A transcription factor, Cbfa1, which belongs to the runt-domain gene family, is expressed restrictively in fetal development. To elucidate the function of Cbfa1, we generated mice with a mutated Cbfa1 locus. Mice with a homozygous mutation in Cbfa1 died just after birth without breathing. Examination of their skeletal systems showed a complete lack of ossification. Although immature osteoblasts, which expressed alkaline phophatase weakly but not Osteopontin and Osteocalcin, and a few immature osteoclasts appeared at the perichondrial region, neither vascular nor mesenchymal cell invasion was observed in the cartilage. Therefore, our data suggest that both intramembranous and endochondral ossification were completely blocked, owing to the maturational arrest of osteoblasts in the mutant mice, and demonstrate that Cbfa1 plays an essential role in osteogenesis.

Mouse myeloid leukemia cells can be induced to differentiate into macrophages in vitro by la,25-dihydroxyvi. tamin D3, the active form of vitamin D3. The minimal concentration of la,25-dihydroxyvitamin D3 to induce the cell differentiation was 0.12 nM. The degree of cell differentiation in various markers induced by 12 nM la,25-dihydroxyvitamin D3 was nearly equivalent to that induced by 1 ,M dexamethasone, the most potent known stimulator. Among several markers of the differentiation by la,25-dihydroxyvitamin D3, phagocytic activity was induced within 24 hr, and this was followed by induction oflysozyme and locomotive activities. Similar changes were also induced by 0.01-1 ,uM la-hydroxyvitamin D3. 25-Hydroxyvitamin D3 and 24R,25-dihydroxyvitamin D.3 showed only weak inducing activity. These results suggest the possibility that, in addition to its wellknown biological activities in enhancing intestinal calcium transport and bone mineral mobilization, la,25-dihydroxyvitamin D3 is involved in the differentiation of bone marrow cells.The myeloid leukemia cell line (Ml), originally established by Ichikawa (1) from an SL mouse with myeloid leukemia, is known to differentiate into mature macrophages and granulocytes in vitro when treated with conditioned media from various cell cultures (2), ascitic fluid of tumor-bearing animals (3), bacterial lipopolysaccharides (4), polyribonucleotides (5), or glucocorticoids (6, 7). Among various inducers, dexamethasone has been found to be the most potent stimulator (8). The differentiation can be detected by changes in cell morphology, adhesion ofcells to the dish surface, increase in lysosomal enzyme activity, induction of phagocytic and locomotive activities, and the appearance of Fc and C3 receptors on the cell surface (2-8).It C02/95% air in Eagle's minimal essential medium supplemented with twice the normal concentrations ofamino acids and vitamins and 10% heat-inactivated calf serum (Chiba Serum Institute, Chiba, Japan). The cells were transferred every 2 to 3 days. Except for the study of cell fractionation, all cells (both adherent and nonadherent) were used for determining parameters of differentiation.Hormone and Vitamin D Derivatives. Dexamethasone was purchased from Sigma and 25(OH)D3 was from Philips-Duphar (Amsterdam). la,25(OH)2D3, 24R,25(OH)2D3, and la-hydroxyvitamin D3 [la(OH)D3] were kindly donated by I. Matsunaga, Chugai Pharmaceutical, Tokyo.Fractionation of the Cells by Discontinuous Density Gradient Centrifugation. For the study of cell fractionation, only adherent cells were used in the cultures treated with dexamethasone, la,25(OH)2D3, or la(OH)D3 whereas all cells were used in the control culture. After the nonadherent cells and loosely adherent cells had been removed by gently rinsing three times with prewarmed culture medium, the cells attached to dishes were collected as adherent cells by pipetting with phosphate-buffered saline lacking Ca2+ and Mg2-(PJNaCl) on ice. Determination of Lysozyme Activity. Lysozyme activity was determined by a mo...

We developed a mouse bone marrow culture system to examine the process of osteoclast-like multinucleated cell formation from its progenitors. When mouse marrow cells were cultured for 8 days with 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3, 10(-10) to 10(-7) M] or human PTH (1-34) (25-100 ng/ml), tartrate-resistant acid phosphatase (TRACP)-positive multinucleated cells formed. No TRACP-positive multinucleated cells appeared in the absence of these hormones. 1 alpha,25-(OH)2D3 and PTH also increased the number of the clusters of TRACP-positive mononuclear cells. Time course studies showed that these TRACP-positive mononuclear cell clusters appeared before the formation of TRACP-positive multinucleated cells, suggesting that the TRACP-positive mononuclear cells are precursors of the multinucleated cells. Salmon calcitonin markedly inhibited the formation of TRACP-positive multinucleated cells but not TRACP-positive mononuclear cell clusters induced by 1 alpha,25-(OH)2D3 or PTH. TRACP-positive mononuclear cells and multinucleated cells were rarely stained for nonspecific esterase, but some mononuclear cells were positively stained for both nonspecific esterase and TRACP. More that 90% of the TRACP-positive mononuclear cell clusters and multinucleated cells were found near colonies of alkaline phosphatase-positive mononuclear cells (possibly osteoblasts). When marrow mononuclear cells were cultured on sperm whale dentine slices in the presence of 1 alpha,25-(OH)2D3 or PTH, numerous resorption lacunae were formed. These results suggest that 1) TRACP-positive multinucleated cells formed in response to osteotropic hormones in mouse marrow cultures satisfy most of the criteria of osteoclasts, and 2) osteoblasts may play an important role in osteoclast formation.