Chrondrogenesis is a multi-step pathway during which multipotential mesenchymals stem cells differentiate into chondrocytes to form articular cartilage. This process is not only essential for cartilage development but also during joint repair. Research on molecular mechanisms of chrondrogenesis will advance our understanding of skeletal development and has the potential to identify new approaches to the treatment of joint diseases. The Sox9 high-mobility group (HMG) domain transcription factor is a key molecule in chondrocyte differentiation. Expression of Sox9 almost parallels that of Col2a1, which encodes cartilage-specific type II collagen during chondrogenesis. In the genital ridge, however, Col2a1 is not expressed, despite high level of endogenous Sox9 expression, suggesting the existence of molecular partner(s) of Sox9 that are required for cartilage-specific Cola1 gene expression. In preliminary studies, we identified PGC-1 as a Sox9 transcriptional co-activator. PGC-1 was previously known as PPAR gamma co-activator and plays a role in adipogenesis and glycogenesis; however, its role in chondrogensis has been unexplored. Whole mount in situ hybridization of day 10-12 mice embryos revealed PGC-1 gene expression only in cells at chondrogenic sites. PGC-1 interacts with Sox9 to enhance Col2a1 promoter activity and promotes chondrogenesis. Furthermore, Sox9 activity on the Col2a1 is tightly regulated by acetylation and deacetylation molecules. CBP (CREB binding protein), one of the chromatin acetylation enzymes, was also shown to interact with Sox9/PGC-1 complex and is involved in this mechanism. These preliminary data provide the basis for our hypothesis that PGC-1 functions as a tissue and developmental stage specific co-activator of Sox9 during chondrogenesis in a mechanism that involves specific chromatin regulation. Toward this end, we propose following Specific Aims: 1. Determine the role of PGC-1 as a co-activator of Sox9 during chondrogenesis and chondrocyte-specific gene expression. 2. Determine the role of Sox9/PGC-1 complex associating HAT activity and subsequent chromatin regulation for tissue specific gene expression and chondrocyte differentiation. 3. Examine mechanisms by which Sox9/PGC-1 complex dependent gene expression is regulated by HDACs and related co-repressor complex during chondrogenesis. 4. Analyze chromatin dependent Sox9/PGC-1 complex transcription mechanism by using in vitro reconstitution of chromatin and transcription assay of the Col2al promoter.
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