Involvement of FcR{gamma} in signal transduction of osteoclast-associated receptor (OSCAR)

Satoru Ishikawa1, Noriko Arase1,3, Tadashi Suenaga1, Yoshitomo Saita4, Masaki Noda4, Takayuki Kuriyama2, Hisashi Arase1,5,6 and Takashi Saito1,3

1 Department of Molecular Genetics and 2 Department of Respirology, Chiba University Graduate School of Medicine, Chiba, Japan 3 Laboratory for Cell Signaling, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan 4 Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan 5 Research Institute for Microbial Diseases, Osaka University, Yamadaoka 3-1, Suita, Osaka 565-0871, Japan 6 PRESTO, Japan Science and Technology Agency, Saitama, Japan

Correspondence to: H. Arase; E-mail: arase{at}biken.osaka-u.ac.jp
Transmitting editor: H. Karasuyama


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
Osteoclasts regulate homeostasis of bone development. A defect in osteoclast development results in osteopetrosis. Recently, the involvement of several molecules in osteoclast development has been found. Osteoclast-associated receptor (OSCAR) is one of such molecules critical for osteoclast differentiation. However, it remains unclear how OSCAR transduces signals for osteoclast differentiation. Here, we found that the FcR{gamma} chain, a signal transducing adaptor molecule for Fc receptors, is associated with OSCAR and is involved in the cell surface expression of OSCAR. Furthermore, FcR{gamma} is required for signal transduction by OSCAR. These findings suggest that the FcR{gamma}-mediated signal transduction by OSCAR is involved in osteoclast differentiation.

Keywords: CD3{zeta}, Fc receptor, Ig-like receptor, ITAM, osteoclast


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
Bone homeostasis is dynamically controlled by the remodeling of new bone by osteoblasts and the resorption of old bone by osteoclasts. Disregulation of the functional balance between osteoblasts and osteoclasts results in severe bone disorder. As osteoclasts are the only population that resorbs bone, a defect in osteoclast function will result in osteopetrosis (1,2). Osteoclasts are derived from bone marrow (BM) precursor cells and belong to a lineage similar to that of macrophages and dendritic cells (1,2).

Recently, several studies have suggested that cell surface molecules such as triggering receptor expressed on myeloid cells 2 (TREM2) (3,4) and OSCAR (5) are involved in osteoclast differentiation. TREM2 is widely expressed on cells that belong to myeloid lineage including osteoclasts (6,7). DAP12 is associated with TREM2 and mediates activation signals through TREM2 (7,8). It has been reported that a defect in TREM2-mediated signal transduction in human results in bone disorder (3,4). On the other hand, Kim et al. (5) have recently cloned OSCAR as a molecule expressed mainly on osteoclasts. They showed that osteoclast differentiation in vitro is significantly inhibited by OSCAR–Ig fusion protein. These findings suggest that OSCAR transduces signals involved in osteoclast differentiation upon recognition of its ligand. However, it remains unclear how OSCAR transduces an activation signal that is required for osteoclast differentiation.

It is known that various activating cell surface receptors on lymphocytes are associated with immunoreceptor tyrosine kinase activation motif (ITAM)-bearing signal transducing adaptor molecules such as CD3{zeta}, FcR{gamma} (Fc{epsilon}RI{gamma}) and DAP12 (9). These ITAM-bearing molecules are required for signal transduction by these receptors. One common feature of these signal transducing adaptor molecules is that they possess negatively charged amino acids at the transmembrane domain (9). On the other hand, activating receptors that are associated with these signal transducing adaptor molecules possess positively charged amino acids at the transmembrane domain. The interaction between the negatively charged amino acids and the positively charged amino acids of these molecules is a primary requisite for the association of these receptors. Indeed, a point mutation of these charged amino acids abrogates this interaction (10,11). In addition to these ITAM-bearing signal transducing adaptor molecules, DAP10, which transduces an ITAM-independent activation signal into lymphocytes, also possesses a negatively charged amino acid at the transmembrane domain and associates with activating NKG2D receptor (12). Interestingly, OSCAR possesses a positively charged amino acid, arginine, within the transmembrane region. In the present study, we analyzed whether OSCAR associates with these ITAM-bearing signal transducing adaptor molecules and found that FcR{gamma} plays a vital role in OSCAR-mediated signal transduction.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
Mice
C57BL/6 mice were purchased from Japan SLC (Hamamatsu, Japan). FcR{gamma}-deficient mice with C57BL/6 background (13) were maintained in our animal facility in the SPF condition.

Induction of BM-derived osteoclasts
BM cells from C57BL/6 mice were cultured in {alpha}-MEM (Sigma) supplemented with 10% FCS (Sigma), 10–7 M dexamethasone (Sigma), and 10–8 M vitamin D3 [1{alpha}, 25(OH)2 D3, Calbiochem] for 10 days. In some experiments, BM cells were cultured in {alpha}-MEM (Sigma) supplemented with 10% FCS (Sigma), 10 ng/ml macrophage colony stimulating factor (M-CSF) (R&D systems), and 30 ng/ml murine soluble receptor activator of NF-{kappa}B ligand (sRANKL) (PeproTech) for 10 days as indicated. Osteoclast development was analyzed using a tartrate-resistant acid phosphatase (TRAP) staining kit (Hokudo).

RT–PCR
RNA was extracted from the BM-derived osteoclasts by means of a QIA quick RNA extraction kit and cDNA was generated using Superscript II (Invitrogen) reverse transcriptase. Primers used for amplification were as follows: CD3{zeta}, sense primer: 5'-ATCATCACAGCCCTGTACCT-3', anti-sense primer: 5'-CCTCTCCGCCTCTCGCCTTT-3'; FcR{gamma}, sense primer: 5'-AAGAATTCCAGCGCC-3', anti-sense primer: 5'-GGA ATTCGCTGCCTTTCGGACCTGGAT-3'; DAP12, sense primer: 5'-AGTGACACTTTCCCAAGATG-3', anti-sense primer: 5'-TGATAAGGCGACTCAGTCTC-3'; DAP10, sense primer: 5'-CCGGATGTGGGACTCTGTCT-3', anti-sense primer: 5'-CTC TGCCAGGCATGTTGATG; and ß-actin, sense primer: 5'-TCT ACAATGAGCTGCGTGTG-3', anti-sense primer: 5'-GGTACG ACCAGAGGCATACA-3'. Sequentially diluted cDNAs were amplified in PCR under the following conditions: 94°C for 1 min, 57°C for 1 min, and 72°C for 1.5 min with 20 cycles for ß-actin and 27 cycles for others.

Transfection of OSCAR and FcR{gamma}
cDNA encoding OSCAR without signal sequence was cloned into a pMx-neo retrovirus expression vector containing human CD8 signal sequence and FLAG sequence. cDNAs encoding CD3{zeta}, FcR{gamma}, DAP12 and DAP10 were subcloned into a pMx-internal ribosome entry site (IRES)-green fluorescent protein (GFP) retrovirus expression vector. Each expression vector was transfected into a Phoenix retrovirus packaging cell line and viral supernatants were added to BM-derived osteoclasts cultured for 5 days in the presence of M-CSF and RANKL, and MA5.8, a CD3{zeta}-negative T-cell hybridoma cell line that lacks CD3{zeta}, FcR{gamma} and DAP12 (14). The transfected cells were selected by G418 (1 mg/ml) and stained with anti-FLAG M2 mAb (Sigma), followed by PE-anti-mouse-IgG antibody (Jackson Immunolaboratories).

Surface biotinylation, immunoprecipitation and western blotting
Cells were surface-biotinylated as previously described (15). Biotinylated cells were lysed with lysis buffer containing 1% digitonin, 50 mM Tris–HCl (pH 7.6), 150 mM NaCl, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM PMSF and 10 mM iodoacetamide, at a concentration of 1 x 107 cell/ml. Cell lysates were immunoprecipitated with anti-Flag mAb, separated on two-dimensional non-reducing and reducing SDS–PAGE, and transferred onto a polyvinylidene difluoride (PVDF) membrane (Immobilon-P; Millipore Corporation, Bedford, MA). The biotinylated proteins were detected using streptavidin–peroxidase (Vectastain Elite ABC kit; Vector Laboratories Incorporated, Burlingame, CA), the ECL system (Amersham International, Buckinghamshire, UK) and autoradiography. After termination of chemiluminescence, the membrane was blotted with anti-FcR{gamma} antibody (Upstate Biotechnology) followed by peroxidase-labeled anti-rabbit antibody (Amersham), and detected by the ECL system.

Stimulation of MA5.8 transfectants by immobilized anti-FLAG mAb
Anti-FLAG M2 (Sigma) or control (anti-human CD3, OKT3) mAbs were immobilized on a 96-well flat-bottomed culture plate (Coaster, Cambridge, MA) by incubating for 2 h at 37°C at a concentration of 10 µg/ml in PBS. MA5.8 transfectants (1 x 105) were stimulated by these immobilized mAbs or phorbol 12-myristate 13-acetate (PMA) (50 ng/ml) plus ionomycin (IM) (500 ng/ml) for 2 days. IL-2 in the culture supernatant was measured by ELISA (Pharmingen).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
Expression of FcR{gamma} and DAP12 but not CD3{zeta} and DAP10 in osteoclasts
Because OSCAR possesses a positively charged amino acid, arginine, within the transmembrane region, we analyzed whether OSCAR associates with such ITAM-bearing signal transducing adaptor molecules as CD3{zeta}, FcR{gamma}, DAP12 and DAP10. First, we analyzed the expression of these adaptor molecules in BM-derived osteoclasts. BM cells were cultured in the presence of dexamethasone and vitamin D3 for 10 days. After the 10 day culture, ~80% of adherent cells were TRAP-positive (data not shown). RNA was extracted from BM cells cultured for 3 and 10 days, and mRNA expressions of OSCAR, CD3{zeta}, FcR{gamma}, DAP12 and DAP10 were analyzed by semi-quantitative RT–PCR. Compared to splenocytes, osteoclast-enriched cultured BM cells expressed a high level of OSCAR mRNA (Fig. 1). TRAP staining and OSCAR expression demonstrated that the osteoclasts were differentiated in the culture. The expressions of CD3{zeta}, FcR{gamma}, DAP12 and DAP10 were clearly detected in splenocytes, although the expression levels of FcR{gamma} and DAP12 were relatively low. In contrast, FcR{gamma} and DAP12 but not CD3{zeta} and DAP10 were detected in this population enriched with BM-derived osteoclasts. Especially, CD3{zeta} was not detected in this population even after 3 days of culture. In addition, CD3{zeta} was not detected in the osteoclast-enriched population from FcR{gamma}-deficient mice. Taken together, although purity of osteoclasts was 80%, neither CD3{zeta} nor DAP10 seems to be expressed on osteoclasts.



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Fig. 1. Semi-quantitative RT–PCR analysis of the expression of OSCAR, CD3{zeta}, FcR{gamma}, DAP12 and DAP10 in splenocytes and BM cells cultured in the presence of M-CSF and vitamin D3 for 3 and 10 days. ß-actin was amplified as a control. Graded amounts of cDNAs (3-fold dilution) generated from cultured BM cells and splenocytes of wild type and FcR{gamma}-deficient mice (FcR{gamma}–/–).

 
Upregulation of cell surface expression of OSCAR by CD3{zeta} and FcR{gamma}
In order to analyze whether OSCAR associates with these ITAM-bearing adaptor molecules, FLAG-tagged OSCAR was transfected into MA5.8, a CD3{zeta}-negative T-cell hybridoma cell line that lacks all of these adaptor molecules (14). Thereafter, CD3{zeta}, FcR{gamma}, DAP12 and DAP10 were further transfected into FLAG–OSCAR-expressing MA5.8, and the cell surface expression of OSCAR was analyzed by means of FLAG staining. When FLAG–OSCAR was transfected into MA5.8, the cells were weakly stained by anti-FLAG mAb, indicating that OSCAR is weakly expressed on the cell surface in the absence of these adaptor molecules (Fig. 2A). Then, CD3{zeta}, FcR{gamma}, DAP12 and DAP10 in the pMx-IRES-GFP retrovirus expression vector were transfected into FLAG–OSCAR-expressing MA5.8 (Fig. 2B). When CD3{zeta} or FcR{gamma} was transfected, the expression of FLAG–OSCAR was significantly augmented in the GFP-positive population. Because the expression of CD3{zeta} and FcR{gamma} is well correlated with GFP expression, this indicates that FLAG–OSCAR expression is upregulated by either CD3{zeta} or FcR{gamma}. On the other hand, the expression of OSCAR was not changed by the transfection of control vector (mock), DAP12 or DAP10. Altogether, these data suggest that CD3{zeta} and FcR{gamma} but not DAP12 or DAP10 are associated with OSCAR and induce the surface expression of OSCAR. However, because the transcripts of CD3{zeta} were not detected in BM-derived osteoclasts (Fig. 1), the OSCAR–CD3{zeta} association may not be relevant to the physiological function of OSCAR.



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Fig. 2. Upregulation of cell surface OSCAR expression by FcR{gamma} and CD3{zeta}. (A) FLAG–OSCAR was transfected into MA5.8 by using pMx-neo retrovirus expression vector. Parental MA5.8 and the FLAG–OSCAR transfectant were stained with anti-FLAG (solid line) or control mAb (dotted line). (B) CD3{zeta}, FcR{gamma}, DAP12 and DAP10 were further transfected by using pMx-IRES-GFP retrovirus expression vector. Transfectants were stained with anti-FLAG mAb, followed by PE-anti-human-IgG antibody. Mean fluorescence intensities of PE on GFP-positive cells are indicated in the figure. (C) Expression of FLAG–OSCAR on BM cells cultured in the presence of M-CSF and RANK-L. BM cells from wild-type and FcR{gamma}-deficient mice were transfected with pMx-FLAG-OSCAR-IRES-GFP (continuous line) or pMx-IRES-GFP (dotted line) vector and FLAG expression was analyzed on GFP-positive cells.

 
Next, in order to see the association of FcR{gamma} with OSCAR in normal cells, we cultured bone marrow cells from wild-type and FcR{gamma}-deficient mice in the presence of M-CSF and RANKL and transfected Flag–OSCAR into them (Fig. 2C). Thereafter, expression of Flag–OSCAR was analyzed. As expected, expression of OSCAR was significantly higher on cells from wild-type mice than those from FcR{gamma}-deficient mice. These data also suggested that FcR{gamma} augments the cell surface expression of OSCAR.

Association of FcR{gamma} with OSCAR
We then analyzed the physical association of FcR{gamma} with OSCAR. MA5.8 cells expressing FLAG–OSCAR alone or FLAG–OSCAR and FcR{gamma} were surface-biotinylated and the cell lysate was immunoprecipitated with anti-Flag mAb, followed by analysis on two-dimensional non-reducing (NR) and reducing (R) SDS–PAGE. When FLAG–OSCAR was precipitated with anti-FLAG mAb from the cell lysate of MA5.8 expressing both FLAG–OSCAR and FcR{gamma}, FLAG–OSCAR was detected as a 29 kDa protein in the diagonal position (Fig. 3A). In addition, a 9 kDa homodimer was co-precipitated with FLAG–OSCAR. In contrast, the 9 kDa homodimer was not detected in the cell lysate of MA5.8 expressing FLAG–OSCAR alone. The 9 kDa homodimer co-precipitated with FLAG–OSCAR was blotted with anti-FcR{gamma} antibody (Fig. 3B), thereby confirming that it is FcR{gamma}. At any rate, these data indicate that FcR{gamma} is physically associated with OSCAR on the cell surface.



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Fig. 3. Physical association between FcR{gamma} and OSCAR. (A) Surface biotinylation analysis. MA5.8 cells transfected with FLAG–OSCAR alone (left) or FLAG–OSCAR and FcR{gamma} (right) were surface-biotinylated and the cell lysate was immunoprecipitated with anti-FLAG mAb and analyzed by two-dimensional non-reducing (NR) and reducing (R) SDS–PAGE. Biotinylated proteins were detected by ECL. (B) Immunoblot analysis of FcR{gamma}. The same membranes used in (A) were further blotted with anti-FcR{gamma} antibody. An open arrow indicates FLAG–OSCAR and closed arrows indicate FcR{gamma} homodimer.

 
Requirement of FcR{gamma} for signal transduction by OSCAR
We next analyzed whether the association of OSCAR with FcR{gamma} is required for the signal transduction by OSCAR. FLAG–OSCAR-expressing MA5.8 cells were further transfected with CD3{zeta}, FcR{gamma} and DAP12, and the transfectants were stimulated with immobilized anti-FLAG mAb (Fig. 4A). When CD3{zeta} and FcR{gamma} were transfected, the transfectants produced a significant amount of IL-2. FcR{gamma} transfection induced greater production of IL-2 than CD3{zeta} transfection. In contrast, FLAG–OSCAR-expressing MA5.8 transfected with DAP12 or DAP10 showed no IL-2 production similar to mock transfectants, although the cells responded to stimulation with PMA plus ionomycin. Furthermore, when the cell surface expression of CD69 as an early activation marker was analyzed by flow cytometry, MA5.8 expressing FLAG–OSCAR and FcR{gamma} or CD3{zeta} but not DAP12 or DAP10 showed upregulation of CD69 expression upon stimulation with anti-FLAG mAb (Fig. 4B). Again, FcR{gamma} induced a higher expression of CD69 than CD3{zeta}. These data demonstrate that both FcR{gamma} and CD3{zeta} transduce activation signals through OSCAR upon crosslinking and that FcR{gamma} transduces stronger signals than CD3{zeta}.



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Fig. 4. IL-2 production by MA5.8 transfected with FLAG–OSCAR. MA5.8 cells transfected with FLAG–OSCAR were further transfected with CD3{zeta}, FcR{gamma}, DAP12 or empty vector (Mock) as indicated in the figure. The transfectants were stimulated with immobilized control, anti-FLAG mAb or PMA plus ionomycine. (A) IL-2 produced in the culture supernatants was measured by ELISA. Data are presented as means ± SD (ng/ml) of triplicate cultures and statistical values were indicated. (B) The transfectants were stimulated with immobilized control, anti-FLAG mAb or PMA plus ionomycine and cells were stained with PE-anti-CD69 mAb. Expression levels of CD69 on the transfectants were calculated.

 
Next, we analyzed osteoclast development from FcR{gamma}-deficient mice. As shown in Fig. 5(A), osteoclasts were differentiated from bone marrow cells from FcR{gamma}-deficient mice. Furthermore, we statistically evaluated the differentiation of osteoclasts (Fig. 5B). Similarly, we could not detect significant difference between wild-type mice and FcR{gamma}-deficient mice. These data suggested that FcR{gamma} is not essential for the differentiation of osteoclasts.



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Fig. 5. Osteoclast differentiation in bone marrow cells from FcR{gamma}-deficient mice. BM cells from wild-type and FcR{gamma}-deficient mice were cultured in the presence of vitamin D3 and M-CSF for 10 days. (A) Osteoclast differentiation was visualized by TRAP staining and representative data are shown. (B) Relative cell size of TRAP-positive cells were calculated. The number of TRAP-positive cells of each cell size was counted and compared between wild-type and FcR{gamma}-deficient mice (B).

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
In the present study, we found that FcR{gamma} is associated with OSCAR and that FcR{gamma} mediates signal transduction by OSCAR. A previous report has shown that osteoclast differentiation induced by co-culture with osteoblasts is significantly impaired when the interaction between OSCAR and a putative OSCAR ligand is blocked by OSCAR–Ig fusion protein (5). Therefore, an FcR{gamma}-mediated signal transduction by OSCAR upon recognition of its putative ligand expressed on osteoblasts may be involved in osteoclast differentiation.

We showed that expression of OSCAR is significantly upregulated in the presence of FcR{gamma} although OSCAR was expressed weakly on the cell surface in the absence of CD3{zeta}, FcR{gamma} and DAP12. We have previously shown that NK1.1 associates with FcR{gamma} and FcR{gamma} is required for signal transduction of NK1.1 (15). In this case, FcR{gamma} was not involved in the expression of NK1.1. Similarly, KIR does not require DAP12 for cell surface expression (9). Therefore, it is likely that OSCAR is expressed on the cell’s surface without adaptors as its intrinsic nature, although we cannot formally exclude the possibility that OSCAR associates with an unknown adaptor molecule in MA5.8.

To date, however, no significant bone disorder has been reported in FcR{gamma}-deficient mice (13,16), and indeed, we did not detect any significant bone disorder in FcR{gamma}-deficient mice in spite of extensive bone analyses (data not shown). Furthermore, we did not observe any obvious defect in osteoclast development when BM cells from FcR{gamma}-deficient mice were cultured in the presence of M-CSF and RANKL (data not shown). Similarly, osteoclasts were well developed from FcR{gamma}-deficient BM cells cultured in the presence of dexamethasone and vitamin D3 wherein the interaction between osteoblasts and osteoclasts can be induced. Taken together, the FcR{gamma}-mediated signal transduction by OSCAR is not essential for osteoclast development. The OSCAR–FcR{gamma}-mediated signal may be redundant with other signals and be involved in an alternative signaling pathway for osteoclast differentiation.

It has been reported that DAP12-deficient human (17) and mouse (18) show defects in osteoclast development. TREM2 utilizes DAP12 for signal transduction and is reported to be involved in osteoclast differentiation (3,4). Indeed, patients lacking DAP12 or TREM2 show bone disorder, the so-called Nasu–Hakola disease (19,20), as well as bone cysts and presenile dementia. Although bone disorder was also observed in DAP12-deficient mice (18), the disorder was very mild compared to that in M-CSF-deficient mice (op/op) that exhibited complete loss of osteoclast differentiation (21). Therefore, a DAP12-independent pathway such as the OSCAR–FcR{gamma} pathway may be involved in osteoclast development. Indeed, FcR{gamma} and DAP12 double-deficient mice show severer bone disorders than DAP12-deficient mice (Dr Takai, Tohoku University, Japan; personal communication). There fore, both the TREM2–DAP12 and OSCAR–FcR{gamma} pathways may be redundantly involved in osteoclast differentiation.

We showed that both CD3{zeta} and FcR{gamma} associate with OSCAR and mediate activation signals by OSCAR. Although the transcription of CD3{zeta} in an osteoclast-enriched population was not detected by RT–PCR, we cannot rule out the possibility that a small amount of CD3{zeta} is expressed on a small number of osteoclast precursor cells in BM, which compensates the defect of FcR{gamma}. In addition, unknown adaptor molecules may also be involved in signal transduction of OSCAR. These possibilities may account for the reason why FcR{gamma}-deficient mice do not show significant osteoclast differentiation. Therefore, further analysis of osteoclast development using double- or triple-deficient mice lacking CD3{zeta}, FcR{gamma} and/or DAP12 is necessary to clarify the redundancy and the physiological function of these molecules in osteoclast differentiation.

Kim et al. (5) showed that osteoblasts are stained well by OSCAR–Ig fusion protein, and suggested that the ligand for OSCAR is expressed on osteoblasts. Identification of the ligand is essential for elucidating the physiological function of OSCAR in osteoclast development. Mouse OSCAR is localized near the proximal end of mouse chromosome 7, the region where the paired immunoglobulin-like receptor (PIR) family resides. In addition, human OSCAR is mapped at the leukocyte receptor complex (LRC) locus on human chromosome 19q13.4 where CD85/immunoglobulin (Ig)-like transcript (ILT)/leukocyte Ig-like receptor (LILR) and killer Ig-like receptor (KIR) are located. Similar to OSCAR, these receptors also contain an Ig-like structure and their activating forms utilize FcR{gamma} or DAP12 for signal transduction. OSCAR may have evolved with these receptors that regulate the immune function. Considering that some of the CD85 and KIR molecules recognize MHC class I, OSCAR might also recognize certain antigens with an MHC class I structure.

FcR{gamma} is associated with not only Fc receptors but also various immuno-receptors such as NK cell receptor (NKR) P1C (15), PIR-A (22), CD85/ILT/LILR (23), Nkp46 (24), platelet glycoprotein IV (25) and dendritic immunoactivating receptor (DCAR) (26). In addition, we have previously reported a defect in the function of antigen presenting cells in FcR{gamma}-deficient mice (27). Therefore, FcR{gamma}-mediated signal transduction seems to be widely involved in immune regulation. Indeed, it is quite interesting that osteoclasts use the same signaling molecule for their function as various receptors in lymphocytes. Further analysis of the function of FcR{gamma}-associated receptors including OSCAR will lead to the elucidation of unknown immune activation mechanisms.


    Acknowledgements
 
We would like to thank Ms Hiroko Yamaguchi and Yoko Kurihara for secretarial assistance.


    Note added in proof
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
Recently, FcR{gamma} and DAP12 double-deficient mice were reported to show severer bone disorders than DAP12-deficient mice. This indicated that FcR{gamma} mediated signals play an important role in regulation of osteoclast function (28).


    Abbreviations
 
BM—bone marrow

DCAR—dendritic immunoactivating receptor

GFP—green fluorescent protein

ILT—Ig-like transcript

IM—ionomycin

IRES—internal ribosome entry site

ITAM—immunoreceptor tyrosine kinase activation motif

KIR—killer Ig-like receptor

LILR—leukocyte Ig-like receptor

LRC—leukocyte receptor complex

M-CSF—macrophage colony stimulating factor

NKR—NK cell receptor

NR—non-reducing

OSCAR—osteoclast-associated receptor

PIR—paired Ig-like receptor

PMA—phorbol 12-myristate 13-acetate

R—reducing

sRANKL—soluble receptor activator of NF-{kappa}B ligand

TRAP—tartrate-resistant acid phosphatase

TREM2—triggering receptor expressed on myeloid cells 2


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 

  1. Teitelbaum, S. L. 2000. Bone resorption by osteoclasts. Science 289:1504.[Abstract/Free Full Text]
  2. Suda, T., Takahashi, N., Udagawa, N., Jimi, E., Gillespie, M. T. and Martin, T. J. 1999. Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr. Rev. 20:345.[Abstract/Free Full Text]
  3. Cella, M., Buonsanti, C., Strader, C., Kondo, T., Salmaggi, A. and Colonna, M. 2003. Impaired differentiation of osteoclasts in TREM-2-deficient individuals. J. Exp. Med. 198:645.[Abstract/Free Full Text]
  4. Paloneva, J., Mandelin, J., Kiialainen, A., Bohling, T., Prudlo, J., Hakola, P., Haltia, M., Konttinen, Y. T. and Peltonen, L. 2003. DAP12/TREM2 deficiency results in impaired osteoclast differentiation and osteoporotic features. J. Exp. Med. 198:669.[Abstract/Free Full Text]
  5. Kim, N., Takami, M., Rho, J., Josien, R. and Choi, Y. 2002. A novel member of the leukocyte receptor complex regulates osteoclast differentiation. J. Exp. Med. 195:201.[Abstract/Free Full Text]
  6. Bouchon, A., Dietrich, J. and Colonna, M. 2000. Cutting edge: inflammatory responses can be triggered by TREM-1, a novel receptor expressed on neutrophils and monocytes. J. Immunol. 164:4991.[Abstract/Free Full Text]
  7. Daws, M. R., Lanier, L. L., Seaman, W. E. and Ryan, J. C. 2001. Cloning and characterization of a novel mouse myeloid DAP12-associated receptor family. Eur. J. Immunol. 31:783.[CrossRef][ISI][Medline]
  8. Bouchon, A., Hernandez-Munain, C., Cella, M. and Colonna, M. 2001. A dap12-mediated pathway regulates expression of cc chemokine receptor 7 and maturation of human dendritic cells. J. Exp. Med. 194:1111.[Abstract/Free Full Text]
  9. Lanier, L. L. 2001. On guard—activating NK cell receptors. Nat. Immunol. 2:23.[CrossRef][ISI][Medline]
  10. Lanier, L. L., Corliss, B., Wu, J. and Phillips, J. H. 1998. Association of DAP12 with activating CD94/NKG2C NK cell receptors. Immunity 8:693.[ISI][Medline]
  11. Wu, J., Cherwinski, H., Spies, T., Phillips, J. H. and Lanier, L. L. 2000. DAP10 and DAP12 form distinct, but functionally cooperative, receptor complexes in natural killer cells. J. Exp. Med. 192:1059.[Abstract/Free Full Text]
  12. Wu, J., Song, Y., Bakker, A. B. H., Bauer, S., Groh, V., Spies, T., Lanier, L. L. and Phillips, J. H. 1999. An activating receptor complex on natural killer and T cells formed by NKG2D and DAP10. Science 285:730.[Abstract/Free Full Text]
  13. Park, S. Y., Ueda, S., Ohno, H., Hamano, Y., Tanaka, M., Shiratori, T., Yamazaki, T., Arase, H., Arase, N., Karasawa, A. et al. 1998. Resistance of Fc receptor-deficient mice to fatal glomerulonephritis. J. Clin. Invest. 102:1229.[Abstract/Free Full Text]
  14. Aoe, T., Goto, S., Ohno, H. and Saito, T. 1994. Different cytoplasmic structure of the CD3 zeta family dimer modulates the activation signal and function of T cells. Int. Immunol. 6:1671.[Abstract]
  15. Arase, N., Arase, H., Park, S. Y., Ohno, H., Ra, C. and Saito, T. 1997. Association with FcR{gamma} is essential for activation signal through NKR-P1 (CD161) in natural killer (NK) cells and NK1.1+ T cells. J. Exp. Med. 186:1957.[Abstract/Free Full Text]
  16. Takai, T., Li, M., Sylvestre, D., Clynes, R. and Ravetch, J. V. 1994. FcR {gamma} chain deletion results in pleiotrophic effector cell defects. Cell 76:519.[ISI][Medline]
  17. Paloneva, J., Kestila, M., Wu, J., Salminen, A., Bohling, T., Ruotsalainen, V., Hakola, P., Bakker, A. B., Phillips, J. H., Pekkarinen, P., Lanier, L. L., Timonen, T. and Peltonen, L. 2000. Loss-of-function mutations in TYROBP (DAP12) result in a presenile dementia with bone cysts. Nat. Genet. 25:357.[CrossRef][ISI][Medline]
  18. Kaifu, T., Nakahara, J., Inui, M., Mishima, K., Momiyama, T., Kaji, M., Sugahara, A., Koito, H., Ujike-Asai, A., Nakamura, A. et al. 2003. Osteopetrosis and thalamic hypomyelinosis with synaptic degeneration in DAP12-deficient mice. J. Clin. Invest. 111:323.[Abstract/Free Full Text]
  19. Hakola, H. P. 1972. Neuropsychiatric and genetic aspects of a new hereditary disease characterized by progressive dementia and lipomembranous polycystic osteodysplasia. Acta. Psychiatr. Scand. (Suppl.) 232:1.
  20. Paloneva, J., Manninen, T., Christman, G., Hovanes, K., Mandelin, J., Adolfsson, R., Bianchin, M., Bird, T., Miranda, R., Salmaggi, A., Tranebjaerg, L., Konttinen, Y. and Peltonen, L. 2002. Mutations in two genes encoding different subunits of a receptor signaling complex result in an identical disease phenotype. Am. J. Hum. Genet. 71:656.[CrossRef][ISI][Medline]
  21. Yoshida, H., Hayashi, S., Kunisada, T., Ogawa, M., Nishikawa, S., Okamura, H., Sudo, T. and Shultz, L. D. 1990. The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature 345:442.[CrossRef][ISI][Medline]
  22. Maeda, A., Kuorsaki, M. and Kurosaki, T. 1998. Paired immunoglobulin-like receptor (PIR)-A is involved in activating mast cells through its association with Fc receptor {gamma} chain. J. Exp. Med. 188:991.[Abstract/Free Full Text]
  23. Nakajima, H., Samaridis, J., Angman, L. and Colonna, M. 1999. Human myeloid cells express an activating ILT receptor (ILT1) that associates with Fc receptor {gamma}-chain. J. Immunol. 162:5.[Abstract/Free Full Text]
  24. Pessino, A., Sivori, S., Bottino, C., Malaspina, A., Morelli, L., Moretta, L., Biassoni, R. and Moretta, A. 1998. Molecular cloning of NKp46: a novel member of the immunoglobulin superfamily involved in triggering of natural cytotoxicity. J. Exp. Med. 188:953.[Abstract/Free Full Text]
  25. Poole, A., Gibbins, J. M., Turner, M., van Vugt, M. J., van de Winkel, J. G., Saito, T., Tybulewicz, V. L. and Watson, S. P. 1997. The Fc receptor {gamma}-chain and the tyrosine kinase Syk are essential for activation of mouse platelets by collagen. EMBO J. 16:2333.[Abstract/Free Full Text]
  26. Kanazawa, N., Tashiro, K., Inaba, K. and Miyachi, Y. 2003. Dendritic cell immunoactivating receptor, a novel C-type lectin immunoreceptor, acts as an activating receptor through association with Fc receptor {gamma} chain. J. Biol. Chem. 278:32645.[Abstract/Free Full Text]
  27. Hamano, Y., Arase, H., Saisho, H. and Saito, T. 2000. Immune complex and Fc receptor-mediated augmentation of antigen presentation for in vivo Th cell responses. J. Immunol. 164:6113.[Abstract/Free Full Text]
  28. Koga, T., Inui, M., Inoue, K. et al. 2004. Costimulatory signals mediated by the ITAM motif cooperate with RANKL for bone homeostasis. Nature 428:758.[CrossRef][ISI][Medline]