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

近年来，小分子分析的新技术进步（例如，脂质组学） 确定了受损伤口愈合的生化表现： 促炎和抗炎类二十烷酸1 -9。类二十烷酸的合成从初始限速步骤开始， 通过磷脂酶A2（PLA 2）的活性产生花生四烯酸（AA）10-12。主要PLA 2之一 参与这一初始步骤的是IVA组胞浆PLA 2（cPLA 2 α）10-12，Chalfant实验室证实了这一点 通过与鞘脂神经酰胺-1-磷酸（C1 P）13-19直接结合而激活。采用新的脂质组学 技术，我们发现，C1 P是时间调节，特别是在炎症阶段增加 人类伤口愈合5.为了评估C1 P诱导的类花生酸在伤口愈合中的作用，我们建立了一个敲入小鼠， 其中cPLA 2中的C1 P位点被切除（KI）。我们的初步数据表明，KI小鼠，不像野生型（WT）和 cPLA 2 β敲除（KO）小鼠表现出显著增强的伤口愈合。这些有益的影响是联系在一起的 导致炎性前列腺素的损失（例如，环氧合酶（考克斯）衍生的PGE 2）和增加的产生 特异性脂质介质（即，脂氧合酶（LOX）衍生的5-HETE），其诱导显著加速的 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 5-来自人类压疮的伤口液中的HETE与更好的愈合结果有关。因此，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至关重要。 初步的机制研究还表明，相关的细胞表型和变异体的产生， 在KI细胞中观察到的类二十烷酸类与C1 P消融突变体的差异细胞定位有关 cPLA 2通过与PIP 2结合而表达。这些发现为以下前提提供了基础：当cPLA 2不能 为了结合C1 P，酶变得自由地与其它脂质调节剂（例如，PIP 2）驱动 产生特异性LOX衍生的类二十烷酸（例如，5-HETE）。这一机制得到了我们初步的支持。 体外研究显示C1 P阻断PIP 2对cPLA 2 α的激活。由于LOX和考克斯产品都是 cPLA 2 β依赖性，但在其生物合成中存在时间对比20.21，我们的数据表明， cPLA 2 β调节的复杂性存在于对炎性激动剂的应答中。因此，我们假设 压力性溃疡伤口愈合的增强将反映一种新的“脂质类开关”， 类花生酸涉及C1 P和PIP 2代谢对cPLA 2 β的复杂拮抗调节。我们也 假设显示无效伤口愈合的老年人将具有缺乏 这些促愈合脂质介质和脂质标记将作为愈合结果的生物标志物。测试这些 假设，我们将采用多学科的团队，新的遗传小鼠模型，和“最先进的” 脂质组学和分子生物学技术，探讨潜在的机制和生物活性的脂质 与衰老和溃疡性伤口的不愈合有关。

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 eicosanoids1-9. 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 healing5. To evaluate C1P-induced eicosanoids in wound healing, we created a knock-in mouse with the C1P site in cPLA2 ablated (KI). Our preliminary data show that KI mice, unlike the wild-type (WT) and cPLA2 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 cPLA2 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 cPLA2-dependent, but temporally contrast in their biosynthesis20.21, our data suggest that an overlooked complexity in cPLA2 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 cPLA2 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.