Phospholipase A2 (PLA2)-activating protein (PLAA) is a novel signaling molecule that regulates the production of prostaglandins (PGE2) and tumor necrosis factor (TNF)-α. To characterize the function of native PLAA in situ, we generated HeLa (Tet-off) cells overexpressing plaa (plaahigh) and control (plaalow) cells, with the plaa gene in opposite orientation in the latter construct. The plaahigh cells produced significantly more PGE2 and interleukin (IL)-6 compared to plaalow cells in response to TNF-α. There was an increased activation and/or expression of cytosolic PLA2, cyclooxgenase-2, and NF-κB after induction of plaahigh cells with TNF-α compared to the respective plaalow cells. Microarray analysis of plaahigh 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 plaalow cells. We demonstrated the role of annexin A4 as an inhibitor of PLA2 and showed that addition of exogeneous clusterin limited the production of PGE2 from plaahigh 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 PLA2 and NF-κB.
- Annexin A4
- Phospholipase A-activating protein
ASJC Scopus subject areas
- Cell Biology