Inflammatory stimuli trigger the expression of the inducible isoform of nitric oxide (NO) synthase (iNOS) in the intestinal epithelium, resulting in the synthesis of high levels of NO, a non-specific mucosal host defense against enteric pathogens. Excessive production of iNOS- derived NO, however, may cause autocrine dysfunction and enterocyte failure, manifested by a loss of intestinal barrier function and the progression to multiple organ failure. We have obtained preliminary data indicating that cytokine-induced expression of the human iNOS gene in cultured enterocytes is regulated in part at the level of transcription, by the interaction of a 5' enhancer and promoter region, and in part at the level of post-transcriptional mRNA processing. Induction of iNOS expression in enterocytes is also stimulated by invasive enteric gram negative microorganisms, suggesting that gut mucosal iNOS expression may represent an innate non-specific component of host defense.
In Specific Aim number 1 we will define the proximal promoter elements and trans-activating factors that regulate transcription of the human iNOS gene in cultured intestinal epithelial cells.
In Specific Aim 2, we will identify the cis- regulatory elements and trans-activating factors responsible for activity of the human iNOS cytokine responsive enhancer.
In Specific Aim 3, we will investigate the role of mRNA turnover and 3' processing in regulating human iNOS gene expression in intestinal epithelial cells.
In Specific Aim 4, we will establish the molecular basis for microbial induction of iNOS expression. All studies will be carried out using a panel of three human enterocytic cell lines to avoid basing conclusions on the potentially idiosyncratic behaviour of a single line. Because culture of primary human intestinal cells is not currently feasible, experiments will be replicated using normal human bronchial epithelial cells to identify regulatory mechanisms that may be restricted to transformed lines. The identification of the cis--regulatory elements and trans-- acting factors that control iNOS expression in intestinal cells will clarify the fundamental basis of human iNOS gene regulation and provide a means of developing novel strategies to precisely manipulate the level of gene expression, both for the benefit of host defense and for maintaining viability of the gut mucosa.
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