Regulation of Intestinal Epithelial Cell Proliferation

描述（由申请人提供）：哺乳动物肠上皮是一个动态系统，在整个生物体的生命周期中，增殖、迁移、分化和凋亡都是精心安排的。多能干细胞和祖细胞（也称为瞬时扩增细胞）的增殖局限于肠隐窝上皮。最近的研究已经确定了几个重要的信号通路参与维持肠上皮细胞的稳态。其中，Wnt通路对多能干细胞的增殖和自我更新至关重要，如Wnt靶点LGR5、OLFM4和ASCL2对多能干细胞的标记。然而，肠道干细胞和祖细胞增殖的确切细胞内介质尚未明确确定。控制隐窝细胞增殖的分子网络的进一步表征将增加对调节肠上皮细胞增殖的精确机制的理解，并有助于阐明某些疾病过程（如炎症性肠病和肠道肿瘤）的分子发病机制。本研究的长期目标是了解调节肠上皮细胞增殖的分子机制。该续期申请所基于的基金（DK052230）支持的项目确定了锌指转录因子Kr¿pel样因子5 （KLF5）在调节肠上皮细胞增殖中起重要作用。KLF5在肠上皮的增殖隐窝上皮细胞区高度富集表达。在体外实验中，KLF5对肠上皮细胞具有促增殖作用。此外，我们发现KLF5是肠上皮细胞对外部应激源（如脂多糖（LPS）、啮齿柠檬酸杆菌病原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质。此外，我们证明了KLF5是ApcMin突变或ApcMin联合激活KRAS突变小鼠肠腺瘤形成的关键介质。基于这些观察结果，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剂。我们提出三个具体目标来验证这一假设：(1)在体内研究肠上皮条件缺失Klf5对肠上皮细胞增殖的影响；(2)在体内验证KLF5在介导活化的KRAS在肠上皮细胞中的致瘤作用中的重要作用；(3)研究Wnt通路调控KLF5蛋白稳定性的机制。这些实验将提供明确的证据，证明KLF5是控制肠上皮细胞增殖的重要体内介质。此外，他们还将确定KLF5介导突变KRAS的致癌活性并激活Wnt信号。结果可能表明KLF5是结肠癌等增殖增加疾病的潜在治疗靶点。

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.