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Physiology and Biophysics (D.S.P., S.E.B., B.W., L.J.S., D.G.), Center for Orthopaedic Research, Orthopaedic Surgery (L.J.S., D.G.), University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205; and Mayo Clinic and Foundation (S.J.A., S.K.), Rochester, Minnesota 55905
Address all correspondence and requests for reprints to: Dana Gaddy, Ph.D., Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, 4301 West Markham, Slot 505, Little Rock, Arkansas 72205. E-mail: gaddydana{at}uams.edu.
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Abstract |
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Objective: The objective of this study was to determine whether decreased serum inhibin A (InhA) and InhB levels correlate with increases in markers of bone turnover in women across the menopause transition and to evaluate serum inhibins as better predictors of bone turnover markers across the menopause transition than FSH or bioavailable E2.
Design: We studied a cross-sectional age-stratified population sample of 188 pre- and postmenopausal women not using oral contraceptives or hormone replacement therapy (age, 21–85 yr).
Results: Serum InhA and InhB levels significantly correlated inversely with markers of bone formation and bone resorption in pre- and perimenopausal women and with markers of bone formation in postmenopausal women (InhA only). FSH was not significantly correlated with bone turnover in either pre- or postmenopausal women; however, FSH was significantly correlated with bone resorption (C-terminal collagen I cross-link) in perimenopausal women (age, 45–54 yr). Using multivariate analyses, serum InhA better predicted bone formation and resorption markers in premenopausal women than either FSH or bioavailable E2.
Conclusions: Decreases in inhibin levels across the menopause transition are associated with increasing bone turnover, regardless of changes in sex steroids or FSH.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Estrogen deficiency has been identified as a major risk factor for osteoporosis in women (1, 5, 6). Recent evidence suggests that estrogen deficiency may be responsible, not only for the rapid bone loss of the early postmenopausal phase, but may also be involved in the later slower phase of bone loss associated with aging (5, 7, 8). However, in late premenopausal women with normal circulating estrogen levels, clinical indices of increased bone turnover are already elevated (9). In fact, the endocrine parameter best correlated with increases in bone turnover in a large cohort of perimenopausal women is elevated serum FSH levels (9). Studies in perimenopausal women have demonstrated that the mechanism involved in this early rise in FSH is a selective decrease in inhibin B (InhB) secretion in the presence of normal levels of estradiol (E2), inhibin A (InhA), GnRH, and LH (10, 11). Because both InhA and InhB isoforms selectively inhibit pituitary FSH secretion, these data suggest that the increased FSH is the result of a loss in feedback inhibition by gonadal InhB in perimenopausal women, resulting in increased bone turnover and bone loss, before the loss of sex steroids. As the loss of gonadal function proceeds in postmenopausal women, the well-established decreases in estrogen accompany the already declining levels of both InhB and InhA, further increasing serum FSH (10) and markedly increasing bone loss (9, 12).
The gonadal proteins InhB and InhA are heterodimeric proteins in the TGFß superfamily composed of 
ßB or ßA subunits, respectively, originally identified based upon their ability to suppress pituitary FSH secretion (13). Although inhibin -subunit expression (required for inhibin dimer formation) is very low in human and rat bone marrow (14, 15), inhibin accumulates in the bone marrow of 25-d-old rats within 10 min of iv injection of [125I]-InhA and is retained for at least 1 h (16). These results support the idea that the effects of inhibin on bone marrow cell differentiation (17, 18, 19, 20) are due to gonadally derived inhibin (21).
In support of this hypothesis, our previous results in murine bone marrow cultures demonstrated that inhibins suppress both osteoblast and osteoclast development (20). Together, these data led us to hypothesize that changes in gonadal inhibin production may directly alter osteoblast and osteoclast development, thereby altering bone turnover. To test this hypothesis, the current study was designed to determine what clinical correlations exist between InhA and/or InhB with systemic markers of bone turnover and then to compare the relative strength of these correlations with those of FSH and E2 with bone turnover in women across the menopause transition.
We determined the levels of circulating InhA and InhB in a well-characterized population-based, age-stratified sample of women, ages 25–80, who were not exposed to either oral contraceptives or estrogen replacement therapy (ERT; n = 188) (7, 8, 22). We then assessed the age-related changes in markers of bone turnover in these women and related these to levels of InhA, InhB, and FSH. The relative importance of InhA and InhB in determining bone turnover in women by multivariate analysis compared with bioavailable E2 (BioE2), previously demonstrated to be a strong predictor of bone turnover markers in this cohort (7), and FSH, previously demonstrated to be a strong predictor of bone turnover markers in a larger cohort of perimenopausal women (9), was also assessed.
In addition, we determined whether inhibins have direct effects on human osteoblast and osteoclast development in vitro, using human mesenchymal stem cells (HMSCs) and peripheral blood mononuclear cells (PBMCs). The results described here demonstrate that significant correlations exist between inhibins and systemic markers of bone turnover. These observations suggest that changes in inhibins, independent of changes in either BioE2 or FSH, significantly contribute to the alterations in bone turnover across the menopause transition and in premenopausal women.
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Subjects and Methods |
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Subjects were recruited from an age-stratified random sample of Rochester, Minnesota, men and women that were selected using the medical records linkage system of the Rochester Epidemiology Project (23). Over half of the Rochester population is identified annually in this system, and the majority of them are seen in any 3-yr period. Thus, the enumerated population approximates the underlying population of the community, including both free-living and institutionalized individuals. Altogether, 938 women aged 20 yr and over were approached, but 126 women were ineligible (89 were demented, 11 were pregnant, nine were radiation workers, eight were participants in an ongoing clinical trial of osteoporosis prophylaxis, and nine died before they could be contacted). Of the 812 eligible women, 351 participated and provided full bone turnover marker data. To exclude the possible influence of exogenous estrogens on inhibin levels, as well as independent estrogen effects on bone turnover, women on birth control pills (BCPs) or ERT were excluded from inhibin analysis. Thus, the remaining 107 premenopausal (defined as having experienced menses any time in the previous 12 months) and 81 postmenopausal women were included in this analysis. All but two women were Caucasian, reflecting the ethnic composition of the population (96% white in 1990). The women ranged in age from 21–85 yr.
Laboratory methods
Fasting serum samples were assayed by RIA for total testosterone (T) [Diagnostic Products Corp., Los Angeles, CA; interassay coefficient of variation (CV), 11%], E2 (Diagnostic Systems Laboratories, Webster, TX; interassay CV, 11%), estrone (E1) (Diagnostic Systems Laboratories; interassay CV, 9%), and SHBG (Wien Laboratories, Succasunna, NJ; interassay CV, 7%). In addition, the non-SHBG-bound (bioavailable) fractions of total T, E2, and E1 were measured as previously described (7). Briefly, tracer amounts of 3H-labeled T, E2, or E1 were added to serum aliquots. An equal volume of saturated solution of ammonium sulfate (final concentration, 50%) was added to precipitate SHBG with its bound steroid. Separation of the SHBG fraction was performed by centrifugation at 1100 x g for 30 min at 4 C. The percentage of labeled steroid remaining in the supernatant (the free and albumin-bound fractions) was then calculated. The bioavailable steroid concentration was obtained by multiplying the total steroid concentration, determined by RIA, by the fraction that was non-SHBG bound. Free T was measured by RIA using a T analog with low affinity for SHBG and albumin (Diagnostic Systems Laboratories; interassay CV, 10%). Serum LH and FSH were measured by immunoradiometric assays (Diagnostic Products Corp.; interassay CV, 14% for LH and 11% for FSH). InhA and InhB levels were measured using two site-specific ELISAs for each hormone (Diagnostics Systems Laboratories; interassay CV, <7%).
Bone formation was assessed by serum alkaline phosphatase (AP) and by serum bone AP (BAP), measured by ELISA (interassay CV, <11%) (7). Bone resorption was evaluated by measurement of 24-h urinary levels of the free pyridinium cross-links, pyridinoline (Pyd) and deoxypyridinoline (Dpd) of type I collagen by ELISA kits (Pyrilinks and Pyrilinks-D kits, Metra Biosystems, Inc., Mountain View, CA) with intraassay CVs of 8.7 and 5.4%, respectively, and assessed as nanomoles per liter glomerular filtrate determined by creatinine clearance (7). Serum carboxyterminal telopeptide of type I collagen [C-terminal collagen I cross-link (CTx)] was measured by one-step ELISA (Osteometer BioTech, Herlev, Denmark; intraassay CV, 2.7–3.7% at 0.36–0.52 ng/ml).
HMSC cultures
HMSCs (Cambrex/Clonetics, Walkersville, MD) were cultured according to manufacturer’s directions in growth medium (mesenchymal stem cell growth medium, Cambrex/Clonetics; 10-cm dishes), with half-feeds twice weekly, and expansion at 1:5 until passage 3. Cells were then plated at 1.5 x 104 cells/well in 12-well plates and cultured to 90% confluence when medium was changed to osteogenic induction medium (Cambrex/Clonetics) containing L-glutamine, antibiotic-antimycotic solution (Cellgro, Mediatech, Inc., Herndon, VA), 0.05 mM L-ascorbic acid 2-phosphate, 10 mM ß-glycerophosphate, and 100 nM dexamethasone (d 0). Four wells per group were treated with vehicle control (0.1 N acetic acid, 0.1% BSA), InhA (50 ng/ml), or InhB (50 ng/ml). Cells were cultured in treatment medium for 8 or 21 d and fed twice weekly by replacing half the medium with fresh induction medium containing 2x concentration of treatment growth factors.
Recruitment into the osteogenic lineage was determined on d 8 by staining for measuring AP as previously described using Sigma kit 86-R (20). Mineralization of differentiated osteoblastic cultures was assessed on d 21 by staining for mineralized extracellular matrix with Alizarin Red, followed by extraction with 20% methanol, 10% acetic acid, and spectrophotometric quantification at 450 nm. Alizarin Red staining was normalized to extracted protein content per well, and the data are expressed as micrograms Alizarin Red per microgram protein.
Isolation and culture of osteoclasts from PBMCs
Peripheral blood was collected from healthy donors approved by the University of Arkansas for Medical Sciences Institutional Review Board with heparin anticoagulant, in the presence of 200 ng/ml RANK-Fc as described (24). Macrophage colony-stimulating factor (mCSF; 25 ng/ml) was present in all treatment groups including control. Receptor activator of nuclear factor 
B (RANKL; 50 ng/ml), recombinant human InhA, and recombinant human InhB (50 ng/ml), RANK-Fc (200 ng/ml), RANKL + InhA, RANKL + InhB, and RANKL + RANK-Fc were used as treatments and were added to respective wells (n = 4 per treatment). All growth factors were purchased from R&D Systems (Minneapolis, MN). On d 10 of culture, staining for tartrate-resistant acid phosphatase (TRAP) was performed (Sigma), and the number of TRAP+ multinucleated cells (MNCs) was measured. TRAP+ cells having more than three nuclei were counted in the entire well with four wells per treatment. Cell counts were averaged, and the results are expressed as the number of TRAP+ MNCs/well per treatment group.
Statistical analysis
Spearman correlations were used to summarize the relationships among the various continuous variables. The relationships among InhA, InhB, and BioE2 with age were studied using least squares regression, where a natural spline was used to allow a nonlinear relationship with age in the regression model. Each model was then compared with a linear relationship, and the simplest model was used for analysis. Changes in variables between ages 20 and 90 yr were based on predicted values from these models. Step-wise model selection was used to determine the relationship of inhibins, pituitary hormones, and sex steroid variables with bone turnover markers. Data for each variable were ranked and divided by the number of women in the sample to create a percentile. Model assumptions were checked by examination of the model residuals.
All cell culture data were obtained in at least four wells per treatment. Data were analyzed by one way ANOVA with Tukey’s or Mann-Whitney U post hoc analyses, where appropriate. P < 0.05 was considered significant.
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Results |
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To determine whether inhibin levels were correlated with markers of bone turnover across the menopause transition, InhA and InhB levels were measured in 188 pre- and postmenopausal Caucasian women from 21–85 yr of age. Serum and urine samples were collected. In premenopausal women, this collection occurred between d 1 and 14 of the cycle, which is during the follicular phase. Figure 1
shows the age-related changes in serum levels of InhA, InhB, and BioE2 by menopause status. Although InhB levels were significantly higher than InhA levels in younger women, serum InhA and InhB levels decreased over lifespan by 96 and 97%, respectively (Fig. 1), whereas BioE2 decreased by 90% (Fig. 1) (7).
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shows the unadjusted and age-adjusted correlation coefficients relating bone formation and bone resorption markers to the levels of serum InhA, InhB, and FSH in premenopausal and postmenopausal women. In premenopausal women, both InhA and InhB were inversely correlated with markers of bone formation as well as bone resorption. Unlike previously published longitudinal studies in perimenopausal women, no associations were found between FSH and bone turnover in either premenopausal or postmenopausal women. In postmenopausal women, the only significant associations identified were negative correlations of InhA with bone formation markers, AP and BAP.
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) levels correlated with markers of bone turnover during the menopause transition. Consistent with results in Table 1
, InhA was inversely correlated with markers of bone formation (AP and BAP) and bone resorption (Dpd) in premenopausal young women (age, 25–34 yr) and women of perimenopausal age (45–54 yr). InhA and bone resorption were inversely correlated in women age 25–34 yr (Dpd) and age 35–44 yr (CTx) (Table 2). However, no significant correlations were found for InhA and bone turnover in older women (>55 yr). Similarly, InhB showed inverse correlations with bone formation in young women (AP, age 25–34 yr) and in women of perimenopausal age (AP and BAP, 45–54 yr). No correlations were found for InhB and bone resorption at any age. Finally, FSH did not significantly correlate with either bone formation or bone resorption at any age (Table 2).
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shows the results of multivariate models constructed to assess the relative contributions of inhibins, BioE2, and other endocrine and skeletal regulators to predict bone turnover markers. After separating the women by menopausal status, models were calculated in which each marker of bone turnover was the dependent variable, and age, InhA, InhB, BioE2, bioavailable T, E1, FSH, and LH were potential independent variables. InhB was not a good predictor of bone formation or bone resorption markers when the women were grouped based upon menopause status. However, InhA was a very good predictor of markers of both bone formation and bone resorption in premenopausal women (Table 3). Interestingly, InhA was the best predictor of bone formation markers in postmenopausal women, whereas age and BioE2 were better predictors of bone resorption markers (Table 3). In contrast, FSH, LH, or E1 were not significant predictors of any bone turnover marker measured.
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Figure 2A demonstrates the in vitro suppression of human osteoblast development by both InhA and InhB, as we have shown previously in murine cells (20). Mesenchymal stem cells cultured in osteogenic medium demonstrated high levels of AP expression on d 8, indicating recruitment into the osteoblast lineage (20); both InhA and InhB suppressed osteoblastogenesis. Similarly, late stage osteoblast differentiation, measured by assessing the extent of mineralization, was also suppressed by both InhA and InhB.
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). The addition of 200 ng/ml RANK-Fc completely suppressed increases in TRAP+ MNC development driven by RANKL. Similarly, both InhA and InhB completely blocked the RANKL-induced increase in TRAP+ MNCs (Fig. 2B). Together, these data using human mesenchymal and hematopoietic progenitors provide compelling evidence that inhibins regulate human osteoblast and osteoclast development. In addition, HMSCs appear to be direct targets of inhibin action during osteoblastogenesis. Although the cellular targets that mediate inhibin suppression of human osteoclast development as well as the molecular mechanism(s) are not well defined, these data provide the cellular basis by which changes in inhibins may regulate bone turnover in vivo.
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Discussion |
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Of the major hormones in the hypogonadal axis, only InhB and FSH levels change significantly during early perimenopause, with decreases in InhB signaling the first event in loss of ovarian function (10, 11). Previously, our laboratory demonstrated that exogenous InhA and InhB suppress development of primary murine osteoblasts and osteoclasts in bone marrow cultures (20). FSH enhancement of RANKL-stimulated osteoclast development in murine bone marrow monocyte cultures has been reported recently (27), although FSH does not appear to affect osteoblast development or mitigate inhibin suppression of osteoblastogenesis (28). Collectively, these reports are entirely consistent with the idea that changes in both inhibins and FSH contribute to the maintenance of skeletal homeostasis. Indeed, this population-based study of 188 pre-, peri-, and postmenopausal women demonstrates that InhA and InhB are strongly and significantly associated with several markers of bone formation and bone resorption across the menopause transition. InhA is an independent predictor of bone formation as well as resorption markers in both pre- and postmenopausal women. The negative correlations of InhA and InhB levels with bone formation markers are more consistent than with bone resorption markers. In contrast, it was determined that serum FSH levels only correlated with bone resorption markers in women of perimenopausal age (age 55–64 yr) and not with bone formation markers. This is consistent with the idea that inhibins, rather than FSH, suppress bone turnover by suppressing osteoblastogenesis, thereby reducing support for osteoclastogenesis (1, 29), even in the presence of RANKL.
BioE2 has previously been documented as a strong predictor of bone turnover in this cohort (7), and FSH has been shown previously in a larger cohort of perimenopausal women to be a better predictor of increased bone resorption markers than E2 (9). Still, in the current study, multivariate analyses demonstrated that InhA was a better predictor of bone turnover markers in premenopausal women than was any other hormone measured. This finding suggests additional diagnostic value for InhA measurements when collected during the early follicular phase of the menstrual cycle.
In contrast, InhB was not a good independent predictor of bone formation or bone resorption markers when the women were grouped by menopause status. The variations in both InhA and InhB during the follicular phase of the cycle are well documented and suggest that inhibin measurement during d 1–7 are more predictive of gonadal function than during d 8–14 (11, 30, 31, 32). Because the serum samples used here were collected between d 1 and 14 of the cycle, the data may actually underestimate the role of inhibins in predicting bone turnover. Thus, the predictive value of serum inhibin levels at more precise phases of the menstrual cycle is an area of intense investigation.
As expected, in postmenopausal women, age and BioE2 were the major predictors of bone resorption markers (7). However, InhA was the best predictor of bone formation markers in postmenopausal women. Interestingly, FSH was not a strong predictor of bone turnover markers in this cross-sectional study, when women were grouped according to age or menopausal status. Previous evidence that FSH is a major predictor of perimenopausal N-telopeptide levels was found in a larger sample of perimenopausal-aged women (281 women aged 45–57 yr) in which individual inhibins were not compared (9). Lack of significant correlations with FSH in the current study may be due to the smaller number of women in each age group in our cohort or the differences in the resorption parameters measured in the two studies. Thus, although future longitudinal studies in this and other cohorts are warranted, our current results indicate that InhA is a better predictor of bone turnover markers than either FSH or BioE2, particularly in pre- and perimenopausal women.
The surprising finding that InhA is consistently negatively correlated with markers of bone formation in women of all ages is likely the result of derepression of inhibin effects on mesenchymal and hematopoietic target cells. This derepression allows the stimulatory effects of locally produced regulators such as activin and bone morphogenetic proteins to stimulate both osteoblastogenesis and osteoclastogenesis (20, 33). In addition, decreased InhA allows elevations in pituitary FSH, which has also been suggested to enhance osteoclastogenesis (27).
The cellular basis by which changes in inhibins alter bone turnover involves suppressive effects of inhibins on osteoblast and osteoclast differentiation (Fig. 2). InhA and InhB blocked the recruitment and full differentiation of human osteoblasts and RANKL-stimulated osteoclastogenesis in vitro. These findings, along with the clinical data described above, suggest that inhibins are direct regulators of bone cell differentiation and bone turnover in vivo. Although the mechanism responsible for the early bone loss in perimenopausal women remains unknown, the data presented here support a model in which diminished ovarian function is first indicated by decreases in InhA or InhB secretion (10, 11, 26). This systemic decrease in InhA initiates an acceleration of osteoblast and osteoclast development leading to increases in bone turnover, in the presence of normal estrogen, that is a characteristic feature of the perimenopause. This process in pre- and perimenopausal women occurs before the loss of sex steroids and may account for the observed decreases in volumetric BMD in these women (25). Collectively, these cross-sectional data lead us to propose that decreases in inhibin levels associated with diminished ovarian function contribute to the initial bone loss observed during the perimenopausal period. It is also likely that decreases in inhibin levels, in the absence of changes in E2, that occur throughout reproductive life may also be associated with increases in bone turnover. Clearly, longitudinal studies are required to determine whether inhibin-related changes in bone turnover lead to long-term changes in bone mass. Such a scenario suggests that inhibin is an important endocrine regulator of normal skeletal health in women.
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Acknowledgments |
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Footnotes |
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Authors D.S.P., S.J.A., S.E.B., B.W., L.J.S., and S.K. have nothing to declare. Author D.G. is an inventor on a U.S. patent pending on this work.
First Published Online January 31, 2006
Abbreviations: AP, Alkaline phosphatase; BAP, bone AP; BCP, birth control pill; BioE2, bioavailable E2; CTx, C-terminal collagen I cross-link; CV, coefficient(s) of variation; Dpd, deoxypyridinoline; E1, estrone; E2, estradiol; ERT, estrogen replacement therapy; HMSC, human mesenchymal stem cell; InhA, inhibin A; InhB, inhibin B; mCSF, macrophage colony-stimulating factor; MNC, multinucleated cell; PBMC, peripheral blood mononuclear cell; Pyd, pyridinoline; RANKL, receptor activator of nuclear factor B; T, testosterone; TRAP, tartrate-resistant acid phosphatase.
Received November 7, 2005.
Accepted January 20, 2006.
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References |
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, ß A, and ß B subunits in various tissues predicts diverse functions. Proc Natl Acad Sci USA 85:247–251This article has been cited by other articles:
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  R. S. Pearsall, E. Canalis, M. Cornwall-Brady, K. W. Underwood, B. Haigis, J. Ucran, R. Kumar, E. Pobre, A. Grinberg, E. D. Werner, et al. A soluble activin Type IIA receptor induces bone formation and improves skeletal integrity PNAS, May 13, 2008; 105(19): 7082 - 7087. [Abstract] [Full Text] [PDF]
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D. S. Perrien, N. S. Akel, P. K. Edwards, A. A. Carver, M. S. Bendre, F. L. Swain, R. A. Skinner, W. R. Hogue, K. M. Nicks, T. M. Pierson, et al. Inhibin A Is an Endocrine Stimulator of Bone Mass and Strength Endocrinology, April 1, 2007; 148(4): 1654 - 1665. [Abstract] [Full Text] [PDF]
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) and postmenopausal (+) women without (w/out) birth control or ERT.