Understanding the mechanisms that govern Bst-1 induction upon caloric restriction

描述(申请人提供)：mTOR途径是一个主要的信号中枢，协调生长和新陈代谢，以响应有机体的营养状态。它的调节失调与一系列常见疾病有关，包括癌症、糖尿病和衰老。有趣的是，两种抑制mTOR信号的干预措施--雷帕霉素和卡路里限制(将卡路里摄入量减少到随意水平的一小部分)--都与抑制肿瘤生长和延长小鼠的寿命密切相关。然而，人们对这些生理和病理生理现象的分子机制知之甚少。生物体衰老被认为在一定程度上是由于干细胞隔间功能的逐渐衰退。我们选择将重点放在肠道上，特别是了解热量限制(CR)和mTOR抑制对肠道干细胞(ISCs)及其生态位的影响。我们发现，CR通过一种非细胞自主机制强烈增强ISCs的自我更新能力，这种机制需要抑制Paneth细胞中的mTORC1和上调Bst1。令人惊讶的是，从CR条件到营养丰富条件的Paneth细胞至少在3天内保持了CR表型，这表明CR状态本身并不是急性信号事件的结果。这个项目的目标是了解CR是如何通过抑制mTORC1上调Bst1的表达的，这是CR表型的一个必要和充分的事件。我们正在考虑的一个假设是，mTORC1抑制通过重塑表观基因组来稳定地调节Bst1的表达。我们正在使用计算和实验相结合的方法来确定可能在mTORC1和Bst1基因之间介导信号级联的候选基因。然后，我们将系统地测试每个候选基因的必要性和充分性，首先是在基于试管的有机化合物共培养中，然后是在小鼠身上，这些小鼠被设计成能够提供专门针对Paneth细胞群体的基因操作。更清楚地了解这一途径可能有助于揭示模仿有益CR状态的新方法。为了确定长期CR调节基因表达的机制，我提出了以下具体目标：I：阐明雷帕霉素对CR或mTORC1抑制反应时调节Bst1mRNA水平的分子机制。II：确定特异性目标I中确定的调节因子(S)在CR和雷帕霉素对ISC自我更新的体内效应中的作用(S)。 与公共卫生相关：该项目将阐明长期抑制mTORC1如何改变肠道Paneth壁龛细胞的基因表达谱。mTORC1是生长和活性的主要调节因子。潘氏壁龛细胞将营养环境中的信号传递给邻近的肠道干细胞，从而增强后者的自我更新能力。阐明这些机制可能有助于深入了解mTOR如何控制衰老过程，并可能进一步揭示抗击衰老的新靶点。

DESCRIPTION (provided by applicant): The mTOR pathway is a major signaling hub that coordinates growth and metabolism in response to the nutritional state of the organism. Its dysregulation has been implicated in a broad spectrum of common disease states, including cancer, diabetes, and aging. Interestingly, two interventions that suppress mTOR signaling - rapamycin and caloric restriction (a reduction in caloric intake to a fraction of ad libitum levels - have both been robustly linked to diminished tumor growth and augmented lifespan in mice. Little is known, however, about the molecular mechanisms responsible for these physiological and pathophysiological phenomena. Organismal aging is thought to be due, in part, to the progressive functional decline in stem cell compartments. We have chosen to focus on the intestine, and, in particular, on understanding the effects of caloric restriction (CR) and mTOR inhibition on intestinal stem cells (ISCs) and their niche. We find that CR strongly augments the self-renewal capacity of ISCs through a non-cell autonomous mechanism requiring the inhibition of mTORC1 and the upregulation of Bst1 in Paneth cells. Surprisingly, Paneth cells transitioned from CR conditions to nutrient-rich conditions maintained the CR phenotypes for at least 3 days, suggesting that the CR state itself is not simply the consequence of an acute signaling event. The goal of this project is to understand how CR, via mTORC1 inhibition, upregulates Bst1 expression, a necessary and sufficient event for the CR phenotype. One hypothesis we are entertaining is that mTORC1 inhibition stably modulates Bst1 expression by remodeling the epigenome. We are using computational and experimental methods in tandem to identify candidate genes that may mediate the signaling cascade between mTORC1 and the Bst1 gene. We will then systematically test the necessity and sufficiency of each candidate gene, first in test tube-based organoid co-culture and then in mice engineered to afford genetic manipulations specifically in the Paneth cell population. A clearer understanding of this pathway may shed light on new ways of mimicking the beneficial CR state. To determine the mechanisms by which long-term CR regulates gene expression, I propose the following specific aims: I: Elucidate the molecular mechanisms that modulate Bst1 mRNA levels in response to CR or mTORC1 inhibition by rapamycin. II: Determine the role(s) of the regulatory factor(s) identified in Specifc Aim I on the in vivo effects of CR and rapamycin on ISC self-renewal. PUBLIC HEALTH RELEVANCE: This project will shed light on how the chronic inhibition of mTORC1, a master regulator of growth and viability, alters the gene expression profile of intestinal Paneth niche cells. Paneth niche cells transduce signals from the nutrient environment to neighboring intestinal stem cells, thereby augmenting the latter's capacity for self- renewal. Elucidation of these mechanisms may lend insights on how mTOR controls the aging process and may further reveal novel targets to combat aging.