Role of Neutral Sphingomyelinase-2 (NSM 2) in the Control of T Cell Plasma Membrane Lipid Composition and Cholesterol Homeostasis.

Charlene Börtlein,Burkhard Kleuser,Fabian Schumacher,Lars Dölken,Elita Avota

doi:10.3389/fcell.2019.00226

Abstract

The activity of neutral sphingomyelinase-2 (NSM2) to catalyze the conversion of sphingomyelin (SM) to ceramide and phosphocholine at the cytosolic leaflet of plasma membrane (PM) is important in T cell receptor (TCR) signaling. We recently identified PKCζ as a major NSM2 downstream effector which regulates microtubular polarization. It remained, however, unclear to what extent NSM2 activity affected overall composition of PM lipids and downstream effector lipids in antigen stimulated T cells. Here, we provide a detailed lipidomics analyses on PM fractions isolated from TCR stimulated wild type and NSM2 deficient (ΔNSM) Jurkat T cells. This revealed that in addition to that of sphingolipids, NSM2 depletion also affected concentrations of many other lipids. In particular, NSM2 ablation resulted in increase of lyso-phosphatidylcholine (LPC) and lyso-phosphatidylethanolamine (LPE) which both govern PM biophysical properties. Crucially, TCR dependent upregulation of the important T cell signaling lipid diacylglycerol (DAG), which is fundamental for activation of conventional and novel PKCs, was abolished in ΔNSM cells. Moreover, NSM2 activity was found to play an important role in PM cholesterol transport to the endoplasmic reticulum (ER) and production of cholesteryl esters (CE) there. Most importantly, CE accumulation was essential to sustain human T cell proliferation. Accordingly, inhibition of CE generating enzymes, the cholesterol acetyltransferases ACAT1/SOAT1 and ACAT2/SOAT2, impaired TCR driven expansion of both CD4+ and CD8+ T cells. In summary, our study reveals an important role of NSM2 in regulating T cell functions by its multiple effects on PM lipids and cholesterol homeostasis.

Highlights

Neutral sphingomyelinase-2 (NSM2) is the best studied NSM of the four mammalian sphingomyelinases that are active at neutral pH
We found mitochondrial proteins in PMs isolated as the giant plasma membrane vesicles (GPMVs) demonstrating objective technical difficulties fully separate organelle and PMs hinting at their close physical communication (Supplementary Figure S1)
Independent measurement of PM ceramide 18:1;2/17:0 (Cer) was performed in parallel at the University of Potsdam and here detected ceramide concentrations and species were similar to those measured by Lipotype (Supplementary Figure S2A)

Summary

Introduction

Neutral sphingomyelinase-2 (NSM2) is the best studied NSM of the four mammalian sphingomyelinases that are active at neutral pH. It is intensively involved in cellular physiology and pathology (Shamseddine et al, 2015). Neutral Sphingomyelinase Regulates Cholesterol Homeostasis catalytic site (Hofmann et al, 2000). NSM2-catalyzed sphingomyelin breakdown commonly occurs in response to cellular stress and regulates bone mineralization. The role of NSM2 in cytokine (IL1-β, TNF-α, and IFN-γ) induced inflammation and bacterial infections is well established (Shamseddine et al, 2015; Wu et al, 2018) as is its role in cytotoxic effects of cancer chemotherapeutics. The involvement in pathogenesis and perpetuation of human tumors seems to be tumor specific (Bhati et al, 2008; Kim et al, 2008; Henry et al, 2013)

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