HRD1 prevents atherosclerosis-mediated endothelial cell apoptosis by promoting LOX-1 degradation

Abstract

The 3-hydroxy-3-methylglutaryl reductase degradation (HRD1) is an E3 ubiquitin ligase that can preserve heart structure and function, but its role in endothelial dysfunction and atherosclerosis (AS) is unclear. The aim of this study was to explore the role and biological function of HRD1 in AS. HRD1 expression was significantly decreased in atherosclerotic intima and ox-LDL led to a decrease of HRD1 level in endothelial cells (ECs). Forced expression of HRD1 inhibited the endothelial apoptosis induced by ox-LDL. The transcription factor KLF2 specifically bound to the HRD1 promoter and positively regulated HRD1 expression. KLF2 up-regulation could reverse the decrease of HRD1 level in ECs treated with ox-LDL. Further analysis showed that HRD1 interacted with LOX-1 and promoted ubiquitination and degradation of LOX-1 by the proteasome. Deletion of LOX-1 attenuated the ECs apoptosis induced by HRD1 downregulation. Pravastatin, which protected EC from damage via a KLF2-dependent mechanism, could dose-dependently enhanced HRD1 expression in EC exposed to ox-LDL. Interestingly, interference of HRD1 abolished the cytoprotective effect of pravastatin. Collectively, our data indicate that decreased HRD1 expression leads to apoptosis of ECs and restoration of HRD1 expression could represent a novel strategy for human AS therapy.

Keywords: HRD1; LOX-1; apoptosis; atherosclerosis; endothelial cell.

Figure 1.

HRD1 expression is decreased in human atherosclerotic intima and ECs treated with ox-LDL. (a) Immunofluorescence staining for HRD1 (green) and CD31 (red) in normal arterial walls. Original magnification, ×100. (b) Immunostaining of HRD1 (red) and the endothelial marker CD31 (green) in normal arterial intima and atherosclerotic arterial intima. Bar = 20 μm. (c) Human umbilical vein endothelial cells (HUVECs) were treated with ox-LDL (0, 20, 40 and 80 μg/ml) for 24 h, followed by measurement of the protein levels of HRD1 and LOX-1. (d) HUVECs were treated with ox-LDL (80 μg/ml) for 0, 6, 12, 24 or 48 h, followed by measurements of the protein levels of HRD1 and LOX-1. Values are means ± SD and are representative of three individual experiments. *P < 0.05 and **P < 0.01, compared to 0 μg/ml or 0 h.

Figure 2.

Ox-LDL-induced apoptosis of endothelial cells can be attenuated by HRD1 overexpression. HUVECs were transfected with Ad-GFP or Ad-HRD1 for 24 h, followed by exposure to ox-LDL (80 μg/ml) for 48 h. HRD1 expression (a), cell viability (b), cleaved caspase-3 levels (c) and HUVEC apoptosis (d) were then measured. Values are means ± SD and are representative of three individual experiments. *P < 0.05 and **P < 0.01, compared to control. #P < 0.05 and ##P < 0.01, compared to ox-LDL + Ad-GFP.

Figure 3.

HRD1 expression is regulated by KLF2 in endothelial cells treated with ox-LDL. (a) Human umbilical vein endothelial cells (HUVECs) were treated with ox-LDL (0, 20, 40 and 80 μg/ml) for 24 h, followed by measurement of the KLF2 expression. (b) HUVECs were transfected with Ad-GFP or Ad-KLF2 for 24 h followed by exposure to ox-LDL (80 μg/ml) for 24 h and measurement of HRD1 mRNA and protein levels. (c) KLF2 bound to the HRD1 promoter in HUVECs in a ChIP analysis. (d) ChIP-qPCR analysis was performed to measure the capacity of KLF2 binding to the HRD1 promoter in HUVECs treated with ox-LDL. (e) HUVECs were transfected with KLF2 siRNA (si-KLF2) for 24 h, followed by measurement of KLF2 and HRD1 expression. Values are means ± SD and are representative of three individual experiments. *P < 0.05 and **P < 0.01, compared to control. ##P < 0.01, compared to ox-LDL + Ad-GFP.

Figure 4.

HRD1 promotes LOX-1 ubiquitination for degradation. (a) The expression of HRD1 and LOX-1 in si-HRD1 transfected human umbilical vein endothelial cells (HUVECs) was measured by immunoblotting. (b) HUVECs were transfected with Ad-GFP or Ad-HRD1 for 24 h, followed by exposure to ox-LDL (80 μg/ml) for 24 h and measurement of LOX-1 expression. (c) HUVECs were transfected with si-HRD1 or si-control for 48 h, followed by exposure to cycloheximide (CHX 50 mg/ml) for 0, 3, or 6 h and measurement of the LOX-1 protein levels in whole cell lysates by immunoblotting. The intensity of the LOX-1 protein bands was analyzed by densitometry, after normalization to the corresponding β-Actin level. (d) HUVECs were pretreated with MG132 (10 μM) for 6 h, followed by determination of endogenous protein-protein interactions between HRD1 and LOX-1 by immunoprecipitation (IP) with HRD1 or LOX-1 antibodies and subsequent immunoblotting. IgG was used as a negative control for IP. HUVECs were stained with Hrd1 (green) and LOX-1 (red). HRD1 and LOX-1 merged appear as orange/yellow. Bar = 10 μm. (e) Ubiquitination of LOX-1 was induced by HRD1. Flag-ubiquitin was coexpressed in HUVECs with myc-HRD1 or vector control with treatment with MG132 (10 μmol/l) for 6 h. Ubiquitinated LOX-1 protein was immunoprecipitated using Flag-Tag antibody and further detected with Anti-LOX-1 antibody. The endogenous levels of LOX-1 and myc-HRD1 in the whole cell lysates were examined by anti-LOX-1 and anti-myc antibodies. Values are means ± SD and are representative of three individual experiments. **P < 0.01, compared to si-control. ##P < 0.01, compared to ox-LDL + Ad-GFP.

Figure 5.

LOX-1 is required for endothelial cell apoptosis induced by HRD1 downregulation. Human umbilical vein endothelial cells (HUVECs) were transfected with si-HRD1 or si-HRD1 plus si-LOX-1 for 48 h, followed by measurement of HRD1 and LOX-1 expression (a), cell viability (b), and HUVEC apoptosis (c). Values are means ± SD and are representative of three individual experiments. *P < 0.05 and **P < 0.01, compared to si-control. #P < 0.05 and ##P < 0.01, compared to si-HRD1.

Figure 6.

Pravastatin stimulated HRD1 expression via a KLF2-dependent mechanism. (a) After pretreatment with pravastatin (0, 5, 10 or 50 mmol/L) for 2 h, human umbilical vein endothelial cells (HUVECs) were treated with ox-LDL for additional 24 h, followed by measurement of HRD1 mRNA and protein levels. (b) HUVECs were transfected with si-KLF2 for 24 h and then treated with ox-LDL and/or pravastatin, followed by measurement of HRD1 mRNA and protein levels. (c) HUVECs were transfected with si-HRD1 for 24 h, followed by treatment with ox-LDL and/or pravastatin and measurement of LOX-1 protein levels. (d) HUVECs were transfected with si-HRD1 for 24 h, followed by treatment with ox-LDL and/or pravastatin and measurement of cell viability. (e) Diagram depicting the role of HRD1 in EC apoptosis and AS development. Values are means ± SD and are representative of three individual experiments. **P < 0.01, compared to control. ##P < 0.01, compared to ox-LDL. &P < 0.01, compared to ox-LDL+pravastatin+ si-control.