Transcriptional Control of the Human Aggrecan Gene

Abstract

Osteoarthritis (OA) is a degenerative joint disease and is the leading cause of physical disability in industrialised nations. Cartilage has received the most attention in the study of OA due to articular chondrocytes acting as potential instigators of disease. These cells are responsible for the anabolic-catabolic balance required for matrix maintenance due to their ability to synthesise the structural components of the extra-cellular matrix along with matrix-degrading proteases. In order to better understand the mechanism by which this balance is shifted in OA, it would be useful to investigate the roles of the genes that are expressed in these cells. One of the most powerful tools to do this would be an inducible and chondrocyte-specific system that utilises a cartilage-specific promoter, such as the aggrecan promoter. Aggrecan, one of the major structural components of cartilage, is a large aggregating chondroitin sulphate proteoglycan. This PhD set out to further our understanding of the transcriptional regulatory mechanisms that govern the chondrocyte-specific expression of the gene. A series of constructs containing various combinations of non-coding aggrecan DNA (mostly upstream of the transcriptional start site and including the first untranslated exon) were used to generate transgenic mouse embryos and an adult line in which the cartilage-specific expression of the transgene indicated the presence of cis-regulatory elements. These studies have identified three putatively important regions. One serves as a basal/core promoter at and around the transcriptional start site (TSS) (containing what could possibly be a non-essential 92bp sequence depending on the developmental stage of the mouse). Another located 10kb upstream of the TSS can serve as an enhancer, and finally another located within a 7kb region further upstream. One or more of these elements seem to respond to injury as indicated by increased promoter activity in the OA-induced knees of the adult line containing all three regulatory regions. Using a combination of our data alongside published work (Han and Lefebvre, 2008), a cartilage-specific Cre recombinase line was generated and tested for specificity and continual gene expression under the induction of OA. The work presented in this thesis can now be used to contribute to knowledge on the pathogenesis of OA