The molecular mechanisms of intestinal homeostasis.

描述(由申请人提供)：在肠道内稳态过程中控制信号事件的分子机制方面，我们的知识有一个严重的空白。这一差距代表着科学进步的障碍，因为在它得到解决之前，对肠道发育障碍引起的疾病的解释将继续超出我们的理解。此外，这种认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细胞动态平衡的分子机制。这项建议的目的是确定肠道特异的NADPH氧化酶(NOx酶)在正常肠道发育中产生生理性ROS的作用。基于我们的初步数据，我们的中心假设是，由Nox1在肠上皮细胞中产生的ROS功能是在肠干细胞(ISC)微环境中刺激宿主基因调控事件。此外，我们还发现，后生动物肠道被特定的共生菌菌株定植后，会在肠细胞内产生ROS。因此，我们还假设，微生物区系的特定成员(和候选益生菌制剂)与肠道细胞接触后，诱导NADPH氧化酶产生ROS，ROS随后作为细菌信号的转导器进入宿主基因调控事件，影响后生动物肠道的内稳态。这一假说的基本原理是基于已有的报道，即ROS，特别是H_2O_2作为信号分子，通过氧化调节蛋白质中的传感器半胱氨酸残基来调节蛋白质的活性。在我们的初步数据中，我们显示肠道特异性Nox1缺失的小鼠和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，是肠上皮细胞中NOX1细胞ROS产生的有效诱导剂，并通过NOX1依赖的机制有效地诱导细胞增殖。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上进行检验：1)确定NADPH氧化酶在肠上皮发育和稳态中的功能，2)确定NADPH氧化酶在损伤后肠上皮再生中的功能，以及3)确定细菌诱导的和NADPH氧化酶依赖的ROS产生对损伤后肠道愈合的影响。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，以及高度创新的、遗传上易驯化的果蝇模型，其生物学可以在比哺乳动物模型更大的程度上进行操纵。此外，果蝇和哺乳动物肠道发育的分子机制也有显著的保守性。这些调查的结果将对公共卫生产生积极影响，因为它直接影响到特发性肠道和全身免疫及发育障碍，并为制定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.