Osteocalcin promotes differentiation of osteoclast progenitors from murine long-term bone marrow cultures.
Murine long-term bone marrow cultures (LTBMCs) were used to generate hematopoietic cells free from marrow stromal cells. These progenitor cells were treated with GM-CSF (5 U/ml) with or without rat bone osteocalcin or rat serum albumin in either alpha-MEM with 2% heat-inactivated horse serum alone (alpha) or supplemented with 10% L-cell-conditioned medium (as a source of M-CSF) (L10). Few substrate-attached cells survived in basal alpha medium, but when treated with L10 medium or GM-CSF, they survived and proliferated. Osteocalcin did not significantly affect survival or proliferation. Subcultures of cells treated with GM-CSF had large numbers of multinucleated cells, more than half of which were tartrate-resistant acid phosphatase-positive (TRAP). Osteocalcin further promoted the development of TRAP-positive multinucleated cells; a dose of 0.7 microgram/ml osteocalcin promoted osteoclastic differentiation by 60%. Using a novel microphotometric assay, we detected significantly more tartrate-resistant acid phosphatase activity in the osteocalcin plus GM-CSF group (75.6 +/- 14.2) than in GM-CSF alone (53.3 +/- 7.3). In the absence of M-CSF, GM-CSF stimulated tartrate-resistant acid phosphatase activity, but osteocalcin did not have an additional effect. These studies indicate that osteocalcin promotes osteoclastic differentiation of a stromal-free subpopulation of hematopoietic progenitors in the presence of GM-CSF and L-cell-conditioned medium. These results are consistent with the hypothesis that this bone-matrix constituent plays a role in bone resorption.
Liggett
W H
WH
Department of Orthopedic Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115.
Lian
J B
JB
Greenberger
J S
JS
Glowacki
J
J
D000135:Acid Phosphatase / Q000378:metabolism; D000818:Animals; D001854:Bone Marrow Cells; D002452:Cell Count; D002454:Cell Differentiation / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D051379:Mice; D008810:Mice, Inbred C57BL; D015675:Osteocalcin / Q000494:pharmacology*; D010010:Osteoclasts / Q000166:cytology*; D013234:Stem Cells / Q000166:cytology*; D013644:Tartrates / Q000494:pharmacology; D013997:Time Factors
D017077:Culture Media, Conditioned; D013644:Tartrates; D015675:Osteocalcin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D000135:Acid Phosphatase; C029768:tartaric acid