Crosstalk between ORMDL3, serine palmitoyltransferase, and 5-lipoxygenase in the sphingolipid and eicosanoid metabolic pathways.

Abstract Leukotrienes and sphingolipids are critical lipid mediators participating in numerous cellular signal transduction events and developing various disorders, such as bronchial hyperactivity leading to asthma. Enzymatic reactions initiating production of these lipid mediators involve 5-lipoxygenase (5-LO)-mediated conversion of arachidonic acid to leukotrienes and serine palmitoyltransferase (SPT)-mediated de novo synthesis of sphingolipids. Previous studies have shown that ER membrane protein ORMDL3 inhibits the activity of SPT and subsequent sphingolipid synthesis. However, the role of ORMDL3 in the synthesis of leukotrienes is not known. In this study, we used peritoneal-derived mast cells (PDMCs) isolated from ORMDL3 knockout (KO) or control mice and examined their calcium mobilization, degranulation, IκB-α phosphorylation, and TNF-α production. We found that PDMCs with ORMDL3 KO exhibited increased responsiveness to antigen. Detailed lipid analysis showed that compared to wild-type cells, ORMDL3-deficient cells exhibited not only enhanced production of sphingolipids, but also of leukotriene (LT) signaling mediators LTB4, 6t-LTB4, LTC4, LTB5, and 6t-LTB5. The crosstalk between ORMDL3 and 5-LO metabolic pathways was supported by the finding that endogenous ORMDL3 and 5-LO are localized in similar ER domains in human mast cells and that ORMDL3 physically interacts with 5-LO. Further experiments showed that 5-LO also interacts with the long-chain (LC)1 and LC2 subunits of SPT. In agreement with these findings, 5-LO knockdown increased ceramide levels and silencing of SPTLC1 decreased arachidonic acid metabolism to leukotrienes to levels observed upon 5-LO knockdown. These results demonstrate functional crosstalk between the leukotriene and sphingolipid metabolic pathways, leading to the production of lipid signaling mediators.