SPDEF in intestinal differentiation.

描述(由申请人提供)： 控制肠道干细胞杯状细胞分化和成熟的具体机制尚未明确。了解杯状细胞是如何发生的很重要，因为如果没有这方面的知识，旨在控制肠道上皮细胞组成的治疗性干预仍然很难实现。我们的长期目标是确定肠上皮内所有细胞类型的分化机制，作为一种手段，开发基于特定细胞类型生产控制的新治疗策略。R03应用的目的是确定控制杯状细胞分化的转录级联的关键成分，并为R01应用探索这一新的研究路线开发额外的初步数据。这一应用的中心假设是SAM指向结构域的ETS转录因子(SPDEF，也称为Pdef或PSE)指导由Atoh1和Gfi1指定的命运限制性分泌前体细胞分化。有两个特定的目的将检验中心假设：1)定义SPDEF指导杯状细胞分化的必要性和充分性。2)将SPDEF定位在控制肠道细胞分化的转录网络中。在目标1中，我们的工作假设是SPDEF是引导双潜能杯状/潘氏祖细胞分化为杯状细胞的必要条件和充分条件。我们将通过评估细胞成分、体内平衡和蛋白质/基因表达的变化来确定在一种新的结肠癌细胞分化模型和转基因小鼠中操纵SPDEF水平的效果。目标2的假设是，转录层次结构控制着逐渐受到限制的祖细胞的命运，以实现个别分泌细胞类型的分化。具体地说，我们假设对于杯状细胞，Hes1抑制Atoh1；在没有Hes1的情况下，Atoh1激活Gfi1的表达，Gfi1调节SPDEF的表达来驱动杯状细胞的分化(在图1中图解，特定的AIMS页)。在这里，我们将确定Hes1、Atoh1和Gfi1控制SPDEF表达的机制。在完成这些研究后，我们希望证明SPDEF作为杯状细胞分化的关键因素的基本作用，并定义SPDEF转录调控的机制框架。这项拟议的研究在定义控制杯状细胞生产的新基因方面具有创新性。此外，这些结果将有助于更广泛地理解不同的上皮细胞类型是如何从全身多能祖细胞分化而来的。有了这些研究提供的额外数据，申请者将处于有利地位，可以获得R01资金，以调查SPDEF在肠道正常和异常功能中的作用。 公共卫生相关性：该项目与美国国立卫生研究院减轻疾病和残疾负担的使命相关，作为研究人类疾病的原因、预防和治疗以及人类成长和发展过程的组成部分。拟议的研究对公众健康很重要，因为控制杯状细胞分化的机制将为控制结直肠癌和炎症性肠病等疾病的肠道病理变化提供靶点。一旦这些方法可用，就有可能根据需要生产或减少特定类型的细胞。

DESCRIPTION (provided by applicant): The specific mechanisms that control differentiation and maturation of goblet cells from intestinal stem cells have not yet been defined. Understanding how goblet cell specification occurs is important because without this knowledge, therapeutic interventions designed to control cellular composition of the intestinal epithelium will remain difficult to achieve. Our long term goal is to define the mechanism of differentiation of all cell types within the intestinal epithelium, as a means to develop novel therapeutic strategies based on controlled production of specific cell types. The objective of this R03 application is to identify the key components of the transcriptional cascade that controls differentiation of goblet cells, and to develop additional preliminary data for an R01 application to pursue this novel line of investigation. The central hypothesis of this application is that the SAM pointed domain-containing ETS transcription factor (Spdef, also called Pdef or PSE) directs differentiation of goblet cells from fate-restricted secretory progenitors specified by Atoh1 and Gfi1.Two specific aims will test the central hypothesis: 1) Define the requirement and sufficiency for Spdef to direct goblet cell differentiation. 2) Position Spdef within the transcriptional network that controls differentiation of intestinal cells. In Aim 1, our working hypothesis is that Spdef is both necessary and sufficient to direct differentiation of bipotential goblet/Paneth progenitor into goblet cells. We will define the effect of manipulating Spdef levels in a novel colon cancer cell model of differentiation and in transgenic mice by assessing changes in cellular composition, homeostasis, and protein/gene expression changes. The hypothesis for Aim 2 is that a transcriptional hierarchy controls the fate of progressively more restricted progenitors to achieve differentiation of individual secretory cell types. Specifically, we hypothesize that for goblet cells, Hes1 represses Atoh1; in the absence of Hes1, Atoh1 activates Gfi1 expression, which regulates expression of Spdef to drive goblet cell differentiation (schematized in Figure 1, Specific Aims page). Here we will identify the mechanisms by which Hes1, Atoh1, and Gfi1 control Spdef expression. Upon completing these studies, we expect to have demonstrated the essential roles for Spdef as a key factor in goblet cell differentiation, and defined the mechanistic framework for transcriptional control of Spdef. The proposed research is innovative in defining a novel gene that controls goblet cell production. Furthermore, these results will contribute to the broader understanding of how distinct epithelial cell types arise from multipotent progenitors throughout the body. With the additional data provided by these studies, the applicant will be well-positioned to obtain R01 funding to investigate the role of Spdef in normal and abnormal function of the intestine. PUBLIC HEALTH RELEVANCE: This project has relevance for the NIH's mission to reduce the burdens of illness and disability, as a component of research into the causes, prevention, and cure of human diseases, and the processes of human growth and development. The proposed studies are important to public health because the mechanisms identified that control goblet cell differentiation will provide targets to control pathologic changes to the intestine in diseases such as colorectal cancer and inflammatory bowel disease. Once these methods are available, it will be possible to produce or reduce specific cell types on demand.