FUNCTIONAL STUDIES OF OSTEOBLASTS

描述(由申请人提供)：在这个项目的期限内，我们已经证明质膜钙ATPase(PMCA)和钠钙交换器(NCX)被部署在成骨细胞的对侧。NCX位于面向基质的细胞表面，具有引导Ca++进入矿化部位的优势。在过去的一年里，我们报道了对NCX的特异性抑制阻止了细胞外基质的矿化(Stains和Gay，J.骨矿物研究，16：1434-1443,2001)。NCX最近被发现以三种不同的亚型(NCX1，NCX2和NCX3)存在，我们已经通过RT-PCR证明了NCX1和NCX3在成骨细胞中表达(Stains等，J.Cell)。《生物化学》，出版中)。在本提案的目的之一，我们计划确定三种NCX亚型中的哪一种对矿化至关重要。反义寡核苷酸将被用来敲除或去除特定的异构体表达。需要检验的假设是，功能性NCX3的缺失会导致矿化受损，而NCX1或NCX2的消融对矿化影响不大。在第二个目标中，我们重点研究了NCX亚型和PMCA的分布与成骨细胞极性的关系。需要检验的假设是，当成骨细胞发展成侧向接触并成为成熟的极化细胞时，质膜结构域相对于NCX和PMCA的分离发生。了解NCX亚型与侧向黏附分子(如钙粘蛋白-11和缝隙连接蛋白-43)表达的时间关系将有助于深入了解矿化的细胞学基础。这也将提供一个框架，以评估矿化可能如何受到监管。NCX是成骨细胞中一种重要的离子转运蛋白。可以预测，NCX基因的排列紊乱(例如突变)会导致骨骼矿化不足。这些研究将揭示骨形成细胞进行细胞外基质矿化的新机制。

DESCRIPTION (provided by applicant): During the tenure of this project we have shown that plasma membrane calcium ATPase (PMCA) and sodium-calcium exchanger (NCX) are deployed on opposing sides of the osteoblast. NCX, being on the matrix-facing cell surface, is advantageously positioned to direct Ca++ into site of mineralization. This past year we reported that specific inhibition of NCX blocks mineralization of extracellular matrix (Stains and Gay, J. Bone Mineral Res. 16:1434-1443,2001). NCX has recently been found to exist as three distinct isoforms (NCX1, NCX2 and NCX3) and we have shown by RT-PCR that NCX1 and NCX3 are expressed in osteoblasts (Stains et al., J. Cell. Biochem., in press). In Aim One of the present proposal we plan to determine which of the three NCX isoforms is critical for mineralization. Antisense oligonucleotides will be used to knockdown or ablate specific isoform expression. The hypothesis to be tested is that loss of functional NCX3 results in impaired mineralization, whereas ablation ofNCX1 or NCX2 has little effect on mineralization. In Aim Two we focus on the distribution of the NCX isoforms and PMCA in relation to the development of osteoblast polarity. The hypothesis to be tested is that segregation of plasma membrane domains with respect to NCX and PMCA occurs when osteoblasts develop lateral contacts and become mature polarized cells. Understanding the timing of expression of NCX isoforms in relation to expression of lateral adhesion devices (e.g. cadherin-1 1 and gap-junction protein, connexin-43) will provide insight into the cellular basis of mineralization. This will also provide a framework to assess how mineralization may be regulated. NCX is emerging as an important ion-translocating protein in osteoblasts. Derangements (e.g. mutations) in NCX can be predicted to result in an undermineralized skeleton. These studies will disclose novel mechanisms by which bone forming cells carry out mineralization of the extracellular matrix.