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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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Nuclear-cytoplasmic distribution of NF-κB p65 determined by western blotting. BV-2 cells were treated for 24 h with 50 ng/mL TNF-α, 25 nM corticosterone, or 50 nM aldosterone in the absence or presence of 1 μM spironolactone or 1 μM RU-486 (A), or in the presence of 250 nM Cay-10512 (B), followed by preparation of nuclear and cytoplasmic fractions, separation of proteins by SDS-PAGE and analysis of p65 and phospho-p65 expression by western blotting. Actin served as a loading control. A representative blot from three independent experiments is shown.
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Figure 7: Nuclear-cytoplasmic distribution of NF-κB p65 determined by western blotting. BV-2 cells were treated for 24 h with 50 ng/mL TNF-α, 25 nM corticosterone, or 50 nM aldosterone in the absence or presence of 1 μM spironolactone or 1 μM RU-486 (A), or in the presence of 250 nM Cay-10512 (B), followed by preparation of nuclear and cytoplasmic fractions, separation of proteins by SDS-PAGE and analysis of p65 and phospho-p65 expression by western blotting. Actin served as a loading control. A representative blot from three independent experiments is shown.

Mentions: The MR-dependent NF-κB activation was further analyzed by determination of the distribution of p65 using Cellomics ArrayScan high-content imaging (Figure 6). Incubation with aldosterone enhanced the presence of p65 in the nucleus, an effect that was blocked by spironolactone. In contrast, incubation with dexamethasone diminished the presence of p65 in the nucleus, and co-incubation with the GR antagonist RU-486 prevented this effect. The subcellular distribution of p65 was further assessed by western blot analysis of cytoplasmic and nuclear fractions of BV-2 cells. Incubation of cells in the presence of corticosterone or aldosterone led to elevated amounts of the phosphorylated form of p65 in the nucleus, an effect that was prevented by co-treatment with spironolactone (Figure 7A) or Cay-10512 (Figure 7B).


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)

Nuclear-cytoplasmic distribution of NF-κB p65 determined by western blotting. BV-2 cells were treated for 24 h with 50 ng/mL TNF-α, 25 nM corticosterone, or 50 nM aldosterone in the absence or presence of 1 μM spironolactone or 1 μM RU-486 (A), or in the presence of 250 nM Cay-10512 (B), followed by preparation of nuclear and cytoplasmic fractions, separation of proteins by SDS-PAGE and analysis of p65 and phospho-p65 expression by western blotting. Actin served as a loading control. A representative blot from three independent experiments is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Nuclear-cytoplasmic distribution of NF-κB p65 determined by western blotting. BV-2 cells were treated for 24 h with 50 ng/mL TNF-α, 25 nM corticosterone, or 50 nM aldosterone in the absence or presence of 1 μM spironolactone or 1 μM RU-486 (A), or in the presence of 250 nM Cay-10512 (B), followed by preparation of nuclear and cytoplasmic fractions, separation of proteins by SDS-PAGE and analysis of p65 and phospho-p65 expression by western blotting. Actin served as a loading control. A representative blot from three independent experiments is shown.
Mentions: The MR-dependent NF-κB activation was further analyzed by determination of the distribution of p65 using Cellomics ArrayScan high-content imaging (Figure 6). Incubation with aldosterone enhanced the presence of p65 in the nucleus, an effect that was blocked by spironolactone. In contrast, incubation with dexamethasone diminished the presence of p65 in the nucleus, and co-incubation with the GR antagonist RU-486 prevented this effect. The subcellular distribution of p65 was further assessed by western blot analysis of cytoplasmic and nuclear fractions of BV-2 cells. Incubation of cells in the presence of corticosterone or aldosterone led to elevated amounts of the phosphorylated form of p65 in the nucleus, an effect that was prevented by co-treatment with spironolactone (Figure 7A) or Cay-10512 (Figure 7B).

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