Keap1/Nrf2 signaling regulates oxidative stress tolerance and lifespan in Drosophila - PubMed (original) (raw)
Keap1/Nrf2 signaling regulates oxidative stress tolerance and lifespan in Drosophila
Gerasimos P Sykiotis et al. Dev Cell. 2008 Jan.
Abstract
Keap1/Nrf2 signaling defends organisms against the detrimental effects of oxidative stress and has been suggested to abate its consequences, including aging-associated diseases like neurodegeneration, chronic inflammation, and cancer. Nrf2 is a prominent target for drug discovery, and Nrf2-activating agents are in clinical trials for cancer chemoprevention. However, aberrant activation of Nrf2 by keap1 somatic mutations may contribute to carcinogenesis and promote resistance to chemotherapy. To evaluate potential functions of Keap1 and Nrf2 for organismal homeostasis, we characterized the pathway in Drosophila. We demonstrate that Keap1/Nrf2 signaling in the fruit fly is activated by oxidants, induces antioxidant and detoxification responses, and confers increased tolerance to oxidative stress. Importantly, keap1 loss-of-function mutations extend the lifespan of Drosophila males, supporting a role for Nrf2 signaling in the regulation of longevity. Interestingly, cancer chemopreventive drugs potently stimulate Drosophila Nrf2 activity, suggesting the fruit fly as an experimental system to identify and characterize such agents.
Figures
Figure 1. Nrf2 and Keap1 homologues are conserved in Drosophila
A. The Keap1/Nrf2 signaling pathway. In basal conditions, Keap1 binds to Nrf2 and inhibits its activity. Oxidative stressors, electrophilic xenobiotics, and cancer chemopreventive agents releave this inhibition. Stabilized Nfr2 then accumulates in the nucleus and dimerizes with a small Maf protein to transcriptionally activate a battery of cell protective genes. B. Nrf2 and Keap1 homologues are present in Drosophila. The cnc locus of Drosophila encodes three protein products. All contain the bZIP region that mediates dimerization and DNA binding. Only the longest isoform, CncC, contains domains predicted to bind Keap1, including the ETGE motif, and is thus potentially a Nrf2 homologue. The Drosophila Keap1 protein shows a high degree of sequence similarity to its vertebrate Keap1 counterparts (sequence alignments for CncC and Keap1 proteins are shown in Supplemental Fig 1). Conserved domains include the BTB/POZ domain required for dimerization and 6 Kelch repeats for binding to Nrf2 and anchoring to actin. C. Over-expression of Drosophila Keap1 can inhibit CncC activity in vivo. Expression of CncC in the developing Drosophila eye from a UAS transgene under the control of sepGal4 causes a reproducible aberrant phenotype. Over-expression of Keap1 under the same conditions (or with GMRGal4, data not shown) has no phenotypic effect, but it can completely suppress the effects of CncC over-expression. The activity of the shorter CncB isoform, which lacks the putative Keap1-interacting domain, is not inhibited by Keap1 co-expression. The GMRGal4 driver was used to express CncB (and Keap1) in the rightmost two panels of C., because expression by sepGal4 did not produce a phenotype at 25°C.
Figure 2. CncC regulates stress response genes and the keap1 gene
A. The over-expression of CncC in the epidermis of the embryo using the enGal4 driver induces the glutathione S-transferase D1 gene, a well known antioxidant and detoxification gene (detected by mRNA in situ hybridization). As negative controls, other members of the gstD gene family were not induced in parallel experiments (not shown). The Drosophila keap1 gene can also be up-regulated by CncC over-expression. B. Validation of gstD1 and keap1 as CncC target genes by real-time RT-PCR. These experiments employ transgenic expression of RNAi targeting either keap1 or cncC mRNA under the control of tubGSGal4, which permits the conditional knockdown of CncC or Keap1 in adult flies. Two separate driver lines with independent transgene insertions were used; tubGS5 (dark blue and dark red bars) consistently yielded slightly stronger effects than tubGS10 (light blue and light red). The expression of UAS keap1RNAi or UAS cncCRNAi (as indicated at the bottom of the histogram) was activated by feeding adults with RU486 as detailed in the methods section. Sibling control flies from the same culture where treated with food containing an equivalent amount of ethanol solvent. The resultant changes in the mRNA levels of gstD1 and keap1 were examined by real time RT-PCR after 4 and 10 days of conditional knockdown.
Figure 3. An oxidative stress-responsive enhancer is regulated by Keap1/CncC
A. Structure of the gstD-GFP reporter transgene. The genomic sequence upstream of the gstD1 gene harbors an ARE sequence, and was used to control the expression of GFP in transgenic reporter flies. The gstD_Δ_ARE-GFP reporter is identical, except that the ARE consensus sequence has been disrupted by base substitutions (see methods). B. The transcriptional activity of the gstD enhancer was potently induced in the gut and in other tissues by oxidants. Animals were exposed to 20 mM Paraquat or 1 mM sodium meta-arsenite in sucrose solution, and GFP fluorescence was monitored after 16 hours. The flies' wings and legs were dissected away to facilitate handling and to expose the abdomen. C. The gstD enhancer responds to Keap1/CncC signaling. The activity of the gstD reporter can be induced by over-expression of CncC or by RNAi-mediated knock-down of Keap1. Keap1 RNAi and CncC expression was driven by the ubiquitously expressed RU486-inducible tubGSGal4 driver; thus the two animals shown in any of the panels are genetically identical and differ only by being fed RU486 or mock treated for 48 hours before analysis. RU486 feeding by itself has no effect on reporter activity (not shown). D. The transcriptional activation of the gstD enhancer by Keap1/CncC signaling is mediated by an ARE. Mutation of the ARE abolishes enhancer activation by both CncC over-expression and Keap1 knock-down.
Figure 4. The cancer chemopreventive agent oltipraz regulates the gstD enhancer via CncC and the ARE
A. Activation of the gstD enhancer by oltipraz requires the ARE. Feeding oltipraz to flies activates the gstD-GFP reporter. However, mutation of the ARE completely abolishes reporter induction by oltipraz. The activity of the gstD-GFP reporter was monitored by inspection of the respective animal under UV light (bottom panel). The same animals are also shown in white light illumination. B. Activation of the gstD enhancer by oltipraz requires CncC. CncC activity was reduced by RU486-inducible knock-down of CncC for 4 days before oltipraz administration. Targeting CncC by RNAi reduced both the basal activity of the gstD enhancer, and its activation by oltipraz.
Figure 5. CncC mediates the resistance of Drosophila to oxidative stress
A. Over-expression of CncC before a Paraquat challenge increases the flies' survival rate. Flies bearing tubGSGal4 and UAS cncC transgenes were maintained on RU486-containing or control food for 4 days before being challenged with a semi-lethal dose of Paraquat. The survival rate of RU486-fed female and male flies 16 hours after the start of Paraquat exposure was significantly higher than that of their sibling controls, suggesting that CncC over-expression increases oxidative stress tolerance. B. RNAi-mediated knock-down of CncC before a Paraquat challenge reduces the flies' survival rate. Flies bearing tubGSGal4 and UAS cncCRNAi transgenes were maintained on RU486-containing or control food for 4 days before being challenged with Paraquat. The survival rate of RU486-fed female and male flies after 16 hours was significantly lower than that of their sibling controls, suggesting that CncC is required for normal oxidative stress tolerance. All data are represented as mean ± SEM of three experiments performed in triplicate. p-values are shown below each histogram. The flies' gender and specific genetic background can profoundly influence their tolerance to oxidative stress. Therefore, the dose of Paraquat for each experiment was adjusted to achieve (approximately) a 50% death rate in each control group after 16 hours. Moreover, only flies within a panel are directly comparable, because they are of the same gender and genetically identical, and differ only in having being fed RU486 for 4 days before the stress. RU486 feeding by itself has no effect on the Paraquat resistance of wild-type w1118 flies, or on any of the tubGSGal4, UAS cncC, and UAS cncCRNAi stocks when out-crossed to the w1118 background (not shown).
Figure 6. keap1 heterozygosity confers oxidative stress resistance and lifespan extension
A. Structure of the Drosophila keap1 gene and nature of loss-of-function alleles. Alternative keap1 transcripts differ at their 5′ ends. Exons are depicted as boxes, with coding segments indicated by dark shading. EY02632 is a homozygous viable allele; keap1036, keap1EY1, and keap1EY5 are larval lethal alleles (see Supplemental Method). B. Elevated gstD1 expression levels in keap1 heterozygotes. mRNA levels of gstD1 in 1 day old flies were quantified separately for males and females by real-time RT-PCR. Control flies were siblings of the heterozygotes and were homozygous wild-type for keap1. Data shown are mean ± SEM of three experiments performed in duplicate. C. Partial loss of Keap1 increases Paraquat resistance. Male heterozygous keap1EY1/+ and keap1EY5/+ flies show a significantly higher survival rate 16 hours after a Paraquat challenge than their otherwise genetically identical wild-type siblings. Data are represented as mean ± SEM of four experiments performed in triplicate. Females do not display this effect. D. keap1 heterozygosity extends lifespan. Under standard culture conditions, male keap1EY1/+ and keap1EY5/+ flies live significantly longer than their sibling controls. Data are represented as the percentage of flies that are alive at each age. 500-700 flies of each genotype and gender were assayed (see methods for experimental details).
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