Osteocalcin fragment in bone matrix enhances osteoclast maturation at a late stage of osteoclast differentiation.
Although 14-day-old mouse embryonic calvarial cells cultured in plastic culture dishes in the presence of 1alpha,25-dihydroxyvitamin D3[1alpha,25-(OH)2D3] for 7 days could barely resorb bone slices, the same calvarial cells cultured with an ethylenediaminetetraacetic acid (EDTA) extract from bovine bone powder under the same conditions stimulated pit formation on bone slices in a dose-dependent manner. Therefore, the present study was conducted to purify and characterize this osteoclast maturation-inducing factor(s) from the bone matrix. The protein having osteoclast maturation-inducing activity in the EDTA extract was purified by gel filtration over Superdex 75 preparation grade and chromatography on hydroxyapatite, Mono Q, and C8 reversed-phase HPLC by monitoring the ability of the eluted fractions to elicit pit formation on bone slices. The molecular weight of the purified protein estimated by high-resolution polyacrylamide gel electrophoresis was 5.7 kDa and 6.8 kDa in the respective absence and presence of 2-mercaptoethanol. The sequence of the 30-amino-acid purified protein corresponded to the 7th to 36th residues of bovine osteocalcin. The osteocalcin fragment, missing the initial 6 residues at the N-terminal region, exhibited higher osteoclast maturation-inducing ability than bovine intact osteocalcin on a per weight basis. The osteocalcin fragment had no effect on the expression of receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) and osteoprotegerin (OPG) genes in calvarial cells, nor did it enhance the bone-resorbing activity of mature osteoclasts. When the osteocalcin fragment was added to late-stage (days 4-7) or to early-stage (days 0-3) cultures of calvarial cells pretreated with 1alpha,25-(OH)2D3, its stimulatory effect was observed in the late-stage cultures rather than in the early-stage ones. In addition, the osteocalcin fragment directly enhanced the formation of osteoclasts with bone-resorbing ability from Mac-1+ c-Fms+ cells in the presence of macrophage colony-stimulating factor (MCSF) and RANKL. These results suggest that the osteocalcin fragment in bone matrix is involved in osteoclast maturation, especially at a late stage of osteoclast differentiation.
Department of Oral Microbiology, Meikai University School of Dentistry, 350-0283, Sakado, Keyakidai, Japan.
Masami
M
Ishida
Shigeru
S
Amano
D015650:24,25-Dihydroxyvitamin D 3 / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001857:Bone Matrix / Q000502:physiology*; D001862:Bone Resorption; D002352:Carrier Proteins / Q000187:drug effects / Q000235:genetics; D002417:Cattle; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D005260:Female; D006023:Glycoproteins / Q000187:drug effects / Q000235:genetics; D016173:Macrophage Colony-Stimulating Factor; D008297:Male; D008562:Membrane Glycoproteins / Q000187:drug effects / Q000235:genetics; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D015675:Osteocalcin / Q000302:isolation & purification / Q000494:pharmacology / Q000502:physiology*; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D053244:Osteoprotegerin; D010446:Peptide Fragments / Q000494:pharmacology; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D018160:Receptors, Cytoplasmic and Nuclear / Q000187:drug effects / Q000235:genetics; D018124:Receptors, Tumor Necrosis Factor; D012886:Skull / Q000166:cytology / Q000187:drug effects / Q000196:embryology; D014020:Tissue Extracts / Q000494:pharmacology
D002352:Carrier Proteins; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D053244:Osteoprotegerin; D010446:Peptide Fragments; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D018160:Receptors, Cytoplasmic and Nuclear; D018124:Receptors, Tumor Necrosis Factor; D014020:Tissue Extracts; C505687:Tnfrsf11a protein, mouse; C506031:Tnfrsf11b protein, mouse; C505689:Tnfsf11 protein, mouse; D015675:Osteocalcin; D015650:24,25-Dihydroxyvitamin D 3; D016173:Macrophage Colony-Stimulating Factor; D004492:Edetic Acid