Identification of the Functional Domain of Osteoclast Inhibitory Peptide-1/hSca

Abstract

Osteoclast (OCL) activity is controlled by local factors produced in the bone microenvironment. We previously identified a novel inhibitor of OCL formation that is produced by OCLs (osteoclast inhibitory peptide‐1/human Sca [OIP‐1/hSca]). OIP‐1/hSca is a glycosylphosphatidylinositol (GPI)‐linked membrane protein (16 kDa) that is cleaved from the OCL surface. Immunocytochemical staining further confirmed the expression of OIP‐1/hSca in OCL formed in mouse bone marrow cultures. However, the structure/function mechanisms responsible for the inhibitory effects of OIP‐1/hSca on OCL formation are unknown. Therefore, we expressed deletion mutants of OIP‐1 in 293 cells and tested their effects on OCL formation. These studies indicated that the carboxy‐terminal peptide (c‐peptide) region is critical for OIP‐1/hSca activity. A 33 amino acid OIP‐1 c‐peptide (10‐100 ng/ml) significantly inhibited 1,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃] induced OCL formation and pit formation capacity of OCL on dentine slices in human bone marrow cultures. Furthermore, the c‐peptide (10‐100 ng/ml) significantly inhibited early human OCL precursor (granulocyte‐macrophage colony‐forming unit [GM‐CFU]) colony formation in methylcellulose cultures. The polyclonal antibody against the OIP‐1 c‐peptide neutralized the inhibitory effect of OIP‐1 c‐peptide on OCL formation in mouse bone marrow cultures in vitro. These results show that the OIP‐1 c‐peptide is the functional domain of OIP‐1 and that availability of neutralizing antibody specific to the OIP‐1 c‐peptide should provide important mechanistic insights into OIP‐1/hSca inhibition of osteoclastogenesis in the bone microenvironment.