The interaction of ceramide 1-phosphate with group IVA cytosolic phospholipase A ₂ coordinates acute wound healing and repair

Abstract

The sphingolipid ceramide 1-phosphate (C1P) directly binds to and activates group IVA cytosolic phospholipase A2 (cPLA2α) to stimulate the production of eicosanoids. Because eicosanoids are important in wound healing, we examined the repair of skin wounds in knockout (KO) mice lacking cPLA2α and in knock-in (KI) mice in which endogenous cPLA2α was replaced with a mutant form having an ablated C1P interaction site. Wound closure rate was not affected in the KO or KI mice, but wound maturation was enhanced in the KI mice compared to that in wild-type controls. Wounds in KI mice displayed increased infiltration of dermal fibroblasts into the wound environment, increased wound tensile strength, and a higher ratio of type I:type III collagen. In vitro, primary dermal fibroblasts (pDFs) from KI mice showed substantially increased collagen deposition and migration velocity compared to pDFs from wild-type and KO mice. KI mice also showed an altered eicosanoid profile of reduced proinflammatory prostaglandins (PGE2 and TXB2) and an increased abundance of certain hydroxyeicosatetraenoic acid (HETE) species. Specifically, an increase in 5-HETE enhanced dermal fibroblast migration and collagen deposition. This gain-of-function role for the mutant cPLA2α was also linked to the relocalization of cPLA2α and 5-HETE biosynthetic enzymes to the cytoplasm and cytoplasmic vesicles. These findings demonstrate the regulation of key wound-healing mechanisms in vivo by a defined protein-lipid interaction and provide insights into the roles that cPLA2α and eicosanoids play in orchestrating wound repair.

Phospholipase A2 enzymes represent a shared pathogenic pathway in psoriasis and pityriasis rubra pilaris

“…The PLA2G2F, PLA2G4D, and PLA2G4E enzymes are part of the phospholipase A2 family, which has over 30 members, consisting of both cytosolic PLA2s (such as PLA2G4D and PLA2G4E), and secreted PLA2s (i.e., PLA2G2F), with variable expression of different members in a broad range of tissues (11,20). Studies have demonstrated that PLA2 enzymes regulate lipid pathways, and that many of these lipid species generated by these enzymes serve as bioactive mediators and essential components of the skin barrier (20,27). In addition, individual PLA2 enzymes exhibit unique tissue and cellular distribution, suggesting that they have distinct biological roles in production of pro-and anti-inflammatory lipid mediators (20,28).…”

Updating Phospholipase A2 Biology

“…Ceramide-1-phosphate (C1P), a sphingolipid-derived bioactive lipid, directly binds to and activates cPLA 2 α to stimulate the production of eicosanoids in vitro [ 86 ], but in vivo evidence for this event has been lacking. Recently, MacKnight et al [ 87 ] addressed this issue by generating knockin mice in which endogenous cPLA 2 α is replaced with a mutant form having an ablated C1P-interaction site. In a skin wound healing model, wound maturation, rather than wound closure, is enhanced in the mutant cPLA 2 α-knockin mice compared to control mice.…”

Evidence for Systemic Reactive Oxygen Species in UVB‐mediated Microvesicle Formation

“…Eicosanoid preparation and analysis. Eicosanoids were extracted using a modified extraction process and analyzed by UPLC ESI-MS/MS as previously described (14)(15)(16). Briefly, urine (4 mL) was combined with an IS mixture comprised of 10% methanol (400 ll) and glacial acetic acid (20 ll) before spiking with internal standard (20 ll) containing the following deuterated eicosanoids (2 pmol ll À1 , 40 pmol total) (all standards purchased from Cayman Chemicals): (d 4 ) Prostaglandin F 2 a, (d 4 ) Prostaglandin E 2 , (d 11 ) 5-iPF2a-VI, (d 4 ) 8-iso Prostaglandin F2a.…”

“…Eicosanoids were separated using a Shimadzu Nexara X2 LC-30AD coupled to a SIL-30AC auto-injector, coupled to a DGU-20A5R degassing unit in the following way as previously described by us (15,16). A 14-minute, reversed-phase LC method utilizing an Acentis Express C18 column (150 mm 9 2.1 mm, 2.7 lm) was used to separate the eicosanoids at a 0.5 ml min À1 flow rate at 40°C as previously described by our group (15,16). The column was equilibrated with 100% Solvent A [acetonitrile: water:formic acid (20:80:0.02, v/v/v)] for 5 min and then 10 ll of sample was injected.…”

“…Eicosanoids were analyzed via mass spec using an AB Sciex Triple Quad 5500 Mass Spectrometer as previously described by us (14)(15)(16). Q1 and Q3 were set to detect distinctive precursor and product ion pairs.…”