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. 2015 Jun;87(6):1021-34.
doi: 10.1124/mol.114.095398. Epub 2015 Apr 9.

The Nurr1 Activator 1,1-Bis(3'-Indolyl)-1-(p-Chlorophenyl)Methane Blocks Inflammatory Gene Expression in BV-2 Microglial Cells by Inhibiting Nuclear Factor κB

Briana R De Miranda  1 , Katriana A Popichak  1 , Sean L Hammond  1 , Bryce A Jorgensen  1 , Aaron T Phillips  1 , Stephen Safe  1 , Ronald B Tjalkens  2
Affiliations

The Nurr1 Activator 1,1-Bis(3'-Indolyl)-1-(p-Chlorophenyl)Methane Blocks Inflammatory Gene Expression in BV-2 Microglial Cells by Inhibiting Nuclear Factor κB

Briana R De Miranda et al. Mol Pharmacol. 2015 Jun.

Abstract

NR4A family orphan nuclear receptors are an important class of transcription factors for development and homeostasis of dopaminergic neurons that also inhibit expression of inflammatory genes in glial cells. The identification of NR4A2 (Nurr1) as a suppressor of nuclear factor κB (NF-κB)-related neuroinflammatory genes in microglia and astrocytes suggests that this receptor could be a target for pharmacologic intervention in neurologic disease, but compounds that promote this activity are lacking. Selected diindolylmethane compounds (C-DIMs) have been shown to activate or inactivate nuclear receptors, including Nurr1, in cancer cells and also suppress astrocyte inflammatory signaling in vitro. Based upon these data, we postulated that C-DIM12 [1,1-bis(3'-indolyl)-1-(p-chlorophenyl) methane] would suppress inflammatory signaling in microglia by a Nurr1-dependent mechanism. C-DIM12 inhibited lipopolysaccharide (LPS)-induced expression of NF-κB-regulated genes in BV-2 microglia including nitric oxide synthase (NOS2), interleukin-6 (IL-6), and chemokine (C-C motif) ligand 2 (CCL2), and the effects were attenuated by Nurr1-RNA interference. Additionally, C-DIM12 decreased NF-κB activation in NF-κB-GFP (green fluorescent protein) reporter cells and enhanced nuclear translocation of Nurr1 primary microglia. Chromatin immunoprecipitation assays indicated that C-DIM12 decreased lipopolysaccharide-induced p65 binding to the NOS2 promoter and concurrently enhanced binding of Nurr1 to the p65-binding site. Consistent with these findings, C-DIM12 also stabilized binding of the Corepressor for Repressor Element 1 Silencing Transcription Factor (CoREST) and the Nuclear Receptor Corepressor 2 (NCOR2). Collectively, these data identify C-DIM12 as a modulator of Nurr1 activity that results in inhibition of NF-κB-dependent gene expression in glial cells by stabilizing nuclear corepressor proteins, which reduces binding of p65 to inflammatory gene promoters.

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Figures

Fig. 1.
Fig. 1.
C-DIM inflammatory gene suppression in glia is structure dependent. Primary murine mixed glial cultures were treated with saline or 1 μg/ml LPS and 1 or 10 μM of DIM-C-pPhOCH3 (C-DIM5), DIM-C-pPhPh (C-DIM9), or DIM-C-pPhCl (C-DIM12) for 8 hours, and assessed for (A) NOS2 or (B) IL-1β expression with real-time PCR. (C) The structure of chloro-substituted C-DIM12. (D–I) BV-2 microglia were treated with saline or 1 μg/ml LPS over a 24-hour time point, and RNA was collected for real-time PCR analysis of cytokine mRNA expression. Data are expressed as mean ± S.E.M. (n = 4); mRNA fold change; internal control (β-actin). Statistical significance is expressed as mean compared with saline control. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Fig. 2.
Fig. 2.
Dose-dependent suppression of inflammatory genes in BV-2 microglia and NF-κB transactivation in reporter cells. (A–F) BV-2 microglia were pretreated for 1 hour with 0.1, 1.0, or 10 μM C-DIM12 followed by 24 hours of LPS (1 μg/ml). The mRNA expression was assessed for the fold change of cytokine expression (β-actin internal control). Data are expressed as mean ± S.E.M. (n = 4); mRNA fold change. (G) NF-κB/GFP/Luc HEK. (H) Dose-response of NF-κB–GFP expression per cell (DAPI counterstain) in NF-κB/GFP/Luc HEK cells with increasing TNFα concentrations. (I) NF-κB/GFP/Luc HEK cells were treated with C-DIM12 (25, 50, 100 μM) or Bay-11 (30, 50 μM) as a positive control (CTL) for NF-κB suppression. (J) Time course data from NF-κB/GFP/Luc HEK cells that received TNFα and were assayed over 24 hours with (gray line) or without (black line) 100 μM C-DIM12. The mRNA were assessed for fold change expression, and data are expressed as mean ± S.E.M. (n = 16). **P < 0.01; ***P < 0.001; ****P < 0.0001.
Fig. 3.
Fig. 3.
C-DIM12–dependent inhibition of inflammatory gene expression requires Nurr1. (A–C) BV-2 microglia were treated with two different sequences of siRNA (denoted RNAi-1, RNAi-2) or scrambled RNAi (control RNAi). (D) NCor2, CoREST, HDAC3 mRNA were measured using real-time PCR after treatment with RNAi-1 (β-actin internal control). (E–F) BV-2 microglia were treated with Nurr1-RNAi-1 or control RNAi-1 for 24 hours followed by saline or 1 μg/ml LPS for 24 hours and assessed for mRNA expression of NOS2 and TNFα. (G–J) BV-2 cells were treated with RNAi-1 or control RNAi-1 for 24 hours followed by 10 μM C-DIM12 (1 hour pretreatment) and 1 μg/ml LPS treatment and assessed for mRNA expression of NOS2, TNFα, IL-1β, and IL-6. Data are expressed as mean ± S.E.M. (n = 4); mRNA fold change. Statistical significance is compared with saline control. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Fig. 4.
Fig. 4.
C-DIM12 does not prevent p65 translocation and blocks NF-κB–transactivation in reporter cells. (A) BV-2 cells were treated with 10 μM C-DIM12 for 1 hour followed by saline or 1 μg/ml LPS for 30 minutes and fixed for immunofluorescence to examine p65 translocation with DAPI (blue), isolectin (green), p65 (red). (B) The p65 nuclear expression was quantified by mean fluorescence intensity encompassing the nuclei (DAPI boundary; background subtracted). (C) Representative protein expression of phosphorylated p65 after LPS and LPS + C-DIM12 treatment. (D) Relative optical density of phospho-p65 immunoblot, mean of three replicate experiments. Protein levels were normalized to β-actin (± S.E.M.). (E and F) BV-2 cells were treated with 30 μM SB216763 (SB21) to inhibit GSK3β for 1 hour, followed by 1 μg/ml LPS and cotreatment with 10 μM C-DIM12 for 24 hours. Data are expressed as mean ± S.E.M. (n = 3). **P < 0.01; ***P < 0.001; ****P < 0.0001. SAL, saline.
Fig. 5.
Fig. 5.
Nurr1 translocation and expression following LPS and C-DIM12 treatment. (A–C) Primary mouse microglia were plated onto cover glass and treated with 10 μM C-DIM12 for 1 hour followed by 1 μg/ml LPS for 24 hours. Cells were fixed and imaged (100×) by confocal microscopy for immunofluorescence of Nurr1 (red), CD11b (green) and DAPI (blue). (D and E) Quantification of Nurr1 protein translocation from the cytoplasm to the nucleus. (F) Nurr1 mRNA levels in BV-2 microglia treated with 1 μg/ml LPS, or 1 μg/ml LPS + 10 μM C-DIM12 for 24 hours. (G and H) IL-6 and Nos2 mRNA levels from the same samples corresponding to Nurr1 mRNA. Data are expressed as mean ± S.E.M.; mRNA fold change (n = 3); *P<0.05; ***P < 0.001; ****P < 0.0001. Scale bar = 10 μm. SAL, saline.
Fig. 6.
Fig. 6.
C-DIM12 enhances Nurr1 recruitment to NOS2 promoter, decreases p65 binding, and stabilizes binding of nuclear corepressors. BV-2 cells were treated with 10 μM C-DIM12 for 1 hour followed by 1 μg/ml LPS over a 24-hour time point and assessed at the NOS2 promoter using ChIP. (A) The amount of p65 bound to the NOS2 promoter was measured in LPS or LPS + C-DIM treatments. (B) The level of Nurr1 bound to the NOS2 promoter with or without C-DIM12 over a 24-hour time course with LPS. (C and D) ChIP assessment of nuclear corepressor NCOR2 and corepressor complex CoREST bound to the NOS2 promoter. (E and F) Representative protein quantitation of total Nurr1 and p65 after LPS and LPS + C-DIM12 treatment. (G) NCOR2, HDAC3, and CoREST protein from 24-hour time course treatment with saline or LPS + 10 μM C-DIM12. (H–J) Relative optical density of total cellular corepressor protein expression, mean of three replicate experiments. Protein levels were normalized to β-actin (±S.E.M.). (K) Hypothesized mechanism by which C-DIM12 promotes Nurr1-dependent transrepression of p65 at the NOS2 promoter in BV-2 microglia. ChIP data are expressed as the percentage of input ± S.E.M. over control IgG (n = 3). **P < 0.01; ***P < 0.001; ****P < 0.0001. SAL, saline.
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