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Original Articles |
INSERM, U-403, Hopital Edouard Herriot (P.S., P.D.D.), 69437 Lyon, France; Division of Gastroenterology, Endocrinology, and Metabolism, Philipps University (L.C.H., A.E.H.), 35033 Marburg, Germany; and Immundiagnostik (S.R.), 64625 Bensheim, Germany
Address all correspondence and requests for reprints to: Pierre D. Delmas, M.D., Ph.D., INSERM, U-403, Hopital Edouard Herriot, place d’Arsonval 3, 69437 Lyon, France. E-mail: delmas{at}lyon151.inserm.fr
Abstract
Previous studies have suggested an important role for androgens and estrogens in bone metabolism in men. However, their local mode of action has not been clearly established. Osteoprotegerin (OPG) is a secreted decoy receptor that inhibits osteoclast formation and activity by neutralizing its cognate ligand. To assess the role of OPG on bone metabolism in men, we conducted a study aimed at evaluating OPG serum levels and their correlation with age, bone mineral density, biochemical markers of bone turnover, and testosterone and estradiol levels in 252 men, aged 19–85 yr. Serum concentrations of OPG increased significantly with age (r = 0.41; P = 0.0001), and were positively correlated with free testosterone index and free estradiol index (r = 0.20; P < 0.002 and r = 0.15; P < 0.03, respectively) after adjustment for age and body weight. Beyond the age of 40 yr, OPG serum concentrations were negatively correlated with urinary excretion of total deoxypyridinoline (r = -0.20; P < 0.01) and PTH serum levels (r = -0.23; P < 0.01). In contrast, there was no correlation with biochemical markers of bone formation, 25-hydroxyvitamin D3 levels, or bone mineral density at any site. Our data reveal that age as well as androgen and estrogen status are significant positive determinants, whereas PTH is a negative determinant, of OPG serum levels in men. These data suggest that OPG may be an important paracrine mediator of bone metabolism in elderly men and highlight the role of estrogens in the homeostasis of the male skeleton.
FOR MANY YEARS, osteoporosis in women has received much attention, whereas scientific and public interest in male osteoporosis has only recently emerged (1, 2, 3). Androgen deficiency has been implicated as a major cause of osteoporosis in men; however, recent epidemiological studies have highlighted the importance of estrogens in regulating bone homeostasis in men. Bone mineral density (BMD) and biochemical markers of bone metabolism were found to better correlate with 17ß-estradiol, rather than testosterone, levels (4, 5, 6). In addition, male subjects with an inactivating gene mutation of the estrogen receptor and of the enzyme aromatase (which converts testosterone into 17ß-estradiol) displayed low BMD, and those with aromatase gene mutations showed marked improvement after treatment with 17ß-estradiol (7, 8, 9). The protective and antiresorptive effects of androgens and estrogens on bone metabolism are thought to be mediated by modulation of stromal cell- and osteoblast-derived cytokines, including interleukin-1ß, interleukin-6, insulin-like growth factors and their binding proteins, PGE2, and transforming growth factor-ß, which regulate osteoclast formation and activity in a paracrine fashion (10).
Recently, osteoprotegerin (OPG), a secreted member of the tumor
necrosis factor receptor superfamily, has been identified as an
osteoblast-derived regulator of bone resorption and bone mass and has
been implicated in the pathogenesis of postmenopausal osteoporosis and
other metabolic bone diseases (11, 12). OPG acts by
neutralizing the receptor activator of nuclear factor-
B ligand
(RANKL), an essential cytokine required for osteoclast formation and
activation (13). Although the emerging knowledge of the
biological effects of the RANKL/OPG system derived from in
vitro and animal studies has changed the paradigm of osteoclast
biology and has generated new insights into the molecular and cellular
basis of various metabolic bone diseases (12, 14), the
contribution of OPG to the pathogenesis of bone diseases in humans has
remained incompletely understood.
Studies designed to assess OPG serum levels in aging women and men with or without osteoporosis have yielded contradictory results (15, 16). Our study was aimed at evaluating OPG serum levels and their correlation with age, BMD, biochemical markers of bone turnover, bioavailable testosterone, and estrogen levels in a well characterized cohort of men, aged 19–85 yr (17).
Materials and Methods
Study cohort
The MINOS project was initiated in 1995 as a prospective study of osteoporosis and its determinants in men. The cohort is composed of 934 healthy men, aged 19–85 yr, without metabolic bone disease or intake of drugs known to affect bone metabolism. The MINOS study cohort has been used for cross-sectional assessment of age-related bone loss in men (17). For the present study we randomly selected an age-stratified sample subset of 252 men.
Laboratory analyses
Fasting serum as well as 24-h urine samples were collected and stored at -80 C until assayed. Serum concentrations of biochemical markers of bone formation were measured as previously described in detail (18). Osteocalcin (OC) was determined using the ELISA-OSTEO from CIS BioInternational (Bagnols/Cèze, France), bone alkaline phosphatase (BAP) using Alkphase-B from Metra Biosystems (Mountain View, CA), and N-terminal extension propeptide of type I collagen (PINP) using Intact PINP from Farmos Diagnostica (Uppsala, Sweden). Urinary excretion of total and free deoxypyridinoline (DPD) was measured using the Pyrilinks-D system from Metra Biosystems (Mountain View, CA) (18).
Total 17ß-estradiol and total testosterone were measured by tritiated RIA after diethyl ether extraction; sex hormone-binding globulin (SHBG) was measured by immunoradiometric assay ([125I]SBP Coatria, Bio-Mérieux, Marcy l’Etoile, France); free testosterone was measured by RIA (Coat-A-Count, Behring, Marburg, Germany), and androstenedione was measured by tritiated RIA after diethyl ether extraction (18). The free testosterone index (FTI) was calculated as the total testosterone to SHBG ratio. The free 17ß-estradiol index (FEI) was calculated as the total 17ß-estradiol to SHBG ratio. Serum levels of 25-hydrocholecalciferol (25OHD) were measured by an RIA (INCSTAR Corp., Stillwater, MN) that excludes any interference from lipids, and serum PTH concentrations were measured by immunochemiluminometric assay (Magic Lite, Ciba Corning, Inc., Medfield, MA) (19).
OPG concentrations were determined using a highly sensitive, commercial sandwich enzyme immunoassay provided by Immundiagnostik (Bensheim, Germany). Measurements were performed in undiluted samples according to the manufacturer’s instructions. In this assay two highly specific antibodies against human OPG are used. As a capture antibody, a monoclonal IgG antibody is used that had been raised from a murine hybridoma cell line after immunizing a mouse with recombinant human OPG (rhOPG). The resulting antibody is able to neutralize the biological activity of rhOPG. The detection antibody is a biotin-labeled polyclonal antihuman OPG antibody derived from a goat after immunization with rhOPG. The lower limit of detection of this assay is 4 pg/mL. The intraassay (n = 16) CV is between 8–10%, and the interassay (n = 7) CV is between 12–15%. Standard curves were generated using rhOPG purchased from Research Diagnostics (Flanders, NJ).
BMD measurement
Bone mineral density (BMD) was measured at the lumbar spine, right hip, and whole body using pencil beam, dual energy x-ray absorptiometry (QDR-1500, Hologic, Inc., Waltham, MA) and at the distal and ultradistal nondominant forearm using single energy x-ray absorptiometry (Osteometer DTX 100; Osteometer MediTech A/S, Horsholm, Denmark). Details concerning measurement and quality assurance have been recently described (17).
Statistical analysis
All analyses were performed using SAS software (SAS Institute, Inc., Cary, NC). Correlations were calculated using the parametric Pearson’s correlation coefficient (for the cohort as a whole) and nonparametric Spearman’s correlation coefficients (for 10-yr age groups). Unless otherwise stated, all values are presented as the mean ± SD. P < 0.05 was considered statistically significant.
Results
Descriptive statistics of 252 men selected for this study are
presented in Table 1
. As a similar number
of probands was selected per decade, young men were overrepresented.
Men in the selected cohort were slightly younger than the men who were
not selected (56 ± 18 vs. 59 ± 13 yr;
P < 0.05). Consequently, their body weight was
slightly lower (78 ± 11 vs. 80 ± 13 kg;
P < 0.03) and their body height was slightly higher
(171 ± 7 vs. 170 ± 7 cm; P <
0.05). After adjustment for these confounding variables, BMD, levels of
biochemical markers of bone turnover, and levels of hormones involved
in bone metabolism in the selected cohort did not differ significantly
from those in the participants who were not selected.
|
). SD calculated
in 10-yr age groups increased with age (r = 0.83;
P < 0.05), suggesting an age-related increase in the
variability in serum OPG concentrations. Serum OPG concentrations were
not correlated with body weight, body height, lean body mass, or fat
mass (all P > 0.2). Partial age- and body
weight-adjusted correlation coefficients between OPG serum levels and
BMD were not statistically significant for any of the sites
measured.
|
). By contrast, OPG serum levels
were not correlated with any of the biochemical markers of bone
formation (OC, BAP, and PINP). BMD and biochemical bone markers did not
differ in elderly men (>50 yr) who had elevated OPG levels (mean
± 2 SD in young men, >73 pg/mL) and those who
had OPG levels comparable to those in young men (<73 pg/mL).
|
After 40 yr of age, OPG serum levels were negatively correlated with PTH (after adjustment for age and body weight: r = -0.23; P < 0.01), but not with 25-hydroxyvitamin D3 or serum creatinine levels.
Discussion
Using a subset of 252 healthy men with an age range of 19–85 yr from the MINOS study cohort, we report an age-dependent increase in circulating serum concentrations of the antiresorptive decoy receptor OPG. Serum OPG levels increase in men by age 40–45 yr, at a time in life when peak bone mass has been achieved and when age-dependent bone loss begins. Our findings are consistent with a previous study from Japan conducted in 56 men and 186 postmenopausal women, in which OPG serum concentrations were positively correlated with age in either gender (15). Our results also confirm the observation that OPG serum concentrations in men below 45 yr of age display a low variability, whereas greater variability was observed in older men (15). The increase in the antiresorptive factor OPG with aging (which is associated with an increase in bone resorption) may reflect a protective mechanism of the skeleton to compensate for increased bone resorption and bone loss. In fact, in the above-mentioned study, serum OPG concentrations were higher in postmenopausal women with a high rate of bone turnover than in those with those with a low rate of bone turnover and were substantially higher in postmenopausal women with the most severe degrees of osteoporosis (15). The researchers hypothesized that the increase in OPG may be only in part capable of counteracting the increased rate of bone resorption and bone loss associated with aging (15). By contrast, a study in the U.S. has failed to confirm these data (16). Moreover, an in vitro study using quantitative competitive RT-PCR revealed that OPG transcripts in bone marrow samples (of 18 subjects undergoing trauma or corrective surgery) decreased in an age-dependent fashion (20), indicating that circulating OPG levels may only in part reflect the local milieu within the bone/bone marrow microenvironment. Because OPG is synthesized by various skeletal and extraskeletal tissues and cell types and is regulated by a variety of osteotropic and nonosteotropic hormones and cytokines (11, 14), our measurements of serum OPG concentrations may have underestimated the changes in local OPG production within the confines of bone. These limitations, however, are inherent to all studies that assess circulating concentrations of paracrine cytokines.
Our study is the first to demonstrate a positive correlation of serum OPG concentrations with bioavailable testosterone (as determined by the free testosterone index) and estradiol levels (as determined by the free estradiol index) after adjustment for age and body weight in men. Arrighi et al. reported that women have slightly higher serum concentrations of OPG than men (16), and that OPG serum concentrations in women were positively correlated with 17ß-estradiol serum concentrations (21). The fact that 17ß-estradiol increases OPG messenger ribonucleic acid steady state levels and protein production in a human estrogen-responsive osteoblastic cell line supports these findings (22). In addition, estrogen replacement therapy increased OPG messenger ribonucleic acid steady state levels in stromal cells of postmenopausal women (20), and parenteral administration of OPG prevented increased bone turnover in postmenopausal women (23). Our data suggest that OPG may be involved in mediating the skeletal actions of testosterone and 17ß-estradiol in elderly men. As testosterone may be converted to 17ß-estradiol by the enzyme aromatase in osteoblasts (2), estrogens may play a key role in bone homeostasis in men. Differences in the absolute serum OPG concentrations between this assay and others (15, 16, 21, 24) could be due to the highly specific and sensitive nature of our assay, because our assay is characterized by a lower detection limit (4 pg/mL).
Although OPG serum levels were positively correlated with age and bioavailable sex steroid hormones, there was a negative correlation with serum PTH concentrations after adjustment for age and body weight in men beyond the age of 40 yr. This inverse correlation between PTH and OPG is consistent with the known suppressive effect of PTH on OPG production in vitro and in vivo (24, 25, 26). PTH decreased OPG production and increased RANKL expression in murine stromal cells (26). Moreover, after systemic administration, PTH inhibited the local production of OPG in the bone of rats (25) and circulating OPG levels in postmenopausal women (24). Thus, inhibition of systemic OPG levels by inappropriately high PTH concentrations (e.g. during secondary hyperparathyroidism due to malnutrition, vitamin D deficiency, or sex hormone deficiency) may contribute to bone loss in aging men.
Beyond age 40 yr, serum OPG levels were negatively correlated with urinary DPD excretion. Negative age- and body weight-adjusted correlation between serum OPG levels and markers of bone resorption suggests a role of OPG as a regulator of bone resorption in aging men. This had been previously suggested for postmenopausal women (23) and ovariectomized rats (11), where parenteral administration of OPG reduced biochemical markers of bone resorption. Our findings do not support the data of Yano et al., who reported positive correlations of serum OPG concentrations with the levels of biochemical markers of bone turnover (OC, BAP, and DPD) (15). However, there are substantial differences between their and our present study in that the findings were derived from women (compared with men), and that simple positive correlations (compared with partial correlation coefficients) were presented (15).
Our data showing a lack of correlation of OPG serum levels and BMD at any site of measurement in men are in contrast with those of Yano et al. (15) and Arrighi et al. (21), both of whom reported a negative correlation of OPG serum concentrations with BMD of lumbar spine, femoral neck, and total body in postmenopausal women (15, 21). By contrast, in experiments on rodents, administration of OPG clearly enhanced BMD (11, 27). However, BMD in adult humans critically depends on peak BMD and the rate of subsequent bone loss, both of which are determined by a variety of genetic and environmental factors not by a single cytokine. A lack of correlation between BMD and OPG serum concentrations was also described by Browner et al. in postmenopausal women (28).
In conclusion, we report an age-related increase in circulating OPG concentrations in men. The significant positive correlations of OPG serum levels with bioavailable testosterone and estrogen levels as well as the negative correlations with PTH serum levels and urinary excretion of DPD suggest that OPG may act as an important paracrine mediator of bone metabolism in elderly men. Our study also underlines the importance of estrogens for bone homeostasis in men.
Acknowledgments
We acknowledge the technical assistance of Ms. Manuela Kauss in performing the OPG assay.
Footnotes
1 This work was supported by Grant 1875/2-1 from Deutsche
Forschungsgemeinschaft (Bonn, Germany; to L.C.H.) and a contract from
INSERM (to P.D.D.). 
Received January 5, 2001.
Revised March 12, 2001.
Accepted March 13, 2001.
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