MOLECULAR REGULATION OF GASTRIC CHIEF (ZYMOGENIC) CELL DIFFERENTIATION

描述(申请人提供)：慢性胃损伤导致易患更严重疾病(如癌症)的关键细胞系的分化改变。慢性损伤的胃上皮以细胞谱系缺失(萎缩)和异常分化(化生)为特征。越来越多的证据表明，消化酶分泌的产酶细胞(ZC)在胃萎缩中丢失，而且ZC谱系的改变在化生中发挥了作用。然而，无论是在正常胃单位还是在慢性损伤中，调控ZC分化的细胞或分子过程知之甚少。我们的长期目标是在分子和细胞水平上了解ZC的分化，希望更好地了解慢性损伤中胃上皮细胞分化是如何出错的。我们最近的工作显示了第一个转录因子Mist1，它在ZC分化中发挥作用。对Mist1缺失小鼠的分析表明，在缺乏Mist1的情况下，ZCS不会正常成熟。相反，Mist1-/-ZCS显著阻碍了顶端细胞质的发育，其特征是顶端质膜投射和细胞骨架蛋白的积累，如肌球蛋白和一般细胞骨架适配器蛋白CD2AP(CD2相关蛋白)。我们还发现，在小鼠萎缩模型的基础上，细胞表现为化生，特征是部分的、异常的ZC分化。有趣的是，这些化生细胞缺乏Mist1，并增加了CD2AP。目前的资助目标集中在确定Mist1在正常胃单位、萎缩模型和体外人胃细胞系中作用的分子和细胞机制。这项资助的目的是：1)确定Mist1如何在分子水平上与CD2AP相互作用(具体地说，CD2AP是否受Mist1转录调控？)目的：1)通过体外培养的人胃癌细胞和CD2AP/Mist1双基因敲除小鼠的体内实验，确定Mist1的表达是否足以驱动细胞骨架和/或增殖性变化；以及3)通过激光捕获显微切割Mist1-/-和野生型ZC细胞，然后用微阵列技术鉴定带有Mist1依赖表达变化的基因，从而确定Mist1的新靶点。我们认为Mist1是胃损伤的先导，即Mist1的表达缺失是胃单位走向异常分化的第一次打击。因此，如果我们了解Mist1是如何工作的，特别是它与哪些基因相互作用，我们最终可能能够治疗或防止化生或萎缩。人们对易患胃癌的分子和基因知之甚少。这个项目旨在加深我们对维持胃壁消化酶分泌细胞正常更新的基因的理解，因为似乎正是这些细胞在肿瘤发展的过程中首先变得异常。

DESCRIPTION (provided by applicant): Chronic gastric injury leads to alterations in the differentiation of key cell lineages that predispose to more severe disease (e.g., cancer). Chronically injured gastric epithelium is characterized by cell lineage drop-out (atrophy) and aberrant differentiation (metaplasia). There is growing evidence that the digestive-enzyme secreting zymogenic cell (ZC) is lost in gastric atrophy, and furthermore, that alterations in the ZC lineage play a role in metaplasia. However, little is known about the cellular or molecular processes regulating ZC differentiation either in normal gastric units or in chronic injury. Our long-term objective is to understand ZC differentiation at the molecular and cellular level in hopes of better understanding how gastric epithelial differentiation goes awry in chronic injury. Our recent work has shown the first transcription factor, Mist1, which plays a role in ZC differentiation. Analysis of Mist1 null mice indicates that, in the absence of Mist1, ZCs do not mature normally. Rather, Mist1-/- ZCs have remarkably stunted apical cytoplasms characterized by apical plasma membrane projections and accumulation of cytoskeletal proteins like myosin and the general cytoskeletal adaptor protein Cd2ap (CD2-associated protein). We also show that the cells in the bases of a mouse model of atrophy show metaplasia, characterized by partial, aberrant ZC differentiation. Interestingly, these metaplastic cells lack Mist1 and have increased Cd2ap. The goals of the current grant are focused on determining the molecular and cellular mechanisms of Mist1 action in normal gastric units, in a model of atrophy, and in vitro in human gastric cell lines. The aims of the grant are to: 1) to determine how Mist1 interacts with Cd2ap at the molecular level (specifically, is Cd2ap transcriptionally regulated by Mist1?) in vitro in human gastric cancer cells and in vivo in Cd2ap/Mist1 double knockout mice; 2) to determine whether Mist1 expression is sufficient to drive cytoskeletal and/or proliferative changes in cells; and 3) to identify new targets of Mist1 by laser-capture microdissecting Mist1-/- and wildtype ZC lineage cells followed by microarray identification of genes with Mist1-dependent expression changes. We propose that Mist1 is the bellwether for gastric injury, i.e., that loss of its expression is a first hit that indicates the gastric unit is on a path to aberrant differentiation. Thus, if we understand how Mist1 works, especially what genes it interacts with, we might be able eventually to treat or prevent metaplasia or atrophy. Little is known about the molecules and genes that predispose people to developing gastric cancer. This project aims to further our understanding of the genes that maintain normal renewal of the digestive enzyme secreting cells in the stomach lining, because it is these cells that seemingly are the first to become aberrant on the way to tumor development.