Ketoconazole and phorbol myristate acetate regulate osteoclast precursor fusion in primary murine marrow culture

Osteoclast formation requires both precursor proliferation and then fusion into a multinuclear cell. These processes can be separated in primary murine marrow culture where osteoclastogenesis is stimulated by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Here we investigate the regulation of precursor fusion. Ketoconazole, an agent known to inhibit cell fusion, added during the fusion period (days 5-6), dose-dependently inhibited formation of tartrate-resistant acid phosphatase+ (TRAP+) multinucleated cells (TRAP+MNCs), maximally at 62 +/- 4% (n = 10). TRAP+MNCs in cultures exposed to 48 h of ketoconazole (1 microM) during fusion had fewer nuclei compared with control (11.7 +/- 0.6 vs. 15.1 +/- 0.9). This inhibitory effect was completely reversed 24 h after removal of ketoconazole from culture. Phorbol myristate acetate (PMA) stimulated TRAP+MNC formation when given during the last 12 h of culture (2.3 +/- 0.2 fold compared with control). This increased formation was unaffected by the addition of hydroxyurea and accompanied by an increase in nuclei per TRAP+MNC (15.5 +/- 0.9 vs. 13.1 +/- 0.6). Finally, staurosporine decreased TRAP+MNC formation in the presence or absence of PMA, implying that protein kinase C is involved in fusogenic processes. Regulation of fusion appears to be another mechanism by which bone remodeling can be modulated in vivo.