Defense Date

2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biochemistry

First Advisor

Charles E. Chalfant

Abstract

In the presented study, we demonstrate that the interaction of group IVA cytosolic phospholipase A2 and ceramide-1-phosphate is crucial for production of eicosanoid synthesis in inflammation. Inflammation is a critical component of many disease states including anaphylaxis, cancer, cardiovascular disease, rheumatoid arthritis, diabetes and asthma. Eicosanoids are well established mediators of inflammation, and the initial rate limiting step in the production of eicosanoids is the liberation of arachidonic acid (AA) from membrane phospholipids by a phospholipase A2 (PLA2). The major phospholipase involved in this liberation of AA during the inflammatory response is group IVA cytosolic phospholipase A2 (cPLA2α). Previous studies from our laboratory demonstrated that the bioactive sphingolipid, ceramide-1-phosphate (C1P), binds cPLA2α at a three amino acid sequence, which is located in the cationic β-groove of the C2 domain of cPLA2α. In this study we examined the effects of the genetic ablation of ceramide kinase (CERK) on eicosanoid synthesis, as CERK is the only known enzyme to produce C1P in mammalian systems. We utilized primary mouse fibroblasts (MEFs) and macrophages isolated from CERK-/- and +/+ mice. The ceramide-1-phosphate and eicosanoid profiles were investigated, and both ceramide-1-phosphate and eicosanoid levels in CERK-/- MEFs were found to be dysregulated. This study also presents the development of a global eicosanoid method to analyze eicosanoids via LC-ESI-MS/MS. Using this new analysis method, we demonstrated that there are significant differences in eicosanoid levels in ex vivo CERK-/- cells when compared to 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.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

2-11-2014

Available for download on Monday, February 11, 2019

Share

COinS