1B) but suppressed reporter gene activation in cells expressing 11-HSD1, an effect that was fully reversed by the selective 11-HSD1 inhibitor T0504 (Fig. in triplicate) represent ratios of 11?-HSD1 mRNA to GAPDH control mRNA from treated cells normalized to the values obtained from cells incubated with vehicle (DMSO). Enzymatic activities were determined in intact HPTC-1E3 cells expressing endogenous 11-HSD1 that were treated with vehicle (DMSO), 2 mM H2O2 (relative gene expression was evaluated in comparison to NQO1, Abcc3 and Hmox1, in each animal, by scatter plot. RNA purified from whole liver tissues of 20 Han Wistar rats (animals listed in Table S1) were hybridized to Rat Genome 230 2.0 chips (Affymetrix, Santa Clara, CA) for characterization of gene expression. Hybridization mixtures were prepared using the 3-IVT Express Kit (Affymetrix) to accommodate 10 g of labeled cDNA in 200 L of hybridization mix. Rat Genome 230 2.0 Arrays were hybridized, revealed and washed according to the Affymetrix protocol. GeneChips were scanned using the Affymetrix 3000 scanner and images were converted to files (*.cel). The raw data were analyzed by GeneSpring GX 11.5.1 (Agilent) using the software default settings for Affymetrix expression chips (Table S2). Raw data were pre-processed by Robust Multi-array Analysis (RMA) algorithm (including Quantile normalization), Log transformed and then pre-filtered within the 20C100 percentile assuming the median as baseline (raw data). Of the 31099 probe-sets in Rat Genome 230 2.0 chip, 27359 have passed the pre-filter. Correlation plot (not shown) shows the correlation analysis (heat map, Pearson correlation factor) across arrays: high correlation degree is indicative of good experimental execution and high reproducibility. The internal quality controls of Affymetrix chips showed no abnormalities in the hybridization Rabbit Polyclonal to GLRB processes (not shown). Both analyses were part of the default quality controls. Linear regression analysis was used to obtain the overall correlation (R2) of expression with other genes (vs. Nqo1 R2?=?0.546, p value?=?2.00E-004; vs. Abcc3 R2?=?0.407, p value?=?2.47E-003; vs. Hmox1 R2?=?0.692, p value?=?5.38E-006). The statistical relevance was assessed by multiple unpaired and was suppressed upon treatment of 11-HSD1 expressing cells with cortisone, an effect that was reversed by 11-HSD1 inhibitors. Furthermore, our results demonstrate that elevated glucocorticoids lowered the ability of cells to detoxify H2O2. Moreover, a comparison of gene expression in male and female rats revealed an opposite sexual dimorphism with an inverse relationship between 11-HSD1 and Nrf2 target gene expression. Conclusions The results demonstrate a suppression of the cellular antioxidant defence capacity by glucocorticoids and suggest that elevated 11-HSD1 activity may lead to impaired Nrf2-dependent antioxidant response. The gender-specific differences in hepatic expression levels of 11-HSD1 and Nrf2 target genes and the impact of pharmacological inhibition of 11-HSD1 on improving cellular capacity to cope with oxidative stress warrants further studies gene), are expressed in hepatocytes to avoid cellular damage by reactive compounds. Nrf2 is the key player of the tightly regulated antioxidant cell defense system [1]. Upon recognition of the antioxidant responsive elements (ARE) on the promoters of its target genes, Nrf2 modulates basal and ligand-induced expression of various cytoprotective enzymes [2]. The importance of Nrf2 is shown in knockout mice, exhibiting an enhanced susceptibility towards CF53 oxidative stress caused by xenobiotics due to diminished expression of cytoprotective genes [3], [4], [5], [6]. Nrf2 target genes include essential phase II detoxification enzymes such as NAD(P)H:quinone oxidoreductases (NQO) [7], heme oxygenase-1 (HO-1, gene) [8] and glutathione S-transferases (GST) [1], [9] that are induced by oxidative stress caused by xenobiotics, antioxidants, UV-light, and ionizing radiation [2]. A recent study reported gender-divergent expression of NQO1 in specific rat strains studied [10]. Hepatic basal NQO1 mRNA expression was two-fold.Of the 31099 probe-sets in Rat Genome 230 2.0 chip, 27359 have passed the pre-filter. mRNA from treated cells normalized to the values obtained from cells incubated with vehicle (DMSO). Enzymatic activities were determined in intact HPTC-1E3 cells expressing endogenous 11-HSD1 that were treated with vehicle (DMSO), 2 mM H2O2 (relative gene CF53 expression was evaluated in comparison to NQO1, Abcc3 and Hmox1, in each animal, by scatter plot. RNA purified from whole liver tissues of 20 Han Wistar rats (animals listed in Table S1) were hybridized to Rat Genome 230 2.0 chips (Affymetrix, Santa Clara, CA) for characterization of gene expression. Hybridization mixtures were prepared using the 3-IVT Express Kit (Affymetrix) to accommodate 10 g of labeled cDNA in 200 L of CF53 hybridization mix. Rat Genome 230 2.0 Arrays were hybridized, revealed and washed according to the Affymetrix protocol. GeneChips were scanned using the Affymetrix 3000 scanner and images were converted to files (*.cel). The raw data were analyzed by GeneSpring GX 11.5.1 (Agilent) using the software default settings for Affymetrix expression chips (Table S2). Raw data were pre-processed by Robust Multi-array Analysis (RMA) algorithm (including Quantile normalization), Log transformed and then pre-filtered within the 20C100 percentile assuming the median as baseline (raw data). Of the 31099 probe-sets in Rat Genome 230 2.0 chip, 27359 have passed the pre-filter. Correlation plot (not shown) shows the correlation analysis (heat map, Pearson correlation factor) across arrays: high correlation degree is indicative of good experimental execution and high reproducibility. The internal quality controls of Affymetrix chips showed no abnormalities in the hybridization processes (not shown). Both analyses were part of the default quality controls. Linear regression analysis was used to obtain the overall correlation (R2) of expression with other genes (vs. Nqo1 R2?=?0.546, p value?=?2.00E-004; vs. Abcc3 R2?=?0.407, p value?=?2.47E-003; vs. Hmox1 R2?=?0.692, p value?=?5.38E-006). The statistical relevance was assessed by multiple unpaired and was suppressed upon treatment of 11-HSD1 expressing cells with cortisone, an effect that was reversed by 11-HSD1 inhibitors. Furthermore, our results demonstrate that elevated glucocorticoids lowered the ability of cells to detoxify H2O2. Moreover, a comparison of gene expression in male and female rats revealed an opposite sexual dimorphism with an inverse relationship between 11-HSD1 and Nrf2 target gene expression. Conclusions The results demonstrate a suppression of the cellular antioxidant defence capacity by glucocorticoids and suggest that elevated 11-HSD1 activity may lead to impaired Nrf2-dependent antioxidant response. The gender-specific differences in hepatic expression levels of 11-HSD1 and Nrf2 target genes and the impact of pharmacological inhibition of 11-HSD1 on improving cellular capacity to cope with oxidative stress warrants further studies gene), are expressed in hepatocytes to avoid cellular damage by reactive compounds. Nrf2 is the key player of the tightly regulated antioxidant cell defense system [1]. Upon recognition of the antioxidant responsive elements (ARE) on the promoters of its target genes, Nrf2 modulates basal and ligand-induced expression of various cytoprotective enzymes [2]. The importance of Nrf2 is shown in knockout mice, exhibiting an enhanced susceptibility towards oxidative stress caused by xenobiotics due to diminished expression of cytoprotective genes [3], [4], [5], [6]. Nrf2 target genes include essential phase II detoxification enzymes such as NAD(P)H:quinone oxidoreductases (NQO) [7], heme oxygenase-1 (HO-1, gene) [8] and glutathione S-transferases (GST) [1], [9] that are induced by oxidative stress caused by xenobiotics, antioxidants, UV-light, and ionizing radiation [2]. A recent study reported gender-divergent manifestation of NQO1 in specific rat strains analyzed [10]. Hepatic basal NQO1 mRNA manifestation was two-fold reduced male compared with female Sprague Dawley rats. Induction of NQO1 manifestation with the classical Nrf2 inducers butylated hydroxyanisole and oltipraz was more pronounced in female compared with male rats. Importantly, it has been reported that male rats have higher susceptibility to carcinogenic xenobiotics [11]. Interestingly, gender-related variations were also found for humans [12]; however, the underlying mechanisms remain unfamiliar. Decreased Nrf2-mediated constitutive and oltipraz- or tert-butylhydroquinone (t-BHQ)-inducible gene manifestation was found in rat H4IIE hepatoma cells upon activation of the glucocorticoid receptor (GR) by dexamethasone [13]. Importantly, the oxidized metabolite of dexamethasone, 11-ketodexamethasone, is also a potent GR agonist; therefore dexamethasone circumvents the 11-hydroxysteroid dehydrogenase (11-HSD) mediated control of GR activation [14]. Under physiological conditions, hepatic GR function.Data (mean SD from two indie experiments measured in triplicate) represent ratios of 11?-HSD1 mRNA to GAPDH control mRNA from treated cells normalized to the values from cells incubated with vehicle (DMSO). in the presence of vehicle (0.05% DMSO) or H2O2 (2 mM) (mRNA expression was performed by RT-PCR on a RotorGene 6000 (Corbett, Australia) using the KAPA SYBR? FAST qPCR Kit (Kapasystems, Boston, MA). Relative gene expression compared with the internal control GAPDH was identified using the delta-delta-CT method. Data (mean SD from two self-employed experiments measured in triplicate) represent ratios of 11?-HSD1 mRNA to GAPDH control mRNA from treated cells normalized to the values from cells incubated with vehicle (DMSO). Enzymatic activities were identified in intact HPTC-1E3 cells expressing endogenous 11-HSD1 that were treated with vehicle (DMSO), 2 mM H2O2 (relative gene manifestation was evaluated in comparison to NQO1, Abcc3 and Hmox1, in each animal, by scatter storyline. RNA purified from whole liver cells of 20 Han Wistar rats (animals listed in Table S1) were hybridized to Rat Genome 230 2.0 chips (Affymetrix, Santa Clara, CA) for characterization of gene manifestation. Hybridization mixtures were prepared using the 3-IVT Express Kit (Affymetrix) to accommodate 10 g of labeled cDNA in 200 L of hybridization blend. Rat Genome 230 2.0 Arrays were hybridized, revealed and washed according to the Affymetrix protocol. GeneChips were scanned using the Affymetrix 3000 scanner and images were converted to documents (*.cel). The uncooked data were analyzed by GeneSpring GX 11.5.1 (Agilent) using the software default settings for Affymetrix expression chips (Table S2). Uncooked data were pre-processed by Robust Multi-array Analysis (RMA) algorithm (including Quantile normalization), Log transformed and then pre-filtered within the 20C100 percentile presuming the median as baseline (uncooked data). Of the 31099 probe-sets in Rat Genome 230 2.0 chip, 27359 have approved the pre-filter. Correlation plot (not shown) shows the correlation analysis (warmth map, Pearson correlation element) across arrays: high correlation degree is definitely indicative of good experimental execution and high reproducibility. The internal quality settings of Affymetrix chips showed no abnormalities in the hybridization processes (not demonstrated). Both analyses were part of the default quality settings. Linear regression analysis was used to obtain the overall correlation (R2) of manifestation with additional genes (vs. Nqo1 R2?=?0.546, p value?=?2.00E-004; vs. Abcc3 R2?=?0.407, p value?=?2.47E-003; vs. Hmox1 R2?=?0.692, p value?=?5.38E-006). The statistical relevance was assessed by multiple unpaired and was suppressed upon treatment of 11-HSD1 expressing cells with cortisone, an effect that was reversed by 11-HSD1 inhibitors. Furthermore, our results demonstrate that elevated glucocorticoids lowered the ability of cells to detoxify H2O2. Moreover, a comparison of gene manifestation in male and female rats exposed an opposite sexual dimorphism with an inverse relationship between 11-HSD1 and Nrf2 target gene manifestation. Conclusions The results demonstrate a suppression of the cellular antioxidant defence capacity by glucocorticoids and suggest that elevated 11-HSD1 activity may lead to impaired Nrf2-dependent antioxidant response. The gender-specific variations in hepatic manifestation levels of 11-HSD1 and Nrf2 target genes and the effect of pharmacological inhibition of 11-HSD1 on improving cellular capacity to cope with oxidative stress warrants further studies gene), are indicated in hepatocytes to avoid cellular damage by reactive compounds. Nrf2 is the important player of the tightly controlled antioxidant cell defense system [1]. Upon acknowledgement of the antioxidant responsive elements (ARE) within the promoters of its target genes, Nrf2 modulates basal and ligand-induced manifestation of various cytoprotective enzymes [2]. The importance of Nrf2 is demonstrated in knockout mice, exhibiting an enhanced susceptibility towards oxidative stress caused by xenobiotics due to diminished manifestation of cytoprotective genes [3], [4], [5], [6]. Nrf2 target genes include essential phase II detoxification enzymes such as NAD(P)H:quinone oxidoreductases (NQO) [7], heme oxygenase-1 (HO-1, gene) [8] and glutathione S-transferases (GST) [1], [9] that are induced by oxidative stress caused by xenobiotics, antioxidants, UV-light, and ionizing radiation [2]. A recent study reported gender-divergent manifestation of NQO1 in specific rat strains analyzed [10]. Hepatic basal NQO1 mRNA.