• Histone acetylation and inflammatory mediators in inflammatory bowel disease

      Tsaprouni, Loukia G. (University of BedfordshireUniversity of Bedfordshire, 2003)
      During cell activation the tightly compacted DNA is made available to DNA-binding proteins allowing the induction of gene transcription. In the resting cell, DNA is packaged into chromatin whose fundamental subunit is the nucleosome, composed of an octamer of four core histones (H) 3, 4, 2A and 2B. During the induction of gene transcription, modification of histones, by acetylation, methylation etc., results in unwinding of the DNA, permitting access of large DNAbinding proteins, such as RNA polymerase II, and subsequent induction of gene transcription. This investigation initially examined the effects of pro-inflammatory stimuli LPS and TNF-a on the production of IL-8 in a macrophage cell line (U937 cells) and in two T-cell lines (Jurkat and HUT-78 cells) as a marker of NF-KB-directed inflammatory gene expression. The ability of dexamethasone (Dex) and triamcinolone acetonide (TA) (synthetic glucocorticoid agonists) to suppress expression of the inflammatory cytokine IL-8 and to regulate histone acetylation was also investigated in these cells. LPS and TNF-a caused an increase in IL-8 expression, which was further enhanced by the histone deacetylases inhibitor trichostatin A (TSA), suggesting a role for histone acetylation in IL-8 production in these cells. Dex and TA, repressed LPS- and TNF-a -induced IL-8 expression in all three cell lines. This effect of both Dex and TA was attenuated by TSA in all cell lines studied, where the effect of TSA was greater in TA stimulated cells. Stimulation of all cell lines with LPS and TNF-a induced acetylation of H4 lysine residues (K5, 8, 12 and 16), the highest elevation of which was for K8 and K12. Also demonstrate is a K5 and K16 specificity of acetylation by glucocorticoids, apparent in all cell lines studied. Dex and, to a greater extent, TA suppressed LPS- and TNFa-induced K8 and K12 acetylation. TSA attenuated the inhibitory effect of the glucocorticoids for all three cell lines. An inCrease in HDAC activity with GCs was observed and ChiP assay showed these events occur on the native IL-8 promoter via histone acetylation. Further studies investigated whether there were any links between histone acetylation and the regulation of apoptosis. It was showed that TSA induced apoptosis in cells previously stimulated with the inducer of oxidative stress hydrogen peroxide (H20 2). Studies into the activation of caspase 3 in LPS- and TNF-a stimulated cells revealed that the combinatory effect of Dex or TA with TSA Significantly enhanced expression of the marker in all three cell lines. In resting cells, Dex, and TA, in the presence of TSA downregulated caspase 3 expression. These findings support the notion that glucocorticoid actions on apoptosis is mediated, at least in part, through an action on histone acetylation. Finally, histone acetylation was investigated in vivo in two rat models of inflammation and in human subjects with inflammatory bowel disease (IBD). The results showed an increase in histone H4 acetylation lysine specificity of acetylation on K8 and K12 in inflamed tissue and Peyer's patches in animal models and in IBD patients. Whereas H3 acetylation was not elevated to the same extent in tissue and was restricted to the mantle zone of Peyer's patches. In general, the present studies on histone acetylation and inflammation (in animal models and IBD patients) underlined the possibility of a general mechanism linking activation of the transcription factor NFKB with histone acetylation. The ultimate objective of this work is to aid in the understanding of the mechanisms of how deregulation of chromosome structure leads to progression of the disease state. This knowledge may aid in the development of new therapeutic approaches or improved glucocorticoids.