Alkaline Ceramidase and Sphingolipid Signaling

描述(由申请人提供)：鞘氨醇-1-磷酸(S1P)介导细胞的多效性反应，如细胞增殖、存活(抗凋亡)和运动。它还与心血管发育、血管生成、炎症和肿瘤发生有关。S1P是G蛋白偶联受体的特异性配体或细胞内信号分子。现在很清楚，S1P只由鞘氨醇产生，而鞘氨醇主要是通过神经酰胺酶的作用来自神经酰胺。然而，关于这一重要信号分子水平的调节仍有许多未知之处。PI最近克隆的三种不同的人类碱性神经酰胺酶之一haCER2的初步数据表明，haCER2通过控制神经酰胺的水解来调节S1P的水平，这是S1P形成的限速步骤，而且重要的是，haCER2能够调节S1P介导的肿瘤细胞存活、血管生成和肿瘤生长。PI的长期目标是确定haCER2在调节S1P和S1P介导的肿瘤生长和血管生成方面的作用，并将这一概念发展为抗癌治疗的新策略。PL的中心假设是，haCER2调节S1P和S1P介导的生物学过程的水平，特别是肿瘤血管生成和生长，这将通过以下特定目标进行测试。目的1为了解haCER2的催化机理，将haCER2在酵母系统中表达、纯化、重组，并进行生化鉴定。目的2验证haCER2在促血管生成细胞因子和生长因子作用下调节S1P生成的假说，将通过表达和活性研究这些激动剂对HeLa肿瘤细胞和人脐静脉内皮细胞的调节作用，鉴定haCER2启动子和顺式作用元件，以响应细胞因子和生长因子。目的3是验证haCER2在体内调节肿瘤生长和血管生成作用的假说。建立表达野生型、上调或下调的haCER2的肿瘤细胞系，以确定haCER2在肿瘤血管生成和生长中的作用。这些研究将坚定地确立haCER2在调节S1P和S1P介导的生物学中的作用，并将为癌症的新治疗方法铺平道路。

DESCRIPTION (provided by applicant): Sphingosine-1-phosphate (S1P) mediates pleiotropic cellular responses such as cell proliferation, survival (anti-apoptosis), and motility. It has also been implicated in cardiovascular development, angiogenesis, inflammation, and tumorigenicity. S1P acts as a specific ligand for G-protein coupled receptors or an intracellular signaling molecule. It is now clear that S1P is only generated from sphingosine, which is in turn essentially derived from ceramide through the action of ceramidases. However, much remains unknown about the regulation of the levels of this important signaling molecule. The Pl's compelling preliminary data suggest that haCER2, one of three distinct human alkaline ceramidases that the PI recently cloned, has an important role in regulating the levels of S1P by controlling hydrolysis of ceramides, a rate-limiting step for the formation of S1P, and importantly, haCER2 is capable of regulating S1P-mediated tumor cell survival, angiogenesis, and tumor growth. The PI's long-term goals are to define the role of haCER2 in regulating the levels of S1P and S1P-mediated tumor growth and angiogenesis and to develop this concept into novel strategies for anti-cancer therapeutics. The Pl's central hypothesis is that haCER2 regulates the levels of S1P and S1P-mediated biological processes, particularly tumor angiogenesis and growth, which will be tested by the following specific aims. Aim 1 is to understand catalytic mechanisms of haCER2, haCER2 will be expressed in the yeast system, purified, reconstituted, and characterized biochemically. Aim 2 is to test the hypothesis that haCER2 regulates the generation of S1P in response to proangiogenic cytokines and growth factors, haCER2 regulation by these agonists will be studied in HeLa tumor cells and human umbilical vein endothelial cells by expression and activity studies, haCER2 promoter will be characterized and cis-acting elements responsive to cytokines, and growth factors will be identified. Aim 3 is to test the hypothesis that haCER2 has a role in regulating tumor growth and angiogenesis in vivo. Tumor cell lines that express the wild type, up-regulated, or down-regulated level of haCER2 will be established to define the roles of haCER2 in tumor angiogenesis and growth using Matrigel implant assay and tumor xenograft mouse models. These studies will firmly establish a role for haCER2 in regulating S1P and S1P-mediated biology and will pave the way for novel therapeutic approaches to cancer.