PXR mediates mifepristone-induced hepatomegaly in mice

Abstract

Mifepristone (Mif), an effective synthetic steroidal antiprogesterone drug, is widely used for medical abortion and pregnancy prevention. Due to its anti-glucocorticoid effect, high-dose Mif is also used to treat Cushing's syndrome. Mif was reported to active pregnane X receptor (PXR) in vitro and PXR can induce hepatomegaly via activation and interaction with yes-associated protein (YAP) pathway. High-dose Mif was reported to induce hepatomegaly in rats and mice, but the underlying mechanism remains unclear. Here, the role of PXR was studied in Mif-induced hepatomegaly in C57BL/6 mice and Pxr-knockout mice. The results demonstrated that high-dose Mif (100 mg · kg^-1 · d^-1, i.p.) treatment for 5 days significantly induced hepatomegaly with enlarged hepatocytes and promoted proliferation, but low dose of Mif (5 mg · kg^-1 · d^-1, i.p.) cannot induce hepatomegaly. The dual-luciferase reporter gene assays showed that Mif can activate human PXR in a concentration-dependent manner. In addition, Mif could promote nuclear translocation of PXR and YAP, and significantly induced the expression of PXR, YAP, and their target proteins such as CYP3A11, CYP2B10, UGT1A1, ANKRD, and CTGF. However, Mif (100 mg · kg^-1 · d^-1, i.p.) failed to induce hepatomegaly in Pxr-knockout mice, as well as hepatocyte enlargement and proliferation, further indicating that Mif-induced hepatomegaly is PXR-dependent. In summary, this study demonstrated that PXR-mediated Mif-induced hepatomegaly in mice probably via activation of YAP pathway. This study provides new insights in Mif-induced hepatomegaly, and provides novel evidence on the crucial function of PXR in liver enlargement and regeneration.

Keywords: hepatomegaly; mifepristone; pregnane X receptor (PXR); yes-associated protein (YAP).

Fig. 1. Mif at 100 mg/kg induces liver enlargement.

a C57BL/6 mice were treated with the vehicle (corn oil, 0.1 mL/10 g, i.p.) or Mif (100 mg · kg⁻¹ · d⁻¹, i.p.) for 5 days. b Representative morphological liver pictures of the vehicle and Mif group. c Liver-to-body-weight ratios of the vehicle and Mif group. d IHC staining of CTNNB1 measuring hepatocyte size around the CV area of the vehicle- or Mif-treated mice. e Quantification of the hepatocyte size around the CV area. f IHC staining of Ki67 of liver sections around the PV area of the vehicle- or Mif-treated mice. g Quantification of Ki67⁺ hepatocytes around the PV area. h IHC staining of PCNA of liver sections around the PV area of the vehicle- or Mif-treated mice. i Quantification of PCNA⁺ hepatocytes around the PV area. The data are shown as mean ± S.D. (n = 5–6) and ^*P < 0.05, ^**P < 0.01 versus the vehicle group.

Fig. 2. Mif-induced hepatomegaly is not associated with inflammation and liver injury.

a Serum biochemical indexes including ALT, AST, ALP, TBA, T-CHO and TG of the vehicle or Mif group. b Representative H&E staining of liver slices of the vehicle or Mif group. c The hepatic mRNA expression of IL-6, IL-10, IL-1β, and TNF-α in livers of the vehicle- or Mif-treated mice. The data are presented as means ± S.D. (n = 5–6) and ^*P < 0.05 versus the vehicle group.

Fig. 3. Low-dose Mif cannot induce hepatomegaly.

a C57BL/6 mice were treated with the vehicle (corn oil, 0.1 mL/10 g, i.p.) or low-dose Mif (Mif, 5 mg · kg⁻¹ · d⁻¹, i.p.) for 5 days. b Representative morphological liver pictures of the vehicle or Mif group. c Liver-to-body-weight ratios of the vehicle or Mif group. d IHC staining of CTNNB1 measuring hepatocyte size around the CV area of the vehicle and Mif group. e Quantification of the hepatocytes size around the CV area. f IHC staining of Ki67 of liver sections of the vehicle and Mif group. g Quantification of Ki67⁺ hepatocytes h IHC staining of PCNA of liver sections of the vehicle and Mif group. i Quantification of PCNA⁺ hepatocytes. The data are shown as mean ± S.D. (n = 5–6) and ^*P < 0.05 versus the vehicle group.

Fig. 4. Mif is a PXR agonist.

a The hepatic Pxr mRNA levels of the vehicle or Mif group. b The hepatic Cyp3a11, Cyp2b10 and Ugt1a1 mRNA levels of the vehicle or Mif group. c The hepatic protein expression levels of PXR downstream targets of the vehicle or Mif group. d Quantification of CYP3A11, CYP2B10 and UGT1A1 protein levels normalized to the expression of β-ACTIN. e Dual-luciferase reporter gene assay was used to determine the effect of RIF or different concentrations of Mif on hPXR activation in HEK293T cells. The data are expressed as mean ± S.D. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 and ^****P < 0.0001 compared with the vehicle group (n = 3–5).

Fig. 5. Mif-induced hepatomegaly is associated with the PXR/YAP activation.

a The hepatic protein levels of nuclear YAP, total YAP and cytosol-p-YAP of the vehicle or Mif group. b Quantification of total YAP, cytoplasmic p-YAP and nuclear YAP normalized to the expression of β-ACTIN. c The hepatic protein levels of YAP downstream targets including ANKRD, CTGF and CYR61 of the vehicle or Mif group. d Quantification of ANKRD, CTGF and CYR61 normalized to the expression of β-ACTIN. e Immunofluorescence double-staining experiment of PXR and YAP in HepG2 cells. The data are expressed as the mean ± S.D. *P < 0.05 compared with the vehicle group (n = 3).

Fig. 6. Mif-induced hepatomegaly is PXR-dependent.

a Pxr-knockout mice were treated with the vehicle (corn oil, 0.1 mL/10 g, i.p.) or Mif (100 mg · kg⁻¹ · d⁻¹, i.p.) for 5 days. b Representative morphological liver pictures of the KO-vehicle and KO-Mif group. c Liver-to-body-weight ratios of the KO-vehicle and KO-Mif group. d IHC staining of CTNNB1 measuring hepatocyte size around the CV area of the KO-vehicle and KO-Mif group. e IHC staining of Ki67 of mice liver of the KO-vehicle and KO-Mif group. f IHC staining of PCNA of mice liver of the KO-vehicle and KO-Mif group. g The hepatic protein levels of PXR downstream targets such as CYP2B10, CYP3A11 and UGT1A1 of Pxr-knockout mice treated with the vehicle or Mif. h Quantification of CYP2B10, CYP3A11 and UGT1A1 normalized to the expression of β-ACTIN. The data are shown as mean ± S.D. (n = 3–6).