Sex hormone-binding globulin (SHBG) mitigates ER stress and improves viability and insulin sensitivity in adipose-derived mesenchymal stem cells (ASC) of equine metabolic syndrome (EMS)-affected horses.

BACKGROUND: Equine metabolic syndrome (EMS), which encompasses insulin resistance, low-grade inflammation and predisposition to laminitis is a critical endocrine disorder among the most prevalent conditions affecting horses from different breeds. According to the most recent research, low human sex hormone-binding globulin (SHBG) serum levels correlate with an increased risk of obesity, insulin resistance and diabetes, and may contribute to overall metabolic dysregulations. This study aimed to test whether exogenous SHBG could protect EMS affected adipose-derived stromal stem cells (EqASCEMS) from apoptosis, oxidative stress, ER stress and thus improve insulin sensitivity. METHODS: EqASCEMS wells were treated with two different concentrations (50 and 100 nM) of exogenous SHBG, whose biocompatibility was tested after 24, 48 and 72 h of incubation. Several parameters including cell viability, apoptosis, cell cycle, reactive oxygen species levels, ER stress, Pi3K/MAPK activation and insulin transducers expression were analysed. RESULTS: Obtained data demonstrated that exogenous SHBG treatment significantly promoted ASCs cells proliferation, cell cycle and survival with reduced expression of p53 and p21 pro-apoptotic mediators. Furthermore, SHBG alleviated the oxidative stress caused by EMS and reduced the overaccumulation of intracellular ROS, by reducing ROS + cell percentage and regulating gene expression of endogenous antioxidant enzymes (Sod 1, Cat, GPx), SHBG treatment exhibited antioxidant activity by modulating total nitric oxide (NO) levels in EMS cells as well. SHBG treatment dampened the activation of ER stress sensors and effectors in EqASCEMS cells via the upregulation of MiR-7a-5p, the decrease in the expression levels of ATF-6, CHOP and eiF2A and the restoration of PDIA3 chaperone protein levels. As a consequence, SHBG application substantially improved insulin sensitivity through the modulation of Pi3K/Akt/Glut4 insulin signalling cascades. CONCLUSION: Our results suggest that the SHBG is endowed with crucial beneficial effects on ASCs metabolic activities and could serve as a valuable therapeutic target for the development of efficient EMS treatment protocols. Video Abstract.

Protective Effect of Sex Hormone-Binding Globulin against Metabolic Syndrome: In Vitro Evidence Showing Anti-Inflammatory and Lipolytic Effects on Adipocytes and Macrophages.

Sex hormone-binding globulin (SHBG) is a serum protein released mainly by the liver, and a low serum level correlates with a risk for metabolic syndrome including diabetes, obesity, and cardiovascular events. However, the underlying molecular mechanism(s) linking SHBG and metabolic syndrome remains unknown. In this study, using adipocytes and macrophages, we focused on the in vitro effects of SHBG on inflammation as well as lipid metabolism. Incubation with 20 nM SHBG markedly suppressed lipopolysaccharide- (LPS-) induced inflammatory cytokines, such as MCP-1, TNFα, and IL-6 in adipocytes and macrophages, along with phosphorylations of JNK and ERK. Anti-inflammatory effects were also observed in 3T3-L1 adipocytes cocultured with LPS-stimulated macrophages. In addition, SHBG treatment for 18 hrs or longer significantly induced the lipid degradation of differentiated 3T3-L1 cells, with alterations in its corresponding gene and protein levels. Notably, these effects of SHBG were not altered by coaddition of large amounts of testosterone or estradiol. In conclusion, SHBG suppresses inflammation and lipid accumulation in macrophages and adipocytes, which might be among the mechanisms underlying the protective effect of SHBG, that is, its actions which reduce the incidence of metabolic syndrome.

Model-Based Dose Selection for Intravaginal Ring Formulations Releasing Anastrozole and Levonorgestrel Intended for the Treatment of Endometriosis Symptoms.

Pharmacokinetics (PK) of anastrozole (ATZ) and levonorgestrel (LNG) released from an intravaginal ring (IVR) intended to treat endometriosis symptoms were characterized, and the exposure-response relationship focusing on the development of large ovarian follicle-like structures was investigated by modeling and simulation to support dose selection for further studies. A population PK analysis and simulations were performed for ATZ and LNG based on clinical phase 1 study data from 66 healthy women. A PK/PD model was developed to predict the probability of a maximum follicle size ≥30 mm and the potential contribution of ATZ beside the known LNG effects. Population PK models for ATZ and LNG were established where the interaction of LNG with sex hormone-binding globulin (SHBG) as well as a stimulating effect of estradiol on SHBG were considered. Furthermore, simulations showed that doses of 40 µg/d LNG combined with 300, 600, or 1050 µg/d ATZ reached anticipated exposure levels for both drugs, facilitating selection of ATZ and LNG doses in the phase 2 dose-finding study. The main driver for the effect on maximum follicle size appears to be unbound LNG exposure. A 50% probability of maximum follicle size ≥30 mm was estimated for 40 µg/d LNG based on the exposure-response analysis. ATZ in the dose range investigated does not increase the risk for ovarian cysts as occurs with LNG at a dose that does not inhibit ovulation.

Complex regulation of GH autofeedback under dual-peptide drive: studies under a pharmacological GH and sex steroid clamp.

To test the postulate that sex difference, sex steroids, and peptidyl secretagogues control GH autofeedback, 11 healthy postmenopausal women and 14 older men were each given 1) a single iv pulse of GH to enforce negative feedback and 2) continuous iv infusion of saline vs. combined GHRH/GHRP-2 to drive feedback escape during pharmacological estradiol (E(2); women) or testosterone (T; men) supplementation vs. placebo in a double-blind, prospectively randomized crossover design. By three-way ANCOVA, sex difference, sex hormone treatment, peptide stimulation, and placebo/saline responses (covariate) controlled total (integrated) GH recovery during feedback (each P < 0.001). Both sex steroid milieu (P = 0.019) and dual-peptide stimulation (P < 0.001) determined nadir (maximally feedback-suppressed) GH concentrations. E(2)/T exposure elevated nadir GH concentrations during saline infusion (P = 0.003), whereas dual-peptide infusion did so independently of T/E(2) and sex difference (P = 0.001). All three of sex difference (P = 0.001), sex steroid treatment (P = 0.005), and double-peptide stimulation (P < 0.001) augmented recovery of peak (maximally feedback-escaped) GH concentrations. Peak GH responses to dual-peptidyl agonists were greater in women than in men (P = 0.016). E(2)/T augmented peak GH recovery during saline infusion (P < 0.001). Approximate entropy analysis corroborated independent effects of sex steroid treatment (P = 0.012) and peptide infusion (P < 0.001) on GH regularity. In summary, sex difference, sex steroid supplementation, and combined peptide drive influence nadir, peak, and entropic measurements of GH release under controlled negative feedback. To the degree that the pharmacological sex steroid, GH, and dual-peptide clamps provide prephysiological regulatory insights, these outcomes suggest major determinants of pulsatile GH secretion in the feedback domain.

Oxytocin and/or steroid hormone binding globulin infused into the ventral tegmental area modulates progestogen-mediated lordosis.

Estradiol (E(2)) and progesterone (P(4)) have classical, steroid receptor-mediated actions in the ventral medial hypothalamus to initiate lordosis of female rodents. P(4) and the P(4) metabolite and neurosteroid, 5 alpha-pregnan-3 alpha-ol-20-one (3 alpha,5 alpha-THP), have non-classical actions in the midbrain ventral tegmental area (VTA) to modulate lordosis. We investigated the role of steroid hormone binding globulin (SHBG) and oxytocin in the VTA as mechanisms for these effects. Rats were ovariectomized and surgically implanted with bilateral guide cannulae aimed at the VTA. Rats were E(2)-primed (10 microg/0.2 ml) at hour 0, and administered 100 (Experiments 1 and 2), 500 (Experiment 3), or 0 (Experiment 1 and 4) microg/0.2 ml P(4) at hour 44. At hour 47.5, rats received bilateral infusions to the VTA, and were tested for lordosis 30 min post-infusion. Experiment 1: rats were infused with sterile saline vehicle or SHBG (4.5 pg/microl) to the VTA. SHBG, compared to vehicle, to the midbrain VTA significantly increased lordosis in E(2)- and P(4)-primed, but not E(2)-primed, rats. Experiment 2: rats were infused with bilateral infusions of sterile saline or oxytocin (1.0 pg/microl). Compared to vehicle, oxytocin to the VTA increased lordosis. Experiment 3: rats were administered bilateral intra-VTA infusions of saline or an oxytocin receptor antagonist, d(CH(2))(5),[TYr(ME)(2),Thr(4),Tyr-NH(9,2)] (1.2 pg/microl). Compared to vehicle, the oxytocin receptor antagonist to the VTA attenuated lordosis of E(2)- and P(4)-primed rats. Experiment 4: rats were E(2)-primed and infused with vehicle, oxytocin, or oxytocin antagonist. There were no effects of these manipulations in E(2)-primed rats. Thus, SHBG and/or oxytocin may have actions in the VTA for progestogen-facilitated lordosis.

Internalization of sex hormone-binding globulin into neurons and brain cells in vitro and in vivo.

BACKGROUND: Sex hormone-binding globulin (SHBG) is a 94-kDa homodimer that binds steroids and is made in the hypothalamus. We have demonstrated that infusions of SHBG into the hypothalami of rats increase their female sexual receptivity except when SHBG is coupled to dihydrotestosterone (DHT) suggesting that SHBG has an active function in behavioral neuroendocrinology. METHODS: This study examines the possibility that SHBG is internalized by neuronal and/or non-neuronal brain cells as one possible mode of action using in vitro and in vivo techniques. RESULTS: First, analysis of the uptake of radiolabeled SHBG ((125)I-SHBG) found (125)I-SHBG uptake in HT22 hippocampal cells stably transfected with cDNA for ER beta (HT22-ER beta). The addition of DHT to (125)I-SHBG significantly inhibited (125)I-SHBG uptake in HT22-ER beta cells but not in HT22-ER alpha or HT22 wild-type cells. SHBG internalization was specific as it did not occur in either the human neuroblastoma cell line SK-N-SH or the glioma cell line C6. Second, SHBG was labeled with a fluor (Alexa-555), and infused into the lateral cerebroventricles of ovariectomized rats. Optimal SHBG uptake was seen 10 min after these infusions. SHBG uptake was seen in specific parts of the choroid plexus and periventricular cells as well as into cells in the paraventricular nucleus, the medial forebrain bundle, and the habenula. CONCLUSIONS: These studies suggest that SHBG is internalized by brain cells, which may be affected by the presence of ER beta. The gonadal steroids have numerous effects in brain and the discovery that the steroid-binding protein SHBG is taken up into neurons and brain cells may demand a change in thinking about how steroids are delivered to brain cells to affect neurophysiology.

Sex hormone-binding globulin selectively modulates estradiol-regulated genes in MCF-7 cells.

Human sex hormone-binding globulin inhibits the effects of estradiol on proliferation and apoptosis of breast cancer cells. We report here the effect of sex hormone-binding globulin on estradiol regulation of gene expression in MCF-7 breast cancer cells using a selected set of genes. Estradiol upregulates genes that are positive regulators of proliferation (e.g., bcl-2, c-fos, c-myc, cyclin D) or/and related to more aggressive form of breast cancer (e.g. BRCA-1, EGF-R) and downregulates two genes (c-jun and ERalpha). Sex hormone-binding globulin modulates only a selected group of estradiol-controlled genes (inhibiting upregulation of bcl-2, c-myc, EGF-R, PR, and downregulation of ERalpha), starting 48 hours after treatment. Our study demonstrates that in breast cancer cells, sex hormone-binding globulin is effective on few selected genes which are involved in cell growth and apoptosis or related to cell estrogen-dependence and that the protein regulation of estradiol effect is selected and specific. Sex hormone-binding globulin action in estrogen breast cancer cells is strongly associated to cell growth and estrogen-sensitivity.

Role of endocytosis in cellular uptake of sex steroids.

Androgens and estrogens are transported bound to the sex hormone binding globulin (SHBG). SHBG is believed to keep sex steroids inactive and to control the amount of free hormones that enter cells by passive diffusion. Contrary to the free hormone hypothesis, we demonstrate that megalin, an endocytic receptor in reproductive tissues, acts as a pathway for cellular uptake of biologically active androgens and estrogens bound to SHBG. In line with this function, lack of receptor expression in megalin knockout mice results in impaired descent of the testes into the scrotum in males and blockade of vagina opening in females. Both processes are critically dependent on sex-steroid signaling, and similar defects are seen in animals treated with androgen- or estrogen-receptor antagonists. Thus, our findings uncover the existence of endocytic pathways for protein bound androgens and estrogens and their crucial role in development of the reproductive organs.

Sex hormone-binding globulin is a major determinant of the lipid profile: the PRIME study.

The prevalence of coronary heart disease is much higher in men than in women and sex hormones might play a role in these differences through their influence on the lipid profile. The aim of this cross-sectional study was to study the relationship between hormonal markers (total testosterone (TT), estradiol (E2), sex-hormone-binding globulin (SHBG)) and plasma lipids in a population-based sample. Subjects were 352 men, 50-59 years old, selected in France (Lille, Strasbourg and Toulouse) and Northern-Ireland (Belfast) who had questionnaires and a medical examination at baseline of the PRIME prospective study (1991-1993). Pearson correlation coefficients and Student's t tests were used to identify factors associated with plasma lipids. Multiple linear regression models were used for multivariate analyses, using triglycerides (TG) (log-transformed) and high density-lipoprotein cholesterol (HDL-C) as dependent variables. SHBG and TT were negatively correlated with TG (p<0.0001 and p<0.05, respectively) and positively correlated with HDL-C (p<0.0001 and p<0.01). E2 was positively correlated with TG (p<0.05). No significant association was found between sex-hormones and LDL-C. In multiple linear regression analyses, SHBG remained independently associated negatively with TG (p<0.01) and positively with HDL-C (p<0.0001) after adjustment for centre of recruitment, age, body mass index, systolic blood pressure, smoking, alcohol intake and physical activity. After further adjustment for insulin, the association between SHBG and HDL-C remained highly significant (p<0.0001). The association between SHBG and TG was weakened but remained also significant. Our results suggest that SHBG might to be a central protein in the hormonal regulation of the lipid profile.

Sex hormone-binding globulin antagonizes the anti-apoptotic effect of estradiol in breast cancer cells.

Sex hormone-binding globulin, the plasma carrier for sex steroids, inhibits the estradiol-induced proliferation of breast cancer cells. Estradiol induces cell proliferation triggering multiple mechanisms. Besides regulating growth factors, it activates Erk-1/-2, thus inhibiting apoptosis. In the present study, we investigated the effect of SHBG on estradiol-mediated anti-apoptotic effect in MCF-7 breast cancer cells. As expected, estradiol reduced the number of cells undergoing apoptosis. Although no modification of estradiol action was observed in cells treated contemporarily with estradiol and SHBG, pre-incubation with SHBG before estradiol treatment contrasted the anti-apoptotic effect completely. A mutant form of SHBG, lacking the O-linked oligosaccharide in Thr(7), displayed no such effect. Moreover, SHBG prevented the estradiol-induced phosphorylation of Erk-1/-2, whereas it had no effect on estradiol-induced transcription. Taken together, our observations suggest that the interaction of SHBG with MCF-7 cell membranes causes inhibition of the anti-apoptotic effect of estradiol which might account for SHBG's inhibitory effect on breast cancer cell growth.

Sex hormone binding globulin facilitates female sexual receptivity except when coupled to dihydrotestosterone.

Sex hormone binding globulin (SHBG) is produced in brain where it is often co-localized with oxytocin. Infusions of SHBG into the medial preoptic area-anterior hypothalamus facilitate female sexual receptivity. SHBG has receptors on plasma membranes of the prostate gland where binding of the 5alpha-reduced androgen dihydrotestosterone (DHT) by SHBG acts as an antagonist on SHBG receptors. This study attempted to determine whether pre-coupling DHT to SHBG would inhibit SHBG-induced facilitation of female sexual receptivity. Ovariectomized rats were injected daily with 0.75 microg estradiol benzoate for 3 days. On the fourth day after a pre-infusion baseline behavioral test animals were infused with 1 microl per side through bilateral cannulae with SHBG (1.77x10(-6) M), SHBG coupled to DHT (SHBG-DHT; 1.66x10(-6) M DHT), with DHT alone or with artificial cerebrospinal fluid vehicle. As before, SHBG significantly increased female sexual receptivity when infused into the medial preoptic area-anterior hypothalamus. Rats infused with SHBG-DHT had significantly lower sexual receptivity. Therefore, whereas SHBG in the medial preoptic area facilitated female sexual behavior, SHBG coupled to DHT did not. DHT itself did not significantly affect sexual receptivity. Pre-coupling DHT to SHBG eliminated the facilitative effect of SHBG on female sexual receptivity just as DHT inhibits SHBG activity at prostate SHBG receptors suggesting that central receptors for SHBG are similar to those demonstrated in the periphery.

O-Glycosylation of human sex hormone-binding globulin is essential for inhibition of estradiol-induced MCF-7 breast cancer cell proliferation.

Human sex hormone-binding globulin (SHBG) is a homodimeric plasma glycoprotein, and each SHBG monomer may have an O-linked oligosaccharide at Thr(7) and up to two N-linked oligosaccharides at Asn(351) and Asn(367). In addition, a common genetic variant of SHBG exists with an extra site for N-glycosylation at residue 327. In the present study, we isolated MCF-7 derived cell lines expressing human SHBG cDNAs encoding the wild type protein or various glycosylation mutants. Estradiol (1 nM) treatment of parental (untransfected) MCF-7 cells or MCF-7 cells transfected with control expression vectors resulted in an increase in proliferation which was fully abrogated by co-incubation with an equimolar amount of human SHBG. In contrast, the same amount of purified SHBG added to MCF-7 cells expressing wild type SHBG partially inhibited the estradiol-induced cell proliferation. A high affinity binding site for SHBG was detectable on untransfected and control cells, but not on MCF-7 cells expressing wild type SHBG. Moreover, the treatment of MCF-7 cells with the conditioned medium containing wild type SHBG caused the disappearance of the SHBG plasma membrane-binding site. Media containing SHBG N-glycosylation mutants exerted the same effect, but mutants lacking the O-linked oligosaccharide at Thr(7) failed to do so. Estradiol-induced proliferation of parental MCF-7 cells was also inhibited by treatment with conditioned medium containing wild type SHBG or SHBG mutants lacking N-linked oligosaccharides, or containing an additional N-linked oligosaccharide at residue 327. However, MCF-7 conditioned medium containing SHBG mutants lacking an O-linked oligosaccharide at Thr(7) failed to exert this effect. These data suggest that O-glycosylation of SHBG is essential for SHBG binding to a membrane receptor that is responsible for inhibiting the estradiol-induced proliferation of MCF-7 breast cancer cells.

Intravenous injection of human sex steroid hormone-binding globulin in mouse decreases blood clearance rate and testicular accumulation of orally administered [2-125I]iodobisphenol A.

Bisphenol A, an environmental compound with estrogenic activity, has been shown to bind human sex steroid hormone-binding globulin (hSHBG), the main plasma transport protein which regulates the metabolism of androgens and estrogens and limits their access to target organs. The present study was conducted to determine whether physiologically relevant concentrations of hSHBG can influence the blood clearance rate of bisphenol A and its accumulation in the testes. A radioactive [2-125I]iodobisphenol tracer was synthesized with an association constant (Ka) for binding to hSHBG of 0.14 +/- 0.01 x 10(6) M(-1) at 37 degrees C, a value much lower than for [2-125I]iodoestradiol, which was also synthesized. We used i.v. injection of immunopurified hSHBG in adult male mice to maintain hSHBG levels within the physiologically possible range for humans (27-267 nM) before gavage administration of [2-125I]iodobisphenol or [2-125I]iodoestradiol, for measuring the blood clearance rate of radioactive signal in blood samples taken during the following 120 min. Testicular accumulation of radioactivity was measured 24 h and 48 h after gavage of [2-125]iodobisphenol A. In mice receiving immunopurified hSHBG or vehicle, the time-dependent blood clearance of radioactivity exhibited a bi-exponential decrease which indicated alpha-diffusion and beta-elimination phases for both radioactive ligands. The presence of circulating hSHBG significantly and dose-dependently lowered the clearance rate of radioactivity. However, much higher circulating levels of hSHBG were required to retard the blood clearance of [2-125I]iodobisphenol A as compared to those required for [2-125I]iodoestradiol, in keeping with the important difference in their respective Ka value for binding to SHBG. In addition, mice treated with hSHBG exhibited significantly (P = 0.036) reduced testicular accumulation of radioactivity 24 h and 48 h after ingestion of [2-125I]iodobisphenol A. Provided that the binding properties of bisphenol A for hSHBG are not substantially different from those measured for [2-125I]iodobisphenol A, these findings suggest that, although hSHBG binds 2-mono-iodobisphenol A with a relatively low binding affinity, high enough concentrations of circulating hSHBG (range concentrations between 85 and 267 nM) are potentially able to exert a protective effect against exposure to bisphenol A.

The control of progesterone receptor expression in MCF-7 breast cancer cells: effects of estradiol and sex hormone-binding globulin (SHBG).

Estradiol controls the gene transcription and expression of many proteins in breast cancer cells, like the progesterone receptor, PR, that is up-regulated by the hormone. Moreover, estradiol is one of the crucial factors inducing the proliferation of breast cancer cells. Sex Hormone-Binding Globulin (SHBG), the plasma carrier for both estradiol and androgens, inhibits the estradiol-induced growth of MCF-7 cells (estrogen-dependent breast cancer cells), through its membrane receptor (SHBG-R), cAMP and PKA. The anti-estrogenic effect of SHBG, which has been described only as far as cell proliferation is concerned, could also play a meaningful role in the estradiol control of other factors in breast cancer cells. In the present study, the effect of SHBG on the estradiol control of PR expression (both mRNA and protein) and function (receptor binding capacity) in MCF-7 cells was examined. SHBG inhibited the estradiol-induced up-regulation of PR mRNA as well as protein level and function. Moreover, the effect of SHBG on estradiol control of PR expression and function was showed to be specific and mediated by PKA. The intermediacy of PKA in the PR expression control, together with the observation that it is effective in the condition in which the SHBG receptor is activated, supports the hypothesis that the anti-estrogenic effect of SHBG could be receptor-mediated. The ability of SHBG to inhibit estradiol action in a specific way in estrogen-dependent breast cancer cells has, therefore, to be taken into account for the development of future therapeutic strategies.

Sex hormone binding globulin stimulates female sexual receptivity.

Sex hormone binding globulin (SHBG) is found in the brain and acts directly on plasma membrane-associated receptors in the prostate gland. Infusing SHBG into the medial preoptic area or medial basal hypothalamus of female rats increases their female sexual receptivity. SHBG, SHBG plus estradiol (SHBG-E), and SHBG-E plus oxytocin all significantly increased female sexual receptivity over vehicle or estradiol plus oxytocin infused controls, as measured by lordosis quotients and receptivity scores, at 40, and 90 min after their infusions into the medial preoptic area. When infused into the medial basal hypothalamus, SHBG-E plus oxytocin resulted in significantly increased sexual receptivity 20 and 40 min after infusion when compared to its estradiol plus oxytocin control group. SHBG produced in the brain may be released endogenously to have immediate effects on reproductive physiology and behavior.

Differential binding of estradiol and testosterone to SHBG. Relation to circulating estradiol levels.

OBJECTIVE: Sex hormone-binding globulin (SHBG) binds testosterone (T) to a greater degree than it does estradiol (E2), acting as an amplifier of E2 action. However, it is not known whether the relative capacity of SHBG for E2 vs. T is altered by the hormonal milieu. We hypothesized that an increase in circulating E2 levels results in a compensatory increase in the relative binding capacity of SHBG for these hormones, dampening the E2 amplification effect in hyperestrogenic conditions. STUDY DESIGN: Retrospective. RESULTS: As expected, during hMG stimulation there was a significant increase in total and free E2 (28 to 1,986 pg/mL, P < .001; and 0.3 to 20.8 pg/mL, P < .001, respectively) and total T levels (40.3 vs. 78.3 ng/dL, P < .001) from basal to late stimulation. Free T levels increased, but the difference did not reach significance. The binding capacity of SHBG for both E2 and T increased in a proportional manner (980 +/- 340 vs. 1,434 +/- 449 nmol/L, P < .009; and 352 +/- 190 vs. 512 +/- 128 nmol/L, P < .02; respectively) since the ratio of SHBG binding to E2 and T was unchanged. Although the SHBG molar concentration appeared increased, the difference did not reach significance (821 +/- 542 to 1,099 +/- 254 nmol/L). CONCLUSION: A short-term, although profound, increase in circulating E2 does not seem to be associated with an increase in the relative binding capacity of the carrier protein for either E2 or T, although an overall increase in binding for both steroids was observed. It is possible that longer periods of exposure to E2 may be necessary to demonstrate a change in the differential binding of this carrier protein with an alteration in the hormonal milieu.

Sex hormone-binding globulin stimulates chorionic gonadotrophin secretion from human cytotrophoblasts in culture.

The effects of sex hormone-binding globulin (SHBG) on the secretion of human chorionic gonadotrophin (HCG) and cAMP by cultured human cytotrophoblasts were investigated. Cytotrophoblasts obtained from normal term placentae were cultured in serum-free medium with or without the addition of human SHBG. The presence of SHBG in the medium increased the release of HCG and the accumulation of cAMP. Ligand-free SHBG was able to raise both HCG and cAMP concentrations and the maximal response was observed with 1 nM of the steroid-binding globulin. Addition of either oestradiol or 5alpha-dihydro-testosterone (DHT) to cultures previously incubated with SHBG in a final molar ratio of 1:10 resulted in a further increase of HCG and cAMP concentrations. This effect was blocked when cultured placental cells were exposed to SHBG that was previously saturated with DHT or when incubated in the presence of steroids only. The results of the present study provide evidence for the in-vitro regulation of HCG secretion by SHBG and further support the concept that this steroid-binding protein may act as a mediator of steroid action at the cellular level. Finally, the increase in cAMP suggests that SHBG receptor located in the surface of syncytiotrophoblast membranes is coupled to adenylate cyclase as part of the G-protein receptor family. Our results may provide new insights into the biological implications of extracellular steroid-binding proteins as well as new perspectives on the endocrinology of pregnancy.

Control of the membrane sex hormone-binding globulin-receptor (SHBG-R) in MCF-7 cells: effect of locally produced SHBG.

The interaction between plasma sex hormone-binding globulin (SHBG) and its receptor (SHBG-R) inhibits estradiol-induced proliferation of MCF-7 cells (human estrogen-dependent breast cancer) through cAMP and PKA. Thus, SHBG can modulate estradiol action in breast cancer, but the implications of this require a more detailed knowledge of the SHBG-R. To this end, we have transfected MCF-7 cells with an expression vector carrying the human SHBG cDNA (S-MCF-7) and studied the effects of this on both SHBG-R binding and cell proliferation. Control cells were parental MCF-7 (P-MCF-7) and MCF-7 cells transfected with the beta-galactosidase gene (B-MCF-7). Transfections were mediated by lipofectin followed by selection of transfected cells with G418. The amounts of SHBG in culture medium were evaluated by IRMA assay, with only S-MCF-7 cells shown to secrete SHBG; SHBG-R levels were evaluated by tracer binding technique. In P-MCF-7 and B-MCF-7 cells, SHBG-R was detectable as a two-binding site receptor, but no binding of SHBG was observed in S-MCF-7 cells. Proliferation of cells treated with estradiol was evaluated by [3H]thymidine incorporation in the three cell lines and in cells pretreated with SHBG (1 nM) purified from human serum or with conditioned medium from S-MCF-7 cells (medium S). In all three lines, cell proliferation increased after estradiol treatment. Preincubation with purified SHBG was effective in reducing estrogen-induced cell proliferation to basal levels in P-MCF-7 and B-MCF-7 but not in S-MCF-7 cells. The estradiol effect was also inhibited in P-MCF-7 cells treated with medium S. In conclusion, 1) SHBG inhibits estradiol-induced proliferation in cells containing a functional SHBG-R, whereas it has no detectable effect in cells in which the SHBG-R is either absent or not available to bind SHBG; and 2) S-MCF-7 cells are insensitive to SHBG (locally produced or exogenous) because their SHBG-R is occupied by SHBG.

Stimulation of Sky tyrosine phosphorylation by bovine protein S--domains involved in the receptor-ligand interaction.

Protein S is an anticoagulant vitamin-K-dependent plasma glycoprotein, which acts as a cofactor to activated protein C in the degradation of coagulation factors Va and VIIIa. It has been proposed that protein S has an additional function as a growth factor. Protein S and a structurally similar protein, Gas6, have been found to stimulate members of the Axl/Sky family of receptor tyrosine kinases. Human Gas6 is able to activate Axl and Sky. In contrast, while bovine protein S activates human Sky and its murine homologue, human protein S activates murine Sky but not the human receptor. In the present investigation, we studied the structural background of this species difference. Using protein S chimeras with domains from human and bovine origin, we found that only those chimeras with the steroid-hormone-binding globulin-like (SHBG) region from bovine protein S activate human Sky, indicating that the SHBG region is essential for the interaction. This observation was confirmed by inhibition of Sky phosphorylation by C4b-binding protein, a plasma protein that interacts tightly with the SHBG region of protein S. Another chimeric molecule, composed of the N-terminal 4-carboxyglutamic-acid-containing domain (Gla domain) and the two epidermal-growth-factor-like domains of human factor IX, and the SHBG region of bovine protein S, stimulated the receptor less efficiently. Antibodies directed against the Gla domain of protein S, inhibited the activation of human Sky by bovine protein S. These results indicate that the N-terminal domains of protein S are not essential for activation of the receptor, but contribute to the affinity of the interaction. Our data suggest that protein S might be a ligand of Sky in some species despite the lack of activity of human protein S on human Sky. The bovine/human protein S species difference will be a useful model to establish the structural requirements for the interaction between Sky and its ligands.

Stimulation of prostate cancer growth by androgens and estrogens through the intermediacy of sex hormone-binding globulin.

Sex hormone-binding globulin (SHBG) not only regulates the free concentration of certain steroid sex hormones in plasma, but is involved in a nongenomic mechanism of steroid hormone action. It binds to a receptor on prostatic cell membranes and is activated by an appropriate steroid to initiate the generation of intracellular cAMP. Using the human prostate cancer cell line ALVA-41, we show that in serum-free medium, both dihydrotestosterone and estradiol increase growth in the presence, but not the absence, of SHBG. The increase in growth also follows the addition of cAMP to the cells and is enhanced by inhibiting protein dephosphorylation with the protein phosphatase inhibitor, okadaic acid. We conclude that cAMP causes increased growth in this prostate cancer cell line, and that both SHBG-dihydrotestosterone and SHBG-estradiol can regulate intracellular cAMP, and hence growth, in these cells.