Istvan Szatmari

Non-DNA binding, dominant-negative, human PPARγ mutations cause lipodystrophic insulin resistance

Summary PPARγ is essential for adipogenesis and metabolic homeostasis. We describe mutations in the DNA and ligand binding domains of human PPARγ in lipodystrophic, severe insulin resistance. These receptor mutants lack DNA binding and transcriptional activity but can translocate to the nucleus, interact with PPARγ coactivators and inhibit coexpressed wild-type receptor. Expression of PPARγ target genes is markedly attenuated in mutation-containing versus receptor haploinsufficent primary cells, indicating that such dominant-negative inhibition operates in vivo. Our observations suggest that these mutants restrict wild-type PPARγ action via a non-DNA binding, transcriptional interference mechanism, which may involve sequestration of functionally limiting coactivators.

Peroxisome Proliferator-activated Receptor γ-regulated ABCG2 Expression Confers Cytoprotection to Human Dendritic Cells

ABCG2, a member of the ATP-binding cassette transporters has been identified as a protective pump against endogenous and exogenous toxic agents. ABCG2 was shown to be expressed at high levels in stem cells and variably regulated during cell differentiation. Here we demonstrate that functional ABCG2 is expressed in human monocyte-derived dendritic cells by the activation of a nuclear hormone receptor, PPARγ. We identified and characterized a 150-base pair long conserved enhancer region, containing three functional PPAR response elements (PPARE), upstream of the human ABCG2 gene. We confirmed the binding of the PPARγ·RXR heterodimer to this enhancer region, suggesting that PPARγ directly regulates the transcription of ABCG2. Consistent with these results, elevated expression of ABCG2 mRNA was coupled to enhanced protein production, resulting in increased xenobiotic extrusion capacity via ABCG2 in PPARγ-activated cells. Furthermore PPARγ instructed dendritic cells showed increased Hoechst dye extrusion and resistance to mitoxantrone. Collectively, these results uncovered a mechanism by which up-regulation of functional ABCG2 expression can be achieved via exogenous or endogenous activation of the lipid-activated transcription factor, PPARγ. The increased expression of the promiscuous ABCG2 transporter can significantly modify the xenobiotic and drug resistance of human myeloid dendritic cells.

Nuclear Hormone Receptors Enable Macrophages and Dendritic Cells to Sense Their Lipid Environment and Shape Their Immune Response

A key issue in the immune system is to generate specific cell types, often with opposing activities. The mechanisms of differentiation and subtype specification of immune cells such as macrophages and dendritic cells are critical to understand the regulatory principles and logic of the immune system. In addition to cytokines and pathogens, it is increasingly appreciated that lipid signaling also has a key role in differentiation and subtype specification. In this review we explore how intracellular lipid signaling via a set of transcription factors regulates cellular differentiation, subtype specification, and immune as well as metabolic homeostasis. We introduce macrophages and dendritic cells and then we focus on a group of transcription factors, nuclear receptors, which regulate gene expression upon receiving lipid signals. The receptors we cover are the ones with a recognized physiological function in these cell types and ones which heterodimerize with the retinoid X receptor. These are as follows: the receptor for a metabolite of vitamin A, retinoic acid: retinoic acid receptor (RAR), the vitamin D receptor (VDR), the fatty acid receptor: peroxisome proliferator-activated receptor γ (PPARγ), the oxysterol receptor liver X receptor (LXR), and their obligate heterodimeric partner, the retinoid X receptor (RXR). We discuss how they can get activated and how ligand is generated and eliminated in these cell types. We also explore how activation of a particular target gene contributes to biological functions and how the regulation of individual target genes adds up to the coordination of gene networks. It appears that RXR heterodimeric nuclear receptors provide these cells with a coordinated and interrelated network of transcriptional regulators for interpreting the lipid milieu and the metabolic changes to bring about gene expression changes leading to subtype and functional specification. We also show that these networks are implicated in various immune diseases and are amenable to therapeutic exploitation.

Transcriptional Regulation of Human CYP27 Integrates Retinoid, Peroxisome Proliferator-Activated Receptor, and Liver X Receptor Signaling in Macrophages

ABSTRACT Cholesterol uptake and efflux are key metabolic processes associated with macrophage physiology and atherosclerosis. Peroxisome proliferator-activated receptor gamma (PPARγ) and liver X receptor alpha (LXRα) have been linked to the regulation of these processes. It remains to be identified how activation of these receptors is connected and regulated by endogenous lipid molecules. We identified CYP27, a p450 enzyme, as a link between retinoid, PPARγ, and LXR signaling. We show that the human CYP27 gene is under coupled regulation by retinoids and ligands of PPARs via a PPAR-retinoic acid receptor response element in its promoter. Induction of the enzymes expression results in an increased level of 27-hydroxycholesterol and upregulation of LXR-mediated processes. Upregulated CYP27 activity also leads to LXR-independent elimination of CYP27 metabolites as an alternative means of cholesterol efflux. Moreover, human macrophage-rich atherosclerotic lesions have an increased level of retinoid-, PPARγ-, and LXR-regulated gene expression and also enhanced CYP27 levels. Our findings suggest that nuclear receptor-regulated CYP27 expression is likely to be a key integrator of retinoic acid receptor-PPARγ-LXR signaling, relying on natural ligands and contributing to lipid metabolism in macrophages.

Nuclear receptor signalling in dendritic cells connects lipids, the genome and immune function

Dendritic cells (DCs) are sentinels of the immune system and represent a heterogeneous cell population. The existence of distinct DC subsets is due to their inherent plasticity and to the changing microenvironment modulating their immunological properties. Numerous signalling pathways have impacts on DCs. It appears that besides cytokines/chemokines, lipid mediators also have profound effects on the immunogenicity of DCs. Some of these lipid mediators exert an effect through nuclear hormone receptors. Interestingly, more recent findings suggest that DCs are able to convert precursors to active hormones, ligands for nuclear receptors. Some of these DC‐derived lipids, in particular retinoic acid (RA), have a central function in shaping T‐cell development and effector functions. In this review, we summarize and highlight the function of a set of nuclear receptors (PPARγ, RA receptor, vitamin D receptor and glucocorticoid receptor) in DC biology. Defining the contribution of nuclear hormone receptor signalling in DCs can help one to understand the regulatory logic of lipid signalling and allow the exploitation of their potential for therapeutic intervention in various immunological diseases.

HoxA3 is an apical regulator of haemogenic endothelium

During development, haemogenesis occurs invariably at sites of vasculogenesis. Between embryonic day (E) 9.5 and E10.5 in mice, endothelial cells in the caudal part of the dorsal aorta generate haematopoietic stem cells and are referred to as haemogenic endothelium. The mechanisms by which haematopoiesis is restricted to this domain, and how the morphological transformation from endothelial to haematopoietic is controlled are unknown. We show here that HoxA3, a gene uniquely expressed in the embryonic but not yolk sac vasculature, restrains haematopoietic differentiation of the earliest endothelial progenitors, and induces reversion of the earliest haematopoietic progenitors into CD41-negative endothelial cells. This reversible modulation of endothelial–haematopoietic state is accomplished by targeting key haematopoietic transcription factors for downregulation, including Runx1, Gata1, Gfi1B, Ikaros, and PU.1. Through loss-of-function, and gain-of-function epistasis experiments, and the identification of antipodally regulated targets, we show that among these factors, Runx1 is uniquely able to erase the endothelial program set up by HoxA3. These results suggest both why a frank endothelium does not precede haematopoiesis in the yolk sac, and why haematopoietic stem cell generation requires Runx1 expression only in endothelial cells.

PPARγ, a Lipid‐Activated Transcription Factor as a Regulator of Dendritic Cell Function

Abstract:  In recent years it became apparent that PPARγ, besides being a key component of adipose tissue development and a target of insulin‐sensitizing drugs, also has a role in immune cell differentiation and function. This receptor has been identified by us and others as a conductor of lipid handling in macrophages, and has roles also in inflammation control. Here we review recent advances on the role of this nuclear receptor in another key cell type of myeloid origin, dendritic cells (DCs). DCs are professional antigen‐presenting cells having essential roles in antigen‐uptake processing and presentation and in initiation of various forms of immune responses. It appears that PPARγ is expressed and is active in myeloid DCs and likely to be a regulator of DC function by altering antigen uptake, maturation, activation, migration, cytokine production, and lipid antigen presentation. Thus PPARγ is at the crossroads of lipid metabolism and innate immune response, and by studying its functions one has a unique opportunity to discern how these two seemingly distant fields (lipid metabolism and immune response) are interrelated. It is also possible that this receptor is a relevant target for pharmacological intervention in immune diseases such as chronic inflammation and autoimmune conditions.

Activation of Liver X Receptor Sensitizes Human Dendritic Cells to Inflammatory Stimuli

Dendritic cells (DCs) respond to changes in their lipid environment by altering gene expression and immunophenotype. Some of these alterations are mediated via the nuclear receptor superfamily. However, little is known about the contribution of liver X receptor (LXR) to DC biology. In this study, we present a systematic analysis of LXR, activated by synthetic ligands or naturally occurring oxysterols in developing human monocyte-derived DCs. We found that LXRs are present and can be activated throughout DC differentiation in monocyte- and blood-derived DCs. Administration of LXR-specific natural or synthetic activators induced target gene expression accompanied by increased expression of DC maturation markers, such as CD80 and CD86. In mature DCs, LXR activation augmented the production of inflammatory cytokines IL-12, TNF-α, IL-6, and IL-8 and resulted in an increased capacity to activate CD4+ T cell proliferation upon ligation with TLR4 or TLR3 ligands. These effects appear to be underpinned by prolonged NF-κB signaling. Supporting such an inflammatory role, we found that LXR positive DCs are present in reactive lymph nodes in vivo. We propose that activation of LXR represents a novel lipid-signaling paradigm that alters the inflammatory response of human DCs.

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

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.

Chronic Obstructive Pulmonary Disease-Specific Gene Expression Signatures of Alveolar Macrophages as well as Peripheral Blood Monocytes Overlap and Correlate with Lung Function

Background: Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease characterized by progressive airflow limitation and significant extrapulmonary (systemic) effects that lead to co-morbid conditions, though the pathomechanism of COPD is largely undetermined. Alveolar macrophages (AM) derived from peripheral monocytes (MO) appear to play a key role in initiating and/or sustaining disease progression. Objectives: To identify disease- and cell type-specific gene expression profiles and potential overlaps in those in order to diagnose COPD, characterize its progression and determine the effect of drug treatment. Method: Global gene expression analysis was used for primary screening in order to obtain expression signatures of AMs and circulating MOs of COPD patients and healthy controls. The results of microarray analyses of AMs (20 controls and 26 COPD patients) and MOs (16 controls and 22 COPD patients) were confirmed and validated by real-time quantitative polymerase chain reaction. Results: We have identified gene sets specifically associated with COPD in AMs and MOs. There were overlapping genes between the two cell types. Our data also show that COPD-specific gene expression signatures in AMs and MOs correlate with percent of predicted FEV1. Conclusion: Disease-specific and overlapping gene expression signatures can be defined in lung-derived macrophages and also in circulating monocytes. Some of the validated expression changes in both cell types correlate with lung function and therefore could serve as biomarkers of disease progression.