Sex hormone binding globulin and corticosteroid binding globulin as major effectors of steroid action.

Contrary to the long-held postulate of steroid-hormone binding globulin action, these protein carriers of steroids are major players in steroid actions in the body. This manuscript will focus on our work with sex hormone binding globulin (SHBG) and corticosteroid binding globulin (CBG) and demonstrate how they are actively involved in the uptake, intracellular transport, and possibly release of steroids from cells. This manuscript will also discuss our own findings that the steroid estradiol is taken up into the cell, as demonstrated by uptake of fluorescence labeled estradiol into Chinese hamster ovary (CHO) cells, and into the cytoplasm where it may have multiple actions that do not seem to involve the cell nucleus. This manuscript will focus mainly on events in two compartments of the cell, the plasma membrane and the cytoplasm.

Co-transport of sex hormone-binding globulin/SHBG with oxytocin in transport vesicles of the hypothalamo-hypophyseal system.

The intrinsic expression of sex hormone binding globulin (SHBG) in magnocellular hypothalamic neurons, in part co-localized with either vasopressin or oxytocin, was recently described. This study is focused on the ultrastructural localization of SHBG in the hypothalamo-neurohypophyseal pathway in rats. Immunostaining for SHBG in the hypothalamic perikarya was increased by colchicine treatment, indicating that the steroid-binding globulin is subject to rapid axoplasmic transport along with the classical posterior lobe peptides. With immunoelectron-microscopic double labeling, we found co-localization of oxytocin and sex hormone binding globulin in a portion of the large dense-core vesicles in paraventricular and supraoptic perikarya and in axonal varicosities in the median eminence and in the posterior lobe. Our observations show that SHBG is processed, transported and stored along with oxytocin suggesting that SHBG is released from nerve terminals in the posterior lobe, the median eminence and possibly the brain similarly to and in conjunction with oxytocin.

Sea bass (Dicentrarchus labrax) sex hormone binding globulin: molecular and biochemical properties and phylogenetic comparison of its orthologues in multiple fish species.

Sex hormone binding globulin (SHBG) binds and transports androgens and estrogens in the blood of vertebrate species including fish. We have used oligonucleotide primers corresponding to highly conserved regions of the SHBG coding sequences within the zebrafish and fugufish genomes to obtain a 1528 bp cDNA encoding SHBG from tissue RNA extracts from the European sea bass. Amino-terminal sequence analysis of recombinant sea bass SHBG indicated that its deduced precursor polypeptide includes a 35-residue secretion signal polypeptide, and the 361-residue mature sea bass SHBG sequence exhibits 45-67% sequence identity with SHBGs from other fish species that have been determined directly (for zebrafish) or deduced (for rainbow trout, medaka and fugufish) from sequences within public databases. The sea bass SHBG (39,894 Da) comprises a tandem repeat of laminin G-like domains typical of SHBG sequences; contains three N-glycosylation sites, and exists as a 118,300 +/- 11,500 Da homodimer. Sea bass SHBG exhibits a high affinity (K(d) = 8.8 nM for 17beta-estradiol) and specificity for gonadal steroids and their precursors (e.g., 17beta-estradiol > testosterone > dehydroepiandrosterone > 5alpha-dihydrotestosterone > androstenedione >11-ketotesterone). Interestingly, the affinity of sea bass SHBG for the synthetic estrogen, 17alpha-ethynylestradiol was found to be essentially identical to that for 17beta-estradiol. The availability of SHBG sequences in sea bass and other fish set the stage for detailed studies of SHBG function in fish reproductive physiology, as well as its potential role as a target of endocrine disruptors.

Monoclonal antibodies to human sex hormone-binding globulin (SHBG): characterization and use in a simple enzyme-linked immunosorbent assay (ELISA) of SHBG in plasma.

Four monoclonal antibodies to human sex hormone-binding globulin were raised and characterized. Three of the four antibodies recognised different antigenic determinants on SHBG. Two of the distinct antibodies were useful for Western blotting and recognized a major 48 kDa band in human plasma as well as a 46 kDa minor component. Carbohydrate residues do not form part of the antigenic determinants of these two antibodies, although one of these showed increased signal following removal of N-linked oligosaccharides. Some of the antibodies were selected to form a basis of a same-day, non-competitive, enzyme-linked immunosorbent assay (ELISA) for SHBG in plasma. The assay employs a purified IgG2a SHBG monoclonal antibody adsorbed to the wells of a microtitre plate. After blocking any further adsorption to the plate, standards or diluted patient samples were added for a 5-h incubation at room temperature, after which the plate was washed and antibody-bound SHBG was detected with an anti-SHBG IgG1 monoclonal antibody followed by peroxidase-labeled antimouse-IgG1 and o-phenylenediamine substrate. The assay correlated well with an existing 2-day ELISA for SHBG in plasma using polyclonal antibodies and also correlated with a dihydrosterone (DHT) ligand-binding assay. The monoclonal antibody-based ELISA shows excellent performance characteristics and is unaffected by added testosterone or estradiol.

Modulating the Th1/Th2 balance in inflammatory arthritis.

The balance between Th1 and Th2 cells regulates the choice between inflammatory and antibody-mediated immune responses. To an increasing extent this balance is thought to involve the participation of antigen-presenting cells, rather than the entirely autonomous activity of T cells and their cytokines. Here we survey current opinion concerning the working of this balance, and its condition in rheumatoid arthritis and the other inflammatory arthritides. The contrast between Lyme arthritis and reactive arthritis is particularly illuminating, since one is triggered by extracellular and the other by intracellular infection. We describe current approaches to the modulation of this balance. Guided by the principles that genetic polymorphism is likely to identify relevant genes, that any cytokine gene picked up by a virus must matter and that natural immunosuppressive activity at mucosal surfaces should be worth exploiting, we identify as particularly worthy of attention: (i) IL-10, (ii) inhibitors of IL-12 production, (iii) inhibitors of CD40 ligand expression and (iv) oral and nasal tolerance. Other protective T cell subsets are touched on, and the impact of oligonucleotide arrays mentioned.

Achievement of near-normal body weight as the prerequisite to normalize sex hormone-binding globulin concentrations in massively obese men.

OBJECTIVE: To investigate the effects of weight loss on sex hormone-binding globulin (SHBG) in massively obese males and whether normal SHBG concentrations could be obtained regardless or not of the achievement of normal body weight values. DESIGN AND SUBJECTS: Sera were collected for SHBG determination from 63 massively obese men, partly before they underwent biliopancreatic diversion (pre-op group = 11) and partly during the post-surgical follow up (post-op group = 52), and twenty normal weight healthy control men. MEASUREMENTS: Serum SHBG was measured using a noncompetitive liquid-phase immunoradiometric assay. RESULTS: Baseline general characteristics were similar in both obese groups. Obese patients in the post-op group had lost 46.4 +/- 2.9 kg since they had undergone operation, namely during a mean period of 14.9 +/- 13.8 (range 1-58) months follow up. Obese groups had significantly lower SHBG than normal weight controls (66.2 +/- 18.6 nmol/l). However, pre-op obese (19.9 +/- 5.5 nmol/l) had significantly lower values than post-op obese subjects (45.5 +/- 24.8 nmol/l; P < 0.001). There were a highly significant correlation between SHBG and individual BMI values (r = -0.629; P < 0.001). Moreover, the post-op obese with BMI values lower or equal to 28 had significantly higher SHBG concentrations than those with BMI greater than 28 (62.8 +/- 22.2 nmol/l vs 32.1 +/- 19.6 nmol/l; P < 0.001), but not significantly different with respect to normal weight controls. CONCLUSIONS: Massively obese men weight loss can completely reverse SHBG abnormalities, which can be restored to the normal range when near-normal body weight is achieved. Since reduced SHBG concentrations can be an independent risk factor for the development of diabetes and cardiovascular disease, this represents an additional benefit of weight loss program in massively obese individuals.

Distribution of immunoreactive androgen-binding protein/sex hormone-binding globulin in tissues of the fetal rat.

Androgen-binding protein/sex hormone-binding globulin (ABP/SHBG) is an extracellular carrier protein that binds androgens and estrogens with high affinity. In the adult, ABP/SHBG is thought to function in the male reproductive system and the general circulation in both sexes to modulate the actions of sex steroids. The ABP/SHBG gene is also expressed in the embryonic rat liver, where SHBG is secreted into the fetal blood of male and female rats. The embryo also expresses an alternative SHBG with a unique N-terminal sequence. In this study, the distribution of immunoreactive SHBG in the 17-day-old male fetal rat was determined with six antisera. In general, all of the antisera reacted with the same structures. Specific tissue immunoreactivity was mostly cytoplasmic and/or extracellular. By far the most prominent immunoreactive structures were the mesoderm-derived tissues: connective tissue, striated and cardiac muscle, cartilage, and the liver hematopoietic system. In addition, all regions of the fetal brain contained immunoreactive neurons. In the developing male reproductive system, there was minor reactivity in the testicular cords, whereas the connective tissue in the differentiating Wolffian duct stained with all of the antisera. The Wolffian duct epithelium and epithelia in other developing organs contained small amounts of immunoreactive SHBG, except for the lung, which stained in the epithelial extracellular matrix. An antibody raised against a unique N-terminal peptide specific for the alternative SHBG protein revealed that it was also present in many tissues. These data suggest that SHBG is important for the differentiation of mesodermal tissues. SHBG may modulate the action of androgens in embryonic stroma, thereby regulating development of the epithelium in hormone-dependent tissues.

Effects of tamoxifen adjuvant therapy and a low-fat diet on serum binding proteins and estradiol bioavailability in postmenopausal breast cancer patients.

Serum was collected at intervals from postmenopausal breast cancer patients to determine the effects of tamoxifen adjuvant therapy and a low-fat dietary intervention, alone and in combination, on sex hormone-binding globulin (SHBG) concentrations and circulating estradiol bioavailability. Serum corticosteroid-binding globulin and follicle-stimulating hormone were also assayed as indicators of patient compliance to tamoxifen therapy. The immunoreactive SHBG concentration was higher (P less than 0.001) in 22 patients who had been treated with tamoxifen for 6-36 weeks when first sampled, compared with 27 who were not receiving tamoxifen therapy. Tamoxifen also produced a reduction in the percentage non-protein-bound estradiol (P less than 0.001) and percentage albumin-bound estradiol (P less than 0.01), the two biologically available fractions, and a corresponding increase in the percentage SHBG-bound estradiol (P less than 0.01). A longitudinal study of 7 patients showed significant reductions in the percentage of albumin-bound estradiol and an increased percentage of SHBG-bound estradiol, after 3-6 months of tamoxifen; after 12-18 months there was also a significant decrease in the non-protein-bound estradiol fraction. We conclude that in postmenopausal breast cancer patients the redistribution of circulating estradiol, with reduced bioavailability, provides an additional mechanism to those demonstrated previously for the therapeutic activity of tamoxifen. Another 12 patients receiving tamoxifen and 8 who were not were followed for 6-12 months on a low-fat diet (fat comprised 20% of the total calories). The dietary intervention had no effect on the serum SHBG concentration or the estradiol distribution. Although tamoxifen increased the serum corticosteroid-binding globulin and partially suppressed the follicle-stimulating hormone concentrations, the responses obtained were less consistent compared with those of the SHBG levels.

A surface plasmon resonance immunosensor based on the streptavidin-biotin complex.

It is shown that a streptavidin monolayer immobilized onto an evaporated gold film with biotin forms the basis of a highly specific sensing element. As an example, we show that by immobilizing the biotinylated antibody sex hormone binding globulin (alpha-SHBG) to the bound streptavidin monolayer a specific sensor for the antigen SHBG is readily fabricated. The interaction between immobilized antibody and corresponding antigen is monitored by surface plasmon resonance spectroscopy and is shown to follow a classic Langmuir isotherm. Detection of SHBG at nanomolar concentrations is demonstrated.

Two-sites immunoradiometric assay using monoclonal antibodies for the determination of serum human sex hormone binding globulin.

An immunoradiometric assay (IRMA) for the determination of human Sex Hormone Binding Globulin (SHBG) in serum is described. This IRMA uses three mouse monoclonal antibodies. Two monoclonals anti-human SHBG are coated on tubes and used as capture antibodies. The third monoclonal labeled with 125I completes the system, allowing the formation of the "sandwich". The detection limit of the assay is 2.5 femtomol SHBG per tube (250 pg/tube). Using this test for the measurement of SHBG and radioimmunoassays for the determination of total Testosterone and Estradiol, we calculated the Free Androgen Index (FAI) and the Free Testosterone. The results obtained were compared with the values of Free Testosterone measured by equilibrium dialysis. There is a close correlation between both calculated parameters and the levels of Free Testosterone, validating this SHBG assay.

Site-specific antibodies to the DNA-binding domain of estrogen receptor distinguish this protein from the 3H-estradiol-binding protein in pancreas.

The estradiol-binding protein (EBP) in extracts of rat and rabbit pancreata was characterized by sucrose density gradient analysis, immunoaffinity adsorption and Western blot (immunoblot) analysis using polyclonal antibodies raised against EBP. Rat pancreatic extracts labeled with 3H-estradiol contained a readily resolvable peak of steroid-binding activity that sedimented as a 4S complex on sucrose density gradients in the presence or absence of 0.4 M KCl. Estrogen receptor (ER) from calf uterine cytosols sedimented as a 4S complex on gradients containing 0.4 M KCl and as an 8S entity on gradients without KCl. Incubation of cytosol fractions from rat pancreas and calf uterus with benzoyl-DL-arginyl-p-nitroanilide (BAN) increased specific binding of 3H-estradiol to EBP but not to ER. Furthermore, two distinct site-specific antibodies to the DNA-binding domain of ER caused a marked increase in sedimentation rate of 3H-estradiol-labeled ER while normal rabbit serum and antibodies against EBP were ineffective in this regard. These data suggest that a significant portion, if not all, of the DNA-binding domain of ER is absent from EBP. Examination of the amino acid sequence of the DNA-binding domain of ER revealed a region of 10 amino acids that is significantly homologous to somatostatin, a tetradecapeptide that is a co-ligand in the binding of 3H-estradiol to EBP. Based on this observation, a possible mode of action of EBP in pancreatic acinar cells is proposed.

Direct effect of plasma sex hormone binding globulin (SHBG) on the metabolic clearance rate of 17 beta-estradiol in the primate.

Sex hormone binding globulin (SHBG) has been shown to be a major determinant of testosterone clearance in the primate. It has also been suggested that SHBG would also be a determinant of estradiol clearance (MCR-E2). However, published studies have suggested that the MCR-E2 do not always vary with changes in the level of SHBG. Therefore, the present study was undertaken to address this issue. The baseline MCR-E2 was determined in adult male pigtail macaques (Macaca nemestrina). Following the baseline determination of MCR-E2 the animals were infused with either purified human (h) SHBG or antibody against hSHBG, which also has a high degree of cross-reactivity with primate SHBG. Following the infusions of either hSHBG or anti-SHBG, MCR-E2 was again determined. In addition, luteinizing hormone (LH) was measured using a mouse Leydig cell bioassay. Following the infusion of hSHBG, a marked increase in serum SHBG was noted and the MCR-E2 decreased. Associated with the increase in SHBG, the SHBG bound T levels decreased and LH increased. Following the infusion of antibody, serum SHBG decreased, and the MCR-E2 also decreased. With the decrease in SHBG following the antibody infusion, non-SHBG bound T increased and serum LH fell. This study demonstrates that an increase in the serum SHBG levels is associated with a decrease in MCR-E2, however, an acute decrease in serum SHBG also decreases the MCR-E2. This later result demonstrates that factors in addition to serum SHBG binding may be important in determining the MCR-E2.

Monoclonal antibodies to rat androgen-binding protein recognize both of its subunits and cross-react with rabbit and human testosterone-binding globulin.

A series of four monoclonal antibodies to rat androgen-binding protein (ABP) has been developed. These antibodies recognize both the heavy (48,400-dalton) and light (43,000-dalton) subunits of the native ABP molecule. In addition, they recognize the subunits from which Asn-linked oligosaccharides have been removed by treatment with N-glycanase, indicating that these moieties do not form the immunological determinants recognized by the antibodies. Two of these antibodies are capable of recognizing both nondenatured ABP, as assessed by dot blot analysis, and denatured ABP, as determined by Western blot analysis of ABP after electrophoresis under denaturing conditions. The immunoreactivity of denatured ABP is decreased with two of the antibodies, suggesting that they more readily recognize the antigenic epitopes when the protein is in its native configuration. The antibodies were capable of immunoprecipitating covalently labeled ABP from solution. All four monoclonal antibodies produced were determined to be immunoglobulins M by both enzyme-linked immunosorbent assay and Ouchterlony immunodiffusion even though the initial serum response of the immunized animals indicated the presence of immunoglobulins G. All of the monoclonal antibodies raised against rat ABP cross-reacted with rabbit and human testosterone-binding globulin (TeBG). They were able to detect two subunits when Western blots of intact rabbit [mol wt (Mr, 43,000 and 40,500] or human (Mr, 47,600 and 44,500) TeBG were probed, but only a single subunit (Mr, 39,300) when deglycosylated samples of rabbit TeBG were analyzed.

An enzyme-linked immunosorbent assay (ELISA) for human sex hormone-binding globulin.

An enzyme-linked immunosorbent assay (ELISA) of the "sandwich-type" for sex hormone binding globulin (SHBG) has been developed. A rabbit anti-SHBG antibody (RAb) is immobilized to the microtitre plate. After incubation with standards and samples a second monospecific rabbit anti-SHBG antibody, labelled with alkaline phosphatase is added (RAb). Following further washing substrate is added, colour developed and the plate read at 405 nm wavelength on a standard ELISA plate reader. The assay is not influenced by the presence of steroids at the binding site, and shows good agreement to SHBG binding capacity assay and commercially available IRMA. Its sensitivity, specificity and precision allows its use in the routine laboratory. The SHBG ELISA has been used to measure SHBG concentrations in sera of normal men, women, pregnant women, and women receiving high-dose medroxyprogesterone acetate as a treatment of metastatic breast cancer.

Comparison of rabbit epididymal androgen binding protein and serum testosterone estradiol binding globulin--II. Immunological properties.

Purified rabbit epididymal androgen binding protein and serum testosterone estradiol binding globulin have been immunologically compared. A comparison using the steady state gel method of Ritzen et al. indicated immunological cross-reactivity. In order to further compare their immunological properties we developed a radioimmunoassay for both rbABP and rbTeBG using specific antisera directed against each. When these assays were compared, the extent or completeness of displacement proved to be the only parameter that was significantly different. This data obtained with homologous and heterologous radioimmunoassays is consistent with the idea that these two proteins contain minor antigenic determinants which are distinct.

Steroid-binding proteins in primate plasma.

We used immunological techniques to compare the serum corticosteroid-binding globulins (CBG) and testosterone-estradiol-binding globulins (TeBG) of Old World primates (man, chimpanzee, cynomologus, and rhesus), New World monkeys (squirrel and owl), and prosimians (galago and lemur). Four different antihuman TeBG antisera could not differentiate human and chimpanzee TeBG and recognized the galago and lemur TeBG as similar as well as the rhesus and cynomologus TeBG, as similar. Western blots of serum subjected to sodium dodecyl sulfate gel electrophoresis, with detection by an anti-TeBG antiserum, showed similar patterns of distribution of the two molecular species of TeBG for all of the New World primates and the owl monkey. The abundance of the two TeBG species was reversed in squirrel monkey serum, while lemur and galago displayed only a single band. Four different antihuman CBG antisera grouped together the CBGs of human and chimpanzee, rhesus and cynomologus, and lemur and galago. The squirrel monkey has a CBG with a markedly decreased affinity for cortisol; all four antisera perceived its CBG as much more immunologically distant from the human protein than that of the owl monkey. Indeed, three of the four antisera grouped squirrel monkey CBG with that of the prosimians, while one antiserum saw squirrel monkey CBG as even more distant from the human protein than the CBG of the primitive primates, the prosimians.

Immunoradiometric assay of sex-hormone binding globulin with use of two different monoclonal antibodies.

Two monoclonal antibodies (MK1 and MK2) reacting with human sex-hormone binding globulin (SHBG) were obtained from mice hybridomas. The dissociation constants for the binding of SHBG to MK1 and MK2 were 7.5 and 75 pmol/L, respectively. MK1 was coupled to polyacrylamide beads with a yield of 37%, resulting in 15 mg of antibody per gram of beads. The maximal binding of SHBG by the MK1-beads was 16% of the theoretical capacity. The amount of 125l-labeled MK2 bound to MK1-beads was related to the amount of SHBG present. The system has been used for the immunoradiometric assay (IRMA) of SHBG in serum, and has been standardized with purified SHBG. Assay sensitivity is 3 micrograms/L; intra- and inter-assay (total variation) CVs were 5% and 10%, respectively. Values obtained with the assay for 100 patients' sera agreed well with those obtained with a conventional radioimmunoassay, and SHBG in a patient's serum subjected to gel chromatography eluted as a symmetrical peak with the expected retention when the effluent was analyzed with the present assay. Analytical recovery of SHBG added to serum from a man, a woman, and a pregnant woman ranged between 93% and 107%. The mean (and SD) concentrations of SHBG in sera from healthy women and men were 3.7 +/- 1.1 and 1.8 +/- 0.9 mg/L, respectively.

Use of a styrene-maleic anhydride copolymer as an adjuvant with testosterone-serum albumin immunogens and its application to increasing ovulation rate in the ewe.

A synthetic poly(styrene-maleic anhydride) copolymer of average molecular weight 470,000 potentiated a testosterone-binding antibody response during immunization of sheep with immunogenic testosterone-3-carboxymethyloxime-serum albumin conjugates. The copolymer had weaker immunostimulatory activity than Freund's complete adjuvant (FCA). The minimum effective dose of the copolymer was about 30 mg at which secondary but not primary immune responses could be detected by radioimmunoassay. There was a generally weak anamnestic response in immune sheep as secondary and teritary responses tended to decline promptly from peak values; tertiary antibody titres usually did not exceed secondaries. The use of the copolymer as a solution or emulsion had no apparent effect on its immunoadjuvant activity when administered intramuscularly but the soluble form was inactive when given intraperitoneally. The testosterone-binding antibody that was produced using either the copolymer or FCA had considerable sensitivity to deactivation by mercaptoethanol. The ovulation rate of a group of 20 Merino ewes following immunization with testosterone-serum albumin using the copolymer adjuvant was significantly higher than an equal group of untreated control ewes.