Alkylglycerol monooxygenase as a potential modulator for PAF synthesis in macrophages

https://doi.org/10.1016/j.bbrc.2013.05.099Get rights and content

Highlights

  • LPS decreases AGMO expression in macrophages.

  • Exogenous expression of AGMO causes reduction of cellular lyso-PAF and PAF levels.

  • AGMO may modulate macrophage PAF production by regulating cellular lyso-PAF levels.

Abstract

Alkylglycerol monooxygenase (AGMO, glyceryl ether monooxygenase) is an enzyme known to catalyze the cleavage of the O-alkyl bond of glyceryl ether lipids. Identification of the gene encoding AGMO was reported recently, however, the involvement of AGMO in modulating cellular lipids has not been reported until now. In this report, we investigate a possible role for AGMO in macrophage platelet-activating factor (PAF) production. AGMO mRNA expression levels decreased with lipopolysaccharide (LPS) treatments in mouse peritoneal macrophages and RAW264.7 cells. Tetrahydrobiopterin-dependent conversion of lyso-PAF to glycerophosphocholine in the microsomal fraction was also reduced in LPS-treated RAW264.7 cells. In the LPS-treated cells, both lyso-PAF and PAF levels increased. Moreover, exogenously expressed AGMO caused a reduction in cellular lyso-PAF and PAF levels in HEK293 cells. Collectively, our results suggest a possible mechanism for AGMO in modulating macrophage PAF production by regulating cellular lyso-PAF levels.

Graphical abstract

Proposed AGMO function in modulation of macrophage PAF levels. Left panel, in resting conditions, AGMO expression is high and lyso-PAF is effectively degraded by AGMO. Right panel, after stimulation with LPS, AGMO levels decrease. Consequently, degradation of lyso-PAF is attenuated and PAF production is enhanced in activated macrophages.

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Introduction

Alkylglycerol monooxygenase (AGMO, glyceryl ether monooxygenase, EC 1.14.16.5) is the only enzyme, so far, known to catalyze the oxidative cleavage of O-alkyl bond of ether lipids. The enzymatic reaction was first described in 1964 as a tetrahydrobiopterin (BH4, (6R)-5,6,7,8-tetrahydro-l-biopterin)-requiring enzyme from rat liver homogenates [1], and later, several detailed studies reported tissue distribution, substrate specificity, and apparent kinetics with the BH4 cofactor [2], [3]. In 2010, using an in silico database search and the recombinant gene expression of selected candidates, Watschinger et al. [4] reported that a gene with an unassigned function, transmembrane protein 195 (TMEM195), encodes AGMO. Alkylglycerols and their various metabolites, including ether phospholipids, are believed to have important biological roles as membrane components and mediators of cell responses [5], [6], [7]. However, the molecular basis of the alkylglycerolipid function, as well as the importance of AGMO in regulating metabolite levels, remain unclear.

Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a phosphatidylcholine (PC) with O-alkyl (typically O-hexadecyl) and acetyl groups at sn-1 and sn-2 positions, respectively. PAF is a potent phospholipid mediator that activates a G protein-coupled receptor (PAFR) and causes pleiotropic biological effects, including inflammation, platelet activation, airway constriction, hypotension, and hypersensitive reactions in allergy [8]. PAF is synthesized in tissues and cells via two distinct pathways, the de novo and remodeling pathways [9]. The de novo pathway is thought to maintain physiological levels of PAF for normal cellular functions, whereas the remodeling pathway is activated by inflammatory stimuli and is thought to be the primary source of PAF under pathological conditions. Induction of PAF synthesis by various extracellular stimuli has been reported in peritoneal and alveolar macrophages, polymorphonuclear neutrophils (PMN), vascular endothelial cells, basophils, and bone marrow-derived mast cells [8].

Watschinger et al. [4] reported that AGMO mRNA expression level and enzymatic activity are high in the murine macrophage cell line RAW264.7. Stimulation of RAW264.7 cells or mouse peritoneal macrophages with lipopolysaccharide (LPS) induces enhanced production of PAF in the remodeling pathway through two enzymes, lysophosphatidylcholine acyltransferase 2 (LPCAT2, EC 2.3.1.23 and EC 2.3.1.67) and phospholipases A2 (PLA2, EC 3.1.1.4) [10], [11]. Among PLA2s, cytosolic PLA2α (cPLA2α, group IVA PLA2) is a key enzyme for lyso-PAF (1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine) production in inflammatory cells, the major source of PAF production [11], [12]. Lyso-PAF may either be converted to PAF or transformed back to PC by LPCAT2 [10]. PAF is then rapidly degraded to lyso-PAF by PAF acetylhydrolases (PAFAH) [13], [14]. AGMO is the only identified enzyme that can irreversibly cleave the O-alkyl bond of lyso-PAF to produce glycerophosphocholine (GPC) [2], [3], [4]. Thus, it is possible that this enzyme essentially eliminates lyso-PAF from the recycling system of lipid pool.

In this study, we investigated a possible role for AGMO in the modulation of macrophage PAF levels. The enzyme is primarily expressed in macrophages, and the expression as well as enzymatic activity is decreased by LPS stimulation, which coincides with increased cellular lyso-PAF and PAF levels. Exogenous expression of AGMO causes a reduction in cellular lyso-PAF and PAF levels. To our knowledge, this is the first report to demonstrate the biological importance of AGMO.

Section snippets

Reagents

PAF, lyso-PAF, PAF-d4 (1-O-hexadecyl-(7,7,8,8-d4)-2-acetyl-sn-glycero-3-phosphorylcholine), and lyso-PAF-d4 (1-O-hexadecyl-(7,7,8,8-d4)-2-lyso-sn-glycero-3-phosphorylcholine) were purchased from Cayman Chemical (Ann Arbor, MI). Oligonucleotide primers were from Sigma–Aldrich (St. Louis, MO) and Greiner Bio-One (Frickenhausen, Germany). LPS, from Salmonella minnesota, was purchased from Sigma–Aldrich. ODN1826 and poly(I:C)-LMW were from InvivoGen (San Diego, CA). Other reagents were from Wako

Downregulation of AGMO following LPS stimulation in mouse peritoneal macrophages and RAW264.7 cells

We first examined the effects of LPS treatment on the expression levels of AGMO in thioglycollate-induced peritoneal macrophages and RAW264.7 cells. LPS, a TLR4 ligand, is one of the major activators of macrophages and induces enhanced production of PAF in the remodeling pathway. We found that AGMO mRNA expression was reduced following LPS-stimulation in both peritoneal macrophages and RAW264.7 cells (Fig. 1A and B). We also examined the effect of the TLR3 ligand (poly(I:C)) and the TLR9 ligand

Discussion

In the present report, we demonstrated a possible role for AGMO in macrophage PAF production. BH4-dependent AGMO activity was first reported in 1964 using batyl alcohol (1-O-octadecylglycerol) as a substrate [1]. Later, substrate specificity of AGMO was investigated in vitro, and lyso-PAF was considered as a biological substrate candidate for AGMO [2], [3]. Although AGMO could cleave the O-alkyl bond of PAF itself in vitro, previous studies showed that BH4-dependent enzymatic activity was much

Acknowledgments

This work was supported by MEXT KAKENHI grant numbers 23790356, 23790324, and 24229003. We are grateful to Dr. H. Ichinose of the Tokyo Institute of Technology and Dr. T. Harayama of the University of Tokyo for valuable suggestions. We thank Dr. M. Yamada, Dr. W. Valentine and Ms. A. Kobayashi for assistance and Dr. J. Miyazaki (Osaka University) for supplying the expression vector, pCXN2.

References (28)

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