Background: Cellular stress leading to cell death induces the formation of lipid droplets. Results: Nutrient deprivation induces LD biogenesis and mobilization, fueling fatty acid oxidation to sustain cell viability. Conclusion: -Oxidation requires biogenesis and mobilization of LD. Significance: The role of LD in cell survival and -oxidation might provide new potential targets for antitumor therapy.
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 ␣). Knocking down cPLA 2 ␣ 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 ␣ 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 ␣ at Ser 505 . Transfection of a S505A mutant cPLA 2 ␣ showed that phosphorylation at Ser 505 is key for enzyme activity and LD formation. cPLA 2 ␣ contribution to LD biogenesis was not because of the generation of arachidonic acid, nor was it related to neutral lipid synthesis. cPLA 2 ␣ 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 ␣ in the formation of nascent LD from the endoplasmic reticulum.
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 ␣). The results demonstrate that iPLA 2 -VIA provides fatty acids for TAG synthesis while cPLA 2 ␣ allows LD biogenesis. LD biogenesis during stress may be a survival strategy, recycling structural phospholipids into energy-generating substrates.
The biogenesis of lipid droplets (LD) induced by serum depends on group IVA phospholipase A 2 (cPLA 2 ␣). This work dissects the pathway leading to cPLA 2 ␣ 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 ␣, 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 ␣ 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 ␣ at Ser-505 and formation of LD, and its down-regulation blocked the phosphorylation of cPLA 2 ␣ and LD biogenesis. These results demonstrate that LD biogenesis induced by serum is regulated by JNK and ceramide kinase.
Although reactive oxygen species (ROS) are conventionally viewed as toxic by-products of cellular metabolism, a growing body of evidence suggests that they may act as signaling molecules. We have studied the effects of hydrogen peroxide (H 2 O 2 )-induced oxidative stress on phospholipid signaling in cultured rat cortical astrocytes. H 2 O 2 stimulated the formation of phosphatidic acid and the accumulation of phosphatidylbutanol, a product of the phospholipase D (PLD)-catalyzed transphosphatidylation reaction. The effect of exogenous H 2 O 2 on the PLD response was mimicked by menadioneinduced production of endogenous H 2 O 2 . Oxidative stress also elicited inositol phosphate accumulation resulting from phosphoinositide phospholipase C (PLC) activation. The PLD response to H 2 O 2 was totally suppressed by chelation of both extracellular and cytosolic Ca 2ϩ with EGTA and BAPTA/AM, respectively. Furthermore, H 2 O 2 -induced PLD stimulation was completely abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide and chelerythrine and by PKC down-regulation. Activation of PLD by H 2 O 2 was also inhibited by the protein-tyrosine kinase inhibitor genistein. Finally, H 2 O 2 also stimulated both PLC and PLD in rat brain cortical slices. These results show for the first time that oxidative stress elicits phospholipid breakdown by both PLC and PLD in rat cultured astrocytes and brain slices.
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