The Role of Ceramide Kinase in Eicosanoid Synthesis

描述(申请人提供)：花生四烯酸(AA)的产物，二十烷类化合物，是公认的炎症介质。在二十烷基类化合物生物合成中，磷脂酶产生AA是最初的限速步骤，而调节二十烷基类化合物合成的主要磷脂酶是IVA胞浆磷脂酶A2(CPLA2a)。我们的实验室首次发现神经酰胺-1-磷酸(C1P)在体外和细胞内都是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，是细胞中二十烷类化合物合成的主要调节因子。我们的实验室现在已经开始深入研究Cerk，因为Cerk可能是抗炎治疗的靶点。初步结果表明，在炎症激动剂(如IL-1)的作用下，Cerk被磷酸化。这些结果还表明，Ser424的磷酸化既调节CERK的酶活性，也调节细胞中二十烷类化合物的合成。我们实验室的其他初步结果表明，Cerk特异性地结合并被磷脂酸(PA)激活。基于这些数据，我们假设CERK是通过磷酸化并与脂质辅助因子PA相互作用来响应炎症介质而激活的。我们的初步结果还表明，LPP家族的成员LPP-3(LPP-3)对C1P的催化降解起作用。因此，在我们以前的工作和这些数据的基础上，我们假设C1P被LPP-3分解代谢，从而有效地“关闭”炎症激动剂对二十烷类化合物合成的长期激活。最后，CERK抑制剂(例如，多个siRNAs)在组织培养系统中有效地阻止二十烷类化合物的合成，以响应炎性激动剂。因此，我们的一个中心假设是，由Cerk磷酸化神经酰胺产生的C1P是炎症表型通过激活cPLA2a诱导二十烷类化合物合成的重要介质。为了验证我们的假设，我们将：1)确定脂质辅助因子在炎症激动剂反应中调节CERK的作用；2)确定磷酸化在炎症激动剂反应中调节CERK的作用；3)确定C1P在炎症细胞因子反应中的分解代谢调节；以及4)确定CERK在体内调节二十烷类化合物合成的作用。*意义：由于对这种对二十烷类化合物合成至关重要的酶的调节几乎一无所知，我们预测这些研究将对这种酶的调节产生巨大的洞察力，并确定更多的二十烷类化合物合成的上游介质。这些研究还将确定CERK在体内介导炎症反应中的作用。这一点不能低估，因为Cerk将被定义为调节二十烷类化合物合成的治疗靶点。事实上，拟议的研究将对炎症中信号转导的新方面产生洞察力，可能导致哮喘、血栓形成、ATH和阿尔茨海默病的新疗法。与公共健康相关：这些研究将对神经酰胺激酶的调控产生重大见解，并确定二十烷类化合物合成的其他上游介体。这些研究还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。这一点不能低估，因为Cerk将被定义为调节二十烷类化合物合成的治疗靶点。因此，这项赠款申请具有很高的潜力(影响很大)，可以建立一个切实的新因素，影响重要的炎症介质--二十烷类化合物。事实上，拟议的研究将对炎症中的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.