Sex steroid binding protein (SBP) receptors in estrogen sensitive tissues

Since the discovery of a specific membrane binding site for sex steroid binding protein (SBP) in human decidual endometrium and in hyperplastic prostate numerous speculations have been raised on the existence of an additional non-receptor-mediated system for steroid hormone action. In the present work SBP cell membrane binding was investigated in human estrogen target tissues other than those previously studied either in the absence of steroids or in the presence of varying amounts (10⁻¹⁰−10⁻⁶M) of estradiol, testosterone and dihydrotestosterone, respectively. Plasma membranes obtained by differential centrifugation from homogenized samples of pre-menopausal endometrium, endometrium adenocarcinoma, normal liver and post-menopausal breast showed a specific binding of highly purified [¹²⁵I]SBP: a major displacement of labeled SBP was elicited by radioinert SBP, while no significant displacement occurred when other human plasma proteins were used as cold competitors (molar excess ranging 500–10,000-fold). A specific, time-dependent binding of [¹²⁵I]SBP was also observed in MCF-7 and in Hep-G2 cell lines. The different patterns of specific binding, observed in membranes from different tissues when SBP was liganded with different sex steroid molecules, leads us to consider the tissue individuality of the receptor as a further entity in the membrane recognition system for SBP.     

Receptor for sex steroid-binding protein of endometrium membranes: solubilization, partial characterization, and role of estradiol in steroid-binding protein-soluble receptor interaction.

The sex steroid-binding protein (SBP) receptor was solubilized from the membranes of human premenopausal endometrium with the zwitterionic detergent CHAPS. The binding activity of the soluble receptor was studied, allowing it to interact with [125I]SBP and precipitating the complex with polyethylene glycol 8,000. The interaction of SBP with the soluble receptor was specific, saturable, and at high affinity. Indeed, the specific binding was definitely improved on the solubilized form of the receptor. The effect exerted by sex steroids on the interaction of SBP with receptor was also examined on both the soluble and membrane-bound forms. At physiologic doses (10(-8) M) estradiol inhibits the binding at a significant extent on the soluble receptor, but not on membrane-bound form. The dose of estradiol required to significantly inhibit the SBP-specific binding was dependent on the form of receptor. In membrane-bound receptor the inhibiting dose of estradiol was higher than its physiologic concentration. Thus, it is likely that, while soluble receptor cannot recognize the complex steroid-SBP, membrane-bound receptor can interact both with "unliganded" SBP and with the estradiol-SBP complex (but not with androgen-SBP complexes) in an estrogen-dependent tissue like human endometrium.     

Regulation of SBP synthesis in human cancer cell lines by steroid and thyroid hormones

The presence of human sex steroid binding protein (SBP) in liver cells, the supposed site of SBP synthesis, and in other target cells for sex steroid hormones such as breast, endometrium and prostate epithelium, have been demonstrated by indirect immunofluorescence. It is not known whether SBP enters endometrial and prostate cells by endocytosis, possibly mediated by a cell membrane receptor process, or if SBP is synthesized in these cells. SBP mRNA has been searched in human cancer cell lines originated from liver (Hep G2/H5A), breast (MCF-7), endometrium (RL95-2) and prostate (LNCaP). It was only found in hepatoma cells where it is regulated by estradiol, antiestrogen tamoxifen and triiodothyronine, in a similar way as secreted SBP. This work provides evidence that human SBP is synthesized in the liver, and it also suggests that its regulation may involve non-steroidal factors.     

Sex steroid-binding protein interacts with a specific receptor on human premenopausal endometrium membrane: modulating effect of estradiol.

Sex steroid-binding protein receptor was detected on membranes prepared from human premenopausal endometrium. The binding of sex steroid-binding protein to membranes was specific, saturable, and high affinity. Scatchard analysis showed the presence of two binding sites at different affinities. The addition of estradiol (10(-8) M) did not produce any inhibition of binding; indeed, it resulted in a modification of binding characteristics. The demonstration of sex steroid-binding protein receptor on membranes of human premenopausal endometrium indicates that the expression of receptor on membranes is not an effect of estrogen over stimulation on target tissues. Estradiol could act as a modulating factor of the binding, probably reflecting the sensitivity of tissues to different steroids.     

A recognition system for sex-hormone-binding protein-estradiol complex in human decidual endometrium plasma membranes

We have studied the specific binding of human serum sex-hormone-binding protein and its complexes with estradiol and testosterone to an enriched membrane fraction isolated from human decidual endometrium. The specific binding of sex-hormone-binding protein to these membranes has been found to be dependent on it being bound with estradiol. When estradiol concentration in the medium is high enough to provide for the saturation of sex-hormone-binding protein with the steroid, the protein exhibits specific binding to the membranes with an apparent equilibrium constant, Kd = (3.5 +/- 2.0) X 10(-12) M. In the absence of estradiol, the binding protein devoid of steroid or complexed with testosterone does not interact with the membranes. At low concentrations of sex-hormone-binding protein and estradiol, when their complex is almost completely dissociated in solution, it can be reconstituted on the membranes with the optimum estradiol to binding protein ratio being 1:1 (mol/mol). It is proposed that, in plasma membranes of the estradiol target cells, there is a recognition system for the sex-hormone-binding protein-estradiol complex which may allow these cells to take up from blood not only free estradiol, but also estradiol complexed with the binding protein.     

A recognition system for sex-hormone-binding protein-estradiol complex in human decidual endometrium plasma membranes

We have studied the specific binding of human serum sex-hormone-binding protein and its complexes with estradiol and testosterone to an enriched membrane fraction isolated from human decidual endometrium. The specific binding of sex-hormone-binding protein to these membranes has been found to be dependent on it being bound with estradiol. When estradiol concentration in the medium is high enough to provide for the saturation of sex-hormone-binding protein with the steroid, the protein exhibits specific binding to the membranes with an apparent equilibrium constant, K_d = (3.5 ± 2.0) · 10⁻¹² M. In the absence of estradiol, the binding protein devoid of steroid or complexed with testosterone does not interact with the membranes. At low concentrations of sex-hormone-binding protein and estradiol, when their complex is almost completely dissociated in solution, it can be reconstituted on the membranes with the optimum estradiol to binding protein ratio being 1:1 (mol/mol). It is proposed that, in plasma membranes of the estradiol target cells, there is a recognition system for the sex-hormone-binding protein-estradiol complex which may allow these cells to take up from blood not only free estradiol, but also estradiol complexed with the binding protein.     

Effects of hormones on SBP mRNA levels in human cancer cells

The human plasma sex steroid binding protein (SBP) has been previously shown to be synthesized in liver cells. The hormonal regulation studies of hepatic SBP mRNA demonstrate that it is controlled by estradiol, antiestrogen tamoxifen, dihydrotestosterone, triiodothyronine and insulin in a similar way as secreted SBP. The metabolic inhibitor cycloheximide was unable to prevent the estrogen or thyroid hormone induced increase in SBP mRNA. The slight stimulation of SBP synthesis by estradiol suggests that non-steroidal factors may be involved in its regulation and that the estrogen regulatory mechanism could also be partly post-transcrptional. In endometrial (Ishikawa cells) and prostatic (LNCaP cells) carcinoma cells, SBP mRNA has been detected suggesting that SBP may play a role in the uptake and intracellular mechanism of action of sex steroid in target cells.     

Structure and dynamics of the estrogen receptor

To evaluate the structure and function of estrogen receptor (ER) in various mammalian systems, the cytosolic forms of receptor from calf uterus and from MCF-7 human breast cancer cells have been purified to virtual homogeneity by sequential selective adsorption to estradiol-Sepharose and heparin-Sepharose. In both cases, the purified steroid-receptor complex appears to exist as an activated 5S homo- or heterodimer of mol. wt 65,000 (4S) steroid-binding subunits. Purified ER has high affinity for DNA and serves as a substrate for phosphorylation by a purified rat brain kinase. Several monoclonal antibodies prepared against affinity-purified MCF-7 cytosol ER have been used to localize receptor by an indirect immunoperoxidase technique in fixed, frozen sections of human breast tumors, human uterus, rabbit uterus and in other mammalian reproductive tissues and cancers, as well as in fixed MCF-7 cell cultures and in paraffin-embedded sections of breast tumors and human endometrium. In all cases, we have observed only nuclear localization of immunoreactive receptor in tissues and whole cells, even under conditions in which virtually all of the receptor is found in a low-salt extract (cytosol) of the target cells. Treatment of cells or tissues in vivo or in vitro with estradiol alters the intensity but not the distribution of specific staining for ER. By immunoelectron microscopy, receptor was localized in the euchromatin, but not in the marginated heterochromatin or nucleoli of MCF-7 nuclei and epithelial and stromal nuclei of postmenopausal human endometrium. These observations suggest that the majority of the unoccupied receptor may actually reside in the nucleus, rather than in the cytoplasm as previously thought. Thus, hormone action may involve binding of the steroid directly to receptor loosely associated with nuclear components, followed by conversion of the steroid-receptor complex to an activated form which becomes more tightly associated with chromatin.     

Binding of sex steroid binding protein to plasma membranes of human testis

The existence of a specific binding site for sex steroid binding protein (SBP or SHBG) was detected on plasma membranes prepared from the testis of a patient affected by a variant form of testicular feminization. A binding technique using [¹²⁵I]SBP as a tracer allowed us to identify a single set of binding sites, characterized by a K_d of 1.917 × 10⁻¹¹ M. The maximum number of binding sites was 5.2 fmol/mg membrane protein. Membranes were also prepared from a sample of genital skin from the same patient, but no binding for [¹²⁵I]SBP was detectable. The evidence of the SBP membrane receptor in the testis of a patient affected by Morris syndrome extends our knowledge about the tissue distribution of the SBP receptor and suggests the possible implication of SBP and its recognition system in a disorder related to peripheral androgen insensitivity.     

Hormonal control of SBP in human hepatoma cells

Since it is currently believed that the biosynthesis of human sex steroid binding plasma protein (SBP) takes place in the liver, the secretion of this protein and its hormonal control were studied in a human hepatoma cell line. The human hepatoma-derived cell line, Hep G2, and a clone, H5A, isolated from Hep G2, were both found to secrete SBP-like protein. This protein had the same dihydrotestosterone binding parameters as plasma SBP, with a Kd ranging from 0.3 to 1 nM at 4 degrees C, and it cross-reacted with a monospecific goat anti-human SBP antiserum. In a chemically defined medium, SBP-like protein secretion was stimulated approx 2-fold by estradiol (1 microM) whereas a smaller concentration of estrogen (100 nM) has only a slight effect. A combined incubation with estradiol (100 nM) and triiodothyronine (10 nM) increased SBP-like protein secretion more than estradiol (1 microM) alone. In response to dexamethasone (100 nM) or tamoxifen (100 nM) treatment, a 3-fold increase is obtained. Therefore, these human parenchymal cells should provide a potent material for investigation of the hormonal regulation of SBP gene.     

Estradiol membrane binding sites on human breast cancer cell lines. Use of a fluorescent estradiol conjugate to demonstrate plasma membrane binding systems

A fluorescent estradiol macromolecular complex was used to study and to characterize steroid binding to membranes of living target cells. Ligand binding to plasma membranes was quantitated with a sensitivity of 0.1 nM. In this way, we found two types of estradiol-binding sites on hormone sensitive MCF-7 cells. Type A sites (8000-16000 sites per cell) were rapidly saturated at low concentrations of the estradiol-bovine serum albumin-fluorescein isothiocyanate macromolecular complex (E2-BSA-FITC). They had a greater affinity for the complex than did the type B sites for which a phenomenon of cooperative fixation was shown. The complex binding was displaced by estrogenic molecules, but not by non-estrogenic compounds, such as cortisol or progesterone. We also studied complex binding on another breast cancer cell line, MDA-MB-231 (MDA), without intracellular estrogen receptors. These cells showed a specific plasma membrane binding system for estrogen, but lacked the high affinity type A binding site. Then, we report the effects of enzyme treatments (trypsin, phospholipase A2 and neuraminidase) on E2-BSA-FITC binding to MCF-7 cell membranes. The quantity of complex bound to membranes decreased after phospholipase and neuraminidase treatments and increased after trypsin. But, in the three cases, the binding was no longer specific because it could not be displaced by E2-BSA or by estradiol. The enzymatic effects were reversible and specific binding was totally restored within 24 h. However, in the presence of the protein synthesis inhibitor, cycloheximide, no restoration of specific binding occurred on trypsin-treated cells. Estrogen binding to MCF-7 and MDA cell plasma membranes thus possesses the three characteristics of all mediated transport processes across biological membranes: saturability, substrate specificity, and specific inhibition. However, the high affinity type A binding site was found only on the estrogen-sensitive cell line, MCF-7.     

Biological function of the carbohydrate component of human sex hormone-binding globulin

The role of the carbohydrate component of sex steroid-binding globulin (SBP) from human blood in the glycoprotein interaction with the recognition system for SBP-estrogen complexes in human decidual endometrium plasma membrane was studied. It was shown that the removal of N-acetylneuraminic acid residues from the oligosaccharide chains of SBP did not affect the steroid-binding or immunochemical properties of the glycoprotein. At the same time, the above modification of the glycoprotein resulted in a loss by SBP of its ability to specifically interact with the membrane recognition system. It is concluded that the oligosaccharide chains of SBP are involved in the formation of determinants needed for recognition of the SBP-estrogen complexes by endometrium cell plasma membranes.     

Modifications of the properties of human sex steroid-binding protein by nonesterified fatty acids

The effect of unsaturated and saturated nonesterified fatty acids (NEFAs) on the electrophoretic, immunological, and steroid-binding properties of human sex hormone-binding protein (SBP) were investigated. Tests were carried out on whole serum from pregnant women and on purified SBP using polyacrylamide gel electrophoresis, crossed immunoelectrophoresis with autoradiography, and equilibrium dialysis. All three methods showed that NEFAs influence the binding of sex steroids to SBP both in whole serum and with the purified protein. Saturated NEFAs caused a 1.5-2-fold increase in binding of dehydrotestosterone, testosterone, and estradiol to SBP, while unsaturated NEFAs, such as oleic (18:1) and docosahexaenoic (22:6) acids inhibited the binding of these steroids to SBP. Thus, unsaturated NEFAs in the concentration range 1-100 microM are more inhibitory for estradiol binding than for testosterone or dehydrotestosterone binding. In addition to these binding changes, polyacrylamide gel electrophoresis and immunoelectrophoretic studies revealed a shift in SBP from the slow-moving active native form to a fast-moving inactive one. There was also a reduction in the apparent SBP concentration by Laurell immunoelectrophoresis in the presence of unsaturated NEFA (5.5 nmol of NEFA/pmol of protein). These studies indicate that unsaturated NEFAs induce conformational changes in human SBP which are reflected in its electrophoretic, immunological, and steroid-binding properties. They suggest that the fatty acid content of the SBP environment may result in lower steroid hormone binding and thus increased free hormone levels.     

Epidermal growth factor binding and steroid receptor content in human benign prostatic hyperplasia

The receptor for epidermal growth factor (EGF-R) was characterized on membrane fractions from human benign prostatic hyperplasia (BPH). Specific binding of [125I]EGF reached equilibrium after 40 min at 25 degrees C and was stable for up to 120 min. Saturation analysis of EGF-R, performed by incubating the membranes with 0.0156-15 nM [125I]EGF in the presence and in the absence of 100-fold excess of cold EGF for 60 min, revealed the presence of two classes of binding sites with high and low affinities (Kd = 0.35 +/- 0.23 and 9.60 +/- 2.87 nM respectively). Competition experiments revealed that FSH, insulin and calcitonin did not compete with [125I]EGF. The simultaneous determination of EGF-R and that of estradiol (ER), progesterone (PR) and androgen receptors (AR) was performed using the same buffer to homogenate the tissues and to obtain cellular membranes. The steroid receptors (SR) were determined by means of the dextran-coated charcoal method. There was a significant negative correlation between nuclear SR and binding capacity of EGF-R. The presence of specific and high affinity binding sites for EGF and the modulation of the level of these sites by steroid receptors suggest a possible role of EGF in prostatic hyperplasia.     

Metabolism of [3H]equilin in normal and malignant human endometrium and in endometrial adenocarcinoma transplanted into nude mice

One of the main components of conjugated equine estrogens is equilin sulfate and this estrogen in postmenopausal women is metabolized to 17 beta-dihydroequilin, 17 beta-dihydroequilenin and equilenin. To investigate the possibility that some of these estrogens may be formed directly in the target tissues, we studied the in vitro metabolism of [3H]equilin in various types of normal and malignant human endometrium, including adenocarcinoma grown in athymic nude mice. The results indicate that normal and neoplastic human endometrium can form the above three metabolites. The highest level of 17 beta-reduced products were isolated from the normal secretory endometrium. Equilenin was the most abundant metabolite isolated from both the normal and malignant endometrium. The formation of [3H]equilenin indicates the presence of a 6,8(9) steroid dehydrogenase-isomerase in the human endometrium. The formation of 17 beta-dihydroequilin in the endometrium may be of importance as this estrogen is 8 times more potent as a uterotrophic agent than equilin and estrone.     

Prolonged cultivation of rat uterine cells with preserved estrogen responsiveness

A rat uterine cell culture was prepared as an experimental system for investigation of mechanisms of steroid hormone actions. Cells frequently supplemented with fresh medium were successfully cultured for 4 weeks through 2 successive passages. Studies of estrogen responsiveness in the primary culture as well as in it's first subculture were performed by a small scale uptake assay for determination of specific steroid binding. Scatchard analysis of specific ovarian hormone binding confirmed that cultured uterine cells preserve both estradiol and progesterone receptors. Characteristics of specific [3H]estradiol binding detected in cells of the first subculture were comparable to those obtained in the initial primary culture. The number of specific estradiol binding sites was diminished to one third of the initial values only in cells of the second subculture, 22 days after isolation of cells from tissue. In the primary culture and in it's first subculture the cells responded to estradiol with a 2-3-fold increase in progesterone receptor level. The subcellular distribution of steroid receptors was also studied; estradiol receptor complexes were detected predominantly in the nuclei whereas progesterone receptors were nearly equally distributed between nuclei and cytosol.     

Plasma sex steroid binding in Chiroptera

Plasma steroid binding was examined in samples obtained from seven species of bats representing four different families. A specific sex steroid-binding protein (SBP) was identified by steady-state polyacrylamide gel electrophoresis in representatives of two families, the phyllostomids and the vespertilionids. In these species, as in primates, SBP not only exhibited high affinity for the androgens testosterone and dihydrotestosterone (DHT), but also for estradiol. A specific SBP was not identified in the tropical American vampire bat or in the two species of pteropodids examined. In all species examined, except for the vampire bat, a specific corticosteroid-binding globulin (CBG) was also identified. In addition to binding glucocorticoids, CBG in these species appeared to bind androgens as well.     

Monoclonal antibodies to the human laminin receptor recognize structurally distinct sites

Laminin, a glycoprotein of basement membranes, binds to a specific receptor on the surface of neoplastic and non-neoplastic cells. The laminin receptor purified from human breast carcinoma plasma membranes was used as an antigen to generate two types of monoclonal antibodies (mAbs). Both types of mAbs bind to (a) the purified receptor coated on a solid phase; (b) isolated breast carcinoma plasma membranes; and (c) the surface of cultured MCF-7 human breast carcinoma cells by immunohistology. Using immunoblotting, both types of mAbs recognize a single 67 000 Dalton protein among all the proteins extracted from breast carcinoma plasma membranes. The mAbs differed in their ability to block binding of laminin to the plasma membrane receptor. Antibody LR1 inhibited virtually 100% of the specific binding of laminin to both the isolated human breast carcinoma plasma membranes or the living MCF-7 cells. In contrast, antibody LR2 had no effect on laminin binding under identical conditions. Thus, the two types of mAbs may recognize structurally distinct sites on the laminin receptor. These mAbs should be useful to dissect the biology and the molecular genetics of the laminin receptor.     

Qualification of estrogen binding variables in tissues of the immature rat.

Serum albumin, alphafetoprotein, and specific receptor binding of estradiol have been shown for the uterus, pituitary, hypothalamus, and brain cortex of the immature rat. Serum proteins were measured by immunologic methods and, following sucrose density gradient fractionation, could be definitively separated from the specific tissue estrogen receptors. The mass of alphafetoprotein in soluble tissue extracts paralleled the amounts of specific antigen detected in the serum. The choice of analytic method for designating a population of specific tissue estrogen receptor, which is free of nonreceptor steroid binding variables must therefore consider the extent of contamination by serum proteins. Excess radioinert steroid is contraindicated in receptor assays where low levels of specific receptor obtain.