[Show abstract][Hide abstract] ABSTRACT: The cytokine receptor activator of nuclear factor kappa B ligand (RANKL), encoded by the Tnfsf11 gene, is essential for osteoclastogenesis and previous studies have shown that deletion of the Tnfsf11 gene using a Dmp1-Cre transgene reduces osteoclast formation in cancellous bone by more than 70%. However, the Dmp1-Cre transgene used in those studies leads to recombination in osteocytes, osteoblasts, and lining cells making it unclear whether one or more of these cell types produce the RANKL required for osteoclast formation in cancellous bone. Because osteoblasts, osteocytes, and lining cells have distinct locations and functions, distinguishing which of these cell types are sources of RANKL is essential for understanding the orchestration of bone remodeling. To distinguish between these possibilities, we have now created transgenic mice expressing the Cre recombinase under the control of regulatory elements of the Sost gene, which is expressed in osteocytes but not osteoblasts or lining cells in murine bone. Activity of the Sost-Cre transgene in osteocytes, but not osteoblast or lining cells, was confirmed by crossing Sost-Cre transgenic mice with tdTomato and R26R Cre-reporter mice, which express tdTomato fluorescent protein or LacZ, respectively, only in cells expressing the Cre recombinase or their descendants. Deletion of the Tnfsf11 gene in Sost-Cre mice led to a threefold decrease in osteoclast number in cancellous bone and increased cancellous bone mass, mimicking the skeletal phenotype of mice in which the Tnfsf11 gene was deleted using the Dmp1-Cre transgene. These results demonstrate that osteocytes, not osteoblasts or lining cells, are the main source of the RANKL required for osteoclast formation in remodeling cancellous bone.
PLoS ONE 09/2015; 10(9):e0138189. DOI:10.1371/journal.pone.0138189 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Activation of Sirtuin1 (Sirt1) - an NAD(+) dependent deacetylase - by natural or synthetic compounds like resveratrol, SRT2104, or SRT3025 attenuates the loss of bone mass caused by ovariectomy, aging, or unloading in mice. Conversely, Sirt1 deletion in osteoclast progenitors increases osteoclast number and bone resorption. Sirt1 deacetylates FoxO1, 3, and 4 and thereby modulates their activity. FoxOs restrain osteoclastogenesis and bone resorption. Here, we tested the hypothesis that the anti-resorptive effects of Sirt1 are mediated by FoxOs. We report that Sirt1 activation by SRT2104 and SRT3025 inhibited murine osteoclast progenitor proliferation and reduced osteoclastogenesis. The effect of Sirt1 stimulators on osteoclastogenesis was abrogated in cells lacking FoxO1, 3, and 4. FoxO1 acetylation was increased by knocking-down Sirt1 or addition of RANKL - the critical cytokine for osteoclast differentiation. Furthermore, acetylation inhibited while deacetylation promoted FoxO-mediated transcription. SRT3025 increased the expression of the FoxO-target genes catalase and hemeoxygenase-1 (HO-1) in osteoclast progenitors, in a FoxO-dependent manner. HO-1 catabolizes heme and attenuates mitochondrial oxidative phosphorylation and ATP production in macrophages. HO-1 levels were strongly reduced and ATP levels increased by RANKL. In contrast, SRT3025 and FoxOs decreased ATP production, and the effect of SRT3025 was mediated by FoxOs. These findings reveal that the anti-osteoclastogenic actions of Sirt1 are mediated by FoxOs and result from impaired mitochondria activity. Along with earlier findings that the osteoblastogenic effects of Sirt1 are also mediated by FoxOs, these results establish that the dual anti-osteoporotic efficacy of Sirt1 stimulators (i.e. decreasing bone resorption and promoting bone formation) is mediated via FoxO deacetylation.
[Show abstract][Hide abstract] ABSTRACT: On May 18, 2015, the Bone and Mineral Community lost Alwyn Michael Parfitt, M.D., one of its giants. Michael's career spanned over fifty years and three different continents and established him as mentor of the field and one of its most transformative figures. His contribution to bone research is unique in its breadth and depth. It is characterized by rigorous scholarship; passion for the subject; refusal to accept established dogma without critical reappraisal; and the ability to generate new concepts that often changed thinking and opened new avenues of exploration.
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Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 06/2015; 30(8). DOI:10.1002/jbmr.2576 · 6.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In men, androgens are critical for the acquisition and maintenance of bone mass in both the cortical and cancellous bone compartment. Male mice with targeted deletion of AR in mature osteoblasts or osteocytes have lower cancellous bone mass, but no cortical bone phenotype. We have investigated the possibility that the effects of androgens on the cortical compartment result from AR signaling in osteo-progenitors or cells of the osteoclast lineage; or via ERα signaling in either or both of these two cell types upon conversion of testosterone to estradiol. To this end, we generated mice with targeted deletion of an AR or an ERα allele in the mesenchymal (AR(f/y) ;Prx1-Cre or ERα(f/f) ;Osx1-Cre) or myeloid cell lineage (AR(f/y) ;LysM-Cre or ERα(f/f) ;LysM-Cre) and their descendants. Male AR(f/y) ;Prx1-Cre mice exhibited decreased bone volume and trabecular number, and increased osteoclast number in the cancellous compartment. Moreover, they did not undergo the loss of cancellous bone volume and trabecular number caused by orchidectomy (ORX) in their littermate controls. In contrast, AR(f/y) ;LysM-Cre, ERα(f/f) ;Osx1-Cre, or ERα(f/f) ;LysM-Cre mice had no cancellous bone phenotype at baseline and lost the same amount of cancellous bone as their controls following ORX. Most unnexpectedly, adult males of all four models had no discernable cortical bone phenotype at baseline, and lost the same amount of cortical bone as their littermate controls following ORX. Recapitulation of the effects of ORX by AR deletion only in the AR(f/y) ;Prx1-Cre mice indicates that the effects of androgens on cancellous bone result from AR-signaling in osteoblasts - not on osteoclasts or via aromatization. The effects of androgens on cortical bone mass, on the other hand, do not require AR or ERα signaling in any cell type across the osteoblast or osteoclast differentiation lineage. Therefore, androgens must exert their effects indirectly by actions on some other cell type(s) or tissue(s). This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 02/2015; 30(7). DOI:10.1002/jbmr.2485 · 6.83 Impact Factor
[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 members of 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 endocortical 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 and resulted from increased
binding of FoxOs to β-catenin. These findings demonstrate that Sirt1-induced deacetylation of FoxOs unleashes Wnt signaling.
A decline in Sirt1 activity in osteoblast progenitors with aging may, therefore, contribute to the age-related loss of bone
mass. Together with evidence that Sirt1 activators increase bone mass in aged mice, our results also suggest that Sirt1 could
be a therapeutic target for osteoporosis.
[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.
Bone 06/2014; 66. DOI:10.1016/j.bone.2014.06.006 · 3.97 Impact Factor
[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: 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 01/2014; 29(1). DOI:10.1002/jbmr.2007 · 6.83 Impact Factor
[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 is essential for osteoclast differentiation, and iron overload in a variety of hematologic diseases is associated with
excessive bone resorption. Iron uptake by osteoclast precursors via the transferrin cycle increases mitochondrial biogenesis,
reactive oxygen species production, and activation of cAMP response element-binding protein, a critical transcription factor
downstream of receptor activator of NF-κB-ligand-induced calcium signaling. These changes are required for the differentiation
of osteoclast precursors to mature bone-resorbing osteoclasts. 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 osteoclasts. 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 oxygen
species, and the activation of cAMP response element-binding protein. These results demonstrate that Steap4 is a critical
enzyme for cellular iron uptake and utilization in osteoclasts and, thus, indispensable for osteoclast development and function.
[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; 123(8). DOI:10.1172/JCI68049 · 13.22 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 knock-out 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 knock-out mice. In addition, oxidative stress was higher in the
bones of conditional knock-out 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
[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. DOI:10.1172/JCI68062 · 13.22 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.