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Abstract
Mammalian Nrf2-Keap1 and the homologous Drosophila CncC-dKeap1 protein complexes regulate both transcriptional responses to xenobiotic compounds as well as native cellular and developmental processes. The relationships between the functions of these proteins in xenobiotic responses and in development were unknown. We investigated the genes regulated by CncC and dKeap1 during development and the signal transduction pathways that modulate their functions. CncC and dKeap1 were enriched within the nuclei in many tissues, in contrast to the reported cytoplasmic localization of Keap1 and Nrf2 in cultured mammalian cells. CncC and dKeap1 occupied ecdysone-regulated early puffs on polytene chromosomes. Depletion of either CncC or dKeap1 in salivary glands selectively reduced early puff gene transcription. CncC and dKeap1 depletion in the prothoracic gland as well as cncCK6/K6 and dKeap1EY5/EY5 loss of function mutations in embryos reduced ecdysone-biosynthetic gene transcription. In contrast, dKeap1 depletion and the dKeap1EY5/EY5 loss of function mutation enhanced xenobiotic response gene transcription in larvae and embryos, respectively. Depletion of CncC or dKeap1 in the prothoracic gland delayed pupation by decreasing larval ecdysteroid levels. CncC depletion suppressed the premature pupation and developmental arrest caused by constitutive Ras signaling in the prothoracic gland; conversely, constitutive Ras signaling altered the loci occupied by CncC on polytene chromosomes and activated transcription of genes at these loci. The effects of CncC and dKeap1 on both ecdysone-biosynthetic and ecdysone-regulated gene transcription, and the roles of CncC in Ras signaling in the prothoracic gland, establish the functions of these proteins in the neuroendocrine axis that coordinates insect metamorphosis.
Highlights
Cellular responses to many xenobiotic compounds, including various toxins and pharmacological agents, are controlled by mammalian Nrf2 and Keap1, and by the homologous Drosophila CncC and dKeap1 proteins [1,2,3]
Nuclear localization of CncC and dKeap1 To investigate if the subcellular localization of CncC was regulated by dKeap1 in the manner that has been reported for mammalian Nrf2 and Keap1, we determined the distributions of CncC and dKeap1
The intensity of anti-dKeap1 immunoreactivity was markedly reduced in dKeap1EY5/EY5 mutant larvae, and the bands corresponding to endogenous dKeap1 and CncC were not detected by immunoblotting of extracts from dKeap1EY5/EY5 and cncK6/K6 mutant larvae, demonstrating the specificity of these antibodies (Figure S1C, S1D). These observations establish that both endogenous as well as ectopically expressed CncC and dKeap1 were localized to the nuclei in many different tissues, in contrast to the predominantly cytoplasmic localization observed for Keap1 and Nrf2 in many cultured mammalian cell lines
Summary
Cellular responses to many xenobiotic compounds, including various toxins and pharmacological agents, are controlled by mammalian Nrf and Keap, and by the homologous Drosophila CncC and dKeap proteins [1,2,3]. The Nrf2-Keap complex has multiple effects on carcinogenesis. Nrf2-deficient mice have increased susceptibility to chemical carcinogens, potentially because of defective activation of cytoprotective genes in response to carcinogen exposure [4]. The deletion of Nrf suppresses pancreatic and lung tumorigenesis in a mouse model with constitutively active K-RasG12D expression [6]. The mechanisms whereby Nrf promotes tumorigenesis in conjunction with K-RasG12D are not known. Nrf and Keap are investigated as potential targets for therapeutic interventions in cancer, neurodegenerative diseases and developmental disorders [1,7]
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