Supplementary Materials and Table 1 from Early Growth Response 1 Acts as a Tumor Suppressor In vivo and In vitro via Regulation of p53

Supplementary Materials and Table 1 from Early Growth Response 1 Acts as a Tumor Suppressor <i>In vivo</i> and <i>In vitro</i> via Regulation of p53

Obesity-related insulin resistance represents the core component of the metabolic syndrome, promoting glucose intolerance, pancreatic beta cell failure and type 2 diabetes. Efficient and safe insulin sensitization and glucose control remain critical therapeutic aims to prevent diabetic late complications Here, we identify transforming growth factor beta-like stimulated clone (TSC) 22 D4 as a molecular determinant of insulin signalling and glucose handling. Hepatic TSC22D4 inhibition both prevents and reverses hyperglycaemia, glucose intolerance and insulin resistance in diabetes mouse models. TSC22D4 exerts its effects on systemic glucose homeostasis—at least in part—through the direct transcriptional regulation of the small secretory protein lipocalin 13 (LCN13). Human diabetic patients display elevated hepatic TSC22D4 expression, which correlates with decreased insulin sensitivity, hyperglycaemia and LCN13 serum levels. Our results establish TSC22D4 as a checkpoint in systemic glucose metabolism in both mice and humans, and propose TSC22D4 inhibition as an insulin sensitizing option in diabetes therapy.

Peroxisome proliferator-activated receptors (PPARs) play major roles in the regulation of hepatic lipid metabolism through the control of numerous genes involved in processes such as lipid uptake and fatty acid oxidation. Here we identify hypoxia-inducible lipid droplet-associated (Hilpda/Hig2) as a novel PPAR target gene and demonstrate its involvement in hepatic lipid metabolism. Microarray analysis revealed that Hilpda is one of the most highly induced genes by the PPARα agonist Wy14643 in mouse precision cut liver slices. Induction of Hilpda mRNA by Wy14643 was confirmed in mouse and human hepatocytes. Oral dosing with Wy14643 similarly induced Hilpda mRNA levels in livers of wild-type mice but not Ppara(-/-) mice. Transactivation studies and chromatin immunoprecipitation showed that Hilpda is a direct PPARα target gene via a conserved PPAR response element located 1200 base pairs upstream of the transcription start site. Hepatic overexpression of HILPDA in mice via adeno-associated virus led to a 4-fold increase in liver triglyceride storage, without any changes in key genes involved in de novo lipogenesis, β-oxidation, or lipolysis. Moreover, intracellular lipase activity was not affected by HILPDA overexpression. Strikingly, HILPDA overexpression significantly impaired hepatic triglyceride secretion. Taken together, our data uncover HILPDA as a novel PPAR target that raises hepatic triglyceride storage via regulation of triglyceride secretion.

In mammals, glucocorticoids (GCs) and their intracellular receptor, the glucocorticoid receptor (GR), represent critical checkpoints in the endocrine control of energy homeostasis. Indeed, aberrant GC action is linked to severe metabolic stress conditions as seen in Cushing's syndrome, GC therapy and certain components of the Metabolic Syndrome, including obesity and insulin resistance. Here, we identify the hepatic induction of the mammalian conserved microRNA (miR)-379/410 genomic cluster as a key component of GC/GR-driven metabolic dysfunction. Particularly, miR-379 was up-regulated in mouse models of hyperglucocorticoidemia and obesity as well as human liver in a GC/GR-dependent manner. Hepatocyte-specific silencing of miR-379 substantially reduced circulating very-low-density lipoprotein (VLDL)-associated triglyceride (TG) levels in healthy mice and normalized aberrant lipid profiles in metabolically challenged animals, mediated through miR-379 effects on key receptors in hepatic TG re-uptake. As hepatic miR-379 levels were also correlated with GC and TG levels in human obese patients, the identification of a GC/GR-controlled miRNA cluster not only defines a novel layer of hormone-dependent metabolic control but also paves the way to alternative miRNA-based therapeutic approaches in metabolic dysfunction.

doi:10.1182/blood-2007-11-121699Prepublished online May 9, 2008;2008 112: 264-276€€€€Yu, Norbert Gretz, Mark Christian, Roger White, Malcolm G. Parker and Stephan HerzigVegiopoulos, Johannes Muggenburg, Dirk Sombroek, Thomas G. Hofmann, Rainer Zawatzky, XiaoleiInka Zschiedrich, Ulrike Hardeland, Anja Krones-Herzig, Mauricio Berriel Diaz, Alexandros€

FOXO3 is a transcription factor involved in the regulation of multiple physiological processes including cell cycle arrest, apoptosis, oxidative stress-response and energy metabolism. Although much is known about its post-translational modification, the transcriptional regulation of FOXO3, as well as the cross-talk between transcription and post-translational events, is still poorly understood. In the present study, we show that FOXO3 is an immediate early glucocorticoid receptor (GR) target, whose transcription is even further enhanced by conditions that mimic metabolic stress. Induction of FOXO3 transcription by GR-binding steroids was reversed by concomitant treatment with the GR antagonist RU-486, but further enhanced by stimuli that activate the AMP-activated protein kinase (AMPK). Analysis of genomic DNA and chromatin immunoprecipitation, as well as luciferase reporter assays, revealed two functional glucocorticoid responsive elements within the FOXO3 promoter. Furthermore, we provide functional evidence for a phosphorylation switch that explains how glucocorticoids induce transcriptional activation of the gene but subsequently inactivate the corresponding protein by site-specific phosphorylation. Only when AMPK is stimulated, pre-existing FOXO3 becomes reverted toward an active form. Energy deprived conditions thus activate FOXO3 on two different levels, namely transcriptional and post-translational. In that way, FOXO3 acts as a metabolic stress sensor that coordinates expression of LKB1, the master upstream kinase involved in metabolic sensing, depending on the energy status of the cell. Additionally, we show that FOXO3 binds and activates its own promoter via a positive autoregulatory feedback loop. In conclusion, our data explain how catabolic glucocorticoid hormones and high intracellular AMP levels cooperate in inducing FOXO3 transcription and in activating the corresponding protein.

Systemic bile acid (BA) homeostasis is a critical determinant of dietary fat digestion, enterohepatic function, and postprandial thermogenesis. However, major checkpoints for the dynamics and the molecular regulation of BA homeostasis remain unknown. Here we show that hypothalamic-pituitary-adrenal (HPA) axis impairment in humans and liver-specific deficiency of the glucocorticoid receptor (GR) in mice disrupts the normal changes in systemic BA distribution during the fasted-to-fed transition. Fasted mice with hepatocyte-specific GR knockdown had smaller gallbladder BA content and were more susceptible to developing cholesterol gallstones when fed a cholesterol-rich diet. Hepatic GR deficiency impaired liver BA uptake/transport via lower expression of the major hepatocyte basolateral BA transporter, Na+-taurocholate transport protein (Ntcp/Slc10a1), which affected dietary fat absorption and brown adipose tissue activation. Our results demonstrate a role of the HPA axis in the endocrine regulation of BA homeostasis through the liver GR control of enterohepatic BA recycling.

After binding to their cognate DNA-binding partner, transcriptional co-factors exert their function through the recruitment of enzymatic, chromatin-modifying activities. In turn, the assembly of co-factor-associated multi-protein complexes efficiently impacts target gene expression. Recent advances have established transcriptional co-factor complexes as a critical regulatory level in energy homeostasis and aberrant co-factor activity has been linked to the pathogenesis of severe metabolic disorders including obesity, type 2 diabetes and other components of the Metabolic Syndrome. The liver represents the key peripheral organ for the maintenance of systemic energy homeostasis, and aberrations in hepatic glucose and lipid metabolism have been causally linked to the manifestation of disorders associated with the Metabolic Syndrome. Therefore, this review focuses on the role of distinct classes of transcriptional co-factors in hepatic glucose and lipid homeostasis, emphasizing pathway-specific functions of these co-factors under physiological and pathophysiological conditions.

OBJECTIVE In mammals, proper storage and distribution of lipids in and between tissues is essential for the maintenance of energy homeostasis. In contrast, aberrantly high levels of triglycerides in the blood (“hypertriglyceridemia”) represent a hallmark of the metabolic syndrome and type 2 diabetes. As hypertriglyceridemia has been identified as an important risk factor for cardiovascular complications, in this study we aimed to identify molecular mechanisms in aberrant triglyceride elevation under these conditions. RESEARCH DESIGN AND METHODS To determine the importance of hepatic lipid handling for systemic dyslipidemia, we profiled the expression patterns of various hepatic lipid transporters and receptors under healthy and type 2 diabetic conditions. A differentially expressed lipoprotein receptor was functionally characterized by generating acute, liver-specific loss- and gain-of-function animal models. RESULTS We show that the hepatic expression of lipid transporter lipolysis-stimulated lipoprotein receptor (LSR) is specifically impaired in mouse models of obesity and type 2 diabetes and can be restored by leptin replacement. Experimental imitation of this pathophysiological situation by liver-specific knockdown of LSR promotes hypertriglyceridemia and elevated apolipoprotein (Apo)B and E serum levels in lean wild-type and ApoE knockout mice. In contrast, genetic restoration of LSR expression in obese animals to wild-type levels improves serum triglyceride levels and serum profiles in these mice. CONCLUSIONS The dysregulation of hepatic LSR under obese and diabetic conditions may provide a molecular rationale for systemic dyslipidemia in type 2 diabetes and the metabolic syndrome and represent a novel target for alternative treatment strategies in these patients.

AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA 1216 The excessive loss of body energy stores represents a hallmark of several tumor entities such as pancreatic, colon, or lung cancer. Inflammatory reactions are intimately linked to wasting hypermetabolism under these conditions. However, the molecular basis for cross-talk between metabolic and inflammatory pathways remains largely enigmatic. Therefore, this study aimed at the identification of transcriptional checkpoints for aberrant energy homeostasis during cancer cachexia. Here we show that the expression of nuclear receptor co-factor RIP140 was induced in livers of tumor-bearing, cachectic mice, correlating with a previously unrecognized invasion of macrophages into cachectic liver tissue, and a loss of circulating lipid energy substrates and body weight. Remarkably, liver-specific disruption of RIP140 expression in tumor-bearing animals prevented these cachectic phenotypes -at least in part- through the inhibition of nuclear factor (NF) kappaB-dependent pro-inflammatory genes, identified here as direct RIP140 targets. By triggering inflammatory dyslipidemia via hepatic macrophage recruitment and pro-inflammatory gene expression, the induction of RIP140 in the liver provides a molecular rationale for inter-tissue communication and wasting metabolism in cancer cachexia. Inhibition of hepatic RIP140 transcriptional activity might, thereby, provide an attractive adjunct scheme in the treatment of this condition.

In mammals, triglycerides (TG) represent the most concentrated form of energy. Aberrant TG storage and availability are intimately linked to the negative energy balance under severe clinical conditions, such as starvation, sepsis, or cancer cachexia. Despite its crucial role for energy homeostasis, molecular key determinants of TG metabolism remain enigmatic. Here we show that the expression of nuclear receptor cofactor receptor interacting protein (RIP) 140 was induced in livers of starved, septic, and tumor-bearing mice. Liver-specific knockdown of RIP140 led to increased hepatic TG release and alleviated hepatic steatosis in tumor-bearing, cachectic animals. Indeed, hepatic RIP140 was found to control the expression of lipid-metabolizing genes in liver. Conclusion: By preventing the mobilization of hepatic TG stores, the induction of RIP140 in liver provides a molecular rationale for hepatic steatosis in starvation, sepsis, or cancer cachexia. Inhibition of hepatic RIP140 transcriptional activity might, thereby, provide an attractive adjunct scheme in the treatment of these conditions.

Inflammatory responses represent a hallmark of numerous pathologies including sepsis, bacterial infection, insulin resistance, and malign obesity. Here we describe an unexpected coactivator function for the nuclear receptor interacting protein 140 (RIP140) for nuclear factor kappaB (NFkappaB), a master transcriptional regulator of inflammation in multiple tissues. Previous work has shown that RIP140 suppresses the expression of metabolic gene networks, but we have found that genetic as well as acute deficiency of RIP140 leads to the inhibition of the proinflammatory program in macrophages. The ability of RIP140 to function as a coactivator for cytokine gene promoter activity relies on direct protein-protein interactions with the NFkappaB subunit RelA and histone acetylase cAMP-responsive element binding protein (CREB)-binding protein (CBP). RIP140-dependent control of proinflammatory gene expression via RelA/CBP may, therefore, represent a molecular rational for the cellular integration of metabolic and inflammatory pathways.

Aberrant accumulation of lipids in the liver ("fatty liver" or hepatic steatosis) represents a hallmark of the metabolic syndrome and is tightly associated with obesity, type II diabetes, starvation, or glucocorticoid (GC) therapy. While fatty liver has been connected with numerous abnormalities of liver function, the molecular mechanisms of fatty liver development remain largely enigmatic. Here we show that liver-specific disruption of glucocorticoid receptor (GR) action improves the steatotic phenotype in fatty liver mouse models and leads to the induction of transcriptional repressor hairy enhancer of split 1 (Hes1) gene expression. The GR directly interferes with Hes1 promoter activity, triggering the recruitment of histone deacetylase (HDAC) activities to the Hes1 gene. Genetic restoration of hepatic Hes1 levels in steatotic animals normalizes hepatic triglyceride (TG) levels. As glucocorticoid action is increased during starvation, myotonic dystrophy, and Cushing's syndrome, the inhibition of Hes1 through the GR might explain the fatty liver phenotype in these subjects.

Hypertriglyceridemia forms a hallmark of the metabolic syndrome and represents an independent risk factor for the development of coronary heart disease. Plasma lipid levels are controlled through a complex interplay between different tissues, the molecular basis of which remains largely unknown. Given the importance of transcriptional regulation in metabolic control, we investigated the role of nuclear receptor co-factor receptor interacting protein 140 (RIP140) in the liver, a key organ in energy homeostasis. Here we show that an acute, liver-specific depletion of RIP140 caused dramatically increased blood triglyceride levels. In contrast, hepatic triglyceride content in these animals was decreased in the absence of RIP140, suggesting that the observed hypertriglyceridemia was not the consequence of increased hepatic VLDL production. Interestingly, RIP140 knockdown in the liver reduced expression of apolipoprotein A5 (ApoA5), the serum levels of which have been shown to be inversely correlated with circulating blood triglycerides. Indeed, RIP140 knockdown in a murine hepatocyte cell line decreased ApoA5 promoter activity in transient transfection studies. Conversely, RIP140 over-expression induced ApoA5 expression in these cells, strengthening the notion that RIP140 acts as a direct transcriptional co-activator on the ApoA5 promoter. ApoA5 has been shown to control blood lipid levels, at least in part, by accelerating the hydrolysis of triglycerides by lipoprotein lipase (LPL) in adipose and muscle tissue. By influencing hepatic ApoA5 release, transcriptional control via RIP140 in the liver might, therefore, provide a novel mechanism of metabolic tissue crosstalk and the development of dyslipidemia associated with the metabolic syndrome.

Glucose homeostasis in mammals is maintained, in large parts, through the hypothalamic-pituitary-hormonal axis (HPA) resulting in the regulation of adrenal glucocorticoid production. Glucocorticoids are critical mediators of liver-specific glucose metabolism the molecular mechanisms of which remain largely unclear and, consequently, represented the focus of this study.

BACKGROUND:The progression of a metastatic disease is a multistep process involving various genetic events. High-throughput technologies such as microarrays offer the unique opportunity of screening the entire gene expression profile of malignant cells. Defining consistent changes in the gene expression pattern of cancer models may lead to detection of genes essential for tumor progression and may ultimately identify new targets for therapy.MATERIALS AND METHODS:Gene expression was determined in multiple progression models of human prostate cancer cell lines derived from tumors after 1 to 5 passages as orthotopic xenografts in nude mice. Samples were analyzed using Affymetrix microarrays determining expression of 7,129 genes. Changes in gene expression were calculated referring to normal epithelial cells and confirmed by quantitative PCR. Data were validated by comparison to expression profiles of tumors induced by these cell lines and were also compared to expression data from clinical samples.RESULTS:Two hundred and fourteen genes were regulated in the same direction in all cell lines compared to RNA of normal epithelial cells. Seventy-nine % of the 214 gene consensus class of the cell culture models were also found to be differentially-regulated in orthotopic tumors induced in mice and 83% also in clinical samples.CONCLUSION:The expression pattern of these genes is consistent with their potential role in cancer progression. The results indicate the relevance of these xenograft models for expression analysis in the progression and development of metastatic cancer.

Together with impaired glucose uptake in skeletal muscle, elevated hepatic gluconeogenesis is largely responsible for the hyperglycemic phenotype in type II diabetic patients. Intracellular glucocorticoid and cyclic adenosine monophosphate (cAMP)/protein kinase A-dependent signaling pathways contribute to aberrant hepatic glucose production through the induction of gluconeogenic enzyme gene expression. Here we show that the coactivator-associated arginine methyltransferase 1 (CARM1) is required for cAMP-mediated activation of ratelimiting gluconeogenic phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) and glucose-6-phosphatase genes. Mutational analysis showed that CARM1 mediates its effect via the cAMP-responsive element within the PEPCK promoter, which is identified here as a CARM1 target in vivo. In hepatocytes, endogenous CARM1 physically interacts with cAMP-responsive element binding factor CREB and is recruited to the PEPCK and glucose-6-phosphatase promoters in a cAMP-dependent manner associated with increased promoter methylation. CARM1 might, therefore, represent a critical component of cAMP-dependent glucose metabolism in the liver.

Abstract The early growth response 1 (Egr1) gene is a transcription factor that acts as both a tumor suppressor and a tumor promoter. Egr1-null mouse embryo fibroblasts bypass replicative senescence and exhibit a loss of DNA damage response and an apparent immortal growth, suggesting loss of p53 functions. Stringent expression analysis revealed 266 transcripts with >2-fold differential expression in Egr1-null mouse embryo fibroblasts, including 143 known genes. Of the 143 genes, program-assisted searching revealed 66 informative genes linked to Egr1. All 66 genes could be placed on a single regulatory network consisting of three branch points of known Egr1 target genes: TGFβ1, IL6, and IGFI. Moreover, 19 additional genes that are known targets of p53 were identified, indicating that p53 is a fourth branch point. Electrophoretic mobility shift assay as well as chromatin immunoprecipitation confirmed that p53 is a direct target of Egr1. Because deficient p53 expression causes tumors in mice, we tested the role of Egr1 in a two-step skin carcinogenesis study (144 mice) that revealed a uniformly accelerated development of skin tumors in Egr1-null mice (P < 0.005). These studies reveal a new role for Egr1 as an in vivo tumor suppressor.