The Role of Ceramide Kinase in Eicosanoid Synthesis

描述（由申请人提供）：花生四烯酸（AA）的产物，二十烷类，是公认的炎症介质。磷脂酶产生AA是类二十烷酸生物合成的初始限速步骤，在炎症激动剂作用下调节类二十烷酸合成的主要磷脂酶是IVA组胞质磷脂酶A2 （cPLA2a）。我们的实验室首次发现神经酰胺-1-磷酸（C1P）在体外和细胞中都是cPLA2a的有效激活剂。在我们之前的资助周期中，PI的实验室证明了CERK及其产物C1P是激活cPLA2a所必需的，并且是细胞中类二十烷合成的主要调节剂。我们的实验室现在已经开始深入研究CERK，因为CERK是抗炎治疗的可能靶点。初步结果表明，CERK在炎症激动剂（如IL-1）的作用下被磷酸化。这些结果还表明，Ser424的磷酸化既调节CERK的酶活性，也调节细胞中类二十烷酸的合成。我们实验室的其他初步结果表明，CERK特异性结合并被磷脂酸（PA）激活。基于这些数据，我们假设CERK是通过磷酸化和与脂质辅助因子PA的相互作用来激活的，以响应炎症介质。我们的初步结果还表明，脂质磷酸磷酸酶-3 （LPP-3）是LPP家族的一员，负责催化C1P的降解。因此，基于我们之前的工作和这些数据，我们假设C1P被LPP-3分解代谢，从而有效地“关闭”炎症激动剂对类二十烷合成的长期激活。最后，CERK抑制剂（例如多个sirna）在组织培养系统中对炎症激动剂的反应中能有效地阻断类二十烷酸合成。因此，我们的中心假设之一是，CERK磷酸化神经酰胺产生的C1P是通过激活cPLA2a诱导炎症表型诱导的类二十烷合成的重要体内介质。为了验证我们的上述假设，我们将：1)确定脂质辅助因子在调节CERK对炎症激动剂的反应中的作用；2)确定磷酸化在调节CERK对炎症激动剂反应中的作用；3)研究C1P对炎性细胞因子的代谢调节；4)确定CERK在体内调节类二十烷合成中的作用。*意义：由于对这种酶的调控几乎一无所知，我们预测这些研究将对这种酶的调控产生重大的见解，并确定额外的类二十烷合成的上游介质。这些研究还将确定CERK在体内介导炎症反应中的作用。这一点不能被低估，因为CERK将被定义为调节类二十烷酸合成的治疗靶点。事实上，拟议的研究将对炎症信号转导的新方面产生见解，可能导致哮喘，血栓形成，ATH和阿尔茨海默病的新疗法。公共卫生相关性：这些研究将对神经酰胺激酶的调控产生重大见解，并确定类二十烷合成的其他上游介质。这些研究也将在很大程度上确定CERK及其产物C1P在体内介导炎症反应中的作用。这一点不能被低估，因为CERK将被定义为调节类二十烷酸合成的治疗靶点。因此，这项资助申请具有很高的潜力（high IMPACT），可以建立一个影响重要炎症介质二十烷类物质的切实的新因子。事实上，拟议的研究将对炎症信号转导的新方面产生重要的见解，可能导致哮喘/AHR，败血症/肺部感染，血栓形成，癌症转移，炎症，ATH和阿尔茨海默病的新疗法。

DESCRIPTION (provided by applicant): Products of arachidonic acid (AA), eicosanoids, are well-established mediators of inflammation. The production of AA by phospholipases is the initial rate-limiting step in eicosanoid biosynthesis, and the major phospholipase that regulates eicosanoid synthesis in response to inflammatory agonists is group IVA cytosolic phospholipase A2 (cPLA2a). Our laboratory first discovered that ceramide-1-phosphate (C1P) is a potent activator of cPLA2a both in vitro and in cells. In our previous funding cycle, the PI's laboratory demonstrated that CERK and its product, C1P, are required for the activation of cPLA2a, and are major regulators of eicosanoid synthesis in cells. Our laboratory has now begun to study CERK in depth since CERK is a possible target for anti-inflammatory therapeutics. Preliminary results demonstrate that CERK is phosphorylated in response to inflammatory agonists (e.g. IL-1¿). These results also demonstrated that phosphorylation of Ser424 regulates both the enzymatic activity of CERK as well as eicosanoid synthesis in cells. Other preliminary results from our laboratory demonstrate that CERK specifically binds and is activated by phosphatidic acid (PA). Based on these data, we hypothesize that CERK is activated by phosphorylation and interaction with the lipid co-factor, PA, in response to inflammatory mediators. Our preliminary results also suggest that lipid phosphate phosphatase-3 (LPP-3), a member of the LPP family, is responsible for the catalytic degradation of C1P. Therefore, building upon our previous work and these data, we hypothesize that C1P is catabolized by LPP-3 to effectively "shut-down" prolonged activation of eicosanoid synthesis in response to inflammatory agonists. Lastly, CERK inhibitors (e.g. multiple siRNAs) are effective in blocking eicosanoid synthesis in response to inflammatory agonists in tissue culture systems. Therefore, one of our central hypotheses is that C1P produced from the phosphorylation of ceramide by CERK is an important in vivo mediator of eicosanoid synthesis induced by inflammatory phenotypes via activation of cPLA2a. To validate our above hypotheses, we will: 1) Determine the role of lipid cofactors in regulating CERK in response to inflammatory agonists; 2) Determine the role of phosphorylation in regulating CERK in response to inflammatory agonists; 3) To determine the catabolic regulation of C1P in response to inflammatory cytokines; and 4) Determine the role of CERK in regulating eicosanoid synthesis in vivo. *Significance: Since almost nothing is known about the regulation of this enzyme critical for eicosanoid synthesis, we predict that these studies will produce great insights into the regulation of this enzyme as well as identify additional upstream mediators of eicosanoid synthesis. These studies will also define a role of CERK in mediating inflammatory responses in vivo. This cannot be understated because CERK will be defined as a therapeutic target for modulation of eicosanoid synthesis. Indeed, the proposed studies will generate insights into novel aspects of signal transduction in inflammation perhaps leading to new therapeutics for asthma, thrombosis, ATH, and Alzheimer's disease. PUBLIC HEALTH RELEVANCE: These studies will produce great insights into the regulation of ceramide kinase as well as identify additional upstream mediators of eicosanoid synthesis. These studies will also largely define a role of CERK and its product, C1P, in mediating inflammatory responses in vivo. This cannot be understated because CERK will be defined as a therapeutic target for modulation of eicosanoid synthesis. Thus, this grant application has the high potential (HIGH IMPACT) for establishing a tangible new factor that impinges on the important inflammatory mediators, eicosanoids. Indeed, the proposed studies will generate significant insights into novel aspects of signal transduction in inflammation perhaps leading to new therapeutics for asthma/AHR, sepsis/pulmonary infection, thrombosis, cancer metastasis, inflammation, ATH, and Alzheimer's disease.