Generation and characterization of keap1a- and keap1b-knockout zebrafish.

The Keap1-Nrf2 pathway is an evolutionarily conserved mechanism that protects cells from oxidative stress and electrophiles. Under homeostatic conditions, Keap1 interacts with Nrf2 and leads to its rapid proteasomal degradation, but when cells are exposed to oxidative stress/electrophiles, Keap1 senses them, resulting in an improper Keap1-Nrf2 interaction and Nrf2 stabilization. Keap1 is therefore considered both an "inhibitor" of and "stress sensor" for Nrf2 activation. Interestingly, fish and amphibians have two Keap1s (Keap1a and Keap1b), while there is only one in mammals, birds and reptiles. A phylogenetic analysis suggested that mammalian Keap1 is an ortholog of fish Keap1b, not Keap1a. In this study, we investigated the differences and similarities between Keap1a and Keap1b using zebrafish genetics. We generated zebrafish knockout lines of keap1a and keap1b. Homozygous mutants of both knockout lines were viable and fertile. In both mutant larvae, the basal expression of Nrf2 target genes and antioxidant activity were up-regulated in an Nrf2-dependent manner, suggesting that both Keap1a and Keap1b can function as Nrf2 inhibitors. We also analyzed the effects of the Nrf2 activator sulforaphane in these mutants and found that keap1a-, but not keap1b-, knockout larvae responded to sulforaphane, suggesting that the stress/chemical-sensing abilities of the two Keap1s are different.