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1.
J Cell Sci ; 133(10)2020 05 27.
Article in English | MEDLINE | ID: mdl-32295848

ABSTRACT

Osteoporosis is associated with vessel diseases attributed to hyperlipidemia, and bone resorption by multinucleated osteoclasts is related to lipid metabolism. In this study, we generated low-density lipoprotein receptor (LDLR)/lectin-like oxidized LDL receptor-1 (LOX-1, also known as Olr1) double knockout (dKO) mice. We found that, like LDLR single KO (sKO), LDLR/LOX-1 dKO impaired cell-cell fusion of osteoclast-like cells (OCLs). LDLR/LOX-1 dKO and LDLR sKO preosteoclasts exhibited decreased uptake of LDL. The cell surface cholesterol levels of both LDLR/LOX-1 dKO and LDLR sKO osteoclasts were lower than the levels of wild-type OCLs. Additionally, the amount of phosphatidylethanolamine (PE) on the cell surface was attenuated in LDLR/LOX-1 dKO and LDLR sKO preosteoclasts, whereas the PE distribution in wild-type OCLs was concentrated on the filopodia in contact with neighboring cells. Abrogation of the ATP binding cassette G1 (ABCG1) transporter, which transfers PE to the cell surface, caused decreased PE translocation to the cell surface and subsequent cell-cell fusion. The findings of this study indicate the involvement of a novel cascade (LDLR∼ABCG1∼PE translocation to cell surface∼cell-cell fusion) in multinucleation of OCLs.


Subject(s)
Atherosclerosis , Osteoclasts , Animals , Cholesterol, LDL , Lipoproteins, LDL , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphatidylethanolamines , Receptors, LDL/genetics
2.
Bone ; 75: 170-82, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25744064

ABSTRACT

Inflammatory bone diseases have been attributed to increased bone resorption by augmented and activated bone-resorbing osteoclasts in response to inflammation. Although the production of diverse proinflammatory cytokines is induced at the inflamed sites, the inflammation also generates reactive oxygen species that modify many biological compounds, including lipids. Among the oxidized low-density lipoprotein (LDL) receptors, lectin-like oxidized LDL receptor-1 (LOX-1), which is a key molecule in the pathogenesis of multifactorial inflammatory atherosclerosis, was downregulated with osteoclast differentiation. Here, we demonstrate that LOX-1 negatively regulates osteoclast differentiation by basically suppressing the cell-cell fusion of preosteoclasts. The LOX-1-deleted (LOX-1(-/-)) mice consistently decreased the trabecular bone mass because of elevated bone resorption during the growing phase. In contrast, when the calvaria was inflamed by a local lipopolysaccharide-injection, the inflammation-induced bone destruction accompanied by the elevated expression of osteoclastogenesis-related genes was reduced by LOX-1 deficiency. Moreover, the expression of receptor activator of NF-κB ligand (RANKL), a trigger molecule for osteoclast differentiation, evoked by the inflammation was also abrogated in the LOX-1(-/-) mice. Osteoblasts, the major producers of RANKL, also expressed LOX-1 in response to proinflammatory agents, interleukin-1ß and prostaglandin E2. In the co-culture of LOX-1(-/-) osteoblasts and wild-type osteoclast precursors, the osteoclastogenesis induced by interleukin-1ß and prostaglandin E2 decreased; this process occurred in parallel with the downregulation of osteoblastic RANKL expression. Collectively, LOX-1 abrogation results in resistance to inflammatory bone destruction, despite promoting osteoclastogenesis in the steady state. Our findings indicate the novel involvement of LOX-1 in physiological bone homeostasis and inflammatory bone diseases.


Subject(s)
Bone Diseases/metabolism , Osteoclasts/cytology , Scavenger Receptors, Class E/metabolism , Animals , Blotting, Western , Bone Diseases/pathology , Bone Resorption/pathology , Cell Differentiation/physiology , Cells, Cultured , Coculture Techniques , Inflammation/metabolism , Inflammation/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Osteoblasts/metabolism , Osteoclasts/metabolism , Polymerase Chain Reaction
3.
J Biol Chem ; 289(16): 11545-11555, 2014 Apr 18.
Article in English | MEDLINE | ID: mdl-24610813

ABSTRACT

Osteoclastogenesis is controlled by osteocytes; osteocytic osteoclastogenesis regulatory molecules are largely unknown. We searched for such factors using newly developed culture methods. Our culture system mimics the three-dimensional cellular structure of bone, consisting of collagen gel-embedded osteocytic MLO-Y4 cells, stromal ST2 cells on the gel as bone lining cells, and bone marrow cells. The gel-embedded MLO-Y4 cells inhibited the osteoclastogenesis induced by 1,25(OH)2D3 without modulating receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) production by ST2 cells, despite MLO-Y4 cells supported osteoclastogenesis in the absence of ST2 cells. In the bone marrow cell culture, the conditioned medium from MLO-Y4 cells decreased the capability of osteoclastic differentiation from the cells induced by macrophage colony-stimulating factor. This decreased capability was concomitant with an increase in protein kinase R mRNA expression and an inhibition of c-Fos translation. These changes were partially normalized by the simultaneous addition of an anti-interferon (IFN)-ß neutralizing antibody to MLO-Y4 cell conditioned medium. To study primary osteocytes, we prepared non-osteocytic cell-free osteocyte-enriched bone fragments (OEBFs). When osteoclast precursors were induced by macrophage colony-stimulating factor in the presence of OEBFs, the generated cells exhibited a diminished capacity for osteoclastogenesis. OEBFs prepared from OPG-knock-out mice exhibited a similar effect, indicating OPG-independent inhibition. The addition of anti-IFN-ß neutralizing antibody during the co-culture with OEBFs partially recovered the osteoclastogenic potential of the generated cells. The MLO-Y4 cells and OEBFs expressed IFN-ß mRNA. Although osteocytic RANKL is known to be important for osteoclastogenesis, our data suggest that osteocytes also produce IFN-ß as an inhibitor of osteoclastogenesis.


Subject(s)
Cell Differentiation/physiology , Interferon-beta/metabolism , Macrophage Colony-Stimulating Factor/metabolism , Osteoclasts/metabolism , Osteocytes/metabolism , RANK Ligand/metabolism , Animals , Antibodies, Neutralizing/pharmacology , Calcitriol/pharmacology , Cell Differentiation/drug effects , Cells, Cultured , Interferon-beta/antagonists & inhibitors , Interferon-beta/genetics , Macrophage Colony-Stimulating Factor/genetics , Male , Mice , Mice, Knockout , Osteoclasts/cytology , Osteocytes/cytology , Osteoprotegerin/genetics , Osteoprotegerin/metabolism , RANK Ligand/genetics
4.
J Biol Chem ; 287(23): 19229-41, 2012 Jun 01.
Article in English | MEDLINE | ID: mdl-22500026

ABSTRACT

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 V(0) 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.


Subject(s)
Bone Resorption/metabolism , Bone and Bones/metabolism , Cell Differentiation , MAP Kinase Signaling System , Osteoclasts/metabolism , Osteoporosis/metabolism , Receptors, LDL/metabolism , Animals , Bone Resorption/genetics , Bone Resorption/pathology , Bone and Bones/pathology , Cell Fusion , Cell Membrane/genetics , Cell Membrane/metabolism , Cells, Cultured , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation/genetics , Humans , Mice , Mice, Knockout , NFATC Transcription Factors/biosynthesis , NFATC Transcription Factors/genetics , Organ Size , Osteoclasts/pathology , Osteoporosis/genetics , Osteoporosis/pathology , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , RANK Ligand/genetics , RANK Ligand/metabolism , Receptors, LDL/genetics , Vacuolar Proton-Translocating ATPases/genetics , Vacuolar Proton-Translocating ATPases/metabolism
5.
Bone ; 50(1): 226-36, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22075210

ABSTRACT

Although extensive studies have done much to clarify the molecular mechanisms of osteoclastogenesis during the last ten years, there may still be unknown molecules associated with osteoclast differentiation. Thus, we used fluorescent differential display to screen for genes whose expression is induced by receptor activator of NF-κB ligand (RANKL), a crucial molecule for osteoclast formation. We identified caveolin-1 (Cav-1) as a RANKL-induced gene. Cav-1 is a major structural protein of caveolae and lipid rafts, cholesterol-enriched microdomains in the plasma membrane (PM). The RANKL-induced Cav-1 was immediately conveyed to lipid rafts. Conversely, expression of flotillin-1 (Flot-1), another scaffolding protein of lipid rafts, was reduced during osteoclastogenesis, indicating conversion of Flot-1-predominant rafts into Cav-1-enriched rafts. However, in vitro osteoclastogenesis of precursor cells from Cav-1-null mice was comparable to that of wild-type mice, while Cav-2 expression in the knockout osteoclasts was maintained. Conversely, Cav-2 gene silencing in Cav-1-null osteoclast precursors using siRNA for Cav-2 increased osteoclast formation, suggesting that the Cav-1/Cav-2 complex may act as a negative regulator for osteoclastogenesis. On the other hand, destruction of lipid rafts by removal of cholesterol from the PM by methyl-ß-cyclodextrin (MCD) treatment caused disordered signal transductions for osteoclastogenesis, such as hyperactivation of Erk1/2 and insensitivity of Akt to RANKL stimulus. The abnormal signaling was reproduced by deleting exogenous lipoproteins from the culture medium, which also resulted in reduced osteoclast formation. In addition, the deletion caused delayed expression of nuclear factor of activated T cells c1 (NFATc1), and depressed its activation in the cytosol and inhibited its translocation into nuclei. Simultaneously, the deletion reduced the level of FcRγ, a trigger protein for initiating the calcium signaling needed to activate NFATc1, and decreased Cav-1 in lipid rafts. These findings indicate that the molecular mechanisms of osteoclastogenesis are highly dependent on extracellular lipoprotein and the integrity of lipid rafts, and suggest possible involvement of cholesterol.


Subject(s)
Bone Resorption/metabolism , Caveolin 1/metabolism , Lipoproteins/metabolism , Osteoclasts/physiology , RANK Ligand/metabolism , Stem Cells/physiology , Animals , Caveolin 1/genetics , Caveolin 2/genetics , Caveolin 2/metabolism , Gene Silencing , Male , Membrane Microdomains/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , NFATC Transcription Factors/genetics , NFATC Transcription Factors/metabolism , Osteoclasts/cytology , RANK Ligand/genetics , Receptor Activator of Nuclear Factor-kappa B/metabolism , Signal Transduction/physiology , Stem Cells/cytology
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