The molecular mechanisms of intestinal homeostasis.

总结 在控制信号事件的分子机制方面，我们的知识存在着一个关键的空白 在肠道内环境稳定期间。这一差距代表了科学进步的障碍，因为， 解决，解释疾病造成的发展障碍，在肠道将继续 超出了我们的理解。此外，这种知识差距阻碍了发展中国家的进步， 促进损伤或损害后肠道恢复的疗法。我们的长期目标是确定 参与上皮内环境稳定的分子机制。本建议的目的是确定以下方面的作用： 在正常肠道发育中，来自肠道特异性NADPH氧化酶（Nox酶）的生理性ROS产生。 基于我们的初步数据，我们的中心假设是，肠上皮细胞中Nox 1产生的ROS 在肠干细胞（ISC）微环境中刺激宿主基因调节事件的功能。在 此外，我们还发现，后生动物肠道中的共生细菌的特定菌株的定植 诱导肠细胞内ROS的产生。因此，我们还假设，特定成员的接触 微生物群（和候选益生菌剂）与肠细胞的结合诱导NADPH氧化酶产生 ROS然后作为细菌信号的转换器进入宿主基因调控事件， 在后生动物的肠道内的自我平衡。这一假设的基本原理是基于已建立的报告，即ROS， 特别是H2O2作为信号分子，通过传感器的氧化来调节蛋白质的活性 调节蛋白内的半胱氨酸残基。在我们的初步数据中，我们表明，这两个泌尿系特异性Nox1- null小鼠和Nox 1水平降低的果蝇改变了肠道生理学。重要的是，我们还 表明通常用作候选益生菌剂乳杆菌是 在肠上皮细胞中，Nox 1细胞产生ROS，并且是通过细胞增殖的有效诱导剂。 Nox1依赖机制。根据我们研究小组得出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进行检验：1）确定NADPH氧化酶在 肠上皮细胞的发育和稳态，2）确定肠道NADPH氧化酶的功能， 损伤后上皮再生，和3）确定细菌诱导的和NADPH氧化酶的影响， 依赖于损伤后肠愈合的ROS产生。我们的方法将采用一种肠道 上皮细胞特异性缺陷型nox 1（B6.Nox 1 <$IEC）小鼠，以及一种高度创新的遗传学上易于处理的 果蝇模型，其生物学可以在更大程度上操纵比哺乳动物模型。还有， 果蝇和果蝇之间在肠道发育的分子机制上存在着惊人的保守性， 哺乳动物这些调查的结果将对公共卫生产生积极影响，因为直接 对特发性肠道和全身性免疫和发育障碍的影响，并提供了一个 这是为这些疾病制定预防干预措施的跳板。

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.