Molecular and cellular mechanisms regulating cross-talk between cyclooxygenase and lipoxygenase biosynthetic axes

Rheumatoid arthritis (RA) is characterized by chronic inflammation, synovial hyperplasia and joint destruction. The pathogenic mechanisms responsible for RA remain poorly understood both systemically and in the microenvironment of diarthrodial joint. Here we hypothesized, based on previous observations, that there is a positive interaction between resident mast cells and synovial fibroblasts (SF) within the rheumatoid synovial compartment. Our principal objectives were to define the cellular and molecular interactions between infiltrating mast cells and resident human synovial fibroblasts (HSF), and we focused our efforts on two genes, cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO), both implicated in the inflammatory response associated with RA. Furthermore, our research led to the discovery of novel epigenetic mechanisms governing the regulation of 5-LO gene expression in RA-affected SF. Epigenetics refers to heritable changes in gene expression regulated by DNA methylation, histone modifications and RNA interference. Epigenetic regulation is critical for normal development and differentiation. But environmental factors can trigger epigenetic dysregulation, leading to the development of many diseases, including cancer and autoimmune diseases.;Genomic bisulfite sequencing was used to detect DNA methylation profiles of 5-LO gene (rate limiting in LTB4 synthesis) in HSF, mast cells and other cell types. The 5-LO gene promoter (DNA CpG islands) was heavily methylated in U937 cells (5-LO negative), but unmethylated in HL-60 cells (5-LO positive). Compared to the 5-LO-positive HMC-1 cells, the 5-LO-negative HMC-1 cells had much higher methylation levels of CpG islands in the promoter region. We found a strong correlation between 5-LO gene expression and DNA methylation in HMC-1 cells. Dexamethasone (DEX) treatment of HMC-1 cells increased the expression of 5-LO, a process associated with reduced methylation of histone H3 on lysines 9 and 27. Interestingly, osteoarthritic (OA)/RA HSF are 5-LO negative; though the promoter region is CpG hypomethylated, histone H3 is hypermethylated at Lys-9 and -27 residues.;The research in this thesis provided innovative insights into the understanding of the pathophysiological mechanisms involved in inflammatory diseases like RA. The elucidation of the cellular and molecular mechanisms involved in these studies may help to establish new therapeutical targets in the treatment of RA patients. Moreover, the bisulfite sequencing and chromatin immunoprecipitation (ChIP) techniques used in this thesis provide reliable procedures for DNA methylation and histone modification studies in various inflammatory diseases and cancers.;Using Western and Northern blot analyses in addition to reporter assays, we demonstrated that mast cell-derived leukotriene B4 (LTB4) contributes to the modulation of inflammatory response in part through stabilization of COX-2 mRNA and protein expression in SF, the key enzyme in prostaglandin biosynthesis and the target of all non-steroidal anti-inflammatory drugs (NSAIDs). Further transient and stable transfection experiments verified that LTB4 exerted this COX-2 stabilization effect at the post-transcriptional level through Ras/c-Raf/MEK1/2/ERK1/2/p42 AUF1 signaling pathway.