IRAK1 mediates TLR4-induced ABCA1 downregulation and lipid accumulation in VSMCs.

L Guo,B-H Li,L-L Zhang,X-J Cao,C-Y Gao,P Deng,Z-P Yu,L Zhang,C-H Chen,Y Pi,G Zhu,J-C Li,Z Zhou,Q-L Ma,Y Liu,Z-C Hu

doi:10.1038/cddis.2015.212

Abstract

The activation of Toll-like receptor 4 (TLR4) signaling has an important role in promoting lipid accumulation and pro-inflammatory effects in vascular smooth muscle cells (VSMCs), which facilitate atherosclerosis development and progression. Previous studies have demonstrated that excess lipid accumulation in VSMCs is due to an inhibition of the expression of ATP-binding cassette transporter A1 (ABCA1), an important molecular mediator of lipid efflux from VSMCs. However, the underlying molecular mechanisms of this process are unclear. The purpose of this study was to disclose the underlying molecular mechanisms of TLR4 signaling in regulating ABCA1 expression. Primary cultured VSMCs were stimulated with 50 μg/ml oxidized low-density lipoprotein (oxLDL). We determined that enhancing TLR4 signaling using oxLDL significantly downregulated ABCA1 expression and induced lipid accumulation in VSMCs. However, TLR4 knockout significantly rescued oxLDL-induced ABCA1 downregulation and lipid accumulation. In addition, IL-1R-associated kinase 1 (IRAK1) was involved in the effects of TLR4 signaling on ABCA1 expression and lipid accumulation. Silencing IRAK1 expression using a specific siRNA reversed TLR4-induced ABCA1 downregulation and lipid accumulation in vitro. These results were further confirmed by our in vivo experiments. We determined that enhancing TLR4 signaling by administering a 12-week-long high-fat diet (HFD) to mice significantly increased IRAK1 expression, which downregulated ABCA1 expression and induced lipid accumulation. In addition, TLR4 knockout in vivo reversed the effects of the HFD on IRAK1 and ABCA1 expression, as well as on lipid accumulation. In conclusion, IRAK1 is involved in TLR4-mediated downregulation of ABCA1 expression and lipid accumulation in VSMCs.

Highlights

Using flow cytometric analysis and immunofluorescence staining methods, human vascular smooth muscle cell (VSMC) have been demonstrated to express Toll-like receptor 4 (TLR4) and the TLR4-related molecules, MD2 and CD14, but not TLR2.5 Upon stimulation, TLR4 activates the nuclear factor-κB (NF-κB) pathway and triggers the production of pro-inflammatory cytokines and chemokines, as well as the upregulation of cell surface molecules.[6]
Real-time PCR demonstrated that mRNA expression levels of IL-1β and tumor necrosis factor (TNF)-α were significantly increased at 24 h post-Oxidized low-density lipoprotein (oxLDL) treatment in WT VSMCs (Supplementary Figures 3C and D)
We demonstrated that VSMC lipid accumulation and ABCA1 downregulation were markedly dependent on TLR4 signaling in vivo and in vitro

Summary

Introduction

Using flow cytometric analysis and immunofluorescence staining methods, human VSMCs have been demonstrated to express Toll-like receptor 4 (TLR4) and the TLR4-related molecules, MD2 and CD14, but not TLR2.5 Upon stimulation, TLR4 activates the nuclear factor-κB (NF-κB) pathway and triggers the production of pro-inflammatory cytokines and chemokines, as well as the upregulation of cell surface molecules.[6]. The signaling pathways that are initiated by the pro-inflammatory cytokines IL-1β and tumor necrosis factor (TNF)-α, which are similar to TLRdependent signaling,[16] have been reported to upregulate low-density lipoprotein receptor-mediated cholesterol influx and to downregulate ABCA1-mediated cholesterol efflux in vivo and in vitro.[17] Likewise, lipopolysaccharide (LPS) impairs 3H-cholesterol efflux from human macrophages to apolipoprotein A-I and significantly reduces macrophage expression of the cholesterol transporter ABCA1 in vitro.[18] By pro-inflammatory and proatherogenic stimuli, NF-κB is involved in suppressing ABCA1 expression in vivo and in vitro, affecting lipid metabolism.[19,20,21] the role and underlying mechanisms of TLR4 in regulating ABCA1 expression are unclear. We tested the hypothesis that IRAK1 was critical for TLR4-induced lipid transporter ABCA1 downregulation, which accelerated lipid accumulation in VSMCs

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Conclusion