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)-alpha. 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-alpha. There was an increased activation and/or expression of cytosolic PLA(2), cyclooxgenase-2, and
NF-kappaB after induction of plaa(high) cells with
TNF-alpha 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-alpha can perpetuate
inflammation by enhancing activation of PLA(2) and
NF-kappaB.