The molecular mechanisms of intestinal homeostasis.

描述（由申请人提供）：关于控制肠道内稳态信号事件的分子机制，我们的知识有一个关键的空白。这一差距代表了科学进步的障碍，因为在解决这一问题之前，对肠道发育障碍导致的疾病的解释将继续超出我们的理解范围。此外，这种知识上的差距阻碍了促进损伤或损伤后肠道恢复的治疗方法的发展。我们的长期目标是确定参与上皮稳态的分子机制。本提案的目的是确定肠道特异性NADPH氧化酶（Nox酶）在正常肠道发育中生理ROS生成的作用。根据我们的初步数据，我们的中心假设是肠上皮中Nox1产生的ROS可以刺激肠干细胞（ISC）微环境中的宿主基因调控事件。此外，我们还发现，特定菌株的共生细菌在后生动物肠道内定植可诱导肠细胞内ROS的产生。因此，我们也假设微生物群的特定成员（和候选益生菌制剂）与肠细胞的接触诱导NADPH氧化酶产生ROS， ROS随后作为细菌信号的转导器进入影响后生动物肠道内稳态的宿主基因调节事件。这一假设的基本原理是基于已有的报道，即ROS，特别是H2O2作为信号分子，通过氧化调节蛋白中的传感器半胱氨酸残基来调节蛋白质活性。在我们的初步数据中，我们发现肠道特异性Nox1- null小鼠和Nox1水平降低的果蝇都改变了肠道生理。重要的是，我们还表明，乳酸菌通常被用作候选益生菌剂，是肠上皮细胞中Nox1细胞ROS生成的有效诱导剂，并且是通过Nox1依赖机制诱导细胞增殖的有效诱导剂。基于这些令人信服的初步数据，我们的研究小组将在三个具体目标上验证中心假设：1)确定NADPH氧化酶在肠上皮发育和稳态中的功能，2)确定NADPH氧化酶在肠上皮损伤后再生中的功能，以及3)确定细菌诱导和NADPH氧化酶依赖的ROS生成对肠损伤后愈合的影响。我们的方法将采用肠上皮细胞特异性缺陷nox1 （B6.Nox1¿IEC）小鼠，以及一种高度创新的遗传可处理的果蝇模型，其生物学可以被操纵到比哺乳动物模型大得多的程度。此外，在果蝇和哺乳动物肠道发育的分子机制上也有惊人的保守性。这些调查的结果将对公共卫生产生积极影响，因为它直接涉及特发性肠道和系统性免疫和发育障碍，并为开发针对这些疾病的预防性干预措施提供了跳板。

DESCRIPTION (provided by applicant): 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.