5ASA REGULATION OF MNSOD IN INTESTINAL CELLS

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Inflammatory bowel disease (ulcerative colitis and Crohn's disease) are chronic inflammatory disease of the gut for which there is no known cause or cure. It has been estimated that 2 million people in the United States have IBD. The peak onset of IBD is between 15 and 25 years of age and the majority of patients with IBD are under age 40. Mesalamine (5-ASA) is the active ingredient in sulfasalazine and is one of the major therapies use to treat active disease and for the maintenance of remission. The mechanism of action of 5-ASA remains unclear, however, we have found that 5-ASA induces a cytoprotective enzyme, manganese superoxide dismutase (MnSOD), at concentrations obtained in the colon of patients taking sulfasalazine orally. MnSOD is the only known 5-ASA regulated gene. The induction of MnSOD by 5-ASA may highlight a new therapeutic mechanism. MnSOD may serve a protective role in the bowel and prevent or reduce cytokine and oxygen radical mediated damage. This may be particularly important in the bowel as it contains low levels of antioxidants. The goal of this research project is to define the mechanisms that control the 5-ASA regulation of MnSOD gene expression in intestinal epithelial cells. The specific aims are designed to answer precise questions on the molecular mechanisms involved in the regulation of MnSOD gene expression by 5-ASA. In this proposal, we are using antisense MnSOD to document the role of MnSOD in 5-ASA induced cytoprotection in cell culture. We will determine how 5-ASA induces MnSOD by defining the sequences involved in the protein-DNA interactions that are responsible for the enhanced transcription of MnSOD. In addition to defining the cis-acting regulatory elements in the promoter region, we will identify and define enhancer elements involved in the regulation of MnSOD. Our studies will involve Dnase I hypersensitivity analysis, promoter deletion analysis, and evaluation of enhancer elements using transient transfection. Although the induction of MnSOD is transcriptional as determined by nuclear run-on experiments, we will also evaluate the role of MnSOD mRNA stabilization as a contributor to the induction of MnSOD mRNA levels. It is our premise that by understanding the mechanisms by which 5-ASA induces MnSOD, we will be able to identify and clone the transcription factors involved as well as design other therapeutic agents and 5-ASA derivatives to act as more potent inducers of MnSOD and possibly other yet to be defined 5-ASA regulated genes. By determining how therapeutic agents such as 5-ASA exert a beneficial influence on the disease activity of IBD, we may gain a further understanding of the pathogenesis of IBD.