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. 2012 Jun 1;287(23):19229-41.
doi: 10.1074/jbc.M111.323600. Epub 2012 Apr 12.

Low-density lipoprotein receptor deficiency causes impaired osteoclastogenesis and increased bone mass in mice because of defect in osteoclastic cell-cell fusion

Mari Okayasu  1 , Mai Nakayachi, Chiyomi Hayashida, Junta Ito, Toshio Kaneda, Masaaki Masuhara, Naoto Suda, Takuya Sato, Yoshiyuki Hakeda
Affiliations

Low-density lipoprotein receptor deficiency causes impaired osteoclastogenesis and increased bone mass in mice because of defect in osteoclastic cell-cell fusion

Mari Okayasu et al. J Biol Chem. .

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.

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Figures

FIGURE 1.
FIGURE 1.
Suppression of in vitro osteoclast formation in the absence of exogenous LDL. Osteoclast precursors obtained from bone marrow cells treated with M-CSF for 3 days were cultured in medium containing normal FBS or lipoprotein-reduced FBS (LR-FBS) with or without various doses of LDL or ox-LDL (A) or a fixed dose (15 μg/ml) of LDL and/or ox-LDL (B) in the presence of M-CSF (20 ng/ml) and sRANKL (10 ng/ml) for 3 days. After culturing, the cells were stained for TRAP activity, and the number of TRAP-positive MNCs per well was counted. The experiments were performed in triplicate, and reproducibility was confirmed. The values presented are the mean ± S.E. of four cultures from a representative experiment. *, p < 0.05 versus cells cultured in medium containing LR-FBS without LDL or ox-LDL; **, p < 0.05 versus cells cultured with LDL or ox-LDL alone. C, photographs of TRAP-stained osteoclastic cells in cultures shown in B. Scale bar = 500 μm. D, osteoclast precursors were cultured in medium containing normal FBS with or without sRANKL in the presence of M-CSF for the indicated times, and total RNA was then prepared and subjected to real-time RT-PCR for LDLR, SR-A, or 18 S rRNA.
FIGURE 2.
FIGURE 2.
Reduced in vitro osteoclastogenesis from LDLR−/− osteoclast precursors. A, osteoclast precursors from LDLR−/− and littermate wild-type mice were treated with sRANKL and M-CSF for the indicated times. After culturing, the cells were stained for TRAP activity, and the number of TRAP-positive MNCs per well was counted. B, the osteoclast precursors from mice of both genotypes were seeded on dentine slices and cultured under the same conditions as indicated in A. After culturing, the osteoclast linage cells formed on the dentine slices were stained for TRAP activity or with acid hematoxylin for resorbed lacunae, and the number of TRAP-positive MNCs and the size of the stained pit area per dentine slice were counted and measured, respectively. The experiments were performed four times, and reproducibility was confirmed. The values presented are the mean ± S.E. of four cultures from a representative experiment. *, p < 0.05 versus culture of wild-type osteoclast precursors. Photographs shown in the right panels of A and B show TRAP-stained osteoclast lineage cells grown in tissue culture wells on day 3.5 and acid hematoxylin-stained pits on dentine slices on day 7, respectively. Scale bar = 500 μm.
FIGURE 3.
FIGURE 3.
Osteoclast differentiation signaling and expression of osteoclast differentiation-related molecules in LDLR−/− osteoclast lineage cells. A, osteoclast precursors and preosteoclasts that had been treated for 2 days with M-CSF or M-CSF/sRANKL, respectively, were starved in serum-free medium for 3 h prior to treatment with sRANKL for the indicated times. The cells were then extracted, and the cell lysates were subjected to Western blot analysis using the indicated antibodies. B and C, the osteoclast precursors were cultured with sRANKL for the indicated times. After culturing, the cells were extracted, and total RNA and cell lysates were subjected to real-time RT-PCR (B) and Western blot analysis (C), respectively. The experiments were performed in triplicate, and reproducibility was confirmed.
FIGURE 4.
FIGURE 4.
Reduced size and cell-cell fusion of TRAP-positive MNCs formed from LDLR−/− osteoclast precursors. LDLR−/− (closed bar) and wild-type (open bar) osteoclast precursors were cultured with sRANKL and M-CSF for 2 or 3 days, and the cells in the cultures were then stained for TRAP activity. Thereafter, more than one hundred TRAP-positive MNCs in each culture were randomly selected, and the maximum diameter (B, double arrow in A) and the area (C, area surrounded by dotted line in A) of the formed TRAP-positive MNCs were measured. D, the fusion index is presented as the average number of nuclei per TRAP-positive MNC formed in the cultures, which indicates the number of cells that participated in fusion. The values presented are the mean ± S.E. for more than 100 cells in each culture. *, p < 0.05 versus culture derived from wild-type mice.
FIGURE 5.
FIGURE 5.
Expression and localization of the osteoclast fusion proteins Atp6v0d2 and DC-STAMP in LDLR−/− osteoclast lineage cells. A, osteoclast precursors derived from LDLR−/− mice and wild-type littermates were cultured with sRANKL for 6 h or the indicated number of days (D). After culturing, the cells were extracted, and total RNA and cell lysates were subjected to real-time RT-PCR (A) and Western blot analysis (B), respectively, to determine the expression levels of Atp6v0d2 and DC-STAMP. The experiments were performed in triplicate, and reproducibility was confirmed. C and D, osteoclast precursors derived from both genotypes were cultured with M-CSF and sRANKL for 2 days. Thereafter, whole-cell lysates, total cellular membranes (TCM) containing proteins from both plasma membranes and cellular organelle membranes, cytosol fractions (C), and plasma membranes (PM) excluding cellular organelle membranes (D) were prepared as described under “Experimental Procedures.” Samples of each fraction were subjected to Western blot analysis for Atp6v0d2, DC-STAMP and Cav-1 protein levels. The experiments were performed four times, and reproducibility was confirmed.
FIGURE 6.
FIGURE 6.
Determination of the localization of Atp6v0d2 in osteoclast lineage cells by confocal laser microscopy. Osteoclast precursors derived from wild-type (WT, a–h and q–s) and LDLR−/− (i–p) mice were cultured in the presence of sRANKL for 2 days. The cells were then treated with (a–p) or without (q–s) Alexa Fluor 555-conjugated CT-B prior to fixation. The fixed cells were incubated with an anti-Atp6v0d2 antibody (a–p) or normal rabbit IgG (q–s) and subsequently with DAPI solution. Fluorescence images for DAPI (blue), anti-Atp6v0d2 (green), and CT-B (red) were merged. Scale bars in (a–d, i–l) and (e–h, m–p, and q–s) = 10 μm and 20 μm, respectively.
FIGURE 7.
FIGURE 7.
Increased bone mass in LDLR-deficient mice. Femora of 8-week-old male LDLR−/− and wild-type mice were radiologically analyzed by μCT (A and B). The two left panels in A show two-dimensional reconstructions of the epiphysis and metaphysis of distal femora from LDLR−/− and control wild-type (+/+) mice, and the two right panels in A show three-dimensional reconstructions of secondary spongy bone of the metaphysis. B, histograms represent bone structural parameters: bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular separation (Tb.Sp), and trabecular thickness (Tb,Th). Mesial tibiae from LDLR−/− and wild-type (+/+) mice were stained with toluidine blue (C) and for TRAP activity (D), and histomorphometric parameters (E), including osteoid volume (OV/BV), osteoblast surface (Ob.S/BS), bone formation rate (BFR/BS), mineral apposition rate (MAR), mineralizing surface (MS/BS), osteoclast number (Oc.N/B.Pm), eroded surface (ES/BS), and osteoclast surface (Oc.S/BS) were determined. Values are the mean ± S.E. for five bones each from LDLR−/− and wild-type mice. *, p < 0.05 versus wild-type mice. NS, not significant. F, the maximum diameter of TRAP-positive osteoclasts (n > 800) and nuclei per TRAP-positive osteoclast (n ≥ 100) on the cross-sections were measured and counted, respectively. *, p < 0.01 versus wild-type mice. Scale bars = 1 mm, 800 μm, 500 μm, and 100 μm in the left panels in A and the right panels in A, C, and D, respectively.
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