The role of Sphingosine Kinase 1 in a Mouse Model of Chronic Inflammation

描述(申请人提供)：项目摘要：类风湿性关节炎(RA)是一种影响世界各地人群的自身免疫性疾病。其潜在的机制尚不清楚，但患者的关节中往往有高水平的肿瘤坏死因子(TNF)。在压力下，肿瘤坏死因子有助于保护身体，但它也可能对未感染的细胞和周围组织造成损害，因为它会引起炎症，导致有害蛋白质的产生和非炎症区域的破坏。尽管医学上取得了进步，但现有的抗炎疗法并不完全有效，而且往往成本高昂，因此需要新的疗法。鞘磷脂存在于细胞膜中，但也被证明参与了促进炎症的细胞途径。一种特殊的鞘磷脂，鞘氨醇-1-磷酸(S1P)，已经在文献中被证明能引起炎症介质的增加。S1P的加入导致前列腺素E2(PGE2)和环氧合酶-2蛋白的增加。同时用肿瘤坏死因子和S1P刺激时，PGE2和COX-2的增加比单独使用S1P更多。因此，S1P的形成减少被假设为导致炎症的减少。S1P是通过鞘氨醇激酶(SphK)1和2的作用形成的，而SphK1是炎症途径中负责S1P形成的主要酶。使用siRNA去除成纤维细胞中的SphK1会减少S1P、COX-2和PGE2的形成。在这些细胞中，肿瘤坏死因子刺激后的效应随着SphK1的消除而被消除，这表明肿瘤坏死因子与SphK1之间存在关系。该项目的目标是研究SphK1对过度表达人肿瘤坏死因子的小鼠(如RA患者)炎症的影响，并且没有SphK1的工作副本。在初步研究中，尽管肿瘤坏死因子α的表达水平保持不变，但缺失SphK1功能拷贝的小鼠的关节炎评分有所降低。本研究的目的是1)确定鞘氨醇激酶1在体内肿瘤坏死因子α诱导的关节炎发病机制中的作用；2)研究带有和不带有SphK1功能副本的肿瘤坏死因子α转基因小鼠关节中的基因表达；3)使用FLS确定去除SphK1影响炎症的机制。这些研究的主要方法将是利用肿瘤坏死因子/SphK1小鼠模型来确定SphK1影响炎症的机制。相关性：该项目旨在确定肿瘤坏死因子α和SphK1在慢性炎症中的意义，可能导致发现一种新的治疗类风湿性关节炎等慢性炎症性疾病的靶点。

DESCRIPTION (provided by applicant): Project Summary: Rheumatoid Arthritis (RA) is an autoimmune disease that affects people world wide. The underlying mechanism is unknown, but patients tend to have high levels of tumor necrosis alpha (TNF) in their joints. TNF helps protect the body when under stress, but it can also be damaging to un-infected cells and the surrounding tissue by causing inflammation leading to production of harmful proteins and destruction of non-inflammed areas. Despite advances in medicine, anti-inflammatory therapies currently available are not totally effective and are often costly, thus, novel therapies are needed. Sphingolipids are found in the cell membrane but have also been shown to be involved in the cellular pathways that promote inflammation. One specific sphingolipid, sphingosine-1-phosphate (S1P), has been shown in the literature to cause an increase in inflammatory mediators. Addition of S1P leads to increased prostaglandin E2 (PGE2) and COX-2 protein. Simultaneous stimulation with TNF and S1P increases PGE2 and COX-2 more than S1P alone. Therefore, decreased formation of S1P has been hypothesized to lead to decreases in inflammation. S1P is formed through the action of sphingosine kinase (SphK) 1 and 2; however SphK1 is the major enzyme responsible for S1P formation in the inflammatory pathway. Using siRNA to remove of SphK1 in fibroblast cells lead to decreases in S1P, COX-2, and PGE2 formation. The effects seen after TNF stimulation in these cells were removed with the elimination of SphK1 suggesting a relationship between TNF and SphK1. The goal of this project is to study the effects of SphK1 on inflammation in mice that over express human TNF, like RA patients, and do not have a working copy of the SphK1. In preliminary investigations, mice that are missing functional copies of SphK1 have decreased arthritis score despite the fact that TNF alpha is being expressed at constant levels. The aims of the study are 1) determine the contribution of sphingosine kinase 1 in the pathogenesis of TNF alpha induced arthritis in vivo; 2) characterize gene expression in joints from TNF alpha transgenic mice with and without functioning copies of SphK1; 3) determine the mechanism in which removal of SphK1 affects inflammation using FLS. The primary methodology of these studies will be to utilize the TNF/SphK1 mouse model to determine the mechanism of SphK1 affects inflammation. Relevance: This project seeks to define the significance of TNF alpha and SphK1 in chronic inflammation, potentially leading to the discovery of a novel therapeutic target for chronic inflammatory diseases like RA.