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
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Abstract |
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Keywords: CD3, Fc receptor, Ig-like receptor, ITAM, osteoclast
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Introduction |
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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 OSCARIg 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, FcR
(Fc
RI
) 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
plays a vital role in OSCAR-mediated signal transduction.
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Methods |
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Induction of BM-derived osteoclasts
BM cells from C57BL/6 mice were cultured in -MEM (Sigma) supplemented with 10% FCS (Sigma), 107 M dexamethasone (Sigma), and 108 M vitamin D3 [1
, 25(OH)2 D3, Calbiochem] for 10 days. In some experiments, BM cells were cultured in
-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-
B ligand (sRANKL) (PeproTech) for 10 days as indicated. Osteoclast development was analyzed using a tartrate-resistant acid phosphatase (TRAP) staining kit (Hokudo).
RTPCR
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, sense primer: 5'-ATCATCACAGCCCTGTACCT-3', anti-sense primer: 5'-CCTCTCCGCCTCTCGCCTTT-3'; FcR
, 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
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, FcR
, 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
-negative T-cell hybridoma cell line that lacks CD3
, FcR
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 TrisHCl (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 SDSPAGE, and transferred onto a polyvinylidene difluoride (PVDF) membrane (Immobilon-P; Millipore Corporation, Bedford, MA). The biotinylated proteins were detected using streptavidinperoxidase (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 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).
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Results |
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Association of FcR with OSCAR
We then analyzed the physical association of FcR with OSCAR. MA5.8 cells expressing FLAGOSCAR alone or FLAGOSCAR and FcR
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) SDSPAGE. When FLAGOSCAR was precipitated with anti-FLAG mAb from the cell lysate of MA5.8 expressing both FLAGOSCAR and FcR
, FLAGOSCAR was detected as a 29 kDa protein in the diagonal position (Fig. 3A). In addition, a 9 kDa homodimer was co-precipitated with FLAGOSCAR. In contrast, the 9 kDa homodimer was not detected in the cell lysate of MA5.8 expressing FLAGOSCAR alone. The 9 kDa homodimer co-precipitated with FLAGOSCAR was blotted with anti-FcR
antibody (Fig. 3B), thereby confirming that it is FcR
. At any rate, these data indicate that FcR
is physically associated with OSCAR on the cell surface.
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Discussion |
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We showed that expression of OSCAR is significantly upregulated in the presence of FcR although OSCAR was expressed weakly on the cell surface in the absence of CD3
, FcR
and DAP12. We have previously shown that NK1.1 associates with FcR
and FcR
is required for signal transduction of NK1.1 (15). In this case, FcR
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 cells 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-deficient mice (13,16), and indeed, we did not detect any significant bone disorder in FcR
-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
-deficient mice were cultured in the presence of M-CSF and RANKL (data not shown). Similarly, osteoclasts were well developed from FcR
-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
-mediated signal transduction by OSCAR is not essential for osteoclast development. The OSCARFcR
-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 NasuHakola 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 OSCARFcR pathway may be involved in osteoclast development. Indeed, FcR
and DAP12 double-deficient mice show severer bone disorders than DAP12-deficient mice (Dr Takai, Tohoku University, Japan; personal communication). There fore, both the TREM2DAP12 and OSCARFcR
pathways may be redundantly involved in osteoclast differentiation.
We showed that both CD3 and FcR
associate with OSCAR and mediate activation signals by OSCAR. Although the transcription of CD3
in an osteoclast-enriched population was not detected by RTPCR, we cannot rule out the possibility that a small amount of CD3
is expressed on a small number of osteoclast precursor cells in BM, which compensates the defect of FcR
. In addition, unknown adaptor molecules may also be involved in signal transduction of OSCAR. These possibilities may account for the reason why FcR
-deficient mice do not show significant osteoclast differentiation. Therefore, further analysis of osteoclast development using double- or triple-deficient mice lacking CD3
, FcR
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 OSCARIg 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 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 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
-deficient mice (27). Therefore, FcR
-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
-associated receptors including OSCAR will lead to the elucidation of unknown immune activation mechanisms.
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Acknowledgements |
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Note added in proof |
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Abbreviations |
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DCARdendritic immunoactivating receptor
GFPgreen fluorescent protein
ILTIg-like transcript
IMionomycin
IRESinternal ribosome entry site
ITAMimmunoreceptor tyrosine kinase activation motif
KIRkiller Ig-like receptor
LILRleukocyte Ig-like receptor
LRCleukocyte receptor complex
M-CSFmacrophage colony stimulating factor
NKRNK cell receptor
NRnon-reducing
OSCARosteoclast-associated receptor
PIRpaired Ig-like receptor
PMAphorbol 12-myristate 13-acetate
Rreducing
sRANKLsoluble receptor activator of NF-B ligand
TRAPtartrate-resistant acid phosphatase
TREM2triggering receptor expressed on myeloid cells 2
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References |
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