ALOX15B controls macrophage cholesterol homeostasis via lipid peroxidation, ERK1/2 and SREBP2

Abstract

Macrophage cholesterol homeostasis is crucial for health and disease and has been linked to the lipid-peroxidizing enzyme arachidonate 15-lipoxygenase type B (ALOX15B), albeit molecular mechanisms remain obscure. We performed global transcriptome and immunofluorescence analysis in ALOX15B-silenced primary human macrophages and observed a reduction of nuclear sterol regulatory element-binding protein (SREBP) 2, the master transcription factor of cellular cholesterol biosynthesis. Consequently, SREBP2-target gene expression was reduced as were the sterol biosynthetic intermediates desmosterol and lathosterol as well as 25- and 27-hydroxycholesterol. Mechanistically, suppression of ALOX15B reduced lipid peroxidation in primary human macrophages and thereby attenuated activation of mitogen-activated protein kinase ERK1/2, which lowered SREBP2 abundance and activity. Low nuclear SREBP2 rendered both, ALOX15B-silenced and ERK1/2-inhibited macrophages refractory to SREBP2 activation upon blocking the NPC intracellular cholesterol transporter 1. These studies suggest a regulatory mechanism controlling macrophage cholesterol homeostasis based on ALOX15B-mediated lipid peroxidation and concomitant ERK1/2 activation.

Keywords: 15-LO2; Arachidonate 15-lipoxygenase type B; Lipid peroxidation; MAPK; Reactive oxygen species; Sterol regulatory element-binding protein 2.

Graphical abstract

Fig. 1

Silencing ALOX15B in primary human macrophages reduces SREBP2-dependent gene expression and nuclear SREBP2 protein (A) Global heatmap showing all downregulated genes in ALOX15B KD macrophages with a log₂FC ≤ −0.58 and all upregulated genes with a log₂FC ≥ 0.58. (B) Enrichment analysis of RNA-seq data comparing control to ALOX15B KD macrophages using Kyoto Encyclopedia of Genes and Genomes (KEGG) and REACTOME gene sets. (C) Heat map displaying differentially expressed SREBP2-target genes in control and ALOX15B KD cells as count-per-million (CPM) with annotated log₂FC and p-adjusted value. (D) Validation of MSMO1, LDLR, HMGCS1, MVK, CYP51A1, and DHCR24 gene expression by real-time qPCR of control and ALOX15B KD macrophages. (E) Schematic representation of the Bloch and Kandutsch-Russell cholesterol biosynthesis pathways. Broken arrows indicate potential crossover sites. Significantly downregulated genes in ALOX15B-silenced macrophages are shown in blue [log₂FC ≤ −0.58]. (F) SREBP2 immunofluorescence microscopy in control and ALOX15B KD macrophages. Densitometry of SREBP2 fluorescence signal in whole cell, nucleus and cytoplasm. Data are presented as mean ± SE from at least five independent experiments. Statistical analysis was performed using one sample t-test for D and F (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 vs siControl).

Fig. 2

ALOX15B-silenced macrophages contain reduced levels of sterol intermediates and oxysterols but not cholesterol (A) Sterol intermediates, (B) cholesterol-derived oxysterols, and (C) total cholesterol in control and ALOX15B KD macrophages measured using GC-MS-SIM for A and B or GC-FID for C, respectively. (D) Free and esterified cholesterol and (E) phytosterols in control and ALOX15B KD macrophages. (F) Flow cytometry determined median fluorescence intensity of uptaken green-fluorescent LDL (10 μg/ml) in control and ALOX15B KD macrophages. (G) Real-time qPCR analysis of MSMO1, HMGCS1, CYP51A1, and DHCR24 gene expression in control and ALOX15B KD cells with or without addition of NPC1 inhibitor U18666A (5 μM) for 24 h. Data are presented as mean ± SE from at least four independent experiments. Statistical analysis was performed using one sample t-test for A-B (*P < 0.05, **P < 0.01, and ****P < 0.0001 vs siControl. ns = not significant), two-tailed student's t-test for C–F (p-values are indicated. ns = not significant), and one-way ANOVA with Sidak's multiple comparisons test for G (**P < 0.01 and ****P < 0.0001 vs siControl).

Fig. 3

ALOX15B KD or inhibition by ML351 reduce cellular lipid peroxidation, oxylipins and SREBP2-target gene expression (A) Representative Western analysis and densitometry of ALOX15 and ALOX15B in macrophages treated with ML351 (10 μM) for 24 h or interleukin 4 (IL-4) (20 ng/ml) for 48 h. (B) Validation of MSMO1, LDLR, HMGCS1, MVK, CYP51A1, and DHCR24 gene expression by real-time qPCR of macrophages treated with DMSO and ML351 (10 μM) for 24 h. Confocal microscopy of BODIPY™ 581/591 C11 lipid peroxidation sensor in (C) control and ALOX15B KD macrophages as well as (D) macrophages treated for 30 min with DMSO or ML351 (10 μM). Graphs depict oxidized BODIPY C11 fluorescence intensity per cell including median. Formation of esterified 15-LOX-specific hydroxy-fatty acids in macrophages treated with (E) control and ALOX15B siRNA as well as (F) DMSO and ML351 (10 μM) for 24 h. Data are presented as mean ± SE from at least three independent experiments. Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparisons test for A (*P < 0.05 vs untreated control), one sample t-test for B (**P < 0.01 and ***P < 0.001 vs DMSO control), two-tailed student's t-test for C-D (***P < 0.001 and ****P < 0.0001 vs siControl or DMSO control), and one-way ANOVA with Sidak's multiple comparisons test for E and F (*P < 0.05 and **P < 0.01 vs siControl or DMSO control).

Fig. 4

Modulating lipid peroxidation by RSL3 and liproxstatin-1 affects SREBP2-dependent gene expression (A) Gene expression of MSMO1, MVK, LDLR, CYP51A1, and DHCR24 via real-time qPCR of macrophages treated with RSL3 (10 μM) for 24 h. (B) Western analysis and corresponding densitometry of DHCR24 and CYP51A1 of macrophages treated with DMSO or RSL3 (10 μM) for 24 h. (C) Gene expression analysis of MSMO1, MVK, LDLR, CYP51A1, and DHCR24 via real-time qPCR of macrophages treated with DMSO or liproxstatin-1 (1 μM) for 6 h. (D) Analysis of SREBP2-target genes in macrophages treated with RSL3 (10 μM) and ML351 (10 μM) for 24 h. For combined treatments, macrophages were exposed to RSL3 for 24 h, while ML351 was added during the last 6 h. Data are presented as mean ± SE from at least ten independent experiments. Statistical analysis was performed using one sample t-test for A, B and C (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 vs DMSO Control) and one-way ANOVA with Tukey's multiple comparisons test for D (***P < 0.001 and ****P < 0.0001 vs RSL3).

Fig. 5

ALOX15B-suppressed macrophages exhibit reduced ERK1/2 activity (A) Schematic illustration of ERK1/2 and AKT as potential signaling hubs that reduce SREBP2 activation in ALOX15B-suppressed macrophages. (B) Gene Set Enrichment Analysis blots of downregulated REACTOME pathways in ALOX15B KD macrophages, including ROS production, signaling by MAPKs as well as ERK1/2. (C) Real-time qPCR of SREBP2-target genes in macrophages treated with ML351 (15-LOX inhibitor, 10 μM), PD98059 (ERK1/2 inhibitor, 10 μM), AKTVIII (AKT inhibitor, 1 μM), and PF4708671 (ribosomal protein S6 kinase B1 inhibitor, 10 μM) for 6 h. Western analysis of total and phosphorylated ERK1 (p44 MAPK) and ERK2 (p42 MAPK) in (D) control and ALOX15B-silenced macrophages as well as (E) DMSO- and ML351-treated macrophages. (F) Western analysis of total and phosphorylated ERK1 (p44 MAPK) and ERK2 (p42 MAPK) of macrophages treated with DMSO, RSL3 (10 μM) or liproxstatin-1 (1 μM) for 24 h. Data are presented as mean ± SE from at least four independent experiments. Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparisons test for C (**P < 0.01 and ****P < 0.0001 vs DMSO control) and one sample t-test for D, E and F (*P < 0.05 and **P < 0.01 vs siControl or DMSO control).

Fig. 6

ERK1/2 inhibition reduces nuclear SREBP2 and prevents SREBP2-target gene expression upon NPC1 blockade (A) SREBP2 immunofluorescence microscopy in macrophages treated with DMSO or the ERK1/2 inhibitor PD98059 (10 μM) for 3 h. Mean pixel intensity of SREBP2 fluorescence signal in whole cell, nucleus and cytoplasm. (B) Real-time qPCR analysis of MSMO1, HMGCS1, CYP51A1, and DHCR24 gene expression in macrophages treated with DMSO and the ERK1/2 inhibitor PD98059 (10 μM) in combination with or without the NPC1 inhibitor U18666A (5 μM) for 24 h. Data are presented as mean ± SE from at least four independent experiments. Statistical analysis was performed using one sample t-test for A (*P < 0.05 and **P < 0.01 vs DMSO control) and one-way ANOVA with Sidak's multiple comparisons test for B (*P < 0.05, **P < 0.01, ***P < 0.001 vs DMSO control).