Characterization of eicosanoid synthesis in a genetic ablation model of ceramide kinase

Jennifer A Mietla,Dayanjan S Wijesinghe,L Alexis Hoeferlin,Michael D Shultz,Ramesh Natarajan,Alpha A Fowler,Charles E Chalfant

doi:10.1194/jlr.m035683

Jennifer A Mietla, Dayanjan S Wijesinghe + Show 5 more

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https://doi.org/10.1194/jlr.m035683

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Abstract

Multiple reports have demonstrated a role for ceramide kinase (CERK) in the production of eicosanoids. To examine the effects of the genetic ablation of CERK on eicosanoid synthesis, primary mouse embryonic fibroblasts (MEFs) and macrophages were isolated from CERK(-/-) and CERK(+/+) mice, and the ceramide-1-phosphate (C1P) and eicosanoid profiles were investigated. Significant decreases were observed in multiple C1P subspecies in CERK-/- cells as compared to CERK(+/+) cells with overall 24% and 48% decreases in total C1P. In baseline experiments, the levels of multiple eicosanoids were significantly lower in the CERK(-/-) cells compared with wild-type cells. Importantly, induction of eicosanoid synthesis by calcium ionophore was significantly reduced in the CERK(-/-) MEFs. Our studies also demonstrate that the CERK(-/-) mouse has adapted to loss of CERK in regards to airway hyper-responsiveness as compared with CERK siRNA treatment. Overall, we demonstrate that there are significant differences in eicosanoid levels in ex vivo CERK(-/-) cells compared with wild-type counterparts, but the effect of the genetic ablation of CERK on eicosanoid synthesis and the serum levels of C1P was not apparent in vivo.

Highlights

Multiple reports have demonstrated a role for ceramide kinase (CERK) in the production of eicosanoids
We examined the effect of genetic ablation of CERK on the levels of various C1P subspecies using immortalized CERKϪ/Ϫ mouse embryonic fibroblast (MEF) in comparison to CERK+/+ MEFs
We have demonstrated that CERK-derived C1P is instrumental in the production of eicosanoids, corroborating our previous studies illustrating that siRNA downregulation of CERK blocks the release arachidonic acid (AA) by inhibiting the production of CERK-derived C1P [15, 22]

Summary

Introduction

Multiple reports have demonstrated a role for ceramide kinase (CERK) in the production of eicosanoids. We demonstrate that there are significant differences in eicosanoid levels in ex vivo CERK؊/؊ cells compared with wild-type counterparts, but the effect of the genetic ablation of CERK on eicosanoid synthesis and the serum levels of C1P was not apparent in vivo.—Mietla, J. The biosynthesis of eicosanoids is triggered in response to a variety of inflammatory signals (e.g., cytokines, growth factors, mechanical trauma, etc.) in which AA is liberated from the sn-2 position of membrane phospholipids by a phospholipase A2 enzyme. In most cases, this initial ratelimiting step in eicosanoid biosynthesis is started by the activation of group IVA phospholipase A2 (cPLA2␣) [10].

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