The Pro-atherogenic Cytokine Interleukin-18 Induces CXCL16 Expression in Rat Aortic Smooth Muscle Cells via MyD88, Interleukin-1 Receptor-associated Kinase, Tumor Necrosis Factor Receptor-associated Factor 6, c-Src, Phosphatidylinositol 3-Kinase, Akt, c-Jun N-terminal Kinase, and Activator Protein-1 Signaling

Devang N Patel,Takeshi Tokuhisa,Liselotte E Jensen,Masahiko Hatano,Srinivas Mummidi,Anthony J Valente,Gregory L Freeman,Steven R Bailey,Bysani Chandrasekar

doi:10.1074/jbc.m502586200

Abstract

We recently demonstrated that the chemokine CXCL16 is expressed in aortic smooth muscle cells (ASMC) and induces ASMC adhesion and proliferation (Chandrasekar, B., Bysani, S., and Mummidi, S. (2004) J. Biol. Chem. 279, 3188-3196). Here we reort that interleukin (IL)-18 positively regulates CXCL16 transcription in rat ASMC. We characterized the cis-regulatory region of CXCL16 and identified a functional activator protein-1 (AP-1) binding motif. Deletion or mutation of this site attenuated IL-18-mediated CXCL16 promoter activity. Gel shift, supershift, and chromatin immunoprecipitation assays confirmed AP-1-dependent CXCL16 expression. CXCL16 promoter-reporter activity was increased by constitutively active c-Fos and c-Jun and decreased by dominant negative or antisense c-Fos and c-Jun. Src kinase inhibitors PP1 and PP2, phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002, Akt inhibitor, the c-Jun N-terminal kinase (JNK) inhibitor SP600125, antisense JNK and dominant negative MyD88, interleukin-1 receptor-associated kinase (IRAK)-1, IRAK4, and phosphatidylinositol 3-kinase expression all attenuated IL-18-mediated AP-1 binding and reporter activity, CXCL16 promoter-reporter activity, and CXCL16 expression. Thus IL-18 induced CXCL16 expression via a MyD88 --> IRAK1-IRAK4-TRAF6 (tumor necrosis factor receptor-associated factor 6) --> c-Src--> PI3K --> Akt --> JNK --> AP-1 pathway. Importantly, IL-18 stimulated ASMC proliferation in a CXCL16-dependent manner. These data provide for the first time a mechanism of IL-18-mediated CXCL16 gene transcription and CXCL16-dependent ASMC proliferation and suggest a role for IL-18-CXCL16 cross-talk in atherogenesis and restenosis following angioplasty.

Highlights

Atherosclerosis is an inflammatory disease responsible for considerable morbidity and mortality in Western societies
Quiescent aortic smooth muscle cells (ASMC) were treated with 25 ng/ml IL-18 for up to 24 h, and total RNA was analyzed for CXCL16 mRNA expression
Quiescent ASMC were treated with IL-18 as in C, and cell extracts were analyzed by Western blotting for CXCL16 protein and ␤-actin

Summary

Introduction

Atherosclerosis is an inflammatory disease responsible for considerable morbidity and mortality in Western societies Both proinflammatory cytokines and chemokines play a central role in atherogenesis. A positive correlation was shown between mutations in CXCL16 gene and the severity of coronary artery stenosis [27], suggesting that CXCL16 may play a causal role in the development and progression of atherosclerosis Taken together, these studies suggest that IL-18 may induce SMC proliferation through a CXCL16-mediated mechanism. IL-18-mediated CXCL16 expression was independent of the classical inflammatory cytokines IL-1␤, TNF-␣, and IFN-␥ These results ascribe a previously unrecognized role for IL-18 in SMC proliferation and suggest that IL-18 and CXCL16 cross-talk may lead to the amplification of an inflammatory cascade in vessel wall-promoting atherosclerosis

Methods

Results

Conclusion