Negative Association Between Sclerostin and INSL3 in Isolated Human Osteocytes and in Klinefelter Syndrome: New Hints for Testis–Bone Crosstalk

Negative Association Between Sclerostin and INSL3 in Isolated Human Osteocytes and in Klinefelter... Abstract Context The regulation of bone mass by the testis is a well-recognized mechanism, but the role of Leydig-specific marker insulin-like 3 peptide (INSL3) on the most abundant bone cell population, osteocytes, is unknown. In this study, we aimed to investigate the relationship between INSL3 and sclerostin, an osteocyte-specific protein that negatively regulates bone formation. Design Serum sclerostin and INSL3 levels were evaluated in Klinefelter syndrome (KS) and healthy controls. In vitro effect of INSL3 on sclerostin production was evaluated in human cultured osteocytes. Patients A total of 103 KS patients and 60 age- and sex-matched controls were recruited. Main Outcome Measures Serum sclerostin and INSL3 levels were assessed by enzyme-linked immunosorbent assay. Osteocytes were isolated by fluorescence-assisted cell sorting. Sclerostin expression was evaluated by western blot, immunofluorescence, and reverse transcription polymerase chain reaction. Measurement of bone mineral density was done by dual-energy X-ray absorptiometry at lumbar spine (L1–L4) and femoral neck. Results Sclerostin levels were significantly increased in KS subjects, and negatively correlated with INSL3 levels in both cohorts and with bone mineral density in the KS group. Stimulation of cultured osteocytes with INSL3 at 10−7 M significantly decreased both sclerostin messenger RNA and protein expression. Conclusions We report a negative association between the testicular hormone INSL3 and the osteocytic negative regulator of bone formation, sclerostin. We further explored this association in vitro and showed that INSL3 was able to reduce sclerostin expression. These results add further knowledge on the emerging role of sclerostin as a therapeutic target for osteoporosis treatment. In recent years, increasing evidence has supported a crosstalk between testis and bone, by the means of the well-known action of testosterone on skeletal growth and bone mass (1). Nevertheless, testosterone replacement therapy does not completely restore bone mass in osteoporotic hypogonadal men (2), which suggests a more complex interaction between bone and testis. In addition to androgens, Leydig cells produce insulin-like 3 (INSL3), a peptide hormone with long-term regulation by luteinizing hormone (LH), which is used as an additional marker of Leydig cell function (3–6). The major endocrine role of INSL3 is related to the regulation of the transabdominal phase of testicular descent during fetal development by acting on gubernaculum through the activation of its specific relaxin family receptor 2 (RXFP2); indeed, mutations in INSL3 and RXFP2 genes lead to increased risk of cryptorchidism in both mice and men (7, 8). However, INSL3 has been shown to exert an anabolic effect on bone metabolism during adulthood by acting directly on osteoblasts (9, 10) through the activation of RXFP2 and subsequent rise in cyclic adenosine monophosphate levels, activation of mitogen-activated protein kinase cascade and finally stimulation of mineralization (10). Importantly, Rxfp2−/− mice showed decreased bone volume and altered trabecular organization at lumbar and femoral sites (9). In particular, the mineralizing surface was reduced in these mice and the bone formation rate was decreased. Interestingly, low levels of INSL3 are observed in conditions characterized by reduced Leydig cell function, such as hypogonadism, Klinefelter syndrome (KS), and aging (3, 4, 11), all of which are typically associated with an increased risk of bone defects and osteoporosis (12). In addition to its anabolic effect on osteoblast activity, INSL3 also positively modulates osteoclastogenesis through dose-dependent increase of macrophage colony-stimulating factor (10). Therefore, the role of INSL3 on bone has two faces: stimulating the bone-forming activity and influencing osteoclastogenesis. Surprisingly, to date, no effort has been made to elucidate any further role of INSL3 on the other bone cell populations. Osteocytes, which make up >95% of bone cells in the adult skeleton, are differentiated osteoblasts with no direct involvement in bone mineralization; however, being embedded in the growing matrix, they play a regulatory role over the other bone cell populations (13). The transition of osteoblasts toward an osteocyte phenotype involves changes in gene expression patterns, including the downregulation of alkaline phosphatase and induction of the SOST gene, which encodes the osteocyte-specific protein sclerostin (14). Sclerostin (SOST), an antagonist of Wnt/b-catenin signaling, is a glycoprotein with an important catabolic action on bone mass through a negative regulation of bone formation (15). In vivo evidence supports the catabolic role of SOST on bone. Sost−/− mice show increased bone mass (16), whereas several studies in humans and mice demonstrated that SOST expression was decreased in conditions that enhanced bone resorption, such as osteoporosis (17). Mechanisms involved in regulation of SOST include: mechanical stimulation, parathyroid hormone, prostaglandin E2, and interleukin-6 members (18–21). Given the emerging evidence linking testis with bone metabolism, our group has recently shown that testosterone downregulate SOST expression (22), adding further evidence to the documented role of androgens on bone mass by the means of a target cell population: osteocytes. On these bases, the aim of this study was to examine SOST levels in men with KS, who usually exhibit hypergonadotropic hypogonadism (23), lower circulating levels of INSL3 (4), and increased risk of osteoporosis (12). To this end, determinations of INSL3, SOST, and sex hormones levels, as well as bone and body composition parameters, were performed. Finally, RXFP2 expression was evaluated in primary osteocytes cultures and the effect of INSL3 administration on SOST expression was evaluated in these cells. Materials and Methods Subjects We retrospectively studied 103 nonmosaic KS patients (mean age, 31.8 ± 9.4 years), diagnosed at the Unit of Andrology and Reproductive Medicine at the University of Padova from January 2014 to June 2017, after referral for fertility problems or testicular hypotrophy. As controls, 60 age-matched healthy and fertile males (mean age, 33.3 ± 9.7 years) were recruited. The study was approved by the Hospital Ethics Committee, and each participant gave his written informed consent. All subjects (patients and controls) underwent peripheral karyotype analysis, evaluating at least 50 peripheral blood lymphocyte metaphases. Patients with more than one supernumerary X chromosome, mosaicisms, or with any endocrine dysfunction different from hypogonadism; any medical disorder of bone minerals, such as primary hyperparathyroidism or hypoparathyroidism; chronic alcoholism; chronic renal insufficiency; and subjects taking any medication known to affect calcium metabolism, such as calcium tablets, bisphosphonate, and corticosteroids, vitamin D supplementation, and testosterone replacement therapy were excluded from the study. Subjects’ evaluation included complete medical history (pubertal history, lifestyle, physical activity, smoking, alcohol misuse), physical examination, hemochrome, serum insulin, and prostate-specific antigen. Hypogonadism was defined as total testosterone <10.4 nmol/L. Measurement of bone densitometry was done by dual-energy X-ray absorptiometry using a Hologic QDR 4500 C densitometer (Hologic, Waltham, MA) in the femoral neck (FN) and lumbar spine (L1 to L4), by the same technician, and a spine phantom was used before each examination. The mean bone mineral density (BMD) index (total BMD coefficient of variation, 1%) and the mean T score were considered. T score was calculated as the number of standard deviations (SDs) that the BMD was above or below the mean for young healthy adults of the same race and sex. Low bone mass was defined based on T score (−2.5 SD < score < −1 SD for osteopenia; ≤ −2.5 for osteoporosis at any site) (24). Hormone determinations Fasting blood withdrawal was collected from each participant between 8:00 and 10:00 am. Serum follicle-stimulating hormone (FSH), LH, and total testosterone were evaluated by commercial electrochemiluminescence immunoassay methods (Elecsys 2010; Roche Diagnostics, Monza, Italy). Parathyroid hormone (PTH) serum levels were determined with a direct, two-site, sandwich-type chemiluminescent immunoassay (LIAISONN-TACTPTH; DiaSorin Inc., Stillwater, MN). 25-Hydroxyvitamin D [25(OH)D] was determined with a direct, competitive chemiluminescent immunoassay [LIAISON 25(OH)D total assay; DiaSorin Inc.] and were evaluated in the same period of the year (between November and March). Serum level of SOST was measured by the enzyme-linked immunosorbent assay quantikine enzyme-linked immunosorbent assay human SOST (R&D Systems, Minneapolis, MN), as previously described (25). For all parameters, the intra-assay and interassay coefficients of variation were at least <8% and 10%, respectively. Serum concentrations of INSL3 in all subjects were measured in duplicate by radioimmunoassay (Phoenix Pharmaceuticals, Burlingame, CA), as previously reported (3). Intra- and interassay coefficients of variation were <5% and 10%, respectively. The lower detection limit of the INSL3 radioimmunoassay kit, determined by the linear range of the standard curve, was established to be 1 pg/tube (10 pg/mL), and the range of the assay is 1 to 128 pg/tube (10 to 1280 pg/mL) (3). All determinations were performed according to the manufacturer’s instructions. Primary osteocyte isolation Human osteocyte cultures were obtained from the femoral head discards of four male subjects undergoing arthroplasty, who gave informed consent. Their use for in vitro scientific research does not require ethics approval from the institutional review board. Primary osteocytes were isolated from human bone as previously described (22, 26). Briefly, bone specimens were finely minced by mechanical grinding in sterile conditions and underwent two sequential 2-hour digestions and a third overnight digestion with type IA collagenase solution (300 U/mL; Sigma-Aldrich, Darmstadt, Germany) and sodium EDTA (5 mM, pH 7.4; Sigma-Aldrich), dissolved in α-MEM (Euroclone, Milano, Italy). After digestions, suspended cells were harvested, washed twice in α-MEM, and then incubated for 1 hour at 4°C with goat antihuman SOST and allophycocyanin-conjugated antihuman alkaline phosphatase (ALP) antibody (both from R&D Systems), followed by incubation with fluorescein isothiocyanate–conjugated donkey antigoat antibody (Santa Cruz Biotechnology, Dallas, TX). Labeled cell suspensions underwent fluorescent cell sorting by the use of the XDP cell sorter (Beckman Coulter, Brea, CA). SOST+/ALP− cells were collected and plated on six-well plates coated with type I rat tail collagen (BD Biosciences, Franklin Lakes, NJ) at a seeding density of ∼20,000 cells/well in α-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. Osteocytes hormonal stimulation in vitro For stimulation experiments, cells were starved in serum-free medium for 16 hours and then stimulated with human 10−7 M PTH (Sigma-Aldrich) and human INSL3 (Phoenix Pharmaceuticals, Burlingame, CA)(1 nM, 10 nM, and 100 nM) for 24 hours. The INSL3 concentrations used bracketed the dissociation constant of INSL3 for its receptor RXFP2 (27). After hormonal stimulation, cultured osteocytes underwent physical detachment by cell scraping. After centrifugation, cell pellet was collected and stored at −80°C for subsequent analysis. Immunofluorescence Bone fragments from femoral heads discards undergoing arthroplasty were fixed in 4% paraformaldehyde in phosphate-buffered saline (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. Sample fixation was performed as previously described (22). After saturation with 5% bovine serum albumin/5% normal donkey serum in PBS for 30 minutes, slides were incubated overnight at 4°C with rabbit antihuman RXFP2 (Antibodies Online, Atlanta, GA) 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 fluorescein isothiocyanate–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, Milano, Italy). 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 optical density at 280 nm with a Nanodrop ND-1000 spectrophotometer (Thermo Fisher, Waltham, MA). Samples were denatured with sodium dodecyl sulfate and 2-β-mercaptoethanol, boiled for 10 minutes, and then fractionated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (Bio-Rad Laboratories, Hercules, CA). After blotting onto a Hybond enhanced chemiluminescence nitrocellulose membrane (PerkinElmer, Waltham, MA) and blocking with 5% nonfat milk in 0.1% PBS-Tween 20 (Bio-Rad Laboratories), blots were incubated overnight at 4°C with the goat antihuman SOST antibody (R&D Systems) or rabbit antihuman RXFP2 (Antibodies Online) at the proper dilution in 5% nonfat milk in 0.1% PBS-Tween 20 buffer. Primary immunoreaction was detected by incubation with goat antirabbit or rabbit antigoat secondary antibodies (KPL) and visualized using an enhanced chemiluminescence reagent (LumiGLO; KPL-SeraCare, Milford, MA) with the Chemidoc XRS System (Bio-Rad Laboratories). β-Actin (sc-47778; Santa Cruz Biotechnology, Inc.) served as housekeeping. For each protein band, the pixel density 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 and the corresponding band density of β-actin, after subtraction of the background signal. Experiments were performed three times in triplicate. Quantitative reverse transcription polymerase chain reaction analysis RNA was extracted from stimulated osteocytes using the RNeasy microkit (QIAGEN, Hilden, Germany), and reverse transcription polymerase chain reaction (RT-PCR) was performed using total RNA (50 ng) and the reverse transcriptase Superscript III (Thermo Fisher Scientific) using random hexamers. The quality of RNA and complementary DNA obtained was tested by a spectrophotometric measurement (NanoDrop; Celbio, Milano, Italy). Quantitative RT-PCR was performed as previously described (22). Specific primers for human RXFP2 and SOST were as follows: RXFP2 forward, 5′-ACCGAGGGCAGTATCAGAAG-3′; RXFP2 reverse, 5′-AGGGGAAGACAATGACCAGG-3′; SOST forward, 5′-CGGAGCTGGAGAACAACAA-3′; SOST reverse, 5′-GGCAGCTGTACTCGGACAC-3′. β-Actin expression (forward, 5′-CACTCTTCCAGCCTTCCTTCC-3′; reverse, 5′-CGGACTCGTCATACTCCTGCTT-3′) was used as housekeeping gene. Experiments were performed three times in triplicate. Data elaboration was performed as relative quantification analysis using the ΔΔCt method. Statistical analyses All statistics were calculated using SPSS (version 23; SPSS Inc., Chicago, IL). P <0.05 was considered statistically significant. 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. Differences between groups or experimental conditions were analyzed using Student t test or analysis of variance for the comparison of multiple parameters. Pearson correlation analysis or Spearman correlation analysis for nonnormally distributed variables, with SOST as the variable of interest, was used to describe correlations between variables and to select principal independent variables for later use in multivariate analyses. Based on the correlation analyses, we then performed stepwise multivariate regression analysis to evaluate the impact of independent variables on SOST levels in the entire cohort. Significance level for entering and for removal of variables from the model was P < 0.05 and P < 0.10, respectively. Results Clinical characteristics of 60 control subjects and 103 KS men are reported in Table 1. With respect to controls, the whole KS group showed significantly lower INSL3, total testosterone, 25(OH)D, and lumbar and femoral BMD (Table 1), but significantly higher body mass index (BMI), LH, FSH, and estradiol (Table 1). Serum SOST levels were also higher in KS subjects than in healthy controls (P < 0.001; Fig. 1). KS subjects were further characterized on the basis of their T score at any site (L1 to L4 or FN), with 53 males (51.5%) having a T score > −1, whereas 50 (48.5%) subjects were diagnosed with osteopenia or osteoporosis (Table 1). Compared with subjects with normal dual-energy X-ray absorptiometry, patients with a T score < −1 had lower circulating levels of INSL3 (P = 0.029; Table 1) and higher SOST levels (P = 0.03; Fig. 1). Correlation coefficients between SOST and serum parameters in the entire cohort and in controls and KS separately were assessed and are reported in Table 2. In both controls and KS, either separately or together, serum SOST levels were negatively correlated with INSL3 and total testosterone and positively correlated with age and BMI (Table 2). In addition, in the entire cohort only, SOST was positively correlated with LH and estradiol and negatively correlated with 25(OH)D and BMD at both L1 to L4 and FN (Table 2). In the same bivariate correlation analysis, INSL3 levels were negatively correlated with age, body mass index, and LH (all P < 0.001) and positively correlated with total testosterone, 25(OH)D, and BMD in both groups (all P < 0.001). Based on correlation analyses, we then performed separate multiple linear stepwise regression analyses using the variables that proved significant in bivariate correlations as possible independent variables in predicting SOST levels. INSL3, testosterone, LH, and age were all independent variables accounting for 57.3% of the variance in SOST levels (Table 3). Table 1. Baseline, Clinical, and Hormonal Parameters in 103 KS Patients Compared With 60 Age-Matched Controls Variable Healthy Controls 
(N = 60) KS Patients (N = 103) All Patients T Score > −1 (N = 53) T Score < −1 (N = 50) Age, y 33.27 ± 9.69 31.80 ± 9.45 31.26 ± 10.38 32.36 ± 8.41 BMI, kg/m2 23.39 ± 2.86 25.10 ± 3.77a 25.44 ± 3.86 24.74 ± 3.68 INSL3, pg/mL 457.96 ± 178.81 225.05 ± 76.40b 240.96 ± 77.59 208.18 ± 72.09c SOST, pg/mL 80.94 ± 20.04 149.39 ± 45.50b 142.04 ± 37.81 159.47 ± 43.29c LH, IU/L 9.52 ± 5.62 19.60 ± 8.14b 18.90 ± 8.62 20.35 ± 7.61 FSH, IU/L 19.72 ± 10.45 35.76 ± 36.54b 35.82 ± 28.42 35.7 ± 17.07 Testosterone, nmol/L 14.07 ± 5.54 10.71 ± 5.48b 11.12 ± 5.72 10.28 ± 5.24 Estradiol, pmol/L 67.73 ± 29.27 101.33 ± 38.90b 104.26 ± 42.47 98.22 ± 34.89 Calcium, nmol/L 2.41 ± 0.08 2.42 ± 0.09 2.45 ± 0.72 2.44 ± 0.11 Phosphorus, nmol/L 0.95 ± 0.16 0.92 ± 0.20 0.91 ± 0.19 0.93 ± 0.21 25(OH)D, nmol/L 64.40 ± 31.59 53.70 ± 26.72a 58.47 ± 31.75 48.64 ± 19.12 PTH, ng/L 50.95 ± 27.39 53.20 ± 30.14 52.18 ± 29.90 54.28 ± 30.66 PSA, g/L 0.60 ± 0.34 0.65 ± 0.51 0.67 ± 0.51 0.63 ± 0.51 L1–L4 BMD, g/cm2 1.25 ± 0.12 1.00 ± 0.14a 1.11 ± 0.09 0.89 ± 0.09c Femoral BMD, g/cm2 1.28 ± 0.21 1.00 ± 0.13a 1.09 ± 0.10 0.91 ± 0.09c Variable Healthy Controls 
(N = 60) KS Patients (N = 103) All Patients T Score > −1 (N = 53) T Score < −1 (N = 50) Age, y 33.27 ± 9.69 31.80 ± 9.45 31.26 ± 10.38 32.36 ± 8.41 BMI, kg/m2 23.39 ± 2.86 25.10 ± 3.77a 25.44 ± 3.86 24.74 ± 3.68 INSL3, pg/mL 457.96 ± 178.81 225.05 ± 76.40b 240.96 ± 77.59 208.18 ± 72.09c SOST, pg/mL 80.94 ± 20.04 149.39 ± 45.50b 142.04 ± 37.81 159.47 ± 43.29c LH, IU/L 9.52 ± 5.62 19.60 ± 8.14b 18.90 ± 8.62 20.35 ± 7.61 FSH, IU/L 19.72 ± 10.45 35.76 ± 36.54b 35.82 ± 28.42 35.7 ± 17.07 Testosterone, nmol/L 14.07 ± 5.54 10.71 ± 5.48b 11.12 ± 5.72 10.28 ± 5.24 Estradiol, pmol/L 67.73 ± 29.27 101.33 ± 38.90b 104.26 ± 42.47 98.22 ± 34.89 Calcium, nmol/L 2.41 ± 0.08 2.42 ± 0.09 2.45 ± 0.72 2.44 ± 0.11 Phosphorus, nmol/L 0.95 ± 0.16 0.92 ± 0.20 0.91 ± 0.19 0.93 ± 0.21 25(OH)D, nmol/L 64.40 ± 31.59 53.70 ± 26.72a 58.47 ± 31.75 48.64 ± 19.12 PTH, ng/L 50.95 ± 27.39 53.20 ± 30.14 52.18 ± 29.90 54.28 ± 30.66 PSA, g/L 0.60 ± 0.34 0.65 ± 0.51 0.67 ± 0.51 0.63 ± 0.51 L1–L4 BMD, g/cm2 1.25 ± 0.12 1.00 ± 0.14a 1.11 ± 0.09 0.89 ± 0.09c Femoral BMD, g/cm2 1.28 ± 0.21 1.00 ± 0.13a 1.09 ± 0.10 0.91 ± 0.09c Data are expressed as means ± SD. Abbreviation: PSA, prostate-specific antigen. a P < 0.05 vs healthy controls. b P < 0.001 vs healthy controls. c P < 0.05 vs T score > −1. View Large Table 1. Baseline, Clinical, and Hormonal Parameters in 103 KS Patients Compared With 60 Age-Matched Controls Variable Healthy Controls 
(N = 60) KS Patients (N = 103) All Patients T Score > −1 (N = 53) T Score < −1 (N = 50) Age, y 33.27 ± 9.69 31.80 ± 9.45 31.26 ± 10.38 32.36 ± 8.41 BMI, kg/m2 23.39 ± 2.86 25.10 ± 3.77a 25.44 ± 3.86 24.74 ± 3.68 INSL3, pg/mL 457.96 ± 178.81 225.05 ± 76.40b 240.96 ± 77.59 208.18 ± 72.09c SOST, pg/mL 80.94 ± 20.04 149.39 ± 45.50b 142.04 ± 37.81 159.47 ± 43.29c LH, IU/L 9.52 ± 5.62 19.60 ± 8.14b 18.90 ± 8.62 20.35 ± 7.61 FSH, IU/L 19.72 ± 10.45 35.76 ± 36.54b 35.82 ± 28.42 35.7 ± 17.07 Testosterone, nmol/L 14.07 ± 5.54 10.71 ± 5.48b 11.12 ± 5.72 10.28 ± 5.24 Estradiol, pmol/L 67.73 ± 29.27 101.33 ± 38.90b 104.26 ± 42.47 98.22 ± 34.89 Calcium, nmol/L 2.41 ± 0.08 2.42 ± 0.09 2.45 ± 0.72 2.44 ± 0.11 Phosphorus, nmol/L 0.95 ± 0.16 0.92 ± 0.20 0.91 ± 0.19 0.93 ± 0.21 25(OH)D, nmol/L 64.40 ± 31.59 53.70 ± 26.72a 58.47 ± 31.75 48.64 ± 19.12 PTH, ng/L 50.95 ± 27.39 53.20 ± 30.14 52.18 ± 29.90 54.28 ± 30.66 PSA, g/L 0.60 ± 0.34 0.65 ± 0.51 0.67 ± 0.51 0.63 ± 0.51 L1–L4 BMD, g/cm2 1.25 ± 0.12 1.00 ± 0.14a 1.11 ± 0.09 0.89 ± 0.09c Femoral BMD, g/cm2 1.28 ± 0.21 1.00 ± 0.13a 1.09 ± 0.10 0.91 ± 0.09c Variable Healthy Controls 
(N = 60) KS Patients (N = 103) All Patients T Score > −1 (N = 53) T Score < −1 (N = 50) Age, y 33.27 ± 9.69 31.80 ± 9.45 31.26 ± 10.38 32.36 ± 8.41 BMI, kg/m2 23.39 ± 2.86 25.10 ± 3.77a 25.44 ± 3.86 24.74 ± 3.68 INSL3, pg/mL 457.96 ± 178.81 225.05 ± 76.40b 240.96 ± 77.59 208.18 ± 72.09c SOST, pg/mL 80.94 ± 20.04 149.39 ± 45.50b 142.04 ± 37.81 159.47 ± 43.29c LH, IU/L 9.52 ± 5.62 19.60 ± 8.14b 18.90 ± 8.62 20.35 ± 7.61 FSH, IU/L 19.72 ± 10.45 35.76 ± 36.54b 35.82 ± 28.42 35.7 ± 17.07 Testosterone, nmol/L 14.07 ± 5.54 10.71 ± 5.48b 11.12 ± 5.72 10.28 ± 5.24 Estradiol, pmol/L 67.73 ± 29.27 101.33 ± 38.90b 104.26 ± 42.47 98.22 ± 34.89 Calcium, nmol/L 2.41 ± 0.08 2.42 ± 0.09 2.45 ± 0.72 2.44 ± 0.11 Phosphorus, nmol/L 0.95 ± 0.16 0.92 ± 0.20 0.91 ± 0.19 0.93 ± 0.21 25(OH)D, nmol/L 64.40 ± 31.59 53.70 ± 26.72a 58.47 ± 31.75 48.64 ± 19.12 PTH, ng/L 50.95 ± 27.39 53.20 ± 30.14 52.18 ± 29.90 54.28 ± 30.66 PSA, g/L 0.60 ± 0.34 0.65 ± 0.51 0.67 ± 0.51 0.63 ± 0.51 L1–L4 BMD, g/cm2 1.25 ± 0.12 1.00 ± 0.14a 1.11 ± 0.09 0.89 ± 0.09c Femoral BMD, g/cm2 1.28 ± 0.21 1.00 ± 0.13a 1.09 ± 0.10 0.91 ± 0.09c Data are expressed as means ± SD. Abbreviation: PSA, prostate-specific antigen. a P < 0.05 vs healthy controls. b P < 0.001 vs healthy controls. c P < 0.05 vs T score > −1. View Large Figure 1. View largeDownload slide Difference in SOST serum levels measured in healthy controls (empty circles) and in KS patients with T score > −1 (blue circles) or T-score < −1 (red circles) at any site. Medians are indicated as horizontal bars. Level of statistical significance is indicated in the figure. Figure 1. View largeDownload slide Difference in SOST serum levels measured in healthy controls (empty circles) and in KS patients with T score > −1 (blue circles) or T-score < −1 (red circles) at any site. Medians are indicated as horizontal bars. Level of statistical significance is indicated in the figure. Table 2. Pearson or Spearman Correlation Coefficients Between SOST and Variables of Interest in the Entire Cohort and in Healthy Controls and KS Patients Separately All Subjects (N = 163) Healthy Controls (N = 60) KS Patients (N = 103) R P Value R P Value R P Value Age 0.286 <0.001 0.413 0.001 0.523 <0.001 BMI 0.440 <0.001 0.419 0.001 0.396 <0.001 INSL3 −0.639 <0.001 −0.359 0.005 −0.521 <0.001 LH 0.296 <0.001 0.047 0.729 −0.133 0.180 FSH 0.128 0.104 0.162 0.218 −0.088 0.377 Total testosterone −0.592 <0.001 −0.572 <0.001 −0.551 <0.001 Estradiol 0.218 0.005 −0.144 0.272 −0.069 0.491 Calcium 0.075 0.345 0.099 0.453 −0.141 0.155 Phosphorus −0.032 0.664 0.140 0.096 −0.110 0.268 25(OH)D −0.333 <0.001 −0.249 0.055 −0.338 <0.001 PTH 0.068 0.391 0.121 0.356 0.045 0.649 PSA −0.016 0.835 −0.008 0.952 −0.086 0.387 L1–L4 BMD −0.196 0.041 −0.178 0.071 −0.216 0.029 Femoral BMD −0.211 0.032 −0.185 0.066 −0.231 0.019 All Subjects (N = 163) Healthy Controls (N = 60) KS Patients (N = 103) R P Value R P Value R P Value Age 0.286 <0.001 0.413 0.001 0.523 <0.001 BMI 0.440 <0.001 0.419 0.001 0.396 <0.001 INSL3 −0.639 <0.001 −0.359 0.005 −0.521 <0.001 LH 0.296 <0.001 0.047 0.729 −0.133 0.180 FSH 0.128 0.104 0.162 0.218 −0.088 0.377 Total testosterone −0.592 <0.001 −0.572 <0.001 −0.551 <0.001 Estradiol 0.218 0.005 −0.144 0.272 −0.069 0.491 Calcium 0.075 0.345 0.099 0.453 −0.141 0.155 Phosphorus −0.032 0.664 0.140 0.096 −0.110 0.268 25(OH)D −0.333 <0.001 −0.249 0.055 −0.338 <0.001 PTH 0.068 0.391 0.121 0.356 0.045 0.649 PSA −0.016 0.835 −0.008 0.952 −0.086 0.387 L1–L4 BMD −0.196 0.041 −0.178 0.071 −0.216 0.029 Femoral BMD −0.211 0.032 −0.185 0.066 −0.231 0.019 Significant correlations are in boldface type. View Large Table 2. Pearson or Spearman Correlation Coefficients Between SOST and Variables of Interest in the Entire Cohort and in Healthy Controls and KS Patients Separately All Subjects (N = 163) Healthy Controls (N = 60) KS Patients (N = 103) R P Value R P Value R P Value Age 0.286 <0.001 0.413 0.001 0.523 <0.001 BMI 0.440 <0.001 0.419 0.001 0.396 <0.001 INSL3 −0.639 <0.001 −0.359 0.005 −0.521 <0.001 LH 0.296 <0.001 0.047 0.729 −0.133 0.180 FSH 0.128 0.104 0.162 0.218 −0.088 0.377 Total testosterone −0.592 <0.001 −0.572 <0.001 −0.551 <0.001 Estradiol 0.218 0.005 −0.144 0.272 −0.069 0.491 Calcium 0.075 0.345 0.099 0.453 −0.141 0.155 Phosphorus −0.032 0.664 0.140 0.096 −0.110 0.268 25(OH)D −0.333 <0.001 −0.249 0.055 −0.338 <0.001 PTH 0.068 0.391 0.121 0.356 0.045 0.649 PSA −0.016 0.835 −0.008 0.952 −0.086 0.387 L1–L4 BMD −0.196 0.041 −0.178 0.071 −0.216 0.029 Femoral BMD −0.211 0.032 −0.185 0.066 −0.231 0.019 All Subjects (N = 163) Healthy Controls (N = 60) KS Patients (N = 103) R P Value R P Value R P Value Age 0.286 <0.001 0.413 0.001 0.523 <0.001 BMI 0.440 <0.001 0.419 0.001 0.396 <0.001 INSL3 −0.639 <0.001 −0.359 0.005 −0.521 <0.001 LH 0.296 <0.001 0.047 0.729 −0.133 0.180 FSH 0.128 0.104 0.162 0.218 −0.088 0.377 Total testosterone −0.592 <0.001 −0.572 <0.001 −0.551 <0.001 Estradiol 0.218 0.005 −0.144 0.272 −0.069 0.491 Calcium 0.075 0.345 0.099 0.453 −0.141 0.155 Phosphorus −0.032 0.664 0.140 0.096 −0.110 0.268 25(OH)D −0.333 <0.001 −0.249 0.055 −0.338 <0.001 PTH 0.068 0.391 0.121 0.356 0.045 0.649 PSA −0.016 0.835 −0.008 0.952 −0.086 0.387 L1–L4 BMD −0.196 0.041 −0.178 0.071 −0.216 0.029 Femoral BMD −0.211 0.032 −0.185 0.066 −0.231 0.019 Significant correlations are in boldface type. View Large Table 3. Parameters Related to SOST Levels in the Entire Cohort (Multiple Stepwise Regression Analysis) Parameters in Final Model (R2 = 0.573) B SE β t P Value INSL3 −0.112 0.02 −0.37 −5.562 <0.001 Total testosterone −3.32 0.551 −0.376 −6.029 <0.001 LH 1.079 0.327 0.188 3.303 0.001 Age 0.628 0.307 0.119 2.043 0.043 Parameters in Final Model (R2 = 0.573) B SE β t P Value INSL3 −0.112 0.02 −0.37 −5.562 <0.001 Total testosterone −3.32 0.551 −0.376 −6.029 <0.001 LH 1.079 0.327 0.188 3.303 0.001 Age 0.628 0.307 0.119 2.043 0.043 Dependent variable: SOST; independent variables: age, BMI, INSL3, LH, testosterone, estradiol, and 25(OH)D. View Large Table 3. Parameters Related to SOST Levels in the Entire Cohort (Multiple Stepwise Regression Analysis) Parameters in Final Model (R2 = 0.573) B SE β t P Value INSL3 −0.112 0.02 −0.37 −5.562 <0.001 Total testosterone −3.32 0.551 −0.376 −6.029 <0.001 LH 1.079 0.327 0.188 3.303 0.001 Age 0.628 0.307 0.119 2.043 0.043 Parameters in Final Model (R2 = 0.573) B SE β t P Value INSL3 −0.112 0.02 −0.37 −5.562 <0.001 Total testosterone −3.32 0.551 −0.376 −6.029 <0.001 LH 1.079 0.327 0.188 3.303 0.001 Age 0.628 0.307 0.119 2.043 0.043 Dependent variable: SOST; independent variables: age, BMI, INSL3, LH, testosterone, estradiol, and 25(OH)D. View Large The expression of RXFP2 receptor in osteocytes was assessed in decalcified human trabecular bone samples (Fig. 2a and 2e) and in human primary osteocyte culture, obtained from digested bones specimens enriched in SOST+/ALP− cells by fluorescence-assisted cell sorting (Fig. 2b–2d and2f–h). Immunofluorescence staining featured by dendrite-like cell extension (bright-field images; Fig. 2b–2d), confirmed membrane RXFP2 expression in sorted osteocytes with positive cytoplasmic staining for SOST. SOST expression in sorted cells was further assessed by both RT-PCR and Western blot analysis (Fig. 3). Figure 2. View largeDownload slide Immunofluorescence analysis of RXFP2 (red) and SOST (green) expression in (a, e) decalcified human bone and in (b–h) isolated cultured human osteocytes. (d, h) In negative control samples, primary antibodies were omitted. (a–d) Bright-field images were captured to ascertain the bone localization of osteocytes and the shape of cultured cells. Figure 2. View largeDownload slide Immunofluorescence analysis of RXFP2 (red) and SOST (green) expression in (a, e) decalcified human bone and in (b–h) isolated cultured human osteocytes. (d, h) In negative control samples, primary antibodies were omitted. (a–d) Bright-field images were captured to ascertain the bone localization of osteocytes and the shape of cultured cells. Figure 3. View largeDownload slide Real-time PCR analysis of (a) Sost and Rxfp2 gene expression and (b, c) western blot analysis of SOST and RXFP2 protein expression in cultured human osteocytes after 24 hours of stimulation with PTH 10−7 M or INSL3 at concentrations ranging from 10−9 to 10−7 M, compared with CTRL. Gene expression data are reported with the ΔΔCt method referred to β-actin as the housekeeping gene and standardized to CTRL. Results are the mean of three independent experiments performed in triplicate. Protein expression data are reported as the ratio between the band density of SOST with the corresponding band density of β-actin (β-Act) as housekeeping. Results are the mean of three independent experiments performed in triplicate. *P < 0.01 vs CTRL. CTRL, unstimulated controls. Figure 3. View largeDownload slide Real-time PCR analysis of (a) Sost and Rxfp2 gene expression and (b, c) western blot analysis of SOST and RXFP2 protein expression in cultured human osteocytes after 24 hours of stimulation with PTH 10−7 M or INSL3 at concentrations ranging from 10−9 to 10−7 M, compared with CTRL. Gene expression data are reported with the ΔΔCt method referred to β-actin as the housekeeping gene and standardized to CTRL. Results are the mean of three independent experiments performed in triplicate. Protein expression data are reported as the ratio between the band density of SOST with the corresponding band density of β-actin (β-Act) as housekeeping. Results are the mean of three independent experiments performed in triplicate. *P < 0.01 vs CTRL. CTRL, unstimulated controls. To assess whether the expression of INSL3 receptor was linked also to a functional regulation of SOST production in osteocytes by INSL3, we stimulated primary cultured osteocytes with INSL3 for 24 hours, at concentrations ranging from 10−9 to 10−7 M, or with 10−7 M human PTH, a reference antagonist of SOST production (28). SOST messenger RNA (mRNA) and protein expressions were assessed, respectively, by RT-PCR and Western blot analysis. After 24 hours, both PTH and INSL3 at 10−7 M, but not at lower concentrations, led to a statistically significant decrease of both SOST mRNA (both P < 0.001 vs control, respectively; Fig. 3a) and protein expression (P = 0.026 and P = 0.004 vs control, respectively; Fig. 3b and 3c). Either PTH or INSL3 did not influence RXFP2 mRNA or protein expression (Fig. 3a–c). Discussion This study shows a strong inverse association between circulating levels of INSL3, a testicular hormone with an important involvement in sexual and bone development, and SOST, an osteocyte-specific protein that favors bone resorption, in a cohort of KS subjects and age-matched healthy men, with the former having significantly higher SOST levels than controls. Along with SOST, body mass index, LH, FSH, and estradiol were significantly increased in KS subjects, whereas testosterone, INSL3, and 25(OH)D were significantly reduced, compared with healthy controls. These hormonal alterations are a typical feature of the hypergonadotropic hypogonadism present in KS, and emphasize that not only steroidogenesis, but also global Leydig cell function is compromised in KS (4, 29). Moreover, in KS patients, SOST levels were negatively associated with BMD at both lumbar spine and FN, with a similar trend in healthy controls, although not statistically significant. Importantly, experimental data using primary cultured osteocytes showed that INSL3, through its specific receptor RXFP2, directly acts on this cell population by reducing SOST protein and gene expression, suggesting that the association between these two hormones may be causal. It should be noted, however, that the effective INSL3 concentration was supraphysiological, although within the dissociation curve range of INSL3 for its receptor RXFP2 (27). Serum SOST levels in our cohort were similar to those previously reported by our group in a population of hypogonadal and eugonadal men (22), in which we showed a negative correlation between testosterone and SOST. Along with INSL3 and testosterone, we also confirmed positive correlations between SOST with age (30) and BMI (17), which we reported in a cohort of KS subjects. When the KS group was split based on the T score, subjects with osteopenia or osteoporosis (T score < −1 at L1 to L4 or FN) had higher levels of SOST and correspondingly lower levels of INSL3, compared with KS subjects with T score > −1 at any site. This result confirms the well-established association of INSL3 with osteoporosis, and suggests a possible role for SOST as a marker of bone remodeling in KS, which is further supported by the negative correlation between SOST and BMD at either lumbar spine or FN in this group. These results are consistent with other studies in different cohorts, reporting associations between increased serum SOST levels with osteoporotic fractures and lower BMD in postmenopausal women and type 2 diabetes patients (17, 31, 32); however, reports on the association between SOST and BMD are inconsistent (33–35). Such discrepancies are most likely from methods used for SOST measurement and/or the selection criteria, as well as the mean age and sex of subjects, the size of the population (36), and the paracrine role of SOST on osteoblasts (37). Because SOST levels increase with age in both men and women (30), it was proposed that SOST levels reflect osteocyte number, which could explain the apparently paradoxical positive association between SOST and total body BMD in postmenopausal women (17). Other factors, including environmental or lifestyle and/or genetic influences acting on bone mass and consequently on SOST production, should not be excluded. In particular, the genetic background of KS subjects should be considered: KS is the most common sex chromosomal disorder and is typically associated with reduced bone mass and osteoporosis because of decreased pubertal peak of bone mass and accelerated bone loss during adulthood (8). Reduced bone mass in KS has been usually ascribed to low testosterone plasma level (39), but its pathogenic role is not well-established. In particular, the prevalence of low BMD is similar in KS men with low and normal T levels (38). Low INSL3 has been suggested as a possible mechanism contributing to bone defects in KS (12) because of its direct regulation of osteoblast maturation (10), mainly acting on trabecular bone component (9). In this framework, our study suggests that SOST is one of the possible pathways dysregulated in KS subjects characterized by low INSL3 levels; this result is further supported by in vitro evidence showing RXFP2 expression in human osteocytes and negative regulation of SOST by relevant INSL3 concentrations, in a manner similar to testosterone action on this cell population (22). Although preliminary, these results could be of importance in clinical practice. The role of SOST in the pathogenesis of bone loss and fracture risk is well-established: on the one hand, subjects with null mutations in the SOST gene and correspondingly undetectable serum SOST levels are identified by osteopetrosis and resistance to fractures, even after severe trauma (40). On the other hand, hypersclerostinemia is a typical feature of long-term immobilized patients characterized by reduced bone formation, suggesting that SOST is a link between mechanical unloading and osteoporosis (41). On these bases, SOST has become a pharmacological target by using specific antibodies to increase bone mass in osteoporotic patients, with successful results (42, 43). However, the association between SOST and INSL3 requires further investigation, possibly in larger cohorts and in different non-KS clinical and pathological conditions, such as male hypogonadism and aging, which are both associated with reduced INSL3 levels and increased risk of osteoporosis (3, 4, 11). Also, bone turnover markers and trabecular bone alterations should be evaluated to better understand the association between SOST and BMD. The results from this study cannot be extended to the general population, and the causal relationship between SOST and INSL3 is not conclusive: further molecular analysis is required to understand the signaling pathways involved in the regulation of SOST production by INSL3. In summary, in this study, we the association between sclerostin, an osteocyte-specific protein with important catabolic action on bone, and INSL3, a testicular hormone with anabolic effects on bone metabolism by its specific receptor RXFP2. In a cohort of KS males and age-matched controls, we found a negative association between SOST and INSL3; in KS subjects further characterized by bone densitometry, we found that SOST levels were negatively correlated with lumbar and femoral BMD and were increased in osteopenic/osteoporotic patients. 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Negative Association Between Sclerostin and INSL3 in Isolated Human Osteocytes and in Klinefelter Syndrome: New Hints for Testis–Bone Crosstalk

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Endocrine Society
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Copyright © 2018 Endocrine Society
ISSN
0021-972X
eISSN
1945-7197
D.O.I.
10.1210/jc.2017-02762
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Abstract

Abstract Context The regulation of bone mass by the testis is a well-recognized mechanism, but the role of Leydig-specific marker insulin-like 3 peptide (INSL3) on the most abundant bone cell population, osteocytes, is unknown. In this study, we aimed to investigate the relationship between INSL3 and sclerostin, an osteocyte-specific protein that negatively regulates bone formation. Design Serum sclerostin and INSL3 levels were evaluated in Klinefelter syndrome (KS) and healthy controls. In vitro effect of INSL3 on sclerostin production was evaluated in human cultured osteocytes. Patients A total of 103 KS patients and 60 age- and sex-matched controls were recruited. Main Outcome Measures Serum sclerostin and INSL3 levels were assessed by enzyme-linked immunosorbent assay. Osteocytes were isolated by fluorescence-assisted cell sorting. Sclerostin expression was evaluated by western blot, immunofluorescence, and reverse transcription polymerase chain reaction. Measurement of bone mineral density was done by dual-energy X-ray absorptiometry at lumbar spine (L1–L4) and femoral neck. Results Sclerostin levels were significantly increased in KS subjects, and negatively correlated with INSL3 levels in both cohorts and with bone mineral density in the KS group. Stimulation of cultured osteocytes with INSL3 at 10−7 M significantly decreased both sclerostin messenger RNA and protein expression. Conclusions We report a negative association between the testicular hormone INSL3 and the osteocytic negative regulator of bone formation, sclerostin. We further explored this association in vitro and showed that INSL3 was able to reduce sclerostin expression. These results add further knowledge on the emerging role of sclerostin as a therapeutic target for osteoporosis treatment. In recent years, increasing evidence has supported a crosstalk between testis and bone, by the means of the well-known action of testosterone on skeletal growth and bone mass (1). Nevertheless, testosterone replacement therapy does not completely restore bone mass in osteoporotic hypogonadal men (2), which suggests a more complex interaction between bone and testis. In addition to androgens, Leydig cells produce insulin-like 3 (INSL3), a peptide hormone with long-term regulation by luteinizing hormone (LH), which is used as an additional marker of Leydig cell function (3–6). The major endocrine role of INSL3 is related to the regulation of the transabdominal phase of testicular descent during fetal development by acting on gubernaculum through the activation of its specific relaxin family receptor 2 (RXFP2); indeed, mutations in INSL3 and RXFP2 genes lead to increased risk of cryptorchidism in both mice and men (7, 8). However, INSL3 has been shown to exert an anabolic effect on bone metabolism during adulthood by acting directly on osteoblasts (9, 10) through the activation of RXFP2 and subsequent rise in cyclic adenosine monophosphate levels, activation of mitogen-activated protein kinase cascade and finally stimulation of mineralization (10). Importantly, Rxfp2−/− mice showed decreased bone volume and altered trabecular organization at lumbar and femoral sites (9). In particular, the mineralizing surface was reduced in these mice and the bone formation rate was decreased. Interestingly, low levels of INSL3 are observed in conditions characterized by reduced Leydig cell function, such as hypogonadism, Klinefelter syndrome (KS), and aging (3, 4, 11), all of which are typically associated with an increased risk of bone defects and osteoporosis (12). In addition to its anabolic effect on osteoblast activity, INSL3 also positively modulates osteoclastogenesis through dose-dependent increase of macrophage colony-stimulating factor (10). Therefore, the role of INSL3 on bone has two faces: stimulating the bone-forming activity and influencing osteoclastogenesis. Surprisingly, to date, no effort has been made to elucidate any further role of INSL3 on the other bone cell populations. Osteocytes, which make up >95% of bone cells in the adult skeleton, are differentiated osteoblasts with no direct involvement in bone mineralization; however, being embedded in the growing matrix, they play a regulatory role over the other bone cell populations (13). The transition of osteoblasts toward an osteocyte phenotype involves changes in gene expression patterns, including the downregulation of alkaline phosphatase and induction of the SOST gene, which encodes the osteocyte-specific protein sclerostin (14). Sclerostin (SOST), an antagonist of Wnt/b-catenin signaling, is a glycoprotein with an important catabolic action on bone mass through a negative regulation of bone formation (15). In vivo evidence supports the catabolic role of SOST on bone. Sost−/− mice show increased bone mass (16), whereas several studies in humans and mice demonstrated that SOST expression was decreased in conditions that enhanced bone resorption, such as osteoporosis (17). Mechanisms involved in regulation of SOST include: mechanical stimulation, parathyroid hormone, prostaglandin E2, and interleukin-6 members (18–21). Given the emerging evidence linking testis with bone metabolism, our group has recently shown that testosterone downregulate SOST expression (22), adding further evidence to the documented role of androgens on bone mass by the means of a target cell population: osteocytes. On these bases, the aim of this study was to examine SOST levels in men with KS, who usually exhibit hypergonadotropic hypogonadism (23), lower circulating levels of INSL3 (4), and increased risk of osteoporosis (12). To this end, determinations of INSL3, SOST, and sex hormones levels, as well as bone and body composition parameters, were performed. Finally, RXFP2 expression was evaluated in primary osteocytes cultures and the effect of INSL3 administration on SOST expression was evaluated in these cells. Materials and Methods Subjects We retrospectively studied 103 nonmosaic KS patients (mean age, 31.8 ± 9.4 years), diagnosed at the Unit of Andrology and Reproductive Medicine at the University of Padova from January 2014 to June 2017, after referral for fertility problems or testicular hypotrophy. As controls, 60 age-matched healthy and fertile males (mean age, 33.3 ± 9.7 years) were recruited. The study was approved by the Hospital Ethics Committee, and each participant gave his written informed consent. All subjects (patients and controls) underwent peripheral karyotype analysis, evaluating at least 50 peripheral blood lymphocyte metaphases. Patients with more than one supernumerary X chromosome, mosaicisms, or with any endocrine dysfunction different from hypogonadism; any medical disorder of bone minerals, such as primary hyperparathyroidism or hypoparathyroidism; chronic alcoholism; chronic renal insufficiency; and subjects taking any medication known to affect calcium metabolism, such as calcium tablets, bisphosphonate, and corticosteroids, vitamin D supplementation, and testosterone replacement therapy were excluded from the study. Subjects’ evaluation included complete medical history (pubertal history, lifestyle, physical activity, smoking, alcohol misuse), physical examination, hemochrome, serum insulin, and prostate-specific antigen. Hypogonadism was defined as total testosterone <10.4 nmol/L. Measurement of bone densitometry was done by dual-energy X-ray absorptiometry using a Hologic QDR 4500 C densitometer (Hologic, Waltham, MA) in the femoral neck (FN) and lumbar spine (L1 to L4), by the same technician, and a spine phantom was used before each examination. The mean bone mineral density (BMD) index (total BMD coefficient of variation, 1%) and the mean T score were considered. T score was calculated as the number of standard deviations (SDs) that the BMD was above or below the mean for young healthy adults of the same race and sex. Low bone mass was defined based on T score (−2.5 SD < score < −1 SD for osteopenia; ≤ −2.5 for osteoporosis at any site) (24). Hormone determinations Fasting blood withdrawal was collected from each participant between 8:00 and 10:00 am. Serum follicle-stimulating hormone (FSH), LH, and total testosterone were evaluated by commercial electrochemiluminescence immunoassay methods (Elecsys 2010; Roche Diagnostics, Monza, Italy). Parathyroid hormone (PTH) serum levels were determined with a direct, two-site, sandwich-type chemiluminescent immunoassay (LIAISONN-TACTPTH; DiaSorin Inc., Stillwater, MN). 25-Hydroxyvitamin D [25(OH)D] was determined with a direct, competitive chemiluminescent immunoassay [LIAISON 25(OH)D total assay; DiaSorin Inc.] and were evaluated in the same period of the year (between November and March). Serum level of SOST was measured by the enzyme-linked immunosorbent assay quantikine enzyme-linked immunosorbent assay human SOST (R&D Systems, Minneapolis, MN), as previously described (25). For all parameters, the intra-assay and interassay coefficients of variation were at least <8% and 10%, respectively. Serum concentrations of INSL3 in all subjects were measured in duplicate by radioimmunoassay (Phoenix Pharmaceuticals, Burlingame, CA), as previously reported (3). Intra- and interassay coefficients of variation were <5% and 10%, respectively. The lower detection limit of the INSL3 radioimmunoassay kit, determined by the linear range of the standard curve, was established to be 1 pg/tube (10 pg/mL), and the range of the assay is 1 to 128 pg/tube (10 to 1280 pg/mL) (3). All determinations were performed according to the manufacturer’s instructions. Primary osteocyte isolation Human osteocyte cultures were obtained from the femoral head discards of four male subjects undergoing arthroplasty, who gave informed consent. Their use for in vitro scientific research does not require ethics approval from the institutional review board. Primary osteocytes were isolated from human bone as previously described (22, 26). Briefly, bone specimens were finely minced by mechanical grinding in sterile conditions and underwent two sequential 2-hour digestions and a third overnight digestion with type IA collagenase solution (300 U/mL; Sigma-Aldrich, Darmstadt, Germany) and sodium EDTA (5 mM, pH 7.4; Sigma-Aldrich), dissolved in α-MEM (Euroclone, Milano, Italy). After digestions, suspended cells were harvested, washed twice in α-MEM, and then incubated for 1 hour at 4°C with goat antihuman SOST and allophycocyanin-conjugated antihuman alkaline phosphatase (ALP) antibody (both from R&D Systems), followed by incubation with fluorescein isothiocyanate–conjugated donkey antigoat antibody (Santa Cruz Biotechnology, Dallas, TX). Labeled cell suspensions underwent fluorescent cell sorting by the use of the XDP cell sorter (Beckman Coulter, Brea, CA). SOST+/ALP− cells were collected and plated on six-well plates coated with type I rat tail collagen (BD Biosciences, Franklin Lakes, NJ) at a seeding density of ∼20,000 cells/well in α-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. Osteocytes hormonal stimulation in vitro For stimulation experiments, cells were starved in serum-free medium for 16 hours and then stimulated with human 10−7 M PTH (Sigma-Aldrich) and human INSL3 (Phoenix Pharmaceuticals, Burlingame, CA)(1 nM, 10 nM, and 100 nM) for 24 hours. The INSL3 concentrations used bracketed the dissociation constant of INSL3 for its receptor RXFP2 (27). After hormonal stimulation, cultured osteocytes underwent physical detachment by cell scraping. After centrifugation, cell pellet was collected and stored at −80°C for subsequent analysis. Immunofluorescence Bone fragments from femoral heads discards undergoing arthroplasty were fixed in 4% paraformaldehyde in phosphate-buffered saline (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. Sample fixation was performed as previously described (22). After saturation with 5% bovine serum albumin/5% normal donkey serum in PBS for 30 minutes, slides were incubated overnight at 4°C with rabbit antihuman RXFP2 (Antibodies Online, Atlanta, GA) 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 fluorescein isothiocyanate–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, Milano, Italy). 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 optical density at 280 nm with a Nanodrop ND-1000 spectrophotometer (Thermo Fisher, Waltham, MA). Samples were denatured with sodium dodecyl sulfate and 2-β-mercaptoethanol, boiled for 10 minutes, and then fractionated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (Bio-Rad Laboratories, Hercules, CA). After blotting onto a Hybond enhanced chemiluminescence nitrocellulose membrane (PerkinElmer, Waltham, MA) and blocking with 5% nonfat milk in 0.1% PBS-Tween 20 (Bio-Rad Laboratories), blots were incubated overnight at 4°C with the goat antihuman SOST antibody (R&D Systems) or rabbit antihuman RXFP2 (Antibodies Online) at the proper dilution in 5% nonfat milk in 0.1% PBS-Tween 20 buffer. Primary immunoreaction was detected by incubation with goat antirabbit or rabbit antigoat secondary antibodies (KPL) and visualized using an enhanced chemiluminescence reagent (LumiGLO; KPL-SeraCare, Milford, MA) with the Chemidoc XRS System (Bio-Rad Laboratories). β-Actin (sc-47778; Santa Cruz Biotechnology, Inc.) served as housekeeping. For each protein band, the pixel density 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 and the corresponding band density of β-actin, after subtraction of the background signal. Experiments were performed three times in triplicate. Quantitative reverse transcription polymerase chain reaction analysis RNA was extracted from stimulated osteocytes using the RNeasy microkit (QIAGEN, Hilden, Germany), and reverse transcription polymerase chain reaction (RT-PCR) was performed using total RNA (50 ng) and the reverse transcriptase Superscript III (Thermo Fisher Scientific) using random hexamers. The quality of RNA and complementary DNA obtained was tested by a spectrophotometric measurement (NanoDrop; Celbio, Milano, Italy). Quantitative RT-PCR was performed as previously described (22). Specific primers for human RXFP2 and SOST were as follows: RXFP2 forward, 5′-ACCGAGGGCAGTATCAGAAG-3′; RXFP2 reverse, 5′-AGGGGAAGACAATGACCAGG-3′; SOST forward, 5′-CGGAGCTGGAGAACAACAA-3′; SOST reverse, 5′-GGCAGCTGTACTCGGACAC-3′. β-Actin expression (forward, 5′-CACTCTTCCAGCCTTCCTTCC-3′; reverse, 5′-CGGACTCGTCATACTCCTGCTT-3′) was used as housekeeping gene. Experiments were performed three times in triplicate. Data elaboration was performed as relative quantification analysis using the ΔΔCt method. Statistical analyses All statistics were calculated using SPSS (version 23; SPSS Inc., Chicago, IL). P <0.05 was considered statistically significant. 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. Differences between groups or experimental conditions were analyzed using Student t test or analysis of variance for the comparison of multiple parameters. Pearson correlation analysis or Spearman correlation analysis for nonnormally distributed variables, with SOST as the variable of interest, was used to describe correlations between variables and to select principal independent variables for later use in multivariate analyses. Based on the correlation analyses, we then performed stepwise multivariate regression analysis to evaluate the impact of independent variables on SOST levels in the entire cohort. Significance level for entering and for removal of variables from the model was P < 0.05 and P < 0.10, respectively. Results Clinical characteristics of 60 control subjects and 103 KS men are reported in Table 1. With respect to controls, the whole KS group showed significantly lower INSL3, total testosterone, 25(OH)D, and lumbar and femoral BMD (Table 1), but significantly higher body mass index (BMI), LH, FSH, and estradiol (Table 1). Serum SOST levels were also higher in KS subjects than in healthy controls (P < 0.001; Fig. 1). KS subjects were further characterized on the basis of their T score at any site (L1 to L4 or FN), with 53 males (51.5%) having a T score > −1, whereas 50 (48.5%) subjects were diagnosed with osteopenia or osteoporosis (Table 1). Compared with subjects with normal dual-energy X-ray absorptiometry, patients with a T score < −1 had lower circulating levels of INSL3 (P = 0.029; Table 1) and higher SOST levels (P = 0.03; Fig. 1). Correlation coefficients between SOST and serum parameters in the entire cohort and in controls and KS separately were assessed and are reported in Table 2. In both controls and KS, either separately or together, serum SOST levels were negatively correlated with INSL3 and total testosterone and positively correlated with age and BMI (Table 2). In addition, in the entire cohort only, SOST was positively correlated with LH and estradiol and negatively correlated with 25(OH)D and BMD at both L1 to L4 and FN (Table 2). In the same bivariate correlation analysis, INSL3 levels were negatively correlated with age, body mass index, and LH (all P < 0.001) and positively correlated with total testosterone, 25(OH)D, and BMD in both groups (all P < 0.001). Based on correlation analyses, we then performed separate multiple linear stepwise regression analyses using the variables that proved significant in bivariate correlations as possible independent variables in predicting SOST levels. INSL3, testosterone, LH, and age were all independent variables accounting for 57.3% of the variance in SOST levels (Table 3). Table 1. Baseline, Clinical, and Hormonal Parameters in 103 KS Patients Compared With 60 Age-Matched Controls Variable Healthy Controls 
(N = 60) KS Patients (N = 103) All Patients T Score > −1 (N = 53) T Score < −1 (N = 50) Age, y 33.27 ± 9.69 31.80 ± 9.45 31.26 ± 10.38 32.36 ± 8.41 BMI, kg/m2 23.39 ± 2.86 25.10 ± 3.77a 25.44 ± 3.86 24.74 ± 3.68 INSL3, pg/mL 457.96 ± 178.81 225.05 ± 76.40b 240.96 ± 77.59 208.18 ± 72.09c SOST, pg/mL 80.94 ± 20.04 149.39 ± 45.50b 142.04 ± 37.81 159.47 ± 43.29c LH, IU/L 9.52 ± 5.62 19.60 ± 8.14b 18.90 ± 8.62 20.35 ± 7.61 FSH, IU/L 19.72 ± 10.45 35.76 ± 36.54b 35.82 ± 28.42 35.7 ± 17.07 Testosterone, nmol/L 14.07 ± 5.54 10.71 ± 5.48b 11.12 ± 5.72 10.28 ± 5.24 Estradiol, pmol/L 67.73 ± 29.27 101.33 ± 38.90b 104.26 ± 42.47 98.22 ± 34.89 Calcium, nmol/L 2.41 ± 0.08 2.42 ± 0.09 2.45 ± 0.72 2.44 ± 0.11 Phosphorus, nmol/L 0.95 ± 0.16 0.92 ± 0.20 0.91 ± 0.19 0.93 ± 0.21 25(OH)D, nmol/L 64.40 ± 31.59 53.70 ± 26.72a 58.47 ± 31.75 48.64 ± 19.12 PTH, ng/L 50.95 ± 27.39 53.20 ± 30.14 52.18 ± 29.90 54.28 ± 30.66 PSA, g/L 0.60 ± 0.34 0.65 ± 0.51 0.67 ± 0.51 0.63 ± 0.51 L1–L4 BMD, g/cm2 1.25 ± 0.12 1.00 ± 0.14a 1.11 ± 0.09 0.89 ± 0.09c Femoral BMD, g/cm2 1.28 ± 0.21 1.00 ± 0.13a 1.09 ± 0.10 0.91 ± 0.09c Variable Healthy Controls 
(N = 60) KS Patients (N = 103) All Patients T Score > −1 (N = 53) T Score < −1 (N = 50) Age, y 33.27 ± 9.69 31.80 ± 9.45 31.26 ± 10.38 32.36 ± 8.41 BMI, kg/m2 23.39 ± 2.86 25.10 ± 3.77a 25.44 ± 3.86 24.74 ± 3.68 INSL3, pg/mL 457.96 ± 178.81 225.05 ± 76.40b 240.96 ± 77.59 208.18 ± 72.09c SOST, pg/mL 80.94 ± 20.04 149.39 ± 45.50b 142.04 ± 37.81 159.47 ± 43.29c LH, IU/L 9.52 ± 5.62 19.60 ± 8.14b 18.90 ± 8.62 20.35 ± 7.61 FSH, IU/L 19.72 ± 10.45 35.76 ± 36.54b 35.82 ± 28.42 35.7 ± 17.07 Testosterone, nmol/L 14.07 ± 5.54 10.71 ± 5.48b 11.12 ± 5.72 10.28 ± 5.24 Estradiol, pmol/L 67.73 ± 29.27 101.33 ± 38.90b 104.26 ± 42.47 98.22 ± 34.89 Calcium, nmol/L 2.41 ± 0.08 2.42 ± 0.09 2.45 ± 0.72 2.44 ± 0.11 Phosphorus, nmol/L 0.95 ± 0.16 0.92 ± 0.20 0.91 ± 0.19 0.93 ± 0.21 25(OH)D, nmol/L 64.40 ± 31.59 53.70 ± 26.72a 58.47 ± 31.75 48.64 ± 19.12 PTH, ng/L 50.95 ± 27.39 53.20 ± 30.14 52.18 ± 29.90 54.28 ± 30.66 PSA, g/L 0.60 ± 0.34 0.65 ± 0.51 0.67 ± 0.51 0.63 ± 0.51 L1–L4 BMD, g/cm2 1.25 ± 0.12 1.00 ± 0.14a 1.11 ± 0.09 0.89 ± 0.09c Femoral BMD, g/cm2 1.28 ± 0.21 1.00 ± 0.13a 1.09 ± 0.10 0.91 ± 0.09c Data are expressed as means ± SD. Abbreviation: PSA, prostate-specific antigen. a P < 0.05 vs healthy controls. b P < 0.001 vs healthy controls. c P < 0.05 vs T score > −1. View Large Table 1. Baseline, Clinical, and Hormonal Parameters in 103 KS Patients Compared With 60 Age-Matched Controls Variable Healthy Controls 
(N = 60) KS Patients (N = 103) All Patients T Score > −1 (N = 53) T Score < −1 (N = 50) Age, y 33.27 ± 9.69 31.80 ± 9.45 31.26 ± 10.38 32.36 ± 8.41 BMI, kg/m2 23.39 ± 2.86 25.10 ± 3.77a 25.44 ± 3.86 24.74 ± 3.68 INSL3, pg/mL 457.96 ± 178.81 225.05 ± 76.40b 240.96 ± 77.59 208.18 ± 72.09c SOST, pg/mL 80.94 ± 20.04 149.39 ± 45.50b 142.04 ± 37.81 159.47 ± 43.29c LH, IU/L 9.52 ± 5.62 19.60 ± 8.14b 18.90 ± 8.62 20.35 ± 7.61 FSH, IU/L 19.72 ± 10.45 35.76 ± 36.54b 35.82 ± 28.42 35.7 ± 17.07 Testosterone, nmol/L 14.07 ± 5.54 10.71 ± 5.48b 11.12 ± 5.72 10.28 ± 5.24 Estradiol, pmol/L 67.73 ± 29.27 101.33 ± 38.90b 104.26 ± 42.47 98.22 ± 34.89 Calcium, nmol/L 2.41 ± 0.08 2.42 ± 0.09 2.45 ± 0.72 2.44 ± 0.11 Phosphorus, nmol/L 0.95 ± 0.16 0.92 ± 0.20 0.91 ± 0.19 0.93 ± 0.21 25(OH)D, nmol/L 64.40 ± 31.59 53.70 ± 26.72a 58.47 ± 31.75 48.64 ± 19.12 PTH, ng/L 50.95 ± 27.39 53.20 ± 30.14 52.18 ± 29.90 54.28 ± 30.66 PSA, g/L 0.60 ± 0.34 0.65 ± 0.51 0.67 ± 0.51 0.63 ± 0.51 L1–L4 BMD, g/cm2 1.25 ± 0.12 1.00 ± 0.14a 1.11 ± 0.09 0.89 ± 0.09c Femoral BMD, g/cm2 1.28 ± 0.21 1.00 ± 0.13a 1.09 ± 0.10 0.91 ± 0.09c Variable Healthy Controls 
(N = 60) KS Patients (N = 103) All Patients T Score > −1 (N = 53) T Score < −1 (N = 50) Age, y 33.27 ± 9.69 31.80 ± 9.45 31.26 ± 10.38 32.36 ± 8.41 BMI, kg/m2 23.39 ± 2.86 25.10 ± 3.77a 25.44 ± 3.86 24.74 ± 3.68 INSL3, pg/mL 457.96 ± 178.81 225.05 ± 76.40b 240.96 ± 77.59 208.18 ± 72.09c SOST, pg/mL 80.94 ± 20.04 149.39 ± 45.50b 142.04 ± 37.81 159.47 ± 43.29c LH, IU/L 9.52 ± 5.62 19.60 ± 8.14b 18.90 ± 8.62 20.35 ± 7.61 FSH, IU/L 19.72 ± 10.45 35.76 ± 36.54b 35.82 ± 28.42 35.7 ± 17.07 Testosterone, nmol/L 14.07 ± 5.54 10.71 ± 5.48b 11.12 ± 5.72 10.28 ± 5.24 Estradiol, pmol/L 67.73 ± 29.27 101.33 ± 38.90b 104.26 ± 42.47 98.22 ± 34.89 Calcium, nmol/L 2.41 ± 0.08 2.42 ± 0.09 2.45 ± 0.72 2.44 ± 0.11 Phosphorus, nmol/L 0.95 ± 0.16 0.92 ± 0.20 0.91 ± 0.19 0.93 ± 0.21 25(OH)D, nmol/L 64.40 ± 31.59 53.70 ± 26.72a 58.47 ± 31.75 48.64 ± 19.12 PTH, ng/L 50.95 ± 27.39 53.20 ± 30.14 52.18 ± 29.90 54.28 ± 30.66 PSA, g/L 0.60 ± 0.34 0.65 ± 0.51 0.67 ± 0.51 0.63 ± 0.51 L1–L4 BMD, g/cm2 1.25 ± 0.12 1.00 ± 0.14a 1.11 ± 0.09 0.89 ± 0.09c Femoral BMD, g/cm2 1.28 ± 0.21 1.00 ± 0.13a 1.09 ± 0.10 0.91 ± 0.09c Data are expressed as means ± SD. Abbreviation: PSA, prostate-specific antigen. a P < 0.05 vs healthy controls. b P < 0.001 vs healthy controls. c P < 0.05 vs T score > −1. View Large Figure 1. View largeDownload slide Difference in SOST serum levels measured in healthy controls (empty circles) and in KS patients with T score > −1 (blue circles) or T-score < −1 (red circles) at any site. Medians are indicated as horizontal bars. Level of statistical significance is indicated in the figure. Figure 1. View largeDownload slide Difference in SOST serum levels measured in healthy controls (empty circles) and in KS patients with T score > −1 (blue circles) or T-score < −1 (red circles) at any site. Medians are indicated as horizontal bars. Level of statistical significance is indicated in the figure. Table 2. Pearson or Spearman Correlation Coefficients Between SOST and Variables of Interest in the Entire Cohort and in Healthy Controls and KS Patients Separately All Subjects (N = 163) Healthy Controls (N = 60) KS Patients (N = 103) R P Value R P Value R P Value Age 0.286 <0.001 0.413 0.001 0.523 <0.001 BMI 0.440 <0.001 0.419 0.001 0.396 <0.001 INSL3 −0.639 <0.001 −0.359 0.005 −0.521 <0.001 LH 0.296 <0.001 0.047 0.729 −0.133 0.180 FSH 0.128 0.104 0.162 0.218 −0.088 0.377 Total testosterone −0.592 <0.001 −0.572 <0.001 −0.551 <0.001 Estradiol 0.218 0.005 −0.144 0.272 −0.069 0.491 Calcium 0.075 0.345 0.099 0.453 −0.141 0.155 Phosphorus −0.032 0.664 0.140 0.096 −0.110 0.268 25(OH)D −0.333 <0.001 −0.249 0.055 −0.338 <0.001 PTH 0.068 0.391 0.121 0.356 0.045 0.649 PSA −0.016 0.835 −0.008 0.952 −0.086 0.387 L1–L4 BMD −0.196 0.041 −0.178 0.071 −0.216 0.029 Femoral BMD −0.211 0.032 −0.185 0.066 −0.231 0.019 All Subjects (N = 163) Healthy Controls (N = 60) KS Patients (N = 103) R P Value R P Value R P Value Age 0.286 <0.001 0.413 0.001 0.523 <0.001 BMI 0.440 <0.001 0.419 0.001 0.396 <0.001 INSL3 −0.639 <0.001 −0.359 0.005 −0.521 <0.001 LH 0.296 <0.001 0.047 0.729 −0.133 0.180 FSH 0.128 0.104 0.162 0.218 −0.088 0.377 Total testosterone −0.592 <0.001 −0.572 <0.001 −0.551 <0.001 Estradiol 0.218 0.005 −0.144 0.272 −0.069 0.491 Calcium 0.075 0.345 0.099 0.453 −0.141 0.155 Phosphorus −0.032 0.664 0.140 0.096 −0.110 0.268 25(OH)D −0.333 <0.001 −0.249 0.055 −0.338 <0.001 PTH 0.068 0.391 0.121 0.356 0.045 0.649 PSA −0.016 0.835 −0.008 0.952 −0.086 0.387 L1–L4 BMD −0.196 0.041 −0.178 0.071 −0.216 0.029 Femoral BMD −0.211 0.032 −0.185 0.066 −0.231 0.019 Significant correlations are in boldface type. View Large Table 2. Pearson or Spearman Correlation Coefficients Between SOST and Variables of Interest in the Entire Cohort and in Healthy Controls and KS Patients Separately All Subjects (N = 163) Healthy Controls (N = 60) KS Patients (N = 103) R P Value R P Value R P Value Age 0.286 <0.001 0.413 0.001 0.523 <0.001 BMI 0.440 <0.001 0.419 0.001 0.396 <0.001 INSL3 −0.639 <0.001 −0.359 0.005 −0.521 <0.001 LH 0.296 <0.001 0.047 0.729 −0.133 0.180 FSH 0.128 0.104 0.162 0.218 −0.088 0.377 Total testosterone −0.592 <0.001 −0.572 <0.001 −0.551 <0.001 Estradiol 0.218 0.005 −0.144 0.272 −0.069 0.491 Calcium 0.075 0.345 0.099 0.453 −0.141 0.155 Phosphorus −0.032 0.664 0.140 0.096 −0.110 0.268 25(OH)D −0.333 <0.001 −0.249 0.055 −0.338 <0.001 PTH 0.068 0.391 0.121 0.356 0.045 0.649 PSA −0.016 0.835 −0.008 0.952 −0.086 0.387 L1–L4 BMD −0.196 0.041 −0.178 0.071 −0.216 0.029 Femoral BMD −0.211 0.032 −0.185 0.066 −0.231 0.019 All Subjects (N = 163) Healthy Controls (N = 60) KS Patients (N = 103) R P Value R P Value R P Value Age 0.286 <0.001 0.413 0.001 0.523 <0.001 BMI 0.440 <0.001 0.419 0.001 0.396 <0.001 INSL3 −0.639 <0.001 −0.359 0.005 −0.521 <0.001 LH 0.296 <0.001 0.047 0.729 −0.133 0.180 FSH 0.128 0.104 0.162 0.218 −0.088 0.377 Total testosterone −0.592 <0.001 −0.572 <0.001 −0.551 <0.001 Estradiol 0.218 0.005 −0.144 0.272 −0.069 0.491 Calcium 0.075 0.345 0.099 0.453 −0.141 0.155 Phosphorus −0.032 0.664 0.140 0.096 −0.110 0.268 25(OH)D −0.333 <0.001 −0.249 0.055 −0.338 <0.001 PTH 0.068 0.391 0.121 0.356 0.045 0.649 PSA −0.016 0.835 −0.008 0.952 −0.086 0.387 L1–L4 BMD −0.196 0.041 −0.178 0.071 −0.216 0.029 Femoral BMD −0.211 0.032 −0.185 0.066 −0.231 0.019 Significant correlations are in boldface type. View Large Table 3. Parameters Related to SOST Levels in the Entire Cohort (Multiple Stepwise Regression Analysis) Parameters in Final Model (R2 = 0.573) B SE β t P Value INSL3 −0.112 0.02 −0.37 −5.562 <0.001 Total testosterone −3.32 0.551 −0.376 −6.029 <0.001 LH 1.079 0.327 0.188 3.303 0.001 Age 0.628 0.307 0.119 2.043 0.043 Parameters in Final Model (R2 = 0.573) B SE β t P Value INSL3 −0.112 0.02 −0.37 −5.562 <0.001 Total testosterone −3.32 0.551 −0.376 −6.029 <0.001 LH 1.079 0.327 0.188 3.303 0.001 Age 0.628 0.307 0.119 2.043 0.043 Dependent variable: SOST; independent variables: age, BMI, INSL3, LH, testosterone, estradiol, and 25(OH)D. View Large Table 3. Parameters Related to SOST Levels in the Entire Cohort (Multiple Stepwise Regression Analysis) Parameters in Final Model (R2 = 0.573) B SE β t P Value INSL3 −0.112 0.02 −0.37 −5.562 <0.001 Total testosterone −3.32 0.551 −0.376 −6.029 <0.001 LH 1.079 0.327 0.188 3.303 0.001 Age 0.628 0.307 0.119 2.043 0.043 Parameters in Final Model (R2 = 0.573) B SE β t P Value INSL3 −0.112 0.02 −0.37 −5.562 <0.001 Total testosterone −3.32 0.551 −0.376 −6.029 <0.001 LH 1.079 0.327 0.188 3.303 0.001 Age 0.628 0.307 0.119 2.043 0.043 Dependent variable: SOST; independent variables: age, BMI, INSL3, LH, testosterone, estradiol, and 25(OH)D. View Large The expression of RXFP2 receptor in osteocytes was assessed in decalcified human trabecular bone samples (Fig. 2a and 2e) and in human primary osteocyte culture, obtained from digested bones specimens enriched in SOST+/ALP− cells by fluorescence-assisted cell sorting (Fig. 2b–2d and2f–h). Immunofluorescence staining featured by dendrite-like cell extension (bright-field images; Fig. 2b–2d), confirmed membrane RXFP2 expression in sorted osteocytes with positive cytoplasmic staining for SOST. SOST expression in sorted cells was further assessed by both RT-PCR and Western blot analysis (Fig. 3). Figure 2. View largeDownload slide Immunofluorescence analysis of RXFP2 (red) and SOST (green) expression in (a, e) decalcified human bone and in (b–h) isolated cultured human osteocytes. (d, h) In negative control samples, primary antibodies were omitted. (a–d) Bright-field images were captured to ascertain the bone localization of osteocytes and the shape of cultured cells. Figure 2. View largeDownload slide Immunofluorescence analysis of RXFP2 (red) and SOST (green) expression in (a, e) decalcified human bone and in (b–h) isolated cultured human osteocytes. (d, h) In negative control samples, primary antibodies were omitted. (a–d) Bright-field images were captured to ascertain the bone localization of osteocytes and the shape of cultured cells. Figure 3. View largeDownload slide Real-time PCR analysis of (a) Sost and Rxfp2 gene expression and (b, c) western blot analysis of SOST and RXFP2 protein expression in cultured human osteocytes after 24 hours of stimulation with PTH 10−7 M or INSL3 at concentrations ranging from 10−9 to 10−7 M, compared with CTRL. Gene expression data are reported with the ΔΔCt method referred to β-actin as the housekeeping gene and standardized to CTRL. Results are the mean of three independent experiments performed in triplicate. Protein expression data are reported as the ratio between the band density of SOST with the corresponding band density of β-actin (β-Act) as housekeeping. Results are the mean of three independent experiments performed in triplicate. *P < 0.01 vs CTRL. CTRL, unstimulated controls. Figure 3. View largeDownload slide Real-time PCR analysis of (a) Sost and Rxfp2 gene expression and (b, c) western blot analysis of SOST and RXFP2 protein expression in cultured human osteocytes after 24 hours of stimulation with PTH 10−7 M or INSL3 at concentrations ranging from 10−9 to 10−7 M, compared with CTRL. Gene expression data are reported with the ΔΔCt method referred to β-actin as the housekeeping gene and standardized to CTRL. Results are the mean of three independent experiments performed in triplicate. Protein expression data are reported as the ratio between the band density of SOST with the corresponding band density of β-actin (β-Act) as housekeeping. Results are the mean of three independent experiments performed in triplicate. *P < 0.01 vs CTRL. CTRL, unstimulated controls. To assess whether the expression of INSL3 receptor was linked also to a functional regulation of SOST production in osteocytes by INSL3, we stimulated primary cultured osteocytes with INSL3 for 24 hours, at concentrations ranging from 10−9 to 10−7 M, or with 10−7 M human PTH, a reference antagonist of SOST production (28). SOST messenger RNA (mRNA) and protein expressions were assessed, respectively, by RT-PCR and Western blot analysis. After 24 hours, both PTH and INSL3 at 10−7 M, but not at lower concentrations, led to a statistically significant decrease of both SOST mRNA (both P < 0.001 vs control, respectively; Fig. 3a) and protein expression (P = 0.026 and P = 0.004 vs control, respectively; Fig. 3b and 3c). Either PTH or INSL3 did not influence RXFP2 mRNA or protein expression (Fig. 3a–c). Discussion This study shows a strong inverse association between circulating levels of INSL3, a testicular hormone with an important involvement in sexual and bone development, and SOST, an osteocyte-specific protein that favors bone resorption, in a cohort of KS subjects and age-matched healthy men, with the former having significantly higher SOST levels than controls. Along with SOST, body mass index, LH, FSH, and estradiol were significantly increased in KS subjects, whereas testosterone, INSL3, and 25(OH)D were significantly reduced, compared with healthy controls. These hormonal alterations are a typical feature of the hypergonadotropic hypogonadism present in KS, and emphasize that not only steroidogenesis, but also global Leydig cell function is compromised in KS (4, 29). Moreover, in KS patients, SOST levels were negatively associated with BMD at both lumbar spine and FN, with a similar trend in healthy controls, although not statistically significant. Importantly, experimental data using primary cultured osteocytes showed that INSL3, through its specific receptor RXFP2, directly acts on this cell population by reducing SOST protein and gene expression, suggesting that the association between these two hormones may be causal. It should be noted, however, that the effective INSL3 concentration was supraphysiological, although within the dissociation curve range of INSL3 for its receptor RXFP2 (27). Serum SOST levels in our cohort were similar to those previously reported by our group in a population of hypogonadal and eugonadal men (22), in which we showed a negative correlation between testosterone and SOST. Along with INSL3 and testosterone, we also confirmed positive correlations between SOST with age (30) and BMI (17), which we reported in a cohort of KS subjects. When the KS group was split based on the T score, subjects with osteopenia or osteoporosis (T score < −1 at L1 to L4 or FN) had higher levels of SOST and correspondingly lower levels of INSL3, compared with KS subjects with T score > −1 at any site. This result confirms the well-established association of INSL3 with osteoporosis, and suggests a possible role for SOST as a marker of bone remodeling in KS, which is further supported by the negative correlation between SOST and BMD at either lumbar spine or FN in this group. These results are consistent with other studies in different cohorts, reporting associations between increased serum SOST levels with osteoporotic fractures and lower BMD in postmenopausal women and type 2 diabetes patients (17, 31, 32); however, reports on the association between SOST and BMD are inconsistent (33–35). Such discrepancies are most likely from methods used for SOST measurement and/or the selection criteria, as well as the mean age and sex of subjects, the size of the population (36), and the paracrine role of SOST on osteoblasts (37). Because SOST levels increase with age in both men and women (30), it was proposed that SOST levels reflect osteocyte number, which could explain the apparently paradoxical positive association between SOST and total body BMD in postmenopausal women (17). Other factors, including environmental or lifestyle and/or genetic influences acting on bone mass and consequently on SOST production, should not be excluded. In particular, the genetic background of KS subjects should be considered: KS is the most common sex chromosomal disorder and is typically associated with reduced bone mass and osteoporosis because of decreased pubertal peak of bone mass and accelerated bone loss during adulthood (8). Reduced bone mass in KS has been usually ascribed to low testosterone plasma level (39), but its pathogenic role is not well-established. In particular, the prevalence of low BMD is similar in KS men with low and normal T levels (38). Low INSL3 has been suggested as a possible mechanism contributing to bone defects in KS (12) because of its direct regulation of osteoblast maturation (10), mainly acting on trabecular bone component (9). In this framework, our study suggests that SOST is one of the possible pathways dysregulated in KS subjects characterized by low INSL3 levels; this result is further supported by in vitro evidence showing RXFP2 expression in human osteocytes and negative regulation of SOST by relevant INSL3 concentrations, in a manner similar to testosterone action on this cell population (22). Although preliminary, these results could be of importance in clinical practice. The role of SOST in the pathogenesis of bone loss and fracture risk is well-established: on the one hand, subjects with null mutations in the SOST gene and correspondingly undetectable serum SOST levels are identified by osteopetrosis and resistance to fractures, even after severe trauma (40). On the other hand, hypersclerostinemia is a typical feature of long-term immobilized patients characterized by reduced bone formation, suggesting that SOST is a link between mechanical unloading and osteoporosis (41). On these bases, SOST has become a pharmacological target by using specific antibodies to increase bone mass in osteoporotic patients, with successful results (42, 43). However, the association between SOST and INSL3 requires further investigation, possibly in larger cohorts and in different non-KS clinical and pathological conditions, such as male hypogonadism and aging, which are both associated with reduced INSL3 levels and increased risk of osteoporosis (3, 4, 11). Also, bone turnover markers and trabecular bone alterations should be evaluated to better understand the association between SOST and BMD. The results from this study cannot be extended to the general population, and the causal relationship between SOST and INSL3 is not conclusive: further molecular analysis is required to understand the signaling pathways involved in the regulation of SOST production by INSL3. In summary, in this study, we the association between sclerostin, an osteocyte-specific protein with important catabolic action on bone, and INSL3, a testicular hormone with anabolic effects on bone metabolism by its specific receptor RXFP2. In a cohort of KS males and age-matched controls, we found a negative association between SOST and INSL3; in KS subjects further characterized by bone densitometry, we found that SOST levels were negatively correlated with lumbar and femoral BMD and were increased in osteopenic/osteoporotic patients. 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Journal of Clinical Endocrinology and MetabolismOxford University Press

Published: Feb 14, 2018

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