Effects of sex hormone-binding globulin (SHBG) on androgen bioactivity in vitro
Introduction
Androgens are endogenous C19 steroid hormones which activate the androgen receptor (AR) and elicit important biological functions in reproductive and other sexually dimorphic tissues (Pihlajamaa et al., 2015). Upon ligand binding in the cell cytoplasm, the AR dimerizes, translocates to the nucleus, binds to specific DNA sequences called androgen response elements (AREs) and recruits co-regulators to activate transcription of its target genes (Helsen and Claessens, 2014, Pihlajamaa et al., 2015). Two types of AREs exist: classical and selective AREs, which are either conserved or partially conserved repeats of the 5′-AGAACA-3′ consensus sequence separated by a three-nucleotide spacer. Classical AREs can be recognized by other nuclear receptors like the glucocorticoid or progesterone receptor, while only AR can bind selective AREs to elicit transcriptional programs specific to e.g. male reproductive organs (Denayer et al., 2010, Kerkhofs et al., 2012, Pihlajamaa et al., 2015, Sahu et al., 2014, Schauwaers et al., 2007).
The AR is mainly a ligand-dependent transcription factor whose biological effects largely depend on the time and degree of AR occupancy by different ligands (Clinckemalie et al., 2012). Serum testosterone (T) concentrations are the primary method to judge androgen status in clinical practice (Snyder et al., 2016). In addition to T and its more active metabolite, 5α-dihydrotestosterone (DHT), androst-4-ene-3,17-dione (Adione) or androst-5-ene-3β,17β-diol (Adiol) are endogenous androgens or androgen precursors that are also present in the circulation, and recent studies suggest that measuring these androgens may also contribute to the assessment of androgen bioactivity (Munzker et al., 2015, O'Reilly et al., 2014). Importantly, the bioavailability of circulating sex steroids is thought to be inhibited by sex hormone-binding globulin (SHBG) (Mendel, 1989). This ∼45 kDa glycoprotein has high (nM Kd) binding affinity for DHT, T and estradiol (E2) (Wu and Hammond, 2014). On average, about 45% of circulating T or E2 is bound to SHBG in normal adult men and women, respectively. This fraction is considered biological unavailable, while non-SHBG-bound steroids constitute the bioavailable fraction. This bioavailable fraction is mainly bound with low affinity to bulk carrier proteins like albumin and only 1–3% circulates freely (i.e. non-protein-bound) (Dunn et al., 1981, Hammond et al., 2012, Rosner et al., 2007, Siiteri et al., 1982, Vermeulen et al., 1999). According to the free hormone hypothesis however, only non-protein-bound or “free” steroid hormones can diffuse into target cells and activate their cognate nuclear receptor (Mendel, 1989). Thus, SHBG is regarded as an important regulator of circulating sex steroid bioactivity. In clinical practice, free T and/or bioavailable T (bioT) and E2 concentrations are routinely calculated from total sex steroid and SHBG concentrations (Rosner et al., 2007, Rosner et al., 2013). However, growing concern surrounds this approach (Heinrich-Balard et al., 2015, Laurent and Vanderschueren, 2014, Zakharov et al., 2015). For example, several non-synonymous single nucleotide polymorphisms (SNPs) in the SHBG gene have been identified recently which may influence androgen binding affinity and thus free T concentrations (Ohlsson et al., 2011, Wu and Hammond, 2014).
The golden standard to measure androgen bioactivity is an in vivo androgen bioassay called the Hershberger test, which involves test compound administration to castrated rats and post-mortem evaluation of the animals' accessory sex organs (Marty and O'Connor, 2014). In vitro bioassays can replace the use of laboratory animals to a certain extent, but their main advantage is that they are more practical for high-throughput screenings (Roy et al., 2008, Voet et al., 2013), screening for steroid abuse in athletes (Bailey et al., 2016, Cooper et al., 2013) or for potentially endocrine-disrupting compounds in environmental samples (Christiaens et al., 2005). In vitro bioassays can be classified as proliferation assays (e.g. proliferation response of an androgen-sensitive prostate cancer cell line) or reporter assays (e.g. based on transactivation of a luciferase construct, which offers more direct evidence of AR-mediated signaling). Furthermore, bioassays have been established using different mammalian or yeast cell lines; the latter may provide an advantage when metabolism and detection of precursor androgens like Adione, Adiol and dehydroepiandrosterone (DHEA) is desired (e.g. for hormone abuse screening in livestock or athletes), although the thick yeast glycocalyx may hinder diffusion into target cells (Soto et al., 2006).
In vitro bioassays are most used for screening compounds in drug development or endocrine disruptor research, but they have also been applied to study circulating androgen bioactivity (Roy et al., 2008). It has been suggested that results from in vitro androgen bioassays also reflect the influence of SHBG (Liimatta et al., 2014, Raivio et al., 2001, Roy et al., 2006), but this has not been tested directly. One concern in this regard is the fact that most AR bioassays are conducted in the presence of diluted serum samples to 1–10% with one notable exception using undiluted serum (Need et al., 2010). Previous studies on equilibrium dialysis have shown that dilution may disturb the equilibrium between SHBG and its ligands (Van Uytfanghe et al., 2004), but whether dilution also affects AR bioassay results is not known. Another important factor identified previously are matrix effects (Paris et al., 2002a, Roy et al., 2006) that are well known in clinical chemistry. Matrix effects can be defined as serum sample components which are unrelated to the analyte of interest per se (in this case, androgen bioactivity) but still produce unwanted alterations of the bioassay read-out. Considering this background, the aim of this study was to establish an androgen reporter bioassay to study the effects of SHBG on androgen bioactivity in vitro, and to validate our bioassay method in pilot studies using patient samples with particular attention to dilution and matrix effects.
Section snippets
Hormones and chemicals
T, DHT, Adiol, Adione, R1881 (methyltrienolone), mibolerone, medroxyprogesterone acetate, E2, progesterone, dexamethasone, hydrocortisone, DHEA and DHEA-sulphate, ethanol, dimethylsulfoxide (DMSO) and ionomycin were purchased from Sigma-Aldrich. MENT (7α-Methyl-19-Nortestosterone) was a kind gift from dr. N. Kumar of the Population Council (New York, NY, U.S.A.). Enzalutamide was purchased from Sequoia Research Products (Pangbourne, U.K.).
Cell lines and culture conditions
Human embryonic kidney (HEK)-293 and VCaP cells were
Bioassay development and characterization
In general, cell lines with the classical ARE (from ADAMTS1,A) showed higher luciferase activity compared to those transfected with selective AREs (from the TMPRSS2 or PDE9A genes) (Supplementary Fig. 1A). The colony with highest sensitivity and high induction factors (arrow in Supplementary Fig. 1B; light green line in Fig. S1A) was selected and termed LARA, for Luciferase Androgen Reporter Assay. Fig. 1A shows the luciferase activity in response to various T concentrations on three
Discussion
Several in vitro androgen bioassays have been previously established and applied to analyze serum from older men (Need et al., 2010, Raivio et al., 2002), women (Chen et al., 2006), children (Paris et al., 2002b, Raivio et al., 2001, Raivio et al., 2004), hyperandrogenic women or children (Hero et al., 2005, Liimatta et al., 2014, Roy et al., 2006) or prostate cancer patients (Raivio et al., 2003, Rannikko et al., 2006), for example. In our hands, optimal assay characteristics were achieved
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgements
We thank H. De Bruyn, I. Jans, E. Van Herck, G. Saelens, R. Bollen and L. Deboel for excellent technical assistance, and E. Goossens, C. D'Haemer, H. Morobé, S. Achten, R. Van Heyste, C. Hulsbosch, H. Van Poppel, T. Devos and the Departments of Urology and Haematology for help with patient recruitment.
This work was supported by the Research Foundation Flanders (FWO grant G085413N) and grant GOA/15/017 from the KU Leuven Research Council. MRL is supported by a PhD Fellowship from the Research
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