Gerstenfeld L C, Gotoh Y, McKee M D, Nanci A, Landis W J, Glimcher M J
Department of Orthopedic Surgery, Harvard Medical School, Children's Hospital, Boston, Massachusetts 02115.
Anat Rec. 1990 Sep;228(1):93-103. doi: 10.1002/ar.1092280113.
Embryonic chicken osteoblasts cultured over a 30 day period were used as a model system for studying the expression of bone phosphoproteins during cellular differentiation and the possible role of these proteins in extracellular matrix mineralization. Accumulation of total phosphoprotein in the cultures, as determined by O-phosphoserine (Ser-P) and O-phosphothreonine (Thr-P) amino acid analysis, revealed a greater than 10-fold increase over the 30 day period. Total phosphoprotein synthesis, as assessed by (32P)-, (3H)-Ser-P, and (14C)-Thr-P protein labeling, showed the highest levels concurrent with initial mineral deposition within the matrix. The major phosphoprotein present in chicken bones and synthesized by the cultured osteoblasts had a molecular weight of approximately 66 kDa. This 66 kDa bone phosphoprotein (66 kDa BPP) was purified to homogeneity and was used for antibody production. Application of this antibody in Western blot analysis revealed that 66 kDa BPP was present only in protein extracts of mineralizing cultured osteoblasts and was absent in cultures of non-mineralizing chondrocytes, myoblasts, and tendon fibroblasts. The 66 kDa BPP in vitro accumulated continuously in the extracellular matrix in a manner that paralleled both phosphoprotein synthesis and total phospho-amino acid production. A comparison of the results obtained in vitro to those from developing embryonic tibiae in vivo demonstrated a similar qualitative and temporal expression of phosphoprotein and a continual accumulation of 66 kDa BPP in the matrix with advancing mineralization and developmental age. Ultrastructural immunocytochemistry using the 66 kDa BPP antibody and the protein A-gold technique revealed specific immunolabeling over electron-dense regions of mineralization in the cultures that appeared identical to the distribution of labeling observed in vivo (McKee et al.: Connect. Tissue Res., 21:21-29, 1989; Anat. Rec., 228:77-92, 1990). These results demonstrate that this major 66 kDa BPP was expressed concurrently with other differentiated osteoblast functions and suggests that it may play a role in the initiation or regulation of mineralization.
将培养30天的鸡胚成骨细胞用作模型系统，以研究细胞分化过程中骨磷蛋白的表达以及这些蛋白在细胞外基质矿化中的可能作用。通过O-磷酸丝氨酸（Ser-P）和O-磷酸苏氨酸（Thr-P）氨基酸分析测定，培养物中总磷蛋白的积累在30天内增加了10倍以上。通过（32P）-、（3H）-Ser-P和（14C）-Thr-P蛋白标记评估的总磷蛋白合成显示，最高水平与基质内最初的矿物质沉积同时出现。鸡骨中存在并由培养的成骨细胞合成的主要磷蛋白分子量约为66 kDa。这种66 kDa的骨磷蛋白（66 kDa BPP）被纯化至同质，并用于制备抗体。将该抗体应用于蛋白质印迹分析表明，66 kDa BPP仅存在于矿化培养的成骨细胞的蛋白质提取物中，而在非矿化软骨细胞、成肌细胞和肌腱成纤维细胞的培养物中不存在。66 kDa BPP在体外以与磷蛋白合成和总磷酸氨基酸产生平行的方式在细胞外基质中持续积累。将体外获得的结果与体内发育中的胚胎胫骨的结果进行比较，结果表明磷蛋白在定性和时间表达上相似，并且随着矿化和发育年龄的增加，66 kDa BPP在基质中持续积累。使用66 kDa BPP抗体和蛋白A-金技术的超微结构免疫细胞化学显示，培养物中矿化的电子致密区域有特异性免疫标记，这与体内观察到的标记分布相同（McKee等人：Connect. Tissue Res., 21:21 - 29, 1989；Anat. Rec., 228:77 - 92,
