Group IVA Phospholipase A2 Is Necessary for the Biogenesis of Lipid Droplets

Miquel Barceló-Torns,Xavier de la Rosa,Fernando Picatoste,Enrique Claro,Jesús Balsinde,David Barneda,María A. Balboa,Roser Masgrau,Albert Gubern,Javier Casas

doi:10.1074/jbc.m800696200

Abstract

Lipid droplets (LD) are organelles present in all cell types, consisting of a hydrophobic core of triacylglycerols and cholesteryl esters, surrounded by a monolayer of phospholipids and cholesterol. This work shows that LD biogenesis induced by serum, by long-chain fatty acids, or the combination of both in CHO-K1 cells was prevented by phospholipase A(2) inhibitors with a pharmacological profile consistent with the implication of group IVA cytosolic phospholipase A(2) (cPLA(2)alpha). Knocking down cPLA(2)alpha expression with short interfering RNA was similar to pharmacological inhibition in terms of enzyme activity and LD biogenesis. A Chinese hamster ovary cell clone stably expressing an enhanced green fluorescent protein-cPLA(2)alpha fusion protein (EGFP-cPLA(2)) displayed higher LD occurrence under basal conditions and upon LD induction. Induction of LD took place with concurrent phosphorylation of cPLA(2)alpha at Ser(505). Transfection of a S505A mutant cPLA(2)alpha showed that phosphorylation at Ser(505) is key for enzyme activity and LD formation. cPLA(2)alpha contribution to LD biogenesis was not because of the generation of arachidonic acid, nor was it related to neutral lipid synthesis. cPLA(2)alpha inhibition in cells induced to form LD resulted in the appearance of tubulo-vesicular profiles of the smooth endoplasmic reticulum, compatible with a role of cPLA(2)alpha in the formation of nascent LD from the endoplasmic reticulum.

Highlights

(TAG) and cholesteryl esters, and are surrounded by a monolayer of phospholipids and cholesterol with which a variety of proteins interact [1,2,3]
We examined various cell models for the occurrence of Lipid droplets (LD), which we labeled with the lipophilic dye Nile red
CPLA2 Is Not Involved in the Synthesis of Neutral Lipids during LD Biogenesis—To test whether the role of Cytosolic phospholipase A2 (cPLA2) in the biogenesis of LD is to provide AA for neutral lipid synthesis (TAG and cholesteryl esters), we assayed the ability of exogeinhibited by methylarachidonyl fluorophosphonate (MAFP) (Fig. 4E). These results show nous AA to induce LD in serum-starved cells and to stimthat overexpression of cPLA2␣ enhances the occurrence of LD. cPLA2␣ Phosphorylation at Ser505 Is Required for the Formation of LD—To determine whether phosphorylation of cPLA2␣ at Ser505 is relevant for enzyme activity and LD biogenesis, we ulate cPLA2␣ (Fig. 6)

Summary

Introduction

(TAG) and cholesteryl esters, and are surrounded by a monolayer of phospholipids and cholesterol with which a variety of proteins interact [1,2,3]. Group IVA PLA2 and Lipid Droplet Biogenesis of these is acyl-CoA synthetase [23,24,25] Inhibition of this enzyme with triacsin C abolishes the formation of LD in cells undergoing apoptosis [15], underlining the need for TAG synthesis in the genesis of new LD. Phospholipase D1 has been found associated with LD [26] and was shown to promote LD budding off from microsomes in a cell-free system, in a manner requiring TAG synthesis and an unidentified cytosolic factor [27]. This factor was later identified as ERK2, working apparently downstream of phospholipase D1 to induce dynein association with LD [28]. Cytosolic phospholipase A2 (cPLA2), on the other hand, has been reported associated to LD [29, 30], its implication in their biogenesis is not clear [31]

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Conclusion