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Osteocalcin fragment in bone matrix enhances osteoclast maturation at a late stage of osteoclast differentiation.
J Bone Miner Metab. 2004; 22(5):415-29.JB

Abstract

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.

Authors+Show Affiliations

Department of Oral Microbiology, Meikai University School of Dentistry, 350-0283, Sakado, Keyakidai, Japan.No affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

15316862

Citation

Ishida, Masami, and Shigeru Amano. "Osteocalcin Fragment in Bone Matrix Enhances Osteoclast Maturation at a Late Stage of Osteoclast Differentiation." Journal of Bone and Mineral Metabolism, vol. 22, no. 5, 2004, pp. 415-29.
Ishida M, Amano S. Osteocalcin fragment in bone matrix enhances osteoclast maturation at a late stage of osteoclast differentiation. J Bone Miner Metab. 2004;22(5):415-29.
Ishida, M., & Amano, S. (2004). Osteocalcin fragment in bone matrix enhances osteoclast maturation at a late stage of osteoclast differentiation. Journal of Bone and Mineral Metabolism, 22(5), 415-29.
Ishida M, Amano S. Osteocalcin Fragment in Bone Matrix Enhances Osteoclast Maturation at a Late Stage of Osteoclast Differentiation. J Bone Miner Metab. 2004;22(5):415-29. PubMed PMID: 15316862.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Osteocalcin fragment in bone matrix enhances osteoclast maturation at a late stage of osteoclast differentiation. AU - Ishida,Masami, AU - Amano,Shigeru, PY - 2003/05/06/received PY - 2004/02/27/accepted PY - 2004/8/19/pubmed PY - 2004/11/4/medline PY - 2004/8/19/entrez SP - 415 EP - 29 JF - Journal of bone and mineral metabolism JO - J Bone Miner Metab VL - 22 IS - 5 N2 - 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. SN - 0914-8779 UR - https://www.unboundmedicine.com/medline/citation/15316862/Osteocalcin_fragment_in_bone_matrix_enhances_osteoclast_maturation_at_a_late_stage_of_osteoclast_differentiation_ L2 - https://dx.doi.org/10.1007/s00774-004-0503-5 DB - PRIME DP - Unbound Medicine ER -