| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Department of Physiology and Biophysics (D.S.P., N.S.A., K.M.N., L.J.S., D.G.) and Department of Orthopaedic Surgery (D.S.P., P.K.E., A.A.C., M.S.B., F.L.S., R.A.S., W.R.H., L.J.S., D.G.), Center for Orthopaedic Research, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205; and Department of Molecular and Cellular Biology (T.M.P.), Baylor College of Medicine, Houston, Texas 77030
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.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In both genders, gonadal function is critical for the maintenance of bone quality. Consequently, hypogonadism is one of the most common causes of osteoporosis (5, 6). In women, this is widely attributed primarily to the loss of gonadal steroids (5, 6) that occurs during the menopausal transition. However, many other gonadally derived factors, including the inhibins, contribute to the regulation of bone turnover and bone quality (7, 8, 9, 10, 11, 12).
Inhibin B (InhB) and inhibin A (InhA) are heterodimeric proteins in the TGFß superfamily composed of 
ßB or ßA subunits, respectively (13). Inhibins were originally identified based on 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 an hour (16). These results are consistent with the idea that the effects of inhibin on marrow cell hematopoiesis (17, 18, 19) are attributable to inhibin derived from gonadal sources (20). Collectively, these data led to our hypothesis that changes in the gonadal inhibins may have direct effects on osteoblast and osteoclast development, thereby regulating increases in bone turnover and bone mass. In support of this hypothesis, we previously demonstrated in vitro effects of inhibins on osteoblast and osteoclast differentiation in both mice (7) and humans (8).
In the study described here, the effect of InhA on bone volume, architecture, and strength was tested using an inducible murine model of human InhA expression (21). Our findings demonstrate that continuous InhA exposure in vivo increases bone volume and strength in intact adult mice and prevents the bone loss associated with gonadectomy. Interestingly, InhA effects are mediated by a mechanism that increases bone formation, with little or no discernable effect on osteoclasts or bone resorption. These data reveal a new role for InhA as a nonsteroidal, gonadally derived endocrine regulator of bone mass.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
- and inhibin ßA-subunits. Subsequently, InhA is selectively and exclusively expressed from the liver of MFP-treated bigenic animals. An internal ribosome entry site sequence inserted between the inhibin and inhibin ßA reading frames dramatically decreases the production of ßA-subunit relative to -subunit, thereby preventing the formation of activin A (21, 22). MFP-induced expression of InhA in bigenic (inh/glvp) mice produces the expected suppression of serum FSH, confirming that the liver-derived InhA is secreted into the circulation and down-regulates pituitary FSH levels (21, 22). The genetic control for the inducible Inh/Glvp mice were monogenic animals expressing only the MFP-activated receptor and not the human InhA transgene (Glvp/–). All animals, identified by unique ear tags, were housed (four per cage) with free access to water and maintained at a constant temperature, on a 12-h light, 12-h dark cycle. The animal treatment and care protocols conformed to National Institutes of Health guidelines, and all studies were performed using a University of Arkansas for Medical Sciences Institutional Animal Care and Use Committee-approved protocol.
Controlled-release pellets (Innovative Research of America, Toledo, OH) designed to deliver vehicle or 6 µg/d of MFP to induce continuous InhA expression were sc implanted in 5.5-month-old male and female mice (10 per group). We previously determined 5.5 months to be the time of peak adult bone mass (data not shown) in these mice. The mice were followed for 4 wk before euthanasia and detailed skeletal analysis.
Similarly, in additional experiments, orchidectomy (ORCH) or sham surgery was performed as described previously (21) and controlled-releases pellets inserted at the time of surgery. The mice (12 per group) were followed for 4 wk after surgery before euthanasia and skeletal analysis.
The skeletal analyses performed were the same for all animals. At the time of killing, blood was collected by cardiac puncture and seminal vesicle or uterine weight determined to verify gonadectomy if performed (data not shown). In all cases, one tibia was excised for decalcified paraffin histology, the other tibia was harvested for microcomputed tomography (µCT) analysis and subsequent non-decalcified methyl methacrylate (MMA) embedded histology. The fifth lumbar vertebrae (L5) were also harvested and used for non-decalcified MMA histology, whereas the 6th lumbar vertebrae (L6) were used for µCT and biomechanical testing. Bone marrow was harvested from both femurs for ex vivo bone marrow culture.
Bone mineral density (BMD)
Total-body BMD and total body bone mineral content were determined in vivo using a PIXImus2 bone densitometer (GE Medical Systems, Madison, WI), as described (23, 24, 25). The precision of this technique in our laboratory is 1.7% (23, 24, 25). BMD and bone mineral content were measured in mice from each genotype at monthly intervals from 10 wk of age to determine the age at which these mice attained peak adult bone mass, which was 5.5 months of age in both genders and both genotypes (data not shown). Subsequently, total-body BMD measurements were obtained at euthanasia, excluding the head as previously described (24, 25). In addition, subregion analysis of the mid-shaft of the tibia and femur of all mice was performed, as we have described previously (23, 24, 25), to determine the effect of InhA treatment on cortical BMD.
Human-specific serum InhA assay
Blood was collected at kill by cardiac puncture, allowed to clot and serum obtained. Human-specific InhA was measured by two-site ELISA according to manufacturer’s protocol (Diagnostics Systems Laboratories, Webster, TX) and as previously described (8).
µCT analysis of bone
Ethanol-fixed tibiae and formalin-fixed L6 vertebrae were imaged using a µCT-40 (Scanco Medical AG, Bassersdorf, Switzerland) using a voxel size of 12 µm in all dimensions. The region of interest selected for tibial analysis comprised 240 transverse CT slices representing the entire medullary volume extending 1.24 mm distal to the end of the primary spongiosa with a border lying approximately 100 µm from the cortex. Vertebrae were evaluated using approximately 250 transverse CT slices encompassing the central trabecular bone in the anterior compartment of the vertebral body between the cranial and caudal end plates, excluding 100 µm near each endplate. Three-dimensional reconstructions were created by stacking the regions of interest from each two-dimensional slice and then applying a gray-scale threshold and Gaussian noise filter (26) specifically optimized for murine trabecular bone. Morphometric variables were computed from the binarized images using direct, three-dimensional techniques that do not rely on any prior assumptions about the underlying structure. Fractional bone volume, i.e. bone volume per tissue volume (BV/TV, %), and architectural properties of trabecular reconstructions [apparent trabecular thickness (µm), trabecular number (TbN, mm–1), and connectivity density (ConnD, mm–3)] were calculated using published methods (26).
Finite element modeling (FEM) of tibial cancellous bone
FEM was used to determine the contribution of bone volume and architecture to bone biomechanical properties. Using FEM version 1.0 (Scanco Medical), the three-dimensional reconstructions of trabecular bone in the proximal tibia were converted to brick element mesh and assigned material properties (with Poisson’s ratio = 0.2 and Young’s modulus = 1.8 GPa). A 1% apparent axial compression was simulated, and the reaction force (or force, in Newtons, required to create the compression) was calculated (27, 28).
Compression testing of L6 vertebrae
The compressive strength of L6 vertebrae was determined in a single load-to-failure compression test as we have previously described (29), using a MTS 858 Bionex Test Systems load frame (MTS, Eden Prairie, MN) with computer control, data logging, and calculations of load to failure using TestWorks version 4.0 (MTS). The load frame was operated at a constant rate of 0.1 mm/sec with load and displacement recorded at 100 Hz. Load to failure was recorded as the load after a 2% drop from peak load.
Histomorphometric analysis of bone
Tibiae were harvested at euthanasia and the muscle dissected away before fixation in Mallonig’s as previously described (24, 30). For static histomorphometric analyses, 4- to 5-µm-thick central sagittal sections of undecalcified MMA-embedded tibiae were stained for tartrate-resistant acid phosphatase and counterstained with hematoxylin to determine osteoclast numbers and eroded surface per cancellous bone surface within the region of interest, or with Masson’s trichrome for all other measurements, as defined by Parfitt et al. (31) using Osteomeasure software (Osteometrics, Atlanta, GA) and as we have previously described (24). Dynamic in vivo measurement of bone formation was performed by injecting each mouse with 30 mg/kg calcein 8 d before killing and 30 mg/kg of either tetracycline or Alizarin Red S 2 d before killing. The proportion of single- and double-labeled surfaces and the interlabel distance between the fluorochrome labels were measured in the cancellous bone of unstained sections adjacent to those used for static measurements. Appropriate measurements were normalized to total cancellous bone perimeter or tissue volume (24, 30). All cancellous bone measurements were made within the area defined by 700-1400 µm distal to the growth plate and 150 µm away from either endocortical surface.
C-terminal telopeptide measurement in serum
Serum levels of the C-terminal telopeptide of collagen I (CTx), a specific marker of bone resorption, were determined using a RatLaps ELISA (Nordic, Herlev, Denmark) according to protocols from the manufacturer.
Osteocalcin measurement in serum
Serum levels of osteocalcin, a specific marker of bone formation, were determined using the mouse osteocalcin single-plex kit assay (Linco Research Inc., St. Charles, MO) according to protocols from the manufacturer.
Ex vivo bone marrow culture
Bone marrow cells were harvested from femurs for osteoblastogenic culture as previously described (7) at specific time points after InhA induction or placebo treatment in vivo. Briefly, cells were flushed from femurs, washed, and cultured in 12-well plates at a density of 2 x 106 cells per well in -MEM, supplemented with 15% fetal calf serum, 50 µg/ml ascorbic acid, and 10 mM ß-glycerol phosphate, in the presence or absence of 50 ng/ml InhA (R&D Systems) or FSH (National Hormone and Peptide Program) in triplicate wells per treatment. Cells were fed every 3 d with half-volumes of medium, until d 28, when cells were fixed and mineral stained with Alizarin Red to facilitate determination of the number of bone nodules (colony-forming unit-osteoblast, CFU-OB) formed per well (7).
Statistical analysis
All experimental data that passed standard normalization tests were analyzed by one-way ANOVA and Student-Neuman-Keuls post hoc test. Data that were not normally distributed were analyzed by Kruskall-Wallis ANOVA on ranks and Dunn’s post hoc tests. Parametric data are presented at mean ± SEM, and nonparametric data are presented as median (25th percentile, 75th percentile). P values < 0.05 were considered statistically significant and are reported as such.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Human InhA increases bone mass and strength in intact mice.
To control for any potential effects of MFP treatment alone on skeletal physiology, Glvp/– mice were analyzed as controls for Glvp/InhA mice. Three-dimensional analysis of trabecular microarchitecture by µCT was performed on the proximal tibial metaphysis (Fig. 1A
) and L6 vertebrae (data not shown), and two-dimensional analysis of total-body BMD was performed using Piximus dual-energy x-ray absorptiometry (Fig. 2A) to exclude the possibility that any observed skeletal effects were site specific (35).
|
|
) or on the total-body BMD (Fig. 2A). Similarly, there was no demonstrable effect of MFP on L6 vertebrae or any other skeletal parameter measured (data not shown). In contrast, in intact MFP-treated Glvp/InhA mice, the expression of human InhA significantly increased bone volume fraction (BV/TV) in both male and female mice (Fig. 1A) and also increased total-body BMD (Fig. 2A).
During the course of our day-to-day management of the mouse colony, we noted variable levels of human InhA levels in female mice, independent of MFP pellet insertion (Fig. 1B). In particular, we repeatedly measured elevated human InhA expression in female Glvp/InhA mice that had received no MFP pellet or had never been housed with mice harboring an MFP pellet, strongly suggesting that the InhA transgene had become non-MFP-inducible in female mice. No male mice were observed to have detectable human InhA levels (assay detection limit is 20 pg/ml) in the absence of MFP induction (Fig. 1B). The uncontrolled expression of human InhA (42–367 pg/ml), independent of MFP pellet insertion, selectively in female Glvp/InhA mice made any subsequent use of female mice unreliable. Females were therefore excluded from further investigation. Nonetheless, measurement of bone volume in the original MFP-inducible intact Glvp/InhA female mice (Fig. 1A) demonstrated that human InhA expression stimulated increases in BV/TV in intact female mice.
InhA prevents ORCH-induced loss of mass and strength.
Vehicle-treated and MFP-treated, Glvp/InhA (bigenic) mice were of similar body weight and did not demonstrate a significant difference in body weight gain over the 4-wk experimental period, regardless of gonadal status.
ORCH initiated at peak adult bone mass induced the expected loss of total-body BMD by dual-energy x-ray absorptiometry (Fig. 2A) and trabecular bone volume fraction (BV/TV) in both the tibiae and vertebrae of both Glvp/– and Glvp/InhA mouse strains, as determined by µCT (Figs. 2B and 3A). These findings demonstrate that the skeletons of both the Glvp/– and Glvp/InhA mice are sensitive to ORCH. In preliminary studies in females, we noted that the skeletons of both the Glvp/– (and Glvp/InhA) mouse strains were insensitive to ovariectomy, as has been reported in a number of mouse strains and transgenic mouse lines (36, 37).
|
), BV/TV (Fig. 2B), or trabecular architecture (data not shown), regardless of gonadal status. These data clearly demonstrate that any effects on the skeleton of Glvp/InhA mice are not the result of effects of MFP on bone but are associated with the induced expression of human InhA.
In contrast to the lack of MFP effect on Glvp/– mice, the effect of human InhA expression in sham (intact) Glvp/InhA mice was to increase bone mass. Total-body BMD was increased (Fig. 2A), and trabecular bone volume in the proximal tibia (Figs. 2B and 3A and Table 1) and the lumbar vertebrae (Fig. 3A and Table 1) were also increased. The µCT analysis of the trabecular microarchitecture revealed that the stimulatory effect of human InhA expression in sham (intact) Glvp/InhA mice was mediated by an increase in TbN and ConnD, leading to a decrease in the structure model index (SMI) (Table 1).
|
) and BV/TV in both the proximal tibiae (Figs. 2B and 3A and Table 1) and L6 vertebrae (Fig. 3A and Table 1) of Glvp/InhA mice as expected. However, human InhA expression in ORCH Glvp/InhA mice prevented the gonadectomy-induced loss of total-body BMD (Fig. 2A) as well as the loss of BV/TV at both tibial and vertebral sites (Figs. 2B and 3A and Table 1). Similar to the observations in the intact Glvp/InhA mice, InhA preserved BV/TV in ORCH mice by maintaining TbN and ConnD (Table 1).
To determine whether InhA maintenance of tibial bone volume and architecture in ORCH mice correlated with an improvement in biomechanical properties, FEM was used to calculate the compressive stiffness of trabecular bone in the proximal tibia (27, 28). Human InhA expression prevented the ORCH-induced loss of bone stiffness in the tibia (Fig. 3B) and, remarkably, even increased stiffness in intact (sham) animals (Fig. 3B). In addition, direct biomechanical compression testing of L6 vertebrae (29) demonstrated that InhA expression also prevented the ORCH-induced loss of bone stiffness that occurs in the axial skeleton (Fig. 3C).
In addition to examining the effects of human InhA expression on trabecular bone, we also examined the effect of InhA on cortical bone. Subregion analysis of the mid-shaft of both the tibia and femur revealed no significant effect of InhA on cortical BMD in intact or ORCH male mice (Fig. 3, D and E), in agreement with the cortical analyses performed using µCT (data not shown). Thus, the protective and stimulatory effects of InhA on cancellous bone mass, architecture, and strength that occur in both the vertebrae and the proximal tibiae are not associated with any demonstrable changes in cortical BMD or architecture.
Inhibin stimulates osteoblast activity
Lack of InhA effects on osteoclastogenesis or bone resorption.
Several quantitative analyses were performed to determine whether the stimulatory effects of InhA on bone mass and strength in Glvp/InhA mice were mediated by decreased bone resorption, increases in bone formation, or a combination of both. As has been previously described, ORCH-stimulated bone turnover was associated with increases in serum CTx (38). However, human InhA expression did not affect bone resorption in either ORCH or sham-operated mice, as measured by serum CTx (Fig. 4A). Similarly, static histomorphometric analysis of the secondary spongiosa of the proximal tibia (30) showed that InhA expression had no effect on osteoclast number, the proportion of osteoclast eroded surfaces, or any measured osteoclast parameter, regardless of gonadal status (Table 2).
|
|
). This effect on a systemic measure of osteoblast activity suggests that InhA stimulates bone formation via changes in osteoblast activity.
To support the notion that the bone-forming effect of InhA is mediated by effects on osteoblastic activity, histomorphometric parameters of osteoblastic activity were also measured in the secondary spongiosa of the proximal tibia as described (24, 30). Surprisingly, InhA did not significantly affect the number of osteoblasts on the trabecular bone surface or the number of osteoblasts per total area, regardless of gonadal status (Table 2). Similarly, InhA expression did not affect osteoblast surface per bone surface, osteoid surface per bone surface, osteoid thickness (Table 2), or any other static osteoblastic parameter measured (data not shown). Interestingly, dynamic histomorphometry demonstrated that human InhA expression in intact sham mice significantly increased (at least 2-fold) osteoblast activity, as measured by an increased mineral apposition rate (MAR) (Fig. 4C and Table 2), increased double-labeled surface per bone surface (Table 2), and the inter-label distance (Fig. 4, D and E), in the absence of an increase in bone formation rate (BFR). The additional increases in MAR observed in response to InhA expression in ORCH mice did not reach significance, presumably because of the increased bone formation and bone turnover rates known to be associated with ORCH alone (38). Thus, in agreement with µCT measurements of BV/TV and microarchitecture, serum osteocalcin levels and dynamic bone histomorphometry confirmed that InhA expression stimulated bone formation in Glvp/InhA mice and demonstrated that the enhanced bone formation occurred predominantly as the result of an increase in osteoblast activity, as measured by MAR and double-labeled surface per bone surface.
InhA in vivo stimulates osteoblast recruitment in ex vivo bone marrow cultures. To better elucidate the effect(s) of InhA on osteoblast progenitors, ex vivo femoral bone marrow cultures were established using bone marrow harvested from vehicle-treated or InhA-overexpressing Glvp/InhA animals. In direct support of the observed stimulatory effects of InhA on osteoblastic cells in vivo, osteogenic differentiation of marrow cells derived from animals exposed to MFP-stimulated InhA for 4 wk in vivo was significantly increased compared with cells from mice not exposed to InhA in vivo (Fig. 5). In addition and entirely consistent with our previously published results (7, 8), direct in vitro treatment of bone marrow cells with exogenous human InhA significantly reduced osteoblast differentiation (Fig. 5), regardless of the in vivo exposure to InhA.
|
). Interestingly, the stimulatory effect of InhA expression on trabecular BV/TV was observed only after 4 wk of InhA treatment in vivo (Fig. 6A). Furthermore, the osteogenic differentiation of marrow cells derived from these same animals exposed to InhA in vivo was significantly increased compared with cells from mice not exposed to InhA in vivo only at wk 3 (Fig. 6B). Most notably, short-time in vivo exposure to InhA (1 wk) resulted in suppression of ex vivo osteogenic differentiation that was comparable to that observed in cells from vehicle-treated mice after exogenous administration of InhA in vitro (Fig. 6B). In addition, as we have shown previously (Fig. 5) (7, 8), the administration of exogenous InhA in vitro significantly inhibited osteoblast differentiation, independent of InhA exposure in vivo (Fig. 6B). These data provide compelling mechanistic information, suggesting that the osteoblasts (and/or their precursors) are the target(s) of InhA action and that the effect of InhA is dependent upon the duration of in vivo exposure.
|
, FSH addition had no effect on the osteogenic differentiation of marrow cells derived from either control animals or animals exposed to InhA in vivo. In addition, FSH had no influence on the osteogenic suppression due to in vitro InhA exposure. These data confirm the effects of InhA on osteoblast differentiation and demonstrate that they are not associated with FSH.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The early rise in FSH levels that occurs in perimenopausal women is attributable to a selective decrease in InhB secretion that occurs in the presence of normal levels of estradiol, InhA, GnRH, and LH (45, 46, 47). Similarly, testicular InhB is a primary regulator of FSH secretion in men (45, 48), although FSH has been demonstrated to be similarly suppressed by exogenous administration of human InhA (49, 50). Because both InhA and InhB isoforms selectively inhibit pituitary FSH secretion, these data suggest that the increased FSH in perimenopausal women is attributable to a loss in feedback inhibition by gonadal InhB (reviewed in Ref. 51). Our recent clinical (8) and in vitro (7, 8) findings suggest that decreases in circulating inhibin levels, caused by reduced ovarian function, contribute to perimenopausal bone loss (44). As the loss of gonadal function progresses in postmenopausal women, the well-established decreases in estradiol accompany declining levels of both InhB and InhA, further increasing serum FSH (45, 46, 47) and markedly increasing bone loss. Recent evidence has implicated elevated FSH as an independent stimulator of the postmenopausal increase in osteoclastic bone resorption (52) (39). However, given that the target of InhA action in our study appears to be cells in the osteoblastic lineage, and the lack of effect of FSH in ex vivo bone marrow cultures, the bone-forming and/or -protective effects of InhA on bone appear independent of the reported FSH-mediated changes in osteoclast function.
Few animal models exist in which to study the action of inhibins on bone. The -inhibin gene has been deleted (53), but these mice develop gonadal tumors at 6 wk of age, during the phase of longitudinal bone growth. After gonadectomy, the resulting lack of sex steroids prevents the mice from reaching peak adult bone mass (53) and precludes the analysis of any selective effects of inhibin on the adult skeleton. The inducible transgenic mouse model used in the study reported here permits the inducible expression of human InhA at any stage of growth and/or adulthood (21). This model is uniquely suited to study the effects of InhA on the adult skeleton. The data demonstrate that InhA increases BV/TV by 36 and 54% in intact male and female mice, respectively, and also prevents the 43% loss of BV/TV that occurs due to ORCH (Fig. 2B). These observations provide additional evidence to support the emerging idea that nonsteroidal gonadal hormones contribute to the regulation of bone homeostasis (8, 12, 44).
The ability of InhA to increase bone mass is somewhat unexpected given inhibin’s well-documented ability to antagonize the effects of activin (54), which has been shown to stimulate bone formation in vivo (55, 56). However, unlike activin, inhibins are not normally produced within the bone microenvironment (57) and are gonadally derived, circulating endocrine hormones (13). Thus, the stimulatory action of InhA on the skeleton significantly differs from those of other TGFß superfamily members such as activin, TGFß, and bone morphogenetic protein. The normal physiological effects on the skeleton of these TGFß superfamily members are entirely a result of their local production in bone (58).
In addition to the stimulatory effects on bone volume, the effects of InhA on trabecular bone microarchitecture demonstrate that InhA is also able to prevent the perforations that occur after ORCH. Such perforations typically raise the SMI and decrease ConnD (26, 59). InhA expression was able to lower the SMI and increase the ConnD in sham-operated mice, suggesting that InhA increased trabecular connections and closed preexisting perforations in the bones of these animals. The stimulatory effect of InhA expression on bone microarchitecture was also associated with similar beneficial effects on both tibial and vertebral biomechanical properties. Furthermore, InhA expression also prevented ORCH-induced losses of bone density, volume, microarchitecture, and strength in both the axial and appendicular skeleton. Thus, the stimulatory effects of InhA in intact sham mice resulted in increased bone quality and strength.
Four weeks of exposure to InhA in vivo is required to induce increases in tibial BV/TV, whereas increases in ex vivo osteoblastogenesis are observed after 3 wk of InhA in vivo. In addition, in vivo exposure to InhA for only 1 wk resulted in significant suppression of osteogenesis in ex vivo marrow cultures. This short-term suppressive effect of osteogenesis is entirely consistent with our clinical observations that serum inhibin levels are inversely correlated with markers of bone formation and bone resorption (8). Moreover, our collective in vivo and in vitro data suggest that inhibins exert a temporal bimodal effect on the regulation of bone metabolism. We propose that the normal physiological role of pulsatile (short-duration) exposure to inhibins is to suppress bone turnover (8), whereas longer-term continuous exposure to inhibins (Glvp/InhA mice) is anabolic. The stimulatory effect of InhA on the skeleton is mediated by increases in mature osteoblast activity and osteoblast differentiation. The significant increases in bone mass and architecture induced by InhA are independent of changes in osteoclast number or function.
Direct bone histomorphometry (Table 2) suggested that InhA expression was anabolic in Glvp/InhA mice and revealed that the increase in bone mass was caused by enhanced bone formation, predominantly as the result of an increase in MAR and double-labeled surface per bone surface, with no effect on BFR. This observation is not new, because others have reported anabolic effects on the skeleton mediated by increased MAR, with or without effects on BFR (60, 61, 62, 63, 64). The InhA-induced increase in MAR and double-labeled surface per bone surface reported here suggests that InhA increases bone formation primarily by stimulating osteoblast activity, an idea supported by the observation that InhA expression also induced significant increases in serum osteocalcin levels. However, these data do not preclude the possibility that InhA also increased BFR, which may have occurred earlier or be more easily detected before the 4-wk time point analyzed here.
As has been observed for other endocrine regulators of bone turnover (1), the effects of InhA in vitro and short continuous duration in vivo are distinct from those of long-term continuous exposure to InhA in vivo. Our data suggest that the endocrine effects of continuous exposure in vivo overrides the direct suppressive effects of exogenous InhA on osteoblastogenesis in vitro. These differential effects of InhA are similar to those demonstrated for estrogens and glucocorticoids.
It is well documented that physiological estrogens suppress bone turnover but that high-dose estrogen treatment increases bone formation and is strongly anabolic (65, 66, 67). Similarly, it is also well accepted that glucocorticoids increase osteoblastogenesis in vitro (68, 69, 70, 71, 72, 73), whereas continuous in vivo exposure decreases osteoblastogenesis, increases osteoblast apoptosis, and increases osteoclast activity leading to bone loss (38, 74, 75, 76, 77, 78). Furthermore, the distinct InhA actions in vitro and in vivo are reminiscent of the well-documented paradigm of continuous vs. pulsatile PTH that have driven mechanistic studies by numerous investigators for decades (79, 80, 81, 82, 83, 84, 85). Thus, we hypothesize that the in vivo effects of InhA, like a host of other endocrine hormones, are pleiotropic. Additional experiments are ongoing to elucidate the differential mechanisms by which inhibins exert their profound effects on the skeleton.
Continuous exposure to physiological levels of InhA in vivo increases MAR, double-labeled surface per bone surface, BV/TV, and bone strength. Ex vivo culture of bone marrow harvested from animals exposed to InhA in vivo demonstrate an increased recruitment of bone marrow cells into the osteoblastic lineage (increasing CFU-OB), which is suppressed by exogenous in vitro treatment with InhA, as we have shown previously (7) (8). These data suggest strongly that the endocrine effects of continuous InhA exposure in vivo overrides the direct suppressive effects of InhA on osteoblastogenesis in vitro. Presumably, the effects of InhA in vivo are mediated by cell types either absent or underrepresented in primary ex vivo murine bone marrow cultures. The identity of the cellular mediators and mechanisms of InhA action on bone mass remains the focus of intensive investigation.
Given that the primary endocrine role of the inhibins is to suppress FSH (86, 87) and that FSH has been recently implicated as a pro-osteoclastogenic agent (52, 39), it is unlikely that decreased FSH is a mediator of the stimulatory effects of InhA on the skeleton. As shown here, FSH treatment did not effect osteoblast differentiation in vitro. In vivo, InhA action increases osteoblast activity, whereas FSH has been shown to exert effects on osteoclasts and their precursors, without effects on osteoblasts (52, 88, 89). The bone-stimulatory effect of InhA demonstrated here resulted from continuous exposure; whether InhA increases bone mass when delivered by pulsatile (cyclic) administration remains to be determined. Unlike other endocrine regulators of bone mass, the lack of InhA effects on bone resorption and osteoclast number suggest the likelihood of an InhA-induced imbalance between bone resorption and bone formation.
Collectively, the data presented here demonstrate that InhA acts as a potent endocrine stimulator of bone mass and strength in vivo that can also prevent the bone loss associated with ORCH, implicating InhA as a bone-anabolic agent. Given the complicated nature of the dimeric inhibin peptides, additional studies elucidating the mechanisms of InhA action are warranted to determine the possible utility of targeting InhA signaling as a therapeutic modality to stimulate bone formation. Together, these data provide direct evidence supporting the emerging idea that gonadal factors other than sex steroids play an important role in regulating both bone mass and bone strength (8, 12). Furthermore, it appears that InhA may be a component of the normal endocrine repertoire that regulates bone volume and strength in both the axial and appendicular skeleton.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Current address for T.M.P.: Children’s Hospital of Philadelphia, Division of Neurology Philadelphia, Pennsylvania 19104.
Current address for D.S.P.: Biomimetic Therapeutics, Franklin, Tennessee 37067.
Disclosure Statement: D.S.P., N.S.A., P.K.E., A.A.C., M.S.B., F.L.S., R.A.S., W.R.H., K.M.N., T.M.P., and L.J.S. have nothing to declare. D.G. is an inventor on a United States patent pending from this work.
First Published Online December 28, 2006
Abbreviations: BFR, Bone formation rate; BMD, bone mineral density; BV/TV, bone volume per tissue volume; CFU-OB, colony-forming unit-osteoblast; µCT, microcomputed tomography; ConnD, connectivity density; CTx, C-terminal telopeptide of collagen I; FEM, finite element modeling; InhA, Inhibin A; MAR, mineral apposition rate; MFP, mifepristone; MMA, methyl methacrylate; ORCH, orchidectomized; SMI, structure model index; TbN, trabecular number.
Received June 22, 2006.
Accepted for publication December 18, 2006.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-subunit gene. Endocrinology 142:4994–5004-Inhibin is a tumour-suppressor gene with gonadal specificity in mice. Nature 360:313–319[CrossRef][Medline]This article has been cited by other articles:
 |
|
 |
|
  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]
|
|
|
|
 |
|
 L. J. Suva, E. Hartman, J. D. Dilley, S. Russell, N. S. Akel, R. A. Skinner, W. R. Hogue, U. Budde, K. I. Varughese, T. Kanaji, et al. Platelet Dysfunction and a High Bone Mass Phenotype in a Murine Model of Platelet-Type von Willebrand Disease Am. J. Pathol., February 1, 2008; 172(2): 430 - 439. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. R. Williams Hypogonadal Bone Loss: Sex Steroids or Gonadotropins? Endocrinology, June 1, 2007; 148(6): 2610 - 2612. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
