Regulation of Sclerostin Production in Human Male Osteocytes by Androgens: Experimental and Clinical Evidence
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 (
). 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 (
) and decreased peak bone mass
associated with prepubertal hypogonadism (
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
). 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 (
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 (
). AR expression has been shown in a
number of bone cells including pluripotent mesenchymal
bone marrow stromal cells, osteoblasts, osteoclasts, and
). 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,
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
). Interestingly, the highest expression of AR has
been observed in osteocytes (
), which make up greater
than 95% of bone cells in the adult skeleton, with this ratio
increasing with age and bone size (
). 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 (
). 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, 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
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
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 (
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 (
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
AR gene silencing was performed by the use of human AR
small interfering RNA (siRNA) kit (
) (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.
Goat; polyclonal 1:100 for IHC,
1:500 for WB
Mouse; monoclonal Undiluted
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
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
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 (
). 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).
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,
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 (
). 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 (
), 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).
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.
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 (
) 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 (
), 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
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).
Androgens play a key role in the
maintenance of male skeletal
integrity through the classical AR
signaling acting on osteoblasts (
recently, selective inactivation of the
AR in osteocytes of male mice has
been shown to accelerate age-related
deterioration of skeletal integrity
), 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 (
). 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 (
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 (
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
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 (
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
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 (
). Interestingly, the
SOST upstream regulatory region shows a core consensus
recognition site for forkhead box A1 (
) and, to this
regard, polymorphisms in this region have been associated
with higher risk of osteoporosis in Chinese and Caucasian
). 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 (
) 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 (
). 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 (
). A clear
inverse correlation between T and SOST has also been
Clinical and Biochemical Data of CTRL and HP
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 (
Mödder et al (
) have shown that androgen administration to
human males did not alter circulating SOST levels. Further
studies are necessary to address these apparent
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 (
). 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 (
). In fact, Sinnesael et al (
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 (
), 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.
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.
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