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

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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References

  1. Motohashi, H., O'Connor, T., Katsuoka, F., Engel, J.D. & Yamamoto, M. Integration and diversity of the regulatory network composed of Maf and CNC families of transcription factors. Gene 294, 1–12 (2002).

    Article  CAS  Google Scholar 

  2. Moi, P., Chan, K., Asunis, I., Cao, A. & Kan, Y.W. Isolation of NF-E2-related factor 2 (Nrf2), a NF-E2-like basic leucine zipper transcriptional activator that binds to the tandem NF-E2/AP1 repeat of the beta-globin locus control region. Proc. Natl. Acad. Sci. USA 91, 9926–9930 (1994).

    Article  CAS  Google Scholar 

  3. Itoh, K., Igarashi, K., Hayashi, N., Nishizawa, M. & Yamamoto, M. Cloning and characterization of a novel erythroid cell-derived CNC family transcription factor heterodimerizing with the small Maf family proteins. Mol. Cell. Biol. 15, 4184–4193 (1995).

    Article  CAS  Google Scholar 

  4. Marini, M.G. et al. hMAF, a small human transcription factor that heterodimerizes specifically with Nrf1 and Nrf2. J. Biol. Chem. 272, 16490–16497 (1997).

    Article  CAS  Google Scholar 

  5. Itoh, K. et al. An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem. Biophys. Res. Commun. 236, 313–322 (1997).

    Article  CAS  Google Scholar 

  6. Itoh, K. et al. Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain. Genes Dev. 13, 76–86 (1999).

    Article  CAS  Google Scholar 

  7. Ishii, T. et al. Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages. J. Biol. Chem. 275, 16023–16029 (2000).

    Article  CAS  Google Scholar 

  8. Enomoto, A. et al. High sensitivity of Nrf2 knockout mice to acetaminophen hepatotoxicity associated with decreased expression of ARE-regulated drug metabolizing enzymes and antioxidant genes. Toxicol. Sci. 59, 169–177 (2001).

    Article  CAS  Google Scholar 

  9. Ramos-Gomez, M. et al. From the Cover: Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice. Proc. Natl. Acad. Sci. USA 98, 3410–3415 (2001).

    Article  CAS  Google Scholar 

  10. Aoki, Y. et al. Accelerated DNA adduct formation in the lung of the Nrf2 knockout mouse exposed to diesel exhaust. Toxicol. Appl. Pharmacol. 173, 154–160 (2001).

    Article  CAS  Google Scholar 

  11. Adams, J., Kelso, R. & Cooley, L. The kelch repeat superfamily of proteins: propellers of cell function. Trends Cell Biol. 10, 17–24 (2000).

    Article  CAS  Google Scholar 

  12. Oyake, T. et al. Bach proteins belong to a novel family of BTB-basic leucine zipper transcription factors that interact with MafK and regulate transcription through the NF-E2 site. Mol. Cell. Biol. 16, 6083–6095 (1996).

    Article  CAS  Google Scholar 

  13. Yoshida, C. et al. Long range interaction of cis-DNA elements mediated by architectural transcription factor Bach1. Genes Cells 4, 643–655 (1999).

    Article  CAS  Google Scholar 

  14. Zipper, L.M. & Mulcahy, R.T. The Keap1 BTB/POZ dimerization function is required to sequester Nrf2 in cytoplasm. J. Biol. Chem. 277, 36544–36552 (2002).

    Article  CAS  Google Scholar 

  15. Robinson, D.N. & Cooley, L. Drosophila kelch is an oligomeric ring canal actin organizer. J. Cell Biol. 138, 799–810 (1997).

    Article  CAS  Google Scholar 

  16. Bork, P. & Doolittle, R.F. Drosophila kelch motif is derived from a common enzyme fold. J. Mol. Biol. 236, 1277–1282 (1994).

    Article  CAS  Google Scholar 

  17. Fulop, V. & Jones, D.T. Beta propellers: structural rigidity and functional diversity. Curr. Opin. Struct. Biol. 9, 715–721 (1999).

    Article  CAS  Google Scholar 

  18. Xue, F. & Cooley, L. kelch encodes a component of intercellular bridges in Drosophila egg chambers. Cell 72, 681–693 (1993).

    Article  CAS  Google Scholar 

  19. Sinha, S., Degenstein, L., Copenhaver, C. & Fuchs, E. Defining the regulatory factors required for epidermal gene expression. Mol. Cell. Biol. 20, 2543–2555 (2000).

    Article  CAS  Google Scholar 

  20. DiSepio, D. et al. Characterization of loricrin regulation in vitro and in transgenic mice. Differentiation 64, 225–235 (1999).

    Article  CAS  Google Scholar 

  21. Takahashi, K., Yan, B., Yamanishi, K., Imamura, S. & Coulombe, P.A. The two functional keratin 6 genes of mouse are differentially regulated and evolved independently from their human orthologs. Genomics 53, 170–183 (1998).

    Article  CAS  Google Scholar 

  22. Itoh, K., Ishii, T., Wakabayashi, N. & Yamamoto, M. Regulatory mechanisms of cellular response to oxidative stress. Free Radic. Res. 31, 319–324 (1999).

    Article  CAS  Google Scholar 

  23. Katoh, Y. et al. Two domains of Nrf2 cooperatively bind CBP, a CREB binding protein, and synergistically activate transcription. Genes Cells 6, 857–868 (2001).

    Article  CAS  Google Scholar 

  24. Li, N. & Karin, M. Signaling pathways leading to nuclear factor-kappa B activation. Methods Enzymol. 319, 273–279 (2000).

    Article  CAS  Google Scholar 

  25. Robbins, D.J. et al. Hedgehog elicits signal transduction by means of a large complex containing the kinesin-related protein costal2. Cell 90, 225–234 (1997).

    Article  CAS  Google Scholar 

  26. Wojcik, S.M., Bundman, D.S. & Roop, D.R. Delayed wound healing in keratin 6a knockout mice. Mol. Cell. Biol. 20, 5248–5255 (2000).

    Article  CAS  Google Scholar 

  27. Wojcik, S.M., Longley, M.A. & Roop, D.R. Discovery of a novel murine keratin 6 (K6) isoform explains the absence of hair and nail defects in mice deficient for K6a and K6b. J. Cell. Biol. 154, 619–630 (2001).

    Article  CAS  Google Scholar 

  28. Koch, P.J. et al. Lessons from loricrin-deficient mice: compensatory mechanisms maintaining skin barrier function in the absence of a major cornified envelope protein. J. Cell. Biol. 151, 389–400 (2000).

    Article  CAS  Google Scholar 

  29. Thiele, J.J., Hsieh, S.N., Briviba, K. & Sies, H. Protein oxidation in human stratum corneum: susceptibility of keratins to oxidation in vitro and presence of a keratin oxidation gradient in vivo. J. Invest. Dermatol. 113, 335–339 (1999).

    Article  CAS  Google Scholar 

  30. Williams, M.L. & Elias, P.M. Heterogeneity in autosomal recessive ichthyosis. Clinical and biochemical differentiation of lamellar ichthyosis and nonbullous congenital ichthyosiform erythroderma. Arch. Dermatol. 121, 477–488 (1985).

    Article  CAS  Google Scholar 

  31. Russell, L.J., DiGiovanna, J.J., Hashem, N., Compton, J.G. & Bale, S.J. Linkage of autosomal recessive lamellar ichthyosis to chromosome 14q. Am. J. Hum. Genet. 55, 1146–1152 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Parmentier, L. et al. Mapping of a second locus for lamellar ichthyosis to chromosome 2q33–35. Hum. Mol. Genet. 5, 555–559 (1996).

    Article  CAS  Google Scholar 

  33. Virolainen, E. et al. Assignment of a novel locus for autosomal recessive congenital ichthyosis to chromosome 19p13.1–p13.2. Am. J. Hum. Genet. 66, 1132–1137 (2000).

    Article  CAS  Google Scholar 

  34. Tiemann, F. & Deppert, W. Immortalization of BALB/c mouse embryo fibroblasts alters SV40 large T-antigen interactions with the tumor suppressor p53 and results in a reduced SV40 transformation-efficiency. Oncogene 9, 1907–1915 (1994).

    CAS  PubMed  Google Scholar 

  35. Yuspa, S.H., Kilkenny, A.E., Steinert, P.M. & Roop, D.R. Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro. J. Cell Biol. 109, 1207–1217 (1989).

    Article  CAS  Google Scholar 

  36. Dignam, J.D. Preparation of extracts from higher eukaryotes. Methods Enzymol. 182, 194–203 (1990).

    Article  CAS  Google Scholar 

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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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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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