REGULATION OF NITRIC OXIDE IN HUMAN ENTEROCYTES

炎症刺激触发肠上皮中一氧化氮（NO）合酶（iNOS）的诱导型亚型的表达，导致高水平NO的合成，这是针对肠道病原体的非特异性粘膜宿主防御。然而，诱导型一氧化氮合酶衍生的NO的过量产生可引起自分泌功能障碍和肠上皮细胞衰竭，表现为肠屏障功能丧失和进展为多器官衰竭。 我们已经获得的初步数据表明，在培养的肠上皮细胞中，精氨酸诱导的人iNOS基因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。肠上皮细胞中诱导型一氧化氮合酶的表达也受到侵入性肠道革兰氏阴性微生物的刺激，这表明肠道粘膜诱导型一氧化氮合酶的表达可能代表宿主防御的先天非特异性组分。 在具体目标1号，我们将确定近端启动子元件和反式激活因子，调节人诱导型一氧化氮合酶基因在培养的肠上皮细胞的转录。 在具体目标2中，我们将鉴定负责人iNOS细胞因子应答增强子活性的顺式调节元件和反式激活因子。 在特定目标3中，我们将研究mRNA周转和3'加工在调节肠上皮细胞中人iNOS基因表达中的作用。 在具体目标4中，我们将建立微生物诱导iNOS表达的分子基础。 所有研究将使用一组三种人肠细胞系进行，以避免基于单一细胞系的潜在特异质行为得出结论。 由于原代人肠细胞的培养目前尚不可行，因此将使用正常人支气管上皮细胞复制实验，以鉴定可能仅限于转化细胞系的调控机制。 确定的顺式调控元件和反式作用因子，控制肠细胞中的iNOS表达，将阐明人类iNOS基因调控的基本基础，并提供了一种手段，开发新的策略，以精确地操纵基因表达水平，既有利于宿主防御和维持肠道粘膜的活力。

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.