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. 2014 Mar;34(5):900-13.
doi: 10.1128/MCB.01384-13. Epub 2013 Dec 30.

Nrf2 enhances cholangiocyte expansion in Pten-deficient livers

Keiko Taguchi  1 , Ikuo Hirano, Tohru Itoh, Minoru Tanaka, Atsushi Miyajima, Akira Suzuki, Hozumi Motohashi, Masayuki Yamamoto
Affiliations

Nrf2 enhances cholangiocyte expansion in Pten-deficient livers

Keiko Taguchi et al. Mol Cell Biol. 2014 Mar.

Abstract

Keap1-Nrf2 system plays a central role in the stress response. While Keap1 ubiquitinates Nrf2 for degradation under unstressed conditions, this Keap1 activity is abrogated in response to oxidative or electrophilic stresses, leading to Nrf2 stabilization and coordinated activation of cytoprotective genes. We recently found that nuclear accumulation of Nrf2 is significantly increased by simultaneous deletion of Pten and Keap1, resulting in the stronger activation of Nrf2 target genes. To clarify the impact of the cross talk between the Keap1-Nrf2 and Pten-phosphatidylinositide 3-kinase-Akt pathways on the liver pathophysiology, in this study we have conducted closer analysis of liver-specific Pten::Keap1 double-mutant mice (Pten::Keap1-Alb mice). The Pten::Keap1-Alb mice were lethal by 1 month after birth and displayed severe hepatomegaly with abnormal expansion of ductal structures comprising cholangiocytes in a Nrf2-dependent manner. Long-term observation of Pten::Keap1-Alb::Nrf2(+/-) mice revealed that the Nrf2-heterozygous mice survived beyond 1 month but developed polycystic liver fibrosis by 6 months. Gsk3 directing the Keap1-independent degradation of Nrf2 was heavily phosphorylated and consequently inactivated by the double deletion of Pten and Keap1 genes. Thus, liver-specific disruption of Keap1 and Pten augments Nrf2 activity through inactivation of Keap1-dependent and -independent degradation of Nrf2 and establishes the Nrf2-dependent molecular network promoting the hepatomegaly and cholangiocyte expansion.

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Figures

FIG 1
FIG 1
Lethality and hepatomegaly in Pten::Keap1-Alb mice. (A) Mating strategy for the generation of compound mutant mice of Pten, Keap1, and Nrf2 genes. A detailed examination was conducted on postnatal day 15 (P15). (B) Survival rates up to P35 (n ≥ 50). (C) Changes in liver-to-body-weight ratios to P28 (n = 11 to 53). (D) Representative macroscopic observation of the livers at P15. The scale bar corresponds to 1 cm. (E) Liver-to-body-weight ratios at P15 (n = 12 to 44). **, P < 0.01.
FIG 2
FIG 2
Increase in cholangiocytes in the Pten::Keap1-Alb mouse livers. (A) Histological analysis of the livers. Results of Masson trichrome staining (a to h) and immunohistochemistry using the anti-CK19 antibody (i to l) at P15 are shown. PV, portal vein; CV, central vein. The scale bars correspond to 1 mm (a to d) and 200 μm (e to l). (B) Serum biochemical test, measuring markers for liver injury (ALT, AST, and LDH), TCHO, DBIL/TBIL ratio, and ALB at P15 (n = 8 to 27). *, P < 0.05; **, P < 0.01. Asterisks without brackets indicate the comparison with control mice.
FIG 3
FIG 3
Expanded cholangiocytes are derived from Pten and Keap1 doubly deficient cells. Liver sections of Pten::Keap::R26R and Pten::Keap1-Alb::R26R mice at P15 were subjected to LacZ staining (A to D), HE staining (E and F), and immunohistochemistry using the anti-CK19 antibody (G and H). Arrowheads indicate tubular structures of cholangiocytes. pv, portal vein. The scale bars correspond to 200 μm (A and B) and 100 μm (C to H).
FIG 4
FIG 4
Pten::Keap1-Alb::Nrf2+/− mice demonstrate progressive hepatomegaly and die by 7 months of age. (A) Compound mutant mice of Pten, Keap1, and Nrf2 genes analyzed at 10 weeks. Pten::Keap1-Alb mice that were lethal by 1 month were not included. (B) Survival rates at up to 240 days after birth (n ≥ 50). (C) Changes in the liver-to-body-weight ratios up to 240 days (n = 2 to 43).
FIG 5
FIG 5
Cholangiocytes expansion in Pten::Keap1-Alb::Nrf2+/− mouse livers at 10 weeks. (A) Liver-to-body-weight ratios at 10 weeks (n = 3 to 10). More than 3 mice were independently examined for males and females of each genotype. *, P < 0.05; **, P < 0.01. Asterisks without brackets indicate the comparison with control mice. (B) Histological analysis of the livers at 10 weeks. Masson trichrome staining (a to j) and immunohistochemistry using the anti-CK19 antibody (k to o) are shown. The scale bars correspond to 1 mm (a to e) and 200 μm (f to o).
FIG 6
FIG 6
Development of polycystic fibrosis in Pten::Keap1-Alb::Nrf2+/− mice at the age of 6 months. (A) Representative macroscopic observation of the livers at 6 months of age. The scale bar corresponds to 1 cm. (B) Polycystic appearance of the Pten::Keap1-Alb::Nrf2+/− mouse liver in a closer view (left and middle panels) and in a section (right panel). Arrowheads indicate grossly recognizable cysts. A red asterisk indicates a cyst. The scale bars correspond to 1 cm (left and middle panels) and 1 mm (right panel). (C) Histological analysis of the livers at 6 months. Masson trichrome staining (a to j) and immunohistochemistry using the anti-CK19 antibody (k to o) are shown. The scale bars correspond to 1 mm (a to e) and 200 μm (f to o).
FIG 7
FIG 7
Expression of cell-specific marker genes in the liver at P15. Cell-specific gene markers were categorized into hepatocyte, cholangiocyte, and oval cell/liver progenitor cells in Pten-Alb, Keap1-Alb, Pten::Keap1-Alb, and Pten::Keap1-Alb::Nrf2−/− mouse livers at P15. The fold change values indicate the base 2 logarithm of the expression ratio to control mouse values.
FIG 8
FIG 8
Gene expression profiles of Pten::Keap1-Alb liver. Relative expression of mRNAs in control, Pten-Alb, Keap1-Alb, Pten::Keap1-Alb, and Pten::Keap1-Alb::Nrf2−/− mouse livers of male mice at P15 (n = 3 to 6). The average values of control mice are set to 1. *, P < 0.05; **, P < 0.01. Asterisks without brackets indicate the comparison with control mice. The full gene names are listed in Fig. 7. (A) Expression levels of cholangiocyte-specific genes. (B) Expression levels of hepatocyte-specific genes. (C) Expression levels of oval cell-specific genes. (D) A representative immunofluorescent image of Pten::Keap1-Alb mouse livers using anti-EpCAM and anti-Trop2 antibodies. Higher magnification of the area surrounded by a white square is shown on the right. Arrowheads indicate the EpCAM-Trop2 double-positive cells. The scale bar corresponds to 50 μm (left panel) and 17 μm (right panel).
FIG 9
FIG 9
Enhancement of Nrf2 activity and the PI3K-Akt pathway in Pten::Keap1-Alb mouse livers. (A) Gene expression levels of representative Nrf2 target genes, Gpx2 and Gclc, in control, Pten-Alb, Keap1-Alb, Pten::Keap1-Alb, and Pten::Keap1-Alb::Nrf2−/− mouse livers at P15 (n = 3 to 6). The average values of control mice are set to 1. *, P < 0.05; **, P < 0.01. Asterisks without brackets indicate the comparison with control mice. (B) Immunoblot analysis of liver extracts from control, Pten-Alb, Keap1-Alb, Pten::Keap1-Alb, and Pten::Keap1-Alb::Nrf2−/− mouse livers measuring the nuclear accumulation of Nrf2, reduction efficiency of Pten and Keap1, and phosphorylation of Akt and Gsk3. Nqo1 is the product of an Nrf2 target gene. Arrowheads indicate Keap1 and two isoforms of Gsk3. Control, Pten-Alb, Keap1-Alb, Pten::Keap1-Alb, and Pten::Keap1-Alb::Nrf2−/− mouse livers were analyzed at P15, and control and Pten-Alb mouse livers were also analyzed at 10 weeks.
FIG 10
FIG 10
Two distinct pathways for Nrf2 degradation. Nrf2 is primarily degraded in a Keap1-Cul3-dependent manner. In Keap1-Alb mouse livers, Nrf2 escapes from the primary degradation but is subjected to secondary degradation mediated through β-TrCP-Cul1-dependent degradation, which limits Nrf2 accumulation. For secondary degradation, Nrf2 needs to be phosphorylated through Gsk3 (left panel). In Pten-Alb mouse livers, Gsk3 is inactivated but does not affect the Nrf2 abundance because the Keap1-Cul3-dependent pathway constantly degrades Nrf2 (middle panel). In Pten::Keap1-Alb mouse livers, Nrf2 escapes from primary and secondary degradation due to Keap1 deletion and Gsk3 inactivation, resulting in a robust increase of Nrf2 accumulation (right panel).
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