Quantitative analysis of heterogeneity of estrogen binding in human breast tumor specimens

A quantitative assessment was made of the heterogeneity of estrogen binding among histologically distinct regions and among nuclei within histologically uniform regions in ten human breast tumors. An autoradiographic method was used that employed sections from fixed tissues and required exposure times of one to two weeks. Grain counting of autoradiograms probably indicated both type I and type II estrogen binding. There were significant decreases in estrogen binding in all specimens after competition with excess diethylstilbestrol, suggesting that a component of binding was specific for estrogen. Significant differences in amount and distribution (nuclear vs cytoplasmic) of estrogen binding were seen not only among tissues with different histologies but also within histologically identical regions of a tissue. In histologically uniform regions, wide ranges of values for nuclear estrogen binding were seen. This method allows for the rapid evaluation of estrogen-binding profiles of tumor specimens and has potential for analyzing the process of neoplastic transformation in mammary epithelia as it affects, or is affected by estrogen binding.     

The autoradiographic demonstration of estrogen binding in normal human cervix and vagina during the menstrual cycle, pregnancy, and the menopause

Using the technique of in vitro steroid autoradiography, the localization and modulation of nuclear estrogen binding sites has been studied in normal human cervix and vagina during the menstrual cycle, pregnancy, and the menopause. Marked differences occur in nuclear estrogen binding between these two organs. Nuclear estrogen binding varies throughout the menstrual cycle in the vaginal epithelium, whereas vaginal stromal cells consistently exhibit nuclear estrogen binding throughout the cycle. In contrast, the cervical squamous and columnar epithelia show much less cyclic variability in nuclear estrogen binding sites. As in the vagina, the cervical stroma consistently binds estrogen. High levels of nuclear estrogen binding sites are found in the vagina of the postmenopausal patient, and lower levels of binding occur postpartum. The implications of these localizations, with special reference to the role of the cervical and vaginal stroma, are discussed.     

Differential Ligand Binding Affinities of Human Estrogen Receptor-α Isoforms

Rapid non-genomic effects of 17β-estradiol are elicited by the activation of different estrogen receptor-α isoforms. Presence of surface binding sites for estrogen have been identified in cells transfected with full-length estrogen receptor-α66 (ER66) and the truncated isoforms, estrogen receptor-α46 (ER46) and estrogen receptor-α36 (ER36). However, the binding affinities of the membrane estrogen receptors (mERs) remain unknown due to the difficulty of developing of stable mER-transfected cell lines with sufficient mER density, which has largely hampered biochemical binding studies. The present study utilized cell-free expression systems to determine the binding affinities of 17β-estradiol to mERs, and the relationship among palmitoylation, membrane insertion and binding affinities. Saturation binding assays of human mERs revealed that [³H]-17β-estradiol bound ER66 and ER46 with K_d values of 68.81 and 60.72 pM, respectively, whereas ER36 displayed no specific binding within the tested concentration range. Inhibition of palmitoylation or removal of the nanolipoprotein particles, used as membrane substitute, reduced the binding affinities of ER66 and ER46 to 17β-estradiol. Moreover, ER66 and ER46 bound differentially with some estrogen receptor agonists and antagonists, and phytoestrogens. In particular, the classical estrogen receptor antagonist, ICI 182,780, had a higher affinity for ER66 than ER46. In summary, the present study defines the binding affinities for human estrogen receptor-α isoforms, and demonstrates that ER66 and ER46 show characteristics of mERs. The present data also indicates that palmitoylation and membrane insertion of mERs are important for proper receptor conformation allowing 17β-estradiol binding. The differential binding of ER66 and ER46 with certain compounds substantiates the prospect of developing mER-selective drugs.     

Modulation of Progestin Binding Activity in Cultured Human Breast Carcinoma Cells: The Effect of Serum Type and Concentration

Abstract

Progesterone receptor levels in MCF-7 human breast cancer cells increase as a specific response to estrogen and to some nonsteroidal antiestrogens. In the present study we demonstrate that the type and quantity of serum present during culture of these cells modifies the level of progestin binding activity, but not the level of estradiol binding activity.

MCF-7 cells maintained in media supplemented with 5% charcoal-dextran treated calf serum (CDCS) contain 0.3 - 0.4 pmol of cytosol progesterone receptor (PR_c) per mg DNA. When cells previously maintained in 5% CDCS-media are shifted to media containing 5% charcoal-dextran treated fetal calf serum (CDFCS), the level of progestin binding increases after day 16, and stabilizes at 2 - 3 pmol/mg DNA at days 30 to 40. Shifting these cells back to 5% CDCS-media, reduces PR_c to 0.2 - 0.4 pmol/mg DNA within 3 days. This reduction is dose dependent with a half-optimal decrease at 1% CDCS, and a full decrease at 2% CDCS (4d incubation). Nuclear progestin binding was uniformly low (0.2 - 0.4 pmol/mg DNA) and unaffected by type or concentration of serum, and no consistent change in cytosol or nuclear estrogen receptor levels was observed. These cytoplasmic progestin binding sites are translocated to the nucleus by progesterone, and are similar to estradiol (E₂) induced sites by Scatchard binding and sucrose gradient analysis. Similar serum-dependent changes are also observed in the T₄₇D human breast cancer cell line where growth in CDFCS-media results in 4-fold higher progestin binidng levels than observed in CDCS-media. Our findings suggest the presence of non-dialyzable stimulatory factor(s) in CDFCS that influence the progestin receptor level the highlight the fact that serum components can alter dramatically the cellular progestin binding activity.     

Identification of estrogen receptors in granulosa cells of immature rats

Nuclear and cytoplasmic binding sites for estradiol (E2-17 beta) in granulosa cells of immature rats were characterized. These binding sites for estrogen were high affinity, low capacity with an affinity constant (Kd) of 1.9 X 10(-10)M (binding capacity, Ro = 80 pM) for nuclear sites and a Kd = 3.5 X 10(-10) M (Ro = 45 pM) for cytosol sites. Binding was specific for biologically active estrogens. The estrogen receptor in granulosa cells is a protein and heat-labile as treatment with protease or pre-incubation at 37 degrees C for 1 h significantly diminished binding. RNase and DNase had no effect on estrogen binding. Sedimentation coefficients for nuclear and cytosol binding components were 5S and 8S respectively, similar to values obtained with uteri. Finally, translocation was demonstrated after a s.c. injection of E2-17 beta. Forty-five minutes post-injection, cytosol binding sites for estradiol were depleted concomitant with accumulation of nuclear binding sites. We concluded that granulosa cells of immature rats have binding sites specific for estradiol which have characteristics similar to the classical estrogen receptor in uteri.     

Cytosol and nuclear estrogen receptor binding in the preoptic area and hypothalamus of female rats during pregnancy and ovariectomized, nulliparous rats after steroid priming: correlation with maternal behavior

In a previous study, high nuclear estrogen receptor concentrations in the preoptic area (POA) were found on Day 16 of pregnancy to prime females to respond to a subsequent low dose of estradiol benzoate (EB) after hysterectomy-ovariectomy by exhibiting maternal behavior in 48 hr. Receptor concentrations in the POA were found to be higher than those in the hypothalamus (HYP). The present study investigated when nuclear estrogen receptors increase during pregnancy in POA and when the difference in receptor concentrations between POA and HYP occurs. An attempt was made to reproduce these pregnancy changes with a 16-day treatment of estrogen and progesterone in ovariectomized (OVX), nulliparous rats. In Experiment 1, we measured cytosol and nuclear estrogen receptor concentrations in the POA and HYP of female rats during pregnancy. Nuclear receptor concentrations in the POA increased beginning on Day 10, increased again on Day 16, and continued at this high level for the remainder of pregnancy. Nuclear estrogen receptor concentrations in the HYP remained at a lower level throughout most of pregnancy until Day 22 when they increased significantly. In Experiment 2, we tested the maternal behavior and measured estrogen receptor concentrations in OVX, steroid-primed, nulliparous rats after hysterectomy (H) and EB treatment. While 90% of estradiol (E) + progesterone (P)-primed females displayed short-latency maternal behavior 48 hr after H and EB treatment, 46% of E + vehicle (V)-treated controls were maternal. At 0 hr (prior to H and EB treatment), there was a significantly larger nuclear receptor accumulation in the POA but significantly attenuated receptor binding in the HYP. P treatment significantly affected cytosol and nuclear estrogen receptor dynamics. Differences in nuclear estrogen receptor concentrations were shown to be based on the number of available binding sites and not to changes in receptor affinity for estradiol.     

The ligand binding profiles of estrogen receptors alpha and beta are species dependent

Estrogens and selective estrogen receptor modulators are used for the treatment and prevention of conditions resulting from menopause. Since estrogens exert their activity by binding to nuclear receptors, there is intense interest in developing new ligands for the two known estrogen receptor subtypes, ER-alpha and ER-beta. Characterization assays used to profile new estrogen receptor ligands often utilize receptors from different species, with the assumption that they behave identically. To test this belief, we have profiled a number of estrogens, other steroids, phytoestrogens and selective estrogen receptor modulators in a solid phase radioligand binding assay using recombinant protein for human, rat, and mouse ER-alpha and ER-beta. Certain compounds show species dependent binding preferences for ER-alpha or ER-beta, leading to differences in receptor subtype selectivity. The amino acids identified by crystallography as lining the ligand binding cavity are the same among the three species, suggesting that as yet unidentified amino acids contribute to the structure of the binding site. We conclude from this analysis that the ability of a compound to selectively bind to a particular ER subtype can be species dependent.     

Cooperation of proto-signals for nuclear accumulation of estrogen and progesterone receptors.

Multiple proto-signals (p-NLSs) for nuclear targeting, none of which suffices on its own, cooperate in the estrogen (ER) and progesterone (PR) receptors. In the ER, an estrogen-inducible p-NLS was found in the hormone binding domain (HBD), in addition to three lysine/arginine-rich motifs resembling prototype constitutive nuclear localization signals (NLSs). The inducible and the constitutive ER p-NLSs cooperate in the presence of estrogen and hydroxy-tamoxifen, but not in the presence of ICI 164,384. In the PR, three p-NLSs, two of which are located within and directly adjacent to the second zinc finger, cooperate with each other and a weak hormone-inducible p-NLS in the PR HBD. No 'masking' of p-NLSs by the HBD was observed for ER and PR, while the ligand-free glucocorticoid receptor HBD inhibited the activity of both homologous and heterologous NLSs. Nuclear co-translocation experiments indicated that in vivo the stability of ER and PR dimers is hormonally controlled, but that, in the absence of the cognate ligand, ER dimers are more stable than PR dimers. This is likely to account for the differential hormone requirement of ER and PR DNA binding in vitro.     

Correlation between PAP-dependent steroid binding activity and substrate specificity of mouse and human estrogen sulfotransferases

Estrogen sulfotransferase (EST) is a cytosolic enzyme that catalyzes the sulfoconjugation and inactivation of estrogens using 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as an activated sulfate donor. A finding of undetermined significance in the study of EST has been that the guinea pig EST is able to bind pregnenolone and estradiol with high affinity in the presence of PAP, the reaction by-product of the sulfate donor PAPS. This finding has raised the possibility that EST may have other physiological functions independent of its enzymatic activity as a sulfotransferase. To determine if the PAP-dependent steroid binding activity is a common property shared by other estrogen sulfotransferases, we have expressed the mouse and human EST in bacteria and used the purified protein to address this question. We found that, in the presence of PAP, both recombinant mouse and human EST were able to bind estradiol with high affinity but only the human EST was able to bind pregnenolone. In addition, we show that human but not the mouse EST was also able to bind dehydroepiandrosterone, a property that was not described for the guinea pig EST. Furthermore, we demonstrate that the promiscuity of human EST in steroid binding is mirrored by a correspondingly low substrate specificity in its enzymatic activity as a sulfotransferase. Reversely, the lack of stable binding of pregnenolone and dehydroepiandrosterone by the mouse EST is paralleled by a lack of sulfotransferase activity of this enzyme toward these two steroids. Mutagenesis of mouse EST within a domain critical for PAPS binding abolished both its sulfotransferase and PAP-dependent estrogen binding activity. These data suggest that stable binding of steroids such as pregnenolone or estrogen is not an independent property of estrogen sulfotransferases but rather is related to their catalytic activity.     

Evidence of type II estrogen receptor in human osteoblast-like cells

Osteoblast-like cells isolated from human bone bioptic specimens were characterized and analysed for the presence of type II estrogen receptor (type II EBS). The amount of type II EBS was measured by a whole-cell assay at 4 degrees C for 2.5 h using [(3)H]-estradiol as tracer. Saturation analysis, used to investigate the binding characteristic of type II EBS, resulted in a sigmoid curve. Scatchard analysis showed the binding affinity of the estrogen receptor, yielding a concave plot. The dissociation constant (K(d)), determined from the [(3)H]-estradiol concentration required for half saturation was about 12+/-2 nM (SD). The number of type II EBS, estimated at maximum binding, was 197,000+/-8800 sites per cell. If the regulation of the receptor by flavonoids would be confirmed, the evidence of type II EBS in osteoblast-like cells could suggest a direct action of ipriflavone and others flavonoids on bone density in postmenopausal osteoporosis.     

Apoprotein-B binding sites in estrogen-treated rabbit liver: quantitative immunoelectron microscopy

Normal and estrogen-treated rabbit livers were perfused with iodinated very low density lipoproteins (125I-VLDL) and binding and cellular distribution of apolipoprotein (Apo)-B rich lipoproteins in hepatocytes was analyzed using quantitative immunoelectron microscopic and autoradiographic techniques. Apo-B containing particles were bound to and internalized through the formation of endocytotic pits and vesicles with considerably more binding to estrogen-treated specimens (2-fold). Thus, estrogen treatment stimulates Apo-B binding and subsequent internalization of the Apo-B containing particles by increasing the number of endocytotic pits. In contrast to the estrogen induced liver distribution change of Apo-E reported previously, the Apo-B distribution was not changed due to estrogen treatment.     

Steroid binding alterations in tissue compartments of the vagina of control and neonatally diethylstilbestrol-treated adult mice

Steroid binding in both the vaginal epithelium and the vaginal fibromuscular wall (FMW) was compared in control and neonatally estrogen-treated mice. Neonatal treatment with a low dose of the estrogen diethylstilbestrol (DES) had no significant effect on adult estrogen binding within the assayed vaginal compartments; however, this treatment caused a 2-fold increase in the level of cytosolic progestin binding in the vaginal FMW over that in vehicle-treated mice. This low neonatal dose did not affect the level of progestin binding in the vaginal epithelium. In contrast, neonatal treatment with a larger dose of DES caused marked increases in cytosolic progestin binding, decreases in cytosolic estrogen binding, and increases in nuclear estrogen binding within the FMW. Furthermore, as a result of the changes in specific binding induced by the neonatal DES treatment, the degree of the estrogen binding within in each tissue shifted from a predominantly cytosolic site to a nuclear one.     

Distribution of nuclear receptors for steroid hormones in the human brain: a preliminary study

BACKGROUND: Expression of the nuclear steroid hormone receptors (SHR) within certain parts of the human brain has been described by many authors. However, a comprehensive analysis of SHR expression in the human brain still has not been performed. AIM: To investigate the expression of SHR in different anatomical areas of the brain, especially within the neocortex. METHOD: Immunohistochemical expression of estrogen receptors (ER), progesterone receptors (PR) and androgen receptors (AR) in different regions of the human brain was examined. RESULTS: Nuclear expression of the AR was found in the mamillary body, praecentral gyrus and hippocampus of males. The same expression in analysed structures of female was not found. The expression of ER and PR was not observed. CONCLUSIONS: The analysis revealed unexpected localization of SHR within the brain cortex, which could be the first step to the explanation of SHR action in brain as an interrelationship to function and behaviour. These results indicate on the possibility of SHR detection in post-mortal brain.     

Reciprocal regulation of estrogen and NGF receptors by their ligands in PC12 cells

Recent work has shown that estrogen receptor mRNA and protein co-localize with neurotrophin receptor systems in the developing basal forebrain. In the present study we examined the potential for reciprocal regulation of estrogen and neurotrophin receptor systems by their ligands in a prototypical neurotrophin target, the PC12 cell. using in situ hybridization histochemistry, RT-PCR and a modified nuclear exchange assay, we found both estrogen receptor mRNA and estrogen binding in PC12 cells. Moreover, while estrogen binding was relatively low in naive PC12 cells, long-term exposure to NGF enhanced estrogen binding in these cells by sixfold. Furthermore, concurrent exposure to estrogen and NGF receptor mRNAs deifferentially regulated the expression of the two NGF receptor mRNAs. The expression of trkA mRNA was up-regulated, while p75^NGFR mRNA was down-regulated transiently. The present data indicate that NGF may increase neuronal sensitivity to estrogen, and that estrogen, by differentially regulating p75^NGFR and trkA mRNA, may alter the ratio fo the two NGF receptors, and, conseuqnetly, neurotrophin responsivity. In view of the widespread co-localization of estrogen and neurotrophin receptor systems in the developing CNS, the reciprocal regulation of these receptor systems by NGF and estrogen may have important implications for processes governing neural maturation and the maintenance of neural funciton. 1994 John Wiley & Sons, Inc.     

Exchange assays using sodium thiocyanate to measure total nuclear content of estrogen receptors in the hypothalamic pituitary axis of mice

We studied the effect of sodium thiocyanate (NaSCN) in the determination of specific nuclear estrogen receptors from the hypothalamic-pituitary axis (HPA) of mice. We compared the results with those of the exchange assay using either suspensions of whole nuclei or KC1-nuclear extracts. Our findings demonstrated that 0.5 M NaSCN was more efficient than 0.5 M KC1 in extracting [³H] E₂ nuclear content from HPA (91% vs 79.3%). Nuclear fractions extracted with 0.5 M NaSCN revealed the presence of a single class of low-capacity-high affinity binding sites that sedimented in the 4.0 S area. Nuclear binding was T° dependent and reached maximum levels of 450 ± 156 (S.E.) fmol/mg DNA after an overnight incubation at 4°C. Such levels were comparable to those observed in whole nuclei suspensions after 1 hour incubation at 37°C (618 ± 71 fmol/mg DNA, p > 0.05) but two-fold higher (p <0.01) than the concentration of binding sites measured in KC1-extracted nuclear fractions under similar experimental conditions. We conclude that NaSCN extracted the total content of nuclear estrogen receptors in HPA of mature mice.