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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.

Supplementary Materials and Table 1 from Early Growth Response 1 Acts as a Tumor Suppressor In vivo and In vitro 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., TSC22D4 regulates hepatic lipoprotein production, but has so far mainly been studied in the context of cancer cachexia. Here, the authors show TSC22D4 inhibition improves insulin sensitivity in several mouse models of diabetes, which they attribute at least in part to the induction of secreted LCN13.

Summary 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.

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.

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. (HEPATOLOGY 2008.)

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.

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.

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 κB (NFκB), 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 NFκB 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.

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 rate-limiting 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.

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.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.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.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.

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.

Expression analysis of 7129 transcripts was carried out in five human prostate cancer cell lines derived from orthotopic xenografts after one to five passages in nude mice and primary cultures of human normal prostate epithelial (NPE) cells. These experiments identified a consensus class of 214 genes (43 up- and 171 downregulated transcripts), expression of which was altered at least twofold in the same direction in all the cell lines relative to NPE cells. To validate the relevance of altered expression behavior of these genes for human prostate cancer, their expression pattern was evaluated in multiple additional experimental and clinical settings. Expression of 170 of these 214 genes (79%) was altered in the same direction in vivo in experimental human prostate tumors in mice. Similarly, the expression of 151 of the 214 genes (71%) was altered in the same direction in M12 cells, a variant of an SV40 large T antigen transformed normal human prostate epithelial cell line selected for increased malignancy in vivo. In clinical samples of human prostate tumors, the changes in transcript expression levels of majority of these genes (85% of downregulated and 76% of upregulated transcripts) are consistent with alterations of their expression pattern in xenograft-derived cancer cell lines. These results imply that the expression pattern of a large class of genes is consistently altered in multiple experimental models and clinical samples of human prostate cancer and underscore the potential relevance of the xenograft models and cell lines derived from them for expression analysis studies relevant to human cancer.

Despite remarkable advances in our understanding of a genetic basis of cancer, the precise molecular definition of the phenotypically relevant genetic features associated with human epithelial malignancies remains a significant and highly relevant challenge. Here we performed a systematic analysis of the chromosomal positions of cancer-associated transcripts for prostate, breast, ovarian, and colon tumors, and identified short segments of human chromosomes that appear to represent a common target for transcriptional activation in major epithelial malignancies in human. These cancer-associated transcriptomeres correspond well to the regions of transient transcriptional activity on chromosomes 1q21-q23 (144-160 Mbp), 12q13 (52-63 Mbp), 17q21 (38-50 Mbp), 17q23-q25 (72-82 Mbp), 19p13 (1-16 Mbp), and Xq28 (132-142 Mbp) during human cell cycle, suggesting a common epigenetic mechanism of transcriptional activation. Consistent with this idea, two of these transcriptomeres (12q13 and 17q21) seemed to be related to the p53-regulated transcriptional clusters, and some of the cancer-associated transcriptomeres appeared to correspond well to the recently identified regions of increased gene expression on human chromosomes.

In the majority of aggressive tumorigenic prostate cancer cells, the transcription factor Egr1 is overexpressed. We provide new insights of Egr1 involvement in proliferation and survival of TRAMP C2 prostate cancer cells by the identification of several new target genes controlling growth, cell cycle progression, and apoptosis such as cyclin D2, P19ink4d, and Fas. Egr1 regulation of these genes, identified by Affymetrix microarray, was confirmed by real-time PCR, immunoblot, and chromatin immunoprecipitation assays. Furthermore we also showed that Egr1 is responsible for cyclin D2 overexpression in tumorigenic DU145 human prostate cells. The regulation of these genes by Egr1 was demonstrated using Egr1 antisense oligonucleotides that further implicated Egr1 in resistance to apoptotic signals. One mechanism was illustrated by the ability of Egr1 to inhibit CD95 (Fas/Apo) expression, leading to insensitivity to FasL. The results provide a mechanistic basis for the oncogenic role of Egr1 in TRAMP C2 prostate cancer cells.

Transcription factor early growth response-1 (Egr-1) is a crucial regulator of cell growth, differentiation and survival. Several observations suggest that Egr-1 is growth promoting in prostate cancer cells and that blocking its function may impede cancer progression. To test this hypothesis, we developed phosphorothioate antisense oligonucleotides that efficiently inhibit Egr-1 expression without altering the expression of other family members Egr-2, Egr-3 and Egr-4. In TRAMP mouse-derived prostate cancer cell lines, our optimal antisense oligonucleotide decreased the expression of the Egr-1 target gene transforming growth factor-beta1 whereas a control oligonucleotide had no effect, indicating that the antisense blocked Egr-1 function as a transcription factor. The antisense oligonucleotide deregulated cell cycle progression and decreased proliferation of the three TRAMP cell lines by an average of 54+/-3%. Both colony formation and growth in soft agar were inhibited by the antisense oligonucleotide. When TRAMP mice were treated systemically for 10 weeks, the incidence of palpable tumors at 32 weeks of age in untreated mice or mice injected with the control scramble oligonucleotide was 87%, whereas incidence of tumors in antisense-Egr-1-treated mice was significantly reduced to 37% (P=0.026). Thus, Egr-1 plays a functional role in the transformed phenotype and may represent a valid target for prostate cancer therapy.

The substrates oxygen and glucose are important for the appropriate regulation of metabolism, angiogenesis, tumorigenesis and embryonic development. The knowledge about an interaction between these two signals is limited. We demonstrated that the regulation of glucagon receptor, insulin receptor and L-type pyruvate kinase (L-PK) gene expression in liver is dependent upon a cross-talk between oxygen and glucose. The periportal to perivenous drop in O2 tension was proposed to be an endocrine key regulator for the zonated gene expression in liver. In primary rat hepatocyte cultures, the expression of the glucagon receptor and the L-PK mRNA was maximally induced by glucose under arterial pO2 whereas the insulin receptor was maximally induced under perivenous pO2. It was demonstrated for the L-PK gene that the modulation by O2 of the glucose-dependent induction occured at the glucose-responsive element (Glc(PK)RE) in the L-PK gene promoter. The reduction of the glucose-dependent induction of the L-PK gene expression under venous pO2 appeared to be mediated via an interference between hypoxia-inducible factor 1 (HIF-1) and the glucose-responsive transcription factors at the Glc(PK)RE. The glucose response element (GlcRE) also functioned as a hypoxia response element and, vice versa, a hypoxia-responsive element was functioning as a GlcRE. Thus, our findings implicate that the cross-talk between oxygen and glucose might have a fundamental role in the regulation of several physiological and pathophysiological processes.