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Low-density Lipoprotein Receptor Deficiency Causes Impaired Osteoclastogenesis and Increased Bone Mass in Mice because of Defect in Osteoclastic Cell-Cell Fusion*

  • Mari Okayasu
    Affiliations
    Division of Oral Anatomy, Meikai University School of Dentistry, Sakado, Saitama 350-0283, Japan

    Division of Orthodontics, Department of Human Development and Fostering, Meikai University School of Dentistry, Sakado, Saitama 350-0283, Japan

    Division of Oral-Maxillofacial Surgery, Dentistry and Orthodontics, Department of Sensory and Motor System Medicine, The University of Tokyo Hospital, Hongo, Tokyo 113-8655, Japan
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  • Mai Nakayachi
    Affiliations
    Division of Oral Anatomy, Meikai University School of Dentistry, Sakado, Saitama 350-0283, Japan

    Division of Orthodontics, Department of Human Development and Fostering, Meikai University School of Dentistry, Sakado, Saitama 350-0283, Japan
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  • Chiyomi Hayashida
    Affiliations
    Division of Oral Anatomy, Meikai University School of Dentistry, Sakado, Saitama 350-0283, Japan
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  • Junta Ito
    Affiliations
    Division of Oral Anatomy, Meikai University School of Dentistry, Sakado, Saitama 350-0283, Japan
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  • Toshio Kaneda
    Affiliations
    Faculty of Pharmaceutical Sciences, Hoshi University, Ebara, Tokyo 142-8501, Japan
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  • Masaaki Masuhara
    Affiliations
    Division of Oral Anatomy, Meikai University School of Dentistry, Sakado, Saitama 350-0283, Japan

    Department of Applied Pharmacology, Kagoshima University Faculty of Dentistry, Kagoshima, Kagoshima 890-8544, Japan
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  • Naoto Suda
    Affiliations
    Division of Orthodontics, Department of Human Development and Fostering, Meikai University School of Dentistry, Sakado, Saitama 350-0283, Japan
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  • Takuya Sato
    Affiliations
    Division of Oral Anatomy, Meikai University School of Dentistry, Sakado, Saitama 350-0283, Japan
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  • Yoshiyuki Hakeda
    Correspondence
    To whom correspondence should be addressed: Division of Oral Anatomy, Meikai University School of Dentistry, Sakado, Saitama 350-283, Japan. Tel.: 81-492-79-2769; Fax: 81-492-71-3523
    Affiliations
    Division of Oral Anatomy, Meikai University School of Dentistry, Sakado, Saitama 350-0283, Japan
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  • Author Footnotes
    * This work was supported by Japanese Ministry of Education, Science, Sports, and Culture Grants-in-aid for scientific research 19592130 and 21592341 (to Y. H.).
    This article contains supplemental Fig. S1.
Open AccessPublished:April 12, 2012DOI:https://doi.org/10.1074/jbc.M111.323600
      Osteoporosis is associated with both atherosclerosis and vascular calcification attributed to hyperlipidemia. However, the cellular and molecular mechanisms explaining the parallel progression of these diseases remain unclear. Here, we used low-density lipoprotein receptor knockout (LDLR−/−) mice to elucidate the role of LDLR in regulating the differentiation of osteoclasts, which are responsible for bone resorption. Culturing wild-type osteoclast precursors in medium containing LDL-depleted serum decreased receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation, and this defect was additively rescued by simultaneous treatment with native and oxidized LDLs. Osteoclast precursors constitutively expressed LDLR in a RANKL-independent manner. Osteoclast formation from LDLR−/− osteoclast precursors was delayed, and the multinucleated cells formed in culture were smaller and contained fewer nuclei than wild-type cells, implying impaired cell-cell fusion. Despite these findings, RANK signaling, including the activation of Erk and Akt, was normal in LDLR−/− preosteoclasts, and RANKL-induced expression of NFATc1 (a master regulator of osteoclastogenesis), cathepsin K, and tartrate-resistant acid phosphatase was equivalent in LDLR-null and wild-type cells. In contrast, the amounts of the osteoclast fusion-related proteins v-ATPase V0 subunit d2 and dendritic cell-specific transmembrane protein in LDLR−/− plasma membranes were reduced when compared with the wild type, suggesting a correlation with impaired cell-cell fusion, which occurs on the plasma membrane. LDLR−/− mice consistently exhibited increased bone mass in vivo. This change was accompanied by decreases in bone resorption parameters, with no changes in bone formation parameters. These findings provide a novel mechanism for osteoclast differentiation and improve the understanding of the correlation between osteoclast formation and lipids.

      Introduction

      Osteoclasts, which are the cells responsible for bone resorption, are of hematopoietic stem cell origin (
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      ). During the last decade, the major molecular mechanisms of osteoclastogenesis have been elucidated. Osteoclast precursors have been demonstrated to share properties with the monocyte/macrophage cell lineage (
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      ), the receptor activator of NF-κB (RANK)
      The abbreviations used are: RANK
      receptor activator of NF-κB
      RANKL
      receptor activator of NF-κB ligand
      M-CSF
      macrophage colony-stimulating factor
      TRAP
      tartrate-resistant acid phosphatase
      Atp6v0d2
      v-ATPase V0 subunit d2
      DC-STAMP
      dendritic cell-specific transmembrane protein
      ox-LDL
      oxidized LDL
      LDLR
      LDL receptor
      LR-FBS
      lipoprotein-reduced FBS
      MNC
      multinucleated cell.
      ligand (RANKL) is the most critical molecule in osteoclastogenesis, acting in cooperation with macrophage colony-stimulating factor (M-CSF) in the interaction between stromal cells and cells of the osteoclast lineage (
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      ). NFATc1, in turn, induces osteoclast differentiation-related molecules, including tartrate-resistant acid phosphatase (TRAP), cathepsin K (
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      v-ATPase V0 subunit d2-deficient mice exhibit impaired osteoclast fusion and increased bone formation.
      ,
      • Kim T.
      • Ha H.I.
      • Kim N.
      • Yi O.
      • Lee S.H.
      • Choi Y.
      Adrm1 interacts with Atp6v0d2 and regulates osteoclast differentiation.
      ,
      • Wu H.
      • Xu G.
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      Atp6v0d2 is an essential component of the osteoclast-specific proton pump that mediates extracellular acidification in bone resorption.
      ) and dendritic cell-specific transmembrane protein (DC-STAMP) (
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      DC-STAMP is essential for cell-cell fusion in osteoclasts and foreign body giant cells.
      ,
      • Mensah K.A.
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      RANKL induces heterogeneous DC-STAMP(lo) and DC-STAMP(hi) osteoclast precursors of which the DC-STAMP(lo) precursors are the master fusogens.
      ,
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      An increasing number of epidemiological studies have demonstrated that dyslipidemia is a risk factor not only for atherosclerosis and vascular calcification, but also for osteoporosis (
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      Atherogenic high-fat diet reduces bone mineralization in mice.
      ). In addition, several in vitro studies have demonstrated that oxidized LDL (ox-LDL) inhibits osteoblast differentiation (
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      Osteoclast formation, survival and morphology are highly dependent on exogenous cholesterol/lipoproteins.
      ). Such coculture systems, however, could not provide a evidence for direct action of LDL on osteoclast precursors. Nevertheless, osteoporosis associated with an abnormal plasma lipid level is likely to be attributed to decreased bone formation, increased bone resorption, or both. In contrast, there are fewer reports on the more detailed molecular mechanisms explaining the parallel progression of these diseases.
      Cholesterol is one of the major components of biological membranes and lipoproteins. It affects the structure and function of biological membranes by defining the physicochemical characteristics of the membrane, such as membrane fluidity (
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      Lipid rafts/caveolae as microdomains of calcium signaling.
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      • Thomas C.M.
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      • Hada N.
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      • Uchida N.
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      Receptor activator of NF-κB ligand-dependent expression of caveolin-1 in osteoclast precursors, and high dependency of osteoclastogenesis on exogenous lipoprotein.
      ), a principal scaffolding protein of lipid rafts and caveolae. Furthermore, depletion of exogenous LDL causes impaired NFATc1 activation and consequently reduces osteoclast formation (
      • Hada N.
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      Receptor activator of NF-κB ligand-dependent expression of caveolin-1 in osteoclast precursors, and high dependency of osteoclastogenesis on exogenous lipoprotein.
      ), consistent with other studies (
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      ,
      • Luegmayr E.
      • Glantschnig H.
      • Wesolowski G.A.
      • Gentile M.A.
      • Fisher J.E.
      • Rodan G.A.
      • Reszka A.A.
      Osteoclast formation, survival and morphology are highly dependent on exogenous cholesterol/lipoproteins.
      ). These results suggest a tight correlation between osteoclast differentiation and cholesterol.
      Intracellular cholesterol homeostasis is strictly controlled by cholesterol uptake from the extracellular space and its intracellular de novo biosynthesis (
      • Brown M.S.
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      Multivalent feedback regulation of HMG CoA reductase, a control mechanism coordinating isoprenoid synthesis and cell growth.
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      The 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductases.
      ), and LDL receptor (LDLR) (
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      ), the stringent requirement for exogenous LDL (
      • Luegmayr E.
      • Glantschnig H.
      • Wesolowski G.A.
      • Gentile M.A.
      • Fisher J.E.
      • Rodan G.A.
      • Reszka A.A.
      Osteoclast formation, survival and morphology are highly dependent on exogenous cholesterol/lipoproteins.
      ,
      • Hada N.
      • Okayasu M.
      • Ito J.
      • Nakayachi M.
      • Hayashida C.
      • Kaneda T.
      • Uchida N.
      • Muramatsu T.
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      Receptor activator of NF-κB ligand-dependent expression of caveolin-1 in osteoclast precursors, and high dependency of osteoclastogenesis on exogenous lipoprotein.
      ) indicates that de novo biosynthesis does not function in osteoclast lineage cells. Indeed, osteoclast lineage cells have been shown to express very low levels of 3-hydroxy-3-methylglutaryl-CoA reductase (
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      • Fisher J.E.
      • Rodan G.A.
      • Reszka A.A.
      Osteoclast formation, survival and morphology are highly dependent on exogenous cholesterol/lipoproteins.
      ). Therefore, the uptake of exogenous cholesterol plays a more important role in regulating osteoclast differentiation than its de novo biosynthesis. Thus, we focused on the involvement of LDLR in osteoclastogenesis.
      In this study, we examined the effect of LDLR deficiency on osteoclast formation using LDLR knockout (LDLR−/−) mice and found that RANKL-induced osteoclast formation from LDLR−/− osteoclast precursors was reduced because of the impaired cell-cell fusion of preosteoclasts, whereas osteoclast differentiation-related transcription factors and functional proteins, such as NFATc1, cathepsin K, and TRAP, were expressed at normal levels in response to RANKL. Similarly, RANKL induced the expression of the osteoclast fusion regulators Atp6v0d2 and DC-STAMP at levels equivalent to those observed in wild-type osteoclast lineage cells. Despite these findings, the amount of Atp6v0d2 and DC-STAMP in the plasma membrane was greatly reduced in LDLR−/− osteoclast lineage cells. The decreased level of the fusion-related proteins in the plasma membrane may explain the observed osteoclastic cell-cell fusion defect. Furthermore, femora and tibiae from LDLR−/− mice exhibited increased bone mass and moderate osteopetrosis. Thus, the results of this study indicate that the uptake of LDL via LDLR is essential for osteoclastogenesis and provide a novel mechanism for osteoclast differentiation, thereby improving our understanding of the correlation between osteoclast formation and lipids.

      DISCUSSION

      In this study, we showed that osteoclastogenesis stringently required exogenous native LDL and modified LDL, which are integral carrier proteins involved in cholesterol uptake into cells. The impairment in osteoclast formation because of the removal of exogenous LDL was additively rescued by simultaneous treatment with LDL and ox-LDL, indicating the existence of distinct incorporation pathways for native and modified LDLs. In support of this hypothesis, osteoclast precursors constitutively expressed both LDLR, which is specific for native LDL, and SR-A, which is specific for modified LDL. In this study, we focused on the physiological roles of LDLR in regulating osteoclastogenesis and attempted to elucidate its roles using LDLR-deficient mice. Deletion of the LDLR gene caused a decrease in osteoclast formation because of the impaired cell-cell fusion of preosteoclasts. Nevertheless, Atp6v0d2 and DC-STAMP, which are essential cell fusion proteins for the production of multinucleated osteoclasts (
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      • Koczon-Jaremko B.
      • Lorenzo J.
      • Choi Y.
      v-ATPase V0 subunit d2-deficient mice exhibit impaired osteoclast fusion and increased bone formation.
      ,
      • Kim T.
      • Ha H.I.
      • Kim N.
      • Yi O.
      • Lee S.H.
      • Choi Y.
      Adrm1 interacts with Atp6v0d2 and regulates osteoclast differentiation.
      ,
      • Wu H.
      • Xu G.
      • Li Y.P.
      Atp6v0d2 is an essential component of the osteoclast-specific proton pump that mediates extracellular acidification in bone resorption.
      ,
      • Yagi M.
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      • Toyama Y.
      • Suda T.
      DC-STAMP is essential for cell-cell fusion in osteoclasts and foreign body giant cells.
      ,
      • Mensah K.A.
      • Ritchlin C.T.
      • Schwarz E.M.
      RANKL induces heterogeneous DC-STAMP(lo) and DC-STAMP(hi) osteoclast precursors of which the DC-STAMP(lo) precursors are the master fusogens.
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      • Keng P.C.
      • Ritchlin C.T.
      ), were expressed at levels equivalent to those observed in wild-type osteoclast lineage cells. Furthermore, no differences were observed in the signal transduction pathways necessary for osteoclast differentiation, such as RANKL-induced activation of Erk and Akt, between LDLR−/− and wild-type preosteoclasts. In addition, the expression of c-Fos and NFATc1, which are integral transcription factors involved in osteoclast differentiation, and the osteoclast differentiation-related proteins TRAP and cathepsin K did not differ between osteoclast lineage cells of the two genotypes. In contrast, the amount of Atp6v0d2 and DC-STAMP in the plasma membranes of LDLR−/− preosteoclasts was clearly less than that observed in wild-type preosteoclasts, suggesting that the reduced amounts of Atp6v0d2 and DC-STAMP in the plasma membrane result in the cell fusion defect observed in the LDLR−/− preosteoclasts. Consistent with the results of in vitro experiments, LDLR−/− mice showed increased bone mass because of a reduction in osteoclast numbers but did not exhibit altered bone formation in vivo.
      Numerous studies have indicated a critical involvement of cholesterol in the differentiation and function of a variety of cell types, including endothelial cells, cardiac muscle cells, macrophages, and osteoblasts (
      • Xu J.
      • Dang Y.
      • Ren Y.R.
      • Liu J.O.
      Cholesterol trafficking is required for mTOR activation in endothelial cells.
      ,
      • Buhagiar K.A.
      • Hansen P.S.
      • Kong B.Y.
      • Clarke R.J.
      • Fernandes C.
      • Rasmussen H.H.
      Dietary cholesterol alters Na+/K+ selectivity at intracellular Na+/K+ pump sites in cardiac myocytes.
      ,
      • Anzinger J.J.
      • Chang J.
      • Xu Q.
      • Buono C.
      • Li Y.
      • Leyva F.J.
      • Park B.C.
      • Greene L.E.
      • Kruth H.S.
      Native low-density lipoprotein uptake by macrophage colony-stimulating factor-differentiated human macrophages is mediated by macropinocytosis and micropinocytosis.
      ). It is well known that cholesterol is a major component of the plasma membrane, and especially of lipid rafts (
      • Syme C.A.
      • Zhang L.
      • Bisello A.
      Caveolin-1 regulates cellular trafficking and function of the glucagon-like Peptide 1 receptor.
      ,
      • Prieto-Sánchez R.M.
      • Berenjeno I.M.
      • Bustelo X.R.
      Involvement of the Rho/Rac family member RhoG in caveolar endocytosis.
      ,
      • Pani B.
      • Singh B.B.
      Lipid rafts/caveolae as microdomains of calcium signaling.
      ,
      • Thomas C.M.
      • Smart E.J.
      Caveolae structure and function.
      ). Cholesterol is intrinsically produced in the liver and transferred to individual cells via the blood (
      • Rudney H.
      • Sexton R.C.
      Regulation of cholesterol biosynthesis.
      ). For individual cells, there are two sources of cholesterol: cholesterol synthesized in the endoplasmic reticulum (
      • Lange Y.
      • Ory D.S.
      • Ye J.
      • Lanier M.H.
      • Hsu F.F.
      • Steck T.L.
      Effectors of rapid homeostatic responses of endoplasmic reticulum cholesterol and 3-hydroxy-3-methylglutaryl-CoA reductase.
      ) and cholesterol acquired from the extracellular space via LDLR-mediated endocytosis (
      • Goldstein J.L.
      • Brown M.S.
      The LDL receptor.
      ). Both pools of cholesterol require proper intracellular transport to reach their final destinations. Therefore, intracellular cholesterol homeostasis is tightly controlled by the de novo synthesis and uptake of cholesterol. However, the facts that intracellular de novo cholesterol biosynthesis did not function well in osteoclast lineage cells (
      • Luegmayr E.
      • Glantschnig H.
      • Wesolowski G.A.
      • Gentile M.A.
      • Fisher J.E.
      • Rodan G.A.
      • Reszka A.A.
      Osteoclast formation, survival and morphology are highly dependent on exogenous cholesterol/lipoproteins.
      ,
      • Bergstrom J.D.
      • Bostedor R.G.
      • Masarachia P.J.
      • Reszka A.A.
      • Rodan G.
      Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase.
      ) and that osteoclastogenesis highly depended on exogenous LDL (as shown in this study) suggest that LDLR plays important roles in the uptake of cholesterol into osteoclast lineage cells and in regulating osteoclast differentiation.
      The study presented here demonstrated constitutive RANKL-independent expression of LDLR mRNA in osteoclast precursors during osteoclastogenesis. Using LDLR−/− mice, we found that the osteoclastogenesis of LDLR−/− osteoclast precursors was obviously delayed compared with that of wild-type osteoclast precursors. In addition, osteoclasts formed from LDLR-deficient precursors were smaller than wild-type osteoclasts, and the LDLR-deficient osteoclasts contained fewer nuclei, indicating impaired cell-cell fusion of osteoclast lineage cells. One possible mechanism underlying this cell fusion defect is that the lack of the LDLR gene results in abnormalities in signal transduction pathways necessary for osteoclast formation, as we reported previously that methyl-β-cyclodextrin-mediated depletion of cholesterol from the plasma membrane and destruction of lipid rafts caused alterations in signal transduction related to osteoclastogenesis, such as hyperactivation of Erk1/2 and insensitivity of Akt to RANKL stimulation (
      • Hada N.
      • Okayasu M.
      • Ito J.
      • Nakayachi M.
      • Hayashida C.
      • Kaneda T.
      • Uchida N.
      • Muramatsu T.
      • Koike C.
      • Masuhara M.
      • Sato T.
      • Hakeda Y.
      Receptor activator of NF-κB ligand-dependent expression of caveolin-1 in osteoclast precursors, and high dependency of osteoclastogenesis on exogenous lipoprotein.
      ). Removal of exogenous lipoproteins from the culture medium recapitulated this abnormal signaling and also resulted in reduced osteoclast formation because of the delayed expression of NFATc1 in preosteoclasts (
      • Hada N.
      • Okayasu M.
      • Ito J.
      • Nakayachi M.
      • Hayashida C.
      • Kaneda T.
      • Uchida N.
      • Muramatsu T.
      • Koike C.
      • Masuhara M.
      • Sato T.
      • Hakeda Y.
      Receptor activator of NF-κB ligand-dependent expression of caveolin-1 in osteoclast precursors, and high dependency of osteoclastogenesis on exogenous lipoprotein.
      ). However, no abnormalities were observed in these signaling pathways in LDLR−/− osteoclast lineage cells. Therefore, the impaired cell-cell fusion observed in LDLR−/− osteoclast lineage cells is not due to this type of abnormality. Although the cause of the discrepancies between studies using LDLR−/− mice and those using MCD-treated cells is not understood at present, it is likely that MCD is able to remove large amounts of cholesterol from the plasma membrane (
      • Rodal S.K.
      • Skretting G.
      • Garred O.
      • Vilhardt F.
      • van Deurs B.
      • Sandvig K.
      Extraction of cholesterol with methyl-β-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles.
      ), and this cholesterol depletion destroys lipid rafts, resulting in intense disorder in the molecular microenvironment around RANK, including certain adaptor proteins or suppressors. In contrast, the uptake, intracellular degradation and reutilization of modified LDL through SR-A remain intact in LDLR−/− osteoclast precursors and preosteoclasts, and thus, the integrity of the plasma membrane might be partially maintained. In addition, NFATc1, a master regulator of osteoclast differentiation, and the osteoclast functional molecules cathepsin K and TRAP were expressed at normal levels in the LDLR−/− cells in response to RANKL, suggesting other mechanisms explaining the impaired cell-cell fusion of preosteoclasts.
      In this study, we found that osteoclast formation was decreased in LDLR−/− mice because of the impaired cell-cell fusion of osteoclast precursors. Similar to our study, Luegmayr et al. (
      • Luegmayr E.
      • Glantschnig H.
      • Wesolowski G.A.
      • Gentile M.A.
      • Fisher J.E.
      • Rodan G.A.
      • Reszka A.A.
      Osteoclast formation, survival and morphology are highly dependent on exogenous cholesterol/lipoproteins.
      ) demonstrated that osteoclast formation was reduced in LDLR−/− mice and that the osteoclasts formed in culture were smaller than those derived from wild-type mice. These authors attributed the reduction in osteoclast formation to a defect in cell spreading. Furthermore, these authors demonstrated that the life spans of multinucleated osteoclasts formed in cocultures of LDLR−/− bone marrow cells and osteoblastic MB1.8 cells were less than the life span of wild-type osteoclasts. However, we observed that the level of activated caspase-3 in LDLR−/− preosteoclasts was very low and was equivalent to the level in wild-type preosteoclasts (data not shown), suggesting that the induction of apoptosis might not be an important cause of the observed decrease in osteoclast formation. In addition, these authors did not report on the impairment of cell-cell fusion of osteoclast lineage cells in LDLR−/− mice. Our finding that the numbers of nuclei per osteoclast formed in vitro and per TRAP-positive osteoclast in cross-sections of tibiae in vivo were reduced in LDLR−/− mice obviously indicates that the impaired cell-cell fusion because of LDLR deficiency plays a more significant role in reducing osteoclast formation.
      Atp6v0d2 and DC-STAMP have been identified as essential regulators of osteoclast fusion on the basis of studies using knockout mice for these genes (
      • Lee S.H.
      • Rho J.
      • Jeong D.
      • Sul J.Y.
      • Kim T.
      • Kim N.
      • Kang J.S.
      • Miyamoto T.
      • Suda T.
      • Lee S.K.
      • Pignolo R.J.
      • Koczon-Jaremko B.
      • Lorenzo J.
      • Choi Y.
      v-ATPase V0 subunit d2-deficient mice exhibit impaired osteoclast fusion and increased bone formation.
      ,
      • Yagi M.
      • Miyamoto T.
      • Sawatani Y.
      • Iwamoto K.
      • Hosogane N.
      • Fujita N.
      • Morita K.
      • Ninomiya K.
      • Suzuki T.
      • Miyamoto K.
      • Oike Y.
      • Takeya M.
      • Toyama Y.
      • Suda T.
      DC-STAMP is essential for cell-cell fusion in osteoclasts and foreign body giant cells.
      ). It has been reported recently that Atp6v0d2 interacts with adhesion-regulating molecule 1 (ADAM1) protein and regulates osteoclast multinucleation (
      • Kim T.
      • Ha H.I.
      • Kim N.
      • Yi O.
      • Lee S.H.
      • Choi Y.
      Adrm1 interacts with Atp6v0d2 and regulates osteoclast differentiation.
      ) and that DC-STAMP cooperates with osteoclast stimulatory transmembrane protein (OC-STAMP) in promoting cell-cell fusion of preosteoclasts (
      • Miyamoto H.
      • Suzuki T.
      • Miyauchi Y.
      • Iwasaki R.
      • Kobayashi T.
      • Sato Y.
      • Miyamoto K.
      • Hoshi H.
      • Hashimoto K.
      • Yoshida S.
      • Hao W.
      • Mori T.
      • Kanagawa H.
      • Katsuyama E.
      • Fujie A.
      • Morioka H.
      • Matsumoto M.
      • Chiba K.
      • Takeya M.
      • Toyama Y.
      • Miyamoto T.
      OC- STAMP and DC-STAMP cooperatively modulate cell-cell fusion to form osteoclasts and foreign body giant cells.
      ). In addition, Atp6v0d2 has been shown to possess the differentiation-specific dual functions as a regulator of cell-cell fusion at the early preosteoclast stage and as an essential component of the osteoclast-specific proton pump in bone resorption (
      • Wu H.
      • Xu G.
      • Li Y.P.
      Atp6v0d2 is an essential component of the osteoclast-specific proton pump that mediates extracellular acidification in bone resorption.
      ). However, the detailed molecular mechanism underlying the involvement of the proteins in the cell-cell fusion of preosteoclasts remains to be clarified.
      Atp6v0d2- and DC-STAMP-deficient mice display a common phenotype with respect to osteoclastogenesis; namely, the multi-nucleation of preosteoclasts is intrinsically impaired, whereas expression of the osteoclast master regulator NFATc1 and the osteoclast differentiation-related proteins TRAP and cathepsin K is comparable with that of wild-type osteoclast lineage cells. Thus, mice deficient in both genes exhibited increased bone mass and osteopetrosis. This phenotype is quite consistent with that of LDLR−/− osteoclast lineage cells, as demonstrated here, although LDLR−/− osteoclast lineage cells expressed levels of Atp6v0d2 and DC-STAMP mRNA and protein in response to RANKL. Despite the equivalent expression of Atp6v0d2 and DC-STAMP, the cell-cell fusion of LDLR−/− osteoclast lineage cells was defective. Cell-cell fusion occurs on the plasma membrane of each cell, and both Atp6v0d2 and DC-STAMP are membrane proteins (
      • Lee S.H.
      • Rho J.
      • Jeong D.
      • Sul J.Y.
      • Kim T.
      • Kim N.
      • Kang J.S.
      • Miyamoto T.
      • Suda T.
      • Lee S.K.
      • Pignolo R.J.
      • Koczon-Jaremko B.
      • Lorenzo J.
      • Choi Y.
      v-ATPase V0 subunit d2-deficient mice exhibit impaired osteoclast fusion and increased bone formation.
      ,
      • Yagi M.
      • Miyamoto T.
      • Sawatani Y.
      • Iwamoto K.
      • Hosogane N.
      • Fujita N.
      • Morita K.
      • Ninomiya K.
      • Suzuki T.
      • Miyamoto K.
      • Oike Y.
      • Takeya M.
      • Toyama Y.
      • Suda T.
      DC-STAMP is essential for cell-cell fusion in osteoclasts and foreign body giant cells.
      ,
      • Smith A.N.
      • Jouret F.
      • Bord S.
      • Borthwick K.J.
      • Al-Lamki R.S.
      • Wagner C.A.
      • Ireland D.C.
      • Cormier-Daire V.
      • Frattini A.
      • Villa A.
      • Kornak U.
      • Devuyst O.
      • Karet F.E.
      Vacuolar H+-ATPase d2 subunit. Molecular characterization, developmental regulation, and localization to specialized proton pumps in kidney and bone.
      ,
      • Hartgers F.C.
      • Vissers J.L.
      • Looman M.W.
      • van Zoelen C.
      • Huffine C.
      • Figdor C.G.
      • Adema G.J.
      DC-STAMP, a novel multimembrane-spanning molecule preferentially expressed by dendritic cells.
      ). Therefore, we hypothesized that the amounts of osteoclastic fusion proteins in LDLR−/− osteoclast lineage cells may be decreased in the plasma membrane, despite the fact that equivalent amounts of these proteins were synthesized in LDLR−/− and wild-type osteoclasts. Indeed, we found that the amount of Atp6v0d2 in the plasma membranes of LDLR−/− osteoclast lineage cells was obviously reduced when compared with that of wild-type osteoclast lineage cells, whereas the amounts of Atp6v0d2 in total membranes, including plasma membranes and intracellular membranous organelles, were comparable between LDLR−/− and wild-type cells.
      With respect to DC-STAMP function as a fusion protein, it has been reported recently that RANKL induces two distinct preosteoclast populations expressing high and low levels of DC-STAMP (DC-STAMPhi and DC-STAMPlo, respectively) on the cell surfaces, that DC-STAMPlo cells act as master fusogens and that DC-STAMPhi cells function as mononuclear donors (
      • Mensah K.A.
      • Ritchlin C.T.
      • Schwarz E.M.
      RANKL induces heterogeneous DC-STAMP(lo) and DC-STAMP(hi) osteoclast precursors of which the DC-STAMP(lo) precursors are the master fusogens.
      ). In addition, the mixed cultures of DC-STAMPlo and DC-STAMPhi cells effectively formed larger and more nucleated osteoclasts than cultures of each pure cell population (
      • Mensah K.A.
      • Ritchlin C.T.
      • Schwarz E.M.
      RANKL induces heterogeneous DC-STAMP(lo) and DC-STAMP(hi) osteoclast precursors of which the DC-STAMP(lo) precursors are the master fusogens.
      ). Furthermore, the level of cell surface DC-STAMP on osteoclast lineage cells was down-regulated during osteoclast differentiation, possibly because of increased internalization of DC-STAMP (
      • Mensah K.A.
      • Ritchlin C.T.
      • Schwarz E.M.
      RANKL induces heterogeneous DC-STAMP(lo) and DC-STAMP(hi) osteoclast precursors of which the DC-STAMP(lo) precursors are the master fusogens.
      ,
      • Chiu Y.H.
      • Mensah K.A.
      • Schwarz E.M.
      • Ju Y.
      • Takahata M.
      • Feng C.
      • McMahon L.A.
      • Hicks D.G.
      • Panepento B.
      • Keng P.C.
      • Ritchlin C.T.
      ). In this study, we did not detect DC-STAMP in the total membrane protein fractions, whereas the proteins were abundant in cytosolic fractions. At present, we do not know the reason why DC-STAMP was undetectable in the total membrane fractions, which included intracellular organelle membranes such as lysosomes and Golgi apparatus. Furthermore, a larger amount of plasma membrane protein was required for the detection of surface DC-STAMP than for the detection of Atp6v0d2 in plasma membrane fractions, implying the down-regulation of cell surface DC-STAMP. Nevertheless, the detectable level of DC-STAMP proteins in the plasma membranes in LDLR−/− preosteoclasts was greatly reduced compared with that of wild-type cells, as was the level of Atp6v0d2 protein. These results suggest that the reduced levels of the fusion proteins in plasma membranes might be a major cause of the impaired cell-cell fusion of the LDLR−/− preosteoclasts. Although the mechanism underlying the reduction remains unclear, the reduction might be caused by the decreased transport of the fusion proteins to the plasma membranes or the increased clearance of the proteins from the plasma membranes, or both. Consequently, the phenotype of LDLR−/− mice mimicked that of Atp6v0d2−/− and DC-STAMP−/− mice (
      • Lee S.H.
      • Rho J.
      • Jeong D.
      • Sul J.Y.
      • Kim T.
      • Kim N.
      • Kang J.S.
      • Miyamoto T.
      • Suda T.
      • Lee S.K.
      • Pignolo R.J.
      • Koczon-Jaremko B.
      • Lorenzo J.
      • Choi Y.
      v-ATPase V0 subunit d2-deficient mice exhibit impaired osteoclast fusion and increased bone formation.
      ,
      • Yagi M.
      • Miyamoto T.
      • Sawatani Y.
      • Iwamoto K.
      • Hosogane N.
      • Fujita N.
      • Morita K.
      • Ninomiya K.
      • Suzuki T.
      • Miyamoto K.
      • Oike Y.
      • Takeya M.
      • Toyama Y.
      • Suda T.
      DC-STAMP is essential for cell-cell fusion in osteoclasts and foreign body giant cells.
      ) with respect to osteoclastogenesis and the impaired cell-cell fusion of osteoclast lineage cells. Furthermore, despite the decrease in Atp6v0d2 and DC-STAMP in the plasma membranes of the knockout mice, the amount of Cav-1 in the membranes did not differ between osteoclast lineage cells of the two genotypes, suggesting a mechanism for Atp6v0d2- and DC-STAMP-specific localization associated with intracellular cholesterol content. Further studies will be required to elucidate the molecular mechanisms.
      In conclusion, LDLR−/− mice exhibited increased bone mass in vivo. This change was accompanied by decreases in bone resorption parameters with no changes in bone formation parameters. Consistently, in vitro osteoclastogenesis was highly dependent on exogenous LDL, and LDLR deficiency impaired osteoclast formation because of the reduced cell-cell fusion of preosteoclasts. However, the osteoclast differentiation-related transcription factor NFATc1, the related functional proteins cathepsin K and TRAP, and the osteoclast cell fusion proteins Atp6v0d2 and DC-STAMP were expressed in LDLR−/− osteoclast lineage cells at levels equivalent to those observed in wild-type cells. In contrast, the levels of Atp6v0d2 and DC-STAMP proteins at the plasma membrane were significantly decreased in the LDLR−/− osteoclast lineage cells. Taken together, the results presented here provide novel insight regarding the correlation between bone and lipid metabolism.

      Acknowledgments

      We thank M. Yamada (Morinaga Milk Industry Co.) for his generous gift of recombinant M-CSF and H. Murayama and N. Yajima for help with the bone histomorphometric analysis.

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