Vitamin D, osteoclastogenesis and bone resorption: from mechanistic insight to the development of new analogs.
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Animals↗
Biomedical subjects
Kyoji Ikeda
Publications and source records attributed to Kyoji Ikeda.
Urinary gamma-glutamyltransferase (GGT) as a potential marker of bone resorption.
We recently identified gamma-glutamyltransferase (GGT) as a novel bone-resorbing factor. The present study was undertaken to determine whether GGT is a marker of bone resorption in two genetic models of hyper- and hypo-function of osteoclasts, as well as in postmenopausal women with accelerated bone resorption, using type I collagen N-telopeptide (NTX) and deoxypyridinoline (DPD) as established biochemical markers. Urinary excretion of GGT, corrected for creatinine, was found to be increased in osteoprotegerin (OPG)-deficient osteoporotic mice as well as in patients with postmenopausal osteoporosis (67-83 years of age); in both cases the urinary level decreased after treatment of patients or mice with alendronate, a selective inhibitor of bone resorption, concomitantly with a reduction in DPD and NTX. Conversely, in osteopetrotic op/op mice, urinary GGT increased in parallel with DPD after induction of osteoclasts with M-CSF injection. Constant infusion of parathyroid hormone (PTH) also increased urinary GGT along with DPD. In a survey of 551 postmenopausal women (50-89 years of age) at their regular health checkup, urinary GGT excretion exhibited a high correlation with DPD (rho = 0.49, p < 0.0001). The calculated sensitivity and specificity for diagnosing elevated bone resorption, as determined by a DPD value higher than 7.6 nM/mM Cr, were 61% and 92%, respectively, when a cut-off value of 40 IU/g Cr was assigned for urinary GGT. Since GGT activity can be measured inexpensively in large numbers in a very short time, the measurement of urinary level may provide a convenient and useful method for mass screening to identify those with increased bone turnover and hence at increased risk for bone fracture.
Aged↗
Effects of a prostaglandin EP4 agonist, ONO-4819, and risedronate on trabecular microstructure and bone strength in mature ovariectomized rats.
The effects of a prostaglandin EP4 agonist, ONO-4819, and risedronate, a representative anti-resorptive drug, on trabecular microarchitecture and biomechanical properties were investigated in mature estrogen-deficient rats; and effects which affected microstructural components that contributed to the improvement of bone strength were also determined. Thirty-three-week-old OVX rats were treated with various doses of ONO-4819, risedronate, or their combination for 11 weeks. Bone mineral density (BMD), trabecular microstructure, and biomechanical strength were determined at the proximal tibia by peripheral quantitative CT, micro CT, and finite element analysis, respectively. Bone histomorphometry was performed at the same site. The results of trabecular structure analysis indicated that whereas risedronate functioned mainly in maintaining trabecular connectivity, ONO-4819 converted the fragile rod-like trabeculae caused by estrogen deficiency to a plate-like structure. In addition, ONO-4819 is one of the few drugs that are capable of increasing trabecular thickness. When the 2 drugs were combined, the beneficial effects of each drug on the trabecular microarchitecture were maintained, resulting in their additive effects on bone strength. The results of bone histomorphometry suggest that ONO-4819 caused an increase in the rate of bone formation by stimulating the differentiation/recruitment of osteoblasts as well as their mineralizing function. ONO-4819 did not stimulate bone resorption, but rather exerted an anti-resorptive function within a certain dose range. ONO-4819 and risedronate increased BMD and improved trabecular structure and biomechanical strength in an additive and independent manner. Thus, EP4 agonist ONO-4819 in combination with risedronate may be an effective treatment for osteoporosis.
Aging↗
A novel ubiquitin-binding protein ZNF216 functioning in muscle atrophy.
The ubiquitin-proteasome system (UPS) is critical for specific degradation of cellular proteins and plays a pivotal role on protein breakdown in muscle atrophy. Here, we show that ZNF216 directly binds polyubiquitin chains through its N-terminal A20-type zinc-finger domain and associates with the 26S proteasome. ZNF216 was colocalized with the aggresome, which contains ubiquitinylated proteins and other UPS components. Expression of Znf216 was increased in both denervation- and fasting-induced muscle atrophy and upregulated by expression of constitutively active FOXO, a master regulator of muscle atrophy. Mice deficient in Znf216 exhibited resistance to denervation-induced atrophy, and ubiquitinylated proteins markedly accumulated in neurectomized muscle compared to wild-type mice. These data suggest that ZNF216 functions in protein degradation via the UPS and plays a crucial role in muscle atrophy.
Animals↗
c-Fos protein as a target of anti-osteoclastogenic action of vitamin D, and synthesis of new analogs.
Although active vitamin D drugs have been used for the treatment of osteoporosis, how the vitamin D receptor (VDR) regulates bone cell function remains largely unknown. Using osteoprotegerin-deficient mice, which exhibit severe osteoporosis due to excessive receptor activator of NF-kappaB ligand/receptor activator of NF-kappaB (RANKL/RANK) stimulation, we show herein that oral treatment of these mice with 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] inhibited bone resorption and prevented bone loss, suggesting that VDR counters RANKL/RANK signaling. In M-CSF-dependent osteoclast precursor cells isolated from mouse bone marrow, 1alpha,25(OH)2D3 potently and dose-dependently inhibited their differentiation into multinucleate osteoclasts induced by RANKL. Among signaling molecules downstream of RANK, 1alpha,25(OH)2D3 inhibited the induction of c-Fos protein after RANKL stimulation, and retroviral expression of c-Fos protein abrogated the suppressive effect of 1alpha,25(OH)2D3 on osteoclast development. By screening vitamin D analogs based on their c-Fos-suppressing activity, we identified a new analog, named DD281, that inhibited bone resorption and prevented bone loss in ovariectomized mice, more potently than 1alpha,25(OH)2D3, with similar levels of calcium absorption. Thus, c-Fos protein is an important target of the skeletal action of VDR-based drugs, and DD281 is a bone-selective analog that may be useful for the treatment of bone diseases with excessive osteoclastic activity.
Animals↗
[Bone and bone related biochemical examinations. Hormone and hormone related substances. Parathyroid hormone related protein (PTHrP)].
Parathyroid hormone related protein (PTHrP) was identified as a PTH-like factor that causes hypercalcemia in cancer patients. A sensitive immunoassay was developed, and is currently used for the diagnosis of hypercalcemia.
Humans↗
VEGF receptor 1 signaling is essential for osteoclast development and bone marrow formation in colony-stimulating factor 1-deficient mice.
VEGF receptor 1 (VEGFR-1/Flt-1) is a high-affinity tyrosine kinase (TK) receptor for VEGF and regulates angiogenesis as well as monocyte/macrophage functions. We previously showed that the osteoclast deficiency in osteopetrotic Csf1op/Csf1op (op/op) mice is gradually restored in an endogenous, VEGF-dependent manner. However, the molecular basis of the recovery is still not clear. To examine which VEGFR is important and to clarify how colony-stimulating factor 1 (CSF-1) and VEGF signals interact in osteoclastogenesis, we introduced a VEGFR-1 signaling deficiency (Flt1(TK)-/-) into op/op mice. The original Flt1(TK)-/- mice showed mild osteoclast reduction without bone marrow suppression. The double mutant (op/opFlt1(TK)-/-) mice, however, exhibited very severe osteoclast deficiency and did not have numbers of osteoclasts sufficient to form the bone marrow cavity. The narrow bone marrow cavity in the op/opFlt1(TK)-/- mice was gradually replaced with fibrous tissue, resulting in severe marrow hypoplasia and extramedullary hematopoiesis. In addition to osteoclasts, osteoblasts also decreased in number in the op/opFlt1(TK)-/- mice. These results strongly suggest that the interaction of signals by means of VEGFR-1 and the CSF-1 receptor plays a predominant role not only in osteoclastogenesis but also in the maintenance of bone marrow functions.
Animals↗
Multi-detector row CT imaging of vertebral microstructure for evaluation of fracture risk.
UNLABELLED: We applied MDCT for in vivo evaluation of the microarchitecture of human vertebrae. Microstructure parameters, such as structure model index, Euler's number, and bone volume fraction, revealed higher relative risk for prevalent vertebral fracture than did BMD obtained by DXA. Thus, microstructure analysis by MDCT, together with simultaneously obtained volumetric BMD values, is useful for clinical assessment of fracture risk. INTRODUCTION: BMD measurement by DXA alone has limitations in predicting fracture, and methods for clinical assessment of bone quality, such as microstructure, are awaited. This study was undertaken to examine the applicability of multidetector row CT (MDCT) for in vivo evaluation of trabecular microstructure. MATERIALS AND METHODS: Optimal conditions for MDCT scanning were determined at a spatial resolution of 250 x 250 x 500 mum, using muCT data of excised human vertebra specimens as a reference. We analyzed the trabecular microstructure of the vertebrae of 82 postmenopausal women (55-76 years old), including 39 women with and 43 without a recent vertebral fracture. RESULTS: Microstructure indices obtained by MDCT scanning revealed higher relative risk for prevalent vertebral fracture (OR: 16.0 for structure model index, 13.6 for bone volume fraction, and 13.1 for Euler's number) than did spinal BMD obtained by DXA (OR: 4.8). MDCT could also provide volumetric BMD data, which had higher diagnostic value (OR: 12.7) than did DXA. CONCLUSION: Vertebral microarchitecture can be visualized by MDCT, and microstructure parameters obtained by MDCT, together with volumetric BMD, provided better diagnostic performance for assessing fracture risk than DXA measurement.
Absorptiometry, Photon↗
Ataxia telangiectasia mutated (Atm) knockout mice as a model of osteopenia due to impaired bone formation.
ATM is a member of the PI-3 kinase protein family, encoded by the gene, ATM, responsible for ataxia telangiectasia (AT). AT is recognized as a genomic instability syndrome, sharing accelerated senescence symptoms in human and mouse. Here, we present evidence that the bone phenotype of Atm knockout (AtmKO) mice is similar to that observed in disuse and/or aging syndromes. A significant decrease in 3-dimensional bone volume fraction (BV/TV) of the fifth lumbar vertebra was observed in AtmKO mice by microCT, compared with heterozygous control mice at 10 weeks of age. Bone histomorphometry revealed that both BFR/BS and Oc.S/BS were significantly decreased in KO mice. To determine the cellular basis of this bone phenotype, we employed in vitro osteoclastogenesis and colony formation assays using bone marrow cells derived from KO and control mice. There was no difference in osteoclast formation in ex vivo cultures. CFU-F was markedly reduced in AtmKO-derived cultures compared with control mice, whereas differentiation of calvaria-derived osteoblasts did not differ between the genotypes. Furthermore, expression levels of IGF1R were significantly decreased, and p38 was aberrantly phosphorylated in marrow stromal cells from AtmKO mice. These results indicate that the pathogenesis of the osteopenic phenotype in AtmKO mice is similar to that of disuse and/or aging syndromes and is caused, at least in part, by a stem cell defect due to lack of IGF signaling.
Animals↗
A RANKL-inducible gene Znf216 in osteoclast differentiation.
Osteoclasts possess catabolic activity in mineralized tissues and are involved in bone remodeling coordinating with osteoblasts. Although the pathway using receptor and activator of NF-kappa B (RANK) and its ligand, RANKL, is known to be essential for osteoclast differentiation, their precise mechanisms are not fully understood. Using DNA microarray technology, we searched for genes that were up-regulated after RANKL stimulation in the macrophage cell line, RAW264.7 cells. A gene, Znf216, which encodes a zinc-finger protein, was detected among those genes up-regulated after RANKL stimulation. Expression of Znf216 was also induced by other cytokines such as TNFalpha and IL-1beta. Although ectopic expression of full-length ZNF216 abrogated osteoclast differentiation, its truncated forms accelerated it. No significant inhibitory effect on the NF-kappa B pathway was observed, however. These results suggest that ZNF216 is a potent inhibitory factor for osteoclast differentiation and that the mechanism is unlikely due to direct attenuation of the NF-kappa B pathway.
Animals↗
SIRT1 is critical regulator of FOXO-mediated transcription in response to oxidative stress.
Forkhead transcription factor, DAF-16, regulates genes that contribute both to longevity and resistance to various stresses in C. elegans. We and others have reported that members of the FOXO, mammalian homologs of DAF-16, also regulate genes related to stress resistance, such as GADD45. The NAD-dependent protein deacetylase, SIR2, is required for life span extension in yeast induced by caloric restriction, which also increases longevity in a wide variety of other organisms, including mammals. Sir2.1, a homolog of yeast SIR2, also extends life span by acting in a DAF-16 signaling pathway in C. elegans. We demonstrate that mammalian SIRT1 (Sir2alpha) physiologically interacts with FOXO. Acetylation of FOXO4, by the transcriptional coactivator p300, counteracted transcriptional activation of FOXO4 by p300. In contrast, mammalian SIRT1 was found to bind to FOXO4, catalyze its deacetylation in an NAD-dependent manner, and thereby increase its transactivation activity. The activity of FOXO4 is suppressed or enhanced by SIRT1 inhibitor, nicotinamide, or its activator, resveratrol, respectively. In response to oxidative stress, FOXO accumulates within the nucleus and induces GADD45 expression. FOXO-mediated GADD45 induction is markedly impaired in the cell, which depleted SIRT1 expression by RNA-interference. These results indicate that mammalian SIRT1 plays a pivotal role for FOXO function via NAD-dependent deacetylation in response to oxidative stress, and thereby may contribute to cellular stress resistance and longevity.
Acetylation↗
[PTHrP: gene, structure, biosynthesis, regulation of expression].
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Animals↗
[Genes causing parathyroid diseases].
Discovery of Gcm2 as an essential transcription factor for parathyroid gland development disclosed another, unexpected source of circulating parathyroid hormone (PTH), the thymus. A recent phylogenetic analysis of Gcm2 has led to a new theory that parathyroid glands in tetrapods were transformed from the gills of fish during evolution. I review genes involved in the pathogenesis of hyper- and hypo-function of parathyroid glands as well as in the development of parathyroid tumors.
Animals↗
Abnormal angiogenesis in Foxo1 (Fkhr)-deficient mice.
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.
Animals↗
Detection of osteoclastic cell-cell fusion through retroviral vector packaging.
Cell-cell fusion generates multinucleated cells such as osteoclasts in bone, myotubes in muscle, and trophoblasts in placenta. Molecular details governing these fusion processes are still largely unknown. As a step toward identification of fusogenic genes, we tested the concept that retroviral vectors can be packaged as a result of cell-cell fusion. First, we introduced replication-deficient retroviral vectors expressing mCAT-1, which mediates fusogenic interaction with the retroviral envelope protein Env, into Chinese hamster ovary (CHO) cells to generate vector cells. Plasmids expressing virion proteins Gag, Pol, and Env were introduced into a separate culture of CHO cells to generate packaging cells. Co-culturing vector and packaging cells resulted in production of infectious retroviruses carrying the mCAT-1 gene as a consequence of cell-cell fusion. Second, we introduced a retroviral vector into primary osteoclast precursors and co-cultured them with established osteoclast precursor RAW264.7 cells, which turned out to harbor packaging activity. Packaged retroviral vector was detected in culture supernatants only where the osteoclast differentiation factor receptor activator for NF-kappaB ligand (RANKL) induced fusion between these two cell types. These data suggest that retrovirus production can occur as a result of cell-cell fusion. This provides a novel approach for isolating and characterizing fusogenic genes using retroviral expression vectors.
Animals↗
[Aging and bone quality].
At the research level, various factors have been correlated with changes in bone quality that underlie age-associated fragility. On the other hand, biochemical markers and imaging technology that can faithfully assess bone quality and are applicable to clinical practice remain to be developed.
Aging↗