The molecular mechanisms of intestinal homeostasis.

摘要 关于控制信号事件的分子机制，我们的知识存在一个严重的缺口 在肠道动态平衡期间。这一差距是科学进步的障碍，因为在它出现之前 关于肠道发育障碍引起的疾病的解释，将继续是 超出了我们的理解。此外，知识上的这种差距阻碍了发展的进步 在受伤或受损后促进肠道恢复的治疗。我们的长期目标是确定 参与上皮细胞动态平衡的分子机制。这项建议的目标是确定以下角色 在正常的肠道发育中，肠道特异的NADPH氧化酶(NOX酶)产生生理性ROS。 根据我们的初步数据，我们的中心假设是Nox1在肠上皮细胞中产生ROS 在肠道干细胞(ISC)微环境中刺激宿主基因调控事件的功能。在……里面 此外，我们还发现，后生动物肠道中特定共生细菌菌株的定植 诱导肠细胞内ROS的产生。因此，我们还假设特定成员的联系 与肠道细胞结合的微生物群(和候选益生菌)诱导NADPH氧化酶生成 ROS随后充当细菌信号的传输者，进入宿主基因调控事件，影响 后生动物肠道中的动态平衡。这一假说的理论基础是基于已有的报告，即ROS， 尤其是过氧化氢作为信号分子，通过感受器的氧化来调节蛋白质的活性 调节蛋白中的半胱氨酸残基。在我们的初步数据中，我们表明肠道特异的Nox1- Null小鼠和Nox1水平降低的果蝇改变了肠道生理。重要的是，我们还 表明乳酸菌，通常用作候选益生菌制剂，是有效的诱导剂 NOX1细胞在肠上皮细胞中产生ROS，是一种有效的细胞增殖诱导剂 Nox1依赖机制。基于我们研究小组产生的这些令人信服的初步数据， 中心假设将在三个具体目标中得到验证：1)确定NADPH氧化酶在 肠上皮发育与动态平衡；2)NADPH氧化酶在肠道中的功能鉴定 损伤后的上皮再生，以及3)确定细菌诱导和NADPH氧化酶的影响。 依赖于损伤后肠道愈合的ROS生成。我们的方法将使用肠道 上皮细胞特异性NOX1(B6.Nox1？IEC)缺陷小鼠，以及一种高度创新的遗传易驯化 果蝇模型，其生物学可操控程度远远超过哺乳动物模型。另外， 果蝇和果蝇肠道发育的分子机制具有显著的保守性 哺乳动物。这些调查的结果将对公共卫生产生积极影响，因为 对特发性肠道和全身免疫和发育障碍的影响，并提供了 为这些情况制定预防性干预措施的跳板。

Summary There is a critical gap in our knowledge regarding the molecular mechanisms that control signaling events during intestinal homeostasis. This gap represents a barrier to scientific progress because, until it is addressed, an explanation for diseases resulting from developmental disorders in the gut will continue to be beyond our understanding. Furthermore, this gap in the knowledge hinders progress in the development of therapies to promote recovery of the intestine following injury or damage. Our long-term goal is to identify molecular mechanisms involved in epithelial homeostasis. The objective of this proposal is to identify roles for physiological ROS generation from gut- specific NADPH oxidases (Nox enzymes) in normal gut development. Based on our preliminary data, our central hypothesis is that ROS generated by Nox1 in the intestinal epithelia functions to stimulate host gene regulatory events within the intestinal stem cell (ISC) microenvironment. In addition, we have discovered that colonization of the metazoan gut with specific strains of symbiotic bacteria induces the generation of ROS within enterocytes. Thus, we also hypothesize that contact of specific members of the microbiota (and candidate probiotic agents) with intestinal cells induces NADPH oxidases to generate ROS which then act as transducers of bacterial signals into host gene regulatory events that influence homeostasis in the metazoan gut. The rationale for this hypothesis is based on established reports that ROS, especially H2O2 function as signaling molecules to modulate protein activity through the oxidation of sensor cysteine residues within regulatory proteins. In our preliminary data, we show that both intestinal-specific Nox1- null mice, and Drosophila with diminished Nox1 levels have altered intestinal physiology. Importantly, we also show that lactobacilli, which are commonly employed as candidate probiotic agents, are potent inducers of Nox1 cellular ROS generation in intestinal epithelial cells, and are potent inducers of cell proliferation by a Nox1-dependent mechanism. Based on these compelling preliminary data generated by our research group, the central hypothesis will be tested in three specific aims: 1) Identify the function of NADPH oxidases in intestinal epithelium development and homeostasis, 2) Identify the function of NADPH oxidases in intestinal epithelium regeneration following injury, and 3) Identify the influence of bacterial-induced and NADPH oxidase- dependent ROS generation on intestinal healing following injury. Our approach will employ an intestinal epithelial cell-specific deficient nox1 (B6.Nox1¿IEC) mouse, and a highly innovative genetically tractable Drosophila model whose biology can be manipulated to a far greater extent than mammalian models. Also, there is striking conservation in the molecular mechanisms of intestinal development between Drosophila and mammals. The outcomes of these investigations will have a positive impact on public health because of direct implications to idiopathic intestinal and systemic immune and developmental disorders and provides a springboard to the development of preventative interventions for these conditions.