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

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 kB (NF-kB)-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(39-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-kB-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-kB activation in NF-kB-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-kBdependent gene expression in glial cells by stabilizing nuclear corepressor proteins, which reduces binding of p65 to inflammatory gene promoters.

Microglia amplify inflammatory activation of astrocytes in manganese neurotoxicity

“…Bay11 was chosen due to its established ability to inhibit of NF-κB including in primary microglia [39, 40]. Media was completely exchanged, and microglia were then treated per standard MCM protocol with either saline or 100 μM MnCl 2 .…”

Adenosine Modulates NR4A Orphan Nuclear Receptors To Attenuate Hyperinflammatory Responses in Monocytic Cells

“…Although NR4A1-3 activity does not appear to be regulated by endogenous ligands, pharmacological modulation of NR4A activity can be achieved with agents including the antineoplastic agent, 6-mercaptopurine, and a series of 1,1-bis(3-indolyl)-1-(p-substituted phenyl) methanes identified as potential NR4A1/2 agonists (52)(53)(54). Recent studies reveal that in microglial cells, 1,1-bis(3-indolyl)-1-(p-substituted phenyl) methanes can increase NR4A2 nuclear localization, stabilization of Co-REST and NCoR2 complexes, and subsequent inhibition of NF-kB/p65 signaling (55). In addition, altering the expression level of these constitutively active receptors may be a feasible approach to modulate NR4A target genes in a cell-specific manner.…”

A novel synthetic activator of Nurr1 induces dopaminergic gene expression and protects against 6-hydroxydopamine neurotoxicity in vitro

“…qPCR was performed as previously described [13] and total RNA was quantified relative to hypoxanthine-guanine phosphoribosyltransferase (N2A) or β-actin (N2A). The sequences of qPCR primers are listed in Supplementary Table 1.…”

“…The sequences of qPCR primers are listed in Supplementary Table 1. Transfections with DsiRNA oligonucletodes or expression plasmids was performed as previously described [13]. …”