Impact of the DNA methyltransferase inhibitor, 5-azacytidine, on chondrocytic phenotype

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

There is increasing amount of data indicating that epigenetics play a role in aging and aging-related diseases. Of the age-related disease, osteoarthritis is of particular importance to current health issues. Osteoarthritis (OA) is a progressive, degenerative disease that affects articular cartilage, the synovial lining and adjacent bone of joints. OA currently affects over 30% of the US population. This figure is almost certain to increase as the American population ages and the childhood obesity ???epidemic??? impacts the consequent development of affected individuals. OA is also an extremely common presenting complaint in veterinary patients such as performance horses and several popular dog breeds. \nDespite the considerable societal burden associated with OA, current medical and surgical therapies for this disease are largely palliative, targeted at controlling the severity of clinical signs without affecting disease progression. Consequently, total joint replacement is the current ???gold standard??? for the treatment of OA; a highly invasive and costly surgical procedure with its own attendant morbidities and complications. Recently, the results of several published studies have linked changes in the chondrocytic phenotype that occur during OA to the epigenetic status of chondrocytes. Previous research done in the Stewart lab suggests that DNA methylation is involved in the regulation of the chondrocytic phenotype. Therefore, the research presented in this dissertation is focused on characterizing the effects of DNA methylation on the chondrocytic phenotype, identifying the mechanisms by which DNA demethylating agents alter the chondrocytic phenotype and defining the role of epigenetics in chondrocyte phenotypic stability, control of differentiation and the pathogenesis of OA. The long-term objective of this research program is to develop strategies using epigenetic modifying-agents for the prevention or mitigation of OA progression. \n Briefly, this thesis is composed by a literature review and three data-based chapters, presented as manuscripts. The literature review covers the topics of osteoarthritis and the different aspects that contribute to disease initiation and perpetuation, as well introducing the concepts of epigenetics and what it is currently known regarding epigenetic influences on chondrocytes and on osteoarthritis. \nThe first study (Chapter 3) addresses the phenotypic effects of 5-azacytidine (5-aza) in human articular chondrocytes and investigates whether upregulation of collagen type II, observed after 5-aza treatment, is associated with demethylation of the promoter and enhancer CpG island. In this chapter, we also mapped the methylation status of the collagen type II CpG island in cartilage, bone, and several chondrogenic and non-chondrogenic cell lines. \nThe second study (Chapter 4) was prompted by the study of Zimmermann and colleagues, published in 2008. This study correlated collagen type X expression during stem cell chondrogenesis with demethylation of two specific CpGs in the collagen type X promoter. We analyzed whether similar demethylation events occurred in articular chondrocytes during osteoarthritis. \nThe third study (Chapter 5) investigated the phenotypic effects of 5-aza on equine articular and growth plate chondrocytes. More specifically, we studied whether 5-aza administration directly stimulated expression of hypertrophic genes in articular and growth plate chondrocytes, and whether 5-aza administration affected the phenotypic responses of articular and growth plate to hypertrophic stimulation with BMP-2.