High-Density Lipoprotein Prevents Endoplasmic Reticulum Stress-Induced Downregulation of Liver LOX-1 Expression

<div><p>Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a specific cell-surface receptor for oxidized-low-density lipoprotein (ox-LDL). The impact of high-density lipoprotein (HDL) on endoplasmic reticulum (ER) stress-mediated alteration of the LOX-1 level in hepatocytes remains unclear. We aimed to investigate the impact on LOX-1 expression by tunicamycin (TM)-induced ER stress and to determine the effect of HDL on TM-affected LOX-1 expression in hepatic L02 cells. Overexpression or silencing of related cellular genes was conducted in TM-treated cells. mRNA expression was evaluated using real-time polymerase chain reaction (PCR). Protein expression was analyzed by western blot and immunocytochemistry. Lipid uptake was examined by DiI-ox-LDL, followed by flow cytometric analysis. The results showed that TM induced the upregulation of ER chaperone GRP78, downregulation of LOX-1 expression, and lipid uptake. Knock down of IRE1 or XBP-1 effectively restored LOX-1 expression and improved lipid uptake in TM-treated cells. HDL treatment prevented the negative impact on LOX-1 expression and lipid uptake induced by TM. Additionally, 1–10 μg/mL HDL significantly reduced the GRP78, IRE1, and XBP-1 expression levels in TM-treated cells. Our findings reveal that HDL could prevent the TM-induced reduction of LOX-1 expression via inhibiting the IRE1/XBP-1 pathway, suggesting a new mechanism for beneficial roles of HDL in improving lipid metabolism.</p></div

Impacts on GRP78 and LOX-1 expression by TM treatment in hepatic L02 cells.

<p>A: Expression levels of GRP78 and LOX-1 in hepatic L02 cells exposed to different concentrations (0.1, 0.5, 1, 1.5, or 2 μg/mL) of TM. Cells were harvested at 24 h after exposure to TM, and the expression levels of GRP78, LOX-1, and β-actin were examined by western blot. B: Impact of the time course of TM treatment on expression. The cells were exposed to TM (1 μg/mL) for 6, 12, or 24 h. Expression levels of GRP78, LOX-1, and β-actin were examined by western blot. C: Immunostaining of GRP78 (red) and LOX-1 (green) in L02 cells before (control) and after TM treatment (1 μg/mL, 24 h). Nuclei were counterstained with DAPI (blue). These data are representative of three independent experiments. *<i>P</i> < 0.05; **<i>P</i> < 0.01, compared with the control. TM: tunicamycin.</p

Impact of LOX-1 overexpression on lipid uptake capability in TM-treated hepatic L02 cells.

<p>A: Flow cytometric analysis of DiI-ox-LDL uptake capability in L02 cells before (control) or after 24 h TM treatment. Mean fluorescence intensity is presented. B: Cells were transfected with pCMV6-XL5-LOX-1 plasmid (LOX-1 overexpression) or pCMV6-XL5 empty vector (N-T mock transfection control) for 24 h before treatment with TM (1 μg/mL). Untransfected cells in the absence of TM were used as a control. These data are representative of three independent experiments. **P < 0.01 vs. control; ## P < 0.01 vs. TM treatment alone. TM: tunicamycin.</p

Involvement of the IRE1/XBP-1 signaling pathway in TM-mediated downregulation of LOX-1 and lipid uptake function.

<p>A: The effects of IRE1 and XBP-1 siRNA on TM-induced IRE1, XBP-1, and LOX-1 protein expression. Cells were transfected with siRNA targeting IRE1 or XBP-1. Protein expression levels were examined by western blotting. B–D: The effects of IRE1 and XBP-1 siRNA on the TM-induced LOX-1 altered expression. Cells were transfected with siRNA targeting IRE1, XBP-1, or negative control (N-T). The mRNA expression of LOX-1 was examined by real-time PCR (B). The LOX-1 protein expression (C) and lipid uptake (D) in L02 cells were examined by immunocytochemistry and DiI-ox-LDL uptake assays, respectively. Nuclei were counterstained with DAPI (blue). These data are representative of the results of three separate experiments. **P < 0.01 vs. control (0 μg/mL TM), ## P < 0.01 vs. TM (1 μg/mL). TM: tunicamycin.</p

The impact of HDL on GRP78, IRE1, and XBP-1 expression levels in TM-treated cells.

<p>L02 cells were exposed to different concentrations (1, 10, and 100 μg/mL) of HDL together with TM (1 μg/mL) for 24 h. Cells treated with TM (1 μg/mL) alone were also included. Cells without drug treatment were used as a control. A: Expression levels of GRP78, IRE1, XBP-1, and β-actin were examined by western blot. B: Immunostaining of GRP78 (red) or IRE1 (red) in cells treated with TM (1 μg/mL), TM (1 μg/mL) plus HDL (1 μg/mL), or TM (1 μg/mL) plus HDL (10 μg/mL) for 24 h. Cells without drug treatment were used as a control. Nuclei were counterstained with DAPI (blue). Data are representative of three independent experiments. **<i>P</i> < 0.01 vs. control; ^##<i>P</i> < 0.01 vs. TM treatment alone. TM: tunicamycin.</p

Proposed mechanism of HDL-mediated prevention of TM-induced downregulation of LOX-1 and lipid uptake function in hepatic cells.

<p>The ER stress inducer TM upregulates the expression of GRP78, thereby activating the IRE1/XBP-1 pathway. Activation of the IRE1/XBP-1 pathway leads to the downregulation of LOX-1 and lipid uptake function in hepatic L02 cells. HDL upregulates the TM-induced decrease of LOX-1 expression and hepatic lipid uptake, which is possibly mediated via inhibiting the IRE1/XBP-1 pathway.</p

The effect of LOX-1 expression and lipid uptake induced by HDL addition to TM-treated hepatic L02 cells.

<p>A: Hepatic L02 cells were exposed to different concentrations (1, 10, and 100 μg/mL) of HDL together with TM (1 μg/mL) for 24 h. Cells without drug treatment were used as a control. Expression levels of LOX-1 and β-actin under different treatments were examined by western blot. B: Immunostaining of LOX-1 (green) in cells treated with TM (1 μg/mL), TM (1 μg/mL) plus HDL (1 μg/mL), or TM (1 μg/mL) plus HDL (10 μg/mL) for 24 h. Cells without drug treatment were used as a control. Nuclei were counterstained with DAPI (blue). C: Flow cytometric analysis of DiI-ox-LDL uptake capability. Mean florescence intensity is presented. These data are representative of three independent experiments. **<i>P</i> < 0.01 vs. control; ^##<i>P</i> < 0.01 vs. TM treatment alone. TM: tunicamycin.</p