Alteration in the activation state of new inflammation-associated targets by phospholipase A2-activating protein (PLAA)

Abstract

Phospholipase A 2 (PLA 2 )-activating protein (PLAA) is a novel signaling molecule that regulates the production of prostaglandins (PGE 2 ) and tumor necrosis factor (TNF)-α. To characterize the function of native PLAA in situ, we generated HeLa (Tet-off) cells overexpressing plaa (plaa high ) and control (plaa low ) cells, with the plaa gene in opposite orientation in the latter construct. The plaa high cells produced significantly more PGE 2 and interleukin (IL)-6 compared to plaa low cells in response to TNF-α. There was increased activation and/or expression of cytosolic PLA 2 , cyclooxgenase-2, and NF-κB after induction of plaa high cells with TNF-α compared to the respective plaa low cells. Microarray analysis of plaa high cells followed by functional assays revealed increased production of proinflammatory cytokine IL-32 and a decrease in the production of annexin A4 and clusterin compared to plaa low cells. We demonstrated the role of annexin A4 as an inhibitor of PLA 2 and showed that addition of exogeneous clusterin limited the production of PGE 2 from plaa high cells. To understand regulation of plaa gene expression, we used a luciferase reporter system in HeLa cells and identified one stimulatory element, with Sp1 binding sites, and one inhibitory element, in exon 1 of the plaa gene. By using decoy DNA oligonucleotides to Sp1 and competitive binding assays, we showed that Sp1 maintains basal expression of the plaa gene and binds to the above-mentioned stimulatory element. We demonstrated for the first time that the induction of native PLAA by TNF-α can perpetuate inflammation by enhancing activation of PLA 2 and NF-κB.

Phospholipase A2-activating protein is associated with a novel form of leukoencephalopathy

“…We previously observed that PLAA regulated NF-kBmediated inflammatory responses, and in particular inducible interleukin (IL)-6 (Zhang et al, 2008). Herein, we observed that p.Leu752Phe variation in PLAA abrogated expression of LPS induced IL-6, IL-8, and macrophage migration inhibitory factor (MIF)-encoding genes in patient fibroblasts when compared to fibroblasts from a representative healthy control based on RT-qPCR ( Fig.…”

“…et al, 1991; Ribardo et al, 2002). Induction of PLA 2 is highly regulated by mitogen-activated protein kinases and NF-kB (Zhang et al, 2008). PLA 2 hydrolyses membrane phospholipids to produce arachidonic acid, which is used as a substrate to produce prostaglandins and leukotrienes (eicosanoids) through cyclooxygenase and lipoxygenase pathways, respectively (Ribardo et al, 2002).…”

“…Cell biology studies NF-iB recruitment to the nucleus is unaffected by the p.Leu752Phe mutation in PLAA We investigated the NF-iB signalling pathway which is known to be regulated by PLAA (Zhang et al, 2008) and showed that it was intact in both fibroblasts from patients and healthy controls ( Supplementary Fig. 1A and B).…”

“…Consequently, we examined other known functions of the PLAA protein. PLAA induces PGE 2 production by increasing levels of PLA 2 and cyclooxygenase (COX)-2 proteins, two major regulators of prostaglandins (Calignano et al, 1991;Zhang et al, 2008). Investigating this function, we measured PGE 2 levels in our patients' fibroblasts.…”

“…Membrane fractions from healthy controls and patients unstimulated and LPS-stimulated fibroblasts were isolated using established procedures (Zhang et al, 2008). Protein concentrations in membrane fractions were determined using Bradford Protein Reagent (Bio-Rad).…”