Regulation of Sclerostin Production in Human Male Osteocytes by Androgens: Experimental and Clinical Evidence

Endocrinology, Dec 2015

In this study we aimed to elucidate a possible role of T in the regulation of sclerostin, a glycoprotein secreted by osteocytes known to regulate bone mass. To this end, we evaluated the effect of T stimulation on sclerostin production and gene expression in human cultured osteocytes. In addition, we evaluated serum sclerostin levels in a cohort of 20 hypogonadal male patients, compared with 20 age-matched eugonadal controls. Stimulation with DHT decreased sclerostin expression in cultured osteocytes in a time- and dose-dependent manner. Confirming a direct androgen receptor-mediated effect on sclerostin production, flutamide coincubation and silencing of androgen receptor gene in osteocytes abolished the DHT effects. In addition, hypogonadal patients showed higher serum sclerostin levels with respect to controls (145.87 ± 50.83 pg/mL vs 84.02 ± 32.15 pg/mL; P < .001) and in both probands and controls, serum T levels were negatively correlated with sclerostin (R = −0.664, P = 0.007, and R = −0.447, P = .045, respectively). Finally, multiple stepwise regression analysis showed that T represented the only independent predictor of sclerostin levels. In conclusion, by showing a direct correlation between T and sclerostin, both in vivo and in vitro, this study adds further support to the emerging clinical and experimental studies focusing on sclerostin as a therapeutic target for osteoporosis treatment.

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Regulation of Sclerostin Production in Human Male Osteocytes by Androgens: Experimental and Clinical Evidence

Endocrine Society Received March Regulation of Sclerostin Production in Human Male Osteocytes by Androgens: Experimental and Clinical Evidence Andrea Di Nisio 0 Luca De Toni 0 Elena Speltra 0 Maria Santa Rocca 0 Giuseppe Taglialavoro 0 Alberto Ferlin 0 Carlo Foresta 0 0 Department of Medicine (A.D.N. , L.D.T., E.S., M.S.R., A.F., C.F.) , Operative Unit of Andrology and Medicine of Human Reproduction, and Department of Surgical , Oncological, and Gastroenterological Sciences (G.T.) , University of Padova , 35128 Padova , Italy In this study we aimed to elucidate a possible role of T in the regulation of sclerostin, a glycoprotein secreted by osteocytes known to regulate bone mass. To this end, we evaluated the effect of T stimulation on sclerostin production and gene expression in human cultured osteocytes. In addition, we evaluated serum sclerostin levels in a cohort of 20 hypogonadal male patients, compared with 20 age-matched eugonadal controls. Stimulation with DHT decreased sclerostin expression in cultured osteocytes in a time- and dose-dependent manner. Confirming a direct androgen receptor-mediated effect on sclerostin production, flutamide coincubation and silencing of androgen receptor gene in osteocytes abolished the DHT effects. In addition, hypogonadal patients showed higher serum sclerostin levels with respect to controls (145.87 50.83 pg/mL vs 84.02 32.15 pg/mL; P .001) and in both probands and controls, serum T levels were negatively correlated with sclerostin (R 0.664, P 0.007, and R 0.447, P .045, respectively). Finally, multiple stepwise regression analysis showed that T represented the only independent predictor of sclerostin levels. In conclusion, by showing a direct correlation between T and sclerostin, both in vivo and in vitro, this study adds further support to the emerging clinical and experimental studies focusing on sclerostin as a therapeutic target for osteoporosis treatment. (Endocrinology 156: 4534 - 4544, 2015) - Smaintenance of the bone apparatus ( 1 ). In this regard, ex steroids are known to influence the growth and androgens are known to have an impact on bone growth, as demonstrated by both the skeletal effects secondary to sex steroid deficiency ( 2 ) and decreased peak bone mass associated with prepubertal hypogonadism ( 3, 4 ). In addition, androgens are believed to be responsible for the sexual dimorphism of the skeleton (5). In fact, androgens have been shown to increase bone size by stimulating periosteal bone formation, as demonstrated in animal models ( 6, 7 ). As a result, male gender by itself is one of the strongest protective factors that approximately halves the risks associated with decreased bone mass, compared with females. In addition, serum levels of free T were positively associated with the cortical cross-sectional area and periosteal circumference at both the tibia and the radius of young male adults (8). Androgens mainly act through androgen receptor (AR), which is a ligand-dependent transcription factor ( 9 ). Evidence for a direct role of AR in male skeletal homeostasis is derived from observations in genetically engineered ubiquitous AR knockout mice and orchidectomized male mice ( 10 ). AR expression has been shown in a number of bone cells including pluripotent mesenchymal bone marrow stromal cells, osteoblasts, osteoclasts, and osteocytes ( 11–13 ). To our knowledge, two bone-specific AR knockouts have been generated. In these two models, both the osteoblast and the osteocyte were targeted, which * A.D.N. and L.D.T. contibuted equally to this work. Abbreviations: ALP, alkaline phosphatase; AR, androgen receptor; AR-KO, AR knockout; BMI, body mass index; E2, 17 -estradiol; FITC, fluorescein isothiocyanate; nRQ, normalized relative quantification; 25(OH)D, 25-hydroxyvitamin D; siRNA, small interfering RNA; SOST, sclerostin. resulted in a peculiar phenotype of trabecular bone loss, confirming that the osteoblast and/or osteocyte is a target cell for AR-mediated maintenance of trabecular bone volume ( 14 ). Interestingly, the highest expression of AR has been observed in osteocytes ( 15 ), which make up greater than 95% of bone cells in the adult skeleton, with this ratio increasing with age and bone size ( 16 ). Osteocytes are differentiated osteoblasts that do not directly participate of bone mineralization but, by being embedded in the growing matrix rather, have a regulatory function on the other bone cell populations ( 17 ). Interestingly, the changes in gene expression that represent a signature for transition of osteoblasts toward an osteocyte phenotype include the down-regulation of alkaline phosphatase and induction of the SOST gene, which encodes the protein sclerostin (SOST) ( 18, 19 ). SOST, a glycoprotein secreted by osteocytes under physiological conditions, is an important negative regulator of bone mass through the inhibition of bone formation by osteoblasts (reviewed in reference 20). Although osteocytes have emerged as key regulators of bone remodeling, the influence of sex steroids on these cells has been poorly studied (21). The aim of this study was to elucidate a possible role of T in the regulation of bone mass through a SOST-dependent pathway in osteocytes. To this end, we investigated whether AR was expressed in primary osteocyte cultures from human male femoral heads. Moreover, we evaluated the effect of T stimulation on SOST production and mRNA expression in human cultured osteocytes. Finally, we evaluated serum SOST levels in a cohort of hypogonadal male patients, compared with age-matched eugonadal controls. Materials and Methods Primary osteocyte isolation Human osteocyte cultures were obtained from the femoral heads discards of three male subjects undergoing arthroplasty, who gave informed consent. Their use for in vitro scientific re Antibody Table search does not require ethics approval from the institutional review board. Primary osteocytes were isolated from human bone following an established protocol from Stern et al ( 22 ) with slight modifications. In detail, bone specimens were finely minced by mechanical grinding in sterile conditions and underwent overnight digestion with type IA collagenase solution (300 U/mL; Sigma-Aldrich) and sodium EDTA (5 mM, pH 7.4; Sigma-Aldrich), dissolved in -MEM (Euroclone). Digestion was performed in a 10-cm petri dish, with a total volume of 8 mL of digestion solution, in a 37°C and 5% CO2 humidified incubator. After the digestion, suspended cells were harvested, washed twice in MEM, and then underwent fluorescent cell sorting. Details on the antibodies used in this study are reported in Table 1. Prior to sorting, a cell pellet from digested bones was incubated 1 hour at 4°C with goat antihuman SOST and allophycocyaninconjugated antihuman alkaline phosphatase antibody (both from R&D Systems), followed by incubation with fluorescein isothiocyanate (FITC)-conjugated donkey antigoat antibody (Santa Cruz Biotechnology). Labeled cell suspensions underwent cell sorting by the use of the XDP cell sorter (Beckman Coulter). Only SOST /alkaline phosphatase (ALP) cells were collected and plated on six-well plates coated with type I rat tail collagen (BD Biosciences) at a seeding density of approximately 20 000 cells/well in MEM supplemented with 5% fetal bovine serum, 5% calf serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. Cells were maintained at 37°C and 5% CO2 ( 22 ). Osteocytes hormonal stimulation in vitro For stimulation experiments, cells were starved in serum-free medium for 16 hours and then exposed for 24 and 48 hours to human 10 7 M PTH (Sigma-Aldrich) and DHT (Sigma-Aldrich) at concentrations ranging from 10 10 to 10 6 M in serum-free medium in the presence and absence of 10 6 M flutamide (Sigma-Aldrich), previously incubated for at least 30 minutes. After hormonal stimulation, cultured osteocytes underwent physical detachment from wells by cell scraping. After centrifugation, the cell pellet was collected and stored at 80°C for subsequent analysis. AR gene silencing was performed by the use of human AR small interfering RNA (siRNA) kit ( 23 ) (sc-29204; Santa Cruz Biotechnology) according to the manufacturer’s instruction. Targeted siRNA transfection with scrambled sequence provided by the kit, known to not lead to the specific degradation of any known cellular mRNA, was used as negative controls. Species Raised (Monoclonal or Polyclonal) Goat; polyclonal 1:100 for IHC, 1:500 for WB Mouse; monoclonal Undiluted Dilution Used Immunofluorescence Bone fragments from femoral heads discards undergoing arthroplasty were fixed in 4% paraformaldehyde in PBS solution for 2 hours at room temperature. Subsequently, trabecular bone specimens underwent decalcification by incubation in 500 mM EDTA for 21 days at 4°C. Samples were then embedded in optimum cutting temperature mounting medium and cut into 5- m slices on Superfrost microscope slides (Menzel-Gläser). After permeabilization with a 1% Triton X-100/PBS solution for 10 minutes at room temperature, the samples were saturated with 5% BSA/5% normal donkey serum in PBS for 30 minutes and then incubated overnight at 4°C with rabbit antihuman C-terminal AR (sc-815; Santa Cruz Biotechnology) and goat antihuman SOST antibody (R&D Systems). In the negative control, primary antibodies were omitted. The following day, primary immunoreaction was detected by incubation with a FITC-conjugated donkey antigoat secondary antibody (Santa Cruz Biotechnology, Inc) and with biotin-conjugated antirabbit secondary antibody followed by streptavidin-Texas Red (both 1:200; Santa Cruz Biotechnology, Inc). Finally, specimens were counterstained with 4 ,6 -diamino-2-phenylindole, mounted with antifade buffer, and analyzed with a video-confocal fluorescence microscope (Nikon). To assess the AR nucleus translocation after DHT stimulation, starved primary cultured osteocytes seeded onto glass slides (BD Biosciences) were stimulated with 10 8 M DHT in the presence and the absence of 10 6 M flutamide for 4 hours at 37°C in serum-free medium. Subsequently, the cells were fixed with a 4% paraformaldehyde/PBS solution for 15 minutes at room temperature, and immunostaining for AR was performed as described previously. Western blotting After hormonal stimulation, cultured osteocytes underwent physical detachment from wells by cell scraping. After centrifugation, the cell pellet was collected and underwent protein extraction by physical procedure (freeze-thaw cycles in liquid nitrogen followed by a 37°C water bath) into lysis buffer (Bio-Rad Laboratories) containing a protease inhibitor (phenylmethylsulfonyl fluoride). Total protein content was assessed in each sample by determination of OD at 280 nm with a Nanodrop ND1000 spectrophotometer (Thermo Fisher). Samples were denatured with sodium dodecyl sulfate and 2- -mercaptoethanol, boiled for 10 minutes, and then fractionated using SDSPAGE gel (Bio-Rad Laboratories). After blotting onto a Hybond enhanced chemiluminescence nitrocellulose membrane (PerkinElmer) and blocking with 5% nonfat milk in 0.1% PBSTween 20 (Bio-Rad Laboratories), blots were incubated overnight at 4°C with the goat antihuman SOST antibody (R&D Systems) or rabbit antihuman C-terminal AR (sc-815; Santa Cruz Biotechnology) at the proper dilution in 5% nonfat milk in 0.1% PBS-Tween 20 buffer. Primary immunoreaction was detected by incubation with goat antirabbit and rabbit antigoat secondary antibodies (KPL) and visualized using an enhanced chemiluminescence reagent (LumiGLO; KPL) with the Chemidoc XRS System (Bio-Rad Laboratories). -Actin (sc-47778; Santa Cruz Biotechnology, Inc) served as housekeeping. For each protein band, the pixel density per square millimeter was calculated by means of Quantity One Software version 4.6.9 (Bio-Rad Laboratories). Results were reported as the ratio between the target band density with the corresponding band density of -actin, after subtraction of the background pixel signal. Experiments were performed three times in triplicate. Quantitative RT-PCR analysis RNA was extracted from stimulated osteocytes using the RNeasy microkit (QIAGEN), and RT-PCR was performed using total RNA (50 ng) and the reverse transcription Sensiscript kit (QIAGEN) by the use of random hexamers. The quality of RNA and cDNA obtained was tested by a spectrophotometric measurement (NanoDrop, Celbio). Quantitative real-time PCR was performed as previously described ( 24 ). Specific intron-spanning primers for human AR, SOST, and ALP were as follows: AR forward, 5 -TAGCCCCCTACGGCTACA-3 ; AR reverse, 5 -TTCCGAAGACGACAAGATGGAC-3 ; SOST forward, 5 -CGGAGCTGGAGAACAACAA-3 ; SOST reverse, 5 -GGCAGCTGTACTCGGACAC-3 ; ALP forward, 5 -CTATCCTGGCTCCGTGCTC-3 ; and ALP reverse, 5 -GCTGGCAGTGGTCAGATGTT-3 . -Actin expression (forward, 5 -CACTCTTCCAGCCTTCCTTCC-3 ; reverse, 5 -CGGACTCGTCATACTCCTGCTT-3) was used as housekeeping gene. Results are reported as normalized relative quantification (nRQ). The RT-PCR products were analyzed electrophoretically through a 1% agarose gel and visualized by SYBRsafe DNA gel staining (Invitrogen) and confirmed by direct sequencing on an ABI Prism sequencer (Applied Biosystems). Patient characteristics The investigation was conformed to the principles of the Declaration of Helsinki, and all subjects gave informed consent to the study, which has been approved by the local ethical committee with protocol number 2406P. Twenty male patients with total T less than 10.4 nmol/L (mean age 22.0 y; range 19.0 –26.0 y) were recruited in the hypogonadal patients group (HP). In details, eight had idiopathic subclinical-hypogonadism (LH 8 UI/L) and 12 had hypergonadotrophic hypogonadism (LH 8 UI/L) with primary posttraumatic testiculopathy. Twenty aged-matched eugonadal male subjects with total T greater than 10.4 nmol/L (mean age 23.0 y; range 19.0 –29.0 y) served as controls. All patients were free of gonadotrophin or T replacement therapy for at least 3 months. Exclusion criteria, assessed by history, clinical examination, and biochemical blood tests, were as follows: diabetes, smoking, hypercholesterolemia, hypertriglyceridemia, hyperhomocysteinemia, obesity, and previous major cardiovascular events. Serum collection and analysis Fasting blood withdrawal was collected from each participant between 8.00 and 10.00 AM. Serum FSH, LH, and total T were evaluated by commercial electrochemiluminescence immunoassay methods (Elecsys 2010; Roche Diagnostics). PTH serum levels were determined with a direct, two-site, sandwich-type chemiluminescent immunoassay (LIAISON N-TACT PTH; DiaSorin Inc). 25-Hydroxyvitamin D (25[OH]D) was determined with a direct, competitive chemiluminescent immunoassay (LIAISON 25(OH)D total assay; DiaSorin Inc). For all parameters the intraassay and interassay coefficients of variation were less than 8% and 10%, respectively. All determinations were performed according to the manufacturer’s instructions. The serum level of SOST was measured by the ELISA quantikine ELISA on the SOST cell population, featured by dendrite-like cell extension (BF inserts in Figure 2C), confirmed cytoplasm AR expression in sorted osteocytes (Figure 2C). SOST expression in sorted cells was further assessed by both RT-PCR and Western blot analysis (Figure 2, D and E). SOST showed a clear expression of both SOST mRNA and protein level with a negligible expression of ALP as osteoblast marker (SOST in Figure 2D). SOST /ALP cells (ALP ) showed almost undetectable levels of SOST (Figure 2, D and E). To evaluate the functional state of AR in osteocytes, we assessed whether AR translocated into the nucleus after stimulation with 10 8 M DHT, a nonaromatizable androgen ( 26 ). To assess the specificity of the androgen action, stimulation was also performed in the presence and absence of flutamide, a typical AR antagonist (Figure 3). As expected, in the control sample, the staining for AR was mainly cytoplasmatic and perinuclear. Stimulation with the sole DHT resulted in most cells showing the AR staining detectable as diffuse nuclear spots and a negligible signal in the cytoplasm (DHT in Figure 3). This staining pattern was not observed by concomitant stimulation with both DHT and flutamide (DHT FLUT in Figure 3). To assess whether AR nucleus translocation induced by DHT in osteocytes may lead to any variation of SOST production by this cell population, we stimulated primary cultured osteocytes with DHT for 24 and 48 hours, at concentrations ranging from 10 10 to 10 6 M. SOST mRNA and protein expressions were assessed, respectively, by RT-PCR and Western blot analysis. After 24 hours, DHT did not influence SOST expression at any of the tested concentrations (Figure 4, A and B). After 48 hours of stimulation, DHT at 10 8 and 10 6 M, but not 10 10 M DHT, led to a statistically significant decrease of both SOST mRNA (P .03, P .008, and P .36 vs control, respectively, Figure 4C) and protein expression (P .01, P .001, and P .49 vs control, respectively, Figure 4D). In a second set of experiments, cultured osteocytes were stimulated for 48 hours with 10 7 M human PTH, a reference antagonist of SOST production ( 27, 28 ), and 10 8 M DHT. The latter stimulation was performed in the presence and absence of 10 6 M flutamide. RT-PCR results showed a decreased expression of SOST mRNA after stimulation with PTH and DHT (P .004 and P .005 vs control, respectively, Figure 5A). Coincubation with human SOST (R&D Systems), according to the manufacturer’s instructions, as previously described (25). Statistical analysis Data analysis was performed using SPSS version 15.0 (SPSS Inc). The results were expressed as means SD. The Kolmogorov-Smirnov test was used to check for normality of distribution. Parameters not showing normal distribution were log transformed. Relationships between continuous variables were assessed using a Pearson’s correlation analysis; for nonnormally distributed variables, nonparametric Spearman’s -correlations coefficients are reported. The Levene’s test was used to test the homogeneity of variance among groups prior to data analysis. If homogeneity of variance assumption was violated, the Welch test was performed and the respective P value was reported. Differences between two groups were analyzed using a Student’s t test or an ANOVA for the comparison of multiple parameters. A multiple stepwise regression analysis was performed to determine the associations between serum SOST levels and age, body mass index (BMI), total T, LH, FSH, 25(OH)D, and PTH concentration after adjusting for potential confounders. Statistical significance was defined at the P .05 level using two-sided tests; highly statistical significance was defined for values of P .01. Results In vitro experiments The expression of the AR on osteocytes was assessed in decalcified human trabecular bone samples, whose hematoxylin/eosin staining is reported in Figure 1, A and B. Double immunofluorescence showed intracellular AR staining in SOST-positive osteocytes embedded in the bone matrix (Figure 1, E and F). This expression pattern was also evaluated in human primary osteocyte culture, obtained from digested bones specimens ( 22 ) enriched in SOST /ALP (SOST ) cells by fluorescence-assisted cell sorting (Figure 2, A and B). Immunofluorescence staining flutamide blunted any observed effect of DHT on SOST mRNA levels (Figure 5A). Gene expression of both AR and ALP levels were also investigated. Either PTH or DHT did not influence AR and ALP mRNA expression and, in particular, ALP levels was very low in all the samples (Figure 5A). In terms of SOST protein expression, both PTH and DHT stimulation led to a significant decrease in SOST levels (P .002 and P .02 vs control, respectively, Figure 4B), whereas samples coincubated with flutamide resulted in SOST levels comparable with controls (P .46, Figure 5B). In addition, AR levels in osteocytes, confirmed by a specific band at 110 kDa ( 29 ), were not influenced by any hormonal stimulation (PTH, DHT, and DHT flutamide all P .05 vs control, Figure 5B). To confirm the AR-mediated role of DHT in the reduction of SOST expression, AR gene expression was silenced by the means of specific AR siRNA in cultured osteocytes, prior to hormonal stimulation as above. Both SOST and AR gene and protein expressions were then evaluated. SOST gene expression was reduced after PTH, but not DHT, stimulation (P .006 and P .28, respectively, Figure 5A). Either PTH or DHT did not influence AR and ALP mRNA expression, which were very low in all samples in which AR silencing was performed (all P .01 vs negative control, Figure 5A). As a counterpart, PTH, but not DHT, significantly reduced SOST protein expression, compared with unstimulated AR siRNA-positive control (P .02 and P .29, respectively, Figure 5A). According to the RT-PCR results, AR expression was blunted in AR siRNA osteocytes, compared with negative controls in which siRNA was omitted (all P .001, Figure 5B). In vivo correlation between SOST and T Clinical characteristics of 20 control subjects and 20 HP are reported in Table 2. With respect to controls, the T levels showed to be the only independent predictor of serum SOST ( .713; t 5.838; P .001). Discussion Androgens play a key role in the maintenance of male skeletal integrity through the classical AR signaling acting on osteoblasts ( 30 ). Very recently, selective inactivation of the AR in osteocytes of male mice has been shown to accelerate age-related deterioration of skeletal integrity ( 21 ), suggesting a main role for this cell population in bone metabolism. In this study we show for the first time, to our knowledge, a direct role for androgens in reducing SOST levels in human cultured osteocytes in vitro. This is supported in vivo by the evidence of increased serum SOST levels in a cohort of hypogonadal men, compared with age-matched healthy controls. SOST is one of the main effectors of osteocyte function, integrating mechanical and endocrine challenges and leading to the inhibition of bone formation by osteoblasts (reviewed in reference 20). Under physiological conditions, osteoblast activation mainly relies on the binding of Wnt ligands to low-density lipoprotein receptor-related protein-5 or -6 ( 31, 32 ). This finally results in the release of axin from its complex with -catenin, which accumulates and translocates to the nucleus, leading to the activation of Wnt target genes (reviewed in reference 33). However, in the presence of SOST, the interaction between Wnt ligands and their receptors is inhibited, and -catenin is phosphorylated by glycogen synthase kinase 3 and finally targeted for degradation via the proteosome pathway, leading to reduced extracellular bone matrix formation and increased osteoblast apoptosis ( 31, 34 –36 ). Because osteocytes are bathed in a unique canalicular fluid that delivers nutrients and humoral information from the systemic circulation, they are sensitive to a variety of systemic hormones with a rapid and efficient communication between osteocytes and systemic circulation ( 37 ). For example, PTH acts on osteocytes by altering the SOST expression: in fact, constitutively active PTH receptor-1 in osteocytes is sufficient to increase bone remodeling due to the inhibition of SOST expression ( 27, 28 ). Furthermore, serum SOST levels are representative of actual SOST release in the bone extracellular milieu (38). whole HP group showed significantly lower total T and 17 -estradiol (E2) (P .001 and P .011, respectively, Table 2) and significantly higher LH, FSH, and PTH (P .003; P .002, and P .001, respectively, Table 2). In addition, the HP group had a significantly lower concentration of serum 25(OH)D compared with the control group (P .001, Table 2). Serum SOST levels were significantly higher in HP with respect to controls (145.87 50.83 pg/mL vs 84.02 32.15 pg/mL; P .001; Table 2), even after correction for age and BMI (P .001). When HP subjects were subdivided into subclinical and hypergonadotropic-hypogonadal (Table 2), a significant reduction compared with controls persisted for total T (both P .001), 25(OH)D (P .001 and P .001, respectively), PTH (P .001 and P .010, respectively), and SOST (P .039P and 0.001, respectively), whereas, as expected, the LH and FSH levels were higher in hypergonadotropic-hypogonadal patients (both P .001 vs controls). Correlation coefficients between SOST and serum parameters in patients were assessed and are reported in Table 3. In both HP and control groups, serum T levels were negatively correlated with SOST levels (HP: R 0.664, P .007; CTRL: R 0.447, P .045; Figure 6). In addition, in the HP group, SOST was negatively correlated with LH and FSH (R 0.566, P .028, and R 0.542, P .037, respectively). To remove the effect of any confounding factors, a multiple stepwise regression analysis was performed (Supplemental Table 1), and total In this study, we showed a direct inhibitory effect of androgen on SOST production in an AR-mediated pathway. In fact, we initially confirmed AR expression in ex vivo and cultured human osteocytes. Moreover, AR was shown to be completely functional as demonstrated by AR nuclear translocation after stimulation with DHT. On this basis, we aimed to test whether AR-mediated stimulation may lead to a decrease in SOST production by osteocytes. Both SOST protein and gene expressions were indeed reduced after 48 hours, but not 24 hours, of stimulation with DHT. In this regard, AR and ALP expressions were not influenced by hormonal stimulation, with the latter showing very low levels, which is in agreement with a typical mature osteocyte phenotype, also excluding any artifact due to dedifferentiation into osteoblasts ( 18, 19 ). Confirming a direct AR-mediated effect on SOST production, flutamide coincubation and silencing of AR gene in osteocytes abolished the DHT effects. In addition, the same inhibitory effect on SOST was observed also after PTH stimulation, which is in agreement with previous studies ( 27, 28 ). A mechanistic hypothesis explaining the effect of DHT/AR on SOST is matter of debate. It has to be considered that the regulation of gene transcription by AR involves numerous AR-interacting coregulator proteins. In addition to androgen response elements in the AR-binding sites, the region of AR chromatin occupancy encompasses cis elements for other transcription factors, such as pioneer transcription factors GATA-binding protein 2, E26 transformation-specific, homeodomain protein homeo box B13, and forkhead box A1 ( 39 ). Interestingly, the SOST upstream regulatory region shows a core consensus recognition site for forkhead box A1 ( 40 ) and, to this regard, polymorphisms in this region have been associated with higher risk of osteoporosis in Chinese and Caucasian populations ( 41 ). On these bases, it could be hypothesized an up-stream modulation SOST expression through an indirect effect mediated by AR. However, other possible pathways, such as through miRNA ( 42 ) cannot be excluded and deserve further insights. To extend in vitro observations to an in vivo scenario, we assessed serum SOST levels in a cohort of hypogonadal men and showed that SOST levels were higher with respect to controls. In addition, we showed a significant negative correlation between SOST levels and total T in both probands and controls. Notably, the impairment of testis function, whether primary or due to reduced levels of gonadotropins, was strictly associated with reduced levels of 25(OH)D and increased PTH. This is in agreement with our recent reports demonstrating a major role of testis in vitamin D 25-hydroxylation ( 43, 44 ). Indeed, the production of SOST by osteocytes is the result of several humoral signals, and we cannot exclude that increased levels of SOST in hypogonadal patients could be the result of a direct effect of PTH on this cell population. However, interestingly, multiple stepwise regression analysis showed that total T represented the only independent predictor of SOST levels, despite the well-recognized role of estradiol and PTH (16). These results are in agreement with previous studies showing an apparent implication of T on circulating SOST levels. In fact, SOST has been recently shown to significantly increase in patients with prostate cancer and particularly in those receiving androgen deprivation therapy, together with a concomitant impairment of bone mineralization parameters ( 45 ). A clear inverse correlation between T and SOST has also been Clinical and Biochemical Data of CTRL and HP CTRL (n Age, y 23.0 BMI, kg/m2 22.85 Total T, nmol/L 18.7 E2, pmol/L 107.7 LH, U/L 5.29 FSH, U/L 5.79 25(OH)D, nmol/L 63.3 PTH, ng/L 28.13 SOST, pg/mL 84.02 Abbreviations: CTRL, control; HP, hypogonadal patients; Hyper-Hypo, hypergonadotropic hypogonadal patients; Subclinical-Hypo, patients with idiopathic subclinical hypogonadism. Data are expressed as means SD. a Significance pertains to significant differences (in bold) considered for values of P .05 according to a Student’s t test. b Significance pertains to significant differences (in bold) considered for values of P .05 according to a one-way ANOVA and Bonferroni correction post hoc test. observed in men with osteoporosis ( 46 ). However, Mödder et al ( 47 ) have shown that androgen administration to human males did not alter circulating SOST levels. Further studies are necessary to address these apparent discrepancies. Studies on murine models are also conflicting with human data, showing little or no alteration of SOST expression in AR knockout (AR-KO) mice ( 14, 48, 49 ). In this field, the generation of conditional genetic mouse models improved the understanding of the mechanisms of action of androgens on osseous tissues ( 50 ). However, Cre-LoxP technology has its own pitfalls, including independent effects of inserting a Cre somewhere in the genome or LoxP sites in a gene, incomplete deletion, and off-target Cre activity in cells and tissues other than those targeted. Moreover, the phenotype of models, even if resulting from embryonic or perinatal imprinting effects, becomes evident at later ages ( 51 ). In fact, Sinnesael et al ( 21 ) described comparable expression of SOST between conditioned osteocytes-AR-KO and control mice, an evidence that might be ascribable to the relatively young age of mice used in the study (12 wk old). Indeed, because SOST production is known to significantly increase with age ( 16 ), the young age of mice might affect the detection of any difference in the SOST levels between controls and AR-KO mice, compared with osteocytes that, in our study, were obtained from bone specimens of elderly men (aged 52–70 y). In summary, our data suggest for the first time a direct role of androgens on SOST production by osteocytes in an AR-dependent manner and increased SOST levels in hypogonadal men. This is further supported by an inverse relationship between SOST and T levels in both hypogonadal patients and age-matched eugonadal controls. This paper adds further support to the emerging clinical and experimental studies focusing on SOST as a therapeutic target for osteoporosis treatment. However, further studies are needed to identify the cell signaling pathways associated with androgen stimulation in this cell population. In addition, to assess the relative weight of this pathway on bone metabolism, a larger cohort of hypogonadal patients, should be evaluated for bone morphometry and serum markers of bone remodeling. Acknowledgments We thank all the staff of the Operative Unit of Andrology and Human Reproductive Medicine for the helpful discussion. Address all correspondence and requests for reprints to: Professor Carlo Foresta, Department of Medicine, Unit of Andrology and Medicine of Human Reproduction, University of Padova, Via Giustiniani 2, 35128 Padova, Italy. E-mail: . Disclosure Summary: The authors have nothing to disclose. 1. Riggs BL , Khosla S , Melton LJ . Sex steroids and the construction and conservation of the adult skeleton . Endocr Rev . 2002 ; 23 : 279 - 302 . 2. Stepan JJ , Lachman M , Zverina J , Pacovsky V , Baylink DJ . Castrated men exhibit bone loss: effect of calcitonin treatment on biochemical indices of bone remodeling . J Clin Endocrinol Metab . 1989 ; 69 : 523 - 527 . 3. 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Di Nisio, Andrea, De Toni, Luca, Speltra, Elena, Rocca, Maria Santa, Taglialavoro, Giuseppe, Ferlin, Alberto, Foresta, Carlo. Regulation of Sclerostin Production in Human Male Osteocytes by Androgens: Experimental and Clinical Evidence, Endocrinology, 2015, 4534-4544, DOI: 10.1210/en.2015-1244