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

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.

Figure 1

TLR4 knockout upregulated ABCA1 expression. Cultured WT or TLR4^−/− VSMCs were stimulated with or without 50 μg/ml oxLDL for 24, 48 and 72 h. (a and b) Impaired expression of ABCA1 mRNA (n=4 experiments in duplicate) and protein (n=3 experiments in duplicate) was found in oxLDL-treated WT VSMCs. (c and d) Elevated expression of ABCA1 mRNA (c), n=4 experiments in duplicate), and protein (d), n=3 experiments in duplicate, were observed in TLR4^−/− VSMCs. For all experiments, the data are represented as the fold change relative to the control and are presented as the mean±S.E.M. *P<0.05, **P<0.01 compared with the untreated control group

Figure 2

TLR4 knockout attenuated lipid accumulation in VSMCs in vitro. Cultured WT and TLR4^−/− VSMCs were treated with or without 50 μg/ml oxLDL for 48 and 72 h. The cells were fixed with 4% paraformaldehyde and stained with Oil Red O or BODIPY 493/503 (green fluorescence). Cell nuclei were counterstained with hematoxylin (blue) or Hoechst 33342 (blue fluorescence). (a and b) Neutral lipid significantly accumulated in WT VSMCs. (c and d) Attenuated neutral lipid accumulation in TLR4^−/− VSMCs. (e) Quantification of lipid accumulation based on the OD values for destained Oil Red O (n=4 experiments in duplicate). Scale bar=20 μm in all images. Data are represented as the fold change relative to the control and are presented as the mean±S.E.M. *P<0.05, **P<0.01 compared with the untreated control group

Figure 3

IRAK1 was involved in the effects of TLR4 signaling on VSMCs lipid accumulation and ABCA1 expression in vitro. WT and TLR4^−/− VSMCs were incubated with or without 50 μg/ml oxLDL for 24, 48 and 72 h. (a and b) Western blot was used to show IRAK1 and p-IRAK1 protein expression in oxLDL-treated or untreated VSMCs. Increased expression of p-IRAK1 protein was detected in oxLDL-treated WT VSMCs (a, n=3 experiments in duplicate), but not in TLR4^−/− VSMCs (b, n=3 experiments in duplicate). (c) IRAK1 kinase activity was significantly elevated in WT VSMCs stimulated with oxLDL, but significantly inhibited in TLR4^−/− VSMCs in response to oxLDL, as determined by an in vitro kinase assay (n=2 experiments in duplicate). (d–f) WT VSMCs were transfected with IRAK1-specific siRNA using a transfection reagent. A non-related scrambled siRNA was used as a negative control (NC-siRNA). At 24 h following transfection, the cells were exposed to 50 μg/ml oxLDL for 48 h. IRAK1 deficiency upregulated ABCA1 expression (d, n=3 experiments in duplicate) and attenuated lipid accumulation in WT VSMCs as determined by BODIPY 493/503 staining (e, green fluorescence, scale bar=20 μm). Quantification of lipid accumulation was based on the OD values for destained Oil Red O in WT VSMCs (f, n=4 experiments in duplicate). All data are represented as the fold change relative to the controls and are expressed as the mean±S.E.M. *P<0.05, **P<0.01 compared with the untreated control group

Figure 4

Effects of TLR4 signaling induced by a high-fat diet (HFD) on IRAK1 activity and ABCA1 expression in vivo. WT mice were either fed a normal chow diet (NCD) or a very high-fat diet for 12 weeks and then their thoracic aortas were harvested. (a) Hematoxylin and eosin (HE) staining on cross-sections from thoracic aorta were presented. Artery sections were double-stained with TLR4 (green fluorescence) and p-IRAK1 (red fluorescence) antibodies and co-localization was determined in the merged images. (b) Immunohistochemical studies were performed by co-staining TLR4 (green fluorescence) and ABCA1 (red fluorescence). The merged panel indicates the co-localization of TLR4 with ABCA1. In images a and b, the nuclei were stained with DAPI (blue fluorescence). Scale bar in HE images=100 μm; scale bar in immunofluorescence images=20 μm. (c and d) The p-IRAK1 or ABCA1 fluorescence intensity mean values were determined by the mean±S.E.M. of n=6 non-consecutive sections from five mice. **P<0.01

Figure 5

In vivo TLR4 knockout inhibited high-fat diet (HFD)-induced IRAK1 activation and rescued ABCA1 downregulation. (a and b) TLR4^−/− mice were fed either a normal chow diet (NCD) or a very HFD for 12 weeks. Following this, their thoracic aortas were obtained. Thoracic aortas were sectioned, and stained for hematoxylin and eosin. IRAK1 activation (red fluorescence) and ABCA1 expression (red fluorescence) were determined using immunofluorescence staining. DAPI was used for nuclear counterstaining (blue fluorescence). (c and d) Determination of p-IRAK1 and ABCA1 fluorescence intensity mean values (n=6 non-consecutive sections from 5 mice). (e–g) WT and TLR4^−/− mice were fed either a normalized chow diet or a very HFD for 12 weeks. Following this, aortic root sections were obtained. A negative control was performed using phosphate-buffered saline (PBS) instead of primary antibodies. BF (bright field) images show cross-sections of the arteries from which the immunofluorescence images were obtained (e). Detection of lipid accumulation in aortic roots using BODIPY 493/503 staining (green fluorescence). Significant lipid accumulation was observed in aortic root sections from WT mice fed a HFD (f), but not from TLR4^−/− mice fed a HFD (g). The white arrows show representative lipid accumulation. (h) Determination of fluorescence (normalized to plaque area, n=6 non-consecutive sections from 5 mice). Scale bar in BF and HE images=100 μm; scale bar in immunofluorescence images=20 μm. Data are expressed as the mean±S.E.M. **P<0.01 compared with NCD group