Understanding the mechanisms that control chondrogenesis is of major clinical importance for improving tissue engineering strategies that can be applied to repair cartilage during osteoarthritis, for example. Studies proposed for the grant renewal period are focused on deciphering the regulation chondrogenesis at the level of gene transcription, precursor messenger RNA (pre-mRNA) alternative splicing and protein synthesis. The following three Specific Aims will directly address these aspects of cartilage development: 1) Investigate the role of transcription factors during chondrogenesis, specifically AP-2, dEF-1 and C/EBP; 2) Investigate the regulation of the type II procollagen pre-mRNA switch that occurs during chondrogenesis and 3) Determine the function of the developmentally-expressed type IIA procollagen protein isoform in vivo. Combined data from these Specific Aims will provide unique insight into the regulation of chondrogenesis as it is becoming apparent that processes of transcription, RNA splicing and translation are tightly coordinated. We also plan to study mechanisms important for cartilage maintenance and repair as described in the fourth Specific Aim: 4) Investigate the mechanism of IL-1b and TNF-a induction of BMP-2 in adult chondrocytes. Processes that occur during chondrogenesis are believed to be recapitulated during tissue repair under conditions of cartilage degradation. Therefore, the final Specific Aim in combination with the first three Specific Aims focused on chondrogenesis will provide invaluable information into the maintenance of cartilage health. The predominant experimental approach to these topics will be to use in vitro systems of chondrogenesis utilizing the ATDC5 cell line and adult stem cells isolated from adipose tissue to investigate state-specific differentiation events. Regulatory factors will be tested by over expression and inhibition studies and correlated with expression in vivo. ? ?
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