Regulation and Function of Cytosolic Phospholipases A2 in Lung Cells

描述（由申请人提供）：本提案的目的是研究三种IV型胞质磷脂酶A2（cPLA 2s），cPLA 2a，cPLA 2？和cPLA 2？的调节和功能。在肺细胞中。这些酶含有钙-磷脂结合结构域和特征性Ser/Asp催化二联体。其创始成员cPLA 2a释放花生四烯酸，用于产生异甘草素和白三烯。这些有效的脂质介质在调节正常和病理过程中具有不同的作用。由于cPLA 2a活化而产生的促炎性脂质介质（白三烯、血栓烷A2）调节肺纤维化、急性肺损伤、过敏反应和关节炎的发展。相反，E2和I2可预防纤维化、高血压和糖尿病。对cPLA 2？和cPLA 2？知之甚少，其在肺上皮细胞和成纤维细胞中与cPLA 2a一起表达。在cPLA 2a敲除小鼠或cPLA 2a缺陷型肺成纤维细胞中，类花生酸的产生没有完全消除。cPLA2？被鉴定为负责从cPLA 2a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产生的PLA 2。cPLA 2a、cPLA 2？和cPLA 2？表现出不同的酶特性和亚细胞定位，表明它们的调节和功能存在差异。在响应细胞内钙离子的增加，cPLA 2a易位从胞质到高尔基体，和cPLA 2？易位到皱褶和核内体。相比之下，cPLA 2 <$constitutively定位于线粒体和早期内体。cPLA 2和cPLA 2？是在核内体上鉴定的第一个PLA 2，我们假设它们调节核内体运输。该提案的具体目标涉及使用纯化的cPLA 2a，cPLA 2？和cPLA 2？以鉴定调节膜结合和水解活性的结构域和残基。钙和polyphosphoinositides在调节这些过程中在体外和细胞中的作用将被调查。cPLA 2和cPLA 2对内体运输的调节？在BEAS-2B细胞和肺成纤维细胞中，将使用shRNA敲低和小分子PLA 2抑制剂来研究。脂质介质产生的结果cPLA 2a和cPLA 2？将全面分析原代肺成纤维细胞和上皮细胞中的活化。自从cPLA 2？以来，与cPLA 2a释放亚油酸不同，我们假设它通过15-脂氧合酶启动13（S）-羟基-十八碳二烯酸的形成。使用条件性基因敲除小鼠模型，cPLA 2？将从显著表达cPLA 2？的肺上皮细胞中诱导删除。小鼠模型将被用来确定cPLA 2的作用？介导脂质介质的产生，以及调节过敏原诱导的炎症和气道高反应性。该提案的结果将提供有关肺细胞中第IV组cPLA 2的调节和功能的详细信息。公共卫生相关性。IV组胞质磷脂酶A2家族的成员启动具有多种功能效应的有效生物活性脂质代谢物的产生。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提供一个更好的了解调节脂质介质的生产，并为控制疾病的机制的发展。

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.