Arachidonic Acid in Platelet Microparticles Up-regulates Cyclooxygenase-2-dependent Prostaglandin Formation via a Protein Kinase C/Mitogen-activated Protein Kinase-dependent Pathway

Orla P Barry,Marcelo G Kazanietz,Domenico Praticò,Garret A Fitzgerald

doi:10.1074/jbc.274.11.7545

Orla P Barry, Marcelo G Kazanietz + Show 2 more

Open Access

https://doi.org/10.1074/jbc.274.11.7545

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Abstract

Activation of platelets results in shedding of membrane microparticles (MP) with potentially bioactive properties. Platelet MP modulate platelet, monocyte, and vascular endothelial cell function, both by direct effects of MP arachidonic acid (AA) and by its metabolism to bioactive prostanoids. We have previously reported that platelet MP induce expression of cyclooxygenase (COX)-2 and prostacyclin production in monocytes and endothelial cells. To elucidate further the molecular mechanisms that underlie MP-induced up-regulation of COX-2 expression, we investigated the response of a human monocytoid (U-937) cell line to platelet MP stimulation. In U-937 cells, MP-induced COX-2 expression and eicosanoid formation is prevented by pharmacological inhibitors of protein kinase C (PKC), PI 3-kinase, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase, and p38 kinase. Treatment with the PI 3-kinase inhibitors wortmannin and LY294002 also blocked MP-induced p42/p44 MAPK, p38, and JNK1 phosphorylation. Conversely, platelet MP stimulation of U-937 cells results in direct activation of PKC, p42/p44 MAPK, p38 kinase, and c-Jun N-terminal kinase (JNK) as well as activation of the transcription factors c-Jun and Elk-1. However, MP failed to activate the cAMP response element. Activation of U-937 cells by MP induces translocation of classical (PKCbeta), novel (PKCdelta) and atypical (PKCzeta and PKClambda) isozymes of PKC from the cytosol to the membrane, with concomitant activation of downstream MAPK. While MP-induced activation of p42/p44 MAPK and p38 kinase is transient, a sustained activation of JNK1 was observed. Although PKC activation is required for MP-induced p42/p44 MAPK, activation of the stress kinases p38 and JNK1 was PKC-independent. The fatty acid fraction of the MP accounted for these effects, which were mimicked by MP AA. Rather than acting directly via nuclear receptors, MP AA activates COX-2-dependent prostaglandin production by a PKC/p42/p44 MAPK/p38 kinase-sensitive pathway in which PI 3-kinase plays a significant role. MP AA also stimulates transcriptional activation of COX-2 as well as c-Jun and Elk-1.

Highlights

In U-937 cells, MP-induced COX-2 expression and eicosanoid formation is prevented by pharmacological inhibitors of protein kinase C (PKC), PI 3-kinase, mitogenactivated protein kinase (MAPK)/extracellular signalregulated kinase, and p38 kinase
We have previously demonstrated that unmetabolized arachidonic acid (AA) in platelet MP modulates adhesive interactions between monocytes and endothelial cells (30), a cellular interaction thought to be of relevance in the early stages of atherogenesis (31)
Induction of Cyclooxygenase Activity by Platelet MP—Treatment of U-937 cells with MP induced the expression of COX-2, which was maximal at 12 h (Fig. 1A)

Summary

Introduction

In U-937 cells, MP-induced COX-2 expression and eicosanoid formation is prevented by pharmacological inhibitors of protein kinase C (PKC), PI 3-kinase, mitogenactivated protein kinase (MAPK)/extracellular signalregulated kinase, and p38 kinase. Platelet MP stimulation of U-937 cells results in direct activation of PKC, p42/p44 MAPK, p38 kinase, and c-Jun N-terminal kinase (JNK) as well as activation of the transcription factors c-Jun and Elk-1. Rather than acting directly via nuclear receptors, MP AA activates COX-2dependent prostaglandin production by a PKC/p42/p44 MAPK/p38 kinase-sensitive pathway in which PI 3-kinase plays a significant role. We have demonstrated that arachidonic acid (AA) in platelet MP can influence platelet activation in a PKC-dependent manner and endothelial cell COX-2 expression via transcellular lipid metabolism (13). The present studies were designed to delineate the signaling pathways involved in MPinduced COX-2 activation and prostaglandin formation. More recent findings link the prostaglandin biosynthetic pathways with activation of MAPK signaling cascades (19 –21)

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