Die Rolle von Signal Transducer and Activator of Transcription 5 (Stat5) und dem Glukokortikoidrezeptor (GR) in der Hepatozytenfunktion

Signal transducer and activator of transcription 5 (Stat5) represents a cytokine- and growth factor-dependent transcription factor regulating important biological processes such as postnatal body growth, erythropoiesis and the expression of milk protein genes. The glucocorticoid receptor (GR) binds Stat5 in a cofactor fashion and was reported to modulate Stat5 target gene specificity. In this study we aimed to elucidate the role of Stat5 in pivotal hepatocyte processes such as the regulation of postnatal body growth, fatty acid metabolism and liver fibrogenesis, taking into account its propensity to interact with the GR. The major findings of this study are summarized in the following: (i) Hepatic loss of Stat5, the GR or both resulted in comparable defects in postnatal body growth. This was accompanied by quantitatively similar changes in transcription profiles, affecting predominantly growth-related Stat5 target genes (e.g. IGF-1 and Socs2). These observation suggested the existence of a linear Stat5-GR signal transduction pathway essential for regulating postnatal body growth. Using protein interaction studies the Stat5 aminoterminus was identified as docking platform for the GR. Luciferase reporter assays confirmed the Stat5-GR-dependent synergism in vitro exemplarily for transactivation of the Socs2 promoter. (ii) Hepatic loss of Stat5 in mice leads to the pathological accumulation of lipids in hepatocytes (steatosis). This coincides with perturbations of hepatic lipid metabolism such as hepatic insulin resistance and induction of de novo lipogenesis mediated by lipogenic master regulators PPARgamma and SREBP-1c. Compound loss of Stat5 and GR in hepatocytes severely aggravates this phenotype, leading to late-stage hepatopathologies such as severe steatosis and liver fibrosis already in young animals. At older ages, these mutants additionally exhibit depletions of peripheral fat stores (lipodystrophy) leading to an increased lipid flux into hepatic tissues and thus exacerbating disease. The two-step pathogenesis observed in double knockout animals closely mimics the onset and progression of human nonalcoholic fatty liver disease (NAFLD). (iii) Hepatic loss of Stat5 in a murine model for proinflammatory liver carcinogenesis (MDR2- model) leads to extrahepatic cholestasis (bile duct obstruction) causing secondary liver fibrosis at young ages. The pathogenesis in these animals comprises major hallmarks observed in fibrotic states such as the activation of hepatic stellate cells and remodelling of the extracellular matrix. Ablation of Stat5 in hepatocytes leads to a direct loss of Stat5-dependent basolateral bile acid transporter transcription in hepatocytes and thus coincides with perturbations in hepatic bile acid circulation and recycling. This may eventually cause hepatocyte damage and, finally, liver fibrosis and cirrhosis. Taken together, two published first-author manuscripts and one first-author manuscript in preparation resulted from this (see section 8).Signal transducer and activator of transcription 5 (Stat5) stellt einen wichtigen, Zytokin- und Wachstumsfaktor-abhängigen Transkriptionsfaktor dar mit essentiellen Funktionen z.B. bei der Regulation des postnatalen Größenwachstums, der Erythropoese sowie der Expression von Milchproteinen während der Laktation. Der Glukocorticoid Rezeptor (GR) bindet als akzessorischer Transkriptionskofaktor an Stat5 und kann dessen Zielgen Spezifität unter bestimmten Umständen modifizieren. In der vorliegenden Arbeit sollte der Einfluss von Stat5 auf Hepatozyten-vermittelte Prozesse wie postnatales Größenwachstum, Lipidstoffwechsel sowie Leberfibrogenese untersucht werden, unter Berücksichtigung der Proteinwechselwirkung mit dem GR. Die wichtigsten Resultate der vorliegenden Studie sind im Folgenden zusammengefasst: (i) Die hepatische Deletion von entweder Stat5, dem GR oder beiden führt zu vergleichbaren Störungen des postnatalen Größenwachstums. Gleichzeitig wurden quantitativ ähnliche Änderungen des Transkriptionsprofils beobachtet, welche besonders solche Stat5-abhängige Gene betraf welche für das postnatal Größenwachsum essentiell sind (z.B. IGF-1 oder Socs2). Diese Beobachtungen ließen auf die Existenz eines linearen Stat5-GR Signaltransduktionsweges schließen, welcher wichtig für die Regulation des Größenwachstums nach der Geburt ist. Der Aminoterminus von Stat5 wurde als Stat5-GR Interaktiondomäne identifiziert. Luciferase Reporter assays bestätigten eine synergistische Transaktivierung des Socs2 Promoters durch Stat5 und den GR in vitro. (ii) Die hepatische Deletion von Stat5 führt zur milden Akkumulation von Fetten in Hepatozyten (Steatose). Dieses wird begleitet von Störungen des hepatischen Lipidstoffwechsels wie z.B. dem Auftreten von hepatischer Insulinresistenz sowie der Induktion der de novo Lipogenese, vermittelt durch die essentiellen prolipogenen Transkriptionsfaktoren PPARgamma und SREBP-1c. Die simultane Deletion von Stat5 und dem GR führt zu einer dramatischen Verschärfung der Steatose sowie zum frühen Auftreten von Leberfibrosen welche normalerweise erst im Endstadium von Steatosen beobachtet werden können. Mit zunehmendem Alter kommt es zu einer Abnahme peripherer Fettspeicher (Lipodystrophie). Dies Zusammenfassung resultiert in einem verstärkten Zustrom von Fetten in die Leber und somit einer Verschlimmerung der bestehenden Steatose. Stat5/GR Doppelmutanten zeigen somit eine zweistufige Pathogenese, welche an den humanen NAFLD Krankheitsverlauf erinnert. (iii) Die hepatische Deletion von Stat5 in einem murinen Modell für inflammatorische Leberkarzinogenese (MDR2- Modell) führt zu extrahepatischer Cholestase (Verschluss der Gallengänge). Diese führt schon in jungen Mäusen zu einer schweren Leberfibrose und Zirrhose. Die Pathogenese in diesen Tieren wird begleitet durch das Auftreten Fibrose-assoziierter Parameter wie z.B. der Aktivierung von hepatischen Sternzellen sowie der Akkumulation von extrazellulären Matrixproteinen. Mechanistisch führt die hepatische Deletion von Stat5 zu einem direkten, transkriptionellen Verlust wichtiger basolateraler Gallentransporter. Dies wiederum resultiert in indirekten Störungen der hepatischen Gallenzirkulation, welche eine konstante Schädigung von Hepatozyten und schließlich Leberfibrose und Leberzirrhose nach sich zieht

Lsd1 ablation triggers metabolic reprogramming of brown adipose tissue

Previous work indicated that lysine-specific demethylase 1 (Lsd1) can positively regulate the oxidative and thermogenic capacities of white and beige adipocytes. Here we investigate the role of Lsd1 in brown adipose tissue (BAT) and find that BAT- selective Lsd1 ablation induces a shift from oxidative to glycolytic metabolism. This shift is associated with downregulation of BAT-specific and upregulation of white adipose tissue (WAT)-selective gene expression. This results in the accumulation of di- and triacylglycerides and culminates in a profound whitening of BAT in aged Lsd1- deficient mice. Further studies show that Lsd1 maintains BAT properties via a dual role. It activates BAT-selective gene expression in concert with the transcription factor Nrf1 and represses WAT-selective genes through recruitment of the CoREST complex. In conclusion, our data uncover Lsd1 as a key regulator of gene expression and metabolic function in BAT

A MAFG-lncRNA axis links systemic nutrient abundance to hepatic glucose metabolism

Obesity and type 2 diabetes mellitus are global emergencies and long noncoding RNAs (lncRNAs) are regulatory transcripts with elusive functions in metabolism. Here we show that a high fraction of lncRNAs, but not protein-coding mRNAs, are repressed during diet-induced obesity (DIO) and refeeding, whilst nutrient deprivation induced lncRNAs in mouse liver. Similarly, lncRNAs are lost in diabetic humans. LncRNA promoter analyses, global cistrome and gain-of-function analyses confirm that increased MAFG signaling during DIO curbs lncRNA expression. Silencing Mafg in mouse hepatocytes and obese mice elicits a fasting-like gene expression profile, improves glucose metabolism, de-represses lncRNAs and impairs mammalian target of rapamycin (mTOR) activation. We find that obesity-repressed LincIRS2 is controlled by MAFG and observe that genetic and RNAi-mediated LincIRS2 loss causes elevated blood glucose, insulin resistance and aberrant glucose output in lean mice. Taken together, we identify a MAFG-lncRNA axis controlling hepatic glucose metabolism in health and metabolic disease

A MAFG-lncRNA axis links systemic nutrient abundance to hepatic glucose metabolism

Obesity and type 2 diabetes mellitus are global emergencies and long noncoding RNAs (lncRNAs) are regulatory transcripts with elusive functions in metabolism. Here we show that a high fraction of lncRNAs, but not protein-coding mRNAs, are repressed during diet-induced obesity (DIO) and refeeding, whilst nutrient deprivation induced lncRNAs in mouse liver. Similarly, lncRNAs are lost in diabetic humans. LncRNA promoter analyses, global cistrome and gain-of-function analyses confirm that increased MAFG signaling during DIO curbs lncRNA expression. Silencing Mafg in mouse hepatocytes and obese mice elicits a fasting-like gene expression profile, improves glucose metabolism, de-represses lncRNAs and impairs mammalian target of rapamycin (mTOR) activation. We find that obesity-repressed LincIRS2 is controlled by MAFG and observe that genetic and RNAi-mediated LincIRS2 loss causes elevated blood glucose, insulin resistance and aberrant glucose output in lean mice. Taken together, we identify a MAFG-lncRNA axis controlling hepatic glucose metabolism in health and metabolic disease

Regulation of metabolism by long, non-coding RNAs

Our understanding of genomic regulation was revolutionized by the discovery that the genome is pervasively transcribed, giving rise to thousands of mostly uncharacterized non-coding ribonucleic acids (ncRNAs). Long, ncRNAs (IncRNAs) have thus emerged as a novel class of functional RNAs that impinge on gene regulation by a broad spectrum of mechanisms such as the recruitment of epigenetic modifier proteins, control of mRNA decay and DNA sequestration of transcription factors. We review those IncRNAs that are implicated in differentiation and homeostasis of metabolic tissues and present novel concepts on how IncRNAs might act on energy and glucose homeostasis. Finally, the control of circadian rhythm by IncRNAs is an emerging principles of IncRNA-mediated gene regulation

s·nr: a visual analytics framework for contextual analyses of private and public RNA-seq data

Abstract Background Next-Generation Sequencing (NGS) has been widely accepted as an essential tool in molecular biology. Reduced costs and automated analysis pipelines make the use of NGS data feasible even for small labs, yet the methods for interpreting the data are not sophisticated enough to account for the amount of information. Results We propose s·nr, a Visual Analytics tool that provides simple yet powerful visual interfaces for displaying and querying NGS data. It allows researchers to explore their own data in the context of experimental data deposited in public repositories, as well as to extract specific data sets with similar gene expression signatures. We tested s·nr on 1543 RNA-Seq based mouse differential expression profiles derived from the public ArrayExpress platform. We provide the repository of processed data with this paper. Conclusion s·nr, easily deployable utilizing its containerized implementation, empowers researchers to analyze and relate their own RNA-Seq as well as to provide interactive and contextual crosstalk with data from public repositories. This allows users to deduce novel and unbiased hypotheses about the underlying molecular processes. Demo Login demo/demo: snr.sf.mpg.de (Tested with Google Chrome

Rapid Generation of Long Noncoding RNA Knockout Mice Using CRISPR/Cas9 Technology

In recent years, long noncoding RNAs (lncRNAs) have emerged as multifaceted regulators of gene expression, controlling key developmental and disease pathogenesis processes. However, due to the paucity of lncRNA loss-of-function mouse models, key questions regarding the involvement of lncRNAs in organism homeostasis and (patho)-physiology remain difficult to address experimentally in vivo. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 platform provides a powerful genome-editing tool and has been successfully applied across model organisms to facilitate targeted genetic mutations, including Caenorhabditis elegans, Drosophila melanogaster, Danio rerio and Mus musculus. However, just a few lncRNA-deficient mouse lines have been created using CRISPR/Cas9-mediated genome engineering, presumably due to the need for lncRNA-specific gene targeting strategies considering the absence of open-reading frames in these loci. Here, we describe a step-wise procedure for the generation and validation of lncRNA loss-of-function mouse models using CRISPR/Cas9-mediated genome engineering. In a proof-of-principle approach, we generated mice deficient for the liver-enriched lncRNA Gm15441, which we found downregulated during development of metabolic disease and induced during the feeding/fasting transition. Further, we discuss guidelines for the selection of lncRNA targets and provide protocols for in vitro single guide RNA (sgRNA) validation, assessment of in vivo gene-targeting efficiency and knockout confirmation. The procedure from target selection to validation of lncRNA knockout mouse lines can be completed in 18–20 weeks, of which <10 days hands-on working time is required

Nanopore sequencing unveils the complexity of the cold-activated murine brown adipose tissue transcriptome

Summary: Alternative transcription increases transcriptome complexity by expression of multiple transcripts per gene. Annotation and quantification of transcripts using short-read sequencing is non-trivial. Long-read sequencing aims at overcoming these problems by sequencing full-length transcripts. Activation of brown adipose tissue (BAT) thermogenesis involves major transcriptomic remodeling and positively affects metabolism via increased energy expenditure. We benchmark Oxford Nanopore Technology (ONT) long-read sequencing protocols to Illumina short-read sequencing assessing alignment characteristics, gene and transcript detection and quantification, differential gene and transcript expression, transcriptome reannotation, and differential transcript usage (DTU). We find ONT sequencing is superior to Illumina for transcriptome reassembly, reducing the risk of false-positive events by unambiguously mapping reads to transcripts. We identified novel isoforms of genes undergoing DTU in cold-activated BAT including Cars2, Adtrp, Acsl5, Scp2, Aldoa, and Pde4d, validated by real-time PCR. The reannotated murine BAT transcriptome established here provides a framework for future investigations into the regulation of BAT

Hepatic growth hormone and glucocorticoid receptor signaling in body growth, steatosis and metabolic liver cancer development

Growth hormone (GH) and glucocorticoids (GCs) are involved in the control of processes that are essential for the maintenance of vital body functions including energy supply and growth control. GH and GCs have been well characterized to regulate systemic energy homeostasis, particular during certain conditions of physical stress. However, dysfunctional signaling in both pathways is linked to various metabolic disorders associated with aberrant carbohydrate and lipid metabolism. In liver, GH-dependent activation of the transcription factor signal transducer and activator of transcription (STAT) 5 controls a variety of physiologic functions within hepatocytes. Similarly, GCs, through activation of the glucocorticoid receptor (GR), influence many important liver functions such as gluconeogenesis. Studies in hepatic Stat5 or GR knockout mice have revealed that they similarly control liver function on their target gene level and indeed, the GR functions often as a cofactor of STAT5 for CH-induced genes. Gene sets, which require physical STAT5-GR interaction, include those controlling body growth and maturation. More recently, it has become evident that impairment of GH-STAT5 signaling in different experimental models correlates with metabolic liver disease, ranging from hepatic steatosis to hepatocellular carcinoma (HCC). While GH-activated STAT5 has a protective role in chronic liver disease, experimental disruption of GC-GR signaling rather seems to ameliorate metabolic disorders under metabolic challenge. In this review, we focus on the current knowledge about hepatic CH-STAT5 and GC-GR signaling in body growth, metabolism, and protection from fatty liver disease and HCC development. (c) 2012 Elsevier Ireland Ltd. All rights reserved

Senescence-Associated Metabolomic Phenotype in Primary and iPSC-Derived Mesenchymal Stromal Cells

Long-term culture of primary cells is characterized by functional and secretory changes, which ultimately result in replicative senescence. It is largely unclear how the metabolome of cells changes during replicative senescence and if such changes are consistent across different cell types. We have directly compared culture expansion of primary mesenchymal stromal cells (MSCs) and induced pluripotent stem cell-derived MSCs (iMSCs) until they reached growth arrest. Both cell types acquired similar changes in morphology, in vitro differentiation potential, senescence-associated beta-galactosidase, and DNA methylation. Furthermore, MSCs and iMSCs revealed overlapping gene expression changes, particularly in functional categories related to metabolic processes. We subsequently compared the metabolomes of MSCs and iMSCs and observed overlapping senescence-associated changes in both cell types, including downregulation of nicotinamide ribonucleotide and upregulation of orotic acid. Taken together, replicative senescence is associated with a highly reproducible senescence-associated metabolomics phenotype, which may be used to monitor the state of cellular aging