REGULATION OF NITRIC OXIDE IN HUMAN ENTEROCYTES

炎症刺激触发肠上皮中一氧化氮（NO）合成酶（iNOS）的诱导异构体的表达，导致高水平一氧化氮的合成，这是一种非特异性粘膜宿主对肠道病原体的防御。然而，过量产生iNOS衍生的NO可能导致自分泌功能障碍和肠细胞衰竭，表现为肠屏障功能丧失和多器官功能衰竭。我们已经获得的初步数据表明，细胞因子诱导的人类iNOS基因在培养肠细胞中的表达部分在转录水平上受到调节，通过5'增强子和启动子区域的相互作用，部分在转录后mRNA加工水平上受到调节。侵袭性肠道革兰氏阴性微生物也会刺激肠细胞中iNOS表达的诱导，这表明肠黏膜iNOS表达可能代表宿主防御的先天非特异性成分。在特异性目标1中，我们将定义在培养的肠上皮细胞中调节人类iNOS基因转录的近端启动子元件和反式激活因子。在特异性目标2中，我们将确定负责人类iNOS细胞因子反应增强子活性的顺式调控元件和反式激活因子。在Specific Aim 3中，我们将研究mRNA转换和3'加工在肠上皮细胞中调节人类iNOS基因表达中的作用。在Specific Aim 4中，我们将建立微生物诱导iNOS表达的分子基础。所有的研究都将使用三种人类肠细胞系来进行，以避免将结论建立在单一细胞系的潜在特殊行为上。由于培养原代人肠细胞目前尚不可行，实验将使用正常的人支气管上皮细胞进行复制，以确定可能仅限于转化系的调节机制。确定肠道细胞中控制iNOS表达的顺式调控元件和反式作用因子将阐明人类iNOS基因调控的基本基础，并为开发精确操纵基因表达水平的新策略提供手段，既有利于宿主防御，又有利于维持肠道黏膜的活力。

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.