cAMP promotion of osteoclast-like cell development from mouse bone marrow cells requires a permissive action of 1,25-(OH)2D3

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Abstract
Recruitment of osteoclasts from monocytic precursors is modulated by local signals. We previously showed that monoblastic differentiation in U937 cells is stimulated by 1,25-(OH)2D3 and cAMP in series. We investigate here the combined effects of these agents to stimulate differentiation of osteoclast-like cells from mouse marrow. Cells from mouse marrow were harvested and cultured in α-MEM with 10% fetal bovine serum. The appearance of tartrate-resistant acid phosphatase-containing multinuclear cells was measured after 8 days in culture by cytochemical staining. Continuous exposure of cultures to 10 nM 1,25-(OH)2D3 positively stimulated development of these cells after 8 days (101 + 3 cells per well, n = 74). No osteoclast-like cells were found when 1,25-(OH)2D3 was added for the first 4 days followed by 4 days more with no treatment. PGE2 (1 μM) as a single agent added during the last 4 days of culture was not able to recruit osteoclast-like cells. However, cultures exposed to 1,25-(OH)2D3 during the first 4 days and 1 μM PGE2 during the second 4 days developed osteoclast-like cells at 8 days [66 + 8% of the formation seen with 1,25-(OH)2D3 alone, p < 0.05]. Dibutyryl cAMP (1 μM to 3 mM) was also not effective used as a single agent, but was able to stimulate formation of TRAP-positive multinuclear cells when 1,25-(OH)2D3 preceded its addition to culture medium. cAMP analogs therefore mimicked the effect of 1 μM PGE2, but these experiments do not allow us to assign the PGE2 action entirely to activation of cAMP second messenger. We postulate that continuous 1,25-(OH)2D3 stimulation is alone sufficient to recruit osteoclasts from precursors but that the effect of cAMP to stimulate osteoclast formation occurs only when combined with a permissive action of 1,25-(OH)2D3.