JNK and ceramide kinase govern the biogenesis of lipid droplets through activation of group IVA phospholipase A2.

The biogenesis of lipid droplets (LD) induced by serum depends on group IVA phospholipase A(2) (cPLA(2)alpha). This work dissects the pathway leading to cPLA(2)alpha activation and LD biogenesis. Both processes were Ca(2+)-independent, as they took place after pharmacological blockade of Ca(2+) transients elicited by serum or chelation with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester). The single mutation D43N in cPLA(2)alpha, which abrogates its Ca(2+) binding capacity and translocation to membranes, did not affect enzyme activation and formation of LD. In contrast, the mutation S505A did not affect membrane relocation of the enzyme in response to Ca(2+) but prevented its phosphorylation, activation, and the appearance of LD. Expression of specific activators of different mitogen-activated protein kinases showed that phosphorylation of cPLA(2)alpha at Ser-505 is due to JNK. This was confirmed by pharmacological inhibition and expression of a dominant-negative form of the upstream activator MEKK1. LD biogenesis was accompanied by increased synthesis of ceramide 1-phosphate. Overexpression of its synthesizing enzyme ceramide kinase increased phosphorylation of cPLA(2)alpha at Ser-505 and formation of LD, and its down-regulation blocked the phosphorylation of cPLA(2)alpha and LD biogenesis. These results demonstrate that LD biogenesis induced by serum is regulated by JNK and ceramide kinase.

NAADP mediates ATP-induced Ca2+ signals in astrocytes.

Intracellular Ca(2+) signals provide astrocytes with a specific form of excitability that enables them to regulate synaptic transmission. In this study, we demonstrate that NAADP-AM, a membrane-permeant analogue of the new second messenger nicotinic acid-adenine dinucleotide phosphate (NAADP), mobilizes Ca(2+) in astrocytes and that the response is blocked by Ned-19, an antagonist of NAADP signalling. We also show that NAADP receptors are expressed in lysosome-related acidic vesicles. Pharmacological disruption of either NAADP or lysosomal signalling reduced Ca(2+) responses induced by ATP and endothelin-1, but not by bradykinin. Furthermore, ATP increased endogenous NAADP levels. Overall, our data provide evidence for NAADP being an intracellular messenger for agonist-mediated calcium signalling in astrocytes.

Lipid droplet biogenesis induced by stress involves triacylglycerol synthesis that depends on group VIA phospholipase A2.

This work investigates the metabolic origin of triacylglycerol (TAG) formed during lipid droplet (LD) biogenesis induced by stress. Cytotoxic inhibitors of fatty acid synthase induced TAG synthesis and LD biogenesis in CHO-K1 cells, in the absence of external sources of fatty acids. TAG synthesis was required for LD biogenesis and was sensitive to inhibition and down-regulation of the expression of group VIA phospholipase A(2) (iPLA(2)-VIA). Induction of stress with acidic pH, C(2)-ceramide, tunicamycin, or deprivation of glucose also stimulated TAG synthesis and LD formation in a manner dependent on iPLA(2)-VIA. Overexpression of the enzyme enhanced TAG synthesis from endogenous fatty acids and LD occurrence. During stress, LD biogenesis but not TAG synthesis required phosphorylation and activation of group IVA PLA(2) (cPLA(2)alpha). The results demonstrate that iPLA(2)-VIA provides fatty acids for TAG synthesis while cPLA(2)alpha allows LD biogenesis. LD biogenesis during stress may be a survival strategy, recycling structural phospholipids into energy-generating substrates.

Group IVA phospholipase A2 is necessary for the biogenesis of lipid droplets.

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.