YAP-TEAD mediates PPAR α-induced hepatomegaly and liver regeneration in mice

Abstract

Background and aims: Peroxisome proliferator-activated receptor α (PPARα, NR1C1) is a ligand-activated nuclear receptor involved in the regulation of lipid catabolism and energy homeostasis. PPARα activation induces hepatomegaly and plays an important role in liver regeneration, but the underlying mechanisms remain unclear.

Approach and results: In this study, the effect of PPARα activation on liver enlargement and regeneration was investigated in several strains of genetically modified mice. PPARα activation by the specific agonist WY-14643 significantly induced hepatomegaly and accelerated liver regeneration after 70% partial hepatectomy (PHx) in wild-type mice and Ppara^fl/fl mice, while these effects were abolished in hepatocyte-specific Ppara-deficient (Ppara^ΔHep ) mice. Moreover, PPARα activation promoted hepatocyte hypertrophy around the central vein area and hepatocyte proliferation around the portal vein area. Mechanistically, PPARα activation regulated expression of yes-associated protein (YAP) and its downstream targets (connective tissue growth factor, cysteine-rich angiogenic inducer 61, and ankyrin repeat domain 1) as well as proliferation-related proteins (cyclins A1, D1, and E1). Binding of YAP with the PPARα E domain was critical for the interaction between YAP and PPARα. PPARα activation further induced nuclear translocation of YAP. Disruption of the YAP-transcriptional enhancer factor domain family member (TEAD) association significantly suppressed PPARα-induced hepatomegaly and hepatocyte enlargement and proliferation. In addition, PPARα failed to induce hepatomegaly in adeno-associated virus-Yap short hairpin RNA-treated mice and liver-specific Yap-deficient mice. Blockade of YAP signaling abolished PPARα-induced hepatocyte hypertrophy around the central vein area and hepatocyte proliferation around the portal vein area.

Conclusions: This study revealed a function of PPARα in regulating liver size and liver regeneration through activation of the YAP-TEAD signaling pathway. These findings have implications for understanding the physiological functions of PPARα and suggest its potential for manipulation of liver size and liver regeneration.

FIGURE 1

PPARα activation induces hepatomegaly in wild-type mice. (A) Wild-type mice were treated with vehicle or WY-14643 (100 mg/kg/day) for 10 days. (B) Liver-to-body weight ratios (n = 5–6). (C) Representative liver photos of vehicle-treated or WY-14643-treated mice. (D) Real-time quantitative PCR analysis of Ppara target gene mRNAs, lipid catabolism–related gene mRNAs, proliferation-related gene mRNAs, and inflammatory gene mRNAs after WY-14643 treatment. (E) H&E staining, CTNNB1 staining for CV areas, and KI67 staining for PV areas of representative mouse liver samples. (F) Quantification of cell size (n = 3) and the number of KI67⁺ cells (n = 3). Data are presented as mean ± SD, **p < 0.01, ****p < 0.0001 compared to the vehicle group. Scale bar, 50 μm. Abbreviations: Ccl2/3/4, C-C motif chemokine ligands 2/3/4; Cd36, cluster of differentiation 36; Cdk4, cyclin-dependent kinase 4; Cpt1a, carnitine palmitoyltransferase 1A; Dgat1/2, diacylglycerol O-acyltransferase 1/2; Hmgcs2, 3-hydroxy-3-methylglutaryl-CoA synthase 2; Ifn-γ, interferon gamma; Krt23, keratin 23; Lipe, lipase E; Pcna1, proliferating cell nuclear antigen 1; Pnpla2, patatin-like phospholipase domain containing 2; Scd1, stearoyl-CoA desaturase 1; Slc27a1, solute carrier family 27 member 1

FIGURE 2

PPARα activation–induced hepatomegaly is abolished in hepatocyte-specific Pparα-deficient (Ppara^ΔHep) mice. (A) Ppara^fl/fl and Ppara^ΔHep mice were treated with vehicle or WY-14643 (100 mg/kg/day) for 10 days. (B) Representative liver photos of vehicle-treated or WY-14643-treated mice. (C) Liver-to-body weight ratios (n = 5). (D) H&E staining, CTNNB1 staining for CV areas, and KI67 staining for PV areas of representative mouse liver samples. (E) Quantification of cell size (n = 3) and the number of KI67⁺ cells (n = 3). Data are presented as mean ± SD, ***p < 0.001, ****p < 0.0001 compared to the vehicle group. Scale bar, 50 μm

FIGURE 3

PPARα activation promotes liver regeneration post-PHx in Ppara^fl/fl mice but not in Ppara^ΔHep mice. (A) Ppara^fl/fl and Ppara^ΔHep mice were treated with vehicle or WY-14643 (100 mg/kg/day) for 2 days following PHx. (B) Liver-to-body weight ratios (n = 3–5). (C) Representative liver photos of vehicle-treated or WY-14643-treated mice. (D) Serum AST, ALT, and ALP levels after WY-14643 treatment in PHx mice. (E) H&E staining, CTNNB1 staining for CV areas, and KI67 staining for PV areas of representative mouse liver samples. (F) Quantification of cell size (n = 3) and the number of KI67⁺ cells (n = 3). Data are presented as mean ± SD, *p < 0.05, **p < 0.01 compared to the vehicle group. Scale bar, 50 μm

FIGURE 4

PPARα regulates and interacts with YAP signaling pathway. (A) Western blot analysis of total YAP, nuclear YAP, cytoplasmic p-YAP, YAP downstream proteins, and proliferation-related proteins in wild-type, Ppara^ΔHep, and PHx mice. (B) Co-IP assays of PPAR α and YAP in HepG2 cells. (C) Immunofluorescence staining of PPARα and YAP in HepG2 cells treated with 20 μM of WY-14643 for 48 hours. Nuclei were counterstained with DAPI. (D) Schematic illustration of the PPARα truncations used for co-IP assays. (E) Co-IP analysis of YAP and PPARα truncations. Abbreviations: ACTB, actin beta; AF-1/2, activation function 1/2; DBD, DNA binding domain; HA, hemagglutinin; IB, immunoblot; IP, immunoprecipitation; LBD, ligand binding domain; LMNB1, lamin B1; NTD, N-terminal domain

FIGURE 5

YAP–TEAD interaction is involved in PPARα-induced hepatomegaly. (A) Verteporfin inhibits YAP–TEAD interaction in Hippo–YAP signaling. (B) Corn oil–treated mice and verteporfin-treated mice were administered vehicle or WY-14643 (100 mg/kg/day) for 5 days. (C) Representative liver photos of vehicle-treated or WY-14643-treated mice. (D) Liver-to-body weight ratios (n = 5). (E) H&E staining, CTNNB1 staining for CV areas, and KI67 staining for PV areas of representative mouse liver samples. (F) Quantification of cell size (n = 3) and the number of KI67⁺ cells (n = 3). Data are presented as mean ± SD, **p < 0.01, ****p < 0.0001 compared to the vehicle group. Scale bar, 50 μm

FIGURE 6

PPARα-induced hepatomegaly is blocked in AAV-Yap shRNA-treated mice. (A) AAV-Control and AAV-Yap shRNA-treated mice were administered vehicle or WY-14643 (100 mg/kg/day) for 10 days. (B) Fluorescence observation of EGFP. (C) Western blot analysis of total YAP. (D) Quantification of YAP expression. (E) Representative liver photos of vehicle-treated or WY-14643-treated mice. (F) Liver-to-body weight ratios (n = 5). (G) H&E staining, CTNNB1 staining for CV areas, and KI67 staining for PV areas of representative mouse liver samples. (H) Quantification of cell size (n = 3) and the number of KI67⁺ cells (n = 3). Data are presented as mean ± SD, *p < 0.05, **p < 0.01, ****p < 0.0001 compared to the vehicle group. Scale bar, 50 μm. Abbreviation: ACTB, actin beta

FIGURE 7

PPARα activation fails to induce hepatomegaly in Yap^ΔHep mice. (A) Yap^fl/fl and Yap^ΔHep mice were treated with vehicle or WY-14643 (100 mg/kg/day) for 10 days. (B) Western blot analysis of total YAP. (C) Quantification of YAP expression. (D) Representative liver photos of vehicle-treated or WY-14643-treated mice. (E) Liver-to-body weight ratios (n = 4–5). (F) H&E staining, CTNNB1 staining for CV areas, and KI67 staining for PV areas of representative mouse liver samples. (G) Quantification of cell size (n = 3) and the number of KI67⁺ cells (n = 3). Data are presented as mean ± SD, **p < 0.01, ****p < 0.0001 compared to the vehicle group. Scale bar, 50 μm. Abbreviation: ACTB, actin beta

FIGURE 8

Proposed mechanisms of YAP–TEAD mediating PPARα-induced hepatomegaly and liver regeneration