Protanoids, Colitis, and Colon Cancer in Gia2-KO mice

描述(申请人提供)：结肠癌是美国男性和女性的第三大杀手，在炎症性肠病(IBD)患者中发病率更高。流行病学数据表明，非类固醇抗炎药物可降低患结肠癌的风险，但其机制(S)尚不清楚。缺乏G蛋白α亚单位Gia2的小鼠会出现过度的、Th1偏斜的炎症反应，并自发发展为结肠炎，然后是非息肉粘液性结肠癌。由于人类IBD的病因尚不清楚，IBD的Giot2-/-模型可用于研究粘膜炎症反应和结肠癌的调节。先前研究人员的体外研究发现，在没有Gia2的情况下，细胞质磷脂酶A2(CPLA2)释放花生四烯酸(AA)的过程中存在缺陷。AA是包括PGE2在内的多种二十烷类化合物的前体，它增强了口服耐受性，并抑制了Th1反应的诱导。我们发现，结肠肌成纤维细胞(CMF)释放的AA减少会导致PGE2合成减少，相应地，结肠组织中PGE2的水平也会降低。外源性PGE2类似物的治疗减轻了结肠炎，论证了花生四烯酸衍生介质在调节结肠炎中的重要性。尽管粘膜中PGE2水平较低，Gia2-/-小鼠仍会患上结肠癌。CPLA2衍生的AA还刺激鞘磷脂酶活性，促进神经酰胺的产生，从而诱导细胞凋亡。因此，我们假设结肠上皮细胞花生四烯酸释放减少可能会干扰细胞凋亡，从而导致Gia2-/-小鼠结肠癌的发生。本项目的长期目标和主要目标是：A)了解缺乏Gioc2抑制cPLA2介导的花生四烯酸释放的机制，以及B)花生四烯酸减少是否影响鞘磷脂代谢，从而抑制上皮细胞凋亡，从而可能导致癌症的发生。在目标1中，我们使用原代小鼠CMF来评估将Gia2信号与cPLA2激活、易位和AA释放联系起来的信号通路。在目标2中，我们研究了花生四烯酸释放减少如何影响不同的结肠鞘磷脂酶活性，量化上皮神经酰胺产生的变化，并确定这些变化如何影响结肠上皮细胞增殖和凋亡的平衡。这些目标的完成将为缺乏GIA2信号如何减少花生四烯酸释放提供机械性的见解，从而对粘膜免疫和结直肠肿瘤产生重要后果。重要的是，这些动物还提供了一个有用的体内模型，用于研究通过抑制COX来预防NSAID是由于抑制前列腺素合成还是提高粘膜游离花生四烯酸水平。

DESCRIPTION (provided by applicant): Colon cancer is the third leading killer of men and women in the U.S, and occurs much more frequently in patients with inflammatory bowel disease (IBD). Epidemiologic data suggests that non-steroidal anti- inflammatory (NSAID) drugs lower the risk of colon cancer, but the mechanism(s) responsible are not known. Mice lacking the G-protein alpha subunit Gia2 mount excessive, Th1-skewed inflammatory responses and spontaneously develop colitis, and then non-polypoid mucinous colon cancers. Since the causes of IBD in humans are unknown, the Giot2-/- model of IBD is useful for studying the regulation of mucosal inflammatory responses and colon cancer. In vitro studies by previous investigators identified a defect in release of arachidonic acid (AA) by cytoplasmic phospholipase A2 (cPLA2) in the absence of Gia2. AA is the precursor for multiple classes of eicosanoids including PGE2, which enhances oral tolerance and suppresses the induction of Th1 responses. We have shown that decreased AA release from colonic myofibroblasts (CMF) leads to less PGE2 synthesis, with corresponding decreases in colonic tissue levels of PGE2. Treatment with exogenous PGE2 analog attenuates colitis, arguing for the importance of arachidonate-derived mediators in regulating colonic inflammation. Gia2-/- mice develop colon cancer despite having lower mucosal levels of PGE2. cPLA2-derived AA also stimulates sphingomyelinase activity, enhancing ceramide production that induces apoptosis. We therefore hypothesize that decreased arachidonate release in colonic epithelium may therefore interfere with apoptosis, contributing to the development of colon cancer in Gia2-/- mice The long-term objective and major aims of this project are: A) to understand the mechanism by which a lack of Gioc2 inhibits cPLA2- mediated arachidonic acid release, and B) whether decreased arachidonate affects sphingolipid metabolism such that there is an inhibition of epithelial apoptosis that may predispose to the development of cancer. In Aim 1 we use primary murine CMF to evaluate the signaling pathways that link Gia2 signaling to cPLA2 activation, translocation, and AA release. In Aim 2 we examine how decreased arachidonate release affects different colonic sphingomyelinase activities, quantify changes in epithelial ceramide production, and determine how these changes affect the balance of colonic epithelial proliferation and apoptosis. Completion of these aims will provide mechanistic insight into how a lack of Gia2 signaling attenuates arachidonic acid release, with important consequences for mucosal immunity and colorectal neoplasia. Importantly, these animals also provide a useful in vivo model for addressing whether NSAID chemoprevention via COX inhibition is due to suppression of prostanoid production or elevation of mucosal free arachidonate levels.