Overexpression of cyclooxygenase-2 (COX-2) in articular tissues is an earmark of arthritis associated with increased chondrocyte apoptosis. Although high fluid shear induces chondrocyte apoptosis, the intracellular signaling pathways regulating this process remain largely unknown. In this application, we will test the hypothesis supported by compelling data that COX-2 activity is responsible for the decreased antioxidant capacity of chondrocytes and their apoptosis in response to shear stimulation. An extensive characterization of the signaling network regulating chondrocyte apoptosis may offer novel avenues for its control. We believe that the most judicious approach is to characterize the signaling mechanisms regulating COX-2 expression, and identify downstream targets of COX-2 activity which in turn regulate the activity of phase 2 antioxidant enzymes, and key pro-/anti- apoptotic genes. As has been argued in the literature, the signaling mechanisms are species-, cell- and stimulus- specific. Thus, Aim 1a will identify the cis-elements and their cognate trans- acting factors of shear-induced COX-2 expression in human chondrocytes. Using bioinformatics tools and cDNA microarrays coupled with selective, individual gene knockdowns via dominant negative/siRNA techno- logy, we will delineate the upstream signaling molecules of COX-2 expression (Aim 1b). This methodology will also enable us to define the downstream targets of COX-2 activity as well as other intermediate gene targets responsible for the differential expression of phase 2 genes (Aim 2).
Aim 3 will provide a mechanistic interpretation for the shear-induced chondrocyte apoptosis by identifying the key pro- and anti- apoptotic genes of the Bcl-2 family as well as their upstream regulatory genes. Comparative studies of the effects of fluid shear and cyclic strain on chondrocyte function will also be performed. This application integrates engineering principles with quantitative biology to reconstruct gene networks in shear-activated chondrocytes. The knowledge gained from these studies will provide a basis for the design of novel cell- based approaches for cartilage repair, and defining ideal bioreactor operating conditions for culturing artificial cartilage. Moreover, this approach will be vital for developing novel therapeutic strategies targeting molecular pathways relevant to arthritic pathogenesis and progression. ? ? ?
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