REGULATION OF CARTILAGE REPAIR-SPECIFIC GENE EXPRESSION

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DESCRIPTION (Adapted from the Applicant's Abstract) In osteoarthritis (OA) , there is evidence for a loss of synchrony between extracellular matrix (ECM) catabolism and anabolism. We have observed quantitative as well as qualitative differences in ECM gene expression when comparing normal cartilage maintenance with cartilage repair. In this proposal, specific aims address the central hypothesis that chondrocyte repair-specific gene expression is regulated by specific cell-matrix interactions involving integrins. In specific aim 1 we will determine the mechanism for the rapid upregulation of link protein and aggrecan mRNA observed during cartilage repair following acute matrix depletion by proteases. Nuclear run-on experiments will be performed to assess the instantaneous rate of link protein and aggrecan gene transcription in response to protease treatment of high density chondrocyte cultures. We will measure link protein and aggrecan mRNA half-life by blocking chondrocyte transcription using actinomycin D. We will determine whether link protein 3' UTR regulatory RNA sequences interact with a cytoplasmic protein (AUBF) that may confer mRNA stabilization. In specific aim 2 , we will define the mechanism for chondrocyte recognition of extracellular matrix alterations leading to altered gene expression. We will identify integrins present on bovine chondrocytes by Northern blot analysis and will determine the distribution of integrin subunit mRNA in bovine and human cartilage by in-situ PCR/in-situ hybridization. Changes in chondrocyte gene expression will be measured following treatment with inhibitors of integrin-matrix interactions and with dihydrocytochalasin B to disrupt microfilament networks. In specific aim 3, we will characterize the cellular pattern of expression of repair genes in bovine cartilage explants following experimental manipulation, and will correlate these findings with human OA cartilage. We will use in-situ PCR/in-situ hybridization determine the cellular pattern of repair-specific gene expression in depleted bovine cartilage explants and human OA cartilage. To confirm and extend these studies, we will also use immunohistochemical techniques to localize repair-specific epitopes in cartilage explants depleted by enzymatic treatment. This research will yield new information that may permit therapeutic manipulation of cell-matrix interactions to stimulate matrix synthesis and repair of OA cartilage, and provide a novel approach to intervention in OA pathophysiology.