Figure 1.
Suppression of Nrf2-dependent reporter gene activation by glucocorticoids in HEK-293 cells.
HEK-293 cells transiently transfected with plasmids for Nrf2, GR, ARE8L-reporter, pCMV-LacZ and either pcDNA3 (A, B) or 11β-HSD1 (C) were incubated for 24 h with vehicle (DMSO), 10 µM sulforaphane, 100 nM cortisone or cortisol, in the presence or absence of 1 µM T0504 or RU-486. Data (mean ± SD) were obtained from three independent experiments each measured in triplicate. *, p<0.05, **, p<0.01, ***, p<0.001, p-value was obtained using one-way ANOVA followed by Bonferroni post-tests compared with vehicle control (DMSO).
Figure 2.
Suppression of Nrf2 transactivation by 11β-HSD1-mediated glucocorticoid activation in H4IIE cells.
The activation of the Nrf2-dependent ARE8L-reporter by 10 µM sulforaphane was measured in rat H4IIE hepatoma cells (A) and in H4IIE cells stably expressing 11β-HSD1 (H4H1 clone) (B) at endogenous Nrf2 expression (black bars) and upon over expression of Nrf2 (white bars). CMV-LacZ plasmid served as a transfection control to normalize luciferase values. Cells were treated with vehicle (DMSO), sulforaphane (10 µM), or sulforaphane and proteasome inhibitor MG132 (10 µM) for 24 h. H4IIE cells transfected with ARE8L-reporter and pCMV-LacZ cells were incubated with sulforaphane and increasing concentrations of glucocorticoids for 24 h, followed by measuring luciferase activity to estimate the effective concentration of cortisol leading to a 50% reduction of the reporter activity (C). Suppression of Nrf2 transactivation by glucocorticoids was further studied in H4IIE cells transiently transfected with ARE8L-reporter and pcDNA3 (D) or with ARE8L-reporter and 11β-HSD1 (E). Cells were incubated with vehicle or sulforaphane, glucocorticoids and vehicle or 11β-HSD1 inhibitor T0504 at the concentrations indicated for 24 h, followed by measuring luciferase activity. The impact of 11β-HSD1 inhibitors and GR antagonists on Nrf2-dependent transactivation was similarly assessed in H4H1 cells transfected with ARE8L-reporter and pCMV-LacZ (F). Data represent mean ± SD from at least two independent experiments performed in triplicate. *, p<0.05, **, p<0.01, ***, p<0.001, p-value was obtained using one-way ANOVA followed by Bonferroni post-tests compared with control (DMSO), ns, not significant.
Figure 3.
Influence of oxidative stress on Nrf2 pathway and GR transactivation.
The activation of the GR-dependent TAT3-TATA-reporter by 100 µM cortisol was measured in rat H4IIE hepatoma cells with endogenous GR expression (A). CMV-LacZ plasmid served as transfection control to normalize luciferase values. Cells were treated with vehicle (DMSO) or cortisol (100 µM), with or without H2O2 (2 mM) for 24 h at 37°C. Data represent mean ± SD from at least three independent experiments performed in triplicate. The influence of oxidative stress induced by H2O2 on Nrf2-dependent transactivation was measured in ARECS3 cells stably expressing the ARE8L-reporter (B). Cells were treated with vehicle (DMSO) or sulforaphane (10 µM) in the presence or absence of H2O2 (2 mM) for 24 h. Data represent mean ± SD from three independent experiments measured in triplicate. Activation of NQO1 mRNA expression by H2O2 was measured in H4IIE cells (C). Cells were incubated for 24 h at 37°C with vehicle (0.05% DMSO) or sulforaphane (10 µM) in the presence or absence of H2O2 (2 mM), followed by determination of NQO1 mRNA levels by real-time RT-PCR. Data (mean ± SD from two independent experiments measured in triplicate) represent ratios of NQO1 mRNA to GAPDH control mRNA from treated cells normalized to the values obtained from cells incubated with vehicle (DMSO).
Figure 4.
Inhibition of Nrf2-induced mRNA expression of NQO1 and GSTA2 by cortisol.
H4IIE cells were incubated for 24 h at 37°C with 10 µM sulforaphane in the absence or presence of 100 nM cortisol or cortisone, respectively, followed by determination of NQO1 (A) and GSTA2 mRNA levels (B) by real-time RT-PCR. Data (mean ± S.D. from three independent experiments performed in triplicate) represent ratios of NQO1 and GSTA2 mRNA to GAPDH control mRNA from treated cells normalized to the values obtained from cells incubated with vehicle (DMSO). *, p<0.05, **, p<0.01, ***, p<0.001, p-values were obtained using one-way ANOVA followed by Bonferroni post-tests compared with vehicle control (DMSO).
Figure 5.
Suppression of Nrf2 and NQO1 protein expression by cortisol in oxidative stress-induced H4IIE cells.
H4IIE cells were treated for 24 h with vehicle (DMSO), cortisone, sulforaphane or cortisone and sulforaphane in the presence or absence of H2O2. Cells were lysed, and equal protein amounts were used for Western blot analysis. Samples were probed for Nrf2 and NQO1 using actin as a loading control. The lower panel shows a densitometric analysis of Nrf2 (left) and NQO1 (right) protein normalized against β-actin. A representative experiment is shown.
Figure 6.
Suppression of NQO1 protein expression by cortisone in 11β-HSD1 expressing H4IIE cells but not in pCDNA3 transfected cells.
H4IIE cells transiently transfected with either pCDNA3 or 11β-HSD1 were treated for 24 h with vehicle (DMSO), cortisone, sulforaphane or cortisone and sulforaphane (upper panel). Cells were lysed, and equal protein amounts were used for Western blot analysis. Samples were probed for NQO1 using actin as a loading control. Lower panel, densitometric analysis of NQO1 bands normalized against b-actin. Graphs are representative of three independent experiments.
Figure 7.
11β-HSD1-mediated suppression of Nrf2-induced NQO1 expression in H4H1 cells.
H4H1 cells were incubated for 24 h at 37°C with 10 µM sulforaphane in the absence or presence of 100 nM cortisone and 1 µM glycyrrhetinic acid (GA), followed by quantification of mRNA levels by real-time RT-PCR. Data (mean ± S.D. from three independent experiments performed in triplicate) represent ratios of NQO1 mRNA to GAPDH control mRNA from treated cells normalized to the values obtained from cells incubated vehicle (DMSO). *, p<0.05, **, p<0.01, ***, p<0.001, p-values were obtained using one-way ANOVA followed by Bonferroni post-tests compared with vehicle control (DMSO).
Figure 8.
Increased susceptibility of 11β-HSD1 expressing cells to H2O2-induced oxidative stress.
H4IIE cells transiently transfected with pCDNA3 (A), pCDNA3 or 11β-HSD1 (B, C and D) were treated for 24 h with vehicle, 100 nM cortisol (A), 100 nM cortisone (C) or simultaneously with cortisone and 1 µM T0504 (B, D). The medium was replaced by assay buffer (HBSS) containing 1 g/L glucose. Single cell real-time measurements were performed on a Leica SP5 confocal microscope. After 5 min baseline adaption, cells were exposed to a final concentration of 10 µM (A, B) or 100 µM (C, D) H2O2 and responses were compared between differentially transfected or vehicle treated cells over a period of 30 min (A, B) or 45 min (C). Data represent mean ± SEM of seven different cells for each transfection. *, p<0.05, **, p<0.01, ***, p<0.001, p-value was obtained using one-way ANOVA followed by Bonferroni post-tests compared with pcDNA3 transfected cells. To analyze the total cell population (D), H4IIE cells 4,000,000 cells/mL were resuspended in assay buffer. Suspensions of cells (100 µL) treated either with cortisone or cortisone and T0504 were transferred into a 96-well plate, centrifuged for 2 min at 180× g and challenged by adding 100 µL assay buffer containing 100 µM H2O2. Fluorescence was immediately measured after adding H2O2 and data were collected every 27 s at 37°C for 9 h. One of three representative experiments is shown (D).
Figure 9.
Rat Genome 230 2.0 Affymetrix chip analysis.
RNA purified from whole liver tissues of ten male and ten female rats was hybridized to Rat Genome 230 2.0 Affymetrix chips. Gender-specific differences in the expression of the HSD11B1, HMOX1, NQO1 and ABCC3 genes were assessed. The data represent fold change in gene expression (male vs. female). The statistical relevance was assessed by multiple unpaired t-tests, with Benjamini Hochberg FDR multiple testing correction, p≤0.01.