Cholera toxin and forskolin stimulate formation of osteoclast-like cells in mouse marrow cultures and cultured mouse calvarial bones

Osteoclasts are hematopoietic in origin and formed by proliferation, differentiation and fusion of osteoclast progenitor cells. However, the signal transducing mechanisms involved in generation of osteoclasts are not clear. We have used two well-known adenylate cyclase stimulators to examine the effect of cyclic AMP (cAMP) on the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in cultured mouse calvarial bones and in mouse bone marrow cultures. The effects of forskolin and cholera toxin were compared with those of parathyroid hormone (PTH) and 1,25(OH)(2)vitaminD(3) (1,25(OH)(2)D-3). PTH, as well as forskolin and cholera toxin, increased the number of osteoclast profiles/mm bone in 24-h and 120-h cultures of mouse calvarial bones. In mouse bone marrow cultures, 1,25(OH)(2)D-3 or PTH stimulated formation of TRAP-positive multinucleated cells. Moreover, forskolin or cholera toxin produced dose-dependent stimulation of these cells at a range of concentrations correlating with their effect on cAMP production. The osteoclastic phenotype of the TRAP-positive cells was demonstrated by autoradiography of I-125-labelled calcitonin binding and by the bone-resorbing activity of the cells. The sustained presence (0-9 d) of forskolin or PTH was required to obtain maximal formation of osteoclasts. However, the presence of 1,25(OH)(2)D-3 was required only for the last 3 d of culture for maximal osteoclast formation. We conclude that PTH may stimulate osteoclast generation using the adenylate cyclase cAMP system as a signal transduction mechanism.