Involvement of Group VI Ca2+-independent Phospholipase A2 in Protein Kinase C-dependent Arachidonic Acid Liberation in Zymosan-stimulated Macrophage-like P388D1 Cells

Satoshi Akiba,Shingo Mizunaga,Keisuke Kume,Misako Hayama,Takashi Sato

doi:10.1074/jbc.274.28.19906

Satoshi Akiba, Shingo Mizunaga + Show 3 more

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https://doi.org/10.1074/jbc.274.28.19906

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Abstract

We investigated the possible involvement of group VI Ca2+-independent phospholipase A2 (iPLA2) in arachidonic acid (AA) liberation in zymosan-stimulated macrophage-like P388D1 cells. Zymosan-induced AA liberation was markedly inhibited by methyl arachidonoyl fluorophosphonate, a dual inhibitor of group IV cytosolic phospholipase A2 (cPLA2) and iPLA2. We found that a relatively specific iPLA2 inhibitor, bromoenol lactone, significantly decreased the zymosan-induced AA liberation in parallel with the decrease in iPLA2 activity, without an effect on diacylglycerol formation. Consistent with this, attenuation of iPLA2 activity by a group VI iPLA2 antisense oligonucleotide resulted in a decrease in zymosan-induced prostaglandin D2 generation. These findings suggest that zymosan-induced AA liberation may be, at least in part, mediated by iPLA2. A protein kinase C (PKC) inhibitor diminished zymosan-induced AA liberation, while a PKC activator, phorbol 12-myristate 13-acetate (PMA), enhanced the liberation. Bromoenol lactone suppressed the PMA-enhanced AA liberation without any effect on PMA-induced PKC activation. Down-regulation of PKCalpha on prolonged exposure to PMA also decreased zymosan-induced AA liberation. Under these conditions, the remaining AA liberation was insensitive to bromoenol lactone. Furthermore, the PKC depletion suppressed increases in iPLA2 proteins and the activity in the membrane fraction of zymosan-stimulated cells. In contrast, the zymosan-induced increases in iPLA2 proteins and the activity in the fraction were facilitated by simultaneous addition of PMA. Although intracellular Ca2+ depletion prevented zymosan-induced AA liberation, the translocation of PKCalpha to membranes was also inhibited. Taken together, we propose that zymosan may stimulate iPLA2-mediated AA liberation, probably through a PKC-dependent mechanism.

Highlights

Effect of an independent phospholipase A2 (iPLA2) Inhibitor on Zymosan-stimulated Lipid Metabolism—As shown in Fig. 1, stimulation of [3H]arachidonic acid (AA)-labeled P388D1 cells with 1 mg/ml zymosan elicited time-dependent AA liberation, of which the time course was similar to that observed in mouse peritoneal macrophages [23]
The iPLA2 activity in the lysate of bromoenol lactone (BEL)-treated P388D1 cells was inhibited in a BEL dose-dependent manner (Fig. 2A), while cytosolic phospholipase A2 (cPLA2) activity was not affected by 10 ␮M BEL (297 pmol/ min/mg protein for controls and 285 pmol/min/mg protein for BEL treatment, means of two experiments)
We explored the possible involvement of iPLA2 in stimulus-induced AA liberation using zymosan-stimulated mouse macrophage-like P388D1 cells, which possess group VI iPLA2, one of the purified and sequenced iPLA2s [11, 13]

Summary

Introduction

IPLA2 in AA liberation in zymosan-stimulated P388D1 cells by evaluating the effects of BEL and a group VI iPLA2 antisense oligonucleotide, which have been shown to attenuate iPLA2 activity in P388D1 cells [16, 20]. Even on treatment of the membrane fraction of PMA-treated cells with 20 ␮M BEL, the PKC activity in the fraction was not affected (data not shown).

Results

Conclusion