Transcription factor myocyte enhancer factor 2D regulates interleukin-10 production in microglia to protect neuronal cells from inflammation-induced death

Abstract

Background: Neuroinflammatory responses have been recognized as an important aspect in the pathogenesis of Parkinson's disease (PD). Transcriptional regulation plays a critical role in the process of inflammation. Transcription factor myocyte enhancer factor 2D (MEF2D) is identified as a central factor in transmission of extracellular signals and activation of the genetic programs in response to a wide range of stimuli in several cell types, including neurons. But its presence and function in microglia have not been reported. We therefore investigated the effect of MEF2D in activated microglia on the progress of neuroinflammation and the survival of neurons.

Methods: BV2 cells and primary cultured glial cells were stimulated with lipopolysaccharide (LPS). Samples from cells were examined for MEF2D expression, interleukin-10 (IL-10), and tumor necrosis factor alpha (TNF-α) by immunoblotting, quantitative real-time PCR (qPCR) or enzyme-linked immunosorbent assay (ELISA). The activity of MEF2D was examined by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP). Recombinant lentivirus expressing shRNA specific to MEF2D was used to silence MEF2D expression in BV2 cells. The role of IL-10 transcriptionally induced by MEF2D on neuronal survival was assessed by anti-IL-10 neutralizing antibody. The survival of neurons was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Male C57bl/6 mice were used to establish an acute PD model. Brain sections and cell slides were tested by immunofluorescence.

Results: We demonstrated that MEF2D was present in microglia. Activation of microglia was associated with an increase in MEF2D level and activity in response to different stimuli in vivo and in vitro. MEF2D bound to a MEF2 consensus site in the promoter region of IL-10 gene and stimulated IL-10 transcription. Silencing MEF2D decreased the level of IL-10, increased the TNF-α mRNA, and promoted inflammation-induced cytotoxicity, consistent with the result of inhibiting IL-10 activity with an anti-IL-10 neutralizing antibody.

Conclusions: Our study identifies MEF2D as a critical regulator of IL-10 gene expression that negatively controls microglia inflammation response and prevents inflammation-mediated cytotoxicity.

Figure 1

Induction of MEF2D expression in the activated microglia. (A) Loss of TH signals in MPTP-treated mouse brain. SNc brain sections from mice injected with MPTP were stained with the antibodies indicated by immunofluorescence (TH, green; Iba-1, red; bar = 200 μm; and n = 3). (B) Expression of MEF2D in activated microglia (MEF2D, green; Iba-1, red; bar = 100 μm; and n = 3).

Figure 2

LPS-induced increase in expression and activity of MEF2D in BV2 cells. (A) LPS-induced increase in MEF2D mRNA expression in BV2 cells. BV2 cells were exposed to 1.0 μg/ml LPS for 6, 12, 18, or 24 h, and the mRNA levels of MEF2D were quantified by qPCR and normalized to β-actin mRNA level as described in ‘Methods’. Data from three independent experiments were expressed as the mean ± SEM and analyzed by one-way ANOVA (**P < 0.01). (B) LPS-induced increase in MEF2D protein in BV2 cells. BV2 cells were treated for the indicated time as described above and immunoblotted for MEF2D. (B bottom graph) Relative quantification of MEF2D. Data were expressed as mean ± SEM from at least three independent experiments and analyzed by one-way ANOVA (**P < 0.01). (C) LPS-induced increase in MEF2D activity. The whole cell extracts were prepared from BV2 cells treated with or without LPS for stimulating 24 h and analyzed by EMSA. Arrow indicates the position of MEF2D-probe complex (unlabelled probe: a 100-fold excess of unlabelled probe was added in the reaction; MT probe: probe with MEF2 binding site mutated).

Figure 3

Regulation of IL-10 gene expression by MEF2D. (A) LPS-induced time-dependent changes of MEF2D protein and cytokines in BV2 cells. BV2 cells treated with LPS as described in Figure 2A were analyzed by immunoblotting for MEF2D and by qPCR for TNF-α and IL-10 mRNA levels. Data from three independent experiments were expressed as the mean ± SEM and analyzed by one-way ANOVA (*P < 0.05; **P < 0.01). (B) Identification of a conserved MEF2 binding site in the IL-10 gene promoter of different species. The underlined sequence indicates the MEF2 binding site. Black-shaded areas show the conversed MEF2 binding site in the IL-10 gene promoter of different species. (C, D) Binding of MEF2D to the IL-10 gene promoter region containing the conserved site in BV2 cells. BV2 cells treated with or without LPS for 24 h were analyzed by ChIP. The result was showed by standard endpoint PCR (C) and qPCR (D). Data from three independent experiments were expressed as the mean ± SEM and analyzed by one-way ANOVA (*P < 0.05; **P < 0.01).

Figure 4

Effect of knocking-down MEF2D on IL-10 expression. (A) BV2 cells were infected with recombinant lentivirus expressing either control shRNA or shRNA to MEF2D (sh-MEF2D) and then treated with LPS for 24 h. The effectiveness of sh-RNA MEF2D was examined by immunoblotting. (A bottom graph) Relative quantification of MEF2D. Data from three independent experiments were expressed as the mean ± SEM and analyzed by one-way ANOVA (**P < 0.01). (B) The effect of sh-MEF2D on IL-10 mRNA in BV2 cells. BV2 cells infected with sh-MEF2D lentivirus were treated with LPS and analyzed for IL-10 mRNA by qPCR. Data from three independent experiments were expressed as the mean ± SEM and analyzed by two-way ANOVA (**P < 0.01). (C) The effect of sh-MEF2D on the level of secreted IL-10. Culture media from BV2 cells treated as descried under (B) were harvested for ELISA. Data from three independent experiments were expressed as the mean ± SEM and analyzed by two-way ANOVA (**P < 0.01). (D) The effect of sh-MEF2D on TNF-α mRNA in BV2 cells. BV2 cells treated as described under (B) and exposed to LPS for different times were analyzed for TNF-α mRNA by qPCR. Data from three independent experiments were expressed as the mean ± SEM and analyzed by two-way ANOVA (**P < 0.01).

Figure 5

The effect of knocking-down MEF2D on microglia-mediated cytotoxicity. (A) Experiment paradigm for the collection of conditioned media (CM). Media collected from BV2 cells treated with LPS for 12 h were labeled as pre-12 h CM. After the first 12 h, cells were washed and placed in fresh media for another 12 h (post-12 h CM). CM was then added to SN4741 cells for 48 h. (B) The effect of silencing MEF2D on CM-mediated toxicity. CM was collected from BV2 cells infected with either control (sh-Control) or sh-MEF2D lentivirus (sh-MEF2D). The viability of SN4741 cells was determined by MTT. Data from three independent experiments were expressed as the mean ± SEM and analyzed by two-way ANOVA (**P < 0.01). (C) The effect of neutralizing IL-10 on CM-mediated toxicity. CM was collected from BV2 cells with (anti-IL-10+) or without (anti-IL-10−) anti-IL-10 neutralizing antibody. The viability of SN4741 cells was determined by MTT. Data from three independent experiments were expressed as the mean ± SEM and analyzed by two-way ANOVA (**P < 0.01). (D) The effect of silencing MEF2D or neutralizing IL-10 on CM-induced toxicity. For (D) left panel, the effect of CM from BV2 cells treated with LPS on SN4741 viability was assessed by TUNEL staining. For (D) middle panel, the effect of post-12 h CM collected from BV2 cells treated as in (B) or (C) were assessed by TUNEL assay in SN4741 cells. For (D) right panel, the quantification of the percentages of TUNEL-positive population. Data from three independent experiments were expressed as the mean ± SEM and analyzed by one-way ANOVA (**P < 0.01).