[Show abstract][Hide abstract] ABSTRACT: A decline of the levels and activity of Sirtuin1 (Sirt1), a NAD+ class III histone deacetylase, with age contributes to the development of several diseases including type 2 diabetes, neurodegeneration, inflammation, and cancer. The anti-aging effects of Sirt1 evidently result from the deacetylation of many transcription factors and co-factors including the Forkhead Box O (FoxO) family and β-catenin. Wnt/β-catenin is indispensable for osteoblast generation. FoxOs, on the other hand, sequester β-catenin and inhibit osteoprogenitor proliferation. Here, we have deleted Sirt1 in osteoprogenitors expressing Osterix1 (Osx1)-Cre and their descendants. Sirt1ΔOsx1 mice had lower cortical thickness in femora and vertebrae because of reduced bone formation at the endosteal surface. In line with this, osteoprogenitor cell cultures from the Sirt1ΔOsx1 mice exhibited lower alkaline phosphatase activity and mineralization, as well as decreased proliferation and increased apoptosis. These changes were associated with decreased Wnt/β-catenin signaling and expression of cyclin D1 resulting from increased binding of FoxOs to β-catenin. Sirt1 deletion also enhanced FoxO-mediated transcription. These findings suggest that a decline in Sirt1 activity in osteoblast progenitors contributes to age-related bone loss. Furthermore, together with evidence that Sirt1 activators increase bone mass in aged mice, our results also suggest that Sirt1 is a potential therapeutic target for osteoporosis.
Journal of Biological Chemistry 07/2014; · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Major advances in understanding basic bone biology and the cellular and molecular mechanisms responsible for the development of osteoporosis, over the last 20 years, have dramatically altered the management of this disease. The purpose of this mini-review is to highlight the seminal role of Wnt signaling in bone homeostasis and disease and the emergence of novel osteoporosis therapies by targeting Wnt signaling with drugs.
[Show abstract][Hide abstract] ABSTRACT: Parathyroid hormone (PTH) excess stimulates bone resorption. This effect is associated with increased expression of the osteoclastogenic cytokine receptor activator of nuclear factor кB ligand (RANKL) in bone. However, several different cell types, including bone marrow stromal cells, osteocytes, and T lymphocytes, express both RANKL and the PTH receptor and it is unclear whether RANKL expression by any of these cell types is required for PTH-induced bone loss. Here we have used mice lacking the RANKL gene in osteocytes to determine whether RANKL produced by this cell type is required for the bone loss caused by secondary hyperparathyroidism induced by dietary calcium deficiency in adult mice. Thirty days of dietary calcium deficiency caused bone loss in control mice, but this effect was blunted in mice lacking RANKL in osteocytes. The increase in RANKL expression in bone and the increase in osteoclast number caused by dietary calcium deficiency were also blunted in mice lacking RANKL in osteocytes. These results demonstrate that RANKL produced by osteocytes contributes to the increased bone resorption and the bone loss caused by secondary hyperparathyroidism, strengthening the evidence that osteocytes are an important target cell for hormonal control of bone remodeling.
[Show abstract][Hide abstract] ABSTRACT: Besides their cell-damaging effects in the setting of oxidative stress, reactive oxygen species (ROS) play an important role in physiological intracellular signalling by triggering proliferation and survival. FoxO transcription factors counteract ROS generation by upregulating antioxidant enzymes. Here we show that intracellular H2O2 accumulation is a critical and purposeful adaptation for the differentiation and survival of osteoclasts, the bone cells responsible for the resorption of mineralized bone matrix. Using mice with conditional loss or gain of FoxO transcription factor function, or mitochondria-targeted catalase in osteoclasts, we demonstrate this is achieved, at least in part, by downregulating the H2O2-inactivating enzyme catalase. Catalase downregulation results from the repression of the transcriptional activity of FoxO1, 3 and 4 by RANKL, the indispensable signal for the generation of osteoclasts, via an Akt-mediated mechanism. Notably, mitochondria-targeted catalase prevented the loss of bone caused by loss of oestrogens, suggesting that decreasing H2O2 production in mitochondria may represent a rational pharmacotherapeutic approach to diseases with increased bone resorption.
[Show abstract][Hide abstract] ABSTRACT: Mouse models with cell-specific deletion of the estrogen receptor (ER) α, the androgen receptor (AR) or the receptor activator of nuclear factor κB ligand (RANKL), as well as cascade-selective estrogenic compounds have provided novel insights into the function and signalling of ERα and AR. The studies reveal that the effects of estrogens on trabecular versus cortical bone mass are mediated by direct effects on osteoclasts and osteoblasts, respectively. The protection of cortical bone mass by estrogens is mediated via ERα, using a non-nucleus-initiated mechanism. By contrast, the AR of mature osteoblasts is indispensable for the maintenance of trabecular bone mass in male mammals, but not required for the anabolic effects of androgens on cortical bone. Most unexpectedly, and independently of estrogens, ERα in osteoblast progenitors stimulates Wnt signalling and periosteal bone accrual in response to mechanical strain. RANKL expression in B lymphocytes, but not T lymphocytes, contributes to the loss of trabecular bone caused by estrogen deficiency. In this Review, we summarize this evidence and discuss its implications for understanding the regulation of trabecular and cortical bone mass; the integration of hormonal and mechanical signals; the relative importance of estrogens versus androgens in the male skeleton; and, finally, the pathogenesis and treatment of osteoporosis.
[Show abstract][Hide abstract] ABSTRACT: Iron uptake through the transferrin cycle is essential for osteoclast differentiation, and iron overload in a variety of hematologic diseases is associated with excessive bone resorption. However, the molecular mechanisms regulating cellular iron metabolism in osteoclasts remain largely unknown. In this report, we provide evidence that Steap4, a member of the six-transmembrane epithelial antigen of prostate (Steap) family proteins, is an endosomal ferrireductase with a critical role in cellular iron utilization in osteoclast lineage cells. Specifically, we show that Steap4 is the only Steap family protein that is up-regulated during osteoclast differentiation. Knocking down Steap4 expression in vitro by lentivirus-mediated short hairpin RNAs inhibits osteoclast formation and decreases cellular ferrous iron, reactive oxidative species, and the activation of CREB, a critical transcription factor downstream of RANKL-induced calcium signaling. We conclude that Steap4, working in concert with transferrin/transferrin receptor pathway, promotes cellular iron uptake and utilization to cope with the increased iron demand during osteoclast development and function.
Journal of Biological Chemistry 08/2013; · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Wnt/β-catenin/TCF signaling stimulates bone formation and suppresses adipogenesis. The hallmarks of skeletal involution with age, on the other hand, are decreased bone formation and increased bone marrow adiposity. These changes are associated with increased oxidative stress and decreased growth factor production, which activates members of the FOXO family of transcription factors. FOXOs in turn attenuate Wnt/β-catenin signaling by diverting β-catenin from TCF- to FOXO-mediated transcription. We show herein that mice lacking Foxo1, -3, and -4 in bipotential progenitors of osteoblast and adipocytes (expressing Osterix1) exhibited increased osteoblast number and high bone mass that was maintained in old age as well as decreased adiposity in the aged bone marrow. The increased bone mass in the Foxo-deficient mice was accounted for by increased proliferation of osteoprogenitor cells and bone formation resulting from upregulation of Wnt/β-catenin signaling and cyclin D1 expression, but not changes in redox balance. Consistent with this mechanism, β-catenin deletion in Foxo null cells abrogated both the increased cyclin D1 expression and proliferation. The elucidation of a restraining effect of FOXOs on Wnt signaling in bipotential progenitors suggests that FOXO activation by accumulation of age-associated cellular stressors may be a seminal pathogenetic mechanism in the development of involutional osteoporosis.
The Journal of clinical investigation 07/2013; · 15.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Skeletal aging is accompanied by decreased cancellous bone mass and increased formation of pores within cortical bone. The latter accounts for a large portion of the increase in non-vertebral fractures after age 65 in humans. We selectively deleted Bak and Bax, two genes essential for apoptosis, in two types of terminally differentiated bone cells: the short-lived osteoblasts that elaborate the bone matrix, and the long-lived osteocytes that are immured within the mineralized matrix and choreograph the regeneration of bone. Attenuation of apoptosis in osteoblasts increased their working lifespan and thereby cancellous bone mass in the femur. In long-lived osteocytes, however, it caused dysfunction with advancing age and greatly magnified intracortical femoral porosity associated with increased production of receptor activator of nuclear factor-κB ligand and vascular endothelial growth factor. Increasing bone mass by artificial prolongation of the inherent lifespan of short-lived osteoblasts, while exaggerating the adverse effects of aging on long-lived osteocytes, highlights the seminal role of cell age in bone homeostasis. In addition, our findings suggest that distress signals produced by old and/or dysfunctional osteocytes are the culprits of the increased intracortical porosity in old age.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 06/2013; · 6.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bone mass declines with age but the mechanisms responsible remain unclear. Here we demonstrate that deletion of a conditional allele for Atg7, a gene essential for autophagy, from osteocytes caused low bone mass in 6-month-old male and female mice. Cancellous bone volume and cortical thickness were decreased, and cortical porosity increased, in conditional knockout mice compared with control littermates. These changes were associated with low osteoclast number, osteoblast number, bone formation rate, and wall width in the cancellous bone of conditional knockout mice. In addition, oxidative stress was higher in the bones of conditional knockout mice as measured by reactive oxygen species levels in the bone marrow and by p66shc phosphorylation in L6 vertebra. Each of these changes has been previously demonstrated in the bones of old versus young adult mice. Thus, these results demonstrate that suppression of autophagy in osteocytes mimics, in many aspects, the impact of aging on the skeleton and suggest that a decline in autophagy with age may contribute to the low bone mass associated with aging.
Journal of Biological Chemistry 05/2013; · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Advances made during the last 35 years have improved our understanding of the mechanisms of steroid hormone action on bone and how physiologic, pathologic, or iatrogenic changes in hormone levels can lead to increased fracture risk. Estrogens, androgens, and glucocorticoids alter the cellular composition of bone by regulating the supply and lifespan of osteoclasts and osteoblasts. Additionally, they influence the survival of osteocytes, long-lived cells that are entombed within the mineralized matrix and mediate the homeostatic adaptation of bone to mechanical forces. Altered redox balance is a proximal underlying mechanism of some of these effects, and sex steroid deficiency or glucocorticoid excess contributes to the aging of the skeleton.
The Journal of clinical investigation 05/2013; 123(5):1919-21. · 15.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Extensive evidence has suggested that at least some of the effects of estrogens on bone are mediated via extranuclear estrogen receptor α signaling. However, definitive proof for this contention and the extent to which such effects may contribute to the overall protective effects of estrogens on bone maintenance have remained elusive. Here, we investigated the ability of a 17β-estradiol (E2) dendrimer conjugate (EDC), incapable of stimulating nuclear-initiated actions of estrogen receptor α, to prevent the effects of ovariectomy (OVX) on the murine skeleton. We report that EDC was as potent as an equimolar dose of E2 in preventing bone loss in the cortical compartment that represents 80% of the entire skeleton, but was ineffective on cancellous bone. In contrast, E2 was effective in both compartments. Consistent with its effect on cortical bone mass, EDC partially prevented the loss of both vertebral and femoral strength. In addition, EDC, as did E2, prevented the OVX-induced increase in osteoclastogenesis, osteoblastogenesis, and oxidative stress. Nonetheless, the OVX-induced decrease in uterine weight was unaltered by EDC but was restored by E2. These results demonstrate that the protection of cortical bone mass by estrogens is mediated, at least in part, via a mechanism that is distinct from the classic mechanism of estrogen action on reproductive organs.
[Show abstract][Hide abstract] ABSTRACT: The detection of estrogen receptor-α (ERα) in osteoblasts and osteoclasts over 20 years ago suggested that direct effects of estrogens on both of these cell types are responsible for their beneficial effects on the skeleton, but the role of ERα in osteoblast lineage cells has remained elusive. In addition, estrogen activation of ERα in osteoclasts can only account for the protective effect of estrogens on the cancellous, but not the cortical, bone compartment that represents 80% of the entire skeleton. Here, we deleted ERα at different stages of differentiation in murine osteoblast lineage cells. We found that ERα in osteoblast progenitors expressing Osterix1 (Osx1) potentiates Wnt/β-catenin signaling, thereby increasing proliferation and differentiation of periosteal cells. Further, this signaling pathway was required for optimal cortical bone accrual at the periosteum in mice. Notably, this function did not require estrogens. The osteoblast progenitor ERα mediated a protective effect of estrogens against endocortical, but not cancellous, bone resorption. ERα in mature osteoblasts or osteocytes did not influence cancellous or cortical bone mass. Hence, the ERα in both osteoblast progenitors and osteoclasts functions to optimize bone mass but at distinct bone compartments and in response to different cues.
The Journal of clinical investigation 12/2012; · 15.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Osteocytes are long-lived and far more numerous than the short-lived osteoblasts and osteoclasts. Immured within the lacunar-canalicular system and mineralized matrix, osteocytes are ideally located throughout the bone to detect the need for, and accordingly choreograph, the bone regeneration process by independently controlling rate limiting steps of bone resorption and formation. Consistent with this role, emerging evidence indicates that signals arising from apoptotic and old/or dysfunctional osteocytes are seminal culprits in the pathogenesis of involutional, post-menopausal, steroid-, and immobilization-induced osteoporosis. Osteocyte-originated signals may also contribute to the increased bone fragility associated with bone matrix disorders like osteogenesis imperfecta, and perhaps the rapid reversal of bone turnover above baseline following discontinuation of anti-resorptive treatments, like denosumab. This article is part of a Special Issue entitled "Osteocyte".
[Show abstract][Hide abstract] ABSTRACT: Production of the cytokine receptor activator of NFκB ligand (RANKL) by lymphocytes has been proposed as a mechanism by which sex steroid deficiency causes bone loss. However, there have been no studies that functionally link RANKL expression in lymphocytes with bone loss in this condition. Herein, we examined whether RANKL expression in either B or T lymphocytes contributes to ovariectomy-induced bone loss in mice. Mice harboring a conditional RANKL allele were crossed with CD19-Cre or Lck-Cre mice to delete RANKL in B or T lymphocytes, respectively. Deletion of RANKL from either cell type had no impact on bone mass in estrogen-replete mice up to 7 months of age. However, mice lacking RANKL in B lymphocytes were partially protected from the bone loss caused by ovariectomy. This protection occurred in cancellous, but not cortical, bone and was associated with a failure to increase osteoclast numbers in the conditional knock-out mice. Deletion of RANKL from T lymphocytes had no impact on ovariectomy-induced bone loss. These results demonstrate that lymphocyte RANKL is not involved in basal bone remodeling, but B cell RANKL does contribute to the increase in osteoclasts and cancellous bone loss that occurs after loss of estrogen.
Journal of Biological Chemistry 07/2012; 287(35):29851-60. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Endogenous glucocorticoids (GCs) and inflammatory cytokines contribute to the age-associated loss of bone mass and strength, but the molecular mechanisms responsible for their deleterious effects on the aging skeleton are unclear. Based on evidence that oxidative stress is a causal mechanism of the insulin resistance produced by either one of these two agents, we tested the hypothesis that their adverse skeletal effects also result from increased oxidative stress. We report that administration of prednisolone to mice increased reactive oxygen species (ROS) and the phosphorylation of p66(shc) (an amplifier of H(2)O(2) generation in mitochondria) in bone. Dexamethasone (Dex) and TNFα had a similar effect on osteoblastic cells in vitro. The generation of ROS by Dex and TNFα required PKCβ/p66(shc) signaling and was responsible for the activation of JNK and induction of apoptosis by both agents. The activity of Forkhead box O (FoxO) transcription factors was also increased in response to ROS; however, FoxO activation opposed apoptosis induced by Dex and TNFα. In addition, both agents suppressed Akt phosphorylation as well as Wnt-induced proliferation and osteoblast differentiation. However, the inhibitory actions on Wnt signaling were independent of PKCβ/p66(shc). Instead, they were mediated by inhibition of Akt and stimulation of FoxOs. These results demonstrate that ROS-induced activation of a PKCβ/p66(shc)/JNK signaling cascade is responsible for the pro-apoptotic effects of Dex and TNFα on osteoblastic cells. Moreover, modulation of Akt and FoxOs by GCs and TNFα are cell-autonomous mechanisms of Wnt/β-catenin antagonism contributing to the adverse effects of GC excess and inflammatory cytokines on bone alike.
Journal of Biological Chemistry 12/2011; 286(52):44326-35. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Osteoclast-mediated bone resorption plays an essential role in calcium homeostasis and lactation. The cytokine receptor activator of nuclear factor κB ligand (RANKL) is one of a number of factors that controls the production, survival, and activity of osteoclasts. Calciotropic hormones, such as PTH, control RANKL transcription in part via an enhancer known as the distal control region (DCR), and mice lacking this enhancer have fewer osteoclasts under normal physiological conditions. Here, we have addressed the role of the DCR in situations in which activation of the PTH receptor is thought to stimulate bone resorption via elevation of RANKL expression. Dietary calcium deficiency stimulated RANKL expression in the bone of young (1 month old) wild-type, but not DCR knockout (KO), mice. Consistent with this, the cancellous bone loss and the increase in osteoclasts caused by dietary calcium deficiency were blunted in young KO mice. DCR deletion also prevented the increase in RANKL expression caused by dietary calcium deficiency in 6-month-old mice. However, the diet-induced bone loss was similar in wild-type and KO mice at this age. The increase in RANKL expression caused by lactation was also blunted in DCR KO mice, but lactation-induced bone loss was similar in both genotypes. These results demonstrate that, even though the DCR is required for the increase in RANKL expression associated with hyperparathyroidism or lactation, this increase is not required for the bone loss caused by these conditions in adult mice, suggesting that changes in other factors, such as osteoprotegerin or estrogen levels, play a dominant role.