Induction of calcitonin receptors by 1 alpha, 25-dihydroxyvitamin D3 in osteoclast-like multinucleated cells formed from mouse bone marrow cells

Abstract

We have developed a mouse marrow culture system, in which multinucleated cells (MNCs) are formed within 6-8 days. These MNCs showed several characteristics of osteoclasts, including tartrate-resistant acid phosphatase (TRACP) and the ability to resorb calcified dentine. 1 alpha, 25-Dihydroxyvitamin D3 [1 alpha, 25 (OH)2D3] stimulated the formation of TRACP-positive MNCs, and salmon calcitonin (CT) inhibited it. In this study, we examined whether the TRACP-positive MNCs formed from mouse marrow cells possess CT receptors, another typical characteristic of osteoclasts. Mouse marrow cells cultured for 8 days with 10 nM 1 alpha, 25(OH)2D3 and freshly isolated authentic mouse osteoclasts were incubated with [125I]-salmon CT in the presence or absence of excess amounts of unlabeled CT, stained for TRACP, and processed for autoradiography. The [125I]-CT exclusively bound to TRACP-positive mononuclear cells and MNCs formed in the 1 alpha, 25(OH)2D3-treated cultures and also to isolated mouse osteoclasts. Both [125I]-CT binding and TRACP activity were induced simultaneously on mononuclear cells on day 3 in the cultures treated with 1 alpha, 25(OH)2D3. CT markedly stimulated cAMP production in the 1 alpha,25(OH)2D3-treated cultures. The CT-dependent cAMP production increased in parallel with the increase in the number of TRACP-positive MNCs formed. Neither freshly isolated marrow cells nor cells cultured without 1 alpha, 25(OH)2D3 showed CT-induced cAMP accumulation. Furthermore, CT induced cytoplasmic contraction of both marrow-derived MNCs and isolated osteoclasts. These results clearly indicate that 1 alpha,25(OH)2D3 induces TRACP activity and CT receptors almost simultaneously in mouse marrow cultures, and the MNCs formed in vitro respond to CT as authentic osteoclasts do.