Figure 1.
Design of ARE and TRE reporter genes.
A. Simplified cartoons of stress signaling by the Nrf2 and JNK signaling pathways. Upon exposure to stress or cancer chemopreventive drugs, Nrf2 is relieved from repression by its cognate inhibitor Keap1. Nrf2 then dimerizes with Maf-S and binds to AREs of its target genes to activate their transcription. Activation of the keap1 gene itself by Nrf2 engages a negative feedback loop that limits Nrf2 responses. The JNK cascade with the stress inducible JNKK and JNK activates the Jun::Fos dimer by phosphorylation. This leads to the activation of genes via Fos::Jun binding sites or TREs. Activation of the gene encoding the JNK phosphatase, Puckered causes negative feedback regulation. B. Sequence of the artificial TRE and ARE elements used for the construction of the Nrf2 and JNK reporters. The sequences correspond to optimal CncC/Maf and Jun/Fos binding sites as reported by Kerppola and Curran [29]. A mutant version of the response elements (mRE) that does not bind either Jun/Fos or Nrf2/Maf dimers was generated as a control construct. C. Modular design of in vivo reporter constructs. Each transgenic cassette carries an attB site for ΦC31 recombinase-catalyzed genomic integration, a minimal heat shock protein 70 (Hsp70) promoter (TATA) and eGFP or DsRed.T4 reporter gene driven by four head-to-tail concatenated copies of the ARE, TRE or mRE elements. The promoter-reporter cassettes are flanked by gypsy insulators to prevent ‘position effects’ of surrounding chromatin. Transgenic flies carry reporter transgenes at predetermined locations, attP 16 on the right arm of chromosome 2 and attP 40 on the left arm of the same chromosome. D. Design of reporters for cell culture experiments. In a similar design as shown in C, the firefly luciferase reporter gene was placed under a minimal heat shock protein 70 (Hsp70) promoter (TATA) and four head-to-tail concatenated copies of the ARE, TRE or mRE elements.
Table 1.
Transgenic reporter fly lines.
Figure 2.
Transgenic ARE reporters and TRE reporters are specifically responsive to Nrf2 and JNK signaling respectively.
Loss of dKeap1 function specifically activates Nrf2 activity in vivo [24]. Consequently, expressing a Keap1-specific RNAi under the control of the ubiquitously active arm-Gal4 driver activated the ARE-green reporter, but not the TRE-red reporter. Conversely, arm-Gal4 driven expression of RNAi targeting Puc, the negative regulator of JNK signaling, stimulated TRE-red reporter activity but did not affect the ARE-green reporter. The upper panels show red and green fluorescence in adult 5-day-old female flies. These fluorescence images were superimposed onto the bright-field images of the same flies, as shown in the lower panels. 15 to 20 flies were analyzed for each genotype and all showed similar results. The images shown show representative, randomly selected specimens.
Figure 3.
ARE and TRE reporters faithfully reproduce known patterns of JNK and Nrf2 signaling.
A. The TRE-red reporter marks the leading edge cells of stage 13 to 15 embryos where JNK is known to be active during dorsal closure. In addition to the TRE-red-2R reporter, the embryos expressed a moesin GFP fusion protein (sGMCA [47]) to visualize the actin cytoskeleton (green). Three embryos at progressively later stages of dorsal closure are shown. B. ARE-green reporter activity was detected in different larval tissues in which Nrf2 function has previously been reported [24]. In 3rd instar larval salivary glands Nrf2 reporter activity was undetectable at basal levels (top panel, genotype TubGSGal4, ARE-green-2L/UAS-dKeap1RNAi, no RU 486 treatment), but was prominent after knockdown of Keap1 expression (same genotype as above, but keap1RNAi expression was induced by RU486). The nuclei are stained with Hoechst (purple). The left panels show a micrograph of the salivary glands. C. Cells in the proventriculus of the digestive tract show prominent Nrf2 activity after Keap1 knockdown. Conditions and genotypes are as described in panel B.
Figure 4.
Signal specificity of cell-based reporters.
A. Artificial stimulation of JNK and Nrf2 signaling in S2 cells specifically activates the TRE-fluc or ARE-fluc reporter, respectively. Expression of a constitutively active mutant of Drosophila JNKK (Hepact) activated a co-transfected TRE-fluc reporter, but not an ARE-fluc reporter. Conversely, overexpression of CncC increased the activity of the ARE and not the TRE reporter. Firefly luciferase activity was normalized using a cotransfected renilla luciferase construct (Act5C rluc) as a reference. The activity under control conditions (reporters co-transfected with pAct-Gal4) was set to 1. B. Signal-dependent activation of TRE and ARE reporters specifically requires JNK or Nrf2 pathway components. S2 cells were transfected with TRE-fluc reporters and JNK activity was stimulated by co-expression of constitutively active Drosophila JNKK (Hepact) as indicated. Cells transfected with the ARE-fluc reporter were treated with the specific Nrf2 activator Oltipraz. dsRNA mediated knockdown of CncC, or MafS compromised only Oltipraz-induced ARE-fluc activity. Conversely, dsRNA against Hep (JNKK) and Bsk (JNK) only reduced TRE activity. Firefly luciferase activity was normalized to renilla luciferase activity driven by Act5C promoter. The activity of the activated reporters under control conditions was set to 1. Error bars indicate standard deviation of triplicate measurements.
Figure 5.
Simultaneous measurement of Nrf2 and JNK responses in vivo.
Exposure of flies that carry two copies each of both the TRE-red-2R and the ARE-green-2L reporters to the oxidative stressor paraquat increased both ARE and TRE activities, however with different spatial specificity. Oral application of the cancer chemopreventive drug oltipraz, on the other hand, only induced ARE and not TRE activity. The upper and the middle panels show green and red fluorescence separately in the same flies. These green and red fluorescence images are merged in the lower panel. The control food for oltipraz treatment was supplemented with 1% DMSO, the solvent used for oltipraz. The control reporter stock mRE-red-2L did not respond to either paraquat or oltipraz.
Figure 6.
Stimulation of cell-based reporters by sulforaphane, oltipraz and stressors.
S2 cells transiently transfected with TRE-fluc, ARE-fluc or mRE-fluc plasmids, as indicated, were exposed to sodium arsenite (NaAsO2), Diethyl Maleate (DEM) or oltipraz. The firefly luciferase activity was measured 24 hrs after drug treatment and normalized to renilla luciferase activity expressed from a co-transfected Act5C renilla luciferase construct. Note that both TRE and ARE reporters respond to the stressors, Arsenite and DEM, but only ARE-fluc responds to Oltipraz. The mRE reporter responds neither to oltipraz nor to the stressors. The activity of mRE-fluc reporter under control conditions was set to 1. Error bars indicate standard deviation of triplicate measurements.