Genetic Evidence that Small Maf Proteins Are Essential for the Activation of Antioxidant Response Element-Dependent Genes

Abstract

While small Maf proteins have been suggested to be essential for the Nrf2-mediated activation of antioxidant response element (ARE)-dependent genes, the extent of their requirement remains to be fully documented. To address this issue, we generated mafG::mafF double-mutant mice possessing MafK as the single available small Maf. Induction of the NAD(P)H:quinone oxidoreductase 1 (NQO1) gene was significantly impaired in double-mutant mice treated with butylated hydroxyanisole, while other ARE-dependent genes were less affected. Similarly, in a keap1-null background, where many of the ARE-dependent genes are constitutively activated in an Nrf2-dependent manner, only a subset of ARE-dependent genes, including NQO1, were sensitive to a simultaneous deficiency in MafG and MafF. Examination of single and double small maf mutant cells revealed that MafK also contributes to the induction of ARE-dependent genes. To obtain decisive evidence, we established mafG::mafK::mafF triple-mutant fibroblasts that completely lack small Mafs and turned out to be highly susceptible to oxidative stress. We found that induction in response to diethyl maleate was abolished in a wider range of ARE-dependent genes in the triple-mutant cells. These data explicitly demonstrate that small Mafs play critical roles in the inducible expression of a significant portion of ARE-dependent genes.

ACKNOWLEDGMENTS

We greatly appreciate the assistance of Y. Meguro in the microarray analysis. We are grateful to K. Itoh, M. Kobayashi, and T. Suzuki for advice and helpful comments. We thank T. O'Connor, Y. Tamagawa, R. Kawai, and A. Sakurai for help.

This work was supported by grants from the NIH (CA80088 and GM28896 to F.K., H.M., and J.D.E.), ERATO-JST (M.Y.), the Ministry of Education, Science, Sports and Culture (H.M. and M.Y.), the Atherosclerosis Foundation (M.Y.), the Yamanouchi Foundation for Research on Metabolic Disorders (H.M.), and the Uehara Memorial Foundation (H.M.). F.K. is a JSPS Research Fellow.