Sex- and age-related response to aromatase deficiency in bone

Deficiency of sex steroids causes osteoporosis, but the relationship between estrogen and androgen is not clear because androgen is converted into estrogen by aromatase. In this study, we characterized bone metabolism in the aromatase-deficient (ArKO) mouse. At 9 weeks old, a marked loss of cancellous bone due to increased bone resorption was observed not only in female ArKO mice but also in males. The degree of bone loss in ArKO males was similar to that in females, and treatment with 17beta-estradiol completely restored the bone mass in both sexes. At 32 weeks old, female ArKO mice showed severe loss of cancellous and cortical bone. Male ArKO mice of this age also showed reduced bone mass, but the degree of bone loss in females was more marked than that in males. Here, we report sex- and age-related responses to aromatase deficiency in bone.     

Aryl hydrocarbon receptor catabolic activity in bone metabolism is osteoclast dependent in vivo

Bone mass is regulated by various molecules including endogenous factors as well as exogenous factors, such as nutrients and pollutants. Aryl hydrocarbon receptor (AhR) is known as a dioxin receptor and is responsible for various pathological and physiological processes. However, the role of AhR in bone homeostasis remains elusive because the cell type specific direct function of AhR has never been explored in vivo. Here, we show the cell type specific function of AhR in vivo in bone homeostasis. Systemic AhR knockout (AhRKO) mice exhibit increased bone mass with decreased resorption and decreased formation. Meanwhile, osteoclast specific AhRKO (AhR^ΔOc/ΔOc) mice have increased bone mass with reduced bone resorption, although the mice lacking AhR in osteoblasts have a normal bone phenotype. Even under pathological conditions, AhR^ΔOc/ΔOc mice are resistant to sex hormone deficiency-induced bone loss resulting from increased bone resorption. Furthermore, 3-methylcholanthrene, an AhR agonist, induces low bone mass with increased bone resorption in control mice, but not in AhR^ΔOc/ΔOc mice. Taken together, cell type specific in vivo evidence for AhR functions indicates that osteoclastic AhR plays a significant role in maintenance of bone homeostasis, suggesting that inhibition of AhR in osteoclasts can be beneficial in the treatment of osteoporosis.     

Reduced bone turnover in mice lacking the P2Y(13) receptor of ADP

Osteoporosis is a condition of excessive and uncoupled bone turnover, in which osteoclastic resorption exceeds osteoblastic bone formation, resulting in an overall net bone loss, bone fragility, and morbidity. Although numerous treatments have been developed to inhibit bone loss by blocking osteoclastic bone resorption, understanding of the mechanisms behind bone loss is incomplete. The purinergic signaling system is emerging to be a pivotal regulator of bone homeostasis, and extracellular ADP has previously been shown to be a powerful osteolytic agent in vitro. We report here that deletion of the P2Y(13) receptor, a G protein-coupled receptor for extracellular ADP, leads to a 40% reduction in trabecular bone mass, 50% reduction in osteoblast and osteoclast numbers in vivo, as well as activity in vitro, and an overall 50% reduction in the rate of bone remodeling in mice in vivo. Down-regulation of RhoA/ROCK I signaling and a reduced ratio of receptor activator of nuclear factor κB ligand/osteoprotegerin observed in osteoblasts from P2Y(13)R(-/-) mice might explain this bone phenotype. Furthermore, because one of the main causes of osteoporosis in older women is lack of estrogen, we examined the effect of ovariectomy of the P2Y(13)R(-/-) mice and found them to be protected from ovariectomy-induced bone loss by up to 65%. These data confirm a role of purinergic ADP signaling in the skeleton, whereby deletion of the P2Y(13) receptor leads to reduced bone turnover rates, which provide a protective advantage in conditions of accelerated bone turnover such as oestrogen deficiency-induced osteoporosis.     

Pathophysiology of bone loss in castrated animals

The pathophysiology of bone loss in castrated animals is reviewed. Both male and female rats rapidly lose metaphyseal trabecular bone from the tibia and the femur due to an imbalance between bone resorption and bone formation. The aetiology of sex hormone deficiency-induced bone loss is not fully understood. It seems unlikely that the bone loss is due to changes in the circulating levels of the calciotropic hormones or to an increase in the spontaneous release from peripheral blood monocytes of the bone resorption stimulating cytokine IL-1. Changes in the sensitivity of bone of castrated rats to calciotropic hormones may play a role as well as the lack of direct stimulatory effects of gonadal oestrogens and androgens on bone cells. In addition several data indicate that prostaglandins may be involved.     

Skeletal sensitivity to dietary calcium deficiency is increased in the female compared with the male rat

We investigated potential sex differences in bone resorption and the conservation of whole body bone mass in 24-week-old Sprague-Dawley rats maintained on a 1.0% calcium diet and then fed diets containing 0.02, 0.5, 1.0, or 1.75% calcium for 31 days. Lowering dietary calcium from 1.00% to 0.02% doubled whole skeleton bone resorption (urinary 3H-tetracycline loss). Female rats were more sensitive to calcium stress, exhibiting the maximal resorptive response when fed the 0.5% calcium diet, whereas the 0.02% calcium diet was required to elicit this response in males. Despite the evidence of increased bone resorption, whole skeleton mass was unchanged in females and was significantly increased in males, indicating that switching to even the 0.02% calcium diet did not result in an overt loss of total body bone mass. Compared with controls, the skeleton mass of females (97+/-1.4%) maintained on the 0.02% calcium diet was significantly lower than males (107+/-2.4%), again suggesting a greater impact of calcium deficiency in females. The calculation of the average percentage growth of selected individual bones in male rats indicated a proportional increase in bone mass between the axial and appendicular skeleton of approximately +4% and +18% in animals maintained on 0.02 and 1.75% diets, respectively. By comparison, female rats consuming the 0.02% calcium diet showed an average 14% loss in axial bone and 7.5% gain in appendicular bone mass. The results indicate increased sensitivity to dietary calcium deficiency in female rats which involves a significant loss in axial bone mass not observed in male rats maintained under similar dietary conditions.     

Annual skeletal changes in the little brown bat, Myotis lucifugus lucifugus, with particular reference to pregnancy and lactation

Studies of bone from summer-active little brown bats, Myotis lucifugus lucifugus, have demonstrated sex differences in the renewal of skeletal mineral reserves following spring-arousal from hibernation. Patterns of bone remodeling in both sexes of bats indicate that new bone formation does not occur during hibernation: All new bone formation occurs during the summer-active season. Results show that a short period of time elapses after hibernation before the initial demands of a large fetus and rapidly growing neonate are expressed on maternal skeletal reserves. Bone loss in summer-active females was associated with pregnancy and lactation, whereas summer-active males did not show evidence of bone loss but, instead, uninterrupted bone accretion throughout the summer-active season. Osteoclasts and bone-forming osteoblasts, absent during the hibernation period, reappeared on bone surfaces following spring-arousal from hibernation. There was no apparent increase in osteoclast numbers or activity during lactation but resorption cavities were found in deep cortical lamellae distant from bone surfaces. The increase in bone resorption in lactating bats appeared to be by osteocytic osteolysis, suggesting that it might be a significant mechanism of bone/calcium regulation in this hibernating mammal throughout the year.     

Bone mass in Guamanian patients with amyotrophic lateral sclerosis and parkinsonism-dementia

Bone mass, as assessed by measurements of total subperiosteal diameter and medullary width of the second metacarpal bone on hand-wrist radiographs, was evaluated for 31 Guamanian patients (15 males, 16 females) with amyotrophic lateral sclerosis (ALS), 67 patients (39 males, 28 females) with parkinsonism-dementia (PD), and 66 (34 males, 32 females) nonaffected Guamanian controls. Comparisons between the two disease groups and between each disease group and the nonaffected controls were made taking into account the sex, age, and disability status of each participant. At all ages, ALS patients of both sexes had significantly lower percent cortical areas (PCA) than did nonaffected controls. The ALS males also had significantly lower PCA than PD males, although no significant differences were observed between female ALS and PD patients. The PD patients of either sex had a lower PCA when compared to controls, but the differences were not statistically significant. The observed differences in PCA were due solely to increased medullary width, suggesting that the diminished cortical bone thickness resulted from greater bone resorption rather than differential bone growth. Longitudinal studies support the cross-sectional findings of accelerated bone loss among ALS patients. It is not possible to determine from the present data whether the observed differences in PCA of the second metacarpal of the ALS patients are due to atrophy of the first interosseous muscle, to a generalized resorption process inherently associated with the development and progression of ALS, or to factors not accounted for by the present analysis.     

Sex differences in bone resorption in the mouse femur

Summary In male and female dd-mice at 4, 7, and 14 weeks of age and in 7 and 14-week-old mice gonadectomized at 4 weeks of age, the number of osteoclasts and the number and size of bone resorption areas along the surface of bone trabeculae in the distal metaphysis of the femur were determined. Osteoclasts were counted at the light-microscopic level in paraffin sections of decalcified femora. The number and size of the bone resorption areas were examined by scanning electron microscopy of femora after removing organic material by means of KOH and NaOCl treatment. In untreated mice, the number of osteoclasts and the number and size of bone resorption areas showed no sex differences at 4 weeks of age but were larger in females than males at 7 and 14 weeks of age. In gonadectomized mice, the number of osteoclasts and the bone resorption areas increased in males and decreased in females. The results of the gonadectomy experiments suggest that bone resorption in young adult mice is stimulated by female sex hormone and inhibited by male sex hormone.     

Imbalance of local bone metabolism in inflammatory arthritis and its reversal upon tumor necrosis factor blockade: direct analysis of bone turnover in murine arthritis

Chronic arthritis typically leads to loss of periarticular bone, which results from an imbalance between bone formation and bone resorption. Recent research has focused on the role of osteoclastogenesis and bone resorption in arthritis. Bone resorption cannot be observed isolated, however, since it is closely linked to bone formation and altered bone formation may also affect inflammatory bone loss. To simultaneously assess bone resorption and bone formation in inflammatory arthritis, we developed a histological technique that allows visualization of osteoblast function by in-situ hybridization for osteocalcin and osteoclast function by histochemistry for tartrate-resistant acid phosphatase. Paw sections from human tumor necrosis factor transgenic mice, which develop an erosive arthritis, were analyzed at three different skeletal sites: subchondral bone erosions, adjacent cortical bone channels, and endosteal regions distant from bone erosions. In subchondral bone erosions, osteoclasts were far more common than osteoblasts. In contrast, cortical bone channels underneath subchondral bone erosions showed an accumulation of osteoclasts but also of functional osteoblasts resembling a status of high bone turnover. In contrast, more distant skeletal sites showed only very low bone turnover with few scattered osteoclasts and osteoblasts. Within subchondral bone erosions, osteoclasts populated the subchondral as well as the inner wall, whereas osteoblasts were almost exclusively found along the cortical surface. Blockade of tumor necrosis factor reversed the negative balance of bone turnover, leading to a reduction of osteoclast numbers and enhanced osteoblast numbers, whereas the blockade of osteoclastogenesis by osteoprotegerin also abrogated the osteoblastic response. These data indicate that bone resorption dominates at skeletal sites close to synovial inflammatory tissue, whereas bone formation is induced at more distant sites attempting to counter-regulate bone resorption.     

Imbalance of local bone metabolism in inflammatory arthritis and its reversal upon tumor necrosis factor blockade: direct analysis of bone turnover in murine arthritis

Chronic arthritis typically leads to loss of periarticular bone, which results from an imbalance between bone formation and bone resorption. Recent research has focused on the role of osteoclastogenesis and bone resorption in arthritis. Bone resorption cannot be observed isolated, however, since it is closely linked to bone formation and altered bone formation may also affect inflammatory bone loss. To simultaneously assess bone resorption and bone formation in inflammatory arthritis, we developed a histological technique that allows visualization of osteoblast function by in-situ hybridization for osteocalcin and osteoclast function by histochemistry for tartrate-resistant acid phosphatase. Paw sections from human tumor necrosis factor transgenic mice, which develop an erosive arthritis, were analyzed at three different skeletal sites: subchondral bone erosions, adjacent cortical bone channels, and endosteal regions distant from bone erosions. In subchondral bone erosions, osteoclasts were far more common than osteoblasts. In contrast, cortical bone channels underneath subchondral bone erosions showed an accumulation of osteoclasts but also of functional osteoblasts resembling a status of high bone turnover. In contrast, more distant skeletal sites showed only very low bone turnover with few scattered osteoclasts and osteoblasts. Within subchondral bone erosions, osteoclasts populated the subchondral as well as the inner wall, whereas osteoblasts were almost exclusively found along the cortical surface. Blockade of tumor necrosis factor reversed the negative balance of bone turnover, leading to a reduction of osteoclast numbers and enhanced osteoblast numbers, whereas the blockade of osteoclastogenesis by osteoprotegerin also abrogated the osteoblastic response. These data indicate that bone resorption dominates at skeletal sites close to synovial inflammatory tissue, whereas bone formation is induced at more distant sites attempting to counter-regulate bone resorption.     

Conditional deletion of hypothalamic Y2 receptors reverts gonadectomy-induced bone loss in adult mice

Reduction in levels of sex hormones at menopause in women is associated with two common, major outcomes, the accumulation of white adipose tissue, and the progressive loss of bone because of excess osteoclastic bone resorption exceeding osteoblastic bone formation. Current antiresorptive therapies can reduce osteoclastic activity but have only limited capacity to stimulate osteoblastic bone formation and restore lost skeletal mass. Likewise, the availability of effective pharmacological weight loss treatments is currently limited. Here we demonstrate that conditional deletion of hypothalamic neuropeptide Y2 receptors can prevent ongoing bone loss in sex hormone-deficient adult male and female mice. This benefit is attributable solely to activation of an anabolic osteoblastic bone formation response that counterbalances persistent elevation of bone resorption, suggesting the Y2-mediated anabolic pathway to be independent of sex hormones. Furthermore, the increase in fat mass that typically occurs after ovariectomy is prevented by germ line deletion of Y2 receptors, whereas in male mice body weight and fat mass were consistently lower than wild-type regardless of sex hormone status. Therefore, this study indicates a role for Y2 receptors in the accumulation of adipose tissue in the hypogonadal state and demonstrates that hypothalamic Y2 receptors constitutively restrain osteoblastic activity even in the absence of sex hormones. The increase in bone formation after release of this tonic inhibition suggests a promising new avenue for osteoporosis treatment.     

Mechanisms of sex steroid effects on bone

Sex steroids play a major role in the regulation of bone turnover. Thus, gonadectomy in either sex is associated with an increase in bone remodeling, increased bone resorption, and a relative deficit in bone formation, resulting in accelerated bone loss. Recent physiological studies have established an important role for estrogen in regulating bone turnover not only in females, but also in males. Studies in mice with knock out of the estrogen receptor, aromatase, or androgen receptor have provided important insights into the in vivo mechanisms of sex steroid action on bone. The cellular and molecular mediators of sex steroid effects on the bone-forming osteoblasts and bone-resorbing osteoclasts are also being increasingly better defined. Estrogen inhibits bone remodeling by concurrently suppressing osteoblastogenesis and osteoclastogenesis from marrow precursors. Both estrogen and androgens inhibit bone resorption via effects on the receptor activator of NF-kappaB ligand (RANKL)/RANK/osteoprotegerin system, as well as by reducing the production of a number of pro-resorptive cytokines, along with direct effects on osteoclast activity and lifespan. Sex steroid effects on bone formation are also likely mediated by multiple mechanisms, including a prolongation of osteoblast lifespan via non-genotropic mechanisms, as well as effects on osteoblast differentiation and function. These pleiotropic actions of sex steroids on virtually all aspects of bone metabolism belie the importance of the skeleton not only in providing structural support for the body and in locomotion, but also as a dynamic tissue responsive, among other things, to the reproductive needs of the organism for calcium.     

Oestrogen action on bone cells

Sex steroids have an important impact on bone physiology. Oestrogen (E) appears to be the most important sex steroid in preventing osteoporosis in women. Despite the overwhelming evidence that oestrogens modulate bone growth and turnover in vivo, oestrogen receptors (ER) were detected only recently. Two forms of ER have been discovered so far, ERalpha and ERbeta. Both have been detected in osteoblasts and osteoclasts as well. A number of growth factors and cytokines appear to modulate bone resorption in vitro and in vivo. Among others, interleukin-1 and -6 and tumor necrosis factor alpha and beta were found to be extremely potent stimulators of bone resorption. Binding of different cytokines to their receptors in osteoblasts result in the release of soluble factors that act directly on osteoclasts to modulate their recruitment or activity. Thus, E, apart from the direct regulation of osteoclasts, which it achieves through its receptors, can inhibit the release of osteoclast stimulatory factors or enhance the release of osteoclast inhibitory factors. In general, E is an inhibitor of bone resorption that decreases both osteoclast numbers and activity. Recently, it has also been shown that it promotes apoptosis. Moreover, it also has anabolic effects on osteoblasts. However, E action on osteoclasts is superior in comparison with that on osteoblasts. Recent data have shown that transforming growth factor beta (TGFbeta) mediates the actions of E in bone. Following the example of raloxifene it may be proved that the role of TGFbeta in the actions of E in bone is central and has not only academic interest. More data are needed to elucidate this issue. Finally, recent data suggest the importance of E for bone maturation and development of peak bone mass in men. It seems likely that both E and androgens are required for the growth and maintenance of the adult male skeleton.     

Inhibition of osteoblastic metalloproteinases in mice prevents bone loss induced by oestrogen deficiency

Matrix metalloproteinases (MMPs) are key mediators in extra-cellular matrix remodelling and implicated primarily in bone growth, and particularly in osteoclastic bone resorption. We hypothesise that MMPs have a role in the increased bone remodelling resulting from oestrogen deficiency. Transgenic (TG) mice overexpressing TIMP-1 in their osteoblastic cells and their wild-type (WT) littermates were ovariectomised. One month after surgery, bone mineral density (BMD) and bone microarchitecture were assessed. Primary cells from WT and TG mice were used to determine how TIMP-1 affects osteoclast and osteoblastic cells. The reduction of BMD induced by ovariectomy in WT mice was not observed in the transgenic mice. The transgene overexpression also dampened the post-ovariectomy increase in bone resorption in contrast to the WT mice. In vivo, osteoclastic surfaces and D-pyridinoline were not increased in TG mice, and ex vivo, the differentiation of osteoclasts from TG bone marrow precursor cells were unaffected by in vivo oestrogen deficiency or treatment. We showed also that TIMP-1 overexpression reduces and delays the osteoblastic proliferation and differentiation respectively, and reduced the generation of the active form of TGFbeta1 in the supernatant of TG osteoblasts. Our findings support the hypothesis that in vivo inhibition of osteoblastic MMPs prevented the bone loss induced by oestrogen deficiency, with a significant decrease in bone resorption. This effect was presumably resulting from (1) a direct inhibition of osteoclastic resorption activity by the TIMP-1 and (2) the modification in the local activation of extra-cellular signalling factors such as TGFbeta1 and the OPG/RANKL ratio.     

Morphometrics of compact bone: an example from Sudanese Nubia.

Morphometric analysis of compact femoral tissue was applied to a prehistoric population from Sudanese Nubia. Microradiographs of thin sections from below the lesser trochanter were examined. A total of 74 adults (40 females, 34 males) from the X-Group population (A.D. 350–550) were used to determine the underlying processes of bone remodeling in skeletal growth and maintenance. The relationship of bone turnover to the age of onset, patterning and frequency of cortical bone loss (osteoporosis) as a function of age and sex was examined. The cortical thickness, cortical area and formation/resorption frequencies were determined. Males exhibit a 4.9% net loss in cortical area, while females show a 10.7% loss. A substantial amount of female loss occurs in the third decade, with a slight gain in the fourth and a steady loss up to the sixth decade. These trends were further explored and refined histologically. The periosteal and endosteal frequencies for males show a variable but definite decrease in mineralization (i.e., increased numbers of osteons in the forming stage) and a slight increase in resorption. Third decade females show a marked difference with high frequencies of resorption spaces and forming osteons. It is suggested that in this population, stress related to childbearing and childrearing may be effecting the frequencies of formation foci and resorption spaces.     

Calcineurin/NFAT signaling in osteoblasts regulates bone mass

Development and repair of the vertebrate skeleton requires the precise coordination of bone-forming osteoblasts and bone-resorbing osteoclasts. In diseases such as osteoporosis, bone resorption dominates over bone formation, suggesting a failure to harmonize osteoclast and osteoblast function. Here, we show that mice expressing a constitutively nuclear NFATc1 variant (NFATc1(nuc)) in osteoblasts develop high bone mass. NFATc1(nuc) mice have massive osteoblast overgrowth, enhanced osteoblast proliferation, and coordinated changes in the expression of Wnt signaling components. In contrast, viable NFATc1-deficient mice have defects in skull bone formation in addition to impaired osteoclast development. NFATc1(nuc) mice have increased osteoclastogenesis despite normal levels of RANKL and OPG, indicating that an additional NFAT-regulated mechanism influences osteoclastogenesis in vivo. Calcineurin/NFATc signaling in osteoblasts controls the expression of chemoattractants that attract monocytic osteoclast precursors, thereby coupling bone formation and bone resorption. Our results indicate that NFATc1 regulates bone mass by functioning in both osteoblasts and osteoclasts.     

Inhibition of TGF-beta receptor signaling in osteoblasts leads to decreased bone remodeling and increased trabecular bone mass.

Transforming growth factor-beta (TGF-beta) is abundant in bone matrix and has been shown to regulate the activity of osteoblasts and osteoclasts in vitro. To explore the role of endogenous TGF-(beta) in osteoblast function in vivo, we have inhibited osteoblastic responsiveness to TGF-beta in transgenic mice by expressing a cytoplasmically truncated type II TGF-beta receptor from the osteocalcin promoter. These transgenic mice develop an age-dependent increase in trabecular bone mass, which progresses up to the age of 6 months, due to an imbalance between bone formation and resorption during bone remodeling. Since the rate of osteoblastic bone formation was not altered, their increased trabecular bone mass is likely due to decreased bone resorption by osteoclasts. Accordingly, direct evidence of reduced osteoclast activity was found in transgenic mouse skulls, which had less cavitation and fewer mature osteoclasts relative to skulls of wild-type mice. These bone remodeling defects resulted in altered biomechanical properties. The femurs of transgenic mice were tougher, and their vertebral bodies were stiffer and stronger than those of wild-type mice. Lastly, osteocyte density was decreased in transgenic mice, suggesting that TGF-beta signaling in osteoblasts is required for normal osteoblast differentiation in vivo. Our results demonstrate that endogenous TGF-beta acts directly on osteoblasts to regulate bone remodeling, structure and biomechanical properties.     

Tumor necrosis factors alpha and beta induce osteoblastic cells to stimulate osteoclastic bone resorption

Antigen- or mitogen-stimulated leukocytes release bone-resorbing activity into culture supernatants in vitro. Among the agents likely to be present in such supernatants are monocyte-derived tumor necrosis factor (TNF-alpha) and lymphocyte-derived tumor necrosis factor (TNF-beta) (lymphotoxin), both of which have recently been shown to stimulate bone resorption in organ culture. To identify the mechanism of action of these agents, we compared bone resorption by isolated osteoclasts with bone resorption by osteoclasts cocultured with osteoblastic cells, and with bone resorption by osteoclasts incubated with supernatants from osteoblastic cells, in the presence and absence of recombinant TNF-alpha and TNF-beta. We found that neither TNF-alpha nor TNF-beta had any significant effect on bone resorption by isolated osteoclasts, but in the presence of osteoblasts the agents caused a twofold to threefold stimulation of bone resorption. A similar degree of stimulation was achieved by supernatants from osteoblasts incubated with TNF before addition to osteoclasts, compared with supernatants to which TNF were added after osteoblast incubation. These experiments suggest that TNF-alpha and TNF-beta stimulate bone resorption through a primary effect on osteoblastic cells, which are induced by TNF to produce a factor that stimulates osteoclastic resorption. Half-maximal stimulation of resorption occurred at 1.5 X 10(-10) M and 2.5 X 10(-10) M for TNF-alpha and TNF-beta, respectively. This degree of potency is comparable to that of parathyroid hormone, the major physiologic systemic regulator of bone resorption, and suggests that the TNF may exert a significant influence on osteoclastic bone resorption in vivo.