Daewon Jeong

v-ATPase V0 subunit d2-deficient mice exhibit impaired osteoclast fusion and increased bone formation.

Matrix-producing osteoblasts and bone-resorbing osteoclasts maintain bone homeostasis. Osteoclasts are multinucleated, giant cells of hematopoietic origin formed by the fusion of mononuclear pre-osteoclasts derived from myeloid cells. Fusion-mediated giant cell formation is critical for osteoclast maturation; without it, bone resorption is inefficient. To understand how osteoclasts differ from other myeloid lineage cells, we previously compared global mRNA expression patterns in these cells and identified genes of unknown function predominantly expressed in osteoclasts, one of which is the d2 isoform of vacuolar (H+) ATPase (v-ATPase) V0 domain (Atp6v0d2). Here we show that inactivation of Atp6v0d2 in mice results in markedly increased bone mass due to defective osteoclasts and enhanced bone formation. Atp6v0d2 deficiency did not affect differentiation or the v-ATPase activity of osteoclasts. Rather, Atp6v0d2 was required for efficient pre-osteoclast fusion. Increased bone formation was probably due to osteoblast-extrinsic factors, as Atp6v02 was not expressed in osteoblasts and their differentiation ex vivo was not altered in the absence of Atp6v02. Our results identify Atp6v0d2 as a regulator of osteoclast fusion and bone formation, and provide genetic data showing that it is possible to simultaneously inhibit osteoclast maturation and stimulate bone formation by therapeutically targeting the function of a single gene.

Microphthalmia transcription factor and PU.1 synergistically induce the leukocyte receptor osteoclast-associated receptor gene expression.

We have recently reported the identification of a novel member of the leukocyte receptor family, osteoclast-associated receptor (OSCAR), which has two Ig-like domains and functions as a bone-specific regulator of osteoclast differentiation. Here, we have cloned the OSCAR promoter region to examine its regulation by transcription factors. The 1.7-kb promoter region of the mouse OSCAR gene contains two potential E-box elements for microphthalmia transcription factor (MITF) and three putative PU.1 sites. MITF or PU.1 alone activates the OSCAR reporter construct 5–6-fold, and the combination of MITF and PU.1 synergistically activates the OSCAR reporter activity up to 110-fold. The mRNA expression patterns of MITF, PU.1, and OSCAR in TRANCE-treated (RAW 264.7) or TRANCE/M-CSF-treated cells (primary osteoclasts) reveal that MITF mRNA expression is induced at a much earlier time point than OSCAR gene expression. In contrast to MITF, PU.1 mRNA levels remain relatively constant at all time points, suggesting that TRANCE-induced MITF, not PU.1 expression, is one of the critical regulatory mechanisms for optimal OSCAR expression during osteoclastogenesis. In addition, we have shown that the combination of MITF and constitutively active MKK6-expressing plasmids synergistically activates OSCAR reporter activity. Taken together, our results strongly suggest that PU.1 and MITF transcription factors synergistically activate OSCAR gene expression. Moreover, the activation of OSCAR gene expression by PU.1/MITF is further enhanced by the TRANCE-induced MKK6/p38 signaling cascade.

The Tec Family Tyrosine Kinase Btk Regulates RANKL-induced Osteoclast Maturation

A spontaneous mutation in Brutons tyrosine kinase (Btk) induces a defect in B-cell development that results in the immunodeficiency diseases X-linked agammaglobulinemia in humans and X-linked immunodeficiency (Xid) in mice. Here we show an unexpected role of Btk in osteoclast formation. When bone marrow cells derived from Xid mice were stimulated with receptor activator of NF-κB ligand, an osteoclast differentiation factor, they did not completely differentiate into mature multinucleated osteoclasts. Moreover, we found that the defects appeared to occur at the stage in which mononuclear preosteoclasts fuse to generate multinucleated cells. Supporting this notion, macrophages from Xid mice also failed to form multinucleated foreign body giant cells. The fusion defect of the Xid mutant osteoclasts was caused by decreased expression of nuclear factor of activated T cells c1 (NFATc1), a master regulator of osteoclast differentiation, as well as reduced expression of various osteoclast fusion-related molecules, such as the d2 isoform of vacuolar H+-ATPase V0 domain and the dendritic cell-specific transmembrane protein. This deficiency was completely rescued by the introduction of a constitutively active form of NFATc1 into bone marrow-derived macrophages. Our data provide strong evidence that Btk plays a critical role in osteoclast multinucleation by modulating the activity of NFATc1.

Selective inhibition of RANK blocks osteoclast maturation and function and prevents bone loss in mice

Regulation of the formation and function of bone-resorbing osteoclasts (OCs) is a key to understanding the pathogenesis of skeletal disorders. Gene-targeting studies have shown that the RANK signaling pathway plays a critical role in OC differentiation and function. Although pharmaceutical blockade of RANK may be a viable strategy for preventing bone destruction, RANK is implicated in multiple biological processes. Recently, a cytoplasmic motif of RANK was identified that may be specifically involved in OC differentiation. Here, we developed a cell-permeable inhibitor termed the RANK receptor inhibitor (RRI), which targets this motif. The RRI peptide blocked RANKL-induced OC formation from murine bone marrow-derived macrophages. Furthermore, RRI inhibited the resorptive function of OCs and induced OC apoptosis. Treatment with the peptide impaired downstream signaling of RANK linked to Vav3, Rac1, and Cdc42 and resulted in disruptions of the actin cytoskeleton in differentiated OCs. In addition, RRI blocked inflammation-induced bone destruction and protected against ovariectomy-induced bone loss in mice. These data may be useful in the development of selective therapeutic agents for the treatment of osteoporosis and other bone diseases.

Discriminative cytotoxicity assessment based on various cellular damages

There are several assays currently available for the assessment of cell cytotoxicity, including trypan blue exclusion, lactate dehydrogenase (LDH) release, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assays. Trypan blue exclusion and LDH release assays are appropriate for evaluating cell membrane damage and a colorimetric MTT assay is available for measuring mitochondrial-related reduction capacity. As these assays were randomly utilized to assess the extent of cell damage, we suggest herein that the assay should be selected in accordance with the prevailing cellular situation. This can be determined by using a variety of cell types with differing reduction status, exogenous and endogenous oxidative stressors, and several different oxidized/reduced molecules. Although the trypan blue exclusion and released LDH assay have proven useful for assessments of necrotic and apoptotic cell death with membrane damage, the LDH assay is not appropriate for the measurement of the number of varied cells without membrane damage. In addition, when the cells were treated with exogenous and endogenous oxidative stressors, MTT reduction was shown to be sensitive to a shift to a more oxidizing cellular environment within a narrow range without loss of membrane integrity, and this effect increased in a linear fashion, dependent on the dosage of cytosolic extracts containing various physiological reductants, small reductive molecules (NADPH and GSH), and artificial DTT reducing agent. Finally, we noted that the MTT assay is available for the determination of small-scale oscillations in cellular reduction status and changes in mitochondrial functional activity, but not for evaluating the cytotoxicity of cells with a higher cellular reduction capacity. Altogether, the findings of this study indicate that tools for the testing of cytotoxicity should be selected differently by considering the correlation between the cellular conditions for various stimuli and the principle underlying the assay system.

NHE10, a novel osteoclast-specific member of the Na+/H+ exchanger family, regulates osteoclast differentiation and survival

Bone homeostasis is tightly regulated by the balanced actions of osteoblasts (OBs) and osteoclasts (OCs). We previously analyzed the gene expression profile of OC differentiation using a cDNA microarray, and identified a novel osteoclastogenic gene candidate, clone OCL-1-E7 [J. Rho, C.R. Altmann, N.D. Socci, L. Merkov, N. Kim, H. So, O. Lee, M. Takami, A.H. Brivanlou, Y. Choi, Gene expression profiling of osteoclast differentiation by combined suppression subtractive hybridization (SSH) and cDNA microarray analysis, DNA Cell Biol. 21 (2002) 541-549]. In this study, we have isolated full-length cDNAs corresponding to this clone from mice and humans to determine the functional roles of this gene in osteoclastogenesis. The full-length cDNA of OCL-1-E7 encodes 12 membrane-spanning domains that are typical of isoforms of the Na(+)/H(+) exchangers (NHEs), indicating that this clone is a novel member of the NHE family (hereafter referred to as NHE10). Here, we show that NHE10 is highly expressed in OCs in response to receptor activator of nuclear factor-kappaB ligand signaling and is required for OC differentiation and survival.

Osteoclast Precursors Display Dynamic Metabolic Shifts toward Accelerated Glucose Metabolism at an Early Stage of RANKL-Stimulated Osteoclast Differentiation

Mature osteoclasts have an increased citric acid cycle and mitochondrial respiration to generate high ATP production and ultimately lead to bone resorption. However, changes in metabolic pathways during osteoclast differentiation have not been fully illustrated. We report that glycolysis and oxidative phosphorylation characterized by glucose and oxygen consumption as well as lactate production were increased during receptor activator of nuclear factor-ĸB ligand (RANKL)-induced osteoclastogenesis from RAW264.7 and bone marrow-derived macrophage cells. Cell proliferation and differentiation varied according to glucose concentrations (0 to 100 mM). Maximal cell growth occurred at 20 mM glucose concentration and differentiation occurred at 5 mM concentration. Despite the similar growth rates exhibited when cultured cells were exposed to either 5 mM or 40 mM glucose, their differentiation was markedly decreased in high glucose concentrations. This finding suggests the possibility that osteoclastogenesis could be regulated by changes in metabolic substrate concentrations. To further address the effect of metabolic shift on osteoclastogenesis, we exposed cultured cells to pyruvate, which is capable of promoting mitochondrial respiration. Treatment of pyruvate synergistically increased osteoclastogenesis through the activation of RANKL-stimulated signals (ERK and JNK). We also found that osteoclastogenesis was retarded by blocking ATP production with either the inhibitors of mitochondrial complexes, such as rotenone and antimycin A, or the inhibitor of ATP synthase, oligomycin. Taken together, these results indicate that glucose metabolism during osteoclast differentiation is accelerated and that a metabolic shift towards mitochondrial respiration allows high ATP production and induces enhanced osteoclast differentiation.

NHE10, a novel osteoclast-specific member of the Na{sup +}/H{sup +} exchanger family, regulates osteoclast differentiation and survival

Bone homeostasis is tightly regulated by the balanced actions of osteoblasts (OBs) and osteoclasts (OCs). We previously analyzed the gene expression profile of OC differentiation using a cDNA microarray, and identified a novel osteoclastogenic gene candidate, clone OCL-1-E7 [J. Rho, C.R. Altmann, N.D. Socci, L. Merkov, N. Kim, H. So, O. Lee, M. Takami, A.H. Brivanlou, Y. Choi, Gene expression profiling of osteoclast differentiation by combined suppression subtractive hybridization (SSH) and cDNA microarray analysis, DNA Cell Biol. 21 (2002) 541-549]. In this study, we have isolated full-length cDNAs corresponding to this clone from mice and humans to determine the functional roles of this gene in osteoclastogenesis. The full-length cDNA of OCL-1-E7 encodes 12 membrane-spanning domains that are typical of isoforms of the Na(+)/H(+) exchangers (NHEs), indicating that this clone is a novel member of the NHE family (hereafter referred to as NHE10). Here, we show that NHE10 is highly expressed in OCs in response to receptor activator of nuclear factor-kappaB ligand signaling and is required for OC differentiation and survival.

Bimodal actions of selenium essential for antioxidant and toxic pro-oxidant activities: The selenium paradox (Review)

Selenium is an essential biological trace element. Adult daily intake of selenium should be approximately 100 µg per day. This compound has a two-sided effect depending on its concentration. A selenium-deficient diet is associated with various endemic diseases, including cardiomuscular malfunctions, osteoarthritis, cancer and viral infections that lead to premature death. These defects are prevented when dietary intake of selenium is adequate. The preventive biological effect of selenium is considered to be due to the antioxidant function of selenoproteins with a selenocysteine in the active site of the catalytic domain. Antioxidant selenoproteins maintain the intracellular redox status and, as a result, normal physiological processes in the cell. Conversely, an overdose of selenium generates oxygen radicals and leads to apoptotic cell death by inducing oxidation and cross-linking of protein thiol groups essential for cell survival. A lower redox state caused by selenium may be implicated in toxic diseases, such as alkali disease and blind staggers. Collectively, selenium seems to have both harmful and beneficial attributes. The aim of this review is to summarize the various biological functions of selenium and to illustrate its opposite roles as a pro-oxidant and an antioxidant.

Inactivation of glycogen synthase kinase-3β is required for osteoclast differentiation.

Background: Bone homeostasis is maintained by balancing the activities of bone-resorbing osteoclasts and bone-forming osteoblasts. Results: GSK-3β is inactivated by receptor activator of NF-κB ligand stimulation via serine phosphorylation during osteoclastogenesis. Conclusion: GSK-3β is crucial for receptor activator of NF-κB ligand-mediated signaling as a negative regulator of osteoclast differentiation. Significance: GSK-3β acts as a novel negative regulator of osteoclast biology. Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine kinase originally identified as a regulator of glycogen deposition. Although the role of GSK-3β in osteoblasts is well characterized as a negative regulator of β-catenin, its effect on osteoclast formation remains largely unidentified. Here, we show that the GSK-3β inactivation upon receptor activator of NF-κB ligand (RANKL) stimulation is crucial for osteoclast differentiation. Regulation of GSK-3β activity in bone marrow macrophages by retroviral expression of the constitutively active GSK-3β (GSK3β-S9A) mutant inhibits RANKL-induced osteoclastogenesis, whereas expression of the catalytically inactive GSK-3β (GSK3β-K85R) or small interfering RNA (siRNA)-mediated GSK-3β silencing enhances osteoclast formation. Pharmacological inhibition of GSK-3β further confirmed the negative role of GSK-3β in osteoclast formation. We also show that overexpression of the GSK3β-S9A mutant in bone marrow macrophages inhibits RANKL-mediated NFATc1 induction and Ca2+ oscillations. Remarkably, transgenic mice expressing the GSK3β-S9A mutant show an osteopetrotic phenotype due to impaired osteoclast differentiation. Further, osteoclast precursor cells from the transgenic mice show defects in expression and nuclear localization of NFATc1. These findings demonstrate a novel role for GSK-3β in the regulation of bone remodeling through modulation of NFATc1 in RANKL signaling.