INTERLEUKIN-1-BETA INDUCES THE SYNTHESIS AND ACTIVITY OF CYTOSOLIC PHOSPHOLIPASE A(2) AND THE RELEASE OF PROSTAGLANDIN E(2) IN HUMAN AMNION-DERIVED WISH CELLS

The objective of this study was to examine the expression and activity of cytosolic phospholipase A(2) (cPLA(2)) in relation to prostaglandi n E(2) (PGE(2)) synthesis in human amnion-derived WISH cells in respon se to stimulation by interleukin-1 beta (IL-1 beta). cPLA(2) activity was characterized by sensitivity to heat and acid treatment, stability to dithiothreitol, and inhibition by the specific inhibitor, arachido nyl trifluoromethyl ketone (AACOCF(3)). Treatment of WISH cells with I L-1 beta (0.01-1 ng/mL) for up to 24 h resulted in a significant incre ase in PGE(2) release in a concentration- and time-dependent manner ac companied by increases both in total cellular cPLA(2) activity and in cPLA(2) protein levels detected by Western blot analysis. The parallel increase in total cellular cPLA(2) activity and cPLA(2) protein level indicates that IL-1 beta may induce the synthesis of cPLA,. Incubatio n of the cells with 10 mu M AACOCF(3) for 24 h significantly inhibited IL-1 beta-induced PGE(2) production strongly suggesting that cPLA(2) mediates IL-1 beta-induced PGE(2) formation. In unstimulated cells, th ere is appreciable total cellular cPLA(2) activity and protein, but th ese cells produce low amounts of PGE, until stimulated by IL-1 beta, s uggesting that cPLA(2) translocation from cytosol to the membrane is n ecessary for its bioactivity. in contrast to IL-1 beta, treatment with phorbol ester (12-O-tetradecanoyl phorbol-13-acetate, TPA, 10(-10)-10 (-6) M) for 24 h significantly inhibited total cellular cPLA(2) activi ty in a concentration-dependent manner. The amount of total cellular c PLA, protein seen on Western blot remained unchanged following TPA tre atment. These data suggest that in WISH cells, IL-1 beta induces both translocation to the membrane and de novo synthesis of cPLA(2) protein to sustain prostaglandin (PG) synthesis. In contrast, TPA may only ca use cPLA(2) translocation but no increase in cPLA(2) protein synthesis , resulting in limited PG synthesis. Our results provide a mechanism f or the effect of IL-1 beta on prostaglandin synthesis in human amnion cells and provide support for a role of cPLA(2) in the mechanism initi ating human parturition.