An evaluation of the role of antiestrogen-binding sites in mediating the growth modulatory effects of antiestrogens: studies using t-butylphenoxyethyl diethylamine, a compound lacking affinity for the estrogen receptor

Tert-butylphenoxyethyl diethylamine (BPEA), a compound synthesized by us, was designed to incorporate features important in binding to antiestrogen-binding sites (AEBS) while lacking features important in binding to the estrogen receptor (ER). With this compound, we have addressed the question of the role of AEBS in mediating the growth modulatory effects of antiestrogens. BPEA has an affinity for AEBS 6% that of tamoxifen and an affinity for ER less than 0.0003% that of estradiol. BPEA (10(-11)-10(-6) M) had no effect on the growth of MCF-7 breast cancer cells and no effect on inhibition of the growth of MCF-7 cells by different concentrations of the antiestrogen tamoxifen. In addition, BPEA (even at doses of 1 mg/day X 50 g rat) exhibited no uterotropic or antiuterotropic activity in immature rats and had no influence on the agonistic or antagonistic activity of varying concentrations of tamoxifen on uterine weight. Hence, we conclude that occupancy of AEBS, at least by BPEA, does not modulate growth of the uterus or breast cancer cells and does not influence the potency of tamoxifen as an antiestrogen. These findings raise serious doubts about the role of the AEBS in mediating directly the growth modulatory effects of antiestrogens.     

Effect of progesterone on the activity of occupied nuclear estrogen receptor in vitro

In previous studies, we have demonstrated that progesterone administration in vivo can selectively alter estrogen receptor levels and distribution in the rat anterior pituitary. The present study represents an attempt to extend these observations to an in vitro model. Cytosolic and nuclear preparations of uterine homogenates from ovariectomized adult rats were shown to be capable of temperature-dependent estrogen-mediated receptor activation and translocation from cytosol to nuclei upon recombination. Addition of progesterone to isolated cytosol did not diminish estrogen receptor binding capacity over at least a 2 h period at 22 degrees C. Preincubation of the subcellular fractions with progesterone, followed by removal of free progesterone prior to cytosol-nuclear recombination, resulted in dramatic reduction in nuclear estrogen receptor activity. This action was equally apparent whether progesterone was introduced to the cytosolic or nuclear fraction, and was confined to the steroid-occupied subpopulation of nuclear receptors. The ability of this in vitro system to mimic the estrogen receptor-suppressive effect of progesterone provides a good model in which to analyze the biochemical basis for a direct estrogen-inhibitory effect of progesterone on estrogen action.     

Ketononestrol aziridine, an agonistic estrogen receptor affinity label: study of its bioactivity and estrogen receptor covalent labeling

Ketononestrol aziridine [(6R,TS)1-(N-aziridinyl)6,7-bis-(4-hydroxyphenyl)5-nonamone (KNA)], an aziridine derivative of hexestrol, is an estrogenic affinity label for the estrogen receptor (ER). It has an apparent relative binding affinity 8% that of estradiol and shows time-dependent irreversible binding to the ER in uterine cytosol preparations and intact human breast cancer cells (MCF-7). The agonistic activity of KNA is evident in MCF-7 cells in culture, where it increases the cell growth rate and elevates the level of progesterone receptor. KNA was prepared in high specific activity tritium-labeled form by iodination of a methanesulfonate precursor, followed by catalytic tritium-iodine exchange and aziridinylation; the material prepared has high radiochemical purity and a specific activity of 67 Ci/mmol. The covalent attachment of [3H]KNA to the ER can be followed directly by a solvent precipitation assay. In cytosol preparations of uterine ER, labeling with [3H]KNA proceeds in a time-, concentration-, and temperature-dependent manner; labeling is efficient and selective and, by competition studies, was shown to be estrogen specific. ER in intact MCF-7 cells can also be covalently labeled by treatment with [3H]KNA. Receptor covalently labeled with [3H]KNA sediments as a 4S species on high salt sucrose gradients, and its sedimentation position is shifted by treatment with monoclonal antireceptor antibodies. On sodium dodecyl sulfate-polyacrylamide gels, the principal labeled species migrates with a mol wt of 66,000. KNA should prove to be a useful probe for studies on receptor structure, function, and chromatin interactions, particularly when the behavior of a receptor-agonist complex is being investigated.     

In vitro evolution of a T cell receptor with high affinity for peptide/MHC

T cell receptors (TCRs) exhibit genetic and structural diversity similar to antibodies, but they have binding affinities that are several orders of magnitude lower. It has been suggested that TCRs undergo selection in vivo to maintain lower affinities. Here, we show that there is not an inherent genetic or structural limitation on higher affinity. Higher-affinity TCR variants were generated in the absence of in vivo selective pressures by using yeast display and selection from a library of Valpha CDR3 mutants. Selected mutants had greater than 100-fold higher affinity (K(D) approximately 9 nM) for the peptide/MHC ligand while retaining a high degree of peptide specificity. Among the high-affinity TCR mutants, a strong preference was found for CDR3alpha that contained Pro or Gly residues. Finally, unlike the wild-type TCR, a soluble monomeric form of a high-affinity TCR was capable of directly detecting peptide/MHC complexes on antigen-presenting cells. These findings prove that affinity maturation of TCRs is possible and suggest a strategy for engineering TCRs that can be used in targeting specific peptide/MHC complexes for diagnostic and therapeutic purposes.     

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.     

Somatostatin analogs: correlation between receptor binding affinity and biological potency in GH pituitary cells

Somatostatin (SRIF) is a hypothalamic tetradecapeptide which acts on several different types of pituitary cells to inhibit hormone release both in vivo and in vitro. We have previously shown that the GH4C1 clonal strain of rat pituitary tumor cells contains a single class of specific high-affinity SRIF receptors and that SRIF is a potent inhibitor of GH and PRL release by these cells. In this study, we have determined the relationship between the apparent binding affinity and biological potency of 19 SRIF analogs in GH4C1 cells. Receptor binding and biological activity were assayed under identical conditions. A good correlation (r = 0.96) was observed over a 10,000-fold range between the receptor binding affinities and biological potencies of SRIF analogs. Modifications at the C- and N-terminal regions of the SRIF molecule had minimal effects on binding to the receptor or potency to inhibit PRL release. However, substitution of residues 6 through 10 or reduction of the disulfide bond resulted in a 100-fold or greater decrease in both activities. The N-terminal extended SRIF analogs, SRIF-28, [D-Trp22]SRIF-28, and SRIF-25, were all somewhat less potent than SRIF. These results strongly support the involvement of the characterized SRIF receptor in initiating the biological actions of SRIF in GH4C1 cells and define the structural features of the SRIF molecule required for both receptor binding and activation.     

Estrogen increases locomotor activity in mice through estrogen receptor alpha: specificity for the type of activity

Estrogens are known to increase running wheel activity of rodents primarily by acting on the medial preoptic area (mPOA). The mechanisms of this estrogenic regulation of running wheel activity are not completely understood. In particular, little is known about the separate roles of two types of estrogen receptors, ERalpha and ERbeta, both of which are expressed in mPOA neurons. In the present study the effects of continuous estrogen treatment on running wheel activity were examined in male and female mice specifically lacking either the ERalpha (alphaERKO) or the ERbeta (betaERKO) gene. Mice were gonadectomized and 1 wk later implanted with either a low dose (16 ng/d) or a high dose (160 ng/d) of estradiol benzoate (EB) or with a placebo control pellet. Home cage running wheel activity was recorded for 9 d starting 10 d after EB implants. The same mice were also tested for open field activity before and after EB implants. In both female and male alphaERKO mice, running wheel activity was not different from that in corresponding wild-type (alphaWT) mice in placebo control groups. In both females and males it was increased by EB only in alphaWT, not alphaERKO, mice. In betaERKO mice, on the other hand, both doses of EB equally increased running wheel activity in both sexes just as they did in betaWT mice. Absolute numbers of daily revolutions of EB-treated groups, however, were significantly lower in betaERKO females compared with betaWT females. Before EB treatment, gonadectomized alphaERKO female were significantly less active than alphaWT mice in open field tests, whereas betaERKO females tended to be more active than betaWT mice. In male mice there were no effect of ERalpha or ERbeta gene knockout on open field activity. Unlike its effect on running wheel activity, EB treatment induced only a small increase in open field activity in female, but not male, mice. These findings indicate that 1) in both sexes estrogenic regulation of running wheel activity is primarily mediated through the ERalpha, not the ERbeta; and 2) hormone/genotype effects are specific to the type of locomotor activity (i.e. home cage running wheel activity and open field activity) measured.     

Increased pituitary response to somatostatin in aging male rats: relationship to somatostatin receptor number and affinity

Previous research has established that growth hormone pulse amplitude declines with increasing age. The purpose of this study was to determine whether this decline is associated with (1) increased pituitary response to somatostatin, and/or (2) increased number or affinity of pituitary somatostatin receptors. In the first study, pituitary slices from young (3-4 months), middle-aged (12-14 months), and old (22-24 months) male Fischer 344 rats were superfused with minimal essential medium (1 ml/min) and fractions collected at 5-min intervals. Tissues were stimulated with 10(-7) M hpGRF (1-44) for 1 min and, 40 min later, with hpGRF in the presence of 5 x 10(-9) M somatostatin-14 or somatostatin-28. Two pituitaries from each age group were superfused simultaneously and the experiment replicated 4 times. Growth hormone release was measured by radioimmunoassay. In a second study, somatostatin receptors in purified pituitary membranes from the three age groups were compared using iodo-[Tyr0]-D-Trp8 somatostatin-14. Animals from each age group were pooled, membranes extracted, and incubated with increasing doses of cold peptide. Binding characteristics were analyzed by Scatchard analysis and Ka and Bmax calculated. Results indicated that (1) basal growth hormone release diminished both with age and somatostatin administration, (2) GRF-induced release of growth hormone was similar in all age groups when data were expressed as percent increase from baseline, and (3) in the presence of somatostatin-14, GRF-induced release of growth hormone was attenuated in old as compared to young or middle-aged rats (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Global transcription profiling of estrogen activity: estrogen receptor alpha regulates gene expression in the kidney

Estrogen receptors (ERs) are expressed in numerous organs, although only a few organs are considered classical targets for estrogens. We have completed a systematic survey of estrogen regulation of approximately 10,000 genes in 13 tissues from wild-type and ERbetaKO mice treated sc with vehicle or 17beta-estradiol (E2) for 6 wk. The uterus and pituitary had the greatest number of genes regulated by E2, whereas the kidney had the third largest number of regulated genes. In situ hybridizations localized E2 regulation in the kidney to the juxtamedullary region of the cortex in both the mouse and rat. The ED(50) for gene inductions in the kidney was 3 micro g/kg.d, comparable with the 2.4 micro g/kg.d ED(50) for c-fos induction in the uterus. E2 regulations in the kidney were intact in ERbetaKO mice, and the ERalpha-selective agonist propylpyrazole triol acted similarly to E2, together suggesting an ERalpha-mediated mechanism. Several genes were induced within 2 h of E2 treatment, suggesting a direct activity of ERalpha within the kidney. Finally, the combination of the activation function (AF)1-selective agonist tamoxifen plus ERalphaKO(CH) mice expressing an AF1-deleted version of ERalpha allowed delineation of genes with differing requirements for AF1 or AF2 activity in the kidney.     

Implications of the binding of tamoxifen to the coactivator recognition site of the estrogen receptor

A number of studies have reported on the unusual pharmacological behavior of type I antiestrogens, such as tamoxifen. These agents display mixed agonist/antagonist activity in a dose-, cell-, and tissue-specific manner. Consequently, many efforts have been made to develop so-called 'pure' antiestrogens that lack mixed agonist/antagonist activity. The recent report of the structure of estrogen receptor (ER) beta with a second molecule of 4-hydroxytamoxifen (HT) bound in the coactivator-binding surface of the ligand-binding domain (LBD) represents the first direct example of a second ER ligand-binding site and provides insight into the possible origin of mixed agonist/antagonist activity of type I antiestrogens. In this review, we summarize the biological reports leading up to the structural conformation of a second ER ligand-binding site, compare the ERbeta LBD structure bound with two HT molecules to other ER structures, and discuss the potential for small molecular inhibitors designed to directly inhibit ER-coactivator and, more generally, nuclear receptor (NR)-coactivator interactions. The studies support a departure from the traditional paradigm of drug targeting to the ligand-binding site, to that of a rational approach targeting a functionally important surface, namely the NR coactivator-binding (activation function-2) surface. Furthermore, we provide evidence supporting a reevaluation of the strict interpretation of the agonist/antagonist state with respect to the position of helix 12 in the NR LBD.     

Estrogen-induced uterine responses and growth: relationship to receptor estrogen binding by uterine nuclei.

The relationship between estrogen receptor(R) binding by uterine nuclei and uterotrophic responses was examined. Immature rats received a single injection of estradiol (E2) or estriol (E3) and the following parameters were measured: accumulation and retention of the estrogen receptor by the nuceus of uterine cells; incorporation of 14C-glucose into CO2 lipid, protein and RNA; RNA polymerase activity; water imbibition and increased dry weight. E2 and E3 were of equal potency with regard to the rapid accumulation of R by the nucleus but differed with respect to long term retention of R. The concentrations of nuclear RE2 and RE3 complexes were equivalent between 1 and 3 hr after estrogen injection; however, by 6 hr RE2 remained significantly elevated while RE3 levels had fallen to control values. E2 and E3 were also of equal potency with respect to the stimulation of enhanced glucose utilization, water imbibition, the incorporation of 14C-glucose into lipid, protein and RNA 3 hours following an injection of the hormone. Likewise the activity of RNA polymerase was equally stimulated by E2 and E3 3 hr after injection. Thus all early uterotropic responses (0-3 hrs) that were measured were equally stimulated by E2 and E3. However, E3 failed to stimulate true uterine growth (increase dry weight 24 hr after injection), whereas E2 produced a significant stimulation of true uterine growth. These data suggest that the RE complex is capable of stimulating early uterotrohic events regardless of which estrogen is present; however, in order to produce true uterine growth the RE complex must be retained in the nucleus for long periods of time. This proposal was tested by the administration of repetitive injections of E3. This treatment resulted in an increase in dry weight that was equivalent to the growth that was produced by repetitive injections of E2. These results demonstrate that E2 and E3 elicit early uterotrophic responses with equal facility following a single injection but that only E2 causes true uterine growth. The ability of E2 to stimulate true uterine growth appears to be related to the time of residence of the RE complex in the nucleus.     

3H]cortivazol: a unique high affinity ligand for the glucocorticoid receptor

Cortivazol (CVZ) and deacylcortivazol (DAC) are pyrazolosteroids with potent glucocorticoid activity. In previous work we showed that DAC is 40-fold more potent than dexamethasone (DEX) in lysing leukemic lymphoblasts. To assess the interaction between these atypical steroids and the glucocorticoid receptor, we examined the binding of [3H]CVZ to cytosol from glucocorticoid-sensitive and -resistant variants of the human leukemic cell line CEM C7. In glucocorticoid-sensitive cells [3H]CVZ causes a 2-fold induction of glutamine synthetase and binds to a protein in the 4.6 S region of high salt sucrose gradients. On DEAE-cellulose chromatography, [3H]CVZ-receptor complexes show a shift from high (0.25 M KP) to low salt (0.09 M KP) eluting forms upon activation. CVZ competes for a 97,000-dalton protein labeled by [3H]dexamethasone mesylate. Scatchard analysis of the binding of [3H]CVZ in glucocorticoid-sensitive cells revealed a curvilinear plot which resolved into high (0.4 nM) and low (11 nM) affinity components. The receptor concentration of the low affinity site (0.30 pmol/mg protein) was approximately twice that of the high affinity site (0.14 pmol/mg protein). Dissociation experiments with dilution and/or excess unlabeled CVZ supported the presence of independent sites. In contrast, the binding of [3H]DEX to C7 cytosol revealed a single class of binding sites (Kd = 1.9 nM; receptor concentration, 0.46 pmol/mg protein). Examination of the binding of [3H]CVZ using 10(-5) M DEX as the competing ligand showed that DEX binds only to the low affinity site detected by [3H]CVZ. In cytosol from a glucocorticoid-resistant cell line with virtually no [3H]DEX binding, [3H]CVZ detected a single high affinity binding site that was similar in dissociation constant (0.8 nM) and receptor concentration (0.13 pmol/mg protein) to the high affinity site detected in the glucocorticoid-sensitive cell line C7.     

A second binding site for hydroxytamoxifen within the coactivator-binding groove of estrogen receptor beta

Evidence is presented that the estrogen antagonist 4-hydroxytamoxifen (HT) can occupy not only the core binding pocket within the ligand-binding domain of estrogen receptor (ER) beta but also a second site on its surface. The crystal structure of the ligand-binding domain (LBD) associated with HT was determined to 2.2 A and revealed two molecules of HT bound to the protein. One was located in the consensus ligand-binding pocket, whereas the other bound to a site that overlaps with the hydrophobic groove of the coactivator recognition surface. Relative to the ERalpha-tamoxifen structure, helix 12 has been displaced from the coactivator recognition surface and occupies a unique position. Although it has been demonstrated that association of the antagonist with the core ligand-binding pocket is sufficient to induce an antagonist ligand-binding domain conformation, this structure suggests that small molecules may directly antagonize receptor-coactivator interactions. These results provide a direct demonstration of two binding sites for HT in ERbeta, as has been previously suggested for ERalpha by using biochemical methods, and represent a crystal structure of a small nonpeptide molecule occupying the coactivator recognition site.     

Activation of estrogen receptor alpha increases and estrogen receptor beta decreases apolipoprotein E expression in hippocampus in vitro and in vivo

Previous evidence indicates that, in carriers of apolipoprotein E4 (ApoE4), estrogen therapy increased the risk of late-onset Alzheimer's disease (AD), whereas in individuals carrying ApoE2/3, estrogen therapy reduced the risk of AD [Cauley JA, Zmuda JM, Yaffe K, Kuller LH, Ferrell RE, Wisniewski SR, Cummings SR (1999) J Bone Miner Res 14:1175-1181; Yaffe K, Haan M, Byers A, Tangen C, Kuller L (2000) Neurology 54:1949-1954]. Estrogen mechanisms of action are mediated by two estrogen receptors (ERs), ERalpha and ERbeta. In this study, we determined the relationship between ER subtype and estrogen regulation of ApoE expression in HT-22 cells ectopically transfected with ERalpha or ERbeta, in primary cultured rat hippocampal neurons in vitro and in rat hippocampus in vivo by both molecular biological and pharmacological analyses. Results of these analyses demonstrated that activation of ERalpha either by 17beta-estradiol or a specific-agonist, propylpyrazole triol, up-regulated ApoE mRNA and protein expression. In contrast, the ERbeta-selective agonist, diarylpropionitrile, down-regulated ApoE mRNA and protein expression. These results demonstrate that, in vitro and in vivo, ApoE expression can be differentially regulated depending on activation of ER subtypes. These data suggest that use of ER-selective ligands could provide therapeutic benefit to reduce the risk of AD by increasing ApoE expression in ApoE2/3 allele carriers and decreasing ApoE expression in ApoE4 allele carriers.     

19-nor analogs of adrenal steroids: mineralocorticoid and glucocorticoid receptor activity

The effect of absence of the C-19 methyl group from five adrenal steroids has been studied in terms of their affinity for mineralocorticoid (MR) and glucocorticoid receptors (GR). In MR assays, 19-nordeoxycorticosterone and 19-norprogesterone showed 3-fold higher affinity for MR than did their respective parent steroids; 19-norcortisol had 1.5 times the affinity of cortisol for MR. In contrast, corticosterone and 19-nororticosterone showed equal affinity, and 19-noraldosterone showed less than 1% the MR activity of aldosterone. In GR assays, the absence of the C-19 methyl group from progesterone increased GR affinity 3-fold and deoxycorticosterone affinity 1.5-fold. In contrast, the other 19-nor steroids showed decreased affinity vis à vis their parent compounds (19-norcorticosterone, 30%; 19-norcortisol, 10%; 19-noraldosterone, < 1%). These findings suggest that while the 19-nor analogs of 11-deoxy steroids are consistently more active than their parent steroid, the 19-nor 11-oxygenated adrenal steroids show no predictable pattern of binding for MR or GR.     

The decline of grip strength in the menopause: relationship to physical activity, estrogen use and anthropometric factors

The focus of this study was the relationship of grip strength to age, physical activity and anthropometric factors, in a population of 255 post-menopausal women not on estrogen therapy (mean age = 57.6) and 55 women currently on estrogen replacement therapy (mean age = 56.9). Grip strength was measured as an indicator of muscular strength in the upper limbs. The grip strength of the estrogen users was significantly higher than that of the estrogen abstainers. Grip strength was related to age (r = -0.25, p less than 0.01), and the body habitus parameters of height (r = 0.36, p less than 0.01) and weight (r = 0.18, p less than 0.01). Although estrogen use was univariately correlated with strength (r = 0.16, p less than 0.05), multiple regression analyses revealed that only the height, age and physical activity were independent determinants of grip strength. These data suggest: height is the major determinant of upper body strength in older women; the reduction in physical activity with advancing age may contribute to strength decline, and modest increase in physical activity may retard the loss of strength that accompanies aging; the loss of ovarian estrogen in menopause may be related to the loss of strength in postmenopausal women.     

Content of nuclear estradiol receptor complex in rat corpora lutea during pregnancy: relationship to estrogen concentrations and cytosol receptor availability.

The content of estradiol receptor in cytosol and nuclear cell fractions of rat corpora lutea changed during pregnancy. The binding of (3-H)) estradiol to luteal cell cytosol was high early in pregnancy between days 3-11, decreased on days 12and15 and was low throughout of the remainder of pregnancy. In contrast, the binding of (3-H) estradiol to nuclear receptor, as measured by nuclear exchange assay, was low early in pregnancy, increased between days 10-15, remained high through day 18 and decreased on days 20 and 22. The administration in vivo of estradiol-17beta (40 mug) 1 hr prior to sacrifice stimulated an increase in nuclear receptor content early in pregnancy (days 3,6,8,10-12) but not later in pregnancy (15,18,20, and 22,).These results suggested that the estradiol binding component(s) present in rat luteal cell cytosol early in pregnancy represented available estradiol receptor capable of being translocated to the nucleus in the presence of sufficient estradiol. However, once available receptor has been saturated by endogenous hormone at midgestation, exogenous hormones hasno further effect on no nuclear receptor content. Importantly,loss of nuclear receptor content late in pregnancy appears to reflect decreased levels of total cellular receptor indicating that corpora lutea at the end of pregnancy have lost the primary mecchanism for responding to estrogens Thus, various stages of luteal cell differentiation are associated with changes in the intra-cellular distribution and total cellular content of estradiol receptor suggesting that luteal cell function may be regulated selectively and separately by hormone concentrations and hormone receptor availability.