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Keap1-null mutation leads to postnatal lethality due to constitutive Nrf2 activation

Nature Genetics volume 35, pages 238–245 (2003)Cite this article

Abstract

Transcription factor Nrf2 (encoded by Nfe2l2) regulates a battery of detoxifying and antioxidant genes, and Keap1 represses Nrf2 function. When we ablated Keap1, Keap1-deficient mice died postnatally, probably from malnutrition resulting from hyperkeratosis in the esophagus and forestomach. Nrf2 activity affects the expression levels of several squamous epithelial genes. Biochemical data show that, without Keap1, Nrf2 constitutively accumulates in the nucleus to stimulate transcription of cytoprotective genes. Breeding to Nrf2-deficient mice reversed the phenotypic Keap1 deficiencies. These experiments show that Keap1 acts upstream of Nrf2 in the cellular response to oxidative and xenobiotic stress.

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Figure 1: Growth retardation and skin abnormalities in Keap1-deficient mice.
Figure 2: Macroscopic observation of stomachs of Keap1-deficient mice.
Figure 3: Histological analysis of stomachs of Keap1-deficient mice during development.
Figure 4: Squamous cell proliferation in esophagus of Keap1 mutant mice.
Figure 5: Aberrant expression of squamous cell genes in Keap1-deficient mice.
Figure 6: Drug metabolizing enzymes and antioxidant proteins are constitutively expressed in Keap1-deficient mice and cells.
Figure 7: Nrf2 loss rescues Keap1 mutant phenotypes.

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Acknowledgements

We are grateful to O. Nakajima, F. Irie, M. Osaki, S. Kawauchi, X. Pan, S. Masuda, N. Kaneko, H. Ohkawa and R. Kawai for help; K-C. Lim, M. Kobayashi, K. Igarashi, T. O'Connor, T. Hosoya, M. Nose, Y. Kawachi and T. W. Kensler for useful suggestions; and J. D. Hayes and K. Satoh for antibodies. This work was supported by a grant from the US National Institutes of Health (J.D.E.) and grants from the Ministry of Education, Science, Sports and Culture (K.I., H.M. and M.Y.), JST-ERATO (M.Y.), JST-CREST (H.M.) and PROBRAIN (H.M. and S.T.). N.W. was a JSPS-RFTF postdoctoral fellow.

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Authors and Affiliations

  1. Center for Tsukuba Advanced Research Alliance, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, 305-8577, Japan

    Nobunao Wakabayashi, Ken Itoh, Junko Wakabayashi, Hozumi Motohashi, Shuhei Noda & Masayuki Yamamoto

  2. ERATO-JST, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, 305-8577, Japan

    Ken Itoh & Masayuki Yamamoto

  3. Institutes of Basic Medical Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, 305-8577, Japan

    Satoru Takahashi & Masayuki Yamamoto

  4. Clinical Medical Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, 305-8577, Japan

    Sumihisa Imakado, Tomoe Kotsuji & Fujio Otsuka

  5. Departments of Molecular and Cellular Biology and Dermatology, Baylor College of Medicine, One Baylor Plaza, Houston, 77030, Texas, USA

    Dennis R Roop

  6. The Institute of Environmental Toxicology 4321 Uchimoriya-cho, Mitsukaido-shi, 303-0043, Ibaraki, Japan

    Takanori Harada

  7. Department of Cell and Developmental Biology, University of Michigan Medical School, 1335 Catherine, Ann Arbor, 48109, Michigan, USA

    James Douglas Engel

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Correspondence to Masayuki Yamamoto.

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Supplementary information

Supplementary Fig. 1. Strategy of Keap1 Gene Disruption by Homologous Recombination.

a. The mouse Keap1 gene targeting vector (top line), the wild type Keap1 locus (middle line) and the mutant allele generated by homologous integration of the targeting vector (bottom line) are depicted. NLS-LacZ, Neo and DTA gene cassettes are shown along with restriction enzyme sites. The middle line indicates the Keap1 exons. Positions of the 5'-probe (blue box) and 3'-probe (green box) used for the genomic Southern analyses are also indicated with the sizes of the predicted fragments. b. Genomic Southern blot analyses of the two independent ES cell clones and heterozygous and homozygous Keap1 knockout mice. Top and bottom panels show detection by the 5'- and 3'-probes, respectively. Genomic DNA samples from wild type ES (+/+, lane 1), ES clone-1 (lane 2) or -2 (lane 3) cells are shown. Genomic DNA samples from Keap1 homozygous mutant (-/-, lane 4), heterozygous mutant (+/-, lane 5), or wild type (lane 6) animals were also analyzed. Sizes of the wild type and mutant DNA fragments are indicated. c. Loss of Keap1 mRNA. RNA samples extracted from MEFs of wild type (lane 1), heterozygous (lane 2) and homozygous (lane 3) Keap1 mutant mice were probed with radiolabeled Keap1 cDNA. GAPDH RNA (middle panel) and rRNA (bottom panel) were used for normalization. d. Expression of the LacZ gene inserted into the Keap1 locus. β-Galactosidase expression was visualized by immunoblotting. Total liver proteins from neonatal wild type (lane 1), heterozygous (lane 2) or homozygous (lane 3) Keap1 mutant mice were interrogated using an anti-β-galactosidase antibody (arrowhead). Nuclear Lamin B was also detected using an antiserum to normalize the amounts of protein loaded in each lane (arrow). (JPG 73 kb)

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Wakabayashi, N., Itoh, K., Wakabayashi, J. et al. Keap1-null mutation leads to postnatal lethality due to constitutive Nrf2 activation. Nat Genet 35, 238–245 (2003). https://doi.org/10.1038/ng1248

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