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: 10614111
• 负责人: CHARLES E. CHALFANT
• 金额: $ 22.28万
• 依托单位: UNIVERSITY OF VIRGINIA
• 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 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10614111
• 关键词: role; mechanistic; regulation; cPLA2alpha; eicosanoid

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（如脂质组学）的新技术取得了进展