FUNCTIONAL STUDIES OF OSTEOBLASTS

描述（由申请人提供）：在本项目的任期内，我们已经证明质膜钙ATP酶（PMCA）和钠钙交换剂（NCX）部署在成骨细胞的相对侧。NCX位于面向基质的细胞表面上，有利地定位成将Ca++引导到矿化位点中。在过去的一年中，我们报道了NCX的特异性抑制阻断了细胞外基质的矿化（Stains和Gay，J. Bone Mineral Res.16：1434- 1443，2001）。最近发现NCX以三种不同的同种型（NCX 1、NCX 2和NCX 3）存在，并且我们已经通过RT-PCR显示NCX 1和NCX 3在成骨细胞中表达（Stains等人，J.细胞。生化、出版中）。在本提案的目的之一，我们计划确定哪三个NCX亚型是矿化的关键。反义寡核苷酸将用于敲低或消除特异性同种型表达。待检验的假设是，功能性NCX 3的丧失会导致矿化受损，而NCX 1或NCX 2的消融对矿化几乎没有影响。在目的二，我们专注于NCX亚型和PMCA的分布与成骨细胞极性的发展。待检验的假设是，当成骨细胞形成横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发生分离。了解NCX同种型的表达与侧向粘附装置（例如钙粘蛋白-11和间隙连接蛋白，连接蛋白-43）的表达相关的时间将提供对矿化的细胞基础的洞察。这也将提供一个框架，以评估如何调节矿化。NCX是成骨细胞中一种重要的离子转运蛋白。可以预测NCX中的紊乱（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.