OSTEOCALCIN PROMOTES DIFFERENTIATION OF OSTEOCLAST PROGENITORS FROM MURINE LONG-TERM BONE-MARROW CULTURES

Murine long-term bone marrow cultures (LTBMCs) were used to generate h ematopoietic cells free from marrow stromal cells. These progenitor ce lls were treated with CM-CSF (5 U/ml) with or without rat bone osteoca lcin 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 sur vived in basal or medium, but when treated with L10 medium or CM-CSF, they survived and proliferated. Osteocalcin did not significantly affe ct survival or proliferation. Subcultures of cells treated with CM-CSF had large numbers of multinucleated cells, more than half of which we re tartrate-resistant acid phosphatase-positive (TRAP). Osteocalcin fu rther promoted the development of TRAP-positive multinucleated cells; a dose of 0.7 mu g/ml osteocalcin promoted osteoclastic differentiatio n by 60%. Using a novel microphotometric assay, we detected significan tly more tartrate-resistant acid phosphatase activity in the osteocalc in 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 effe ct. These studies indicate that osteocalcin promotes osteoclastic diff erentiation of a stromal-free subpopulation of hematopoietic progenito rs in the presence of CM-CSF and L-cell-conditioned medium. These resu lts are consistent with the hypothesis that this bone-matrix constitue nt plays a role in bone resorption. (C) 1994 Wiley-Liss, Inc.