Cystein, Intestinal Thiols and Goblet Cell Development

半胱氨酸、肠硫醇和杯状细胞发育

• 批准号: 6678652
• 负责人: Rex Gaskins
• 金额: $ 29.4万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6678652
• 关键词: cell growth regulation; cysteine; diet route /schedule; gastrointestinal epithelium; gel mobility shift assay; gene expression; genetic transcription; glutathione; goblet cells; high performance liquid chromatography; inflammation; intestinal mucosa; intravenous administration; laser capture microdissection; microarray technology; miniature swine; nutrition related tag; oxidation reduction reaction; parenteral feedings; polymerase chain reaction; small intestines; terminal nick end labeling; thiols; thioredoxin; tissue /cell culture; western blottings

DESCRIPTION (provided by applicant): The proposal originates from our observations of a three to four fold increase in the number of acidomucin-positive goblet cells accompanied by glutathione (GSH) depletion in the small intestinal mucosa of neonatal pigs being nourished parenterally. We hypothesize that the lack of luminal cysteine is the primary insult perceived by the parenterally nourished epithelium, and that the functional endpoint of this nutritional deficiency is a more oxidized intracellular redox potential The identification of this amino acid as a key nutritional factor is based on (a) the high rate of first-pass consumption of dietary cysteine and the low rate of arterial cysteine utilization by the intestine; (b) evidence that cellular redox status is a key determinant of cellular differentiation, and (c) the fact that cysteine is structurally and functionally involved in both intracellular redox status and mucosal defense via its inherent contribution to the thiol buffers GSH and thioredoxin (Trx) on one hand, and the goblet cell-specific secretory mucins and intestinal trefoil factor (ITF) on the other. We hypothesize that parenteral nutrition results in a preferential activation of the mucous cell lineage as well as selective sparing of goblet cells leading to the repopulation (at a reduced rate) of the villus with goblet cells relative to absorptive enterocytes. The alteration in goblet cell development is initiated by hyperoxia that stems from compromised cysteine status. In other words, we hypothesize that goblet cells are the guardians of mucosal redox homeostasis. Our hypothesis will be tested in three specific aims: 1) Determine if a causal relationship exists between epithelial redox status and goblet cell expansion, independent of TPN-associated inflammation. 2) Quantify, in a goblet cell culture model system, the hierarchy of cysteine usage for the synthesis of GSH and Trx versus ITF and secretory mucins, as well as the role of the transsulfuration pathway in the formation of cysteine and sulfate from methionine, as affected by apical versus basal provision of cysteine, and in response to graded levels of hyperoxia. Simultaneously, determine if the source (inorganic vs. cysteine-derived) and flux of sulfate used for sulfomucin biosynthesis is affected by cysteine availability and hyperoxic stress. 3) Test the hypothesis that Cysl31 and/or Cys165, found within the activation domain of Cdx2, are redox sensitive and upon post-translational modification alter the specificity of Cdx2 transcriptional regulation.

描述（由申请人提供）：该提案源于我们对肠外喂养的新生猪小肠粘膜中酸粘蛋白阳性杯状细胞数量增加三到四倍并伴随谷胱甘肽（GSH）消耗的观察。我们假设管腔半胱氨酸的缺乏是肠外营养上皮所感受到的主要损害，并且这种营养缺乏的功能终点是细胞内氧化还原电位的氧化程度更高。将该氨基酸鉴定为关键营养因素是基于（a）膳食半胱氨酸的首过消耗率高和动脉半胱氨酸利用率低 肠道； (b) 细胞氧化还原状态是​​细胞分化的关键决定因素的证据，以及 (c) 半胱氨酸一方面通过其对硫醇缓冲液 GSH 和硫氧还蛋白 (Trx) 的固有贡献，另一方面对杯状细胞特异性分泌粘蛋白和肠三叶因子 (ITF) 的固有贡献，在结构和功能上参与细胞内氧化还原状态和粘膜防御。我们假设肠外营养导致粘液细胞谱系的优先激活以及杯状细胞的选择性保留，导致杯状细胞相对于吸收性肠上皮细胞的绒毛重新增殖（以降低的速率）。杯状细胞发育的改变是由半胱氨酸状态受损引起的高氧引发的。换句话说，我们假设杯状细胞是粘膜氧化还原稳态的守护者。我们的假设将在三个具体目标上进行检验：1）确定上皮氧化还原状态和杯状细胞扩张之间是否存在因果关系，独立于 TPN 相关炎症。 2) 在杯状细胞培养模型系统中，量化 GSH 和 Trx 与 ITF 和分泌性粘蛋白合成中半胱氨酸使用的层次结构，以及转硫途径在从蛋氨酸形成半胱氨酸和硫酸盐中的作用，受半胱氨酸顶端与基底提供的影响，以及对分级高氧水平的反应。同时，确定用于磺粘蛋白生物合成的硫酸盐的来源（无机与半胱氨酸衍生）和通量是否受到半胱氨酸可用性和高氧应激的影响。 3) 检验以下假设：Cdx2 激活域内发现的 Cysl31 和/或 Cys165 具有氧化还原敏感性，并且翻译后修饰会改变 Cdx2 转录调控的特异性。