Regulation and Function of Cytosolic Phospholipases A2 in Lung Cells

描述(申请人提供)：本方案的目的是研究三种第四类胞浆磷脂酶A2(CPLA2)、cPLA2a、cPLA2和cPLA2？在肺细胞中。这些酶含有一个钙-磷脂结合域和一个特有的丝氨酸/天冬氨酸催化二聚体。创始成员cPLA2a释放花生四烯酸来生产前列腺素和白三烯。这些强效的脂质介体在调节正常和病理过程中有不同的作用。由cPLA2a激活产生的促炎性脂质介质(白三烯、血栓素A2)调节肺纤维化、急性肺损伤、过敏反应和关节炎的发展。相比之下，前列腺素E2和I2可以预防纤维化、高血压和糖尿病。在肺上皮细胞和成纤维细胞中与cPLA2a一起表达的cPLA2和cPLA2？知之甚少。在cPLA2a基因敲除小鼠或cPLA2a缺陷的肺成纤维细胞中，二十烷类化合物的产生并未完全消融。CPLA2？被鉴定为PLA2，负责从cPLA2a基因敲除小鼠分离的肺成纤维细胞中钙诱导的脂肪酸释放和前列腺素E2的产生。CPLA2a、cPLA2和cPLA2？表现出不同的酶性质和亚细胞定位，表明它们的调节和功能不同。随着细胞内钙离子浓度的增加，cPLA2a从胞浆向高尔基体转移，cPLA2？移位到褶皱和内小体。相反，cPLA2在组成上定位于线粒体和早期内吞体内。CPLA2？和cPLA2？是第一批在内体上发现的PLA2，我们假设它们调控内体的运输。这项提案的具体目标涉及使用纯化的cPLA2a、cPLA2和cPLA2？确定调节膜结合和水解性的结构域和残基。钙和多磷酸肌醇在体外和细胞中调节这些过程的作用将被研究。CPLA2和cPLA2对内体转运的调节？在BEAS-2B细胞和肺成纤维细胞中，将使用shRNA敲除和小分子PLA2抑制剂进行研究。CPLA2a和cPLA2？将全面分析原代肺成纤维细胞和上皮细胞的激活情况。由于cPLA2？与cPLA2a不同，它释放亚油酸，我们推测它通过15-脂氧合酶启动13(S)-羟基十八碳二烯酸的形成。使用条件基因敲除小鼠模型cPLA2？将被诱导从显著表达cPLA2？的肺上皮细胞中删除。将小鼠模型用于确定cPLA2？在调节脂质介质的产生，以及调节过敏原诱导的炎症和呼吸道高反应性方面。这项研究的结果将提供有关肺细胞中IV组cPLA2的调节和功能的详细信息。与公共卫生相关。第四组胞浆磷脂酶A2家族的成员开始产生具有多种功能的有效生物活性脂类代谢物。脂质介质调节生理过程，但也有助于炎症性疾病的病理后果。阐明这些酶的性质将有助于更好地了解脂质介体产生的调节，并为控制疾病的机制的发展提供帮助。

DESCRIPTION (provided by applicant): The objective of this proposal is to study the regulation and function of three Group IV cytosolic phospholipases A2 (cPLA2s), cPLA2a, cPLA2¿ and cPLA2? in lung cells. These enzymes contain a calcium- phospholipid binding domain and a characteristic Ser/Asp catalytic dyad. The founding member, cPLA2a, releases arachidonic acid for the production of prostaglandins and leukotrienes. These potent lipid mediators have diverse roles in regulating both normal and pathological processes. Pro-inflammatory lipid mediators (leukotrienes, thromboxane A2) produced as a result of cPLA2a activation regulate the development of pulmonary fibrosis, acute lung injury, allergic reactions and arthritis. In contrast prostaglandins E2 and I2 protect against fibrosis, hypertension and diabetes. Little is known about cPLA2¿ and cPLA2?, which are expressed together with cPLA2a in lung epithelial cells and fibroblasts. Eicosanoid production is not completely ablated in cPLA2a knockout mice or in cPLA2a-deficient lung fibroblasts. cPLA2? was identified as the PLA2 responsible for calcium-induced fatty acid release and prostaglandin E2 production in lung fibroblasts isolated from the cPLA2a knockout mouse. cPLA2a, cPLA2¿ and cPLA2? exhibit distinct enzymatic properties and subcellular localization suggesting differences in their regulation and function. In response to increases in intracellular calcium, cPLA2a translocates from the cytosol to Golgi, and cPLA2? translocates to ruffles and endosomes. In contrast, cPLA2¿ constitutively localizes to mitochondria and early endosomes. cPLA2¿ and cPLA2? are the first PLA2s identified on endosomes, where we hypothesize they regulate endosome trafficking. The specific aims of this proposal involve using purified cPLA2a, cPLA2¿ and cPLA2? to identify domains and residues that regulate membrane binding and hydrolytic activity. The role of calcium and polyphosphoinositides in regulating these processes in vitro and in cells will be investigated. The regulation of endosome trafficking by cPLA2¿ and cPLA2? in BEAS-2B cells and lung fibroblasts will be investigated using shRNA knockdown and small molecule PLA2 inhibitors. The lipid mediators produced as a result of cPLA2a and cPLA2? activation in primary lung fibroblasts and epithelial cells will be comprehensively analyzed. Since cPLA2?, unlike cPLA2a, releases linoleic acid, we hypothesize that it initiates the formation of 13(S)-hydroxy-octadecadienoic acid through 15- lipoxygenase. Using a conditional knockout mouse model, cPLA2? will be inducibly deleted from lung epithelial cells, which prominently express cPLA2?. The mouse model will be used to determine the role of cPLA2? in mediating lipid mediator production, and in regulating allergen-induced inflammation and airway hyper-responsiveness. Results from this proposal will provide detailed information about the regulation and function of Group IV cPLA2s in lung cells. PUBLIC HEALTH RELEVANCE. Members of the Group IV cytosolic phospholipase A2 family initiate the production of potent biologically active lipid metabolites that have diverse functional effects. Lipid mediators regulate physiological processes but also contribute to the pathological consequences of inflammatory diseases. Elucidating the properties of these enzymes will provide a better understanding of the regulation of lipid mediator production, and for development of mechanisms for controlling disease.