VESICLES AND ENDOCHONDRAL BONE FORMATION

该项目的主要目标是研究导致 矿化组织的形成。 这些研究将侧重于 碱性磷酸酶（AP）在基质囊泡，软骨细胞和 成骨细胞介导羟基磷灰石形成。 假设， 作为磷灰石的成核剂和无机磷酸盐的促进剂， 测试. 两种体外矿化系统的可用性， 利用分离的基质囊泡和另一种使用成骨细胞 表达不同水平AP的细胞系提供了两种互补的 研究矿物形成的实验方法。 通过比较 对比MV矿化和成骨细胞矿化，我们预期 分离AP依赖性因子，从而证明 AP对这些进程的贡献。 有两个很好的实验系统 研究AP依赖的矿化，并获得AP 基因克隆、AP晶体结构坐标和分子生物学工具 生物学和蛋白质工程，我们希望确定不同的领域， AP分子和选择性修饰它们。 这些变异的AP可以 在AP成骨细胞瘤细胞（ROS 25/1）中表达或掺入 脂质体，并测试其矿化潜力。 最后，我们可以用 这些相同的技术与传统的细胞生物学相结合， 关于AP功能的新假设。 这些假设是基于非常 最近对细胞表面的研究表明， 许多其他的外蛋白通过一种 磷脂酰肌醇（PIG）锚。 我们建议调查 生物学和生物化学后果，AP和细胞功能， 成熟AP通过PIG锚结构域锚定到膜上的事实。 通过 通过基因和化学修饰锚结构域，我们可以测试 AP释放后锚组件的命运，它们对 细胞功能，以及全身激素对AP释放的调节。

The major goal of this project is to study the initial events leading to the formation of mineralized tissues. These studies will focus on the role of alkaline phosphatase (AP) in matrix vesicles, in chondrocytes and in osteoblasts to mediate hydroxyapatite formation. Hypotheses which posit AP as a nucleator of apatite and a booster of inorganic phosphate will be tested. The availability of two in vitro mineralization systems, one utilizing isolated matrix vesicles and the other using osteoblast cell lines which express differing levels of AP provides two complementary experimental approaches for investigating mineral formation. By comparing and contrasting MV mineralization with osteoblast mineralization we expect to isolate the AP dependent factors, and thus demonstrate the unique contribution of AP to these processes. With two good experimental systems for studying AP dependent mineralization in hand, and having access to AP gene clones, AP crystal structure coordinates and the tools of molecular biology and protein engineering, we hope to identify distinct domains in the AP molecule, and selectivity modify them. These mutant AP's can then be expressed in AP osteoblastoma cells (ROS 25/1) or incorporated into liposomes, and their mineralization potential tested. Finally,l we can use these same techniques in combination with conventional cell biology to test new hypotheses concerning AP function. These hypotheses are based on very recent studies of the cell surface in which it has been shown that AP and a number of other ectoproteins are attached to the cell membrane by a phosphatidylinositol (PIG) anchor. We propose to investigate the biological and biochemical consequences, for AP and cell function, of the fact that mature AP is anchored to the membrane by a PIG anchor domain. By modifying the anchor domain both genetically and chemically, we can test the fate of anchor components following release of AP, their effects on cell function, and the regulation of AP release by systemic hormones.