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

描述（由申请人提供）：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，非甾体抗炎药（NSAID）可降低结肠癌的风险，但其作用机制尚不清楚。缺乏g蛋白α亚基Gia2的小鼠会产生过度的、th1扭曲的炎症反应，并自发发展为结肠炎，然后发展为非息肉样粘液性结肠癌。由于人类IBD的病因尚不清楚，因此IBD的Giot2-/-模型对于研究粘膜炎症反应和结肠癌的调控是有用的。先前的研究人员在体外研究中发现，在缺乏Gia2的情况下，细胞质磷脂酶A2 （cPLA2）释放花生四烯酸（AA）存在缺陷。AA是包括PGE2在内的多种类二十烷酸的前体，可增强口服耐受性并抑制Th1反应的诱导。我们已经证明，结肠肌成纤维细胞（CMF） AA释放减少导致PGE2合成减少，结肠组织中PGE2水平相应降低。外源性PGE2类似物治疗可减轻结肠炎，论证花生四烯酯衍生介质在调节结肠炎症中的重要性。尽管具有较低的粘膜PGE2水平，Gia2-/-小鼠仍会发生结肠癌。cpla2衍生的AA也刺激鞘磷脂酶活性，增强神经酰胺的产生，诱导细胞凋亡。因此，我们假设结肠上皮中花生四烯酸酯释放减少可能因此干扰细胞凋亡，从而促进Gia2-/-小鼠结肠癌的发展。A)了解缺乏Gioc2抑制cPLA2介导的花生四烯酸释放的机制，B)花生四烯酸的减少是否会影响鞘脂代谢，从而抑制上皮细胞凋亡，从而可能导致癌症的发生。在Aim 1中，我们使用原代小鼠CMF来评估连接Gia2信号与cPLA2激活、易位和AA释放的信号通路。在Aim 2中，我们研究了花生四烯酸酯释放减少如何影响不同结肠鞘磷脂酶活性，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信号如何减弱花生四烯酸释放提供机制见解，对粘膜免疫和结直肠肿瘤产生重要影响。重要的是，这些动物也提供了一个有用的体内模型，用于解决通过COX抑制非甾体抗炎药的化学预防是由于抑制前列腺素的产生还是由于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.