SHEAR STRESS AND CHONDROCYTE GENE EXPRESSION

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While the precise etiology of the degenerative joint disease, osteoarthritis, is unknown, clinical manifestations of pain and disability are most often associated with inappropriate mechanical loading. As evident at surgery, focal erosion of articular cartilage culminates in loss of joint function and remains the final common pathway in all cases of osteoarthritis irrespective of cause. The goal of this study is to determine the cellular mechanisms by which mechanical loading influences cartilage matrix synthesis and degradation. In the joint, cartilage cells, chondrocytes, are subject to a complex array of stresses and strains. Our work shows that normal chondrocytes in culture react metabolically to univariate mechanical stimulation applied either as intermittent hydrostatic pressure or as fluid- induced shear stress. The hypothesis to be tested here is that distinct intracellular signaling pathways underlie the articular cartilage response to the two forms of mechanical stimulation. Fundamental knowledge exists regarding the effects of the proinflammatory cytokine, interleukin-1, on inhibition of cartilage extracellular matrix macromolecule synthesis and induction of cartilage degrading enzyme synthesis by chondrocytes. However, the effects of mechanical loading on the expression of articular chondrocyte degradative enzymes in the presence of IL-1 remain unclear. The specific aims will quantify effects of shear stress (SS) and intermittent hydrostatic pressure (IHP) on human osteoarthritic articular chondrocytes in vitro to: (1) Test the hypothesis that IHP and SS differentially modulate extracellular matrix macromolecule expression in OA versus normal chondrocytes; (2) Test the hypothesis that IHP and SS modulate IL-1beta induced inhibition of proteoglycan and type II collagen synthesis in OA and normal chondrocytes; (3) Test the hypothesis that IHP and SS alter endogenous MMPs and aggrecanase expression in OA and normal chondrocytes; (4) Test the hypothesis that IHP and SS alter IL-1beta induced expression of MMPs and aggrecanse in OA and normal chondrocytes. The expected result is that SS and IHP will show dissimilar capacities to overcome the IL-1 induced shift of chondrocyte metabolism from cartilage maintenance to matrix destruction. The results of this study will be of importance to the fields of orthopaedics, rheumatology and rehabilitation medicine. The techniques will involve analysis of proteoglycan and collagen synthesis by incorporation of radiolabeled precursors. mRNA signal levels will be quantified by Northern blotting and RT-PCR analysis. Cytokines will be quantified by bioassays and commercially available ELISA. MMPs will be identified by zymography and Western blotting and quantified by