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Mineralocorticoid and glucocorticoid receptors differentially regulate NF-kappaB activity and pro-inflammatory cytokine production in murine BV-2 microglial cells.

Chantong B, Kratschmar DV, Nashev LG, Balazs Z, Odermatt A - J Neuroinflammation (2012)

Bottom Line: Corticosteroids are potent modulators of inflammation and mediate their effects by binding to mineralocorticoid receptors (MR) and glucocorticoid receptors (GR).Biphasic effects were observed for 11-dehydrocorticosterone and corticosterone, with an MR-dependent potentiation of IL-6 and tumor necrosis factor-α (TNF-α) expression and NF-κB activation at low/moderate concentrations and a GR-dependent suppression at high concentrations.The respective effects were confirmed using the MR ligand aldosterone and the antagonist spironolactone as well as the GR ligand dexamethasone and the antagonist RU-486.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland.

ABSTRACT

Background: Microglia, the resident macrophage-like cells in the brain, regulate innate immune responses in the CNS to protect neurons. However, excessive activation of microglia contributes to neurodegenerative diseases. Corticosteroids are potent modulators of inflammation and mediate their effects by binding to mineralocorticoid receptors (MR) and glucocorticoid receptors (GR). Here, the coordinated activities of GR and MR on the modulation of the nuclear factor-κB (NF-κB) pathway in murine BV-2 microglial cells were studied.

Methods: BV-2 cells were treated with different corticosteroids in the presence or absence of MR and GR antagonists. The impact of the glucocorticoid-activating enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) was determined by incubating cells with 11-dehydrocorticosterone, with or without selective inhibitors. Expression of interleukin-6 (IL-6), tumor necrosis factor receptor 2 (TNFR2), and 11β-HSD1 mRNA was analyzed by RT-PCR and IL-6 protein expression by ELISA. NF-κB activation and translocation upon treatment with various corticosteroids were visualized by western blotting, immunofluorescence microscopy, and translocation assays.

Results: GR and MR differentially regulate NF-κB activation and neuroinflammatory parameters in BV-2 cells. By converting inactive 11-dehydrocorticosterone to active corticosterone, 11β-HSD1 essentially modulates the coordinated action of GR and MR. Biphasic effects were observed for 11-dehydrocorticosterone and corticosterone, with an MR-dependent potentiation of IL-6 and tumor necrosis factor-α (TNF-α) expression and NF-κB activation at low/moderate concentrations and a GR-dependent suppression at high concentrations. The respective effects were confirmed using the MR ligand aldosterone and the antagonist spironolactone as well as the GR ligand dexamethasone and the antagonist RU-486. NF-κB activation could be blocked by spironolactone and the inhibitor of NF-κB translocation Cay-10512. Moreover, an increased expression of TNFR2 was observed upon treatment with 11-dehydrocorticosterone and aldosterone, which was reversed by 11β-HSD1 inhibitors and/or spironolactone and Cay-10512.

Conclusions: A tightly coordinated GR and MR activity regulates the NF-κB pathway and the control of inflammatory mediators in microglia cells. The balance of GR and MR activity is locally modulated by the action of 11β-HSD1, which is upregulated by pro-inflammatory mediators and may represent an important feedback mechanism involved in resolution of inflammation.

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Potentiation of the IL-6-dependent increase of 11β-HSD1 mRNA expression by low concentrations of 11-dehydrocorticosterone. BV-cells were pretreated with 1 μM 11β-HSD1 inhibitor T0504 where indicated for 1 h, followed by addition of 50 nM 11-dehydrocorticosterone with or without 20 ng/mL IL-6 and incubation for another 24 h. 11β-HSD1 mRNA was determined by real-time RT-PCR. Data (mean ± SD from three independent experiments) represent ratios of 11β-HSD1 mRNA to GAPDH control mRNA from treated cells normalized to the values obtained from cells incubated with vehicle (DMSO). **P <0.01, ***P <0.005.
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Figure 9: Potentiation of the IL-6-dependent increase of 11β-HSD1 mRNA expression by low concentrations of 11-dehydrocorticosterone. BV-cells were pretreated with 1 μM 11β-HSD1 inhibitor T0504 where indicated for 1 h, followed by addition of 50 nM 11-dehydrocorticosterone with or without 20 ng/mL IL-6 and incubation for another 24 h. 11β-HSD1 mRNA was determined by real-time RT-PCR. Data (mean ± SD from three independent experiments) represent ratios of 11β-HSD1 mRNA to GAPDH control mRNA from treated cells normalized to the values obtained from cells incubated with vehicle (DMSO). **P <0.01, ***P <0.005.

Mentions: Glucocorticoids showed a bi-phasic effect on the production of pro-inflammatory cytokines, depending on activation of MR only, at low concentrations, or both MR and GR at higher concentrations. Thus, an elevation of the intracellular concentration of active glucocorticoids by 11β-HSD1 may play an important role in the resolution of inflammation by causing a shift from MR- to GR-mediated regulation of pro-inflammatory cytokine expression. As shown in Figure 9, incubation of BV-2 cells with 20 ng/mL of IL-6 resulted in increased 11β-HSD1 mRNA expression, which was strongly enhanced by co-incubation with 50 nM 11-dehydrocorticosterone. Pharmacological inhibition of 11β-HSD1 prevented the effects of 11-dehydrocorticosterone. To test whether incubation with IL-6 would enhance 11β-HSD1 enzyme activity, we measured the conversion of radiolabeled cortisone to cortisol. Due to technical reasons cortisone instead of 11-dehydrocorticosterone was used to study the effect on enzyme activity. Both IL-6 and cortisone resulted in increased 11β-HSD1 reductase activity, which was strongly enhanced by co-incubation with IL-6 and cortisone (data not shown), indicating that the changes observed on the mRNA level are translated to enzyme activity.


Mineralocorticoid and glucocorticoid receptors differentially regulate NF-kappaB activity and pro-inflammatory cytokine production in murine BV-2 microglial cells.

Chantong B, Kratschmar DV, Nashev LG, Balazs Z, Odermatt A - J Neuroinflammation (2012)

Potentiation of the IL-6-dependent increase of 11β-HSD1 mRNA expression by low concentrations of 11-dehydrocorticosterone. BV-cells were pretreated with 1 μM 11β-HSD1 inhibitor T0504 where indicated for 1 h, followed by addition of 50 nM 11-dehydrocorticosterone with or without 20 ng/mL IL-6 and incubation for another 24 h. 11β-HSD1 mRNA was determined by real-time RT-PCR. Data (mean ± SD from three independent experiments) represent ratios of 11β-HSD1 mRNA to GAPDH control mRNA from treated cells normalized to the values obtained from cells incubated with vehicle (DMSO). **P <0.01, ***P <0.005.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3526453&req=5

Figure 9: Potentiation of the IL-6-dependent increase of 11β-HSD1 mRNA expression by low concentrations of 11-dehydrocorticosterone. BV-cells were pretreated with 1 μM 11β-HSD1 inhibitor T0504 where indicated for 1 h, followed by addition of 50 nM 11-dehydrocorticosterone with or without 20 ng/mL IL-6 and incubation for another 24 h. 11β-HSD1 mRNA was determined by real-time RT-PCR. Data (mean ± SD from three independent experiments) represent ratios of 11β-HSD1 mRNA to GAPDH control mRNA from treated cells normalized to the values obtained from cells incubated with vehicle (DMSO). **P <0.01, ***P <0.005.
Mentions: Glucocorticoids showed a bi-phasic effect on the production of pro-inflammatory cytokines, depending on activation of MR only, at low concentrations, or both MR and GR at higher concentrations. Thus, an elevation of the intracellular concentration of active glucocorticoids by 11β-HSD1 may play an important role in the resolution of inflammation by causing a shift from MR- to GR-mediated regulation of pro-inflammatory cytokine expression. As shown in Figure 9, incubation of BV-2 cells with 20 ng/mL of IL-6 resulted in increased 11β-HSD1 mRNA expression, which was strongly enhanced by co-incubation with 50 nM 11-dehydrocorticosterone. Pharmacological inhibition of 11β-HSD1 prevented the effects of 11-dehydrocorticosterone. To test whether incubation with IL-6 would enhance 11β-HSD1 enzyme activity, we measured the conversion of radiolabeled cortisone to cortisol. Due to technical reasons cortisone instead of 11-dehydrocorticosterone was used to study the effect on enzyme activity. Both IL-6 and cortisone resulted in increased 11β-HSD1 reductase activity, which was strongly enhanced by co-incubation with IL-6 and cortisone (data not shown), indicating that the changes observed on the mRNA level are translated to enzyme activity.

Bottom Line: Corticosteroids are potent modulators of inflammation and mediate their effects by binding to mineralocorticoid receptors (MR) and glucocorticoid receptors (GR).Biphasic effects were observed for 11-dehydrocorticosterone and corticosterone, with an MR-dependent potentiation of IL-6 and tumor necrosis factor-α (TNF-α) expression and NF-κB activation at low/moderate concentrations and a GR-dependent suppression at high concentrations.The respective effects were confirmed using the MR ligand aldosterone and the antagonist spironolactone as well as the GR ligand dexamethasone and the antagonist RU-486.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland.

ABSTRACT

Background: Microglia, the resident macrophage-like cells in the brain, regulate innate immune responses in the CNS to protect neurons. However, excessive activation of microglia contributes to neurodegenerative diseases. Corticosteroids are potent modulators of inflammation and mediate their effects by binding to mineralocorticoid receptors (MR) and glucocorticoid receptors (GR). Here, the coordinated activities of GR and MR on the modulation of the nuclear factor-κB (NF-κB) pathway in murine BV-2 microglial cells were studied.

Methods: BV-2 cells were treated with different corticosteroids in the presence or absence of MR and GR antagonists. The impact of the glucocorticoid-activating enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) was determined by incubating cells with 11-dehydrocorticosterone, with or without selective inhibitors. Expression of interleukin-6 (IL-6), tumor necrosis factor receptor 2 (TNFR2), and 11β-HSD1 mRNA was analyzed by RT-PCR and IL-6 protein expression by ELISA. NF-κB activation and translocation upon treatment with various corticosteroids were visualized by western blotting, immunofluorescence microscopy, and translocation assays.

Results: GR and MR differentially regulate NF-κB activation and neuroinflammatory parameters in BV-2 cells. By converting inactive 11-dehydrocorticosterone to active corticosterone, 11β-HSD1 essentially modulates the coordinated action of GR and MR. Biphasic effects were observed for 11-dehydrocorticosterone and corticosterone, with an MR-dependent potentiation of IL-6 and tumor necrosis factor-α (TNF-α) expression and NF-κB activation at low/moderate concentrations and a GR-dependent suppression at high concentrations. The respective effects were confirmed using the MR ligand aldosterone and the antagonist spironolactone as well as the GR ligand dexamethasone and the antagonist RU-486. NF-κB activation could be blocked by spironolactone and the inhibitor of NF-κB translocation Cay-10512. Moreover, an increased expression of TNFR2 was observed upon treatment with 11-dehydrocorticosterone and aldosterone, which was reversed by 11β-HSD1 inhibitors and/or spironolactone and Cay-10512.

Conclusions: A tightly coordinated GR and MR activity regulates the NF-κB pathway and the control of inflammatory mediators in microglia cells. The balance of GR and MR activity is locally modulated by the action of 11β-HSD1, which is upregulated by pro-inflammatory mediators and may represent an important feedback mechanism involved in resolution of inflammation.

Show MeSH
Related in: MedlinePlus