Sun Jin Choi

ADAM8: A Novel Osteoclast Stimulating Factor

We used polymerase chain reaction (PCR)‐selective complementary DNA (cDNA) subtraction hybridization with an immortalized murine osteoclast (OCL) precursor cell line to identify genes that are highly expressed in OCLs compared with OCL precursors and which may be involved in the OCL differentiation process. ADAM8 was one of the 50 genes identified. ADAM (a disintegrin and metalloproteinase) peptides are membrane‐bound proteins that can act as cell‐to‐cell and cell‐to‐matrix adhesion molecules, degrade the extracellular matrix, and play a role in tissue morphogenesis. Addition of antisense (AS) S‐oligonucleotides for ADAM8 (1‐10 nM) to mouse bone marrow cultures treated with 10−9 M 1,25‐dihydroxyvitamin D3 [1,25‐(OH)2D3] significantly inhibited OCL formation compared with treatment with the control S‐oligonucleotide. Furthermore, conditioned media from 293 cells transiently transfected with a secretable form of the ADAM8 cDNA increased OCL formation in a dose‐dependent manner. In addition, treatment of OCLs with soluble ADAM8 conditioned media significantly increased pit formation per dentin slice compared with control OCLs. Time course studies indicated that ADAM8 increased OCL formation only when it was present during days 4‐7 of the 7‐day culture period. Structural analysis, using truncated constructs of ADAM8, showed that the cysteine‐rich/disintegrin domain was responsible for its OCL stimulatory activity. Western blot analysis confirmed that the soluble form of ADAM8 is present in normal marrow cultures. These data suggest that ADAM8 plays an important role in OCL formation and acts primarily at the later stages of OCL differentiation.

Annexin II increases osteoclast formation by stimulating the proliferation of osteoclast precursors in human marrow cultures

Annexin II (AXII), a calcium-dependent phospholipid-binding protein, has been recently found to be an osteoclast (OCL) stimulatory factor that is also secreted by OCLs. In vitro studies showed that AXII induced OCL formation and bone resorption. However, the mechanism of action by which AXII acts as a soluble extracellular protein to induce OCL formation is unknown. In this paper, we demonstrate that AXII gene expression is upregulated by 1,25-dihydroxyvitamin D3 [1, 25-(OH)2D3] and that addition of AXII significantly increased OCL-like multinucleated cell formation. Time-course studies suggested that AXII acted on the proliferative stage of OCL precursors and that AXII increased thymidine incorporation in OCL precursors. Moreover, AXII enhanced the growth of CFU-GM, the earliest identifiable OCL precursor, when bone marrow cultures were treated with low concentrations of GM-CSF. This capacity of AXII to induce OCL precursor proliferation was due to induction of GM-CSF expression, because the addition of neutralizing antibodies to GM-CSF blocked the stimulatory effect of AXII on OCL formation. RT-PCR analysis using RNA from highly purified subpopulations of marrow cells demonstrated that T cells, especially CD4(+) T cells, produced GM-CSF in response to AXII. Furthermore, FACS(R) analysis of T-cell subpopulations treated with fluorescein-labeled AXII suggested that the CD4(+), but not CD8(+), subpopulation of T cells express an AXII receptor. Taken together, these data suggest that AXII stimulates OCL formation by activating T cells through a putative receptor to secrete GM-CSF. GM-CSF then expands the OCL precursor pool to enhance OCL formation.

Identification of Human Asparaginyl Endopeptidase (Legumain) as an Inhibitor of Osteoclast Formation and Bone Resorption

We screened a human osteoclast (OCL) cDNA expression library for OCL inhibitory factors and identified a clone that blocked both human and murine OCL formation and bone resorption by more than 60%. This clone was identical to human legumain, a cysteine endopeptidase. Legumain significantly inhibited OCL-like multinucleated cell formation induced by 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and parathyroid hormone-related protein (PTHrP) in mouse and human bone marrow cultures, and bone resorption in the fetal rat long bone assay in a dose-dependent manner. Legumain was detected in freshly isolated marrow plasma from normal donors and conditioned media from human marrow cultures. Furthermore, treatment of human marrow cultures with an antibody to legumain induced OCL formation to levels that were as high as those induced by 1,25-(OH)2D3. Implantation in nude mice of 293 cells transfected with the legumain cDNA and constitutively expressing high levels of the protein significantly reduced hypercalcemia induced by PTHrP by about 50%, and significantly inhibited the increase in OCL surface and in OCL number expressed per mm2 bone area and per mm bone surface induced by PTHrP. These results suggest that legumain may be a physiologic local regulator of OCL activity that can negatively modulate OCL formation and activity.

Isolation and characterization of a cDNA clone encoding a novel peptide (OSF) that enhances osteoclast formation and bone resorption.

Using an expression cloning approach, we identified and cloned a novel intracellular protein produced by osteoclasts that indirectly induces osteoclast formation and bone resorption, termed OSF. Conditioned media from 293 cells transiently transfected with the 0.9 kb OSF cDNA clone stimulated osteoclast‐like cell formation in both human and murine marrow cultures in the presence or absence 10−9 M 1,25‐dihydroxyvitamin D3. In addition, conditioned media from 293 cells transfected with the OSF cDNA clone enhanced the stimulatory effects of 1,25‐(OH)2D3 on bone resorption in the fetal rat long bone assay. In situ hybridization studies using antisense oligomers showed expression of OSF mRNA in highly purified osteoclast‐like cells from human giant cell tumors of the bone. Northern blot analysis demonstrated ubiquitous expression of a 1.3 kb mRNA that encodes OSF in multiple human tissues. Sequence analysis showed the OSF cDNA encoded a 28 kD peptide that contains a c‐Src homology 3 domain (SH3) and ankyrin repeats, suggesting that it was not a secreted protein, but that it was potentially involved in cell signaling. Consistent with these data, immunoblot analysis using rabbit antisera against recombinant OSF demonstrated OSF expression in cell lysates but not in the culture media. Furthermore, recombinant OSF had a high affinity for c‐Src, an important regulator of osteoclast activity. Taken together, these data suggest that OSF is a novel intracellular protein that indirectly enhances osteoclast formation and osteoclastic bone resorption through the cellular signal transduction cascade, possibly through its interactions with c‐Src or other Src‐related proteins. J Cell Physiol 177:636–645, 1998.

Osteoclast Inhibitory Peptide 2 Inhibits Osteoclast Formation via Its C‐Terminal Fragment

Osteoclast inhibitory peptide 2 (OIP‐2) is a novel autocrine/paracrine factor produced by osteoclasts (OCLs) that inhibits bone resorption and OCL formation in vitro and in vivo. It is identical to the asparaginyl endopeptidase legumain. During maturation of OIP‐2, a signal peptide and a 17‐kDa C‐terminal fragment (CTF) are cleaved to produce the mature enzyme. To determine if enzyme activity is required for inhibition of OCL formation or if only the CTF is responsible for these effects, we synthesized His‐tagged complementary DNA (cDNA) constructs for the CTF of OIP‐2, the proform of OIP‐2, and the “mature enzyme” form of OIP‐2. The proform or the CTF portion of OIP‐2 inhibited OCL formation in a dose‐dependent manner in murine bone marrow cultures stimulated with 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3]. The mature form of OIP‐2, which was enzymatically active, did not inhibit OCL formation. In addition, OIP‐2 inhibited OCL formation in cultures of highly purified human OCL precursor cells or RAW264.7 cells stimulated with 10 ng/ml of receptor activator of NF‐κB (RANK) ligand. Binding studies with His‐tagged OIP‐2 showed expression of a putative OIP‐2 receptor on RAW264.7 cells treated with RANK ligand for 4 days and human marrow cultures treated with 1,25(OH)2D3 for 3 weeks. These data show that the CTF of OIP‐2, rather than the mature enzyme, mediates the inhibitory effects of OIP‐2 through a putative receptor on OCL precursors.

Characterization of immortalized osteoclast precursors developed from mice transgenic for both bcl-X(L) and simian virus 40 large T antigen.

We recently developed an immortalized osteoclast (OCL) precursor cell line that forms large numbers of OCLs. This cell line was derived from mice doubly transgenic for bcl-XL and large T antigen that was targeted to cells in the OCL lineage (bcl-XL/Tag cells). We have now characterized these cells in terms of their surface and enzymatic phenotype, responsiveness to osteotropic factors, and differentiation potential. The bcl-XL/Tag cells expressed interleukin-1 receptors 1 and 2, gelatinase B (MMP9), as well as Mac-1, CD16/CD32 (Fcγ receptors), CD45.2 (common leukocyte marker), CD86 (costimulatory molecule expressed on B cells, follicular dendritic cells, and thymic epithelium), major histocompatibility complex I, and nonspecific esterase when cocultured with MC3T3E1 cells. However, they did not express the antigens for F4/80 (mature macrophage/dendritic cell marker) by immunostaining. Treatment of bcl-XL/Tag cells, cocultured with MC3T3E1 cells, with the combination of 1,25-dihydroxyvitamin D3 and dexamet...

Cloning and identification of human Sca as a novel inhibitor of osteoclast formation and bone resorption.

Increased osteoclast activity is responsible for the enhanced bone destruction in postmenopausal osteoporosis, Pagets disease, bone metastasis, and hypercalcemia of malignancy. However, the number of known inhibitory factors that block osteoclast formation and bone resorption are limited. Therefore, we used an expression-cloning approach to identify novel factors produced by osteoclasts that inhibit osteoclast activity. A candidate clone was identified and isolated from a human osteoclast-like multinucleated cell (MNC) cDNA library, named osteoclast inhibitory peptide-1 (OIP-1), and the cDNA sequence was determined. This sequence matched that of the recently identified human stem cell antigen, was structurally similar to the mouse Ly-6 gene family, and the sequence predicted it was a glycosyl phosphatidyl inositol (GPI)-anchored protein that had a cleavable COOH-terminal peptide. Western blot analysis of conditioned media from 293 cells transfected with the OIP-1 cDNA clone confirmed that OIP-1 was released into the media as a membrane-bound GPI-linked protein. Interestingly, both recombinant OIP-1 expressed in Escherichia coli (which does not have GPI linker) and OIP-1 expressed by mammalian cells significantly reduced osteoclast-like MNC formation induced by 1,25-dihydroxyvitamin D3 or PTH-related protein in mouse and human bone marrow cultures, and inhibited 45Ca release from prelabeled bone in fetal rat organ cultures. In contrast, recombinant OIP-1 did not inhibit the growth of a variety of other cell types. These data indicate that OIP-1 is a novel, specific inhibitor of osteoclast formation and bone resorption.