EXPRESSION OF THE CALCITONIN RECEPTOR IN BONE-MARROW CELL-CULTURES AND IN BONE - A SPECIFIC MARKER OF THE DIFFERENTIATED OSTEOCLAST THAT ISREGULATED BY CALCITONIN

We studied the temporal sequence of osteoclast (OC) differentiation fr om precursor cells in murine marrow cultures. Two markers of the OC ph enotype, calcitonin (CT) receptor (CTR) and tartrate resistant acid ph osphatase (TRAP), were assessed. Marrow cells from C57BL/6 mice were c ultured for 3, 5, 7, and 9 days with or without 1,25-(OH)(2) vitamin D -3 (10(-8) M). In controls only small numbers of osteoclastic multinuc leated cells (MNCs) formed per well (<15 per well). In contrast, 1,25( OH)(2)D-3 strongly stimulated MNC formation (>80 per well on day 7). M essenger RNA (mRNA) for TRAP was detectable by reverse transcription-p olymerase chain reaction amplification in both control and 1,25-(OH)(2 )D-3 treated groups at all times. However, TRAP mRNA was detectable in MNCs by the less sensitive in situ hybridization only on days 5, 7, a nd 9 and only in 1,25-(OH)(2)D-3 treated cells. In control cultures, C TR mRNA was present on day 3 only in nonadherent cells and was not pre sent in adherent cells (where MNCs formed) at any time point. In 1,25( OH)(2)D-3 treated cultures CTR mRNA was detectable in nonadherent cell s on day 3 and in adherent cells on day 5 and thereafter. Peak levels of CTR mRNA were seen in adherent cells on day 7 (15-fold more than da y 5 and 4-fold more than day 9). CT (10(-7) M) treatment of 7 day cult ures, which had been stimulated to express the osteoclastic phenotype, caused a marked decrease in CTR mRNA expression at 24 h. There was no effect of CT treatment on CTR mRNA expression at 3 h or on TRAP mRNA expression at 3 or 24 h. In neonatal mouse calvaria cultures, CTR mRNA expression was constitutively present and was markedly decreased by 4 8 h of CT treatment. Similarly, bone resorption in these cultures was inhibited at 24 h by CT treatment, but at 48 and 72 h there was escape from the inhibitory effects of CT on resorption. In the marrow cultur es, MNCs were greater than 98% positive for [I-125]-salmon calcitonin (sCT) binding and this binding was completely competed away by excess cold sCT (10(-7) M). All primary isolated osteoclasts from 1- to 3-day -old mouse long bones exhibited [I-125]-sCT binding and TRAP activity and were strongly positive for CTR and TRAP mRNA. by in situ hybridiza tion. Both MNCs that formed in bone marrow cultures and isolated prima ry osteoclasts formed resorption pits on bone slices. These studies de monstrate that CTR and TRAP functional activity and mRNA are expressed in marrow cultures that were induced to differentiate into OC by 1,25 -(OH)(2)D-3. The expression of CT receptor mRNA was coincident with th e development of the osteoclast-like phenotype in the marrow cultures and was more specific than expression of TRAP mRNA. Treatment of eithe r marrow or bane organ cultures with CT down-regulated the expression of the CTR mRNA. Hence, calcitonin-induced down-regulation of CTR mRNA and the resultant decrease in functional CTR activity provide a mecha nism for the phenomenon of CT escape.