Dexamethasone-Induced Liver Enlargement Is Related to PXR/YAP Activation and Lipid Accumulation but Not Hepatocyte Proliferation

Abstract

Dexamethasone (Dex), a widely prescribed anti-inflammatory drug, was reported to induce liver enlargement (hepatomegaly) in clinical practice and in animal models. However, the underlying mechanisms are not elucidated. Dex is a known activator of pregnane X receptor (PXR). Yes-associated protein (YAP) has been implicated in chemically induced liver enlargement. Here, the roles of PXR and YAP pathways were investigated in Dex-induced hepatomegaly. Upregulation of PXR downstream proteins, including cytochrome P450 (CYP) 3A11, 2B10, and organic anion transporter polypeptide 2 (OATP2), indicated PXR signaling was activated after high dose of Dex (50 mg/kg, i.p.), and Dex at 100 μM activated PXR in the dual-luciferase reporter gene assay. Dex also increased the expression of total YAP, nuclear YAP, and YAP downstream proteins, including connective tissue growth factor and cysteine-rich angiogenic inducer 61, indicating activation of the YAP pathway. Furthermore, nuclear translocation of YAP was promoted by activation of PXR. However, hepatocyte proliferation was inhibited with significant decrease in the expression of proliferation-related proteins cyclin D1 and proliferating cell nuclear antigen as well as other regulatory factors, such as forkhead box protein M1, c-MYC, and epidermal growth factor receptor. The inhibitory effect of Dex on hepatocyte proliferation was likely due to its anti-inflammation effect of suppression of inflammation factors. β-catenin staining revealed enlarged hepatocytes, which were mostly attributable to the accumulation of lipids, such as triglycerides. In summary, high-dose Dex increased liver size accompanied by enlarged hepatocytes, and this was due to the activation of PXR/YAP and their effects on lipid accumulation but not hepatocyte proliferation. These findings provide new insights for understanding the mechanism of Dex-induced hepatomegaly. SIGNIFICANCE STATEMENT: This study identified the roles of pregnane X receptor (PXR) and yes-associated protein (YAP) pathways in dexamethasone (Dex)-induced hepatomegaly. Dex induced PXR/YAP activation, enlarged hepatocytes, and promoted liver enlargement with lipid accumulation, such as triglycerides. However, hepatocyte proliferation was inhibited by the anti-inflammatory effect of Dex. These findings provide new insights for understanding the mechanism of Dex-induced hepatomegaly.

Graphical abstract

Fig. 1.

Dex significantly induces liver enlargement in mice. (A) Mice were intraperitoneally treated with vehicle (corn oil, 0.1 ml/10 g) or Dex (50 mg/kg per day) for 5 days. (B) Representative morphologic pictures of mice livers of the vehicle or Dex group. (C) Liver-to-body-weight ratios. Data are expressed as mean ± S.D. (n = 5). (D) CTNNB1 staining of representative liver samples measuring the size of hepatocytes around the CV area. Scale bar, 50 μm. (E) Quantification of the size of hepatocytes around the CV area. Data are expressed as mean ± S.D. (n = 3). *P < 0.05 compared with the vehicle group.

Fig. 2.

Dex activates mPXR and hPXR. (A and B) Western blot and quantification of mPXR downstream proteins from vehicle- or Dex-treated mice livers. Data are expressed as mean ± S.D. (n = 3). *P < 0.05; **P < 0.01 compared with the vehicle group. (C) Dual-luciferase reporter gene assay was used to determine the effect of RIF or Dex on hPXR activation in HEK293T cells. Data are expressed as mean ± S.D. (n = 5). **P < 0.01; ***P < 0.001; ****P < 0.0001 compared with the control group.

Fig. 3.

Effect of Dex on YAPsignaling pathway. (A and B) Western blot analysis and quantification of total YAP, nuclear YAP and cytoplasmic phosphorylated YAP protein of liver samples after a 5-day treatment with the vehicle or Dex. (C and D) Western blot analysis and quantification of YAP downstream proteins of liver samples after the vehicle or Dex treatment. Data are expressed as mean ± S.D. (n = 3). *P < 0.05; **P < 0.01 compared with the vehicle group. (E) Confocal microscopy displaying PXR and YAP distribution in HepG2 cells treated with DMSO or 100 μM of Dex for 6 hours. Scale bar, 40 μm. (F) Quantification of immunofluorescence double staining of YAP and PXR. Data are expressed as mean ± S.D. (n = 3). *P < 0.05 compared with the control group. ANKRD1, ankyrin repeat domain 1; CTGF, connective tissue growth factor; CYR61, cysteine-rich angiogenic inducer 61; DAPI, 4′,6-diamidino-2-phenylindole; p-YAP, phosphorylated YAP.

Fig. 4.

Effect of Dex on hepatocyte proliferation. (A and B) Immunohistochemistry staining of Ki67 and PCNA in mice treated with the vehicle or Dex. Scale bar, 100 μm. (C–F) Western blot analysis and quantification of proliferation-related protein in mice after a 5-day treatment with the vehicle or Dex. Data are expressed as mean ± S.D. (n = 3). *P < 0.05; **P < 0.01; ****P < 0.0001 compared with the vehicle group. (G) qRT-PCR analysis of inflammatory factors in mice in response to the vehicle or Dex treatment. Data are expressed as mean ± S.D. (n = 5). *P < 0.05; ***P < 0.001 compared with the vehicle group.Ifng, interferon γ.

Fig. 5.

Effect of Dex on the lipid profiles and lipid homeostasis. (A) H&E staining of representative liver samples from the vehicle- or Dex-treated mice. Scale bar, 100 μm. (B) Oil red O staining of representative liver samples of mice treated with the vehicle or Dex. Scale bar, 100 μm. (C and D) PCA scatter plots, (E and F) OPLS-DA score plots, and (G and H) S-plots under positive and negative ion modes of lipid profiles from livers of mice treated with the vehicle or Dex. For PCA and OPLS-DA plots (C–F), blue diamonds represent the vehicle-treated mice, red diamonds represent the Dex-treated mice, and pink diamonds represent the quality controls. For S-plots (G and H), the specific calculated lipid components with P value <0.05, variable importance in projection (VIP) >1 were highlighted by red diamonds. (I–M) Altered lipid contents and species in liver samples of mice treated with the vehicle or Dex. (I and J) TG; (K) phosphatidyl ethanolamine (PE); (L) phosphatidylcholine (PC); and (M) phosphatidylinositol (PI). (N) qRT-PCR analysis of TG metabolic genes in mice treated with the vehicle or Dex. Data are expressed as mean ± S.D. (n = 4 or 5). *P < 0.05; **P < 0.01; ***P < 0.001 compared with the vehicle group.