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酶的神经酰胺代谢酶家族在炎症中的作用，并靶向这些酶用于新的抗炎治疗。PI的实验室在鞘脂代谢和功能方面具有既定的专业知识记录。上一个资助期的研究使我们进入了一个新的令人兴奋的方向，即神经酰胺酶及其代谢产物在炎症中的作用和调节。神经酰胺酶分解神经酰胺以产生鞘氨醇和鞘氨醇-1-磷酸（S1 P），鞘氨醇和鞘氨醇-1-磷酸又介导若干生物活性，包括炎症反应。在这一竞争性更新中，我们有令人信服的新数据，据此我们暗示该途径是环加氧酶-2（考克斯-2）表达和前列腺素E2（PGE 2）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TNF炎症反应。因此，该提议将检验神经酰胺酶调节精氨酸介导的慢性炎症以及抑制该神经酰胺酶活性抑制炎症反应的假设。 为了验证这一假设，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。这将通过确定哪种神经酰胺酶（酸性、中性和碱性）在炎症中被上调并研究炎症细胞模型中这种调节的机制，以及通过确定来自人类和动物炎症模型的炎症组织中神经酰胺酶的表达和细胞类型分布来完成。2)建立神经酰胺酶在炎症调节中的功能，并确定其产物的作用机制。通过评价神经酰胺酶在细胞和体内炎症模型中过度表达的作用，并确定神经酰胺酶调节炎症途径（S1 P、NF-κ B、ERK、考克斯-2）的机制，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)确定每种神经酰胺酶在炎症中的相对作用，并分析其机制。这将通过使用针对不同神经酰胺酶的小干扰RNA阻断细胞中和体内的神经酰胺酶活性来完成。此外，我们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TNF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通路在炎症反应中的作用，也可能为炎症提供新的治疗方法。 公共卫生相关性： 叙述性。与退伍军人健康相关。VA患者人群的慢性炎症性疾病，特别是关节炎和结肠炎的发病率较高。这些病症难以治疗，并且通常对许多可用的治疗方式不敏感。此外，越来越多的人认为慢性炎症会导致癌症。肿瘤坏死因子（TNF）和考克斯-2与大多数这些慢性炎性病症有关，并且最近高效的抗炎治疗适合于阻断它们的作用。然而，这些抗炎药已被证明具有心血管和其他副作用。我们的初步研究表明，鞘脂分子是TNF对考克斯-2作用的中间体。因此，我们将利用类风湿性关节炎和炎症性结肠炎模型来研究这些炎症模型中的鞘脂调节酶神经酰胺酶，并将这些酶靶向用于治疗慢性炎症的新疗法。

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. PUBLIC HEALTH RELEVANCE: NARRATIVE. Relevance to Veterans Health. The VA patient population has a high incidence of chronic inflammatory conditions, in particular, arthritis and colitis. These conditions are difficult to treat and generally are not sensitive to many available modalities of treatment. Moreover, chronic inflammatory conditions are increasingly thought to lead to cancer. Tumor necrosis factor (TNF) and COX-2 are implicated in most of these chronic inflammatory conditions and recent highly effective anti-inflammatory therapy is geared at blocking their action. However, these anti-inflammatory agents have proven to have cardiovascular and other side effects. Our preliminary studies demonstrate that sphingolipid molecules are intermediates in the action of TNF on COX-2. We will therefore utilize models of rheumatoid arthritis and inflammatory colitis to study the sphingolipid regulating enzymes ceramidases in these models of inflammation and target these enzymes for novel therapies in the treatment of chronic inflammation.