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Cited by 892Bone Metabolism and DiseasesPhagocytosis and Immune RegulationOsteomyelitis and Bone Disorders Research

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DC-STAMP is essential for cell–cell fusion in osteoclasts and foreign body giant cells

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Yagi, Mitsuru, Miyamoto, Takeshi, Sawatani, Yumi, Iwamoto, Katsuya, Hosogane, Naobumi, Fujita, Nobuyuki, Morita, Kozo, Ninomiya, Ken, Suzuki, Toru, Miyamoto, Kana, Oike, Yuichi, Takeya, Motohiro, Toyama, Yoshiaki, Suda, Toshio·2005·The Journal of Experimental Medicine
DOI 10.1084/jem.20050645PMC PMC2213087
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Abstract

Osteoclasts are bone-resorbing cells that play a pivotal role in bone remodeling. Osteoclasts form large multinuclear giant cells by fusion of mononuclear osteoclasts. How cell fusion is mediated, however, is unclear. We identify the dendritic cell–specific transmembrane protein (DC-STAMP), a putative seven-transmembrane protein, by a DNA subtraction screen between multinuclear osteoclasts and mononuclear macrophages. DC-STAMP is highly expressed in osteoclasts but not in macrophages. DC-STAMP–deficient mice were generated, and osteoclast cell fusion was completely abrogated in homozygotes despite normal expression of osteoclast markers and cytoskeletal structure. As osteoclast multinucleation was restored by retroviral introduction of DC-STAMP, loss of cell fusion was directly attributable to a lack of DC-STAMP. Defects in osteoclast multinucleation reduce bone-resorbing activity, leading to osteopetrosis. Similar to osteoclasts, foreign body giant cell formation by macrophage cell fusion was also completely abrogated in DC-STAMP–deficient mice. We have thus identified an essential regulator of osteoclast and macrophage cell fusion, DC-STAMP, and an essential role of osteoclast multinucleation in bone homeostasis.

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Figure 1. Identification and gene targeting of DC-STAMP. (A) Specific expression of DC-STAMP (arrow) in osteoclasts (M-CSF + RANKL) was determined among macrophages (M-CSF alone), immature dendritic cells (M-CSF + RANKL + GM-CSF), and osteoclasts derived from common precursor cells by Northern blot analysis. (B) The endogenous DC-STAMP locus (DC-STAMP gene), targeting vector, and targeted locus (mutant allele) are shown. Exons, represented by black boxes, are numbered. The ATG start codon is located in exon 2. The neomycin resistance gene (Neo) and EGFP-polyA sequence are indicated. The probe used for Southern hybridization is indicated (P). The EGFP-polyA sequence and Neo cassette were inserted into exon 2 to yield a construct encoding a DC-STAMP (1–55)–EGFP chimeric protein. (C) RT-PCR analysis of the expression of DC-STAMP and the chimeric DC-STAMP-EGFP genes in osteoclasts and bones from mice of the indicated genotypes. (D) TRAP staining in tibias of 8-wk-old DC-STAMP+/+ or DC-STAMP−/− mice (a) and the number of multinuclear TRAP-positive cells (b). Multinuclear osteoclast formation was abrogated in DC-STAMP−/− mice. Values represent SD.
Figure 1. Identification and gene targeting of DC-STAMP. (A) Specific expression of DC-STAMP (arrow) in osteoclasts (M-CSF + RANKL) was determined among macrophages (M-CSF alone), immature dendritic cells (M-CSF + RANKL + GM-CSF), and osteoclasts derived from common precursor cells by Northern blot analysis. (B) The endogenous DC-STAMP locus (DC-STAMP gene), targeting vector, and targeted locus (mutant allele) are shown. Exons, represented by black boxes, are numbered. The ATG start codon is located in exon 2. The neomycin resistance gene (Neo) and EGFP-polyA sequence are indicated. The probe used for Southern hybridization is indicated (P). The EGFP-polyA sequence and Neo cassette were inserted into exon 2 to yield a construct encoding a DC-STAMP (1–55)–EGFP chimeric protein. (C) RT-PCR analysis of the expression of DC-STAMP and the chimeric DC-STAMP-EGFP genes in osteoclasts and bones from mice of the indicated genotypes. (D) TRAP staining in tibias of 8-wk-old DC-STAMP+/+ or DC-STAMP−/− mice (a) and the number of multinuclear TRAP-positive cells (b). Multinuclear osteoclast formation was abrogated in DC-STAMP−/− mice. Values represent SD.CC BY-NC-SA · PubMed Central
Figure 2. Lack of multinucleation in DC-STAMP−/−osteoclasts. (A) TRAP expression was induced, but multinucleation was completely abrogated in DC-STAMP−/− osteoclasts. TRAP staining (a) and the number of multinuclear TRAP-positive cells (b) are shown. Values represent SD. Bar, 50 μm. (B) TRAP solution assay of macrophages (M-CSF alone) and osteoclasts (M-CSF + RANKL). (C) Expression of c-fos, NFATc1, or cathepsin K in macrophages (M-CSF alone) or osteoclasts (M-CSF + RANKL) derived from DC-STAMP+/+, DC-STAMP+/−, or DC-STAMP−/− mice was analyzed by RT-PCR. NC, no template control. (D) Ruffled border formation was detected in osteoclasts of DC-STAMP−/− tibial sections under electron microscopy. RB, ruffled border. (E) Total number of nuclei in cultured osteoclasts. Values represent SD. (F) The percent frequency of a population of osteoclast precursor cells (boxes, c-Fms+c-Kit+Mac-1low) is shown as the mean ± SD. (G) Resorbing lacunae were visualized by toluidine blue O staining (a), and relative resorbing areas were scored (b). Values represent SD.
Figure 2. Lack of multinucleation in DC-STAMP−/−osteoclasts. (A) TRAP expression was induced, but multinucleation was completely abrogated in DC-STAMP−/− osteoclasts. TRAP staining (a) and the number of multinuclear TRAP-positive cells (b) are shown. Values represent SD. Bar, 50 μm. (B) TRAP solution assay of macrophages (M-CSF alone) and osteoclasts (M-CSF + RANKL). (C) Expression of c-fos, NFATc1, or cathepsin K in macrophages (M-CSF alone) or osteoclasts (M-CSF + RANKL) derived from DC-STAMP+/+, DC-STAMP+/−, or DC-STAMP−/− mice was analyzed by RT-PCR. NC, no template control. (D) Ruffled border formation was detected in osteoclasts of DC-STAMP−/− tibial sections under electron microscopy. RB, ruffled border. (E) Total number of nuclei in cultured osteoclasts. Values represent SD. (F) The percent frequency of a population of osteoclast precursor cells (boxes, c-Fms+c-Kit+Mac-1low) is shown as the mean ± SD. (G) Resorbing lacunae were visualized by toluidine blue O staining (a), and relative resorbing areas were scored (b). Values represent SD.CC BY-NC-SA · PubMed Central
Figure 3. Increased bone mass in DC-STAMP−/− mice. (A) Soft x-ray analysis. Elevated radioopacity was observed in DC-STAMP−/− mice. (B) Increased BMD (mg/cm2) was detected in DC-STAMP−/− mice (closed circles) compared with DC-STAMP+/+ mice (open circles) by the dual energy x-ray absorptiometry method measured in 20 longitudinal divisions of femurs from 8-wk-old female mice. (C) Bone morphometric analysis of tibia from 8-wk-old mice. The percent BV/TV was elevated in DC-STAMP−/− mice compared with wild-type mice (*, P < 0.01). Values represent SD. (D) Increased numbers of trabeculae were observed in tibial sections of DC-STAMP−/− mice by toluidine blue staining.
Figure 3. Increased bone mass in DC-STAMP−/− mice. (A) Soft x-ray analysis. Elevated radioopacity was observed in DC-STAMP−/− mice. (B) Increased BMD (mg/cm2) was detected in DC-STAMP−/− mice (closed circles) compared with DC-STAMP+/+ mice (open circles) by the dual energy x-ray absorptiometry method measured in 20 longitudinal divisions of femurs from 8-wk-old female mice. (C) Bone morphometric analysis of tibia from 8-wk-old mice. The percent BV/TV was elevated in DC-STAMP−/− mice compared with wild-type mice (*, P < 0.01). Values represent SD. (D) Increased numbers of trabeculae were observed in tibial sections of DC-STAMP−/− mice by toluidine blue staining.CC BY-NC-SA · PubMed Central
Figure 4. Rescue of cell fusion in DC-STAMP−/− osteoclasts. (A) Osteoclast precursors from DC-STAMP−/− mice were transduced by retrovirus expressing full-length (full) or a splice variant form (splice) of DC-STAMP (a). The indicated form of DC-STAMP expressed via retroviral infection was detected in DC-STAMP−/−osteoclasts by RT-PCR analysis (b). NC, no template control. Bar, 50 μm. (B) No multinuclear TRAP-positive cells were induced in DC-STAMP−/− cells by high density culture (a) or cocultivation with osteoblasts (b). Bar, 50 μm. (C) 2 × 104 cells/well of M-CSF–dependent osteoclast precursors from DC-STAMP−/− and DC-STAMP+/+ mice were mixed and cultured in the presence of M-CSF and RANKL for 4 d in 96-well culture plates. Cells were stained by rabbit anti-EGFP antibody followed by Alexa 488 (EGFP)–conjugated anti–rabbit IgG antibody with rhodamine-conjugated phalloidin for F-actin staining and TOTO3 for nuclear staining. Note the formation of EGFP-positive multinuclear cells. *, EGFP-negative multinuclear cells; **, EGFP-positive multinuclear cells.
Figure 4. Rescue of cell fusion in DC-STAMP−/− osteoclasts. (A) Osteoclast precursors from DC-STAMP−/− mice were transduced by retrovirus expressing full-length (full) or a splice variant form (splice) of DC-STAMP (a). The indicated form of DC-STAMP expressed via retroviral infection was detected in DC-STAMP−/−osteoclasts by RT-PCR analysis (b). NC, no template control. Bar, 50 μm. (B) No multinuclear TRAP-positive cells were induced in DC-STAMP−/− cells by high density culture (a) or cocultivation with osteoblasts (b). Bar, 50 μm. (C) 2 × 104 cells/well of M-CSF–dependent osteoclast precursors from DC-STAMP−/− and DC-STAMP+/+ mice were mixed and cultured in the presence of M-CSF and RANKL for 4 d in 96-well culture plates. Cells were stained by rabbit anti-EGFP antibody followed by Alexa 488 (EGFP)–conjugated anti–rabbit IgG antibody with rhodamine-conjugated phalloidin for F-actin staining and TOTO3 for nuclear staining. Note the formation of EGFP-positive multinuclear cells. *, EGFP-negative multinuclear cells; **, EGFP-positive multinuclear cells.CC BY-NC-SA · PubMed Central
Figure 5. Macrophage cell fusion is abrogated in DC-STAMP−/− mice. (A) Histological analysis of implants in DC-STAMP−/− and DC-STAMP+/+ mice. Hematoxylin and eosin staining (a) and the number of FBGCs (b) are shown. Arrows indicate FBGCs. (B) FBGCs were induced and stained with May-Gruenwald Giemsa (a), and the number of FBGCs containing more than three nuclei was scored (b). Values represent SD. Bar, 50 μm.
Figure 5. Macrophage cell fusion is abrogated in DC-STAMP−/− mice. (A) Histological analysis of implants in DC-STAMP−/− and DC-STAMP+/+ mice. Hematoxylin and eosin staining (a) and the number of FBGCs (b) are shown. Arrows indicate FBGCs. (B) FBGCs were induced and stained with May-Gruenwald Giemsa (a), and the number of FBGCs containing more than three nuclei was scored (b). Values represent SD. Bar, 50 μm.CC BY-NC-SA · PubMed Central

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  1. 1995, Am. J. Pathol., 147, 1487

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Metadata

Authors
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.
Affiliations
  • Keio University (JP)
  • Kumamoto University (JP)
  • Kyushu University (JP)
  • 1Department of Cell Differentiation, The Sakaguchi Laboratory
ORCID
Venue
The Journal of Experimental Medicine
Publisher
Rockefeller University Press
Published
2005-08-01
Volume
202
Issue
3
Pages
345-351
ISSN
  • 0022-1007 (ISSN-L)
  • 1540-9538
DOI
10.1084/jem.20050645
PMC
PMC2213087
OpenAlex
W2111239144
Cited by
890
References
24
Citations by year
2026: 36 · 2025: 35 · 2024: 33 · 2023: 36 · 2022: 56 · 2021: 48 · 2020: 60 · 2019: 39 · 2018: 40 · 2017: 52 · 2016: 48 · 2015: 48 · 2014: 50 · 2013: 37 · 2012: 46
FWCI
11.80
Type
journal article
Language
en
Open access
Locations
  • The Journal of Experimental Medicine (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-sa)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Bone Metabolism and Diseases
  • Life Sciences › Immunology and Microbiology › Immunology › Phagocytosis and Immune Regulation
  • Health Sciences › Medicine › Rheumatology › Osteomyelitis and Bone Disorders Research
Keywords
DC-STAMP, cell fusion, osteoclasts, foreign body giant cells, multinucleation, osteoclastogenesis, bone resorption, osteopetrosis, macrophages, bone homeostasis, seven-transmembrane proteins
MeSH terms
Animals, Bone and Bones, Bone Resorption, Cell Fusion, Homeostasis, Macrophages, Membrane Proteins, Nerve Tissue Proteins, Osteoclasts, Giant Cells, Mice, Knockout, Mice

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