Disrupting LXRα phosphorylation promotes FoxM1 expression and modulates atherosclerosis by inducing macrophage proliferation

Abstract

Macrophages are key immune cells for the initiation and development of atherosclerotic lesions. However, the macrophage regulatory nodes that determine how lesions progress in response to dietary challenges are not fully understood. Liver X receptors (LXRs) are sterol-regulated transcription factors that play a central role in atherosclerosis by integrating cholesterol homeostasis and immunity. LXR pharmacological activation elicits a robust antiatherosclerotic transcriptional program in macrophages that can be affected by LXRα S196 phosphorylation in vitro. To investigate the impact of these transcriptional changes in atherosclerosis development, we have generated mice carrying a Ser-to-Ala mutation in myeloid cells in the LDL receptor (LDLR)-deficient atherosclerotic background (M-S196A^Ldlr-KO). M-S196A^Ldlr-KO mice fed a high-fat diet exhibit increased atherosclerotic plaque burden and lesions with smaller necrotic cores and thinner fibrous caps. These diet-induced phenotypic changes are consistent with a reprogramed macrophage transcriptome promoted by LXRα-S196A during atherosclerosis development. Remarkably, expression of several proliferation-promoting factors, including the protooncogene FoxM1 and its targets, is induced by LXRα-S196A. This is consistent with increased proliferation of plaque-resident cells in M-S196A^Ldlr-KO mice. Moreover, disrupted LXRα phosphorylation increases expression of phagocytic molecules, resulting in increased apoptotic cell removal by macrophages, explaining the reduced necrotic cores. Finally, the macrophage transcriptome promoted by LXRα-S196A under dietary perturbation is markedly distinct from that revealed by LXR ligand activation, highlighting the singularity of this posttranslational modification. Overall, our findings demonstrate that LXRα phosphorylation at S196 is an important determinant of atherosclerotic plaque development through selective changes in gene transcription that affect multiple pathways.

Keywords: FoxM1; atherosclerosis; liver X receptor; macrophages; proliferation.

Fig. 1.

M-S196A^Ldlr-KO mice develop increased atherosclerosis on a WD. (A, Left) Representative images of en face Oil Red O-stained whole aortas (n = 8–11 per group). (Original magnification: 8×.) (A, Right) Quantification of stained areas as percent plaque coverage for each genotype. (B) H&E-stained aortic roots (n = 9–10 per group). (Scale bars: 500 μm.) (C) Quantification of stained areas as percent plaque coverage for each genotype. (D) CD68 staining of aortic roots (n = 7–8 per group). Data are mean ± SEM (*P < 0.05, relative to WT^Ldlr-KO mice). NS, not significant.

Fig. 2.

Changes in LXRα phosphorylation reprogram macrophage gene expression. (A) Volcano plot of log₂ ratio vs. P value of differentially expressed genes comparing 12-wk-old, WD-fed M-S196A^Ldlr-KO and WT^Ldlr-KO bone marrow-derived macrophages (n = 3 per group). The blue line indicates an adjusted P value threshold of 0.04 (Wald test for logistic regression). (B) Clustered heat map of RNA-seq gene counts in WD-fed macrophages (n = 3 mice per group). (C) Gene set enrichment analysis showing enriched pathways in M-S196A^Ldlr-KO macrophages derived from hallmark gene sets. (D) Fold change of RNA-seq gene counts in M-S196A^Ldlr-KO compared with WT^Ldlr-KO macrophages (set as 1) (n = 3 per genotype) for the top induced genes (≥twofold expression, P ≤ 0.01) involved in cell proliferation. (E) Heat map of RNA-seq gene counts of immune response genes down-regulated by S196A in WD-fed macrophages (n = 3 mice per group). (F) Heat map of RNA-seq gene counts of chemokine receptor (Top) and chemokine ligand genes (Bottom) showing differentially expressed genes in S196A WD-fed macrophages (n = 3 mice per group). For all heat maps, blue and orange depict up-regulated and down-regulated genes, respectively, and only genes showing ≥1.3-fold change with P ≤ 0.01 are shown.

Fig. 3.

Impaired macrophage LXRα phosphorylation induces FoxM1 expression and increases plaque cell proliferation. (A) Fold-change of RNA-seq gene counts in WD-fed M-S196A^Ldlr-KO compared with WD-fed WT^Ldlr-KO macrophages (set as 1) for FoxM1 and FoxM1 target genes with ≥1.3-fold expression (P ≤ 0.01; n = 3 per genotype). LXR occupancy and H3K27 acetylation (HK27Ac) at the Srebf1 reported LXRE (B) and FoxM1 (C) and Cenpf (D) identified DR4 sequences (LXR-binding sites) in bone marrow-derived macrophages from M-S196A^Ldlr-KO and WT^Ldlr-KO WD-fed mice are shown. Data shown are normalized to input compared with a region within in a gene desert (Neg S) as a negative control. One representative experiment of three (each using n = 2 mice per genotype) is shown. (E) Representative images of plaques exhibiting Ki67⁺ nuclei. L, lumen; P, plaque. (Scale bars: 250 μm.) (F) Quantification of Ki67⁺ nuclei in WD-fed WT^Ldlr-KO and M-S196A^Ldlr-KO plaques (n = 6–10 mice per group). **P ≤ 0.01. (G) Flow cytometry histograms of Ki67 expression in F4/80⁺ macrophages (Left) and bar chart of percentage of Ki67⁺ cells in WT^LdlrKO and M-S196A^Ldlr-KO macrophages exposed to 20 μM FDI-6 inhibitor (Right). Data shown are representative of three independent experiments (n = 2 mice per genotype each). (H) Histogram (Left) and bar chart of percentage of Ki67⁺ M-S196A^Ldlr-KO macrophages in response to indicated concentrations of FDI-6 (Right). Data shown are representative of three independent experiments (n = 2 mice per genotype each). (I) RT-qPCR analysis of FoxM1 target genes in WT^Ldlr-KO and M-S196A^Ldlr-KO macrophages. Normalized data are shown relative to WT^Ldlr-KO as mean ± SD (n = 3) compared with WT-DMSO (a) and with M-S196A-DMSO (b).

Fig. 4.

M-S196A^Ldlr-KO mice show decreased plaque necrotic cores and increased efferocytosis capacity. (A, Left) H&E-stained mature plaques depict necrotic core (NC; n = 4–6 per group); representative images are shown. (A, Right) Quantification of H&E-stained areas for each genotype. (Scale bars: 250 μm.) (B) Engulfment of apoptotic Jurkat cells (*P ≤ 0.05; n = 6 per group). (C) Fold change of RNA-seq gene counts in WD-fed M-S196A^Ldlr-KO compared with WD-fed WT^Ldlr-KO macrophages (set as 1) for pro- and antiphagocytic genes (n = 3 per genotype).

Fig. 5.

Macrophage transcriptional reprogramming in response to changes in LXRα phosphorylation is fundamentally different from ligand activation responses. (A and B) RNA-seq analysis on M-S196A^Ldlr-KO and WT^Ldlr-KO macrophages from WD-fed mice exposed to 1 μM GW3965 (GW) (n = 3 per group). Venn diagrams of genes induced (A) or reduced (B) in S196A compared with WT cells are shown. (C) Comparison of ligand responses in M-S196A^Ldlr-KO and WT^Ldlr-KO macrophages. (Middle) Venn diagram of genes induced (UP) or reduced (DOWN) by GW in WT LXRα cells compared with differentially expressed genes (DEGs) in S196A vs. WT in vehicle-treated (DMSO) conditions. Bar graphs show fold changes in gene expression for genes induced (Top) or reduced (Bottom) by GW that are also differentially regulated by S196A. #, not significant. (D) Clustered heat map of RNA-seq gene counts for FoxM1 and its target genes in macrophages from WD-fed mice (n = 3 per group) treated as indicated. (E) RT-qPCR analysis of FoxM1 and its target genes in WD-fed WT^Ldlr-KO and M-S196A^Ldlr-KO macrophages. Normalized data are shown relative to WT^Ldlr-KO macrophages as mean ± SD (n = 3) [P ≤ 0.001 compared with WT-DMSO (a), P ≤ 0.001 compared with S196A-DMSO (b), and P ≤ 0.001 compared with WT-GW (c)]. (F) mRNA expression of FoxM1 regulators. Data and statistical analysis are as in E.