Korean J Bone Metab.  2012 May;19(1):11-20. 10.11005/kjbm.2012.19.1.11.

Identification of Alternatively Spliced Forms of human OSCAR in Osteoclasts

• Kim N
• Park HY
• Kim HD

• KMID: 1464231
• DOI: http://doi.org/10.11005/kjbm.2012.19.1.11

Abstract

OBJECTIVES
Osteoclasts are multinucleated giant cells which can resorb bone and differentiated from hematopoietic cells. We have previously reported murine osteoclast-associated receptor (OSCAR) may be an important bone-specific regulator of osteoclast differentiation. We have cloned soluble form of human OSCAR (hOSCAR) and examined the role of hOSCAR on osteoclast differentiation.
METHODS
Osteoclast differentiation was induced by treatment with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa B ligand (RANKL) and tartrate-resistant acid phosphatase (TRAP) staining and pit formation were performed. Expression was measured by flow cytometry analysis, Northern and Western blot analysis.
RESULTS
hOSCAR is expressed in osteoclast cells and involved in the differentiation of osteoclasts from peripheral blood mononuclear cells (PBMC). Two alternatively spliced forms (soluble hOSCAR [hOSCAR-S]) of hOSCAR were identified from osteoclasts complementary deoxyribonucleic acid (cDNA) library derived from PBMC. Putative transmembrane domain was not found in hOSCAR-S forms and it suggested that these forms might be secreted from osteoclast cells. These secreted forms of hOSCAR attenuated RANKL-induced osteoclast formation and bone resorption.
CONCLUSIONS
Human osteoclasts express at least five different OSCAR messenger ribonucleic acid (mRNA) isoforms which could play different regulatory roles for differentiation. The secreted forms of hOSCAR might be a negative regulator of membrane-bounded forms of OSCAR.

Keyword

Alternative splicing; Differentiation; OSCAR; Osteoclast

MeSH Terms

Acid Phosphatase
Alternative Splicing
Blotting, Western
Clone Cells
DNA
Flow Cytometry
Giant Cells
Humans
Isoenzymes
Macrophage Colony-Stimulating Factor
Osteoclasts
Protein Isoforms
RANK Ligand
RNA
Acid Phosphatase
DNA
Isoenzymes
Macrophage Colony-Stimulating Factor
Protein Isoforms
RANK Ligand
RNA

Figure

• 

  Fig. 1 Genomic structure and isoforms of human osteoclast-associated receptor (hOSCAR). Four exons and three introns are shown as boxes and lines, respectively. The numbers of nucleotide sequences are indicated in the parentheses. The potential transmembrane domain (TM) and stop codons are indicated with triangle. The numbers of amino acid and transcript sizes of hOSCAR isoforms are indicated. hOSCAR-M, membrane-bound human osteoclast-associated receptor; hOSCAR-S, soluble human osteoclast-associated receptor.

• 

  Fig. 2 Sequence analysis of human osteoclast-associated receptor (hOSCAR). The amino acid sequences of hOSCAR isoforms are shown as one letter code. Sequences are divided into each putative region: N-termini, domain 1, domain 2, and C-termini. The potential transmembrane domain is in boldface and underlined letters. All isoforms have common domain 1 and 2. GeneBank accession numbers for hOSCAR isoforms are AF391162, AF391163, AF391164, AF474152, and AF474153.

• 

  Fig. 3 Human osteoclast-associated receptor (hOSCAR) messenger ribonucleic acid (mRNA) expression. (A) hOSCAR mRNA expression during the differentiation of peripheral blood mononuclear cells (PBMC) to mature osteoclasts by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa B ligand (RANKL). Northern blot analysis was performed using total RNAs for 0 to 8 days. OSCAR exon 3 and 4b were used for probes. (B) Northern blot analysis of RNAs from PBMC (lane 1), PBMC-derived osteoclasts (lane 2), macrophages (lane 3), and dendritic cells (lane 4). (C) Northern blot analysis of OSCAR in various human tissues and PBMC-derived osteoclasts. Twenty-eight S and 18S of ribosomal RNAs are shown as control.

• 

  Fig. 4 Transient expression of human osteoclast-associated receptor (hOSCAR) isoforms in 293T cells. (A) FLAG-tagged human OSCAR isoforms (hOSCAR-I, II, and V) were synthesized from hOSCAR-M1 or -S1 forms. (B) Western blot analysis of 293T cells transiently transfected with human OSCAR isoforms, Lane 1; control vector, 2; hOSCAR-I, 3; hOSCAR-II, and 4; hOSCAR-V. Cell lysates and supernatants were collected and immunoprecipitated with anti-FLAG antibody. Samples were resolved on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed with anti-FLAG antibody. (C) Flow cytomety analysis of 293T cells transfected with human OSCAR isoforms. The transfected cells were stained with biotinylated anti-FLAG antibody, followed streptoavidin-conjugated adenomatous polyposis coli (APC) and analyzed by flow cytometry. Dot line indicates vector control transfectant and solid lines indicate hOSCAR-I, -II, and -V isoforms transfectants.

• 

  Fig. 5 Role of human osteoclast-associated receptor (hOSCAR) in human osteoclasts differentiation. (A) Human peripheral blood mononuclear cells (PBMC) were cultured in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa B ligand (RANKL) with 30 µg/mL hOSCAR-Fc, 30 µg/mL human IgG, or 5 µg/mL RANK-Fc in 96-well culture plates (top) or on dentine slices placed in 96-well culture plates (bottom). Cultured cells were fixed and stained for TRAP (top). Cells on dentine slices were removed, and dentine slices were stained with Mayer's hematoxylin. Pits appeared as dark spots (bottom). (B) Number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) in A was counted as osteoclasts (OCs). (C) Number of pits formed on dentine slices in A was counted. Data represent means ± SDs of triplicate samples. The results are representative of at least three independent sets of similar experiments.

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