Research resource: transcriptome profiling of genes regulated by RXR and its permissive and nonpermissive partners in differentiating monocyte-derived dendritic cells

Abstract

Retinoid X receptors (RXRs) are heterodimerization partners for many nuclear receptors and also act as homodimers. Heterodimers formed by RXR and a nonpermissive partner, e.g. retinoic acid receptor (RAR) and vitamin D receptor (VDR), can be activated only by the agonist of the partner receptor. In contrast, heterodimers that contain permissive partners, e.g. liver X receptor (LXR) and peroxisome proliferator-activated receptor (PPAR), can be activated by agonists for either the partner receptor or RXR, raising the possibility of pleiotropic RXR signaling. However, it is not known to what extent the receptor's activation results in triggering mechanisms dependent or independent of permissive heterodimers. In this study, we systematically and quantitatively characterized all probable RXR-signaling pathways in differentiating human monocyte-derived dendritic cells (Mo-DCs). Using pharmacological, microarray and quantitative RT-PCR techniques, we identified and characterized gene sets regulated by RXR agonists (LG100268 and 9-cis retinoic acid) and agonists for LXRs, PPARs, RARα, and VDR. Our results demonstrated that permissiveness was partially impaired in Mo-DCs, because a large number of genes regulated by PPAR or LXR agonists was not affected by RXR-specific agonists or was regulated to a lesser extent. As expected, we found that RXR agonists regulated only small portions of RARα or VDR targets. Importantly, we could identify and characterize PPAR- and LXR-independent pathways in Mo-DCs most likely mediated by RXR homodimers. These data suggested that RXR signaling in Mo-DCs was mediated via multiple permissive heterodimers and also by mechanism(s) independent of permissive heterodimers, and it was controlled in a cell-type and gene-specific manner.

Figure 1

RXRα is highly expressed and transcriptionally active in Mo-DC. A, The expression of genes coding RXRα, RXRβ, and RXRγ in human Mo-DCs was compared with other nuclear receptors (NRs) and to all probe sets of Affymetrix microarrays. Normalized signal intensities of probe sets specific for RXR isotypes are shown. If more than one probe set represent a certain gene, the probe set having the highest signal intensity is shown. B, RXRα is the dominant isotype in all tested DC types as determined by microarray. C, The RXRα protein (red fluorescence) is localized in the nuclei [visualized by 4′,6-diamidino-2-phenylindole (DAPI)] of Mo-DCs identified as DCSign positive cells (green cell membrane fluorescence). Original magnification, ×40. D, Cell surface expression of the indicated DC markers are regulated by RXR agonists, 9cisRA, and rexinoid LG268 determined by flow cytometry after 5 d of ligand treatment. E, Induction of direct target genes of LXR, PPAR, and RAR by agonists for RXR (9cis RA and LG268), LXRα/β (GW3965), and PPARγ (RSG) was detected by RT-qPCR. Monocytes were conditioned in the presence of IL-4 and GM-CSF, agonists were added 18 h after plating. Values are expressed as mean of technical triplicates ± sd of the mean. ABCG1, ATP-binding cassette, subfamily G, member 1.

Figure 2

Expression patterns of genes coding RXRs and RXR partners during monocyte to DC differentiation. Human monocytes were isolated and cultured in the presence of IL-4 and GM-CSF for 24 h and for 5 d. RNA samples were isolated from monocytes (0.0) from differentiating DCs (24.0) and immature DCs (120.0). Microarray experiment was performed, and expression patterns of RXR partners were evaluated. Per chip normalized signal intensities of probe sets specific for indicated nuclear receptors are shown. If more than 1 probe set represent a certain gene, the probe set having the highest signal intensity is shown. Raw signal intensities were normalized to the median of all transcripts (per chip normalization). RXR partners were considered to be expressed on median or high level if normalized expression levels were above 10 (dashed line). The genes that were given low significant attribute (absent flag) for all time points were marked as absent (A). CAR, Constitutive active/androstane receptor.

Figure 3

Gene sets regulated by LXRα/β, PPARγ, PPARδ, and RXR agonists only partially overlap in Mo-DCs. A, Experimental setup for Affymetrix microarray analysis. Eighteen hours after isolation of human monocyte from healthy donors (n = 3), cells were treated for 12 h with LG268 (RXR agonist) and 9cisRA (agonist for RAR and RXR), GW3965 (LXRα/β panagonist), RSG (PPARγ agonist), and GW1516 (PPARδ agonist). Cells were cultured in presence of IL-4 and GM-CSF from zero time point. B, Area-proportional Venn diagram shows the overlap between probe sets regulated by RXR agonists. The intersection of gene sets regulated by RXR agonists is labeled as “LG268 AND 9cisRA.” C, Overlap between probe sets regulated the agonist specific for LXRα/β (GW3965), PPARγ (RSG), and PPARδ (GW1516) is shown on an area-proportional Venn diagram. The union of these genes is labeled as “GW3965 AND/OR RSG AND/OR GW1516.” D, Area-proportional Venn diagram shows the overlap between gene sets regulated by both RXR agonists and by “GW3965 AND/OR RSG AND/OR GW1516.” Lists of genes/probe sets representing different sets of Venn diagram (intersection and two complement sets) are shown in heat maps. Color intensities reflect the ratios of signal intensities as shown.

Figure 4

RXR agonists are less effective than their LXR and PPAR counterparts in the regulation of direct LXR and PPAR target genes. A and B, The fold changes induced by LG268, 9cisRA, GW3965, RSG, and GW1516 were determined by Affymetrix microarrays. Bar graphs show the effect of agonists on LXR direct target genes (A) and PPAR direct target genes (B). Fold inductions were calculated from mean values of biological triplicates for agonist-treated and vehicle-treated samples. C and D, Dose response curves of selected LXR (C) and PPAR (D) direct target genes measured by RT-qPCR. Differentiating DCs were treated with various concentrations of RXR, LXR, or PPAR agonists 18 h after plating. Cells were harvested 12 h thereafter. One representative experiment of three performed is shown. Values are expressed as mean of technical triplicates ± sd of the mean. ABCG1, ATP-binding cassette, subfamily G, member 1.

Figure 5

Regulation of FABP4 by agonists for PPARγ and RXR in (A) differentiating Mo-DC and (B) PMA-treated THP1 cells. Cells were harvested 12 h after ligand treatment, and mRNA level of FABP4 was determined by RT-qPCR. Increasing concentration of LG268 or RSG alone or increasing amount of RSG with a fixed concentration of LG268 (100 nm) were used. PMA was added at 20 nm 3 h before ligand treatment. One representative experiment of three performed is shown. Values are expressed as mean of technical triplicates ± sd of the mean.

Figure 6

Comprehensive analysis of transcriptional changes induced by agonists for RXR and its partners in Mo-DCs. A, Genes regulated by LG268 and 9cisRA were classified into two clusters based on their response to agonists for permissive partners, LXR and PPAR. Cluster 2 contains genes that are not regulated by LXR and/or PPAR agonists. B and C, Probe sets regulated by agonists of nonpermissive partners: RARα (AM580) and VDR (1,25-vitD) were also identified and compared with the entire list of RXR-regulated probe sets as well as to cluster 2. Area-proportional Venn diagrams show the overlap between the indicated probe sets. Note the proportion (65%) of probe sets of cluster 2 that are regulated by RARα agonist AM580. D, Comparisons of the effect of agonists of RXR partners and RXR-specific LG268. RXR partners were activated by GW3965 (LXRα/β), RSG (PPARγ), GW1516 (PPARδ), AM580 (RARα), and 1,25-vitD (VDR). Twenty genes exhibiting the highest fold-induction values were identified for each agonist by microarray analysis. Fold inductions were calculated from mean values of biological triplicates of agonist-treated vs. vehicle-treated microarray samples.

Figure 7

RARα agonist AM580 up-regulates many genes that are induced by RXR agonists but not by agonists for LXR or PPARs. A, Six genes from cluster 2 were chosen for RT-qPCR validation. Monocytes differentiating into DCs were treated with RXR, LXR, PPARs, or RARα agonists 18 h after plating. Cells were harvested 12 h thereafter. B, Schematic illustration shows that RXR agonist can regulate transcriptional targets of RAR-RXR via different mechanisms. Note that these genes were not regulated by agonists for LXRs and PPARs. HRE, Hormone responsive element. C, Concentration of ATRA and 9cisRA were determined by an LC-MS method in differentiating DCs (diff.DC; 30 h) and immature DCs (IDC; 5 d). Chromatograms of one representative experiment with external 9cisRA and ATRA standards are shown. E, LG268 and 9cisRA regulate most selected genes in the late phase of DC differentiation as determined by RT-qPCR. Cells were treated with LG268, 9cisRA, or 1 μm XCT0135908 (XCT; Nurr1-RXR-selective RXR agonist) at d 4.5 for 12 h. One representative experiment of three performed is shown. Values are expressed as mean of technical triplicates ± sd of the mean (A and D).