MECHANOTRANSDUCTION PATHWAYS IN CHONDROCYTE BIOSYNTHESIS

软骨细胞负责维持软骨细胞外 矩阵，在正常的关节，在动态平衡之间 降解和形成。在关节炎疾病中， 矩阵就会崩溃机械对…有深远的影响 软骨细胞的生物合成行为，这个项目的中心目标是 以表征软骨细胞检测生理学的机制， 机械信号，特别是流体流动，并将它们转换为 适当的生物合成反应。我们的核心假设是流体 在牛关节软骨细胞中流动诱导的Ca 2 +/i尖峰，导致 从细胞外Ca 2+的流入和从IP/3-释放Ca 2+， 敏感的细胞内储存，介导软骨细胞的生物合成。我们将 使用电生理学， 荧光成像和分子生物学技术来假设 使用电生理学、荧光成像和 分子生物学技术，以完成四个具体目标。在 具体目标1和2，我们将表征膜Ca 2+通道， 软骨细胞及其在流体流动诱导的Ca 2 +/i尖峰中的作用。在 具体目标3我们将确定G蛋白激活的作用，IP/3 在流体流动诱导的细胞内钙库中刺激钙释放 Ca 2 +i升高。在最后的具体目标，我们将确定是否流体- 流动诱导的蛋白聚糖（PG）合成和聚集蛋白聚糖mRNA表达是 通过软骨细胞内Ca ~（2+）i峰化介导。为实现这些目标将 在存在和不存在以下物质的情况下定量流动诱导的PG合成： 破坏Ca 2+依赖性机械- 目的1至3中鉴定的软骨细胞中的转导途径。这 信息将给我们开发药理学工具的潜力， 调节软骨细胞Ca 2+途径，其生物合成行为， 最终破坏软骨基质的完整性

Chondrocytes are responsible for maintaining cartilage extracellular matrix which, in the normal joint, is in dynamic equilibrium between degradation and formation. In arthritic diseases, this balance is upset and the matrix breaks down. Mechanical have a profound influence on chondrocyte biosynthetic behavior, and the central goal of this project is to characterize the mechanism by which chondrocytes detect physiological mechanical signals, in particular fluid flow, and transduce them into an appropriate biosynthetic response. Our central hypothesis is that fluid flow-induced Ca2+/i spiking in bovine articular chondrocytes, resulting from both influx of extracellular Ca2+, and release of Ca2+ from IP/3- sensitive intracellular stores, mediates chondrocyte biosynthesis. We will test this hypothesis using a combination of electrophysiological, fluorescent imaging and molecular biological techniques to hypothesis using a combination of electrophysiological, fluorescent imaging and molecular biological techniques to complete four specific aims. In specific aims 1 and 2 we will characterize membrane Ca2+ channels in chondrocytes and their roles in fluid flow-induced Ca2+/i spiking. In specific aim 3 we will determine the role of G-protein activated, IP/3 stimulated release of Ca2+ from intracellular stores in fluid flow-induced Ca2+i spiking. In the final specific aim we will determine whether fluid- flow induced proteoglycan (PG) synthesis and aggrecan mRNA expression is mediated by Ca2+i spiking in chondrocytes. This will be achieved by quantifying flow-induced PG synthesis in the presence and absence of pharmacological agents which disrupt the Ca2+ dependent mechano- transduction pathways in chondrocytes identified in aims 1 to 3. This information will give us the potential to develop pharmacological tools to modulate chondrocyte Ca2+ pathways, their biosynthetic behavior and ultimately the integrity of the cartilage matrix.