Abstract
The sphingolipid, ceramide-1-phosphate (C1P), has been shown to promote the inflammatory phase and inhibit the proliferation and remodeling stages of wound repair via direct interaction with group IVA cytosolic phospholipase A2, a regulator of eicosanoid biosynthesis that fine-tunes the behaviors of various cell types during wound healing. However, the anabolic enzyme responsible for the production of C1P that suppresses wound healing as well as bioactive eicosanoids and target receptors that drive enhanced wound remodeling have not been characterized. Herein, we determined that decreasing C1P activity via inhibitors or genetic ablation of the anabolic enzyme ceramide kinase (CERK) significantly enhanced wound healing phenotypes. Importantly, postwounding inhibition of CERK enhanced the closure rate of acute wounds, improved the quality of healing, and increased fibroblast migration via a “class switch” in the eicosanoid profile. This switch reduced pro-inflammatory prostaglandins (e.g., prostaglandin E2) and increased levels of 5-hydroxyeicosatetraenoic acid and the downstream metabolite 5-oxo-eicosatetraenoic acid (5-oxo-ETE). Moreover, dermal fibroblasts from mice with genetically ablated CERK showed enhanced wound healing markers, while blockage of the murine 5-oxo-ETE receptor (oxoeicosanoid receptor 1) inhibited the enhanced migration phenotype of these cell models. Together, these studies reinforce the vital roles eicosanoids play in the wound healing process and demonstrate a novel role for CERK-derived C1P as a negative regulator of 5-oxo-ETE biosynthesis and the activation of oxoeicosanoid receptor 1 in wound healing. These findings provide foundational preclinical results for the use of CERK inhibitors to shift the balance from inflammation to resolution and increase the wound healing rate.
Highlights
Activation of oxoeicosanoid receptor 1 in wound healing
To evaluate the ability of SYR382141 to inhibit ceramide kinase (CERK)-derived C1P production in cells, Primary mouse dermal fibroblasts (pDFs)) and human umbilical vein endothelial cell (HUVEC) were treated with SYR382141 or the positive control, the CERK inhibitor N-[2-(Benzoylamino)-6-benzothiazolyl] tricyclo[3.3.1.13 (NVP-231), and analyzed via ultra performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry [22, 36] (Fig. 1)
We showed that inhibition of CERKderived C1P could recapitulate the finding that genetic ablation of the C1P/cytosolic phospholipase A2 alpha (cPLA2α) interaction site enhances acute wound healing through increased dermal fibroblast migration and accelerated type I collagen deposition characteristic of nonfibrotic healed wounds [22]
Summary
Activation of oxoeicosanoid receptor 1 in wound healing. These findings provide foundational preclinical results for the use of CERK inhibitors to shift the balance from inflammation to resolution and increase the wound healing rate. Mutagenesis of critical amino acids for C1P interaction within this site inhibited the ability of cPLA2α to translocate in response to inflammatory agonists [21] These data suggest that CERK and its product, C1P, are required for the activation of cPLA2α, and are major regulators of eicosanoid synthesis in cells. Additional work by our laboratory has recently shown that the C1P:cPLA2α interaction negatively regulates the migration of dermal fibroblasts and 5-HETE production, and genetic ablation of this interaction enhanced acute wound healing in mice [23] (e.g. enhanced wound tensile strength, increased collagen I deposition, reduced collagen III deposition, and increased fibroblast wound infiltration)
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