FUNCTIONAL STUDIES OF OSTEOBLASTS

成骨细胞的功能研究

• 批准号: 6606130
• 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/6606130
• 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）：在该项目的任期期间，我们表明质膜钙ATPase（PMCA）和钠钙兑换剂（NCX）被部署在成骨细胞的相对侧。 NCX处于面向基质细胞表面的位置，可有利地将Ca ++引导到矿化部位。在过去的一年中，我们报道了NCX的特异性抑制是细胞外基质的矿化（STAINS和GAY，J。BONE MINARERRES。16：1434-1443,2001）。最近发现NCX作为三种不同的同工型（NCX1，NCX2和NCX3）存在，我们已通过RT-PCR表明NCX1和NCX3在成骨细胞中表达（Stains等，J。Cell。J.Cell。Biochem。在印刷中）。在目的中，我们计划确定三种NCX同工型中的哪一个对于矿化至关重要。反义寡核苷酸将用于敲低或消耗特定的同工型表达。要检验的假设是功能性NCX3的损失会导致矿化受损，而ncx1或ncx2的消融对矿化影响不大。在目标二中，我们专注于与成骨细胞极性发展有关的NCX同工型和PMCA的分布。要测试的假设是，当成骨细胞发展出侧向接触并成为成熟的极化细胞时，质膜域的分离就会发生。了解NCX同工型表达与横向粘附装置的表达有关（例如Cadherin-1 1和GAP - 结蛋白Connexin-43）将提供有关矿化细胞基础的见解。这还将提供一个评估如何调节矿化的框架。 NCX成为成骨细胞中重要的离子转移蛋白。可以预测NCX中的紊乱（例如突变）会导致骨骼破裂。这些研究将披露新的机制，通过这些机制，骨形成细胞进行细胞外基质的矿化。