The Role of Ceramide Kinase in Eicosanoid Synthesis

神经酰胺激酶在类二十烷酸合成中的作用

• 批准号: 7525444
• 负责人: CHARLES E. CHALFANT
• 金额: $ 36.75万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7525444
• 关键词: Address; Affinity; Agonist; Alzheimer' s Disease; Amino Acids; Anabolism; Anti-Inflammatory Agents; Anti-inflammatory; Antibiotic A23187; Applications Grants; Arachidonic Acids; Asthma; Atherosclerosis; Binding; Binding Sites; Biochemical; Biomedical Research; C2 Domain; Calcium; Calcium Binding; Catabolism; Cells; Ceramides; Chronic Phase; Cytosolic Phospholipase A2; Data; Disease; Dose; Down-Regulation; Eicosanoid Modulation; Eicosanoid Production; Eicosanoids; Enzymes; Event; Family; Funding; Generations; Hydrolysis; In Vitro; Infection; Inflammation; Inflammation Mediators; Inflammatory; Inflammatory Response; Ionophores; Laboratories; Light; Lipids; Lung; Malignant Neoplasms; Mediating; Mediator of activation protein; Modeling; Molecular; Mus; Mutagenesis; Neoplasm Metastasis; Ovalbumin; Ovum; Pathogenesis; Phase; Phenotype; Phosphatidic Acid; Phospholipase; Phosphorylation; Play; Principal Investigator; Production; Protein-Serine-Threonine Kinases; Regulation; Research; Role; Sepsis; Signal Transduction; Site; Site-Directed Mutagenesis; Small Interfering RNA; Source; Sphingolipids; System; Technology; Testing; Therapeutic; Thrombosis; Time; Validation; Work; airway hyperresponsiveness; base; career; cell type; ceramide 1-phosphate; ceramide kinase; cofactor; cytokine; enzyme activity; enzyme mechanism; in vivo; insight; kinase inhibitor; lipid phosphate phosphatase; member; mutant; novel; novel therapeutics; public health relevance; research study; response; therapeutic target; tissue culture; tool

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.

性状（由申请方提供）：花生四烯酸（AA）的产物，类花生酸，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，调节类二十烷酸合成以响应炎症激动剂的主要磷脂酶是IVA组细胞质磷脂酶A2（cPLA 2a）。本实验室首次发现1-磷酸神经酰胺（C1 P）在体外和细胞内都是cPLA 2a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C1 P是cPLA 2a激活所必需的，并且是细胞中类花生酸合成的主要调节剂。我们的实验室现已开始深入研究CERK，因为CERK是抗炎治疗的可能靶点。初步结果表明，CERK是磷酸化的炎症激动剂（如IL-1）的反应。这些结果还表明，Ser 424的磷酸化调节细胞中CERK的酶活性以及类花生酸合成。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是激活的磷酸化和相互作用的脂质辅因子，PA，在炎症介质的反应。我们的初步结果还表明，脂质磷酸磷酸酶-3（LPP-3），LPP家族的成员，是负责C1 P的催化降解。因此，基于我们以前的工作和这些数据，我们假设C1 P被LPP-3分解代谢，以有效地“关闭”响应于炎症激动剂的类花生酸合成的延长激活。最后，在组织培养系统中，CERK抑制剂（例如多种siRNA）有效阻断响应于炎性激动剂的类花生酸合成。因此，我们的中心假设之一是，C1 P产生的神经酰胺的磷酸化的CERK是一个重要的体内介导的类花生酸合成诱导的炎症表型通过激活cPLA 2a。为了验证我们的上述假设，我们将：1）确定脂质辅因子在调节CERK响应炎症激动剂中的作用; 2）确定磷酸化在调节CERK响应炎症激动剂中的作用; 3）确定C1 P响应炎症细胞因子的分解代谢调节;和4）确定CERK在体内调节类花生酸合成中的作用。* 重要性：由于几乎没有什么是已知的这种酶的调节类花生酸合成的关键，我们预测，这些研究将产生很大的见解，这种酶的调节，以及确定额外的上游介质类花生酸合成。这些研究还将确定CERK在体内介导炎症反应中的作用。这不能被低估，因为CERK将被定义为调节类花生酸合成的治疗靶点。事实上，拟议的研究将产生对炎症信号转导新方面的见解，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节产生深刻的见解，并确定类花生酸合成的其他上游介质。这些研究还将在很大程度上确定CERK及其产物C1 P在体内介导炎症反应中的作用。这不能被低估，因为CERK将被定义为调节类花生酸合成的治疗靶点。因此，这项拨款申请具有很高的潜力（高影响），建立一个有形的新因素，影响重要的炎症介质，类花生酸。事实上，所提出的研究将对炎症中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。