The Nuclear Receptor Nurr1 Modulates the Expression and Activity of PPARγ in Human Pro-Inflammatory Macrophages

Abstract

Nurr1 is a member of the nuclear receptor family NR4A that modulates inflammation typically by inhibiting the NF-κB signalling pathway. In vitro, Nurr1 can interact with the peroxisome proliferator-activated receptor (PPAR)γ, as well as be recruited to the Pparg promoter in microglial cells; however, their functional relationship is not established. Here, we aimed to investigate the role of Nurr1 on PPARγ activity in human macrophages. Blood monocytes were cultured with GM-CSF or M-CSF to generate pro- (GM-MDMs) and anti-inflammatory (M-MDMs) macrophages, respectively. The protein levels of PPARγ and Nurr1 were elevated in GM-MDMs compared to M-MDMs, and their expression was positively correlated. PPARγ activation in GM-MDMs with the agonist rosiglitazone did not modify Nurr1 expression. However, Nurr1 activation with the agonist C-DIM12 increased PPARγ levels through protein stabilisation. Further, agonistic activation of Nurr1 decreased the proportion of PPARγ molecules phosphorylated at Ser84, which is a repressive mark for PPARγ transcriptional activity. Accordingly, exposure of GM-MDMs to C-DIM12 enhanced the expression of two PPARγ target genes induced by rosiglitazone, CD36 and PLIN2. Both PPARγ and Nurr1 agonists exhibited anti-inflammatory effects on LPS-stimulated GM-MDMs when administered alone, but C-DIM12 did not globally increase the effectiveness of rosiglitazone under that condition. These findings suggest that Nurr1 enhances PPARγ transcriptional activity, potentially through stabilising PPARγ protein levels and decreasing its repressive phosphorylation at Ser84. This novel mechanism highlights the role of Nurr1 in targeting not only inflammation but additional pathways regulated by PPARγ in macrophages.

Keywords: Nurr1; PPARγ; macrophages.

FIGURE 1

Nurr1 and PPARγ expression positively correlated in human macrophages. (A) Western blot (left) and densitometric analysis (right) of Nurr1 and PPARγ expression in GM‐MDMs (GM) and M‐MDMs (M). Western blot quantification was normalised with the housekeeping protein GAPDH. Mean ± SEM of 4 independent donors is shown. (B) M‐MDMs were incubated with IL‐4 or left unstimulated (−) for 24 h. Nurr1 and PPARγ expression was determined by Western blot. Shown are the results from two independent donors in the Western blot and from 5 donors in the graph. (C) Correlation analysis of PPARγ and Nurr1 expression in GM‐MDMs (left, n = 32) and M‐MDMs (right, n = 13). Data from M‐MDMs included unstimulated and IL‐4‐treated cells. (D) Samples from visceral AT were triple‐stained with antibodies to CD163L1, PPARγ and Nurr1. Nuclei were counterstained with DAPI (blue). Shown are representative images from a lean individual stained with PPARγ (green) and Nurr1 (purple), presented together with micrographs showing CD163L1 staining (red) to delimit the ATM fraction. The RFI of each marker was quantified in single cells in different AT sections/sample/marker at X20 magnification with the ImageJ software, and expressed in arbitrary units. The correlation data between the expression (RFI) of Nurr1 and PPARγ in lean and obese individuals is shown. Scale bar, 50 μm. Statistics: *p < 0.05; **p < 0.005.

FIGURE 2

Nurr1 activation stabilises PPARγ expression at the protein level. (A) Representative Western blot showing the expression of Nurr1 and PPARγ in GM‐MDMs treated for 8 h with C‐DIM12, IP7e, Rosi, or vehicle (DMSO). (B) Densitometric analysis of the regulation of PPARγ (left) and Nurr1 (right) by Rosi (1 and 10 μM), C‐DIM12 (1 and 10 μM) and IP7e (1 and 10 μM), as in (A) (n = 4–13). Fold change of Nurr1 and PPARγ expression is shown relative to DMSO‐treated cells. (C) Expression of NR4A2 (left) and PPARG (right) genes in GM‐MDMs treated with Rosi (10 μM), CDIM‐12 (10 μM), or IP7e (10 μM) for 4 h, evaluated by RT‐qPCR (Rosi, n = 4; C‐DIM12, n = 7; IP7e, n = 2). The NR4A2 and PPARG gene levels are shown relative to cells cultured with DMSO. Data are presented as the mean ± SEM. Statistics (DMSO vs. Agonists): *p < 0.05; **p < 0.005.

FIGURE 3

The nuclear localisation of PPARγ is not affected by Nurr1 agonists. (A) Subcellular localisation of PPARγ and Nurr1 in GM‐MDMs. Representative Confocal microscopy images of GM‐MDMs stained with Abs against PPARγ (green) and Nurr1 (blue). Nuclei were counterstained with DAPI (red). Scale bar, 50 μm. (B) Subcellular localisation of Nurr1 and PPARγ in cytoplasmic and nuclear extracts evaluated by Western blot (upper panel). GM‐MDMs were treated with Rosi (R, 10 μM), C‐DIM12 (C, 10 μM), IP7e (I, 10 μM) or vehicle (DMSO, D) for 4 h. Lower panel: Densitometric analysis of PPARγ expression in nuclear extracts. Western blot quantification was normalised with the housekeeping protein lamin B, and values are shown relative to cells cultured with DMSO. Mean ± SEM of 6–10 independent donors is shown. Statistics (DMSO vs. Agonists): *p < 0.05; **p < 0.005.

FIGURE 4

The Nurr1 agonists stabilised PPARγ protein expression. (A) GM‐MDMs were incubated for 8 h under control conditions (DMSO) or in the presence of Rosi (10 μM), C‐DIM12 (10 μM), or IP7e (10 μM) and then treated with CHX for 1–8 h. Cells cultured with the agonists or DMSO in the absence of CHX are marked as 0 h. Shown are representative Western blots of PPARγ expression (upper panels) and the quantitation of PPARγ decay (lower panels). Mean ± SEM of 5 independent donors is shown. (B) GM‐MDMs were pretreated for 1 h with the proteasome inhibitor MG132 and then exposed for 8 h to DMSO, Rosi, or C‐DIM12 as in (A). Nuclear cell extracts were harvested and immunoprecipitation was performed with anti‐PPARγ Ab (PPAR) or mouse IgG as a control (IgG). The immunoprecipitated proteins were analyzed by Western blot using anti‐PPARγ (left) and anti‐ubiquitin (right) Abs (n = 4). The graph shows the intensity of the ubiquitin signal relative to the immunoprecipitated PPARγ. Statistics (DMSO vs. Agonists): *p < 0.05; **p < 0.005; ***p < 0.0005.

FIGURE 5

Nurr1 agonists inhibit the suppressive Ser84 phosphorylation of PPARγ. (A) GM‐MDMs were treated with Rosi (10 μM), C‐DIM12 (10 μM), IP7e (10 μM), or vehicle (DMSO) for 4 h. Whole cell extracts were harvested and analyzed for the expression of phospho‐PPARγ (Ser84) and total PPARγ. Shown are representative Western blots (left) and the densitometric quantification of phospho‐PPARγ expression relative to the expression of PPARγ (right, n = 3–8). (B) GM‐MDMs were treated as in (A). Whole cell extracts were prepared and the expression of phospho‐Erk1/2 and total Erk1/2 was evaluated. A representative Western blot (left) and the densitometric quantification of phospho‐Erk1/2 expression relative to the expression of Erk1/2 (right) is shown (n = 6–13). In (A, B) data are presented as fold change relative to DMSO‐treated cells (mean ± SEM). Statistics (DMSO vs. Agonists): *p < 0.05; **p < 0.005; ***p < 0.0005; ****p < 0.0001.

FIGURE 6

C‐DIM12 synergistically promotes the transcriptional activity of PPARγ induced by Rosi. (A) PPAR‐dependent transcriptional activity in GM‐MDMs treated with Rosi (1 μM), C‐DIM12 (10 μM), or vehicle (DMSO) for 4 h. The relative PPAR‐dependent firefly luciferase activity (compared to Renilla luciferase activity) is shown (n = 6). (B) GM‐MDMs were treated with Rosi (10 μM), C‐DIM12 (10 μM), or vehicle (DMSO) for 4 h. Relative expression of CD36 and PLIN2 transcripts evaluated by RT‐qPCR. Results are expressed relative to the mRNA levels in the presence of DMSO (n = 5–6). (C) GM‐MDMs were treated for 24 h with Rosi (10 μM), C‐DIM12 (10 μM), or vehicle (DMSO) and then the expression of CD36 by flow cytometry was assessed. Representative histograms (left) and quantitation of CD36 expression (right). Results are expressed relative to the values in the presence of DMSO (n = 4). (D) GM‐MDMs were treated for 24 h with Rosi, C‐DIM12, or vehicle at the indicated concentrations and then exposed to Dil‐oxLDL for 4 h. Representative histograms (left) and quantitation of Dil‐oxLDL labeling (right) expressed as relative values in the presence of DMSO (n = 6–7) are shown. Data are presented as the mean ± SEM. Statistics [DMSO vs. Agonists (*), or Agonists alone vs. Agonists in combination (#)]: *, #p < 0.05; **, ##p < 0.005; ***p < 0.0005; ****p < 0.0001.

FIGURE 7

Anti‐inflammatory activities of C‐DIM12 and Rosi in LPS‐activated GM‐MDMs. Macrophages were pre‐incubated with Rosi (10 μM), C‐DIM12 (10 μM), or vehicle (DMSO) for 4 h and then exposed to LPS for 1 h. Shown is the quantification by RT‐qPCR of the indicated cytokine/chemokine transcripts. Values are shown relative to those obtained in GM‐MDMs treated with DMSO. Data are expressed as the mean ± SEM (n = 4–5). Statistics: DMSO vs. Agonists: *p < 0.05; **p < 0.005; ****p < 0.0001.

FIGURE 8

C‐DIM12 and Rosi did not show synergy in the suppression of inflammatory cytokine/chemokine secretion. GM‐MDMs were treated with Rosi (10 μM), C‐DIM12 (10 μM), or vehicle (DMSO) for 4 h and then exposed to LPS for 18 h. Then, cell supernatants were harvested and analyzed for the indicated cytokines/chemokines by ELISA. Data are expressed as the mean ± SEM (n = 3–5). Statistics [DMSO vs. Agonists (*), or Agonists alone vs. Agonists in combination (#)]: *, #p < 0.05; **p < 0.005; ***, ###p < 0.0005.