Berdal A, Hotton D, Saffar J L, Thomasset M, Nanci A
INSERM U120, Hôpital Robert Debré, Paris, France.
J Bone Miner Res. 1996 Jun;11(6):768-79. doi: 10.1002/jbmr.5650110608.
Following their terminal differentiation, highly specialized cells, ameloblasts, odontoblasts, and osteoblasts sequentially elaborate mineralized tissues. While the developmental expression pattern of matrix proteins has been studied extensively, less attention has been paid to the molecules involved in calcium handling, such as calcium-binding proteins. This shortcoming, as well as previous conflicting data, led us to conduct studies on calbindin-D9k and calbindin-D28k in rat mandibular bone and incisor based on several methods established on rat ameloblasts in vivo. Radioimmunoassays showed that calbindin-D28k accounts for approximately 0.1% of cytosolic proteins in the ectomesenchymal fraction and 1% in the epithelial fraction of the rat incisor and is 100-fold more concentrated than calbindin-D9k in both tissue types. Western blot analysis confirmed that the anticalbindin-D28k reactive species corresponded to the well characterized renal calbindin-D28k in the ectomesenchyme. In this tissue, calbindin-D28k was ultrastructurally immunolocalized in the odontoblasts. Quantitative immunocytochemistry showed that labeling was distributed throughout their nucleus and cytoplasm. The similar cytoplasmic distribution of both calbindin-D proteins and mRNAs suggests that their expression is regulated at the subcellular level. In particular, immunoreactive calbindin-D28k appeared to be associated with rough endoplasmic reticulum. Calbindin-D9k antisense probe showed negligible labeling in odontoblasts, in parallel with the protein quantities measured (approximately 10 ng/mg of total protein). Finally, in situ hybridization showed transcripts for both calbindins-D in ameloblasts and also in osteoblasts. In summary, the present results support the concept that an elevated expression of these vitamin D-dependent calcium-binding proteins may characterize the phenotype of cells directly involved in the elaboration of mineralized tissues, enamel, dentine, and bone.
高度特化的细胞——成釉细胞、成牙本质细胞和成骨细胞在终末分化后依次形成矿化组织。虽然基质蛋白的发育表达模式已得到广泛研究，但对参与钙处理的分子，如钙结合蛋白的关注较少。这一缺陷以及先前相互矛盾的数据，促使我们基于在大鼠成釉细胞体内建立的几种方法，对大鼠下颌骨和切牙中的钙结合蛋白-D9k和钙结合蛋白-D28k进行研究。放射免疫分析表明，钙结合蛋白-D28k在大鼠切牙的外胚间充质部分的胞质蛋白中约占0.1%，在上皮部分占1%，在两种组织类型中其浓度均比钙结合蛋白-D9k高100倍。蛋白质印迹分析证实，抗钙结合蛋白-D28k反应性物质与外胚间充质中特征明确的肾钙结合蛋白-D28k相对应。在该组织中，钙结合蛋白-D28k通过超微结构免疫定位在成牙本质细胞中。定量免疫细胞化学显示，标记物分布于整个细胞核和细胞质。两种钙结合蛋白-D的蛋白质和mRNA在细胞质中的相似分布表明，它们的表达在亚细胞水平受到调控。特别是，免疫反应性钙结合蛋白-D28k似乎与粗面内质网相关。钙结合蛋白-D9k反义探针在成牙本质细胞中的标记可忽略不计，这与所测蛋白质数量（约10 ng/mg总蛋白）一致。最后，原位杂交显示成釉细胞和成骨细胞中均有两种钙结合蛋白-D的转录本。总之，目前的结果支持这样一种概念，即这些维生素D依赖性钙结合蛋白的高表达可能是直接参与矿化组织、牙釉质、牙本质和骨形成的细胞表型的特征。
