Lipin-1 controls phospholipase A2 activation in human macrophages

Abstract

[Introduction]: The lipin family of proteins plays multiple roles in cells by regulating lipid biosynthesis and cellular signaling. Lipins possess phosphatidic acid (PA) phosphatase activity that hydrolyzes PA to yield diacylglycerol (DAG). Both PA and DAG are important signaling lipids, having the capacity to modulate key signaling events. Most cellular studies on lipin have been conducted in metabolically active tissues such as adipose tissue. As a matter of fact, Lpin-1 is known as an obesity-related gene because its absence promotes the lack of adipose tissue and its overexpression promotes obesity in mice. However, little is known on the possible roles of lipin in inflammation. Before the nucleotide sequence of the PAP1 enzymes was revealed, studies with pharmacological inhibitors showed that PAP1 activity is involved in the regulation of the mobilization of free arachidonic acid (AA), the precursor of the inflammatory mediators known as the eicosanoids. However, identification of the actual PAP1/lipin form involved in AA mobilization has not been elucidated. [Objectives]: The aim of the present study was evaluate the role of lipin-1 in Phospholipase A2 (PLA2) activation.[Methods]: Human macrophages derived from blood monocytes, siRNA technology, mass-spectrometry and confocal microscopy was used.

[Results]: Human macrophages made deficient in lipin-1 by siRNA mobilized significantly less AA than did control cells upon treatment with innate immunity and metabolic stimulus. AA mobilization under these conditions was abolished by treating the cells with the selective inhibitor for Group IVA PLA2, pyrrophenone (1 mM). Furthermore, Group IVA PLA2 phosphorylation was found to be significantly reduced (p<0.05) in homogenates from cells deficient in lipin-1, as measured by immunoblot using an Ab specific for the phosphorylated protein. In keeping with these observations, measurements of PGE2 formation demonstrated that cells deficient in lipin-1 manifest a marked defect in eicosanoid generation. The distribution of AA among glycerophospholipids in untreated versus lipin-1–deficient cells was studied by mass spectrometry. The results demonstrated that lipin-1 deficiency did not significantly alter the distribution of AA among phospholipids in resting cells, thus suggesting that diminished Group IVA PLA2 activation in the lipin-1–deficient cells is unlikely due to altered availability of substrate. [Conclusions]: Collectively, these data suggest that lipin-1 regulates the activation of cytosolic group IVA phospholipase A2 in human monocyte-derived macrophages.