1c1c7 mutant (class 1 and class 2) cell lines which have been characterized previously as TCDD non-responsive due to either decreased aryl hydrocarbon ( Ah) receptor levels or a defect in the accumulation of transcriptionally active nuclear Ah receptor complexes, respectively. Treatment of these mutant cell lines with TCDD and [ 3H]-17β-estradiol (as described above) caused only a minimum (class 1) or non-detectable (class 2) decrease in nuclear estrogen receptor binding activity or immunodetectable protein levels. These results, coupled with the structure-dependent differences in the activities of TCDD (a strong Ah receptor agonist) and 2,8-dichlorodibenzo- p-dioxin (a weak Ah receptor agonist) in this assay system, support a role for the Ah receptor in the TCDD-mediated decrease of the nuclear estrogen receptor in mouse Hepa 1c1c7 cells. In addition, actinomycin D and cycloheximide both inhibited the TCDD-mediated decrease of nuclear estrogen receptor levels in the Hepa 1c1c7 wild-type cells, and these results suggest that TCDD may induce specific gene products which are involved in this process.

Effects of Charcoal on Dissociation Kinetics of Nuclear and Cytosolic Steroid-Receptor Complexes from Hen Oviduct

The observed rate of dissociation of radioactive estradiol from nuclear estrogen-receptor complexes from hen oviduct has been shown to depend to a large extent on the method used to initiate dissociation. The present study indicates that nuclear progesterone receptors display the same pattern of behavior. Dissociation kinetics of nuclear progesterone receptor extracted from hen oviducts were affected by the method of initiation of dissociation; the rate of dissociation at 25 degrees C when dissociation was initiated with unlabeled steroid was three times that observed when dissociation was initiated by addition of a charcoal/dextran suspension. In contrast to nuclear receptors, both estrogen and progesterone receptors prepared from cytosol displayed only a single rate of dissociation, no matter what the method of initiation of dissociation. These results strengthen the idea that nuclear receptors contain a factor or subunit which may be removed by charcoal, which alters the rate of dissociation of steroid from the complex.

Effect of clomiphene on nuclear estrogen receptor of the fallopian tube during ovum transport in rabbits.

The effect of clomiphene on nuclear estrogen receptors of the Fallopian tube during ovum transport in the rabbit has been studied. Nuclear binding capacity was observed in ampulla (A), ampullary-isthmic junction (AIJ), isthmus (I), uterine-isthmic junction (UIJ) and uterus (U). Receptor concentration decreased in all segments of the tube after administration of clomiphene in mated animals. The bindings are of high affinity and low capacity. Important alterations were observed during transport when compared to that of 14, 24, 34, 48, 72, 144 and 168 hr post-coitum (p.c). At 24 hr p.c binding increased only in I and decreased in A and AIJ. Retention of eggs at I at 24 hr p.c showed as increase in binding at I. Egg transport was accelerated and eggs reached prematurely in the uterus due to the influence of clomiphene. Binding in I remained constant from 48 hr p.c to 144 hr p.c but concurrently the binding level increased in U from 34 hr p.c. The elevation of nuclear estrogen receptor level was maximum at 24 hr p.c which coincided with increased plasma estrogen level. The result of such study showed that clomiphene depleted nuclear estrogen receptor complex in the fallopian tube before transfer to the uterus. Further, observation indicated that clomiphene acted directly on the rate of egg transport because of the variations in estrogen receptors during different time periods. Thus, clomiphene reduced the quantity of estrogen receptor i.e., insensitiveness to estrogen. The variations in estrogen binding to its receptor and plasma level at different post-coital periods are modulated by clomiphene resulting in the acceleration of egg transport and prevention of pregnancy.

Estrogen induces expression of c-fos and c-myc protooncogenes in rat uterus.

Estrogen stimulates DNA synthesis and cell proliferation in the luminal and glandular epithelia of rodent uterus. We tested the hypothesis that the mitogenic effect of estrogen occurs via activation of the expression of cellular proto-oncogenes by measuring the rate of transcription of 20 proto-oncogenes (abl, bas, erb-A, erb-B, ets, fms, fos, fps/fes, mos, myb, myc, N-myc, raf, Ha-ras, Ki-ras, N-ras, rel, sis, src, and B-lym) in the uterus of ovariectomized rats before and after injection of estrogen. c-onc transcriptional activity was monitored both by an in vitro transcription assay on isolated nuclei (run-on) and by analysis of mature mRNA. c-fos and c-myc proto-oncogenes were found to respond to estrogen with increased expression: c-fos within 30 min, with a first, sharp peak at 2 h and c-myc within 1.5 h, with a first, broad peak at 4-6 h. DNA synthesis start to increase in the uterus 13 h after estrogen injection and show a first peak at 24 h. In the liver and muscle of the same animals there is neither elevation of c-fos and c-myc expression nor increase of DNA synthesis. The kinetics of the induction by estrogen of c-fos gene expression in the uterus parallels the rate of formation of active nuclear estrogen-receptor complex. Furthermore, the ability of estrogen to induce c-fos mRNA was not abolished by the protein synthesis inhibitor cycloheximide.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the subunit nature of nuclear estrogen receptors by chemical cross-linking and dense amino acid labeling.

We have used chemical cross-linking and dense amino acid labeling of estrogen receptors to characterize the subunit nature and rate of turnover of nuclear 5S estrogen-receptor complexes. When MCF-7 human breast cancer cells are incubated with [3H]estradiol or [3H]antiestrogen [alpha-[4-pyrrolidinoethoxy]phenyl-4-hydroxy-alpha'-nitrostilbe ne (CI628M) or (Z)-1-[4-(2-[N-aziridinyl]ethoxy)phenyl] 1,2-diphenyl-1-butene (tamoxifen aziridine)] and nuclear estrogen-receptor complexes are extracted with 0.6 M KCl and then chemically cross-linked with the cross-linker 2-iminothiolane, the cross-linked receptor complexes sediment as a 5.4S species on 3 M urea-containing sucrose gradients, while the noncross-linked species are 4S. Sodium dodecyl sulfate-polyacrylamide gel analyses of these cross-linked nuclear receptor complexes labeled with the covalently attaching ligand [3H]tamoxifen aziridine reveal a species of about 130,000 mol wt, while the noncross-linked or the cross-linked but mercaptan-cleaved receptor is 65,000 mol wt. Both receptor species are also detectable by interaction with an immunoadsorbent column containing antireceptor monoclonal antibody. For analyses of receptor turnover rates, cells exposed for different time periods to medium containing dense (15N, 13C, and 2H) amino acids were labeled with [3H]antiestrogen [1-[4-(2-dimethylaminoethoxy)phenyl]1-[4-hydroxyphenyl] 2-phenylbut-1-(2)ene (trans-hydroxytamoxifen) or CI628M] or [3H]estradiol, and salt-extracted nuclear estrogen receptors were analyzed on sucrose gradients. The normal density 5S form shifted to a broader, more dense peak at 2 and 4 h and finally, by 8-10 h, to a more dense, sharply sedimenting species. The time course of this shift is the same as that seen for the 4S urea-dissociated nuclear receptor form (t1/2 approximately 4h), suggesting that the 5.4S nuclear receptor is composed of two species which turn over at the same rate. We conclude from these cross-linking and density shift experiments that the nuclear 5S receptor complex consists of two similarly sized units, which turn over with similar half-lives. These data provide strong evidence that the 5S nuclear receptor complex is a homodimer of two 4S, 65,000 mol wt monomers.

Estrogen receptor replenishment. Evidence for receptor recycling.

Replenishment of uterine estrogen receptor was examined in immature rats following injection of 16 alpha-estradiol. 16 alpha-Estradiol is a "short-acting" estrogen that, after a single injection, stimulates early estrogenic responses (water imbibition, induced protein synthesis, etc.), but not long term responses (DNA synthesis). Replenishment after 16 alpha-estradiol injection was complete within 4 h. Furthermore, disappearance of receptor from the nucleus closely corresponded to a reappearance of receptor in the cytoplasm. In contrast to this, receptor replenishment following injection of 1 microgram of either diethylstilbesterol or 17 beta-estradiol was very slow and lagged behind the disappearance of nuclear receptor, leading to an apparent decrease in total receptor content. Half-lives for the clearance of nuclear estrogen-receptor complexes were estimated to be 30 min for 16 alpha-estradiol and 2 h for 17 beta-estradiol, respectively. Inhibition of protein synthesis by cycloheximide did not inhibit replenishment after 16 alpha-estradiol injection. Studies on replenishment after 17 beta-estradiol injection in the presence of cycloheximide could not be interpreted due to a decrease in total receptor content caused by long term cycloheximide treatment. Multiple injections of 17 alpha-estradiol did not lead to a lag in replenishment time or a decrease in total receptor content. This represents a case in which estrogen receptor replenishment appears to be due entirely to receptor recycling.

Modulation of rat uterine steroid hormone receptors by estrogen and antiestrogen.

The effect of sc injections (25 microgram) of estradiol benzoate, monohydroxytamoxifen [1-(4 beta-dimethylaminoethoxyphenyl)1-(4-hydroxyphenyl)-2-phenylbut-1-ene] and tamoxifen [trans-1-(4 beta-dimethylaminoethoxyphenyl)1,2-diphenylbut-1-ene] every 12 h on uterine wet weight, DNA, and cytoplasmic estrogen and progesterone receptor levels has been studied in the immature rat for up to 90 h. Estradiol benzoate produced a 4-fold rise in uterine wet weight, with a doubling of uterine DNA within 48 h. Tamoxifen and monohydroxytamoxifen doubled uterine weight, with a small rise in uterine DNA. Cytoplasmic estrogen receptor levels were reduced within 24 h by all treatments. Estradiol benzoate and monohydroxytamoxifen produced a maximal rise in nuclear estrogen receptor levels within 8 h, followed by a rapid decline to control levels within 80 h. Tamoxifen produced a slower rise in nuclear estrogen receptor levels and never reached the levels achieved by estradiol or monohydroxytamoxifen. The level of nuclear tamoxifen-estrogen receptor complexes slowly decreased with time. In each case, the cytoplasmic progesterone receptor levels increased as nuclear estrogen receptor levels decreased. Cycloheximide (5 microgram/2 h for 8 h before and 20 h after the first estrogen or antiestrogen injection) was used to determine the effect of protein synthesis inhibition on the hormone receptor profiles. Progesterone receptor synthesis was inhibited by cycloheximide. Cycloheximide did not affect translocation, but produced a rapid decrease in nuclear estrogen receptor levels. The results suggest that without the continual translocation of estrogen receptors from the cytoplasm, the antiestrogen-estrogen receptor levels in the nuclear compartment decrease because of destruction or processing. The nuclear antiestrogen-estrogen receptor pool is therefore not static but dynamic. High affinity ligand-estrogen receptor complexes are readily processed in the nucleus to effect progesterone receptor synthesis in the cytoplasm; however, this series of biochemical reactions is only secondary to the fundamental events essential for cell division. After an initial increase in cytoplasmic steroid receptor synthesis by estrogen or antiestrogen, there is a gradual reduction in total cytoplasmic and nuclear estrogen receptor complexes in response to continual nuclear stimulation. This reduction, in turn, reduces progesterone receptor synthesis.

The development of estrogen receptor systems in the rat brain: Perinatal development

The estrogen receptor system of the rat brain is not detected at 15 and 19 days of gestation, even though estrogen receptors can be measured in the maternal hypothalamus. Estrogen receptors are first detected around the 21st day of gestation after which they increase rapidly in the perinatal period and reach a plateau around postnatal day 6. Estrogen receptors are found predominantly in the limbic brain (hypothalamus, preoptic area, amygdala) and in the cerebral cortex, and they are present in very low levels in a sample consisting of the midbrain, brain stem and cerebellum. Development of cortical estrogen receptors is retarded by a few days compared to those in the limbic brain, but then is extremely rapid so that cortical receptor levels are equal to those in the limbic block on or about postnatal day 6. As early as estrogen receptors can be detected in the fetus (day 21 of gestation), they are occupied by endogenous estradiol in male but not in female fetuses. This is measured by the occurrence of cell nuclear estrogen receptor complexes which can be detected by an exchange assay. The presence of such complexes in male limbic brain tissue is consistent with the presence of testicular androgen in the calculation and with the existence in the brain of aromatizing enzymes capable of converting testosterone to estradiol. The absence of such complexes from the female limbic brain is consistent with the protective role of alpha-fetoprotein in scavenging circulating maternal estradiol and keeping it from reaching intracellular receptor sites. The results are discussed with respect to the timing of the critical period for brain sexual differentiation and related maturational events.

Pure estrogen receptor complexes stimulate RNA transcription [formula omitted

The high-affinity species (K d = 0.2 nM) of the two estrogen receptors present in hen oviduct nuclei has been isolated, characterized, and purified to apparent homogeneity by affinity chromatography. An in vitro transcription system using isolated chick oviduct nuclei was developed and optimized to examine the effects of estrogen-receptor complexes on gene transcription. The capacity of nuclei to synthesize [ 3H]RNA in vitro was enhanced by the addition of pure nuclear estrogen-receptor complexes.

Effect of intercalating drugs on the release of uterine nuclear estrogen receptors.

Ethidium bromide has been reported to extract estrogen receptors from uterine nuclei whereas actinomycin D appears to block nuclear receptor “processing” in MCF-7 cells. We have examined the effect of these drugs on the nuclear estrogen receptor during the [3H]estradiol exchange assay. Uterine nuclear fractions were prepared from ovariectomized rats that had received a 5-pg subcutaneous injection of e&radio1 1 h prior to killing. Incubation of nuclear fractions with [3H]estradiol at 37°C resulted in a rapid labeling of nuclear estrogen receptor within 30 min which was followed by a loss of receptor sites that qualitatively resembled nuclear estrogen receptor processing. The addition of actinomycin D or ethidium bromide blocked this loss of nuclear estrogen receptor, resulting in a prolonged elevation of specific nuclear [3H]estradiol binding. Examination of the DNA by polyacrylamide-agarose gel electrophoresis showed extensive fragmentation that could be inhibited by actinomycin D in a dose-dependent manner. These findings suggest that a nuclease(s) present in crude nuclear fractions was responsible for the DNA fragmentation and loss of nuclear estrogen receptor complexes. Nuclear estrogen receptor release and DNA hydrolysis did not occur in highly purified nuclei. Nuclear estrogen receptor lost at 37°C could be recovered in the supernatant fraction as a family of soluble macromolecular complexes. Low salt sucrose gradients of this fraction showed specifically bound [3H]estradiol in an aggregate fraction that sedimented to the bottom of the gradient, and a free 8 S form. Both of these were converted to a 6 S form in gradients containing 0.4

Isolation of estrogen receptor in complex with a discrete nuclear subfraction from hen oviduct.

A nuclear subfraction containing bound estrogen receptor in presumed complex with its nuclear acceptor site has been partially purified from hen oviduct. Sucrose density gradient ultracentrifugation was used to separate mechanically sheared chromatin (i.e. lysed nuclei) into several fractions which differed in protein to DNA ratio as well as in vitro template activity. Gradient fractions were then examined for the presence of bound estrogen receptors. Care was taken to use physiological ionic strength buffers when preparing nuclei since the number of estrogen receptors per nucleus decreased from 5600 to 1600 when nuclei prepared in low ionic strength (mu = 0.013 M) were compared with nuclei prepared in physiological ionic strength (mu = 0.2 M). [3H]Estradiol was introduced into nuclear estrogen receptors by exposing minced oviduct to labeled hormone in tissue culture or by exchanging nuclear estrogen receptor complexes formed in vivo with labeled hormone. In all cases, receptor was found in a fast sedimenting nuclear subfraction of low in vitro template activity. Sodium dodecyl sulfate-gel electrophoresis revealed no differences between proteins from receptor-containing and slower sedimenting fractions. Hybrdization experiments using a cDNA probe made from ovalbumin mRNA indicated no enrichment of this gene in DNA from receptor-containing nuclear material. Salt-extracted nuclear estrogen receptor was shown to partially aggregate to fast sedimenting species of heterogeneous size when sedimented in gradients containing low salt concentrations. Bound receptors were distinguished from such receptor aggregates using a novel electrophoresis technique. In addition, receptor aggregates could be disrupted in high salt, while bound receptors were resistant to this treatment. The number of exchangeable nuclear estrogen receptors in immature chicks given secondary estrogen stimulation was compared with birds that had been withdrawn from hormone. The number of receptors per nucleus was shown to be higher in animals given secondary stimulation, and these receptors were associated exclusively with fast sedimenting nuclear material.

The Testicular Estrogen Receptor System in Two Strains of Mice Differing in Susceptibility to Estrogen-Induced Ley dig Cell Tumors*

Cytosol obtained from cryptorchid testes of tumor-susceptible BALB/c and resistant C3HBi (Z) mice both bound 17 beta-estradiol (E2) and diethylstilbestrol (DES) specifically. The dissociation constant (Kd) of this binding component (RE) for E2 was determined to approximate 5 x 10(-9) M. Gel filtration of cytosols resulted in a significant increase in the binding constant (Kd approximately 3 x 10(-10) M) with the majority of the complex migrating in the 7-8S area after sucrose gradient centrifugation. Incubation of either untreated or gel-filtered cytosol with [3H]DES resulted in considerable nonspecific binding appearing in the 4S region in a low salt sucrose gradient. This 4S binding of [3H]DES was not inhibited by the addition to the incubation mixtures of a 100-fold excess of either E2 or DES, while the lesser peak at 7-8S as well as the major 7-8S peak formed with E2 were inhibited by both. In vitro translocation of the cytosol RE to the nucleus was demonstrated in both mouse strains using either estrogen. Quantitation of the in vivo translocation, employing the exchange method after a single injection of 2.5 micrograms E2/mouse, revealed a rapid increase in cytoplasmic receptor content accompanied by a concomitant increase in nuclear receptor content. Greater nuclear receptor content was identified in nuclei from BALB/c mice than in those from Z animals 45 min after injection of E2. The binding behavior of E2-RE complexes to nuclei was studied by the KCl extraction method. The percent extracted from the nuclei in the Z strain was significantly greater than that in the BALB/c at all concentrations of KCl tested. Essentially 100% of the RE was extracted from nuclei of Z animals at 0.4 M KCl, while nuclei of BALB/c mice retained 35-40% even in 2 M KCl. Cross-over experiments in a cell-free system suggested that the difference in binding was due to differences in chromatins rather than in nuclear estrogen-receptor complexes. The greater nuclear receptor content and stronger binding of nuclear receptor to chromatin might explain why estrogen-induced phenomena, including neoplastic transformation occur to a much greater degree in the BALB/c strain than in the Z strain of the mouse.

Nuclear estrogen receptor in the avian liver: Correlation with biologic response

Diethylstilbestrol (DES) administration to cockerels resulted in an elevation (7-fold) in the number of high affinity binding sites measured in isolated liver nuclei by the [ 3H]-estradiol exchange method. Increases were noted within 60 min, reached a maximum by 4 h and by 48 h had returned to control values. Elevation in the quantity of nuclear binding sites was estrogen-specific and 20% of the [ 3H]-estradiol bound could be extracted by 0.4 M KC1. When isolated chromatin was used to evaluate estrogen-receptor interaction, the binding data were similar to those obtained with intact nuclei. To correlate estrogen binding with a specific hepatic response to this hormone, very low density lipoproteins (VLDL) synthesis was measured by immunochemical techniques in liver slices incubated in vitro in the presence of [ 3H]-lysine following a single injection of DES. An increase in immunoprecipitable VLDL in the liver slices was first noted 2 h after estrogen administration, suggesting that nuclear receptor occupancy occurs prior to the earliest detectable stimulation of protein synthesis. To determine whether receptor binding and protein synthesis were temporally separated by estrogen-induced changes in transcription. RNA polymerase activities were measured in isolated nuclei and RNA synthesis initiation sites were quantified in chromatin preparations. Stimulation of RNA polymerase I and II activities as well as an 85% increase in chromatin initiation sites were demonstrable within 1 h following DES treatment. Thus. these effects were closely correlated in time with increases in nuclear estrogen receptor levels and again preceded enhanced VLDL synthesis. Finally, a dose-related correlation was noted between accumulation of nuclear estrogen-receptor complexes and elevation of plasma triglycerides and VLDL following DES treatment. Collectively, these data provide evidence that estrogen receptor interaction may underwrite the physiologically important regulation of plasma lipoproteins by a mechanism involving both transcription and translation.

Fluctuations in Levels of Cytosol and Nuclear Estrogen Receptors in Rat Mammary Tumors during the Estrous Cycle*

Levels of cytosol estrogen receptors (R) and nuclear estrogen-receptor complexes (ER) in rat mammary tumors induced by 7,12-dimethylbenz-(a)anthracene were estimated during the host's estrous cycle. Cytosol R was measured by the method of charcoal absorption employing the principle of Scatchard. Nuclear ER was determined by a [3H]estradiol exchange method. Levels of cytosol R were low at proestrus and estrus and increased significantly during metestrus-diestrus. Levels of nuclear ER were slightly elevated at diestrus-2, substantially higher at proestrus, and relatively low during other stages of the cycle. The high levels of nuclear ER during diestrus-2 and proestrus coincided with the reported rising levels of circulating estrogen in the host animals. Furthermore, rates of [3H]leucine incorporation by the tumor tissue correlated significantly with levels of nuclear ER in the corresponding tumors. The low level of cytosol R observed at proestrus could be explained by a depletion of R following the high le...

Effect of nafoxidine (U-11,100A) on the induction of uterine peroxidase

The relationship between the quantity of nuclear estrogen receptor complex formed and the amount of peroxidase induced by estradiol in immature rat uteri was determined. A good correlation was obtained between the quantity of 3H-estradiol specifically bound to the nuclear pellet and the amount of enzyme present 12 h after treatment with physiological doses of estrogen. Nafoxidine (U-11,100A) administered together with estrogen reduced significantly the increase in peroxidase activity brought about by estradiol. Pretreatment with nafoxidine was less effective. The possible mechanism of action of this antiestrogen is discussed.

Estrogen‐receptor binding: Relationship to estrogen‐induced responses

The relationship between nuclear binding of the receptor-estrogen complex (R-E) and estrogenic responses was examined. Nuclear-bound R-E was measured by the [3H] estradiol exchange assay, which permits the quantification of R-E as a function of either endogenous or non labeled estrogen. The quantity of nuclear R-E fluctuated as a function of blood levels of estrogen during the estrous cycle in the rat. This indicates that the accumulation of the R-E complex by the nucleus of uterine cells may be of physiologic significance and under the control of endogenous estrogen.

Temporal Relationships Between Hormone Receptor Binding and Biological Responses inthe Uterus: Studies with Short- and Long-Acting Derivatives of Estriol

The temporal relationships between hormone receptor binding and early and late biological responses in the uterus were examined using estriol (E3), a weak estrogen, and several more long-acting estriol derivatives, namely ethinyl estriol (EE3), estriol cyclopentyl ether (E3CPE), and ethinyl estriol cyclopentyl ether (EE3CPE). Dose-response curves of 3-day uterotrophic assays indicate that biological potency follows the order EE3CPE greater than EE3 or estradiol greater than E3CPE greater than E3. After a single injection of 5 mug of compound, E3 elicits the early uterotrophic responses (increased uterine wet weight and 2-deoxyglucose phosphorylation at 2-6 h) but gives only weak stimulation of later uterotrophic responses (enhanced rates of 2-deoxyglucose phosphorylation at 20-24 h and increased DNA synthesis rate and uterine weight over a 72 h period). E3, EE3, and estradiol all elicit a rapid (maximal by 1/2-1 h) uptake of receptor into the nucleus and show an equivalent wet weight response at 3 h. After E3, nuclear receptor levels and uterine weight decline rapidly; however, after EE3 or estradiol, nuclear receptor levels decline less rapidly remaining at least two-fold above the control until 24-48 hr, and uterine weight also remains elevated for at least 48-72 h. EE3CPE elicits both the early (4 h) and later (20-24 h) waves of glucose metabolism, shows a prolonged effect on DNA synthesis rate, and shows the most dramatic and prolonged (beyond 72 h) maintenance of elevated uterine weight and high nuclear receptor (beyond 24 h). Thus, chemical modifications of the estriol molecule which result in a prolonged stimulation of uterine growth and metabolism also result in a long-term maintenance of hormone-receptor complex in the uterine nucleus. These studies give strong support to the concept that true uterine growth requires the direct and prolonged influence of the nuclear estrogen-receptor complex.