MECHANOTRANSDUCTION PATHWAYS IN CHONDROCYTE BIOSYNTHESIS

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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.