Oxidized Phospholipids Increase Interleukin 8 (IL-8) Synthesis by Activation of the c-src/Signal Transducers and Activators of Transcription (STAT)3 Pathway

Michael Yeh,Nima M Gharavi,Jenny Choi,Xavier Hsieh,Erin Reed,Kevin P Mouillesseaux,Amy L Cole,Srinivasa T Reddy,Judith A Berliner

doi:10.1074/jbc.m312198200

Abstract

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC) and its component phospholipids 1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine (PEIPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine induce endothelial cells to synthesize chemotactic factors, such as interleukin 8 (IL-8). We have shown recently that Ox-PAPC-mediated induction of IL-8 transcription is independent of NF-kappaB activation, a major transcription factor utilized by cytokines and lipopolysaccharide for the induction of IL-8 transcription. In this study, we provide evidence for the role of c-src in Ox-PAPC and, specifically, PEIPC-mediated IL-8 induction. Ox-PAPC and its component phospholipids induced a rapid and transient phosphorylation of c-src Tyr418, a hallmark of c-src activation, in human aortic endothelial cells (HAEC). Ox-PAPC-mediated IL-8 protein synthesis in HAEC was inhibited by Src family kinase inhibitors, PP1 and PP2, but not by an inactive analog, PP3. Transient expression of plasmids containing C-terminal Src kinase or kinase-deficient dominant-negative c-src resulted in a 72 and 50% reduction in Ox-PAPC-induced IL-8 promoter activation in human microvascular endothelial cells, respectively. In contrast, overexpression of v-src kinase resulted in a 4-fold increase in IL-8 promoter activation, without inducing NF-kappaB promoter activation. Furthermore, treatment of HAEC with Ox-PAPC and its component PEIPC induced the activation of STAT3 by phosphorylating Tyr705, a feature of STAT3 activation. STAT3 is a known downstream effector of c-src. Ox-PAPC-induced activation of STAT3 resulted in the translocation of STAT3 from the cytoplasm of HAEC into their nuclear compartment. Transient expression of a dominant-negative STAT3beta construct in HMEC strongly inhibited IL-8 induction by Ox-PAPC. Taken together, these data demonstrate the role of the c-src kinase/STAT3 pathway in Ox-PAPC-mediated IL-8 expression in endothelial cells.

Highlights

Atherosclerosis is a chronic inflammatory condition involving monocyte infiltration into the subendothelial space [1]
We demonstrated that Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3phosphorylcholine (Ox-PAPC) activates peroxisome proliferator-activated receptor (PPAR)␣ in murine aortic EC and fails to induce monocyte chemoattractant protein 1 protein synthesis in PPAR␣-null murine endothelial cells, suggesting that PPAR␣ is involved in Ox-PAPC signaling of monocyte chemoattractant protein 1 synthesis in mouse aortic endothelial cells [7]
PEIPC was the most active in promoting c-src Tyr418 phosphorylation (Fig. 1C); POVPC induced phosphorylation of Tyr418 to a lesser degree, whereas PGPC was minimally active (Fig. 1C). These results demonstrate that Ox-PAPC and its active components, PEIPC and POVPC, rapidly activate c-src kinase in human aortic endothelial cells (HAEC) by phosphorylating Tyr418

Summary

Introduction

Atherosclerosis is a chronic inflammatory condition involving monocyte infiltration into the subendothelial space [1]. Ox-PAPC increases monocyte binding to EC by activating ␤1-integrins on the surface of EC by means of an inside-out signaling mechanism, mediated through R-Ras activation and elevated intracellular cyclic AMP levels [3]. This activated integrin binds CS-1/fibronectin, which in turn, interacts with monocyte-specific integrins [6]. Ox-PAPC-induced IL-8 synthesis is more sustained than that described for lipopolysaccharide and cytokines and is independent of NF-␬B and AP-1, transcription factors regulating the IL-8 promoter by lipopolysaccharide and cytokines [8] These findings suggest that Ox-PAPC induction of IL-8 synthesis is mediated through a novel, alternative signal transduction pathway. The current study demonstrates an important role for c-Src kinase as the initial activator of IL-8 induction by Ox-PAPC treatment in human EC

Methods

Results

Conclusion