Regulation and Role of Ceramidase in Inflammation

描述(由申请人提供)： 该项目的长期目标是确定神经酰胺代谢酶家族神经酰胺酶在炎症中的作用，并将这些酶作为新的抗炎治疗的靶点。PI的实验室在鞘脂新陈代谢和功能方面拥有成熟的专业记录。来自前一个资金时期的研究将我们带入了一个新的令人兴奋的方向，即神经酰胺酶在炎症中的作用和调节及其代谢产物。神经酰胺酶能分解神经酰胺生成鞘氨醇和鞘氨醇-1-磷酸(S1P)，而神经鞘氨醇和鞘氨醇-1-磷酸(S1P)又参与多种生物学活动，包括炎症反应。在这一竞争更新中，我们有了令人信服的新数据，由此我们发现这一途径是环氧合酶-2(COX-2)表达和前列腺素E2(PGE2)产生的关键调节因子。此外，我们发现至少有一种神经酰胺酶参与了肿瘤坏死因子的炎症反应。因此，这项提议将检验神经酰胺酶调节细胞因子介导的慢性炎症的假设，以及抑制这种神经酰胺酶活性抑制炎症反应的假设。为了验证这一假说，我们提出了以下目标：1)确定哪些神经酰胺酶在炎症中受到调节，并确定这种调节的机制。这将通过确定哪些神经酰胺酶(酸性、中性和碱性)在炎症中上调，并在炎症细胞模型中研究这种调节机制，以及通过确定神经酰胺酶在人类和动物炎症模型中的表达和细胞类型分布来实现这一点。2)明确神经酰胺酶在炎症反应中的调节作用，并确定其产物的作用机制。这将通过评估神经酰胺酶在细胞和体内炎症模型中的过度表达的影响，以及确定神经酰胺酶调节炎症途径(S1P、NF-KB、ERKs、COX-2)的机制来证明神经酰胺酶及其代谢产物在调节炎症中具有重要作用。3)确定各神经酰胺酶在炎症反应中的相对作用，并剖析其作用机制。这将通过在细胞和体内使用针对不同神经酰胺酶的小干扰RNA来阻断神经酰胺酶的活性来实现。此外，我们还将测试我们合成的不同化合物在细胞和体内炎症模型中抑制神经酰胺酶的能力。我们还将测试神经酰胺酶K/O小鼠在IBD结肠炎和肿瘤坏死因子诱导的关节炎模型中是否受到炎症保护。这些研究将使我们能够深入了解神经酰胺酶途径在炎症反应中的作用，并可能提供治疗炎症的新方法。

DESCRIPTION (provided by applicant): The long-term goal of this project is to define the role of the ceramide metabolizing enzyme family of ceramidases in inflammation and to target these enzymes for novel anti-inflammatory therapy. The PI's laboratory has an established track record of expertise in sphingolipid metabolism and function. Studies from the previous funding period have led us into a novel exciting direction on the role and regulation of ceramidases in and their metabolic products in inflammation. Ceramidases breakdown ceramide to generate sphingosine and sphingosine-1-phosphate (S1P) which in turn mediates several biologic activities, including inflammatory responses. In this competing renewal we have compelling new data, whereby we have implicated this pathway as a key regulator of cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production. In addition we find that at least one ceramidase is involved in the TNF inflammatory response. This proposal will therefore, test the hypothesis that ceramidases regulate cytokine-mediated chronic inflammation, and that inhibiting this ceramidase activity inhibits inflammatory responses. To test this hypothesis we propose the following aims: 1) Determine which ceramidases are regulated in inflammation and determine the mechanisms of this regulation. This will be done by determining which of the ceramidases (acid, neutral and alkaline) is/are upregulated in inflammation and studying the mechanisms of this regulation in cell models of inflammation, and by determining the expression and cell-type distribution of ceramidases in inflammatory tissues from humans and from animal models of inflammation. 2) Establish the function of ceramidases in regulation of inflammation and determine the mechanisms of action of their products. This will be done by demonstrating that ceramidases and their metabolic products have a significant role in regulating inflammation by evaluating the effect of over expression of ceramidases in cells and in vivo models of inflammation, and determining the mechanisms by which ceramidases regulate inflammatory pathways (S1P, NF-KB, ERKs, COX-2). 3) Determine the relative contribution of each of the ceramidases in inflammation and dissect the mechanisms involved. This will be done by blocking ceramidase activity in cells and in vivo using small interfering RNA to the different ceramidases. In addition we will test different compounds that we synthesized for their ability to inhibit ceramidases in cells and in vivo models of inflammation. We will also test if ceramidase K/O mice are protected from inflammation in a model of IBD colitis and TNF-induced arthritis. These studies will enable us to gain important insight into the role of the ceramidase pathway in inflammatory responses and may also provide novel therapeutic approaches to inflammation.