Hepatocyte-specific deletion of the keap1 gene activates Nrf2 and confers potent resistance against acute drug toxicity

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Abstract

Nrf2 is a key regulator of many detoxifying enzyme genes, and cytoplasmic protein Keap1 represses the Nrf2 activity under quiescent conditions. Germ line deletion of the keap1 gene results in constitutive activation of Nrf2, but the pups unexpectedly died before weaning. To investigate how constitutive activation of Nrf2 influences the detoxification system in adult mice, we generated mice bearing a hepatocyte-specific disruption of the keap1 gene. Homozygous mice were viable and their livers displayed no apparent abnormalities, but nuclear accumulation of Nrf2 is elevated. Microarray analysis revealed that, while many detoxifying enzyme genes are highly expressed, some of the typical Nrf2-dependent genes are only marginally increased in the Keap1-deficient liver. The mutant mice were significantly more resistant to toxic doses of acetaminophen than control animals. These results demonstrate that chronic activation of Nrf2 confers animals with resistance to xenobiotics without affecting the morphological and physiological integrity of hepatocytes.

Section snippets

Materials and methods

Generation of mice with floxed keap1-targeted allele and hepatocyte-specific disruption of the keap1 gene. Mouse keap1 genomic DNA was isolated from mouse 129SvJ genomic library (Stratagene). To construct a targeting vector, we included cDNAs encoding Diphtheria toxin A (DTA) and neomycin resistance (Neo) for negative and positive selection, respectively. We constructed a triple-loxP keap1-targeting vector to generate a floxed mouse keap1-targeted locus consisting of a loxP site inserted into

Generation of hepatocyte-specific keap1-null mutant mice

To examine how constitutive activation of Nrf2 modulates the defense mechanisms against xenobiotic toxicity, we generated hepatocyte-specific keap1-null mutant mice using the Cre-loxP system (Fig. 1). A targeting vector was constructed in which exons 4–6 of the keap1 locus were flanked with loxP sequences (Fig. 1A). This region encodes the double glycine repeat (DGR) domain, a region that is important for Keap1 interaction with Nrf2 [29], [30]. It was expected therefore that Cre-loxP

Discussion

In this study, we observed that somatic disruption of keap1 does not interfere with the development or the morphological and physiological integrity of the liver. Our microarray analysis showed that genetic disruption of the keap1 gene induced overlapping but clearly distinct sets of drug-metabolizing enzymes from those induced by exogenous chemicals. Through the induction of detoxifying enzymes, specific knockout of the keap1 gene confers hepatocytes with a strong resistance to drug-induced

Acknowledgments

We thank Drs. K. Araki and K. Yamamura for providing us AYU1-Cre transgenic mice and Dr. S. Yamamoto for giving us advice on microarray data interpretation. We also thank Ms. N. Kaneko and R. Kawai for experimental assistance and Dr. V. Kelley for English edition. This work was supported by grants from the Ministry of Education, Science, Sports and Culture (H.M. and M.Y.), the Uehara Memorial Foundation (H.M.), Yamanouchi Foundation (H.M.), Atherosclerosis Foundation (M.Y.), and ERATO-JST

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