Role of the JAK/STAT Pathway in the Regulation of Interleukin-8 Transcription by Oxidized Phospholipids in Vitro and in Atherosclerosis in Vivo

Nima M Gharavi,Jackelyn A Alva,Kevin P Mouillesseaux,Chi Lai,Michael Yeh,Winnie Yeung,Jaclyn Johnson,Wan Lam Szeto,Longsheng Hong,Michael Fishbein,Lai Wei,Lawrence M Pfeffer,Judith A Berliner

doi:10.1074/jbc.m704267200

Abstract

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC) and its component phospholipid, 1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine, induce endothelial cells (EC) to synthesize chemotactic factors, such as interleukin 8 (IL-8). Previously, we demonstrated a role for c-Src kinase activation in Ox-PAPC-induced IL-8 transcription. In this study, we have examined the mechanism regulating IL-8 transcription by Ox-PAPC downstream of c-Src. Our findings demonstrate an important role for JAK2 in the regulation of IL-8 transcription by Ox-PAPC. Treatment of human aortic EC with Ox-PAPC and 1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine induced a rapid yet sustained activation of JAK2; activation of JAK2 by Ox-PAPC was dependent on c-Src kinase activity. Furthermore, pretreatment with selective JAK2 inhibitors significantly reduced Ox-PAPC-induced IL-8 transcription. In previous studies, we also demonstrated activation of STAT3 by Ox-PAPC. Here we provide evidence that STAT3 activation by Ox-PAPC is dependent on JAK2 activation and that STAT3 activation regulates IL-8 transcription by Ox-PAPC in human EC. Transfection with small interfering RNA against STAT3 significantly reduced Ox-PAPC-induced IL-8 transcription. Using chromatin immunoprecipitation assays, we demonstrated binding of activated STAT3 to the sequence flanking the consensus gamma-interferon activation sequence (GAS) in the IL-8 promoter; site-directed mutagenesis of GAS inhibited IL-8 transcription by Ox-PAPC. Finally, these studies demonstrate a role for STAT3 activation in atherosclerosis in vivo. We found increased staining for activated STAT3 in the inflammatory regions of human atherosclerotic lesions and reduced fatty streak formation in EC-specific STAT3 knock-out mice on the atherogenic diet. Taken together, these data demonstrate an important role for the JAK2/STAT3 pathway in Ox-PAPC-induced IL-8 transcription in vitro and in atherosclerosis in vivo.

Highlights

Cardiovascular disease (CVD)2 is a major cause of morbidity and mortality in Western nations
This study suggests an important role for the c-Src/JAK2/ STAT3 pathway in atherosclerosis
We demonstrate that treatment of human aortic endothelial cells (HAEC) with Oxidized PAPC (Ox-PAPC) and PEIPC activates JAK2 rapidly, and that this activation is sustained for up to 4 h (Fig. 1, A and B)

Summary

Introduction

Cardiovascular disease (CVD)2 is a major cause of morbidity and mortality in Western nations. We demonstrated a role for c-Src kinase activation in Ox-PAPC-induced IL-8 transcription. Total levels of JAK2 remained unchanged at all time points observed no effect on phosphorylation of Tyr-418 of c-Src in following treatment with Ox-PAPC (Fig. 1A, middle panel) and response to OxPAPC (data not shown).

Results

Conclusion