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. 2005 Mar 15;102(11):4120-5.
doi: 10.1073/pnas.0500660102. Epub 2005 Feb 28.

Liver-specific inactivation of the Nrf1 gene in adult mouse leads to nonalcoholic steatohepatitis and hepatic neoplasia

Zhenrong Xu  1 , Linyun Chen, Laura Leung, T S Benedict Yen, Candy Lee, Jefferson Y Chan
Affiliations

Liver-specific inactivation of the Nrf1 gene in adult mouse leads to nonalcoholic steatohepatitis and hepatic neoplasia

Zhenrong Xu et al. Proc Natl Acad Sci U S A. .

Abstract

Knockout studies have shown that the transcription factor Nrf1 is essential for embryonic development. Nrf1 has been implicated to play a role in mediating activation of oxidative stress response genes through the antioxidant response element (ARE). Because of embryonic lethality in knockout mice, analysis of this function in the adult knockout mouse was not possible. We report here that mice with somatic inactivation of nrf1 in the liver developed hepatic cancer. Before cancer development, mutant livers exhibited steatosis, apoptosis, necrosis, inflammation, and fibrosis. In addition, hepatocytes lacking Nrf1 showed oxidative stress, and gene expression analysis showed decreased expression of various ARE-containing genes, and up-regulation of CYP4A genes. These results suggest that reactive oxygen species generated from CYP4A-mediated fatty acid oxidation work synergistically with diminished expression of ARE-responsive genes to cause oxidative stress in mutant hepatocytes. Thus, Nrf1 has a protective function against oxidative stress and, potentially, a function in lipid homeostasis in the liver. Because the phenotype is similar to nonalcoholic steatohepatitis, these animals may prove useful as a model for investigating molecular mechanisms of nonalcoholic steatohepatitis and liver cancer.

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Figures

Fig. 1.
Fig. 1.
Targeting of the nrf1 locus. (a Top) Map of the targeting construct containing loxP elements (dark ovals) flanking the terminal exon of nrf1 and the positive (PGKNEO) selection cassette. The targeting construct also contains an internal ribosome entry site (IRES)-EGFP cassette and a negative (DT) selection cassette. (Middle) The terminal portion of the wild-type nrf1 gene, and the 5′ external probe used to detect targeted clones. (Bottom) The targeted allele. Sites for restriction enzymes EcoRI (R) and NsiI (N) are shown. The predicted sizes of NsiI fragments of wild-type (14-kb) and targeted allele (11.5-kb) are shown. (b) Southern blot analysis of ES clones digested with NsiI and probed with the 5′ external probe. Asterisks indicate targeted clones. (c Top) The nrf1-neo knockout allele described previously (11). (Middle) The floxed nrf1 allele. (Bottom) The Cre-mediated recombined allele. Restriction enzyme sites for EcoRI (R), and NsiI (N) are shown. Primers used to detect the nonrecombined floxed nrf1 allele (250-bp product) and the recombined allele (500-bp) are indicated by short straight arrows and wiggly arrow, respectively. (Right) Southern blot analysis of liver DNA obtained from control and Nrf1LKO mice. The top band represents the 4kb EcoRI fragment of both the nrf1-neo and floxed nrf1 alleles detected by the indicated probe. The lower band represents the 2.1kb EcoRI fragment of the recombined allele. (d) Analysis of recombined and nonrecombined floxed nrf1 allele in liver DNA. The top band represents the 550-bp product amplified from the recombined floxed nrf1 allele. The bottom band represents the 250-bp product amplified from the nonrecombined floxed nrf1 allele.
Fig. 4.
Fig. 4.
Expression of ARE-regulated genes in Nrf1LKO livers. (a) Western blot analysis of control and Nrf1LKO livers. Equal loading of protein in each lane was confirmed by actin. (b) RT-PCR analysis of mRNA encoding various ARE-dependent genes and lipid metabolism. 18s levels were used as control. Figures are representative of six control and six Nrf1LKO liver samples.
Fig. 2.
Fig. 2.
Steatohepatitis and liver cancer in Nrf1LKO mice. (a) Serum concentration of alanine aminotransferase (ALT) in control and Nrf1LKO mice at 4 weeks of age. Bar graph shows mean ± SD of 5–7 animals per group. *, P < 0.05. (b) Liver triglyceride levels in control and Nrf1LKO mice at 6–8 weeks of age. Bar graph shows mean ± SD of 6–8 animals per group. *, P < 0.05. (c) A representative Nrf1LKO liver at 4–8 weeks of age stained with hematoxylin/eosin showing apoptosis (chevrons), necrosis (black arrow), inflammatory infiltrate (Inset), and vacuolated cells. (d) Immunohistochemical staining for active caspase-3 in Nrf1LKO liver showing multiple apoptotic hepatocytes. (e) Frozen section of Nrf1LKO liver stained with oil red O showing increased number of lipid droplets. (f) Cultured primary Nrf1LKO hepatocytes stained with oil red O showing increased number of lipid droplets. (g) Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) showing increased number of actively dividing hepatocytes in Nrf1LKO liver. (h) Masson's trichrome staining showing fibrosis in a 6-month-old Nrf1LKO animal. (i) Gross appearance of a liver from a 4-month-old Nrf1LKO mouse showing multiple small nodules. (j) Representative liver from a control mouse at 12 months of age. (k) Liver from an Nrf1LKO mouse at 12 months of age showing multiple large, vascularized nodules. (l) Hematoxylin/eosin-stained sections showing hepatocellular adenomas with distinctive borders between tumors and parenchyma. (m) Hematoxylin/eosin stained sections showing HCC with trabecullar features. (n) Section showing HCC-containing clear cells.
Fig. 3.
Fig. 3.
Oxidative stress in Nrf1LKO livers. (a) Liver TBARS levels in control and Nrf1LKO mice. Values are expressed as mean ± SD for six control (open bar) and five Nrf1LKO (filled bar); P < 0.05. (b) Immunohistochemical staining for 8-oxoG showing no reactivity in normal liver. (c) Immunohistochemical detection of 8-oxoG-positive cells in Nrf1LKO liver. Shown are flow cytometric determination of dichlorofluorescein (DCF) fluorescence reporter DCF dye in untreated hepatocytes (d) and in hepatocytes treated with 100 μM tert-butylhydroperoxide (tBHP) (e). The x axis shows a 4-decade log scale representing fluorescent emission, and the y axis represents relative number of cells. Genotypes are indicated. Figures are representative overlays.
Fig. 5.
Fig. 5.
Microsome proliferation and induction of ω-fatty acid oxidation in Nrf1LKO livers. (a) RT-PCR analysis of mRNA encoding various enzymes associated with β- and ω-fatty acid oxidation. 18s levels were used as control. Representative electron micrograph of control (b) and Nrf1LKO (c) livers at 2 months. Note lipid droplets, smaller and darker mitochondria, and proliferation of smooth ER in the Nrf1LKO liver.
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