Identification of the functional domain of osteoclast inhibitory peptide-1/hSca.
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.
Koide
Masanori
M
Department of Medicine/Hematology, University of Texas Health Science Center, San Antonio, USA.
Kurihara
Noriyoshi
N
Maeda
Hidefumi
H
Reddy
Sakamuri V
SV
D000074183:ATPases Associated with Diverse Cellular Activities; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D001483:Base Sequence; D002117:Calcitriol / Q000494:pharmacology; D002460:Cell Line; D003114:Colony-Forming Units Assay; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D060588:LIM Domain Proteins; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D010449:Peptide Mapping; D046988:Proteasome Endopeptidase Complex; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D017384:Sequence Deletion; D014157:Transcription Factors
D048868:Adaptor Proteins, Signal Transducing; D018076:DNA, Complementary; D060588:LIM Domain Proteins; C092282:PSMC5 protein, human; D011506:Proteins; D014157:Transcription Factors; D046988:Proteasome Endopeptidase Complex; D000074183:ATPases Associated with Diverse Cellular Activities; D002117:Calcitriol