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

项目概述：类风湿关节炎（RA）是一种影响全球人群的自身免疫性疾病。潜在的机制尚不清楚，但患者的关节往往有高水平的肿瘤坏死α (TNF)。TNF有助于在压力下保护身体，但它也可以通过引起炎症导致有害蛋白质的产生和非炎症区域的破坏，从而损害未感染的细胞和周围组织。尽管医学取得了进步，但目前可用的抗炎疗法并不完全有效，而且往往价格昂贵，因此需要新的疗法。鞘脂存在于细胞膜中，但也被证明参与了促进炎症的细胞途径。一种特殊的鞘脂，鞘鞘醇-1-磷酸（S1P），已在文献中显示引起炎症介质的增加。添加S1P导致前列腺素E2 （PGE2）和COX-2蛋白升高。与单独刺激S1P相比，TNF和S1P同时刺激PGE2和COX-2增加更多。因此，假设S1P的形成减少导致炎症减少。S1P是通过鞘氨酸激酶（SphK） 1和2的作用形成的；然而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导致S1P、COX-2和PGE2的形成减少。随着SphK1的消除，这些细胞中TNF刺激后的效果被去除，这表明TNF和SphK1之间存在关系。该项目的目标是研究SphK1对过度表达人类TNF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，缺失SphK1功能拷贝的小鼠关节炎评分降低，尽管TNF α在恒定水平表达。本研究的目的是：1)确定鞘氨醇激酶1在体内TNF α诱导的关节炎发病机制中的作用；2)观察具有和不具有SphK1功能拷贝的TNF - α转基因小鼠关节中的基因表达；3)利用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TNF/SphK1小鼠模型来确定SphK1影响炎症的机制。相关性：本项目旨在确定TNF α和SphK1在慢性炎症中的意义，可能导致发现慢性炎症性疾病（如RA）的新治疗靶点。

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.