Chromatin recruitment of activated AMPK drives fasting response genes co-controlled by GR and PPARα

Adaptation to fasting involves both Glucocorticoid Receptor (GRα) and Peroxisome Proliferator-Activated Receptor α (PPARα) activation. Given both receptors can physically interact we investigated the possibility of a genome-wide cross-talk between activated GR and PPARα, using ChIP- and RNA-seq in primary hepatocytes. Our data reveal extensive chromatin co-localization of both factors with cooperative induction of genes controlling lipid/glucose metabolism. Key GR/PPAR co-controlled genes switched from transcriptional antagonism to cooperativity when moving from short to prolonged hepatocyte fasting, a phenomenon coinciding with gene promoter recruitment of phosphorylated AMP-activated protein kinase (AMPK) and blocked by its pharmacological inhibition. In vitro interaction studies support trimeric complex formation between GR, PPARα and phospho-AMPK. Long-term fasting in mice showed enhanced phosphorylation of liver AMPK and GRα Ser211. Phospho-AMPK chromatin recruitment at liver target genes, observed upon prolonged fasting in mice, is dampened by refeeding. Taken together, our results identify phospho-AMPK as a molecular switch able to cooperate with nuclear receptors at the chromatin level and reveal a novel adaptation mechanism to prolonged fasting

The logic of transcriptional regulator recruitment architecture at cis-regulatory modules controlling liver functions.

Control of gene transcription relies on concomitant regulation by multiple transcriptional regulators (TRs). However, how recruitment of a myriad of TRs is orchestrated at cis-regulatory modules (CRMs) to account for coregulation of specific biological pathways is only partially understood. Here, we have used mouse liver CRMs involved in regulatory activities of the hepatic TR, NR1H4 (FXR; farnesoid X receptor), as our model system to tackle this question. Using integrative cistromic, epigenomic, transcriptomic, and interactomic analyses, we reveal a logical organization where trans-regulatory modules (TRMs), which consist of subsets of preferentially and coordinately corecruited TRs, assemble into hierarchical combinations at hepatic CRMs. Different combinations of TRMs add to a core TRM, broadly found across the whole landscape of CRMs, to discriminate promoters from enhancers. These combinations also specify distinct sets of CRM differentially organized along the genome and involved in regulation of either housekeeping/cellular maintenance genes or liver-specific functions. In addition to these TRMs which we define as obligatory, we show that facultative TRMs, such as one comprising core circadian TRs, are further recruited to selective subsets of CRMs to modulate their activities. TRMs transcend TR classification into ubiquitous versus liver-identity factors, as well as TR grouping into functional families. Hence, hierarchical superimpositions of obligatory and facultative TRMs bring about independent transcriptional regulatory inputs defining different sets of CRMs with logical connection to regulation of specific gene sets and biological pathways. Altogether, our study reveals novel principles of concerted transcriptional regulation by multiple TRs at CRMs

Voies de signalisation induites par l'HGF/SF impliquant les MAPKs ERK et JNK dans les cellules épithéliales MDCK

L'hgf/sf est un facteur de croissance d'origine mesenchymateuse, dont le recepteur tyrosine kinase met est present majoritairement sur les cellules d'origine epitheliale. L'implication essentielle du couple hgf/sf-met dans les interactions epithelium/ mesenchyme a ete demontree au cours du developpement normal et pathologique. En accord avec sa caracterisation et son profil d'expression, l'hgf/sf induit la proliferation et la survie, mais aussi des reponses plus specifiques de dispersion et de morphogenese dans les cellules epitheliales. Cependant, les mecanismes conduisant a ces reponses biologiques sont mal connus. Le laboratoire a montre que l'hgf/sf induit l'expression du facteur de transcription ets1 lorsque les cellules epitheliales mdck se dissocient. Ces travaux ont ete suivis de la demonstration que l'hgf/sf, via ras et ets1, induit l'activation de promoteurs de proteases, impliquant des elements de reponse ebs/ap1. Ainsi, cette voie de signalisation conduit a l'expression de proteases facilitant l'action d'essaimage de l'hgf/sf (fafeur et al, 1997). Nous avons tire parti de cette voie de signalisation de l'hgf/sf conduisant a une reponse transcriptionnelle ras-dependante, pour comparer le role fonctionnel des residus tyrosine de met. Les resultats montrent que les residus tyrosine du site de recrutement multisubstrat du recepteur met sont facultatifs pour induire cette reponse transcriptionnelle et la dispersion, alors qu'ils sont indispensables pour le recrutement de gab1, grb2, shc et pi3k, et pour induire la morphogenese. Ces resultats montrent que la signalisation du recepteur met ne passe pas uniquement par le site de recrutement multisubstrat, et contredisent le mode de fonctionnement generalement admis du recepteur met (tulasne et al, 1999).Puis nous avons etudie les cascades de map kinases mises en jeu par l'hgf/sf pour conduire a l'activation de cette reponse transcriptionnelle. Nous avons montre que l'hgf/sf : 1 - induit la kinase erk de maniere rapide et soutenue. Cette kinase est activee par la voie ras-raf-mek et permet la phosphorylation et l'activation de ets1. Cette voie mene a la dispersion et a la morphogenese (paumelle et al, manuscrit en preparation). 2 - induit la kinase jnk de maniere rapide et transitoire, puis la reprime en quelques heures de maniere soutenue. L'activation transitoire de jnk est sans doute impliquee dans l'etalement, qui precede la dispersion des cellules. La repression soutenue de jnk, via l'activation de la voie ras et de la phosphatase mkp2, permet a l'hgf/sf de lever un effet represseur de la voie cdc42-jnk-jun sur la transcription, et permet de rendre compte d'une plus grande efficacite de dispersion de l'hgf/sf a basse densite cellulaire (paumelle et al, 2000). L'ensemble de nos resultats montrent que l'hgf/sf agit par les voies de signalisation ras-erk et cdc42-jnk qui peuvent cooperer ou s'opposer pour induire des reponses de dispersion, de morphogenese et de survie. Nos resultats amenent a prendre en compte, non seulement un aspect qualitatif de la signalisation, mais aussi un aspect quantitatif, puisque l'intensite et la duree de la mise en jeu des map kinases erk et jnk a ete associee a des reponses biologiques distinctes.LILLE1-BU (590092102) / SudocSudocFranceF

Rôle du suppresseur de tumeur p16INK4a dans l'activation des macrophages primaires murins et humains (implication dans le développement de maladies inflammatoires chroniques telles que l'infection parasitaire, l'athérosclérose et l'obésité)

Le locus CDKN2A/B contient le gène codant le suppresseur de tumeur p16INK4a. Des études d association de gènes ont identifiées que le locus CDN2A/B est associé à un risque de développement de maladies inflammatoires, dans lesquelles les macrophages jouent un rôle important. Les macrophages constituent une population cellulaire he térogène dont la forme d activation et les fonctions sont déterminées par les signaux environnants. Les cytokines Th1, tel que l interféron gamma, et les antigènes bactériens, tel que le lipopolysaccharide, induisent une activation classique ou M1 des macrophages alors que les cytokines Th2, telles que les interleukines 4 et 13, induisent une activation alternative ou M2 des macrophages. Cependant, les mécanismes moléculaires impliqués dans l acquisition de ces phénotypes sont encore mal connus. Cette étude montre que l absence d expression de p16INK4a (p16-/-) inhibe la réponse inflammatoire et favorise l activation alternative des macrophages primaires murins in vitro. Afin de déterminer la relevance de ces observations in vivo, la fonction de p16INK4a dans les macrophages a été étudiée chez la souris lors d une infection parasitaire, dans laquelle le rôle des macrophages alternatifs est connu, puis au cours du développement de l athérosclérose dans lequel le rôle des macrophages alternatifs reste à déterminer, en utilisant la transplantation de moelle osseuse. Les souris transplantées avec de la moelle osseuse p16-/- présentent une induction de l expression génique des marqueurs de macrophages alternatifs dans le foie après infection parasitaire, démontrant que p16INK4a influence l activation des macrophages in vivo. Cependant, l absence de p16INK4a dans les cellules hématopoïétiques n influence pas le développement de l athérosclérose dans nos conditions expérimentales. Enfin, l invalidation de l expression de p16INK4a par ARN interférence dans les macrophages primaires humains et la mesure d expression de p16INK4a dans les macrophages isolés du tissu adipeux de patient obèses montrent que l absence de p16INK4a favorise un phénotype alternatif des macrophages proche de celui des macrophages du tissu adipeux. Ces travaux de recherche identifient p16INK4a comme modulateur de l activation des macrophages murins et humains. Mots clés : CDKI-activation alternative-inflammation-infection parasitaire-athérosclérose-obésité.The CDKN2A/B locus, which contains the tumor suppressor gene p16INK4a, is associated with an increased risk of age-related inflammatory diseases, such as cardiovascular disease and type 2 diabetes, in which macrophages play a crucial role. Activation state and functions of macrophages are profoundly affected by environmental cytokines and microbial products. Indeed, Th1 cytokines, such as interferon gamma, and lipopolysaccharide induce a classical activation phenotype whereas Th2 cytokines, such as inteleukins 4 and 13, induce an alternative activation phenotype. However, the molecular mechanisms underlying the acquisition of these phenotypes are not well defined. In this study, we show that p16INK4a-deficiency (p16-/-) skews murine macrophages towards an alternative activation in vitro. The influence of p16INK4a-deficiency on macrophage activation in vivo was investigated using bone marrow transplantation, first in a parasite infectious model in which the role of alternatively activated macrophages is well characterized, then during atherosclerosis development, in which the role of alternatively activated macrophages is undefined. While mice transplanted with p16-/- bone marrow displayed higher hepatic marker expression levels of alternative activation upon parasite infection, no effect was observed on atherosclerosis development in our experimental conditions. Finally, confirming the data obtained in p16-/- macrophages, silencing of p16INK4a with siRNA in human blood-monocyte-derived macrophages also resulted in the induction of alternative activation. In addition, analysis of human adipose tissue macrophages from obese patients, which are typically alternatively activated, showed that p16ink4a expression levels were lower in ATM than in monocyte-derived macrophages of the same subjects. These findings identify a novel role for the tumor suppressor p16INK4a as modulator of murine and human macrophage activation. Keywords: CDKI-alternative activation-inflammation-parasite infectious-atherosclerosis-obesity.LILLE2-BU Santé-Recherche (593502101) / SudocSudocFranceF

Downregulation of the tumour suppressor p16INK4A contributes to the polarisation of human macrophages toward an adipose tissue macrophage (ATM)-like phenotype.

International audienceAIMS/HYPOTHESIS: Human adipose tissue macrophages (ATMs) display an alternatively activated (M2) phenotype, but are still able to produce excessive inflammatory mediators. However, the processes driving this particular ATM phenotype are not understood. Genome-wide association studies associated the CDKN2A locus, encoding the tumour suppressor p16(INK4A), with the development of type 2 diabetes. In the present study, p16(INK4A) levels in human ATMs and the role of p16(INK4A) in acquiring the ATM phenotype were assessed. METHODS: Gene expression of p16 ( INK4A ) in ATMs was analysed and compared with that in monocyte-derived macrophages (MDMs) from obese patients or with macrophages from human atherosclerotic plaques (AMs). Additionally, p16(INK4A) levels were studied during macrophage differentiation and polarisation of monocytes isolated from healthy donors. The role of p16(INK4A) in MDMs from healthy donors was investigated by small interfering (si)RNA-mediated silencing or adenovirus-mediated overproduction of p16(INK4A). RESULTS: Compared with MDMs and AMs, ATMs from obese patients expressed lower levels of p16 ( INK4A ). In vitro, IL-4-induced M2 polarisation resulted in lower p16(INK4A) protein levels after differentiation of monocytes from healthy donors in macrophages. Silencing of p16(INK4A) in MDMs mediated by siRNA increased the expression of M2 marker genes and enhanced the response to lipopolysaccharide (LPS), to give a phenotype resembling that of ATM. By contrast, adenovirus-mediated overproduction of p16(INK4A) in MDMs diminished M2 marker gene expression and the response to LPS. Western blot analysis revealed that p16(INK4A) overproduction inhibits LPS- and palmitate-induced Toll-like receptor 4 (TLR4)-nuclear factor of κ light polypeptide gene enhancer in B cells (NF-κB) signalling. CONCLUSIONS/INTERPRETATION: These results show that p16(INK4A) inhibits the acquisition of the ATM phenotype. The age-related increase in p16(INK4A) level may thus influence normal ATM function and contribute to type 2 diabetes risk

Plasma BCAA changes in Patients with NAFLD are Sex Dependent 1

International audienceContext: Plasma branched chain amino acid (BCAA) concentrations correlate positively with body mass index (BMI), measures of insulin resistance (IR), and severity of nonalcoholic fatty liver disease (NAFLD). Moreover, plasma BCAA concentrations also differ between the sexes, which display different susceptibilities to cardio-metabolic diseases.Objective: Assess whether plasma BCAA concentrations associate with NAFLD severity independently of BMI, IR, and sex.Patients: Patients visiting the obesity clinic of the Antwerp University Hospital were consecutively recruited from 2006 to 2014.Design and setting: A cross-sectional study cohort of 112 obese patients (59 women and 53 men) was divided into 4 groups according to NAFLD severity. Groups were matched for sex, age, BMI, homeostatic model assessment of IR, and hemoglobin A1c.Main outcome measures: Fasting plasma BCAA concentrations were measured by tandem mass spectrometry using the aTRAQ™ method.Results: In the study cohort, a modest positive correlation was observed between plasma BCAA concentrations and NAFLD severity, as well as a strong effect of sex on plasma BCAA levels. Subgroup analysis by sex revealed that while plasma BCAA concentrations increased with severity of NAFLD in women, they tended to decrease in men. Additionally, only women displayed significantly increased plasma BCAAs with increasing fibrosis.Conclusion: Plasma BCAA concentrations display sex-dimorphic changes with increasing severity of NAFLD, independently of BMI, IR, and age. Additionally, plasma BCAA are associated with significant fibrosis in women, but not in men. These results highlight the importance of a careful consideration of sex as a major confounding factor in cross-sectional studies of NAFLD

Control of cell death/survival balance by the MET dependence receptor

International audienceControl of cell death/survival balance is an important feature to maintain tissue homeostasis. Dependence receptors are able to induce either survival or cell death in presence or absence of their ligand, respectively. However, their precise mechanism of action and their physiological importance are still elusive for most of them including the MET receptor. We evidence that pro-apoptotic fragment generated by caspase cleavage of MET localizes to the mitochondria-associated membrane region. This fragment triggers a calcium transfer from endoplasmic reticulum to mitochondria, which is instrumental for the apoptotic action of the receptor. Knock-in mice bearing a mutation of MET caspase cleavage site highlighted that p40MET production is important for FAS-driven hepatocyte apoptosis, and demonstrate that MET acts as a dependence receptor in vivo. Our data shed light on new signaling mechanisms for dependence receptors' control of cell survival/death balance, which may offer new clues for the pathophysiology of epithelial structures