E2F1 inhibits circulating cholesterol clearance by regulating Pcsk9 expression in the liver
Qiuwen Lai, Albert Giralt, Cédric Le May, Lianjun Zhang, Bertrand Cariou, Pierre-Damien Denechaud, Lluis Fajas
Qiuwen Lai, Albert Giralt, Cédric Le May, Lianjun Zhang, Bertrand Cariou, Pierre-Damien Denechaud, Lluis Fajas
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Research Article Hepatology Metabolism

E2F1 inhibits circulating cholesterol clearance by regulating Pcsk9 expression in the liver

Abstract

Cholesterol accumulation in the liver is an early event in nonalcoholic fatty liver disease (NAFLD). Here, we demonstrate that E2F1 plays a crucial role in maintaining cellular cholesterol homeostasis by regulating cholesterol uptake via proprotein convertase subtilisin/kexin 9 (PCSK9), an enzyme that promotes low-density lipoprotein receptor (LDLR) degradation upon activation. E2f1–/– mice display reduced total plasma cholesterol levels and increased cholesterol content in the liver. In this study, we show that E2f1 deletion in cellular and mouse models leads to a marked decrease in Pcsk9 expression and an increase in LDLR expression. In addition to the upregulation of LDLR, we report that E2f1–/– hepatocytes exhibit increased LDL uptake. ChIP-Seq and PCSK9 promoter reporter experiments confirmed that E2F1 binds to and transactivates the PCSK9 promoter. Interestingly, E2f1–/– mice fed a high-cholesterol diet (HCD) display a fatty liver phenotype and liver fibrosis, which is reversed by reexpression of PCSK9 in the liver. Collectively, these data indicate that E2F1 regulates cholesterol uptake and that the loss of E2F1 leads to abnormal cholesterol accumulation in the liver and the development of fibrosis in response to an HCD.

Authors

Qiuwen Lai, Albert Giralt, Cédric Le May, Lianjun Zhang, Bertrand Cariou, Pierre-Damien Denechaud, Lluis Fajas

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Figure 3

Loss of E2f1 decreases Pcsk9 expression and increases LDLR protein expression in hepatocytes.

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Loss of E2f1 decreases Pcsk9 expression and increases LDLR protein expre...
(A) Relative expression of Pcsk9 mRNA in livers from E2f1+/+ and E2f1–/– mice that were fed a normal chow diet or fasted overnight. (B) Western blot of PCSK9 and LDLR in the livers of E2f1+/+ and E2f1–/– mice. Ponceau staining of the membrane are shown as a loading control (LC). Normalizations of the quantified values are represented. Differences between E2f1+/+ and E2f1–/– were determined by 2-tailed unpaired t test. *P < 0.05. (C) Relative expression of Pcsk9 mRNA in primary E2f1+/+ and E2f1–/– mouse hepatocytes treated with medium supplemented with DMSO (control, ctrl) or 0.065 mg/ml cholesterol (chol). Three independent experiments in triplicate. (D) Representative images of PCSK9 Western blots from primary E2f1+/+ and E2f1–/– mouse hepatocytes in medium with cholesterol. Tubulin was used as a loading control (n = 2). Normalization of the quantified values is represented. Differences between E2f1+/+ and E2f1–/– were determined by 2-tailed unpaired t test. *P < 0.05. (E) Representative images of LDLR immunostaining in primary E2f1+/+ and E2f1–/– mouse hepatocytes (n = 3). Experiments were independently repeated 3 times. LDLR is stained in green; nuclei are stained in blue. Scale bars: 100 μm. (F) Filipin staining of E2f1+/+ and E2f1–/– mouse hepatocytes (n = 3). Quantification of the intensity of filipin staining are represented. Scale bars: 100 μm. (G) Quantification of the rate of cholesterol biosynthesis in E2f1+/+ and E2f1–/– mouse hepatocytes treated with medium supplemented with DMSO (ctrl) or 0.065 mg/ml cholesterol (chol). All data are presented as the mean ± SEM. Differences between E2f1+/+ and E2f1–/– were determined by 2-way ANOVA unless indicated otherwise. *P < 0.05, **P < 0.01, ***P < 0.005.