Regulation of Intestinal Epithelial Cell Proliferation

描述(申请人提供)：哺乳动物的肠道上皮是一个动态的系统，在这个系统中，增殖、迁移、分化和凋亡在生物体的整个生命周期中都得到了精心的安排。多能干细胞和祖细胞(也称为瞬时扩增)的增殖仅限于肠道的隐窝上皮。最近的研究已经确定了几个重要的信号通路，这些信号通路参与维持肠上皮细胞的动态平衡。其中，Wnt途径对多能干细胞的增殖和自我更新至关重要，Wnt靶标如LGR5、OLFM4和ASCL2对这些细胞的标记就证明了这一点。然而，确切的肠道干细胞和祖细胞增殖的细胞内介质还没有明确的确定。控制隐窝细胞增殖的分子网络的进一步表征将增加对调控肠上皮细胞增殖的确切机制的理解，并有助于阐明某些疾病过程的分子发病机制，如炎症性肠病和肠道肿瘤。本研究项目的长期目标是了解调控肠上皮细胞增殖的分子机制。这一更新申请所基于的资助项目(DK052230)证实，锌指转录因子Kr？ppel-like factor5(KLF5)在调节肠上皮细胞的增殖中发挥重要作用。KLF5的表达高度集中在肠上皮的增殖性隐窝上皮细胞室。在体外，KLF5对肠上皮细胞有促增殖作用。此外，我们还发现KLF5是肠上皮细胞对外界应激源(如脂多糖(LPS)、轮状柠檬酸杆菌致病菌感染和葡聚糖硫酸钠(DSS)诱导的结肠炎)的增殖或再生反应的介体。此外，我们还证明了KLF5是ApcMin突变或ApcMin和激活KRAS突变联合突变的小鼠肠腺瘤形成的关键介质。基于这些观察，我们提出了一个中心假设，即KLF5是肠隐窝干细胞和祖细胞增殖的重要细胞内调节因子。我们提出了三个具体的目的来验证这一假说：(1)研究KLF5有条件地从肠上皮细胞中删除对在体肠道上皮细胞增殖的影响；(2)在体内建立KLF5在介导激活的KRAS在体内的致癌作用中的重要作用；(3)探讨Wnt途径调节KLF5蛋白稳定性的机制。这些实验将提供确凿的证据，证明KLF5是体内控制肠上皮细胞增殖的重要介质。此外，他们还将确定KLF5介导突变的KRAS的致癌活性和激活的Wnt信号。这一结果可能表明，KLF5是一种潜在的治疗靶点，用于治疗结肠癌等增殖增加的疾病。 公共卫生相关性：胃肠道疾病是一个重大的卫生保健负担。它们通常是由肠上皮细胞动态平衡的紊乱或失衡引起的。从分子和细胞水平研究调控肠上皮细胞增殖的机制将有助于了解炎症性肠病和包括结直肠癌在内的胃肠道肿瘤等多种胃肠道疾病的病理生理机制。本文所述的项目将有助于获得有关肠道上皮细胞增殖如何被调控的新知识，并可能有助于建立潜在的治疗胃肠道疾病的新方法。

DESCRIPTION (provided by applicant): The mammalian intestinal epithelium is a dynamic system in which proliferation, migration, differentiation, and apoptosis are carefully orchestrated throughout the lifespan of an organism. Proliferation of multipotent stem cells and progenitor (also called transient amplifying) cells is confined to the crypt epithelium of the intestine. Recent studies have identified several important signaling pathways that are involved in maintaining intestinal epithelial cell homeostasis. Among these, the Wnt pathway is critical for the proliferation and self-renewal of multipotent stem cells, as demonstrated by the marking of these cells by Wnt targets such as LGR5, OLFM4 and ASCL2. However, the exact intracellular mediators of proliferation of intestinal stem cells and progenitor cells have not been definitively identified. Further characterization of the molecular network that controls crypt cell proliferation will increase the understanding of the precise mechanism that regulates intestinal epithelial cell proliferation and help elucidate the molecular pathogenesis underlying certain disease processes such as inflammatory bowel disease and neoplasm of the intestinal tract. The long term goal of this research project is to understand the molecular mechanisms regulating intestinal epithelial cell proliferation. The project supported by the grant (DK052230) on which this renewal application is based established that the zinc finger transcription factor, Kr¿ppel-like factor 5 (KLF5), plays an important role in regulating proliferation of intestinal epithelial cells. Expression of KLF5 is highly enriched in the proliferating crypt epithelial cell compartment of the intestinal epithelium. In vitro, KLF5 exhibits a pro-proliferative effect on intestinal epithelial cells. In addition, we showed that KLF5 is a mediator for the proliferative or regenerative response of intestinal epithelial cells to external stressors such as lipopolysaccharide (LPS), pathogenic bacterial infection by Citrobacter rodentium, and dextran sodium sulfate (DSS)-induced colitis. Furthermore, we demonstrated that KLF5 is a crucial mediator for formation of intestinal adenomas in mice with ApcMin mutation or combined ApcMin and activating KRAS mutations. Based on these observations, we propose the central hypothesis that KLF5 is an essential intracellular regulator of proliferation of intestinal crypt stem cells and progenitor cells. We propose three specific aims to test this hypothesis: (1) To investigate the effect of conditional deletion of Klf5 from the intestinal epithelium on intestinal epithelial cell proliferation in vivo; (2) To establish an essential role for KLF5 in mediating the oncogenic effect of activated KRAS in intestinal epithelial cells in vivo; and (3) To investigate the mechanism by which the Wnt pathway regulates KLF5 protein stability. These experiments will provide definitive evidence that KLF5 is an essential in vivo mediator controlling proliferation of intestinal epithelial cells. In addition, they will also establish that KLF5 mediates the oncogenic activity of mutated KRAS and activated Wnt signaling. The results may demonstrate that KLF5 is a potential therapeutic target for diseases with increased proliferation such as colon cancer. PUBLIC HEALTH RELEVANCE: Diseases of the gastrointestinal (GI) tract represent a significant health care burden. They are often caused by perturbation or imbalance in intestinal epithelial cell homeostasis. Investigating the mechanisms that regulate proliferation of intestinal epithelial cells at a molecular and cellular level will help understand the pathophysiology of many GI diseases such as inflammatory bowel disease and cancers of the GI tract, including colorectal cancer. The project stated herein will help gain new knowledge on how proliferation of intestinal epithelial cells is regulated and may help establish potentially novel therapeutic approaches to GI diseases.