Benoit Pourcet

SUMOylation of Human Peroxisome Proliferator-activated Receptor alpha Inhibits Its Trans-activity through the Recruitment of the Nuclear Corepressor NCoR

The nuclear receptor peroxisome proliferator-activated receptor α (PPARα) is a key regulator of genes implicated in lipid homeostasis and inflammation. PPARα trans-activity is enhanced by recruitment of coactivators such as SRC1 and CBP/p300 and is inhibited by binding of corepressors such as NCoR and SMRT. In addition to ligand binding, PPARα activity is regulated by post-translational modifications such as phosphorylation and ubiquitination. In this report, we demonstrate that hPPARα is SUMOylated by SUMO-1 on lysine 185 in the hinge region. The E2-conjugating enzyme Ubc9 and the SUMO E3- ligase PIASy are implicated in this process. In addition, ligand treatment decreases the SUMOylation rate of hPPARα. Finally, our results demonstrate that SUMO-1 modification of hPPARα down-regulates its trans-activity through the specific recruitment of corepressor NCoR but not SMRT leading to the differential expression of a subset of PPARα target genes. In conclusion, hPPARα SUMOylation on lysine 185 down-regulates its trans-activity through the selective recruitment of NCoR.

LXRα Regulates Macrophage Arginase 1 Through PU.1 and Interferon Regulatory Factor 8

Rationale: Activation of liver X receptors (LXRs) inhibits the progression of atherosclerosis and promotes regression of existing lesions. In addition, LXR&agr; levels are high in regressive plaques. Macrophage arginase 1 (Arg1) expression is inversely correlated with atherosclerosis progression and is markedly decreased in foam cells within the lesion. Objective: To investigate LXR&agr; regulation of Arg1 expression in cultured macrophages and atherosclerotic regressive lesions. Methods and Results: We found that Arg1 expression is enhanced in CD68+ cells from regressive versus progressive lesions in a murine aortic arch transplant model. In cultured macrophages, ligand-activated LXR&agr; markedly enhances basal and interleukin-4–induced Arg1 mRNA and protein expression as well as promoter activity. This LXR&agr;-enhanced Arg1 expression correlates with a reduction in nitric oxide levels. Moreover, Arg1 expression within regressive atherosclerotic plaques is LXR&agr;-dependent, as enhanced expression of Arg1 in regressive lesions is impaired in LXR&agr;-deficient CD68+ cells. LXR&agr; does not bind to the Arg1 promoter but instead promotes the interaction between PU.1 and interferon regulatory factor (IRF)8 transcription factors and induces their binding of a novel composite element. Accordingly, knockdown of either IRF8 or PU.1 strongly impairs LXR&agr; regulation of Arg1 expression in macrophage cells. Finally, we demonstrate that LXR&agr; binds the IRF8 locus and its activation increases IRF8 mRNA and protein levels in these cells. Conclusions: This work implicates Arg1 in atherosclerosis regression and identifies LXR&agr; as a novel regulator of Arg1 and IRF8 in macrophages. Furthermore, it provides a unique molecular mechanism by which LXR&agr; regulates macrophage target gene expression through PU.1 and IRF8.Rationale Activation of Liver X Receptors (LXRs) inhibits the progression of atherosclerosis and promotes regression of existing lesions. In addition, LXRα levels are high in regressive plaques. Macrophage arginase 1 (Arg1) expression is inversely correlated with atherosclerosis progression and is markedly decreased in foam cells within the lesion.

The Nuclear Receptor Rev-erbα Is a Liver X Receptor (LXR) Target Gene Driving a Negative Feedback Loop on Select LXR-Induced Pathways in Human Macrophages

A role of the nuclear receptor Rev-erbalpha in the regulation of transcription pathways involving other nuclear receptors is emerging. Indeed, Rev-erbalpha is a negative regulator of transcription by binding to overlapping response elements shared with various nuclear receptors, including the peroxisome proliferator-activated receptors and the retinoid-related orphan receptor alpha (RORalpha). Here, we show that Rev-erbalpha is expressed in primary human macrophages and that its expression is induced by synthetic ligands for the liver X receptors (LXRs), which control cholesterol homeostasis, inflammation, and the immune response in macrophages. LXRalpha binds to a specific response element in the human Rev-erbalpha promoter, thus inducing Rev-erbalpha transcriptional expression. Interestingly, Rev-erbalpha does not influence basal or LXR-regulated cholesterol homeostasis. However, Rev-erbalpha overexpression represses the induction of toll-like receptor (TLR)-4 by LXR agonists, whereas Rev-erbalpha silencing by short interfering RNA results in enhanced TLR-4 expression upon LXR activation. Electrophoretic mobility shift, chromatin immunoprecipitation, and transient transfection experiments demonstrate that Rev-erbalpha represses human TLR-4 promoter activity by binding as a monomer to a RevRE site overlapping with the LXR response element site in the TLR-4 promoter. These data identify Rev-erbalpha as a new LXR target gene, inhibiting LXR-induction of TLR-4 in a negative transcriptional feedback loop, but not cholesterol homeostasis gene expression.

Transcriptional regulation of macrophage arginase 1 expression and its role in atherosclerosis

Atherosclerosis results from a metabolic imbalance and chronic arterial inflammation and macrophages are key during the initiation and progression of atherosclerotic lesions. A number of macrophage subsets have been identified in atherosclerotic plaques. Arginase 1 (Arg1), a marker for the M2 anti-inflammatory subset, hydrolyzes l-arginine into urea and ornithine, a precursor to l-proline and polyamines, which are implicated in tissue repair and wound healing. Additionally, Arg1 inhibits nitric oxide-mediated inflammatory pathways by competing with iNOS for the same substrate, l-arginine. Therefore, changes in Arg1 expression in macrophages may affect the development of atherosclerosis. Here, we present an overview of the transcriptional regulation of macrophage Arg1, focusing on the nuclear receptor family of ligand-activated transcription factors, and the relevance of this regulation to atherosclerosis.

Modulation of Macrophage Gene Expression via Liver X Receptor α Serine 198 Phosphorylation

ABSTRACT In mouse models of atherosclerosis, normalization of hyperlipidemia promotes macrophage emigration and regression of atherosclerotic plaques in part by liver X receptor (LXR)-mediated induction of the chemokine receptor CCR7. Here we report that LXRα serine 198 (S198) phosphorylation modulates CCR7 expression. Low levels of S198 phosphorylation are observed in plaque macrophages in the regression environment where high levels of CCR7 expression are observed. Consistent with these findings, CCR7 gene expression in human and mouse macrophages cell lines is induced when LXRα at S198 is nonphosphorylated. In bone marrow-derived macrophages (BMDMs), we also observed induction of CCR7 by ligands that promote nonphosphorylated LXRα S198, and this was lost in LXR-deficient BMDMs. LXRα occupancy at the CCR7 promoter is enhanced and histone modifications associated with gene repression are reduced in RAW264.7 cells expressing nonphosphorylated LXRα (RAW-LXRα S198A) compared to RAW264.7 cells expressing wild-type (WT) phosphorylated LXRα (RAW-LXRα WT). Expression profiling of ligand-treated RAW-LXRα S198A cells compared to RAW-LXRα WT cells revealed induction of cell migratory and anti-inflammatory genes and repression of proinflammatory genes. Modeling of LXRα S198 in the nonphosphorylated and phosphorylated states identified phosphorylation-dependent conformational changes in the hinge region commensurate with the presence of sites for protein interaction. Therefore, gene transcription is regulated by LXRα S198 phosphorylation, including that of antiatherogenic genes such as CCR7.

The nuclear receptor LXR modulates interleukin-18 levels in macrophages through multiple mechanisms.

IL-18 is a member of the IL-1 family involved in innate immunity and inflammation. Deregulated levels of IL-18 are involved in the pathogenesis of multiple disorders including inflammatory and metabolic diseases, yet relatively little is known regarding its regulation. Liver X receptors or LXRs are key modulators of macrophage cholesterol homeostasis and immune responses. Here we show that LXR ligands negatively regulate LPS-induced mRNA and protein expression of IL-18 in bone marrow-derived macrophages. Consistent with this being an LXR-mediated process, inhibition is abolished in the presence of a specific LXR antagonist and in LXR-deficient macrophages. Additionally, IL-18 processing of its precursor inactive form to its bioactive state is inhibited by LXR through negative regulation of both pro-caspase 1 expression and activation. Finally, LXR ligands further modulate IL-18 levels by inducing the expression of IL-18BP, a potent endogenous inhibitor of IL-18. This regulation occurs via the transcription factor IRF8, thus identifying IL-18BP as a novel LXR and IRF8 target gene. In conclusion, LXR activation inhibits IL-18 production through regulation of its transcription and maturation into an active pro-inflammatory cytokine. This novel regulation of IL-18 by LXR could be applied to modulate the severity of IL-18 driven metabolic and inflammatory disorders.

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

Significance To date, the importance of liver X receptors (LXRs) in atherosclerosis development has been gleaned from their pharmacological or genetic manipulation. Here, we show that altering LXRα phosphorylation can shape proatherogenic responses to fat-rich diets, uncovering previously unrecognized mechanisms. Disrupting LXRα phosphorylation in myeloid cells triggers global changes in gene expression in macrophages, including the up-regulation of proliferation-promoting factors, consistent with increased proliferation of lesion-resident cells. This leads to an enhanced atherosclerotic plaque burden and plaques with altered phenotypic features. Notably, novel LXRα-regulated targets revealed by impaired LXRα phosphorylation are markedly distinct from those promoted by LXR ligand activation. Overall, this work reveals LXRα phosphorylation as an important determinant of atherosclerosis development. This could be exploited for the design of novel antiatherosclerotic strategies. 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-S196ALdlr-KO). M-S196ALdlr-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-S196ALdlr-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.

Alternative macrophages in atherosclerosis: not always protective!

Atherosclerosis is a chronic inflammatory disease of the vasculature that is initiated by cholesterol deposition into the arterial wall, which triggers the infiltration of immune and inflammatory cells, including monocytes and macrophages. As atherosclerotic plaques progress, localized hypoxia promotes compensatory angiogenesis from the vasa vasorum. Immature neovessels are prone to leakage, thus destabilizing the plaque and leading to intraplaque hemorrhage. Macrophages with different phenotypes, ranging from classical inflammatory subtypes to alternatively activated antiinflammatory macrophages, have been identified in atherosclerotic lesions. Antiinflammatory hemoglobin-scavenging CD163+ macrophages are present in neovessel- and hemorrhage-rich areas; however, the role of these macrophages in atherogenesis has been unclear. In this issue of the JCI, Guo, Akahori, and colleagues show that CD163+ macrophages promote angiogenesis, vessel permeability, and leucocyte infiltration in human and mouse atherosclerotic lesions through a mechanism involving hemoglobin:haptoglobin/CD163/HIF1&agr;-mediated VEGF induction. This study thus identifies proatherogenic properties of CD163+ macrophages, which previously were thought to be beneficial.

Endospanin-2 enhances skeletal muscle energy metabolism and running endurance capacity

Metabolic stresses such as dietary energy restriction or physical activity exert beneficial metabolic effects. In the liver, endospanin-1 and endospanin-2 cooperatively modulate calorie restriction-mediated (CR-mediated) liver adaptations by controlling growth hormone sensitivity. Since we found CR to induce endospanin protein expression in skeletal muscle, we investigated their role in this tissue. In vivo and in vitro endospanin-2 triggers ERK phosphorylation in skeletal muscle through an autophagy-dependent pathway. Furthermore, endospanin-2, but not endospanin-1, overexpression decreases muscle mitochondrial ROS production, induces fast-to-slow fiber-type switch, increases skeletal muscle glycogen content, and improves glucose homeostasis, ultimately promoting running endurance capacity. In line, endospanin-2-/- mice display higher lipid peroxidation levels, increased mitochondrial ROS production under mitochondrial stress, decreased ERK phosphorylation, and reduced endurance capacity. In conclusion, our results identify endospanin-2 as a potentially novel player in skeletal muscle metabolism, plasticity, and function.

Luciferase Reporter Assays to Assess Liver X Receptor Transcriptional Activity.

Luciferase reporter assays are sensitive and accurate tests that enable the analysis of regulatory sequences, the magnitude of transcriptional activity by transcription factors, and the discovery of gene regulatory elements and small-molecule modulators with high levels of precision. This is made possible through detection of bioluminescence produced by luciferase-coding reporters in a wide range of cellular environments. These assays are routinely used to analyze the activity of transcription factors, including the lipid-activated liver X receptor (LXR), in response to different stimuli as well as for the identification of their ligands. In this chapter we describe in detail the assays performed to investigate LXR activity in a macrophage-like cell line (RAW 267.4). These can be easily adapted to other nuclear receptors and transcription factors.