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

We studied the temporal sequence of osteoclast (OC) differentiation from precursor cells in murine marrow cultures. Two markers of the OC phenotype, calcitonin (CT) receptor (CTR) and tartrate resistant acid phosphatase (TRAP), were assessed. Marrow cells from C57BL/6 mice were cultured 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 multinucleated 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). Messenger RNA (mRNA) for TRAP was detectable by reverse transcription-polymerase 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, and 9 and only in 1,25-(OH)(2)D-3 treated cells. In control cultures, CTR mRNA was present on day 3 only in nonadherent cells and was not present 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 cells 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 day 5 and 4-fold more than day 9). CT (10(-7) M) treatment of 7 day cultures, 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 48 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 cultures, 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 hybridization. Both MNCs that formed in bone marrow cultures and isolated primary osteoclasts formed resorption pits on bone slices. These studies demonstrate 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 the development of the osteoclast-like phenotype in the marrow cultures and was more specific than expression of TRAP mRNA. Treatment of either 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 mechanism for the phenomenon of CT escape.