All cell culture media and supplements were obtained from Gibco (Grand Island, NY, USA). Soluble recombinant mouse RANKL was purified from insect cells as described3 and recombinant human M-CSF was obtained from Genetics Institute (Cambridge, MA, USA). TRIzol and Oligotex poly A⁺ RNA column were purchased from Life Technologies (Grand Island, NY, USA) and QIAGEN (Valencia, CA, USA), respectively. Ready-to-Go labeling kit and ProbeQuant G-50 purification kit were from Amersham Pharmacia Biotech (Piscataway, NJ, USA). ZAP cDNA synthesis kit, Pfu DNA polymerase, and pIRES-hrGFP-1a mammalian expression vector were purchased from Agilent Technologies Inc. (Santa Clara, CA, USA). Antibodies specific for FLAG epitope (M2), biotinylated anti-FLAG epitope (BioM2), and anti-FLAG-M2 agarose bead were from Sigma-Aldrich Co. (St. Louis, MO, USA); mouse IgG-conjugated horseradish peroxidase from Amersham Pharmacia Biotech; and streptoavidin-conjugated APC from BD PharMingen (San Diego, CA, USA).

Human monocytes were isolated from peripheral blood by counter flow centrifugation. Cells from fraction 180 and 190 (more than 90% are CD14⁺) were used for generating macrophage (M), osteoclast (OC), and dendritic cells (DC). The fractionated cells (5 × 10⁶) were incubated with M-CSF (30 ng/mL) alone for M, with M-CSF (30 ng/mL) and RANKL (200 ng/mL) for OC in 100 mm culture dish using alpha minimum essential medium (α-MEM) containing 10% fetal bovine serum (FBS) for 6 days. After 3 days culture, media containing macrophage-stem cell factor (M-SCF) or M-CSF and RANKL were changed freshly. The generation of DC from peripheral blood mononuclear cells (PBMC) used the method originally as described.15 To generate DC, the purified human monocytes (2 × 10⁸) in 30 mL AIM-V (Invitrogen, Carlsbad, CA, USA) were plated in a T-150 flask, and incubated for 2 hr at 37℃ in 5% carbon dioxide (CO₂) incubator. Then, non- or semiadherent cells were discarded by gentle pipetting. Adherent monocytes were cultured for 7 days with 30 mL AIM-V medium, supplemented with human granulocyte-macrophage colony-stimulating factor (hGM-CSF, 5 ng/mL) and human interleukin-4 (hIL-4, 10 ng/mL; R&D system, Minneapolis, MN, USA).

Total ribonucleic acid (RNA) was harvested from human monocyte-drived osteoclast cells using TRIzol after incubation with M-CSF and RANKL for 6 days. Poly A⁺ RNA was prepared from total RNA using Oligotex poly A⁺ RNA column. Human osteoclast complementary deoxyribonucleic acid (cDNA) library was synthesized using ZAP cDNA synthesis kit according to the manufacturer's instructions. The ecto domain of hOSCAR was amplified by polymerase chain reaction (PCR) to screen the full length cDNA clones and 13 clones of hOSCAR cDNA were picked up and analyzed.

Total RNA samples were separated and transferred to nylon membranes as described.16 Hybridization was performed at 42℃ for 16 hr with ³²P-deoxycytidine triphosphate (dCTP) DNA probe prepared using Ready-to-Go labeling kit and ProbeQuant G-50 purification kit. After washing with 0.1 × saline sodium citrate (SSC) and 0.1% sodium dodecyl sulfate (SDS) at 60℃ for 1 hr, membrane was exposed to X-ray film.

Three different FLAG epitope-tagged hOSCAR constructs were amplified by PCR using Pfu DNA polymerase. To make each construct, the following primers were used: FLAG, 5'-ATA AGA ATG CGG CCG CAC CAT GTC TGC ACT TCT G-3' (sense) and 5'-CGG GAT CCG AAG CTT GTC GTC ATC GTC TTT-3' (antisense); hOSCAR-I, 5'-CGG GAT CCC CCC CAG CTT CAT ACC ACC CTA A-3' (sense) and 5'-CCG CTC GAG CGG GGG GCG GAT ACC AGC AGG AGC-3' (antisense); hOSCAR-II, 5'-CGG GAT CCC CCC CAG CTT CAT ACC ACC CTA A-3' (sense) and 5'-CCG CTC GAG CGG GAC TCC TGG ATC TGA GGG AGG A-3' (antisense); hOSCAR-V, 5'-CGG GAT CCC CCC CAG CTT CAT ACC ACC CTA A-3' (sense) and 5'-CCG CTC GAG ATT CAG CAG GAC TGT GGG GCT GCA GGA-3' (antisense). The amplified FLAG epitope fragment was digested with NotI and BamHI. The PCR products of hOSCAR-I, -II, and -V were digested with BamHI and XhoI, respectively. The digested FLAG epitope and each hOSCAR fragment were ligated into pIRES-hrGFP vector using NotI and XhoI sites. The FLAG epitope and full length of each hOSCAR were confirmed by sequencing.

Two hundred ninety-three T cells were transfected using FuGENE 6 (Roche Applied Sciences, Indianapolis, IN, USA). All cells and media were harvested after 36 hr incubation. A half of the cells were lysed in extraction buffer (10 mM Tris, potential of hydrogen [pH] 7.5, 150 mM sodium chloride [NaCl], 0.4 mM etylenediaminetetraacetic acid [EDTA], 1% Triton X-100, 1 mM phenylmethylsulfonylfluoride, 1 µg/mL leupeptin, and 0.1 U/mL aprotinin) and cleared by centrifugation to obtain the whole-cell extracts. Whole cell extracts and filtered media were incubated with anti-FLAG M2 agarose beads at 4℃ for 1 hr on rocker table and washed 3 times with extraction buffer. The immunoprecipitates were then subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride membrane. The membrane was blocked with 5% nonfat milk in phosphate-buffered saline containing 0.1% Tween 20 at room temperature for 1 hr and incubated with 1 µg/mL of anti-FLAG M2 antibody at 4℃ for overnight. After thorough washing, the membrane was incubated with anti-mouse IgG-conjugated horseradish peroxidase antibody at room temperature for 1 hr. Membrane was developed with enhanced chemiluminescence (ECL) western blotting detection systems.

The other half of the transfected cells were used for flow cytometry analysis. The expression of FLAG epitope-tagged hOSCAR on the surface of the cells was assessed using biotinylated anti-FLAG antibody (5 µg/mL) and followed by streptoavidin-conjugated APC.

The extracellular domain of hOSCAR (amino acid 1-219 of soluble hOSCAR [hOSCAR-S]1) was fused to Fc region of hIgG1. The soluble hOSCAR-Fc was expressed and purified from insect cells as described.16

Human peripheral blood monocytes were cultured in the presence of M-CSF (30 ng/mL) and RANKL (200 ng/mL) with hOSCAR-Fc (30 µg/mL), hIgG1 (30 µg/mL), or RANK-Fc (5 µg/mL) in 96 well culture plates or dentine slices. TRAP staining and pit formation assay were performed as described.16

We have shown that mouse OSCAR (mOSCAR) and hOSCAR have three different transcripts by alternative splicing.16 hOSCAR as well as mOSCAR has a putative signal sequence, an extracellular region consisting of two Ig-like domains, a single transmembrane region, and a short intracellular domain (Fig. 1, 2). Here we report that there are two more transcripts by alternative splicing in case of hOSCAR. The previously reported three hOSCAR transcripts are membrane-bounded forms (membrane-bound hOSCAR [hOSCAR-M]1, M2, and M3) which leader sequence region has a variation.16 The two new transcripts (hOSCAR-S1 and S2) showed variations in a leader sequence region and C-terminus region (transmembrane and cytoplasmic domains) (Fig. 1, 2). These new transcripts have exon 4b region which are spliced out in the hOSCAR-M.

Among these new transcripts, hOSCAR-S2 form represents a deletion in exon 2 like as hOSCAR-M2 form, but hOSCAR-S1, a major transcript of hOSCAR, has a deletion in only exon 2b region like as hOSCAR-M1 form.

By searching the human genomic database with hOSCAR cDNA sequence, we found a human bacterial artificial chromosome (BAC) clone, AC009968.6, containing the full span of hOSCAR gene. hOSCAR gene was located in chromosome 19q13.4, which contains in the leukocyte receptor complex (LRC). The full hOSCAR gene consists of 4 exons spaning over 6 Kbps (Fig. 1). Thus, we could determine the genomic structure of hOSCAR and its 5' flanking region. All sequences at the exon-intron junctions followed to the GT/AG rules. Exon1 and 2 encoded a leader sequence, where exon 3 and 4a encoded two Ig-like domains. Exon 4c encoded a transmembrane domain and contained a stop codon and a 3'-untranslated region. Exon 4b was spliced out by alternative splicing in hOSCAR-M forms, not in hOSCAR-S forms. We were unable to find a putative transmembrane domain in exon 4b. In case of new transcripts, there is another stop codon in exon 4b region which upstream of the transmembrane domain of membrane-bounded forms (Fig. 1).

To investigate the expression of hOSCAR gene, Northern blot analyses were performed (Fig. 3). Human monocyte cells were isolated from peripheral blood by counter flow centrifugation. More than 90% of the cells from fractions between 180 and 190 were CD14⁺ (data not shown). These cells were incubated with M-CSF and RANKL for the indicated days until day 8. Two major transcripts, 2.0 Kb and 1.5 Kb, were detected by probe exon 3, which is a common extracellular domain. The expression levels of those transcripts were increased until day 6 and decreased (Fig. 3A). It implicated that hOSCAR is highly expressed in mature osteoclasts, even though it is expressed in low level in human monocyte precursor cells. Since there hOSCAR-M forms don't have exon 4b, we used exon 4b region as a probe. It showed that exon 4b is specific for large transcript of hOSCAR. To check the specificity of OSCAR gene expression, we used mRNA from human monocytes, monocyte-derived mature osteoclast, macrophage, and dendritic cells for Northern blot analysis. OSCAR as well as TRAP was highly expressed in mature osteoclast (Fig. 3B). When we used mRNA from the various tissues, the expression of hOSCAR and TRAP genes were not detected (Fig. 3C).

To characterize the hOSCAR-S isoforms, we performed a transient transfection experiment in 293T cells using three different constructs of hOSCAR-I, -II, and -V forms (Fig. 4). Cell lysates and supernatants were pulled down with anti-FLAG antibody and probed with anti-FLAG antibody after SDS-PAGE. The strong signal of hOSCAR-I form (FLAG-tagged hOSCAR-M form) was detected in cell lysate and hOSCAR-II and -V forms (FLAG-tagged hOSCAR-S form) showed bands in supernatants. In consistency, FACS analysis with those transiently transfected 293T cells revealed that OSCAR is highly expressed on the cell surface in hOSCAR-I form-transfected but not in hOSCAR-II and -V forms, although some minor expression was detected on the surface of hOSCAR-V. These data implicate that hOSCAR-S forms can be mainly secreted from osteoclasts and hOSCAR-M forms are expressed on the surface of osteoclasts.

Since we already showed mOSCAR is involved in the differentiation of osteoclast precursors to mature, multinucleated osteoclasts. We examined the role of hOSCAR in human monocyte culture using a soluble form of OSCAR, hOSCAR-Fc, which is made by fusing the extracellular domain of OSCAR to the Fc portion of human IgG1. When hOSCAR-Fc was added to human monocyte culture in the presence of M-CSF and RANKL, the formation of TRAP (+) multinucleated osteoclasts (MNCs) was significantly inhibited (Fig. 5). Furthermore, when cultures were made on dentin slice, hOSCAR-Fc inhibited significantly bone resorption (Fig. 5). These results indicate that OSCAR is required for the differentiation of mature osteoclasts derived from PBMC in the presence of M-CSF and RANKL.