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

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.

Figure 1

Potentiation of LPS-induced TNF-α mRNA expression by low concentrations of corticosterone. (A) BV-2 cells were incubated with 10, 50, and 100 ng/mL LPS for 24 h. (B) Cells were exposed to 50 nM 11-dehydrocorticosterone, which requires conversion to corticosterone by 11β-HSD1, or 25 nM corticosterone for 24 h prior to incubation with 50 ng/mL LPS for another 24 h. TNF-α mRNA expression was measured by real-time RT-PCR. Data (mean ± SD of three independent experiments) represent ratios of TNF-α mRNA to GAPDH control mRNA from treated cells normalized to the values obtained from cells incubated with vehicle (DMSO). *P <0.05, ***P <0.005.

Figure 2

Inhibition of 11β-HSD1 reversed the 11-dehydrocorticosterone-dependent stimulation of LPS induced IL-6 expression. (A, B) IL-6 mRNA expression was measured by real-time RT-PCR. Ratios of IL-6 mRNA to GAPDH control mRNA of treated cells were normalized to values obtained from cells incubated with vehicle (DMSO). (A) BV-2 cells were treated for 24 h with LPS. (B) Cells were pretreated for 24 h with 25 nM corticosterone or with 50 nM 11-dehydrocorticosterone in the presence or absence of 1 μM of the 11β-HSD1 inhibitor T0504, followed by incubation with 50 ng/mL LPS for an additional 24 h. (C) IL-6 protein levels were quantified by ELISA. Cells were treated for 24 h with 25 nM corticosterone or with 50 nM 11-dehydrocorticosterone in the presence or absence of 1 μM T0504, followed by 10 ng/mL LPS for an additional 24 h. Data normalized to control represent mean ± SD from three independent experiments. *P <0.05, ***P <0.005.

Figure 3

Differential modulation of IL-6 expression by MR and GR. BV-2 cells were treated with various concentrations of 11-dehydrocorticosterone (A, B), corticosterone (A-D), dexamethasone (E, F), or aldosterone (G, H) in the presence or absence of 1 μM of MR antagonist spironolactone (C, D, G, H) or 1 μM of GR antagonist RU-486 (C-F) for 24 h, as indicated. (A, C, G, E) Quantification of IL-6 mRNA expression by real-time RT-PCR. Data represent ratios of IL-6 mRNA to GAPDH control mRNA from treated cells normalized to the values obtained from cells incubated with vehicle (DMSO). (B, D, F, H) Quantification of IL-6 protein expression by ELISA. Data represent mean ± SD from three independent experiments. **P <0.01, ***P <0.005.

Figure 4

Blockage of corticosteroid-dependent IL-6 induction by inhibition of NF-κB. BV-2 cells were pretreated with 250 nM of the NF-κB inhibitor Cay-10512 for 1 h, followed by incubation with 25 nM corticosterone, 50 nM 11-dehydrocorticosterone, or aldosterone for another 24 h. IL-6 protein was measured by ELISA. Results represent mean ± SD from three independent experiments. **P <0.01 and ***P <0.005.

Figure 5

Corticosterone- and aldosterone-mediated NF-κB translocation is dependent on MR. BV-2 cells were treated for 24 h with vehicle (A), 50 ng/mL TNF-α (B), 25 nM corticosterone (C), 25 nM corticosterone and 1 μM spironolactone (D), 25 nM corticosterone and 250 nM Cay-10512 (E), 50 nM aldosterone (F), 50 nM aldosterone and 1 μM spironolactone (G), or 50 nM aldosterone and 250 nM Cay-10512 (H). The localization of the p65 subunit of NF-κB was visualized by immunofluorescence microscopy using a primary antibody against p65 and a secondary ALEXA fluor 488 labeled antibody (magnification 400×).

Figure 6

Differential effects of MR and GR agonists on NF-κB translocation. BV-2 cells were treated with aldosterone or dexamethasone in the presence or absence of 1 μM RU-486 or 1 μM spironolactone for 24 h, followed by analysis of the intracellular localization of p65 by Cellomics ArrayScan HCS imaging system. The ratio between the intensity of nuclear p65 fluorescence and total cellular p65 fluorescence was quantitated. Results represent mean ± SD from three independent experiments. *P <0.05 and ***P <0.005.

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.

Figure 8

Activation of MR but not GR enhanced TNFR2 expression by a NF-κB-dependent mechanism. BV-2 cells were treated for 24 h with corticosterone (A, C, D, E), 11-dehydrocorticosterone (F), dexamethasone (B), or aldosterone (C-F), in the presence or absence of 1 μM spironolactone (A, C, D), 1 μM RU-486 (A, B), or 250 nM NF-κB inhibitor Cay-10512 (C, E). The impact of 11β-HSD1 was assessed by co-incubation of cells with 1 μM T0504. TNFR2 mRNA was quantitated by real-time RT-PCR. Data (mean ± SD from three independent experiments) represent ratios of TNFR2 mRNA to GAPDH control mRNA from treated cells normalized to the values obtained from cells incubated with vehicle (DMSO). For western blot analysis (D, E, F) equal protein amounts were loaded and probed for TNFR2 with actin as a loading control. A representative blot from three independent experiments is shown. ***P <0.005.

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.

Figure 10

11β-HSD1 mRNA expression is enhanced by both MR and GR activation. BV-2 cells were treated for 24 h with corticosterone (A), aldosterone (B), or dexamethasone (C) in the presence or absence of 1 μM spironolactone (A, B) or 1 μM RU-486 (A, C). 11β-HSD1 mRNA was quantitated 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.

Figure 11

Model for the role of MR and GR in regulating neuroinflammation in BV-2 cells. This study suggests that MR promotes a neuroinflammatory response mediated through NF-κB activation, which can be blocked by activation GR. The control of local glucocorticoid availability by 11β-HSD1 is important in regulating the fine-tuning of the balance between MR and GR activity. Corticosterone binds with high affinity to MR, followed by binding to GR with lower affinity. 11β-HSD1 inhibitors (such as T0504) block the differential effects of 11-dehydrocorticosterone on MR and GR. IL-6 stimulated 11β-HSD1 expression, suggesting that IL-6 is involved in a regulatory feed-forward mechanism to adjust the local levels of active glucocorticoids and therefore the balance between MR and GR. IL-6 and TNFR2 activation both lead to the activation of NF-κB, and their own expression is upregulated upon NF-κB activation.