The role of kinase signaling in intestinal epithelial homeostasis

描述(申请人提供)：肠上皮的自我更新需要对组织动态平衡至关重要的多层精确控制。肠上皮层是动态平衡系统的一个主要例子，每5天就会完全更新一次。令人惊讶的是，在连续的机械、化学和病原体来源的损伤下，肠道干细胞在整个生物体的整个生命周期中以正确的比例产生多种细胞类型。缺乏这种精细的体内平衡控制是各种疾病的基础，包括克罗恩病、肠易激综合征、溃疡性结肠炎和胃肠道癌症。我们对这个复杂的上皮系统如何维持如此强大的组织以及在疾病中如何出错的理解是可悲的不完整的。最近的研究表明，肠上皮细胞在体外三维培养中具有惊人的增殖和自组织行为。尤其是，小鼠的小肠干细胞产生了一系列千篇一律的细胞，准确地极化成类似组织的结构。我的目标是通过单细胞和化学方法识别和了解肠道内环境平衡的调控机制。我已经开发了高通量形式的未转化的小鼠和人的肠道上皮的2-D和3-D培养。这使我能够利用我们实验室开发的独特的单细胞图谱和分析平台，以前所未有的速度和细胞分辨率研究肠道动态平衡的机制。众所周知，蛋白激酶在许多正常和病理的细胞生理学中扮演着重要的角色。关于单个激酶在分子和结构水平上的丰富信息是已知的，但它们在复杂组织(如肠上皮)如何更新和自组织中的作用尚不清楚。我的假设是，关键的激酶网络调节组织的动态平衡和治疗反应。这项研究计划试图破译激酶网络如何调节肠道内环境平衡(目标1)，中央激酶中心GSK-3如何控制细胞命运(目标2)，并确定调节肠道上皮中激酶活性的新方法(目标3)。为了实现这些目标，在指导K99阶段，我将与我的导师Steven Altschuler博士和合作者Melanie Cobb博士密切合作，他们分别是单细胞分析和激酶生物学方面的公认专家，以实施我的研究和职业规划。通过利用肠道干细胞培养和高含量成像方面的令人兴奋的进展，这个奖项将使我能够建立一个尖端研究计划，长期目标是解决上皮系统的精细控制机制。

DESCRIPTION (provided by applicant): Self-renewal of the intestinal epithelium requires layers of precise control that are critical for tissue homeostasis. The epithelial lining of the intestine, a prime example of a homeostatic system, completely renews itself every 5 days. Amazingly, in response to continuous mechanical, chemical and pathogen-derived wounding, intestinal stems cells produce multiple cell types at just the right ratios throughout the entire lfe of the organism. Lack of this exquisite homeostatic control is the basis for a variety of disease including Crohn's disease, Irritable Bowel Syndrome, Ulcerative Colitis, and gastrointestinal cancers. Our understanding of how this complex epithelial system maintains such robust organization and how is goes wrong in disease is woefully incomplete. Recent studies have shown that intestinal epithelial cells are capable of a surprising degree of proliferative and self organizing behavior in 3-demensional cultures in vitro. In particular, mouse small intestinal stem cells give rise to ensembles of cells in stereotyped lineages, accurately polarized into tissue-lik structures. My goal is to identify and understand control mechanism of intestinal homeostasis through single cell and chemical approaches. I have developed the 2-D and 3-D culture of non-transformed mouse and human intestinal epithelium in high-throughput format. This allows me to utilize the unique single-cell profiling and analysis platform developed by our lab to study mechanism of gut homeostasis with a level of speed and cellular resolution never before achieved. Protein kinases are well known to play prominent roles in much of normal and pathological cell physiology. A wealth of information is known about individual kinase at the molecular and structural level, yet their role in how complex tissues, such as intestinal epithelium, renew and self-organize is unclear. My hypothesis is that key kinase networks modulate tissue homeostasis and therapeutic response. This Research Plan seeks to decipher how the kinase networks regulates gut homeostasis (Aim 1), how a central kinase hub, GSK-3, controls cell fate (Aim 2), and identify novel approaches to modulate kinase activity in gut epithelium (Aim 3). To achieve these aims, during the mentored K99 phase, I will work closely with my mentor Dr. Steven Altschuler and collaborator Dr. Melanie Cobb, recognized experts in single cells analysis and kinase biology respectively, to implement my research and career plan. By taking advantage of exciting advances in intestinal stem cell culture and high-content imaging, this award will enable me to establish a cutting edge research program with the long-term goal of addressing the exquisite control mechanisms of epithelial systems.