Korean J Gastroenterol.  2021 Mar;77(3):123-131. 10.4166/kjg.2020.156.

Atorvastatin Induces FXR and CYP7A1 Activation as a Result of the Sequential Action of PPARγ/PGC-1α/HNF-4α in Hep3B Cells

• Lee J
• Hong EM
• Jung JH
• Park SW
• Lee SP
• Koh DH
• Jang HJ
• Kae SH

• KMID: 2514088
• DOI: http://doi.org/10.4166/kjg.2020.156

Abstract

Background/Aims
PPARγ, farnesoid X receptor (FXR) and CYP7A1 are associated with solubility of bile. This study was performed to understand a mechanism and interactions of statin-induced PPARγ, PGC-1α and HNF-4α related to the statin-induced activation of FXR and CYP7A1, and verify whether the mevalonate pathway is involved in the mechanism.
Methods
MTT assays were performed using cultured human Hep3B cells to determine the effect of atorvastatin on the cell proliferation. Expression levels of indicated proteins were measured using Western blotting assays by inhibiting the protein expression or not.
Results
Atorvastatin increased expression of PPARγ, PGC-1α, HNF-4α, FXR, and CYP7A1 in Hep3B cells. PPARγ ligand of troglitazone upregulated the expression of PGC-1α, HNF-4α, FXR, and CYP7A1 in Hep3B cells. Silencing of PPARγ, PGC1α, and HNF4α using respective siRNA demonstrated that atorvastatin-induced FXR and CYP7A1 activation required sequential action of PPARγ /PGC-1α/HNF-4α. The silencing of PPARγ completely inhibited atorvastatin-induced PGC-1α expression, and the PGC1α silencing partially inhibited atorvastatin-induced PPARγ expression. The inhibition of HNF4α did not affect atorvastatin-induced PPARγ expression, but partially inhibited atorvastatin-induced PGC-1α expression. Besides, mevalonate completely reversed the effect of atorvastatin on PPARγ, PGC-1α, HNF-4α, FXR, and CYP7A1.
Conclusions
Atorvastatin induces FXR and CYP7A1 activation as a result of sequential action of PPARγ/PGC-1α/HNF-4α in human hepatocytes. We propose that atorvastatin enhances solubility of cholesterol in bile by simultaneously activating of FXR and CYP7A1.

Keyword

Gallstones; Statin; PGC-1α; HNF-4α; FXR; CYP7A1

Figure

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  Fig. 1 Atorvastatin suppresses proliferation of Hep3B cells in a dose-dependent manner. Hep3B cells were treated with indicated concentrations of atorvastatin in DMEM with SFM for 24 hours. Cell viability was evaluated using the MTT assay. Data are expressed as the means±SD. Atorvastatin suppressed Hep3B cell proliferation dose-dependently. Concentrations over 25 μM significantly affected cell growth in 24 hours. DMEM, Dulbecco's modified eagle medium; SFM, serum-free medium; SD, standard deviation. ap<0.05, compared to untreated control cells and the cells treated with the lower concentrations of atorvastatin; bp<0.001, compared to untreated control cells and cells treated with lower concentrations of atorvastatin.

• 

  Fig. 2 Atorvastatin increases the expressions of PPARγ, PGC-1α, HNF-4α, FXR, and CyP7A1 in Hep3B cells. Hep3B cells were treated with indicated concentrations of atorvastatin in DMEM with SFM for 24 hours. Expression levels of PPARγ, PGC-1α, HNF-4α, FXR, and CyP7A1 were analyzed using Western blotting. All results are representative of at least three separate experiments. Atorvastatin induced activation of PPARγ, PGC-1α, HNF-4α, and CYP7A1 in a dose-dependent or independent manner. ap<0.01, compared to untreated control cells.

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  Fig. 3 PPARγ ligand increases expressions of PGC-1α, HNF-4α, FXR, and CyP7A1 in Hep3B cells. Hep3B cells were treated with indicated concentrations of troglitazone or WY-14643 in DMEM with SFM for 24 hours. Expression levels of PGC-1α, HNF-4α, FXR, and CyP7A1 were analyzed using Western blotting. All results are representative of at least three separate experiments. Troglitazone induced activation of PGC-1 α, HNF-4α, FXR, and CyP7A1. However, WY-14643 did not induce significant change in expression of PGC-1α, HNF-4α, FXR and CyP7A1. ap<0.01, compared to untreated control cells.

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  Fig. 4 PGC-1α is necessary for complete PPARγ activation whereas, PPARγ actiavtion is essential for activation of PGC-1α, and HNF-4α activation is dependent on PGC-1α. Hep3B cells were treated with siRNA against PPARγ, PGC-1α, HNF-4α or scrambled siRNA, and incubated for 48 hours. Afterwards, cells were treated with 10 μM atorvastatin in DMEM with SFM for 24 hours. Expression levels of PPARγ, PGC-1α, and HNF-4α were analyzed using Western blotting. All results are representative of at least three separate experiments. PGC-1α silencing partially inhibited atorvastatin-induced PPARγ expression, and HNF-4α silencing did not affect atorvastatininduced PPARγ expression. In addition, PPARγ silencing completely inhibited atorvastatin-induced PGC-1α expression, and HNF-4α silencing partially inhibited atorvastatin-induced PGC-1α expression. si-NC, scrambled siRNA transfection without atorvastatin treatment (negative control); Cont, atorvastatin treatment only without siRNA transfection; si-, treated with siRNA against specific target. ap<0.01, compared to Cont; bp<0.001, compared to Cont and si-HNF-4α.

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  Fig. 5 Atorvastatin-induced FXR and CYP7A1 activation requires sequential actions of PPARγ/PGC-1α/HNF-4α. Hep3B cells were treated with siRNA against PPARγ, PGC-1α, HNF-4α or scrambled siRNA, and incubated for 24 hours. Afterwards, cells were treated with or without 10 μM atorvastatin in DMEM with SFM for 24 hours. Expression levels of FXR and CYP7A1 were analyzed by using Western blotting. All results are representative of at least three separate experiments. Silencing of PPARγ, PGC1α, and HNF4α using respective siRNA inhibits FXR and CYP7A1 expression. si-NC, scrambled siRNA transfection without atorvastatin treatment (negative control); Cont, atorvastatin treatment only without siRNA transfection; si-, treated with siRNA against specific target. ap<0.001, compared to Cont.

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  Fig. 6 Inhibition of the mevalonate-isoprenoid pathway through the HMG-CoA reductase block by statin is associated with activation of PPARγ, PGC-1α, HNF-4α, FXR, and CYP7A1. Hep3B cells were pre-treated with or without 100 mM mevalonate for 2 hours, and then co-treated with 10 μM atorvastatin with SFM for 24 hours. Expression levels of PPARγ, PGC-1α, HNF-4α, FXR, and CYP7A1were analyzed using Western blotting. All results are representative of at least three separate experiments. Mevalonate completely reversed the effect of atorvastatin on PPARγ, PGC-1α, HNF-4α, FXR, and CYP7A1. ap<0.01, compared to untreated control cells; bp<0.01, compared to only atorvastatin treatment cells.

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  Fig. 7 Suggestive mechanism of statin that activates and regulates FXR and CYP7A1 in terms of bile acid metabolism in hepatocytes. 1) Statin activates PPARγ and PGC-1α in order (PGC-1α is necessary for complete PPARγ activation, and PPARγ is essential for the activation of PGC-1α), inducing increase of HNF-4α expression (PGC-1α is an essential factor required to activate HNF-4α), and sequentially resulting in activation of FXR. Activated FXR stimulates BSEP (ABCB11) in hepatocytes, inducing increase of bile acid excretion.20 2) FXR suppresses CYP7A1 via SHP activation that inhibits LRH-1 to activates CYP7A1 (purple arrows, SHP-dependent pathway). However, statin increase expression of CYP7A1, despite the fact that statins activate FXR. Bile acids stimulate PKC which deactivates HNF-4α via the c-Jun kinase pathway, resulting in failure of HNF-4α-induced CYP7A1 gene activation (red arrows, SHP-independent pathway).21,22 Accordingly, the activation of CYP7A1 via HNF-4α, the SHP-independent pathway, is predominant compared with the SHP-dependent pathway, in terms of increased statin-induced-CYP7A1 expression. In this study, HNF-4α silencing using siRNA did not show activation of CYP7A1 upon treatment with atorvastatin. Taken together, statin increases hepatic bile acid synthesis that can contribute to lowering CSI. SHP, small heterodimer partner; FXR, farnesoid X receptor; LRH, liver homologue receptor; PKC, protein kinase C.

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