Takeshita S, Arai S, Kudo A
Department of Life Science, Tokyo Institute of Technology, Yokohama, Japan.
Bone. 2001 Sep;29(3):236-41. doi: 10.1016/s8756-3282(01)00505-1.
Osteoblasts are derived from mesenchymal/stromal cells in bone marrow, and gain the ability to support osteoclastogenesis during differentiation though the expression of receptor activator of NF-kappaB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF). However, the properties (differentiation stage and expression of osteoblast marker genes) of stromal or osteoblastic cells that have the capacity to support osteoclast differentiation are unclear. Therefore, we sought to establish and characterize bone marrow-derived stromal cell lines (TSB) from temperature-sensitive SV40 T-antigen transgenic mice to define them at the clonal level. Of the 24 randomly selected cell lines, only 2 cell lines, TSB13 and TSB20, could support osteoclast differentiation in the presence of 1alpha,25(OH)(2)D(3). In both cell lines, RANKL mRNA was induced and osteoprotegerin (OPG) mRNA was decreased in response to treatment with 1alpha,25(OH)(2)D(3) for 2 days. Other RNA expression analyses of osteoblast-specific marker genes demonstrated the following characteristics of TSB13 and TSB20: (1) alkaline phosphatase (ALP) and type I collagen genes are expressed; (2) osteocalcin and osteopontin genes are expressed at low levels, and their expression levels are upregulated after induction of differentiation by a temperature shift from 33 degrees C to 37 degrees C, or 1alpha,25(OH)(2)D(3) treatment. Consequently, the long-term culture of TSB13 and TSB20 cell lines strongly stimulated osteocalcin expression and effectively induced calcified nodule formation in the presence of phosphate. The results suggest that the supportive cells for osteoclastogenesis are restricted to a specialized population of bone marrow stromal cells, and the high ratio of RANKL vs. OPG expression found in this population after 1alpha,25(OH)(2)D(3) treatment might be a general property of osteoclast-supporting cells.
成骨细胞源自骨髓中的间充质/基质细胞，在分化过程中通过表达核因子κB受体激活剂配体（RANKL）和巨噬细胞集落刺激因子（M-CSF）获得支持破骨细胞生成的能力。然而，具有支持破骨细胞分化能力的基质细胞或成骨细胞的特性（分化阶段和成骨细胞标志物基因的表达）尚不清楚。因此，我们试图从温度敏感的SV40 T抗原转基因小鼠建立并鉴定骨髓来源的基质细胞系（TSB），以便在克隆水平上对它们进行定义。在随机选择的24个细胞系中，只有TSB13和TSB20这两个细胞系在1α,25(OH)₂D₃存在的情况下能够支持破骨细胞分化。在这两个细胞系中，用1α,25(OH)₂D₃处理2天后，RANKL mRNA被诱导，而骨保护素（OPG）mRNA减少。对成骨细胞特异性标志物基因的其他RNA表达分析显示了TSB13和TSB20的以下特征：（1）碱性磷酸酶（ALP）和I型胶原基因表达；（2）骨钙素和骨桥蛋白基因表达水平较低，在从33℃温度转变为37℃诱导分化后或1α,25(OH)₂D₃处理后其表达水平上调。因此，TSB13和TSB20细胞系的长期培养在有磷酸盐存在的情况下强烈刺激骨钙素表达并有效诱导钙化结节形成。结果表明，破骨细胞生成的支持细胞仅限于骨髓基质细胞的一个特定群体，并且在1α,25(OH)₂D₃处理后该群体中发现的RANKL与OPG表达的高比例可能是破骨细胞支持细胞的一个普遍特性。
