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.

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.

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.

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.

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.

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.

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.

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.

Hormonal and immunological aspects of the phylogeny of sex steroid binding plasma protein.

Sex steoid binding plasma protein (Sbp) in man and in monkeys binds the androgens dihydrotestosterone and testosterone and the estrogen estradiol with high affinity (Kd approximately 0.5, 1, and 2 nM, respectively). Detailed studies of steroid binding specificity give the same results in all primates, except that in humans and chimpanzees estrone does not compete for dihydrotestosterone binding. In other mammals, Sbps of Artiodactyla and Lagomorpha have the same range of affinities for androgens but they do not bind estradiol to any significant extent (Kd > 280 nM). The dog has an unusual Sbp (Kd for dihydrotestosterone, 7.1 nM; for estradiol, 125 nM), and rodents do not have a specific dihydrotestosterone-binding plasma protein. Gel filtration and immunoelectrophoretic experiments have been performed with a monospecific antiserum against human Sbp. The results indicate variable crossreactivities with Sbps of primates (from complete in chimpanzee and gorilla to weak in Prosimii). No crossreaction was observed with specific androgen-binding plasma proteins of other species. These results suggest the evolutionary emergence of bifunctional Sbp.

Hormonal and immunological aspects of sex steroid-binding plasma protein of primates.

Sex steroid-binding plasma protein binds dihydrotestosterone, testosterone, and oestradiol with high affinity (Kd,eq. approximately 0.5, 1 and 2 nM, respectively), in man (h-SBP) and in monkeys. Steroid-binding specificity is identical in all primates. However oestrone does not displace dihydrotestosterone binding in man or chimpanzee. Gel filtration experiments and immunoelectrophoretic analysis with a mono-specific antiserum raised in the rabbit against h-SBP indicate cross-reactivity with primate SBPs (from complete in chimpanzee and gorilla to weak in prosimians), but no cross-reaction with other mammalian androgen-binding plasma proteins.

Isolation of highly purified sex hormone binding globulin (SHBG): evidence for microheterogeneity.

Highly purified sex hormone binding globulin (SHBG) was isolated in milligram amounts from a human serum fraction (Cohn IV-4). The final preparation was homogeneous by the criteria of polyacrylamide-gel electrophoresis. Immunological evidence for purity could be given by double diffusion according to Ouchterlony. However, following gel isoelectric focusing highly purified SHBG displayed four different bands, as could be demonstrated by staining as well as by a photoscan of the [3H]5alpha-dihydrotestosterone-SHBG complex. After incubation with neuraminidase the microheterogeneity of SHBG disappeared and the asialo-SHBG showed only one band.