Mechanical stimulation of cultured osteocytic cells attenuates their apoptosis. We report here that, conversely, reduced mechanical forces in the murine model of unloading by tail suspension increases the prevalence of osteocyte apoptosis, followed by bone resorption and loss of mineral and strength.Introduction: Mechanical loading is critical for the maintenance of bone mass; weightlessness, as with reduced physical activity in old age, bed rest, or space flight, invariably leads to bone loss. However, the cellular and molecular mechanisms responsible for these phenomena are poorly understood. Based on our earlier findings that physiologic levels of mechanical strain prevent apoptosis of osteocytic cells in vitro, we examined here whether, conversely, reduced mechanical forces increase the prevalence of osteocyte apoptosis in vivo and whether this event is linked to bone loss. Materials and Methods: Swiss Webster mice or OG2-11␤-hydroxysteroid dehydrogenase type 2 (OG2-11␤-HSD2) transgenic mice and wildtype littermates were tail-suspended or kept under ambulatory conditions. Static and dynamic histomorphometry and osteocyte and osteoblast apoptosis by in situ end-labeling (ISEL) were assessed in lumbar vertebra; spinal BMD was measured by DXA; and bone strength was measured by vertebral compression. Results: We show that within 3 days of tail suspension, mice exhibited an increased incidence of osteocyte apoptosis in both trabecular and cortical bone. This change was followed 2 weeks later by increased osteoclast number and cortical porosity, reduced trabecular and cortical width, and decreased spinal BMD and vertebral strength. Importantly, whereas in ambulatory animals, apoptotic osteocytes were randomly distributed, in unloaded mice, apoptotic osteocytes were preferentially sequestered in endosteal cortical bone-the site that was subsequently resorbed. The effect of unloading on osteocyte apoptosis and bone resorption was reproduced in transgenic mice in which osteocytes are refractory to glucocorticoid action, indicating that stressinduced hypercortisolemia cannot account for these effects. Conclusions: We conclude that diminished mechanical forces eliminate signals that maintain osteocyte viability, thereby leading to apoptosis. Dying osteocytes in turn become the beacons for osteoclast recruitment to the vicinity and the resulting increase in bone resorption and bone loss.

Osteocytes, former osteoblasts entombed in the bone matrix, form an extensive cell communication network that is thought to detect microdamage and mechanical strains and to transmit signals leading to repair and compensatory bone augmentation or reduction. Bone active hormones and drugs control the integrity of this network by regulating osteocyte apoptosis, which might be a determinant of bone strength. Herein we demonstrate that mechanical stimulation by stretching activates the ERKs, which in turn are responsible for the attenuation of osteocyte apoptosis. The effect of osteocyte stretching is transmitted by integrins and cytoskeletal and catalytic molecules, such as Src kinases. Stretch-induced antiapoptosis also requires nuclear translocation of ERKs and new gene transcription. The evidence linking mechanical stimulation, activation of an integrin/cytoskeleton/Src/ERK signaling pathway, and osteocyte survival provides a mechanistic basis for the profound role of mechanical forces, or lack thereof, on skeletal health and disease.

Nitric oxide (NO) has been implicated in the local regulation of bone metabolism. However, the contribution made by specific NO synthase (NOS) enzymes is unclear. Here we show that endothelial NOS gene knockout mice (eNOS؊/؊) have marked abnormalities in bone formation. Histomorphometric analysis of eNOS؊/؊ femurs showed bone volume and bone formation rate was reduced by up to 45% (P < 0.01) and 52% (P < 0.01), respectively. These abnormalities were prevalent in young (6 to 9 weeks old) adults but by 12 to 18 weeks bone phenotype was restored toward wild-type. Dual energy X-ray absorptiometry analysis confirmed the age-related bone abnormalities revealing significant reductions in femoral (P < 0.05) and spinal bone mineral densities (P < 0.01) at 8 weeks that were normalized at 12 weeks. Reduction in bone formation and volume was not related to increased osteoclast numbers or activity but rather to dysfunctional osteoblasts. Osteoblast numbers and mineralizing activity were reduced in eNOS؊/؊ mice. In vitro, osteoblasts from calvarial explants showed retarded proliferation and differentiation (alkaline phosphatase activity and mineral deposition) that could be restored by exogenous administration of a NO donor. These cells were also unresponsive to 17␤-estradiol and had an attenuated chemotactic response to transforming growth factor-␤. Bone is a vital dynamic connective tissue that has evolved to maintain a balance between its two major functions: provision of mechanical integrity for locomotion and modulation and control of mineral homeostasis. 1 Mineralized bone is continuously resorbed by osteoclasts and new bone is formed by osteoblasts. This process, known as bone remodeling, is highly regulated with maintenance of normal integrity and structure. 2 Systemic hormones including calcitonin, parathyroid hormone, and sex steroids, particularly estrogen, are known to be important regulators of bone cell function. Their effects on bone turnover are in general exerted by activation of local mediators and second messengers present within bone cells. 3 Recent investigations have focused on the role of nitric oxide (NO) as one of these possible local regulators of bone metabolism and bone cell activity. NO is a shortlived radical gas generated from L-arginine by nitric oxide synthase (NOS) isoenzymes. 4 Three distinct isoforms of NOS have been identified: a neuronal form (type I; nNOS) originally isolated from brain, 5 an endothelial form (type III; eNOS) originally isolated from bovine aortic endothelial cells, 6 and an inducible form (type II; iNOS) originally isolated from murine macrophages. 7 Both eNOS and nNOS are expressed constitutively and are characterized by highly regulated rapid but low-output NO production. 4 In contrast the iNOS pathway is generally only activated after stimulation by certain pro-inflammatory cytokines such as interferon-␥, interleukin-1␤, and tumor necrosis factor-␣. The inducible NOS isoform is characterized by production of persistent and high concentrations of NO. 8 There is now am...

Though osteonecrosis of the jaw (ONJ) is temporally-associated with the use of nitrogen-containing bisphosphonates (N-BPs), a cause/effect relationship has not yet been established. We hypothesize that ONJ is a two-stage process in which: a) risk factors initiate pathologic processes in the oral cavity that lead to a supranormal rate of hard tissue necrosis, and b) powerful anti-resorptives reduce the rate of removal of necrotic bone sufficiently to allow its net accumulation in the jaw. To test this hypothesis, we used the rice rat model of periodontitis. At age 28 days, rats (n=15/group) were placed on a high sucrose and casein diet to exacerbate the development of periodontitis. Animals were injected SC biweekly with vehicle or alendronate (ALN, 15μg/kg), or IV once monthly with vehicle, a low dose (LD), or a high dose (HD) of zoledronic acid (ZOL) and sacrificed after 6, 12, 18, and 24 wks. Mandibles and maxillae were analyzed to determine the effects on the: a) progression of periodontitis, b) integrity of alveolar bone, c) status of bone resorption and formation, d) vascularity, and e) osteocyte viability. We found that only HD-ZOL induced ONJ-like lesions in mandibles of rice rats after 18 and 24 wks of treatment. These lesions were characterized by areas of exposed necrotic alveolar bone, osteolysis, a honey comb-like appearance of the alveolar bone, presence of bacterial colonies, and periodontal tissue destruction. In addition, inhibition of bone formation, a paradoxical abolition of the antiresorptive effect of only HD-ZOL, increased osteocyte necrosis/apoptosis, and decreased blood vessel number were found after 18 and/or 24 wks. Our study suggests that only HD-ZOL exacerbates the inflammatory response and periodontal tissue damage in rice rats, inducing bone lesions that resemble ONJ.

Both estrogens and bisphosphonates attenuate osteocyte apoptosis by activating the extracellular signalregulated kinases (ERKs). However, whereas estrogens activate ERKs via an extranuclear function of the estrogen receptor, bisphosphonates do so by opening connexin 43 hemichannels. Here, we demonstrated that the signaling events downstream of ERKs induced by these two stimuli are also distinct. Inhibition of osteocyte apoptosis by estrogens requires nuclear accumulation of ERKs and activation of downstream transcription factors. On the other hand, anti-apoptosis induced by bisphosphonates requires neither transcription nor ERKdependent transcription factors. Instead, the effect of bisphosphonates is abolished when ERKs are restricted to the nucleus by blocking CRM1/exportin1-mediated nuclear protein export or by expressing nuclear-anchored ERKs, but it is unaffected in cells expressing cytoplasmic-anchored ERKs. Connexin 43/ERK-mediated anti-apoptosis induced by bisphosphonates requires the kinase activity of the cytoplasmic target of ERKs, p90 RSK , which in turn phosphorylates the proapoptotic protein BAD and C/EBP␤. Phosphorylation of BAD renders it inactive, whereas phosphorylation of C/EBP␤ leads to binding of pro-caspases, thus inhibiting apoptosis independently of the transcriptional activity of this transcription factor. Consistent with the evidence that estrogens and bisphosphonates phosphorylate diverse targets of ERKs, probably resulting from activation of spatially distinct pools of these kinases, the two agents had additive effects on osteocyte survival.Previous work from our group (1, 2) demonstrates that bisphosphonates prevent apoptosis of osteocytes and other cells of the osteoblastic lineage via the opening of hemichannels formed by connexin (Cx) 1 43 leading to activation of Src and the extracellular signal-regulated kinases (ERKs). Remarkably, Cx43 (but not other connexins) confers de novo responsiveness to these agents to connexin-naïve cells. The ability of Cx43 to transduce pro-survival signals requires both the pore-forming domain of Cx43 and the C-terminal portion of the protein, which is physically associated with the kinase Src (3, 4). Src activation and its interaction with Cx43 are indispensable for the anti-apoptotic effects of bisphosphonates (2).Similar to bisphosphonates, ligand-induced activation of sex steroid receptors also attenuates osteocyte and osteoblast apoptosis by a mechanism that requires activation of ERKs and Src. However, the molecular events that lead to activation of the Src/ERK pathway by estrogens or androgens are different from the ones involved in bisphosphonate action. Thus, either class of sex steroids activate ERKs via an extranuclear function of their receptors that can be mediated by the ligand binding domain of the proteins (5). In addition, kinase-dependent activation of transcription factors and gene transcription is required for the anti-apoptotic effect of sex steroids on osteoblastic cells (6). Heretofore, the mechanism by which bisphosphonates pr...

Sex steroids exert anti-apoptotic effects on osteoblasts/osteocytes but exert pro-apoptotic effects on osteoclasts, in both cases requiring activation of the extracellular signal-regulated kinases (ERKs). To explain the mechanistic basis of this divergence, we searched for differences in the kinetics of phosphorylation and/or in the subcellular localization of ERKs in response to 17␤-estradiol in the two cell types. In contrast to its transient effect on ERK phosphorylation in osteocytic cells (return to base line by 30 min), 17␤-estradiol-induced ERK phosphorylation in osteoclasts was sustained for at least 24 h following exposure to the hormone. Conversion of sustained ERK phosphorylation to transient, by means of cholera toxin-induced activation of the adenylate cyclase/cAMP/protein kinase A pathway, abrogated the pro-apoptotic effect of 17␤-estradiol on osteoclasts. Conversely, prolongation of ERK activation in osteocytes, by means of leptomycin B-induced inhibition of ERK export from the nucleus or overexpression of a green fluorescent protein-ERK2 mutant that resides permanently in the nucleus, converted the anti-apoptotic effect of 17␤-estradiol to a pro-apoptotic one. These findings indicate that the kinetics of ERK phosphorylation and the length of time that phospho-ERKs are retained in the nucleus are responsible for pro-versus anti-apoptotic effects of estrogen on different cell types of bone and perhaps their many other target tissues.Sex steroids prevent bone loss by suppressing the rate of bone turnover and maintaining a focal balance between bone formation and resorption (1-3). Suppression of bone turnover results from the attenuating effects of sex steroids on the birth rate of osteoblast and osteoclast progenitors. Maintenance of a focal balance between formation and resorption results from opposing effects on the life span of osteoblasts and osteoclasts, an anti-apoptotic effect on the former and a pro-apoptotic effect on the latter cell type (1, 4). As in osteoblasts, sex steroids exert anti-apoptotic effects on osteocytes, which are former osteoblasts entombed in the mineralized bone matrix and forming an extensive cell communication network that perceives and responds to mechanical strains by compensatory bone augmentation and reduction (5). A shortening of the life span of boneforming cells in combination with prolongation of the life span of bone-resorbing cells represent critical pathophysiologic changes in most acquired metabolic bone diseases, including the osteoporosis that results not only from sex steroid deficiency but also from glucocorticoid excess or old age (4, 6 -9). Interestingly, other agents, such as parathyroid hormone and bisphosphonates, used commonly for the treatment of metabolic bone diseases, exert their beneficial effects on bone by regulating the rate of birth of new osteoblasts or osteoclasts or their apoptosis (10 -12).Earlier work from our group has elucidated that the antiapoptotic effect of estrogens or androgens on osteoblasts results from nongenotropic mechan...

Inhibition of sclerostin with sclerostin antibody (Scl-Ab) has been shown to stimulate bone formation, decrease bone resorption, and increase bone mass in both animals and humans. To obtain insight into the temporal cellular and transcriptional changes in the osteoblast (OB) lineage associated with long-term Scl-Ab treatment, stereological and transcriptional analyses of the OB lineage were performed on lumbar vertebrae from aged ovariectomized rats. Animals were administered Scl-Ab 3 or 50mg/kg/wk or vehicle (VEH) for up to 26weeks (d183), followed by a treatment-free period (TFP). At 50mg/kg/wk, bone volume (BV/total volume [TV]) increased through d183 and declined during the TFP. Bone formation rate (BFR/bone surface [BS]) and total OB number increased through d29, then progressively declined, coincident with a decrease in total osteoprogenitor (OP) numbers from d29 through d183. Analysis of differentially expressed genes (DEGs) from microarray analysis of mRNA isolated from laser capture microdissection samples enriched for OB, lining cells, and osteocytes (OCy) revealed modules of genes that correlated with BFR/BS, BV/TV, and osteoblastic surface (Ob.S)/BS. Expression change of canonical Wnt target genes was similar in all three cell types at d8, including upregulation of Twist1 and Wisp1. At d29, the pattern of Wnt target gene expression changed in the OCy, with Twist1 returning to VEH level, sustained upregulation of Wisp1, and upregulation of several other Wnt targets that continued into the TFP. Predicted activation of pathways recognized to integrate with and regulate canonical Wnt signaling were also activated at d29 in the OCy. The most significantly affected pathways represented transcription factor signaling known to inhibit cell cycle progression (notably p53) and mitogenesis (notably c-Myc). These changes occurred at the time of peak BFR/BS and continued as BFR/BS declined during treatment, then trended toward VEH level in the TFP. Concurrent with this transcriptional switch was a reduction in OP numbers, an effect that would ultimately limit bone formation. This study confirms that the initial transcriptional response in response to Scl-Ab is activation of canonical Wnt signaling and the data demonstrate that there is induction of additional regulatory pathways in OCy with long-term treatment. The interactions between Wnt and p53/c-Myc signaling may be key in limiting OP populations, thus contributing to self-regulation of bone formation with continued Scl-Ab administration.

Alendronate transiently decreases bone formation and vascularity in the extraction socket and delays the removal of interdental alveolar bone after tooth extraction in rats.