The role and mechanistic regulation of cPLA2alpha in eicosanoid biosynthesis and wound healing

cPLA2α在类二十烷酸生物合成和伤口愈合中的作用和机制调节

• 批准号: 10156764
• 负责人: CHARLES E. CHALFANT
• 金额: $ 25.74万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2021-11-11
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10156764
• 关键词: Ablation; Age; Aging; Agonist; Anabolism; Anti-Inflammatory Agents; Arachidonic Acids; Binding; Biochemical; Biological Markers; CRISPR/Cas technology; Cells; Complex; Coupled; Cytosolic Phospholipase A2; Data; Dermal; Development; Dinoprostone; Eicosanoid Production; Eicosanoids; Endothelium; Enzymes; Equilibrium; Etiology; Exhibits; Fibroblasts; Foundations; Generations; Genetic; Genetically Engineered Mouse; Human; Hydroxyeicosatetraenoic Acids; Immunoglobulin Class Switching; Impaired wound healing; In Vitro; Inflammatory; Knock-in Mouse; Knock-out; Knockout Mice; Laboratories; Link; Lipids; Lipoxygenase; Liquid substance; Mediating; Metabolism; Methods; Modeling; Molecular; Molecular Biology; Mus; Outcome; Pathology; Patients; Phase; Phenotype; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipase A2; Phospholipases A; Physiological; Physiology; Plasma; Population; Pre-Clinical Model; Process; Production; Prostaglandin-Endoperoxide Synthase; Prostaglandins; Regulation; Role; Signal Pathway; Site; Sphingolipids; Technology; Testing; Therapeutic; Tissues; Variant; ceramide 1-phosphate; ceramide kinase; chronic ulcer; chronic wound; decubitus ulcer; genetic manipulation; healing; in vivo; inhibitor/antagonist; lipid mediator; lipidomics; migration; mortality; mouse model; multidisciplinary; mutant; neutrophil; non-healing wounds; novel; patient population; phosphatidylinositol phosphate, PtdIns(4,5)P2; pre-clinical; response; skin ulcer; small molecule; small molecule inhibitor; wound; wound healing

In recent years, new technological advancements in small molecule analyses (e.g., lipidomics) have identified a biochemical manifestation of impaired wound healing: the development of an imbalance between pro- and anti-inflammatory eicosanoids 1-9. The synthesis of eicosanoids begins with the initial rate-limiting step, the generation of arachidonic acid (AA) via the activity of a phospholipase A2 (PLA2) 10-12. One of the major PLA2s involved in this initial step is group IVA cytosolic PLA2 (cPLA2α) 10-12, which the Chalfant laboratory demonstrated is activated by direct binding to the sphingolipid, ceramide-1-phosphate (C1P) 13-19. Employing newer lipidomic technology, we discovered that C1P is temporally regulated and specifically increases in the inflammatory phase of human wound healing 5. To evaluate C1P-induced eicosanoids in wound healing, we created a knock-in mouse with the C1P site in cPLA2α ablated (KI). Our preliminary data show that KI mice, unlike the wild-type (WT) and cPLA2α knockout (KO) mice, exhibit dramatically enhanced wound healing. These beneficial effects were linked to the loss of inflammatory prostaglandins (e.g., cyclooxygenase (COX)-derived PGE2) and increased production of specific lipid mediators (i.e., lipoxygenase (LOX)-derived 5-HETE), which induced significantly accelerated migration of dermal fibroblasts and neutrophils. Importantly, in an initial study, we also found that high levels of 5-HETE in wound fluid from human pressure ulcers are linked to a better healing outcome. Thus, a balancing act between LOX- and COX-derived lipid mediators is critical in the wound healing process. Initial mechanistic studies also showed that relevant cellular phenotypes and variant production of eicosanoid classes observed in KI cells are linked to a differential cellular localization of the C1P-ablated mutant cPLA2α via association with PIP2. The findings provide a foundation for the premise that, when cPLA2 is unable to bind C1P, the enzyme becomes free to associate with other lipid regulators (e.g., PIP2) that drive the production of specific LOX-derived eicosanoids (e.g., 5- HETE). This mechanism is supported by our preliminary in vitro studies showing that C1P blocks the activation of cPLA2α by PIP2. As LOX and COX products are both cPLA2α-dependent, but temporally contrast in their biosynthesis 20.21, our data suggest that an overlooked complexity in cPLA2α regulation exists in response to inflammatory agonists. Thus, we hypothesize that the enhanced wound healing of pressure ulcers will reflect a novel “lipid-class switch” producing pro-healing eicosanoids involving the complex, antagonistic regulation of cPLA2α by C1P and PIP2 metabolism. We also hypothesize that aging humans, who display ineffective wound healing, will have ulcerative wounds lacking these pro-healing lipid mediators, and a lipid signature will act as biomarker of healing outcome. To test these hypotheses, we will employ a multi-disciplinary team, novel genetic mouse models, and “state of the art” lipidomics and molecular biology technologies to explore the underlying mechanisms and bioactive lipids associated with aging and the non-healing of ulcerative wounds.

近年来，小分子分析方面的新技术进步(例如，脂质组学)已经 确定了伤口愈合受损的一种生化表现： 促炎和抗炎二十烷类化合物1-9。二十烷类化合物的合成从最初的限速步骤开始， 通过磷脂酶A2(PLA2)10-12的活性产生花生四烯酸(AA)。主要的PLA2之一 参与这第一步的是查尔方特实验室证明的IVA族胞浆PLA2(CPLA2α)10-12 通过与神经鞘脂神经酰胺-1-磷酸(C1P)13-19直接结合而激活。采用较新的脂肪制剂 技术，我们发现C1P在时间上受到调节，并在炎症阶段特异性增加 为了评估C1P诱导的二十烷基类化合物在伤口愈合中的作用，我们创建了一只敲入小鼠 CPLA2α中的C1P位点被消融(KI)。我们的初步数据显示，Ki小鼠不同于野生型(WT)和 CPLA2α基因敲除(KO)小鼠的伤口愈合能力显著增强。这些有益的影响是相互关联的 与炎性前列腺素(例如，环氧合酶(COX)衍生的PGE2)的丧失和产量的增加有关 特异性脂质介质(即脂氧合酶(LOX)衍生的5-HETE)的表达，导致显著加速 真皮成纤维细胞和中性粒细胞的迁移。重要的是，在最初的研究中，我们还发现高水平的 人体压疮伤口液中的5-HETE与更好的愈合结果有关。因此，一种平衡 LOX和COX衍生的脂质介质之间的作用在伤口愈合过程中是至关重要的。 初步的机制研究还表明，相关的细胞表型和变异产物 在Ki细胞中观察到的二十烷类化合物与C1P消融突变体的不同细胞定位有关 CplA2α通过与PIP2的结合。这些发现为假设提供了基础，即当cPLA2不能 为了结合C1P，该酶变得可以自由地与其他脂质调节剂(例如，PIP2)结合，从而驱动 生产特定的LOX衍生二十烷类化合物(例如5-HETE)。这个机制得到了我们初步的支持 体外研究表明，C1P可阻断PIP2对CPLA2α的激活。因为LOX和COX产品都是 CPLA2依赖于α，但在它们的生物合成上形成了时间对比20.21，我们的数据表明一个被忽视的 炎性激动剂对cPLA2α的调控存在复杂性。因此，我们假设 促进压疮的伤口愈合将反映出一种新的能促进愈合的“脂类开关” 二十烷酸类化合物涉及C1P和PIP2代谢对cpla2α的复杂、拮抗调节。我们也 假设上了年纪的人，伤口愈合无效，会有溃烂伤口缺失 这些促进愈合的脂质介体和脂质信号将作为愈合结果的生物标志物。为了测试这些 假设，我们将使用一个多学科的团队，新的遗传小鼠模型，以及“最先进的技术” 脂质组学和分子生物学技术探索其潜在机制和生物活性脂类 与衰老和溃烂创面无法愈合有关。