FUNCTIONAL STUDIES OF OSTEOBLASTS

描述（由申请人提供）：在这个项目的任期内，我们已经证明质膜钙atp酶（PMCA）和钠钙交换器（NCX）部署在成骨细胞的两侧。NCX位于面向基质的细胞表面，有利于引导Ca++进入矿化部位。在过去的一年里，我们报道了NCX的特异性抑制可阻断细胞外基质的矿化（Stains and Gay， J. Bone Mineral Res. 16:1434-1443,2001）。最近发现NCX以三种不同的亚型（NCX1、NCX2和NCX3）存在，我们通过RT-PCR证明NCX1和NCX3在成骨细胞中表达(stainet al.， J. Cell。物化学。（待印）。在本提案的目的一中，我们计划确定三种NCX亚型中哪一种对矿化至关重要。反义寡核苷酸将被用来敲除或清除特定的异构体表达。需要验证的假设是，功能性NCX3的丢失会导致矿化受损，而ncx1或NCX2的消融对矿化几乎没有影响。在目标二中，我们关注NCX亚型和PMCA的分布与成骨细胞极性发展的关系。待验证的假设是，当成骨细胞形成横向接触并成为成熟的极化细胞时，NCX和PMCA的质膜结构域发生分离。了解NCX亚型的表达时间与侧粘附装置（如cadherin- 11和缝隙连接蛋白connexin-43）的表达有关，将有助于深入了解矿化的细胞基础。这也将提供一个框架来评估如何调节矿化。NCX是成骨细胞中一种重要的离子转运蛋白。NCX的紊乱（例如突变）可以预测导致骨骼矿化不足。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.