FUNCTIONAL STUDIES OF OSTEOBLASTS

成骨细胞的功能研究

• 批准号: 6707510
• 负责人: Carol V Gay
• 金额: $ 24.68万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6707510
• 关键词: SDS polyacrylamide gel electrophoresis; calcium channel; calcium flux; calcium ion; calcium transporting ATPase; cell adhesion molecules; cell membrane; cellular polarity; chickens; extracellular matrix; immunocytochemistry; ion transport; membrane transport proteins; normal ossification; osteoblasts; osteocytes; polymerase chain reaction; sodium channel; sodium potassium exchanging ATPase; tissue /cell culture; western blottings

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.

描述（由申请人提供）：在该项目期间，我们已经证明质膜钙 ATP 酶 (PMCA) 和钠钙交换器 (NCX) 部署在成骨细胞的相对两侧。 NCX 位于面向基质的细胞表面，其位置有利，可将 Ca++ 引导至矿化位点。去年，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 以三种不同的亚型（NCX1、NCX2 和 NCX3）的形式存在，并且我们通过 RT-PCR 表明 NCX1 和 NCX3 在成骨细胞中表达（Stains 等人，J. Cell. Biochem.，出版中） 。在本提案的目标之一中，我们计划确定三种 NCX 亚型中的哪一种对于矿化至关重要。反义寡核苷酸将用于敲低或消除特定亚型表达。要测试的假设是功能性 NCX3 的丧失会导致矿化受损，而 NCX1 或 NCX2 的消融对矿化影响很小。在目标二中，我们重点关注 NCX 亚型和 PMCA 的分布与成骨细胞极性发育的关系。要测试的假设是，当成骨细胞形成横向接触并成为成熟的极化细胞时，质膜域相对于 NCX 和 PMCA 的分离就会发生。了解 NCX 亚型的表达时间与横向粘附装置（例如 cadherin-1 1 和间隙连接蛋白 connexin-43）的表达相关，将有助于深入了解矿化的细胞基础。这也将为评估如何监管矿化提供一个框架。 NCX 正在成为成骨细胞中重要的离子转位蛋白。 NCX 的紊乱（例如突变）预计会导致骨骼破坏。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。