Abstract 2: Elucidation of the Mechanism of Apolipoprotein(a)/Lipoprotein(a)-Induced Interleukin 8 Gene Expression: Insight into Potential Receptors and Signaling Pathways and the Role of Oxidized Phospholipids

Abstract

Elevated plasma Lipoprotein(a) (Lp(a)) levels are a risk factor for a variety of atherosclerotic disorders, although the exact role(s) of Lp(a) in atherogenesis remains poorly understood. It is clear, however, that the unique pathogenic effects attributed to Lp(a) are mediated by its apolipoprotein(a) moiety. Over the past decade, a growing body of evidence suggests that the harmful effects of apo(a) involve stimulation of inflammatory responses such as monocyte chemotaxis. It was previously reported that Lp(a), through apo(a), induces IL-8 gene expression by macrophages. The kringle V domain of apo(a) was implicated in this effect, along with oxidized phospholipids potentially present in this domain. In the current study, we found that a recombinant apo(a) species containing 17 kringle-IV domians (17K) elicited a dose-dependant increase in IL-8 mRNA abundance and protein secretion in THP-1 macrophages. This effect was significantly reduced by mutation of the strong lysine binding site (sLBS) in kringle IV type 10, but not by treatment with lysine analogue ε-ACA. Notably, this mutation eliminates the EO6-reactive oxidized phsopholipid in apo(a). Removal of kringle V did not reduce the ability of apo(a) to enhance IL-8 expression. Therefore, to examine the potential role of the modification of apo(a) by oxidized phospholipids, we used an siRNA knockdown approach to screen candidate receptors. We found that CD36 and TLR2, but not TLR4, are required for apo(a)-induced IL-8 expression. Specific MAPK pathway inhibitors were used to investigate the mechanism of IL-8 induction by apo(a): inhibitors of JNK and ERK1/2 abolished the effect of apo(a) whereas an inhibitor of p38 had no effect. Accordingly, apo(a) also stimulated an increase in JNK phosphorylation. To assess the role of downstream transcription factors, luciferase reporter gene experiments were conducted using the IL-8 promoter. Apo(a) induced expression of this reporter construct, as expected, and this induction was eliminated by mutation of known binding sites for NFκB or AP-1. Our results provide tantalizing mechanistic links between apo(a) lysine binding function, oxidative modification, and stimulation of intracellular signaling pathways through inflammation-linked receptors.