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
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
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DOI：
10.1210/en.136.10.4572
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发表时间：
1995-10-01
期刊：
影响因子：
4.8
通讯作者：
LORENZO, JA
中科院分区：
文献类型：
--
作者：
LEE, SK;GOLDRING, SR;LORENZO, JA
我们研究了小鼠骨髓培养中破骨细胞（OC）从前体细胞分化的时间序列。评估了OC表型的两个标志物，降钙素（CT）受体（CTR）和抗酒石酸酸性磷酸酶（TRAP）。C57BL/6小鼠骨髓细胞分别在添加或不添加1,25-(OH)(2)维生素D-3 （10(-8) M）的情况下培养3、5、7和9天。在对照组中，每孔仅形成少量破骨细胞多核细胞（MNCs）（第7天每孔80个）。在对照组和1,25-(OH)(2)D-3处理组中，通过逆转录聚合酶链反应扩增检测TRAP信使RNA （mRNA）。然而，TRAP mRNA仅在第5、7和9天通过低灵敏度的原位杂交在MNCs中检测到，并且仅在1,25-(OH)(2)D-3处理的细胞中检测到。在对照培养中，CTR mRNA仅在第3天出现在非贴壁细胞中，而在贴壁细胞（形成MNCs的地方）的任何时间点都不存在。在1,25(OH)(2)D-3处理的培养物中，第3天在非贴壁细胞中检测到CTR mRNA，第5天及以后在贴壁细胞中检测到CTR mRNA。第7天，贴壁细胞中CTR mRNA水平达到峰值（比第5天高15倍，比第9天高4倍）。CT （10(-7) M）处理7天的培养物，刺激表达破骨表型，在24小时时CTR mRNA表达明显下降。CT处理对3小时时CTR mRNA表达和3或24小时时TRAP mRNA表达没有影响。在新生小鼠颅骨培养物中，CTR mRNA表达组成性存在，并在CT处理48小时时显著降低。同样，这些培养物的骨吸收在24 h时被CT抑制，但在48和72 h时，CT对骨吸收的抑制作用消失了。在骨髓培养中，跨国公司的[I-125]-鲑鱼降钙素（sCT）结合阳性超过98%，这种结合被过量的冷sCT完全竞争掉（10(-7)M）。从1- 3日龄小鼠长骨中分离的所有原代破骨细胞均表现出[I-125]- sct结合和TRAP活性，并且CTR和TRAP mRNA呈强阳性。通过原位杂交。骨髓培养形成的MNCs和分离的原代破骨细胞在骨片上形成吸收坑。这些研究表明，CTR和TRAP的功能活性和mRNA在被1,25-(OH)(2)D-3诱导分化为OC的骨髓培养物中表达。CT受体mRNA的表达与骨髓培养中破骨细胞样表型的发生一致，比TRAP mRNA的表达更具特异性。用CT处理骨髓或骨髓器官培养均可下调CTR mRNA的表达。因此，降钙素诱导的CTR mRNA下调以及由此导致的CTR功能性活性降低为CT逃逸现象提供了一种机制。
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 (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.