Functional analysis of osteocyte-specific acidic phosphoprotein by gene transfection

牙本质基质蛋白1（Dentin matrix protein 1，DMP1）是从大鼠切牙cDNA文库中分离到的一种酸性磷酸化蛋白。DMP1具有丰富的酸性结构域和大量的酪蛋白激酶I和II的磷酸化共有位点，这些位点在生理pH下带负电荷。因此，DMP1可能与钙结合并可能调节基质矿化。我们证明，DMP1 mRNA主要表达于骨细胞而不是成骨细胞，并且DMP1蛋白存在于骨细胞的细胞周骨基质中，而其它骨基质蛋白包括骨素、骨桥蛋白和骨唾液蛋白在成骨细胞中表达。我们确定了牙本质和牙骨质中DMP1 mRNA的精确表达模式及其蛋白质的定位。为了阐明DMPI在体内的功能，我们利用I型胶原启动子建立了DMP1在成骨细胞和成牙本质细胞中过表达的转基因小鼠。在DMP1转基因小鼠中，干骺端骨小梁显著减少。然而，破骨细胞的数量并没有增加，虽然破骨细胞的功能水平没有研究。近干骺端皮质骨骨密度增加。北方印迹分析表明骨基质的产生没有变化。这些结果表明，DMP1促进骨矿化过程的速度，虽然DMP1表达的骨细胞特异性的意义尚不清楚。

Dentin matrix protein 1 (DMP1) is one of the acidic phosphoproteins originally identified from a rat incisor cDNA library. DMP1 has abundant acidic domains and a large number of phosphorylation consensus sites for casein kinase I and II, which are negatively charged at physiological pH. Therefore, DMP1 likely binds with calcium and may regulate matrix mineralization.We demonstrated that DMP1 mRNA was predominantly expressed in osteocytes but not in osteoblasts and that DMP1 protein was in the pericellular bone matrix of osteocytes, while other bone matrix proteins including osteohalcin, osteopontin and bone sialoprotein were expressed in osteoblasts. We determined the precise expression pattern of DMP1 mRNA and localization of its protein in dentin and cementum. The localization of DMP1 mRNA and DMP1 protein in bone and tooth was closely related to their mineralization, suggesting that DMP1 plays an important role in mineraliztion.To elucidate the function of DMPI in vivo, we generated transenic mice that overexpressed DMP1 in osteoblasts and odontoblasts using type I collagen promoter. In DMP1 transgenic mice, trabecular bone the metaphysis was dramatically decrease. However, the number of osteoclasts was not increased, although the functional level of osteoclasts was not studied. The bone density of cortical bone near the metaphysis was increased. Northern blot analysis demonstrated no change of the production of the bone matrix. These findings suggested that DMP1 promotes the rate of bone mineralization process, although the significance of DMP1 expression specific for osteocytes was not yet known.