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

Viktor Bugajev,Tomas Paulenda,Pavol Utekal,Michal Mrkacek,Ivana Halova,Ladislav Kuchar,Ondrej Kuda,Petra Vavrova,Björn Schuster,Sergio Fuentes-Liso,Lucie Potuckova,Daniel Smrz,Sara Cernohouzova,Lubica Draberova,Monika Bambouskova,Petr Draber

doi:10.1016/j.jlr.2021.100121

Abstract

Leukotrienes (LTs) 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 LTs and serine palmitoyltransferase (SPT)-mediated de novo synthesis of sphingolipids. Previous studies have shown that endoplasmic reticulum membrane protein ORM1-like protein 3 (ORMDL3) inhibits the activity of SPT and subsequent sphingolipid synthesis. However, the role of ORMDL3 in the synthesis of LTs is not known. In this study, we used peritoneal-derived mast cells isolated from ORMDL3 KO or control mice and examined their calcium mobilization, degranulation, NF-κB inhibitor-α phosphorylation, and TNF-α production. We found that peritoneal-derived mast cells with ORMDL3 KO exhibited increased responsiveness to antigen. Detailed lipid analysis showed that compared with WT cells, ORMDL3-deficient cells exhibited not only enhanced production of sphingolipids but also of 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 endoplasmic reticulum 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 1 and long-chain 2 subunits of SPT. In agreement with these findings, 5-LO knockdown increased ceramide levels, and silencing of SPTLC1 decreased arachidonic acid metabolism to LTs to levels observed upon 5-LO knockdown. These results demonstrate functional crosstalk between the LT and sphingolipid metabolic pathways, leading to the production of lipid signaling mediators.

Highlights

L crosstalk between the LT and sphingolipid metabolic pathways, leading to the production of lipid signaling mediators
Antigen-mediated high-affinity IgE receptor (FcεRI) triggering in peritoneal-derived mast cell (PDMC) with ORM1-like protein 3 (ORMDL3) KO resulted in enhanced calcium mobilization (Fig. 1F) and degranulation measured by β-glucuronidase release (Fig. 1G)
We used PDMCs from mice with ORMDL3 KO and cell lines with enhanced or reduced expression of ORMDL3, 5-LO, or SPTLC1 to determine the role of these proteins in the metabolic pathways leading to the production of sphingolipids and eicosanoids

Summary

Introduction

L crosstalk between the LT and sphingolipid metabolic pathways, leading to the production of lipid signaling mediators. In vivo experiments with mice showed that ORMDL3 deficiency prevented development of Alternariainduced allergic airway response [24] and that enhanced expression of ORMDL3 resulted in increased levels of airway remodeling followed by an enhanced spontaneous response to methacholine [25]. These findings were confronted by Debeuf et al [26] using mice with enhanced or reduced ORMDL3 expression levels, exhibiting the expected changes in ceramides, but with no effects on experimental allergic asthma models. The complexity of ORMDL3 function in vivo was underlined by the study showing that selective deletion of ORMDL3 in the airway epithelium challenged with allergen unexpectedly exhibit increased airway hyperresponsiveness [27]

Methods

Results

Conclusion