Bone remodeling is performed by osteoclasts and osteoblasts at the bone surface. Inside of bone is a network of numerous osteocytes, whose specific function has remained an enigma. Here we describe a transgenic mouse model in which inducible and specific ablation of osteocytes is achieved in vivo through targeted expression of diphtheria toxin (DT) receptor. Following a single injection of DT, approximately 70%-80% of the osteocytes, but apparently no osteoblasts, were killed. Osteocyte-ablated mice exhibited fragile bone with intracortical porosity and microfractures, osteoblastic dysfunction, and trabecular bone loss with microstructural deterioration and adipose tissue proliferation in the marrow space, all of which are hallmarks of the aging skeleton. Strikingly, these "osteocyte-less" mice were resistant to unloading-induced bone loss, providing evidence for the role of osteocytes in mechanotransduction. Thus, osteocytes represent an attractive target for the development of diagnostics and therapeutics for bone diseases, such as osteoporosis.
The mammalian Chk2 kinase is thought to mediate ATM‐dependent signaling in response to DNA damage. The physiological role of mammalian Chk2 has now been investigated by the generation of Chk2‐deficient mice. Although Chk2−/− mice appeared normal, they were resistant to ionizing radiation (IR) as a result of the preservation of splenic lymphocytes. Thymocytes and neurons of the developing brain were also resistant to IR‐induced apoptosis. The IR‐induced G1/S cell cycle checkpoint, but not the G2/M or S phase checkpoints, was impaired in embryonic fibroblasts derived from Chk2−/− mice. IR‐induced stabilization of p53 in Chk2−/− cells was 50–70% of that in wild‐type cells. Caffeine further reduced p53 accumulation, suggesting the existence of an ATM/ATR‐dependent but Chk2‐independent pathway for p53 stabilization. In spite of p53 protein stabilization and phosphorylation of Ser23, p53‐dependent transcriptional induction of target genes, such as p21 and Noxa, was not observed in Chk2−/− cells. Our results show that Chk2 plays a critical role in p53 function in response to IR by regulating its transcriptional activity as well as its stability.
Osteoclasts are acid-secreting polykaryons that have high energy demands and contain abundant mitochondria. How mitochondrial biogenesis is integrated with osteoclast differentiation is unknown. We found that the transcription of Ppargc1b, which encodes peroxisome proliferator-activated receptor-gamma coactivator 1beta (PGC-1beta), was induced during osteoclast differentiation by cAMP response element-binding protein (CREB) as a result of reactive oxygen species. Knockdown of Ppargc1b in vitro inhibited osteoclast differentiation and mitochondria biogenesis, whereas deletion of the Ppargc1b gene in mice resulted in increased bone mass due to impaired osteoclast function. We also observed defects in PGC-1beta-deficient osteoblasts. Owing to the heightened iron demand in osteoclast development, transferrin receptor 1 (TfR1) expression was induced post-transcriptionally via iron regulatory protein 2. TfR1-mediated iron uptake promoted osteoclast differentiation and bone-resorbing activity, associated with the induction of mitochondrial respiration, production of reactive oxygen species and accelerated Ppargc1b transcription. Iron chelation inhibited osteoclastic bone resorption and protected against bone loss following estrogen deficiency resulting from ovariectomy. These data establish mitochondrial biogenesis orchestrated by PGC-1beta, coupled with iron uptake through TfR1 and iron supply to mitochondrial respiratory proteins, as a fundamental pathway linked to osteoclast activation and bone metabolism.
Phosphorylation of IkappaB by the IkappaB kinase (IKK) complex is a critical step leading to IkappaB degradation and activation of transcription factor NF-kappaB. The IKK complex contains two catalytic subunits, IKKalpha and IKKbeta, the latter being indispensable for NF-kappaB activation by pro-inflammatory cytokines. Although IKK is activated by phosphorylation of the IKKbeta activation loop, the physiological IKK kinases that mediate responses to extracellular stimuli remain obscure. Here we describe an IKK-related kinase, named NAK (NF-kappaB-activating kinase), that can activate IKK through direct phosphorylation. NAK induces IkappaB degradation and NF-kappaB activity through IKKbeta. Endogenous NAK is activated by phorbol ester tumour promoters and growth factors, whereas catalytically inactive NAK specifically inhibits activation of NF-kappaB by protein kinase C-epsilon (PKCepsilon). Thus, NAK is an IKK kinase that may mediate IKK and NF-kappaB activation in response to growth factors that stimulate PKCepsilon activity.
Members of the Foxo family, Foxo1 (Fkhr), Foxo3 (Fkhrl1), and Foxo4 (Afx), are mammalian homologs of daf-16, which influences life span and energy metabolism in Caenorhabditis elegans. Mammalian FOXO proteins also play important roles in cell cycle arrest, apoptosis, stress resistance, and energy metabolism. In this study, we generated Foxo1-deficient mice to investigate the physiological role of FOXO1. The Foxo1-deficient mice died around embryonic day 11 because of defects in the branchial arches and remarkably impaired vascular development of embryos and yolk sacs. In vitro differentiation of embryonic stem cells demonstrated that endothelial cells derived from wild-type and Foxo1-deficient embryonic stem cells were able to produce comparable numbers of colonies supported by a layer of OP9 stromal cells. Although the morphology of the endothelial cell colonies was identical in both genotypes in the absence of exogenous vascular endothelial growth factor (VEGF), Foxo1-deficient endothelial cells showed a markedly different morphological response compared with wild-type endothelial cells in the presence of exogenous VEGF. These results suggest that Foxo1 is essential to the ability of endothelial cells to respond properly to a high dose of VEGF, thereby playing a critical role in normal vascular development.
Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) is a soluble member of the tumor necrosis factor receptor family of proteins and plays an important role in the negative regulation of osteoclastic bone resorption. Whether OPG/OCIF circulates in human blood and how its level changes under pathological conditions is not known. To address these issues, a panel of monoclonal antibodies was generated against recombinant OPG/OCIF and screened for reactivity with solid-phase monomeric and homodimeric forms of the recombinant protein. Antibodies that showed high affinity for both forms of OPG/OCIF and those that selectively recognized the homodimer were identified, enabling development of two types of sensitive enzyme-linked immunosorbent assay (ELISA): one that detects both forms of OPG/OCIF equally and one specific for the homodimer. Characterization of circulating OPG/OCIF with these ELISAs revealed that the protein exists in human serum mainly in the monomeric form. The serum concentration of OPG/OCIF increased with age in both healthy Japanese men and women, and was significantly higher in postmenopausal women with osteoporosis than in age-matched controls. Within the osteoporotic group, serum OPG/OCIF concentrations were higher in patients with low bone mass. Serum OPG/OCIF concentrations were also significantly increased in those postmenopausal women with a high rate of bone turnover, as determined by increased serum bone-specific alkaline phosphatase and urinary excretion of pyridinoline and deoxypyridinoline. The results suggested that circulating OPG/OCIF levels are regulated by an age-related factor(s) and that the increased serum concentration may reflect a compensative response to enhanced osteoclastic bone resorption and the resultant bone loss rather than a cause of osteoporosis. (J Bone Miner Res 1999;14:518-527)
The osteoclast is a giant cell that resorbs calcified matrix by secreting acids and collagenolytic enzymes. The molecular mechanisms underlying metabolic adaptation to the increased biomass and energetic demands of osteoclastic bone resorption remain elusive. Here we show that during osteoclastogenesis the expression of both glucose transporter 1 (Glut1) and glycolytic genes is increased, whereas the knockdown of hypoxia-inducible factor 1-alpha (Hif1a), as well as glucose deprivation, inhibits the bone-resorbing function of osteoclasts, along with a suppression of Glut1 and glycolytic gene expression. Furthermore, the expression of the glutamine transporter solute carrier family 1 (neutral amino acid transporter), member 5 (Slc1a5) and glutaminase 1 was increased early in differentiation, and a depletion of L-glutamine or pharmacological inhibition of the Slc1a5 transporter suppressed osteoclast differentiation and function. Inhibition of c-Myc function abrogated osteoclast differentiation and function, along with a suppression of Slc1a5 and glutaminase 1 gene expression. Genetic and pharmacological inhibition of mammalian target of rapamycin (mTOR), as well as the activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), inhibited osteoclastogenesis. Thus, the uptake of glucose and glutamine and utilization of the carbon sources derived from them, coordinated by HIF1a and c-Myc, are essential for osteoclast development and bone-resorbing activity through a balanced regulation of the nutrient and energy sensors, mTOR and AMPK.
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