Alkaline Ceramidase and Sphingolipid Signaling

描述（由申请人提供）：鞘氨醇-1-磷酸（S1 P）介导多效性细胞反应，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、炎症和致瘤性有关。S1 P作为G蛋白偶联受体或细胞内信号分子的特异性配体。现在清楚的是，S1 P仅由鞘氨醇产生，而鞘氨醇又基本上通过神经酰胺酶的作用衍生自神经酰胺。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的令人信服的初步数据表明，PI最近克隆的三种不同的人碱性神经酰胺酶之一的haCER 2通过控制神经酰胺的水解（S1 P形成的限速步骤）在调节S1 P水平中具有重要作用，并且重要的是，haCER 2能够调节S1 P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haCER 2在调节S1 P和S1 P介导的肿瘤生长和血管生成水平中的作用，并将此概念发展为抗癌治疗的新策略。PI的中心假设是haCER 2调节S1 P和S1 P介导的生物过程的水平，特别是肿瘤血管生成和生长，这将通过以下具体目的进行测试。目的1为了解haCER 2的催化机制，在酵母系统中表达haCER 2，纯化，重组，并进行生物化学表征。目的二是验证haCER 2在促血管生成的细胞因子和生长因子的作用下调节S1 P的生成这一假设，通过表达和活性研究，在HeLa肿瘤细胞和人脐静脉内皮细胞中研究这些激动剂对haCER 2的调节作用，表征haCER 2启动子，并鉴定对细胞因子和生长因子起反应的顺式作用元件。目的3是检验haCER 2在体内调节肿瘤生长和血管生成中具有作用的假设。将建立表达野生型、上调或下调水平的haCER 2的肿瘤细胞系，以使用Matrigel植入试验和肿瘤异种移植小鼠模型确定haCER 2在肿瘤血管生成和生长中的作用。这些研究将坚定地确立haCER 2在调节S1 P和S1 P介导的生物学中的作用，并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.