Selective cleavage of human sex hormone-binding globulin by kallikrein-related peptidases and effects on androgen action in LNCaP prostate cancer cells

Stimulation of the androgen receptor via bioavailable androgens, including testosterone and testosterone metabolites, is a key driver of prostate development and the early stages of prostate cancer. Androgens are hydrophobic and as such require carrier proteins, including sex hormone-binding globulin (SHBG), to enable efficient distribution from sites of biosynthesis to target tissues. The similarly hydrophobic corticosteroids also require a carrier protein whose affinity for steroid is modulated by proteolysis. However, proteolytic mechanisms regulating the SHBG/androgen complex have not been reported. Here, we show that the cancer-associated serine proteases, kallikrein-related peptidase (KLK)4 and KLK14, bind strongly to SHBG in glutathione S-transferase interaction analyses. Further, we demonstrate that active KLK4 and KLK14 cleave human SHBG at unique sites and in an androgen-dependent manner. KLK4 separated androgen-free SHBG into its two laminin G-like (LG) domains that were subsequently proteolytically stable even after prolonged digestion, whereas a catalytically equivalent amount of KLK14 reduced SHBG to small peptide fragments over the same period. Conversely, proteolysis of 5α-dihydrotestosterone (DHT)-bound SHBG was similar for both KLKs and left the steroid binding LG4 domain intact. Characterization of this proteolysis fragment by [(3)H]-labeled DHT binding assays revealed that it retained identical affinity for androgen compared with full-length SHBG (dissociation constant = 1.92 nM). Consistent with this, both full-length SHBG and SHBG-LG4 significantly increased DHT-mediated transcriptional activity of the androgen receptor compared with DHT delivered without carrier protein. Collectively, these data provide the first evidence that SHBG is a target for proteolysis and demonstrate that a stable fragment derived from proteolysis of steroid-bound SHBG retains binding function in vitro.

Biosensor-based detection of reduced sex hormone-binding globulin binding capacities in response to growth-promoter administrations

Growth-promoting agents are illicitly used during animal rearing processes and the detection of their use is limited by new compounds and dosing practices that limit the efficiency of current testing which is based on residue analysis by liquid chromatography–tandem mass spectrometry (LC–MS/MS) and gas chromatography–mass spectrometry (GC–MS) methodology. An alternative approach is to use indirect biological evidence as a screening tool to identify growth-promoter treated animals thus improving the effectiveness of residue testing through the targeted sampling of these animals. Sex hormone-binding globulin (SHBG) is a glycoprotein which binds and controls the levels of sex-hormones within the circulation. Using a biosensor assay based on measurement of binding to an immobilised 1a-dihydrotestosterone (1a-DHT) derivative, reduced SHBG binding capacities were detected in growth-promoter treated animals. During the course of a veal treatment regime based on repeated oestradiol benzoate, nortestosterone decanoate and dexamethasone administrations, treated male and female calves were shown to have significantly lower SHBG capacities. To assess the effectiveness of using SHBG binding capacities as a biomarker of treatment and to investigate the role of individual growth-promoter components to the SHBG capacity lowering effects, adult heifer animals were subjected to repeated doses of nortestosterone decanoate. These animals also demonstrated a reduction in SHBG capacity levels at Day 39 of the study, in contrast to oestradiol benzoate treated adult steers who were found to have unaltered levels. These findings suggest that the measurement of SHBG binding capacities using a biosensor assay has potential in the identification of illegally treated animals, particularly those exposed to androgens.

Sex hormone binding globulin and insulin resistance

Sex hormone binding globulin (SHBG) is a glycoprotein composed of two 373-amino-acid subunits. The SHBG gene and a promotor region have been identified. The SHBG receptor has yet to be cloned but is known to act through a G-protein-linked second-messenger system following plasma membrane binding. The principal function of SHBG has traditionally been considered to be that of a transport protein for sex steroids, regulating circulating concentrations of free (unbound) hormones and their transport to target tissues. Recent research suggests that SHBG has functions in addition to the binding and transport of sex steroids. Observational studies have associated a low SHBG concentration with an increased incidence of type 2 diabetes mellitus (DM) independent of sex hormone levels in men and women. Genetic studies using Mendelian randomization analysis linking three single nucleotide polymorphisms of the SHBG gene to risk of developing type 2 DM suggest SHBG may have a role in the pathogenesis of type 2 DM. The correlation between SHBG and insulin resistance that is evident in a number of cross-sectional studies is in keeping with the suggestion that the association between SHBG and incidence of type 2 DM is explained by insulin resistance. Several potential mechanisms may account for this association, including the identification of dietary factors that influence SHBG gene transcription. Further research to characterize the SHBG-receptor and the SHBG second messenger system is required. An interventional study examining the effects on insulin resistance of altering SHBG concentrations may help in determining whether this association is causal.

The effects of bed-rest and countermeasure exercise on the endocrine system in male adults: evidence for immobilization-induced reduction in sex hormone-binding globulin levels

BACKGROUND AND AIM: There is limited data on the effects of inactivity (prolonged bed-rest) on parameters of endocrine and metabolic function; we therefore aimed to examine changes in these systems during and after prolonged (56- day) bed-rest in male adults. SUBJECTS AND METHODS: Twenty healthy male subjects underwent 8 weeks of strict bed-rest and 12 months of follow-up as part of the Berlin Bed Rest Study. Subjects were randomized to an inactive group or a group that performed resistive vibration exercise (RVE) during bed-rest. All outcome parameters were measured before, during and after bed-rest. These included body composition (by whole body dual X-ray absorptiometry), SHBG, testosterone (T), estradiol (E2), PRL, cortisol (C), TSH and free T3 (FT3). RESULTS: Serum SHBG levels decreased in inactive subjects but remained unchanged in the RVE group (p<0.001). Serum T concentrations increased during the first 3 weeks of bed-rest in both groups (p<0.0001), while E2 levels sharply rose with re-mobilization (p<0.0001). Serum PRL decreased in the control group but increased in the RVE group (p=0.021). C levels did not change over time (p≥0.10). TSH increased whilst FT3 decreased during bed-rest (p all ≤0.0013). CONCLUSIONS: Prolonged bed-rest has significant effects on parameters of endocrine and metabolic function, some of which are related to, or counteracted by physical activity.

Primordial germ cells (spermatogonial stem cells) of bullfrogs express sex hormone-binding globulin and steroid receptors during seasonal spermatogenesis

In vertebrate species, testosterone seems to inhibit spermatogonial differentiation and proliferation. However, this androgen can also be converted, via aromatase, into estrogen which stimulates spermatogonial differentiation and mitotic activity. During seasonal spermatogenesis of adult bullfrogs Lithobates catesbeianus, primordial germ cells (PGCs) show enhanced testosterone cytoplasm immunoexpression in winter; however, in summer, weak or no testosterone immunolabelling was observed. The aim of this study was to confirm if PGCs express stem cell markers-alkaline phosphatase (AP) activity and GFRα1 (glial-cell-line-derived neurotrophic factor)-and verify whether testosterone is maintained in these cells by androgen receptors (ARs) and/or sex hormone-binding globulin (SHBG) in winter. Furthermore, regarding the possibility that testosterone is converted into estrogen by PGCs in summer, the immunoexpression of estrogen receptor (ER)β was investigated. Bullfrog testes were collected in winter and in summer and were embedded in glycol methacrylate for morphological analyses or in paraffin for the histochemical detection of AP activity. GFRα1, AR, SHBG and ERβ expression were detected by Western blot and immunohistochemical analyses. The expression of AP activity and GFRα1 in the PGCs suggest that these cells are spermatogonial stem cells. In winter, the cytoplasmic immunoexpression of ARs and SHBG in the PGCs indicates that testosterone is maintained by these proteins in these cells. The cytoplasmic immunoexpression of ERβ, in summer, also points to an ER-mediated action of estrogen in PGCs. The results indicate a participation of testosterone and estrogen in the control of the primordial spermatogonia during the seasonal spermatogenesis of L. catesbeianus. © 2012 S. Karger AG, Basel.

Corticosteroid-binding globulin: a possible early predictor of infection in acute necrotizing pancreatitis

OBJECTIVE: Infected pancreatic necrosis is the main cause of death in patients with acute pancreatitis, and therefore its early prediction is of utmost importance. Endogenous cortisol metabolism plays a basic role both in the course of acute pancreatitis and in the process of infection. The purpose of this study was to analyze corticosteroid-binding globulin (CBG), total cortisol, calculated free cortisol and adrenocorticotropic hormone as potential early predictors in order to differentiate between infected pancreatic necrosis and sterile pancreatic necrosis in patients with acute pancreatitis. MATERIAL AND METHODS: Serum levels of CBG, total cortisol, calculated free cortisol, and plasma levels of adrenocorticotropic hormone were determined in 109 consecutive patients with acute pancreatitis. C-reactive protein was measured as the control parameter. Thirty-five patients developed necrotizing pancreatitis and 10 developed infection of the necrosis. Blood was monitored for 6 days after the onset of pain; 30 healthy individuals served as controls. RESULTS: Of all parameters only CBG showed a significant difference (p = 0.0318) in its peak levels measured in the first 48 h in patients with sterile (26.5 microg/ml, range 21.3-34.7) and infected (16.0 microg/ml, range 15.2-25.0) necrosis at a cut-off level of 16.8 microg/ml. That difference was further preserved for the first 6 days after onset of pain. CONCLUSIONS: In our group of patients, a decreased CBG level below 16.8 g/ml within the initial 48 h of acute pancreatitis was an early predictor of later infected pancreatic necrosis, with a positive predictive value of 100% and a negative predictive value of 87.5%.

Postmenopausal hot flushes, vascular health and hormone therapy

Vasomotor hot flushes are complained of by approximately 75% of postmenopausal women, but their frequency and severity show great individual variation. Hot flushes have been present in women attending observational studies showing cardiovascular benefit associated with hormone therapy use, whereas they have been absent or very mild in randomized hormone therapy trials showing cardiovascular harm. Therefore, if hot flushes are a factor connected with vascular health, they could perhaps be one explanation for the divergence of cardiovascular data in observational versus randomized studies. For the present study 150 healthy, recently postmenopausal women showing a large variation in hot flushes were studied in regard to cardiovascular health by way of pulse wave analysis, ambulatory blood pressure and several biochemical vascular markers. In addition, the possible impact of hot flushes on outcomes of hormone therapy was studied. This study shows that women with severe hot flushes exhibit a greater vasodilatory reactivity as assessed by pulse wave analysis than do women without vasomotor symptoms. This can be seen as a hot flush-related vascular benefit. Although severe night-time hot flushes seem to be accompanied by transient increases in blood pressure and heart rate, the diurnal blood pressure and heart rate profiles show no significant differences between women without and with mild, moderate or severe hot flushes. The levels of vascular markers, such as lipids, lipoproteins, C-reactive protein and sex hormone-binding globulin show no association with hot flush status. In the 6-month hormone therapy trial the women were classified as having either tolerable or intolerable hot flushes. These groups were treated in a randomized order with transdermal estradiol gel, oral estradiol alone or in combination with medroxyprogesterone acetate, or with placebo. In women with only tolerable hot flushes, oral estradiol leads to a reduced vasodilatory response and increases in 24-hour and daytime blood pressures as compared to women with intolerable hot flushes receiving the same therapy. No such effects were observed with the other treatment regimes or in women with intolerable hot flushes. The responses of vascular biomarkers to hormone therapy are unaffected by hot flush status. In conclusion, hot flush status contributes to cardiovascular health before and during hormone therapy. Severe hot flushes are associated with an increased vasodilatory, and thus, a beneficial vascular status. Oral estradiol leads to vasoconstrictive changes and increases in blood pressure, and thus to possible vascular harm, but only in women whose hot flushes are so mild that they would probably not lead to the initiation of hormone therapy in clinical practice. Healthy, recently postmenopausal women with moderate to severe hot flushes should be given the opportunity to use hormone therapy alleviate hot flushes, and if estrogen is prescribed for indications other than for the control of hot flushes, transdermal route of administration should be favored.

The Hinge Region of Human Thyroid-Stimulating Hormone (TSH) Receptor Operates as a Tunable Switch between Hormone Binding and Receptor Activation

The mechanism by which the hinge regions of glycoprotein hormone receptors couple hormone binding to activation of downstream effecters is not clearly understood. In the present study, agonistic (311.62) and antagonistic (311.87) monoclonal antibodies (MAbs) directed against the TSH receptor extracellular domain were used to elucidate role of the hinge region in receptor activation. MAb 311.62 which identifies the LRR/Cb-2 junction (aa 265-275), increased the affinity of TSHR for the hormone while concomitantly decreasing its efficacy, whereas MAb 311.87 recognizing LRR 7-9 (aa 201-259) acted as a non-competitive inhibitor of Thyroid stimulating hormone (TSH) binding. Binding of MAbs was sensitive to the conformational changes caused by the activating and inactivating mutations and exhibited differential effects on hormone binding and response of these mutants. By studying the effects of these MAbs on truncation and chimeric mutants of thyroid stimulating hormone receptor (TSHR), this study confirms the tethered inverse agonistic role played by the hinge region and maps the interactions between TSHR hinge region and exoloops responsible for maintenance of the receptor in its basal state. Mechanistic studies on the antibody-receptor interactions suggest that MAb 311.87 is an allosteric insurmountable antagonist and inhibits initiation of the hormone induced conformational changes in the hinge region, whereas MAb 311.62 acts as a partial agonist that recognizes a conformational epitope critical for coupling of hormone binding to receptor activation. The hinge region, probably in close proximity with the alpha-subunit in the hormone-receptor complex, acts as a tunable switch between hormone binding and receptor activation.

Fasting differentially regulates plasma corticosterone-binding globulin, glucocorticoid receptor, and cell cycle in the gastric mucosa of pups and adult rats

Ogias D, de Andrade Sa ER, Kasai A, Moisan M, Alvares EP, Gama P. Fasting differentially regulates plasma corticosterone-binding globulin, glucocorticoid receptor, and cell cycle in the gastric mucosa of pups and adult rats. Am J Physiol Gastrointest Liver Physiol 298: G117-G125, 2010. First published October 15, 2009; doi:10.1152/ajpgi.00245.2009.-The nutritional status influences gastric growth, and interestingly, whereas cell proliferation is stimulated by fasting in suckling rats, it is inhibited in adult animals. Corticosterone takes part in the mechanisms that govern development, and its effects are regulated in particular by corticosterone-binding globulin (CBG) and glucocorticoid receptor (GR). To investigate whether corticosterone activity responds to fasting and how possible changes might control gastric epithelial cell cycle, we evaluated different parameters during the progression of fasting in 18- and 40-day-old rats. Food restriction induced higher corticosterone plasma concentration at both ages, but only in pups did CBG binding increase after short-and long-term treatments. Fasting also increased gastric GR at transcriptional and protein levels, but the effect was more pronounced in 40-day-old animals. Moreover, in pups, GR was observed in the cytoplasm, whereas, in adults, it accumulated in the nucleus after the onset of fasting. Heat shock protein (HSP) 70 and HSP 90 were differentially regulated and might contribute to the stability of GR and to the high cytoplasmic levels in pups and elevated shuttling in adult rats. As for gastric epithelial cell cycle, whereas cyclin D1 and p21 increased during fasting in pups, in adults, cyclin E slowly decreased, concomitant with higher p27. In summary, we demonstrated that corticosterone function is differentially regulated by fasting in 18-and 40-day-old rats, and such variation might attenuate any possible suppressive effects during postnatal development. We suggest that this mechanism could ultimately increase cell proliferation and allow regular gastric growth during adverse nutritional conditions.

Effects of EDTA and Sodium Citrate on hormone measurements by fluorometric (FIA) and immunofluorometric (IFMA) methods

Abstract Background Measurements of hormonal concentrations by immunoassays using fluorescent tracer substance (Eu3+) are susceptible to the action of chemical agents that may cause alterations in its original structure. Our goal was to verify the effect of two types of anticoagulants in the hormone assays performed by fluorometric (FIA) or immunofluorometric (IFMA) methods. Methods Blood samples were obtained from 30 outpatients and were drawn in EDTA, sodium citrate, and serum separation Vacutainer®Blood Collection Tubes. Samples were analyzed in automatized equipment AutoDelfia™ (Perkin Elmer Brazil, Wallac, Finland) for the following hormones: Luteinizing hormone (LH), Follicle stimulating homone (FSH), prolactin (PRL), growth hormone (GH), Sex hormone binding globulin (SHBG), thyroid stimulating hormone (TSH), insulin, C peptide, total T3, total T4, free T4, estradiol, progesterone, testosterone, and cortisol. Statistical analysis was carried out by Kruskal-Wallis method and Dunn's test. Results No significant differences were seen between samples for LH, FSH, PRL and free T4. Results from GH, TSH, insulin, C peptide, SHBG, total T3, total T4, estradiol, testosterone, cortisol, and progesterone were significant different between serum and EDTA-treated samples groups. Differences were also identified between serum and sodium citrate-treated samples in the analysis for TSH, insulin, total T3, estradiol, testosterone and progesterone. Conclusions We conclude that the hormonal analysis carried through by FIA or IFMA are susceptible to the effects of anticoagulants in the biological material collected that vary depending on the type of assay.