The role and mechanistic regulation of cPLA2alpha in eicosanoid biosynthesis and 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 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.

Grant Number: 5R01GM137394-05
NIH Institute/Center: NIH
Principal Investigator: CHARLES CHALFANT