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Khetan S, Bulyk ML. Overlapping binding sites underlie TF genomic occupancy. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.03.05.583629. [PMID: 38496549 PMCID: PMC10942454 DOI: 10.1101/2024.03.05.583629] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/19/2024]
Abstract
Sequence-specific DNA binding by transcription factors (TFs) is a crucial step in gene regulation. However, current high-throughput in vitro approaches cannot reliably detect lower affinity TF-DNA interactions, which play key roles in gene regulation. Here, we developed PADIT-seq ( p rotein a ffinity to D NA by in vitro transcription and RNA seq uencing) to assay TF binding preferences to all 10-bp DNA sequences at far greater sensitivity than prior approaches. The expanded catalogs of low affinity DNA binding sites for the human TFs HOXD13 and EGR1 revealed that nucleotides flanking high affinity DNA binding sites create overlapping lower affinity sites that together modulate TF genomic occupancy in vivo . Formation of such extended recognition sequences stems from an inherent property of TF binding sites to interweave each other and expands the genomic sequence space for identifying noncoding variants that directly alter TF binding. One-Sentence Summary Overlapping DNA binding sites underlie TF genomic occupancy through their inherent propensity to interweave each other.
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2
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Ahi EP, Tsakoumis E, Brunel M, Schmitz M. Transcriptional study reveals a potential leptin-dependent gene regulatory network in zebrafish brain. FISH PHYSIOLOGY AND BIOCHEMISTRY 2021; 47:1283-1298. [PMID: 34236575 PMCID: PMC8302498 DOI: 10.1007/s10695-021-00967-0] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/15/2020] [Accepted: 05/12/2021] [Indexed: 06/01/2023]
Abstract
The signal mediated by leptin hormone and its receptor is a major regulator of body weight, food intake and metabolism. In mammals and many teleost fish species, leptin has an anorexigenic role and inhibits food intake by influencing the appetite centres in the hypothalamus. However, the regulatory connections between leptin and downstream genes mediating its appetite-regulating effects are still not fully explored in teleost fish. In this study, we used a loss of function leptin receptor zebrafish mutant and real-time quantitative PCR to assess brain expression patterns of several previously identified anorexigenic genes downstream of leptin signal under different feeding conditions (normal feeding, 7-day fasting, 2 and 6-h refeeding). These downstream factors include members of cart genes, crhb and gnrh2, as well as selected genes co-expressed with them based on a zebrafish co-expression database. Here, we found a potential gene expression network (GRN) comprising the abovementioned genes by a stepwise approach of identifying co-expression modules and predicting their upstream regulators. Among the transcription factors (TFs) predicted as potential upstream regulators of this GRN, we found expression pattern of sp3a to be correlated with transcriptional changes of the downstream gene network. Interestingly, the expression and transcriptional activity of Sp3 orthologous gene in mammals have already been implicated to be under the influence of leptin signal. These findings suggest a potentially conserved regulatory connection between leptin and sp3a, which is predicted to act as a transcriptional driver of a downstream gene network in the zebrafish brain.
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Affiliation(s)
- Ehsan Pashay Ahi
- Department of Organismal Biology, Comparative Physiology, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18A, SE-752 36 Uppsala, Sweden
- Organismal and Evolutionary Biology Research Programme, University of Helsinki, Viikinkaari 9, 00014 Helsinki, Finland
| | - Emmanouil Tsakoumis
- Department of Organismal Biology, Comparative Physiology, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18A, SE-752 36 Uppsala, Sweden
| | - Mathilde Brunel
- Department of Molecular Sciences, Swedish University of Agricultural Sciences, Allmas Allé 5, SE-750 07 Uppsala, Sweden
| | - Monika Schmitz
- Department of Organismal Biology, Comparative Physiology, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18A, SE-752 36 Uppsala, Sweden
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3
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Abstract
The liver is an essential organ for nutrient and drug metabolism - possessing the remarkable ability to sense environmental and metabolic stimuli and provide an optimally adaptive response. Early growth response 1 (Egr1), an immediate early transcriptional factor which acts as a coordinator of the complex response to stress, is induced during liver injury and controls the expression of a wide range of genes involved in metabolism, cell proliferation, and role of Egr1 in liver injury and repair, deficiency of Egr1 delays liver regeneration process. The known upstream regulators of Egr1 include, but are not limited to, growth factors (e.g. transforming growth factor β1, platelet-derived growth factor, epidermal growth factor, hepatocyte growth factor), nuclear receptors (e.g. hepatocyte nuclear factor 4α, small heterodimer partner, peroxisome proliferator-activated receptor-γ), and other transcription factors (e.g. Sp1, E2F transcription factor 1). Research efforts using various animal models such as fatty liver, liver injury, and liver fibrosis contribute greatly to the elucidation of Egr1 function in the liver. Hepatocellular carcinoma (HCC) represents the second leading cause of cancer mortality worldwide due to the heterogeneity and the late stage at which cancer is generally diagnosed. Recent studies highlight the involvement of Egr1 in HCC development. The purpose of this review is to summarize current studies pertaining to the role of Egr1 in liver metabolism and liver diseases including liver cancer.
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Affiliation(s)
- Nancy Magee
- Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160, USA
| | - Yuxia Zhang
- Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160, USA
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4
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Jeong K, Kwon H, Lee J, Jang D, Pak Y. Insulin-response epigenetic activation of Egr-1 and JunB genes at the nuclear periphery by A-type lamin-associated pY19-Caveolin-2 in the inner nuclear membrane. Nucleic Acids Res 2015; 43:3114-3127. [PMID: 25753664 PMCID: PMC4381080 DOI: 10.1093/nar/gkv181] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2014] [Revised: 02/16/2015] [Accepted: 02/20/2015] [Indexed: 11/15/2022] Open
Abstract
Insulin controls transcription to sustain its physiologic effects for the organism to adapt to environmental changes added to genetic predisposition. Nevertheless, insulin-induced transcriptional regulation by epigenetic factors and in defined nuclear territory remains elusive. Here we show that inner nuclear membrane (INM)-integrated caveolin-2 (Cav-2) regulates insulin-response epigenetic activation of Egr-1 and JunB genes at the nuclear periphery. INM-targeted pY19-Cav-2 in response to insulin associates specifically with the A-type lamin, disengages the repressed Egr-1 and JunB promoters from lamin A/C through disassembly of H3K9me3, and facilitates assembly of H3K9ac, H3K18ac and H3K27ac by recruitment of GCN5 and p300 and the subsequent enrichment of RNA polymerase II (Pol II) on the promoters at the nuclear periphery. Our findings show that Cav-2 is an epigenetic regulator of histone H3 modifications, and provide novel mechanisms of insulin-response epigenetic activation at the nuclear periphery.
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Affiliation(s)
- Kyuho Jeong
- Department of Biochemistry, Division of Applied Life Science (BK21 Plus Program), PMBBRC, Gyeongsang National University, Jinju 660-701, Korea
| | - Hayeong Kwon
- Department of Biochemistry, Division of Applied Life Science (BK21 Plus Program), PMBBRC, Gyeongsang National University, Jinju 660-701, Korea
| | - Jaewoong Lee
- Department of Biochemistry, Division of Applied Life Science (BK21 Plus Program), PMBBRC, Gyeongsang National University, Jinju 660-701, Korea
| | - Donghwan Jang
- Department of Biochemistry, Division of Applied Life Science (BK21 Plus Program), PMBBRC, Gyeongsang National University, Jinju 660-701, Korea
| | - Yunbae Pak
- Department of Biochemistry, Division of Applied Life Science (BK21 Plus Program), PMBBRC, Gyeongsang National University, Jinju 660-701, Korea
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5
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Gokey NG, Lopez-Anido C, Gillian-Daniel AL, Svaren J. Early growth response 1 (Egr1) regulates cholesterol biosynthetic gene expression. J Biol Chem 2011; 286:29501-10. [PMID: 21712389 DOI: 10.1074/jbc.m111.263509] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
The early growth response (EGR) family of transcription factors has been implicated in control of lipid biosynthetic genes. Egr1 is induced by insulin both in vitro and in vivo and is the most highly expressed family member in liver. In this study, we investigated whether Egr1 regulates cholesterol biosynthetic genes in liver. Using an insulin-sensitive liver cell line, we show that localization of Egr1 to cholesterol biosynthetic genes is induced by insulin treatment and that this localization precedes the induction of the genes. Reduction in Egr1 expression using targeted siRNA blunted the insulin-dependent induction of cholesterol genes. A similar reduction in squalene epoxidase expression was also observed in Egr1 null mice. In addition, application of chromatin immunoprecipitation (ChIP) samples to tiled gene microarrays revealed localization of Egr1 in promoter regions of many cholesterol gene loci. In vivo ChIP assays using liver tissue show that Egr1 localization to several cholesterol biosynthetic gene promoters is induced by feeding. Finally, analysis of plasma cholesterol in Egr1(-/-) mice indicated a significant decrease in serum cholesterol when compared with wild-type mice. Together these data point to Egr1 as a modulator of the cholesterol biosynthetic gene family in liver.
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Affiliation(s)
- Nolan G Gokey
- Comparative Biomedical Sciences Graduate Program, School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin 53705, USA
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6
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Tur G, Georgieva EI, Gagete A, López-Rodas G, Rodríguez JL, Franco L. Factor binding and chromatin modification in the promoter of murine Egr1 gene upon induction. Cell Mol Life Sci 2010; 67:4065-77. [PMID: 20582451 PMCID: PMC11115556 DOI: 10.1007/s00018-010-0426-3] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2009] [Revised: 05/12/2010] [Accepted: 05/31/2010] [Indexed: 12/22/2022]
Abstract
The influence of chromatin on immediate-early gene expression has been studied in a model of Egr1 induction in intact mouse cells. ChIP analysis of factor and RNA polymerase binding reveals that the gene is constitutively poised for transcription in nonstimulated cells, but a repressing chromatin structure hampers productive transcription. Stimulation with phorbol esters results in a transient activation, which starts at 5 min and peaks at 30 min. Quantitative mapping of promoter occupancy by the different factors shows for the first time that no direct competition between SP1 and EGR1 occurs. The phosphorylation of ELK1 and CREB, which involves both the cascades of MEK1/2 and p38 kinases, is required for gene expression, which ceases following the binding of NAB1 and NAB2 to the promoter. The changes in histone acetylation and the differential recruitment of histone-modifying complexes further show the role of chromatin in the activation of this immediate-early gene.
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Affiliation(s)
- Gema Tur
- The Chromatin Laboratory, Department of Biochemistry and Molecular Biology, University of Valencia, Dr Moliner, 50, 46100 Burjassot, Valencia Spain
| | - Elena I. Georgieva
- The Chromatin Laboratory, Department of Biochemistry and Molecular Biology, University of Valencia, Dr Moliner, 50, 46100 Burjassot, Valencia Spain
| | - Andrés Gagete
- The Chromatin Laboratory, Department of Biochemistry and Molecular Biology, University of Valencia, Dr Moliner, 50, 46100 Burjassot, Valencia Spain
| | - Gerardo López-Rodas
- The Chromatin Laboratory, Department of Biochemistry and Molecular Biology, University of Valencia, Dr Moliner, 50, 46100 Burjassot, Valencia Spain
| | - José L. Rodríguez
- The Chromatin Laboratory, Department of Biochemistry and Molecular Biology, University of Valencia, Dr Moliner, 50, 46100 Burjassot, Valencia Spain
| | - Luis Franco
- The Chromatin Laboratory, Department of Biochemistry and Molecular Biology, University of Valencia, Dr Moliner, 50, 46100 Burjassot, Valencia Spain
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7
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Dimova EY, Kietzmann T. The MAPK pathway and HIF-1 are involved in the induction of the human PAI-1 gene expression by insulin in the human hepatoma cell line HepG2. Ann N Y Acad Sci 2007; 1090:355-67. [PMID: 17384280 DOI: 10.1196/annals.1378.039] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Enhanced levels of plasminogen activator inhibitor-1 (PAI-1) are considered to be a risk factor for pathological conditions associated with hypoxia or hyperinsulinemia. The expression of the PAI-1 gene is increased by insulin in different cells, although, the molecular mechanisms behind insulin-induced PAI-1 expression are not fully known yet. Here, we show that insulin upregulates human PAI-1 gene expression and promoter activity in HepG2 cells and that mutation of the hypoxia-responsive element (HRE)-binding hypoxia-inducible factor-1 (HIF-1) abolished the insulin effects. Mutation of E-boxes E4 and E5 abolished the insulin-dependent activation of the PAI-1 promoter only under normoxia, but did not affect it under hypoxia. Furthermore, the insulin effect was associated with activation of HIF-1alpha via mitogen-activated protein kinases (MAPKs) but not PDK1 and PKB in HepG2 cells. Furthermore, mutation of a putative FoxO1 binding site which was supposed to be involved in insulin-dependent PAI-1 gene expression influenced the insulin-dependent activation only under normoxia. Thus, insulin-dependent PAI-1 gene expression might be regulated by the action of both HIF-1 and FoxO1 transcription factors.
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Affiliation(s)
- Elitsa Y Dimova
- University of Kaiserslautern, Faculty of Chemistry, Department of Biochemistry, Erwin-Schroedinger Strasse 54, 67663 Kaiserslautern, Germany.
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8
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Mounier C, Posner BI. Transcriptional regulation by insulin: from the receptor to the gene. Can J Physiol Pharmacol 2007; 84:713-24. [PMID: 16998535 DOI: 10.1139/y05-152] [Citation(s) in RCA: 70] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Insulin, after binding to its receptor, regulates many cellular processes and the expression of several genes. For a subset of genes, insulin exerts a negative effect on transcription; for others, the effect is positive. Insulin controls gene transcription by modifying the binding of transcription factors on insulin-response elements or by regulating their transcriptional activities. Different insulin-signaling cascades have been characterized as mediating the insulin effect on gene transcription. In this review, we analyze recent data on the molecular mechanisms, mostly in the liver, through which insulin exerts its effect. We first focus on the key transcription factors (viz. Foxo, sterol-response-element-binding protein family (SREBP), and Sp1) involved in the regulation of gene transcription by insulin. We then present current information on the way insulin downregulates and upregulates gene transcription, using as examples of downregulation phosphoenolpyruvate carboxykinase (PEPCK) and insulin-like growth factor binding protein 1 (IGFBP-1) genes and of upregulation the fatty acid synthase and malic enzyme genes. The last part of the paper focuses on the signaling cascades activated by insulin in the liver, leading to the modulation of gene transcription.
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Affiliation(s)
- Catherine Mounier
- BioMed, Department of Biological Science, University of Quebec in Montreal, 141 President Kennedy, Montreal, QC H2X 3Y7, Canada
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9
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Egr-1 binds the GnRH promoter to mediate the increase in gene expression by insulin. Mol Cell Endocrinol 2007; 270:64-72. [PMID: 17379398 DOI: 10.1016/j.mce.2007.02.007] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/29/2006] [Revised: 02/15/2007] [Accepted: 02/21/2007] [Indexed: 12/26/2022]
Abstract
Insulin increases gonadotropin-releasing hormone (GnRH) gene expression in in vitro models of GnRH neurons. Early growth response-1 (Egr-1) is a transcription factor that mediates the effect of insulin on target genes. In the GN11 cell line--an immortalized GnRH-secreting neuronal cell line--insulin maximally increases Egr-1 mRNA after 30min of treatment and Egr-1 protein and GnRH mRNA after 60min of treatment. Egr-1 small interfering RNA blocks the insulin-induced increase in GnRH promoter activity, measured as luciferase expression. Chromatin immunoprecipitation using Egr-1 antibody precipitates DNA in a proximal region of the GnRH promoter but not DNA in a distal region. Mutagenesis of a putative Egr-1 binding site within the proximal region blocks the insulin-induced increase in GnRH promoter activity. Thus, Egr-1 binds the GnRH promoter at a site between -67 and -76bp from the transcriptional start site to mediate the insulin-induced increase in GnRH gene transcription.
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10
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Böhmer S, Carapito C, Wilzewski B, Leize E, Van Dorsselaer A, Bernhardt R. Analysis of aldosterone-induced differential receptor-independent protein patterns using 2D-electrophoresis and mass spectrometry. Biol Chem 2006; 387:917-29. [PMID: 16913842 DOI: 10.1515/bc.2006.116] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
In the human body the mineralocorticoid aldosterone is responsible for maintaining water and electrolyte homeostasis and therefore controlling blood pressure. In addition, aldosterone has recently been associated with severe heart failure. Besides receptor-dependent action, the damaging effects of aldosterone may also be partly mediated through non-genomic mechanisms. The present study focuses on the mineralocorticoid receptor-independent action of aldosterone at the protein level. We chose the fission yeast Schizosaccharomyces pombe as a model organism, since this yeast does not contain nuclear steroid receptors, but many genes and regulatory mechanisms that are close to those of mammals. Using 2D-electrophoresis we identified for the first time protein spots affected by aldosterone in a nuclear receptor-free system. Mass spectrometry analysis using MALDI-TOF MS and nanoLC-MS/MS approaches allowed the unambiguous identification of 11 proteins that showed increased or decreased levels, which may represent newly identified players and pathways of aldosterone-induced action. Two proteins with a connection to osmotic regulation (NAD-dependent malic enzyme and glycerol-3-phosphate-dehydrogenase), as well as two proteins involved in the overall organization of the cytoskeleton, vip1 and glyceraldehyde-3-phosphate dehydrogenase, which was also found to be specifically affected by aldosterone in human HCT116 cells, are discussed.
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Affiliation(s)
- Susanne Böhmer
- Universität des Saarlandes, FR 8.3 Biochemie, Postfach 151150, D-66041 Saarbrücken, Germany
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11
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Duttaroy A, Kanakaraj P, Osborn BL, Schneider H, Pickeral OK, Chen C, Zhang G, Kaithamana S, Singh M, Schulingkamp R, Crossan D, Bock J, Kaufman TE, Reavey P, Carey-Barber M, Krishnan SR, Garcia A, Murphy K, Siskind JK, McLean MA, Cheng S, Ruben S, Birse CE, Blondel O. Development of a long-acting insulin analog using albumin fusion technology. Diabetes 2005; 54:251-8. [PMID: 15616036 DOI: 10.2337/diabetes.54.1.251] [Citation(s) in RCA: 87] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
The primary therapeutic goal for the treatment of diabetes is maintenance of a long-term, near-normoglycemic condition and prevention of the onset or progression of the complications associated with the disease. Although several analogs of human insulin have been developed, the currently prescribed long-acting insulin analogs do not provide a stable basal glycemia for more than a few hours. Here, we report the development of Albulin, a long-acting insulin analog obtained by direct gene fusion of a single-chain human insulin to human serum albumin. Albulin showed an elimination t(1/2) of approximately 7 h in normoglycemic mice. In vitro pharmacodynamic profiles for Albulin characterized by receptor binding, inhibition of gluconeogenesis, induction of glucose uptake, and global regulation of gene expression in relevant cell types showed that Albulin produced similar activity profiles compared with that of recombinant human insulin. A single Albulin administration in vivo normalized blood glucose level in diabetic mice in a relatively peakless and sustained (24-h) fashion. A further reduction in glucose levels was achieved by administering a recombinant human insulin a few hours after Albulin injection in mice, indicating the potential for Albulin therapy in combination with available fast-acting insulin derivatives. In summary, Albulin displays characteristics of a potent long-acting insulin analog that can be evaluated for use as a novel insulin therapy for patients with insulin-dependent diabetes.
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Affiliation(s)
- Alokesh Duttaroy
- Division of Diabetes, Endocrinology and Metabolic Diseases, NIDDK/NIH, 6707 Democracy Blvd., Rm. 606, MSC5460, Bethesda, MD 20892-5460, USA
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12
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Hansen L, Gaster M, Oakeley EJ, Brusgaard K, Damsgaard Nielsen EM, Beck-Nielsen H, Pedersen O, Hemmings BA. Expression profiling of insulin action in human myotubes: induction of inflammatory and pro-angiogenic pathways in relationship with glycogen synthesis and type 2 diabetes. Biochem Biophys Res Commun 2004; 323:685-95. [PMID: 15369805 DOI: 10.1016/j.bbrc.2004.08.146] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2004] [Indexed: 11/19/2022]
Abstract
Myotube cultures from patients with type 2 diabetes mellitus (T2DM) represent an experimental in vitro model of T2DM that offers a possibility to perform gene expression studies under standardized conditions. During a time-course of insulin stimulation (1 microM) at 5.5 mM glucose for 0 (no insulin), 0.5, 1, 2, 4, 8, and 24 h, mRNA contents were analyzed in human myotubes for each time point using Affymetrix DNA chip technology. Insulin treatment induced an inflammatory and pro-angiogenic response in the myotubes, with expression of early response factors followed by inflammatory chemokines, metabolic enzymes, and finally cell cycle regulating genes. One-hundred-forty-four genes were differentially expressed in myotubes from donors with type 2 diabetes compared with control subjects, including HSP70, apolipoprotein D/E, tropomyosin, myosin, and actin previously reported from in vivo studies of diabetic skeletal muscle. We conclude, (i) that insulin induces a time-dependent inflammatory and pro-angiogenic transcriptional response in cultured human myotubes, (ii) that myotubes in vitro retain a gene expression pattern specific for type 2 diabetes and sharing five genes with that of type 2 diabetic skeletal muscle in vivo, and (iii) that insulin, despite similar metabolic effects of glucose uptake and glycogen synthesis, regulates different pools of genes in skeletal muscle during in vivo and in vitro conditions.
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Affiliation(s)
- Lars Hansen
- Steno Diabetes Center, 2820 Gentofte, Denmark.
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Yang F, Agulian T, Sudati JE, Rhoads DB, Levitsky LL. Developmental regulation of galactokinase in suckling mouse liver by the Egr-1 transcription factor. Pediatr Res 2004; 55:822-9. [PMID: 14973178 DOI: 10.1203/01.pdr.0000120682.05408.79] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
The numerous changes in metabolic pathways that accompany liver development entail associated changes in gene expression. Egr-1 is a zinc-finger transcription factor that regulates genes involved in cellular growth, differentiation, stress response, and apoptosis in many cell types. Egr-1 is induced in liver regeneration in rodents, but its role in normal hepatocyte function has not been characterized. We examined the developmental expression of Egr-1 in mouse liver and found that its expression increased during the suckling period. In screening the sequences of the genes involved in lactose assimilation, we found that the galactokinase gene Glk contains four potential Egr-1 binding sites in its proximal promoter. A minimal promoter of 155 nucleotides encompassing the four Egr-1 sites exhibited activity in hepatoma cell lines by transient transfection assays. Moreover, co-transfection of an Egr-1 expression plasmid increased promoter activity. Finally, mutations introduced into three of the four Egr-1 binding sites decreased activity, whereas mutation of the remaining site increased promoter activity. These data tie Egr-1 and galactokinase together in a developmentally regulated chain to prepare the neonate for suckling.
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Affiliation(s)
- Fang Yang
- Pediatric Endocrine Unit, MassGeneral Hospital for Children, Harvard Medical School, Boston, MA 02114, USA
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14
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Chen C, Grzegorzewski KJ, Barash S, Zhao Q, Schneider H, Wang Q, Singh M, Pukac L, Bell AC, Duan R, Coleman T, Duttaroy A, Cheng S, Hirsch J, Zhang L, Lazard Y, Fischer C, Barber MC, Ma ZD, Zhang YQ, Reavey P, Zhong L, Teng B, Sanyal I, Ruben SM, Blondel O, Birse CE. An integrated functional genomics screening program reveals a role for BMP-9 in glucose homeostasis. Nat Biotechnol 2003; 21:294-301. [PMID: 12598908 DOI: 10.1038/nbt795] [Citation(s) in RCA: 150] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2002] [Accepted: 12/12/2002] [Indexed: 11/09/2022]
Abstract
A coordinated functional genomics program was implemented to identify secreted polypeptides with therapeutic applications in the treatment of diabetes. Secreted factors were predicted from a diverse expressed-sequence tags (EST) database, representing >1,000 cDNA libraries, using a combination of bioinformatic algorithms. Subsequently, approximately 8,000 human proteins were screened in high-throughput cell-based assays designed to monitor key physiological transitions known to be centrally involved in the physiology of type 2 diabetes. Bone morphogenetic protein-9 (BMP-9) gave a positive response in two independent assays: reducing phosphoenolpyruvate carboxykinase (PEPCK) expression in hepatocytes and activating Akt kinase in differentiated myotubes. Purified recombinant BMP-9 potently inhibited hepatic glucose production and activated expression of key enzymes of lipid metabolism. In freely fed diabetic mice, a single subcutaneous injection of BMP-9 reduced glycemia to near-normal levels, with maximal reduction observed 30 hours after treatment. BMP-9 represents the first hepatic factor shown to regulate blood glucose concentration. Using a combination of bioinformatic and high-throughput functional analyses, we have identified a factor that may be exploited for the treatment of diabetes.
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Affiliation(s)
- Cecil Chen
- Department of Lead Development and Characterization, Human Genome Sciences, Inc., 9800 Medical Center Dr., Rockville, MD 20850, USA
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15
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Raychowdhury R, Schäfer G, Fleming J, Rosewicz S, Wiedenmann B, Wang TC, Höcker M. Interaction of early growth response protein 1 (Egr-1), specificity protein 1 (Sp1), and cyclic adenosine 3'5'-monophosphate response element binding protein (CREB) at a proximal response element is critical for gastrin-dependent activation of the chromogranin A promoter. Mol Endocrinol 2002; 16:2802-18. [PMID: 12456801 DOI: 10.1210/me.2001-0292] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Recently, binding of specific protein 1 (Sp1) and cAMP response element binding protein (CREB) to a GC-rich element at -92/-62 has been identified as a critical step in gastrin-dependent regulation of the chromogranin A (CgA) gene in gastric epithelial cells. Here we demonstrate that binding of early growth response protein 1 (Egr-1) to the distal part of the -92/-62 site is also required for gastrin-dependent CgA transactivation. Gastrin elevated cellular and nuclear Egr-1 levels in a time-dependent manner and also increased Egr-1 binding to the CgA -92/-73 region. Disruption of this site reduced gastrin responsiveness without influencing basal promoter activity, while loss of Sp1 and/or CREB binding sites diminished basal and gastrin-stimulated CgA promoter activity. Ectopic Egr-1 overexpression potently stimulated the CgA promoter, whereas coexpression of Egr-1 with Sp1 and/or CREB resulted in additive effects. Functional analysis of Sp1-, Egr-1-, or CREB-specific promoter mutations in transfection studies confirmed the tripartite organization of the CgA -92/-62 element. Signaling studies revealed that MAPK kinase 1 (MEK1)/ERK1/2 cascades are critical for gastrin-dependent Egr-1 protein accumulation as well as Egr-1 binding to the CgA promoter. Our studies for the first time identify Egr-1 as a nuclear target of gastrin and show that functional interplay of Egr-1, Sp1, and CREB is indispensable for gastrin-dependent CgA transactivation in gastric epithelial cells.
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Affiliation(s)
- Raktima Raychowdhury
- Medizinische Klink mit Schwerpunkt Gastroenterologie, Hepatologie, Endokrinologie und Stoffwechsel, Universitätsklinikum Charité, Campus Virchow-Klinikum, Humboldt Universität, 13353 Berlin, Germany
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16
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Irie T, Kawai A. Studies on the different conditions for rabies virus neutralization by monoclonal antibodies #1-46-12 and #7-1-9. J Gen Virol 2002; 83:3045-3053. [PMID: 12466481 DOI: 10.1099/0022-1317-83-12-3045] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Virus-neutralizing activity of two monoclonal antibodies (mAbs), #7-1-9 and #1-46-12, against rabies virus glycoprotein (G) was compared. Although these mAbs affected the virion's ability to bind to host cells similarly, a big difference was found in the titres of virus neutralization (1:7132 and 1:32 for mAbs #1-46-12 and #7-1-9, respectively, at a concentration of 10 micro g protein/ml). Although no big difference in virion-binding affinity between the two mAbs was found, the number of antibodies required for virus neutralization was very low, </=20 molecules for mAb #1-46-12 and >/=250 molecules for mAb #7-1-9. In the latter case, the mAbs cover a major part of the virion surface and cause steric hindrance of viral receptor-binding activity. The infectivity of an epitope-preserved escape mutant virus (R-61) was not affected by the binding of high numbers of mAb #1-46-12 to the virion, which implies that mAb binding does not mask the receptor-binding site of the viral spikes. Based on these results, it is hypothesized that mAb #1-46-12 affected virus infectivity by a mechanism different from covering the virion spikes. Possible virus-neutralizing mechanisms by low numbers of mAb #1-46-12 in comparison to that of mAb #7-1-9 are discussed.
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Affiliation(s)
- Takashi Irie
- Department of Molecular Microbiology, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan1
| | - Akihiko Kawai
- Department of Molecular Microbiology, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan1
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17
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Ayala JE, Streeper RS, Svitek CA, Goldman JK, Oeser JK, O'Brien RM. Accessory elements, flanking DNA sequence, and promoter context play key roles in determining the efficacy of insulin and phorbol ester signaling through the malic enzyme and collagenase-1 AP-1 motifs. J Biol Chem 2002; 277:27935-44. [PMID: 12032154 DOI: 10.1074/jbc.m203682200] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023] Open
Abstract
Insulin stimulates malic enzyme (ME)-chloramphenicol acetyltransferase (CAT) and collagenase-1-CAT fusion gene expression in H4IIE cells through identical activator protein-1 (AP-1) motifs. In contrast, insulin and phorbol esters only stimulate collagenase-1-CAT and not ME-CAT fusion gene expression in HeLa cells. The experiments in this article were designed to explore the molecular basis for this differential cell type- and gene-specific regulation. The results highlight the influence of three variables, namely promoter context, AP-1 flanking sequence, and accessory elements that modulate insulin and phorbol ester signaling through the AP-1 motif. Thus, fusion gene transfection and proteolytic clipping gel retardation assays suggest that the AP-1 flanking sequence affects the conformation of AP-1 binding to the collagenase-1 and ME AP-1 motifs such that it selectively binds the latter in a fully activated state. However, this influence of ME AP-1 flanking sequence is dependent on promoter context. Thus, the ME AP-1 motif will mediate both an insulin and phorbol ester response in HeLa cells when introduced into either the collagenase-1 promoter or a specific heterologous promoter. But even in the context of the collagenase-1 promoter, the effects of both insulin and phorbol esters, mediated through the ME AP-1 motif are dependent on accessory factors.
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Affiliation(s)
- Julio E Ayala
- Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, Tennessee 37232, USA
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18
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Wu MY, Chen MH, Liang YR, Meng GZ, Yang HX, Zhuang CX. Experimental and clinic-opathologic study on the relationship between transcription factor Egr-1 and esophageal carcinoma. World J Gastroenterol 2001; 7:490-5. [PMID: 11819815 PMCID: PMC4688659 DOI: 10.3748/wjg.v7.i4.490] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To observe the growth suppression effect of exogenous introduction of early growth response gene-1 (Egr-1 gene) on esophageal carcinoma tissue as well as on esophageal carcinoma cell line Eca109 and to explore the potential application of Egr-1 gene in gene therapy of tumor.
METHODS: Eukaryotic expression vector of PCMV-Egr-1 plasmid was introduced into Eca109 cell line which expressed no Egr-1 protein originally with lipofectamine transfection method. The introduction and expression of PCMV-Egr-1 plasmid into Eca109 cell line was confirmed by G418 selection culture, PCR amplification of neogene contained in the vector, Western blot analysis and immunocytochemical analysis. The cell growth curve, soft agar colony formation rate and tumorigenicity in SCID mice were examined to demonstrate the growth suppression effect of exogenous Egr-1 gene on Eca109 cell line. The Egr-1 mRNA and Egr-1 protein were also detected in 50 surgical specimens of esophageal carcinoma by in situ hybridization and immunohistochemistry.
RESULTS: Exogenous Egr-1 gene was introduced successfully into Eca109 cell line and expressed Egr-1 protein stably. The transfected Eca109 cell line grew more slowly than control Eca109 as shown by cell growth curves, the soft agar colony formation rate (4.0% vs 6.9%, P < 0.01) and the average growth rate of tumor in SCID mice (35.5 ± 7.6 vs 65.8 ± 7.6, P < 0.05). The expression level of Egr-1 mRNA and protein significantly increased in dysplastic epithelia adjacent to cancer rather than in cancer tissues (65.8% vs 20.0% by ISH and 57.9% vs 14.0% by IHC, P < 0.01).
CONCLUSION: Exogenous Egr-1 gene shows the strong effect of growth inhibition in Eca109 cell line. Egr-1 in the cancer tissue shows down-regulated expression that supports the inhibited function of Egr-1 in cancer growth and suggests Egr-1 may have an important role in gene therapy of esophageal carcinoma.
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Affiliation(s)
- M Y Wu
- Department of Pathology, Shantou University Medical College, 22 Xinling Road, Shantou 515031, Guangdong Province, China.
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19
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Ragoczy T, Miller G. Autostimulation of the Epstein-Barr virus BRLF1 promoter is mediated through consensus Sp1 and Sp3 binding sites. J Virol 2001; 75:5240-51. [PMID: 11333906 PMCID: PMC114930 DOI: 10.1128/jvi.75.11.5240-5251.2001] [Citation(s) in RCA: 78] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
As an essential step in the lytic cascade, the Rta homologues of gammaherpesviruses all activate their own expression. Consistent with this biologic function, the Epstein-Barr virus (EBV) Rta protein powerfully stimulates the promoter of its own gene, Rp, in EBV-positive B cells in transient-transfection reporter-based assays. We analyzed the activity of RpCAT in response to Rta by deletional and site-directed mutagenesis. Two cognate Sp1 binding sites located at -279 and -45 relative to the transcriptional start site proved crucial for Rta-mediated activation. Previously described binding sites for the cellular transcription factor Zif268 and the viral transactivator ZEBRA were found to be dispensable for activation of RpCAT by Rta. Gel shift analysis, using extracts of B cells in latency or induced into the lytic cycle, identified Sp1 and Sp3 as the predominant cellular proteins bound to Rp near -45. During the lytic cycle, ZEBRA bound Rp near the Sp1/Sp3 site. The binding of Sp1 and Sp3 to Rp correlated with the reporter activities in the mutagenesis study, establishing a direct link between transcriptional activation of Rp by Rta and DNA binding by Sp1 and/or Sp3. The relative abundance or functional state of the cellular Sp1 and Sp3 transcription factors may be altered in response to stimuli that induce the BRLF1 promoter and thereby contribute to the activation of the viral lytic cycle.
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Affiliation(s)
- T Ragoczy
- Department of Molecular Biophysics, Yale School of Medicine, New Haven, Connecticut 06520, USA
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20
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Harada S, Esch GL, Holgado-Madruga M, Wong AJ. Grb-2-associated binder-1 is involved in insulin-induced egr-1 gene expression through its phosphatidylinositol 3'-kinase binding site. DNA Cell Biol 2001; 20:223-9. [PMID: 11403719 DOI: 10.1089/104454901750219107] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
The Grb2-associated binder-1 (Gab1) is one of the major adapter molecules downstream of growth factor receptor signaling. Even though insulin causes tyrosine phosphorylation of Gab1, its role in insulin signaling has not been identified yet. We have demonstrated that insulin increased expression of early growth response gene-1 (egr-1), which is one of the most important transcription factors involved in cell proliferation and differentiation. In the present study, the possible role of Gab1 in insulin-induced egr-1 expression was studied using Rat1 fibroblasts expressing human insulin receptors and wildtype Gab1 (HIRc/Gab1(WT)), Gab1 with three tyrosines in the phosphatidylinositol (PI) 3'-kinase binding domain mutated to phenylalanine (HIRc/Gab1(DeltaPI3K)), or histidinol resistance only (HIRc/HIS). Insulin-induced egr-1 expression in HIRc/Gab1(DeltaPI3K) cells was much lower than in the other cells, as determined by Northern blot analysis. These results suggest that Gab1 is involved in the signaling pathway for insulin-induced egr-1 expression through increasing PI3'-kinase activity. The MAP kinase activity increased less with insulin treatment in HIRc/Gab1(DeltaPI3K) cells than in other cells. Inhibition of MAP kinase by the MEK inhibitor completely abolished insulin-induced egr-1 expression. These results suggest that Gab1 increases MAP kinase activity through its PI3'-kinase binding site, which then leads to egr-1 expression. Our results indicate that Gab1 is involved in the control of egr-1 expression regulated by insulin.
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Affiliation(s)
- S Harada
- Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
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21
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Tsuruzoe K, Emkey R, Kriauciunas KM, Ueki K, Kahn CR. Insulin receptor substrate 3 (IRS-3) and IRS-4 impair IRS-1- and IRS-2-mediated signaling. Mol Cell Biol 2001; 21:26-38. [PMID: 11113178 PMCID: PMC86565 DOI: 10.1128/mcb.21.1.26-38.2001] [Citation(s) in RCA: 75] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
To investigate the roles of insulin receptor substrate 3 (IRS-3) and IRS-4 in the insulin-like growth factor 1 (IGF-1) signaling cascade, we introduced these proteins into 3T3 embryonic fibroblast cell lines prepared from wild-type (WT) and IRS-1 knockout (KO) mice by using a retroviral system. Following transduction of IRS-3 or IRS-4, the cells showed a significant decrease in IRS-2 mRNA and protein levels without any change in the IRS-1 protein level. In these cell lines, IGF-1 caused the rapid tyrosine phosphorylation of all four IRS proteins. However, IRS-3- or IRS-4-expressing cells also showed a marked decrease in IRS-1 and IRS-2 phosphorylation compared to the host cells. This decrease was accounted for in part by a decrease in the level of IRS-2 protein but occurred with no significant change in the IRS-1 protein level. IRS-3- or IRS-4-overexpressing cells showed an increase in basal phosphatidylinositol 3-kinase activity and basal Akt phosphorylation, while the IGF-1-stimulated levels correlated well with total tyrosine phosphorylation level of all IRS proteins in each cell line. IRS-3 expression in WT cells also caused an increase in IGF-1-induced mitogen-activated protein kinase phosphorylation and egr-1 expression ( approximately 1.8- and approximately 2.4-fold with respect to WT). In the IRS-1 KO cells, the impaired mitogenic response to IGF-1 was reconstituted with IRS-1 to supranormal levels and was returned to almost normal by IRS-2 or IRS-3 but was not improved by overexpression of IRS-4. These data suggest that IRS-3 and IRS-4 may act as negative regulators of the IGF-1 signaling pathway by suppressing the function of other IRS proteins at several steps.
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Affiliation(s)
- K Tsuruzoe
- Research Division, Joslin Diabetes Center, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, USA
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22
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Thiel G, Kaufmann K, Magin A, Lietz M, Bach K, Cramer M. The human transcriptional repressor protein NAB1: expression and biological activity. BIOCHIMICA ET BIOPHYSICA ACTA 2000; 1493:289-301. [PMID: 11018254 DOI: 10.1016/s0167-4781(00)00207-4] [Citation(s) in RCA: 92] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
Abstract
The zinc finger protein early growth response 1 (Egr-1) is a transcriptional activator involved in the regulation of growth and differentiation. Egr-1 has a large activating domain and three zinc finger motifs that function as a DNA binding region. We show here that a third functional domain of the Egr-1 protein, localized between the extended activation domain and the zinc finger DNA binding region, acts as a transcriptional repressor domain when fused to a heterologous DNA binding domain (DBD). Through protein-protein interaction this inhibitory domain of Egr-1 brings the transcriptional corepressor NAB1 in close proximity to the transcription unit. NAB1 is expressed ubiquitously in human cell lines as shown by RNase protection mapping. Overexpression studies revealed that NAB1 is able to completely block transcription mediated by Egr-1. In addition, the transcriptional repression activity of a fusion protein containing the inhibitory domain of Egr-1 and the DBD of the yeast transcription factor GAL4 was increased by overexpression of NAB1. A fusion protein consisting of the DBD of GAL4 and the coding region of human NAB1 repressed transcription from model promoters with engineered upstream GAL4 binding sites. The GAL4-NAB1 fusion protein functioned from proximal and distal positions indicating that NAB1 displays transcriptional repressor activity at any position within the transcription unit. Thus, the biological function of the inhibitory domain of Egr-1 is solely to provide a docking site for NAB1 via protein-protein interaction.
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Affiliation(s)
- G Thiel
- Medical Biochemistry and Molecular Biology, University of Saarland, Homburg, Germany.
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23
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Grzeskowiak R, Amin J, Oetjen E, Knepel W. Insulin responsiveness of the glucagon gene conferred by interactions between proximal promoter and more distal enhancer-like elements involving the paired-domain transcription factor Pax6. J Biol Chem 2000; 275:30037-45. [PMID: 10862760 DOI: 10.1074/jbc.m000984200] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Regulation of gene transcription is an important aspect of insulin's action. However, the mechanisms involved are poorly understood. Insulin inhibits glucagon gene transcription, and insulin deficiency is associated with hyperglucagonemia that contributes to hyperglycemia in diabetes mellitus. Transfecting glucagon-reporter fusion genes into a glucagon-producing pancreatic islet cell line, a 5'-, 3'-, and internal deletion analysis, and oligonucleotide cassette insertions failed in the present study to identify a single insulin-responsive element in the glucagon gene. They rather indicate that insulin responsiveness depends on the presence of both proximal promoter elements and more distal enhancer-like elements. When the paired domain transcription factor Pax6 binding sites within the proximal promoter element G1 and the enhancer-like element G3 were mutated into GAL4 binding sites, the expression of GAL4-Pax6 and GAL4-VP16 restored basal activity, whereas only GAL4-Pax6 restored also insulin responsiveness. Likewise, GAL4-CBP activity was inhibited by insulin within the glucagon promoter context. The results suggest that insulin responsiveness is conferred to the glucagon gene by the synergistic interaction of proximal promoter and more distal enhancer-like elements, with Pax6 and its potential coactivator the CREB-binding protein being critical components. These data thereby support concepts of insulin-responsive element-independent mechanisms of insulin action to inhibit gene transcription.
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Affiliation(s)
- R Grzeskowiak
- Department of Molecular Pharmacology, University of Göttingen, 37070 Göttingen, Germany
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24
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Stoner M, Wang F, Wormke M, Nguyen T, Samudio I, Vyhlidal C, Marme D, Finkenzeller G, Safe S. Inhibition of vascular endothelial growth factor expression in HEC1A endometrial cancer cells through interactions of estrogen receptor alpha and Sp3 proteins. J Biol Chem 2000; 275:22769-79. [PMID: 10816575 DOI: 10.1074/jbc.m002188200] [Citation(s) in RCA: 64] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
Treatment of HEC1A endometrial cancer cells with 10 nm 17beta-estradiol (E2) resulted in decreased vascular endothelial growth factor (VEGF) mRNA expression, and a similar response was observed using a construct, pVEGF1, containing a VEGF gene promoter insert from -2018 to +50. In HEC1A cells transiently transfected with pVEGF1 and a series of deletion plasmids, it was shown that E2-dependent down-regulation was dependent on wild-type estrogen receptor alpha (ERalpha) and reversed by the anti-estrogen ICI 182, 780, and this response was not affected by progestins. Deletion analysis of the VEGF gene promoter identified an overlapping G/GC-rich site between -66 to -47 that was required for decreased transactivation by E2. Protein-DNA binding studies using electrophoretic mobility shift and DNA footprinting assays showed that both Sp1 and Sp3 proteins bound this region of the VEGF promoter. Coimmunoprecipitation and pull-down assays demonstrated that Sp3 and ERalpha proteins physically interact, and the interacting domains of both proteins are different from those previously observed for interactions between Sp1 and ERalpha proteins. Using a dominant negative form of Sp3 and transcriptional activation assays in Schneider SL-2 insect cells, it was confirmed that ERalpha-Sp3 interactions define a pathway for E2-mediated inhibition of gene expression, and this represents a new mechanism for decreased gene expression by E2.
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Affiliation(s)
- M Stoner
- Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas 77843-4466, USA
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Abstract
The leptin and lipogenic enzyme genes contain the common DNA sequences of binding sites for Sp1 proteins. These sites appear to be responsible for glucose/insulin stimulation and polyunsaturated fatty acid suppression. In rat adipose tissue leptin and lipogenic gene expression is similarly regulated by nutritional manipulation. Interestingly, leptin has the ability to down-regulate lipogenic enzyme expression.
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Affiliation(s)
- N Iritani
- Department of Human and Cultural Studies, Tezukayama Gakuin University, Sakai, Osaka, Japan.
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Zhao AZ, Shinohara MM, Huang D, Shimizu M, Eldar-Finkelman H, Krebs EG, Beavo JA, Bornfeldt KE. Leptin induces insulin-like signaling that antagonizes cAMP elevation by glucagon in hepatocytes. J Biol Chem 2000; 275:11348-54. [PMID: 10753948 DOI: 10.1074/jbc.275.15.11348] [Citation(s) in RCA: 189] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Although many effects of leptin are mediated through the central nervous system, leptin can regulate metabolism through a direct action on peripheral tissues, such as fat and liver. We show here that leptin, at physiological concentrations, acts through an intracellular signaling pathway similar to that activated by insulin in isolated primary rat hepatocytes. This pathway involves stimulation of phosphatidylinositol 3-kinase (PI3K) binding to insulin receptor substrate-1 and insulin receptor substrate-2, activation of PI3K and protein kinase B (AKT), and PI3K-dependent activation of cyclic nucleotide phosphodiesterase 3B, a cAMP-degrading enzyme. One important function of this signaling pathway is to reduce levels of cAMP, because leptin-mediated activation of both protein kinase B and phosphodiesterase 3B is most marked following elevation of cAMP by glucagon, and because leptin suppresses glucagon-induced cAMP elevation in a PI3K-dependent manner. There is little or no expression of the long form leptin receptor in primary rat hepatocytes, and these signaling events are probably mediated through the short forms of the leptin receptor. Thus, leptin, like insulin, induces an intracellular signaling pathway in hepatocytes that culminates in cAMP degradation and an antagonism of the actions of glucagon.
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Affiliation(s)
- A Z Zhao
- Department of Pharmacology, University of Washington, Seattle, Washington 98195, USA
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McCaffrey TA, Fu C, Du B, Eksinar S, Kent KC, Bush H, Kreiger K, Rosengart T, Cybulsky MI, Silverman ES, Collins T. High-level expression of Egr-1 and Egr-1-inducible genes in mouse and human atherosclerosis. J Clin Invest 2000; 105:653-62. [PMID: 10712437 PMCID: PMC289183 DOI: 10.1172/jci8592] [Citation(s) in RCA: 237] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
To understand the mRNA transcript profile in the human atherosclerotic lesion, RNA was prepared from the fibrous cap versus adjacent media of 13 patients undergoing carotid endarterectomy. cDNA expression arrays bearing 588 known genes indicated that lesions express unexpectedly high levels of the early growth response gene, Egr-1 (NGFI-A), a zinc-finger transcription factor that modulates a cluster of stress-responsive genes including PDGF and TGF-beta. Expression of Egr-1 was an average of 5-fold higher in the lesion than in the adjacent media, a result confirmed by RT-PCR, and many Egr-1-inducible genes were also strongly elevated in the lesion. Time-course analyses revealed that Egr-1 was not induced ex vivo. Immunocytochemistry indicated that Egr-1 was expressed prominently in the smooth muscle-actin positive cells, particularly in areas of macrophage infiltration, and in other cell types, including endothelial cells. Induction of atherosclerosis in LDL receptor-null mice by feeding them a high-fat diet resulted in a progressive increase in Egr-1 expression in the aorta. Thus, induction of Egr-1 by atherogenic factors may be a key step in coordinating the cellular events that result in vascular lesions.
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Affiliation(s)
- T A McCaffrey
- Department of Medicine, Division of Hematology/Oncology, Weill Medical College of Cornell University, New York, New York 10021, USA.
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Liu SC, Wang Q, Lienhard GE, Keller SR. Insulin receptor substrate 3 is not essential for growth or glucose homeostasis. J Biol Chem 1999; 274:18093-9. [PMID: 10364263 DOI: 10.1074/jbc.274.25.18093] [Citation(s) in RCA: 125] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
The insulin receptor substrates (IRS) 1 and 2 are required for normal growth and glucose homeostasis in mice. To determine whether IRS-3, a recently cloned member of the IRS family, is also involved in the regulation of these, we have generated mice with a targeted disruption of the IRS-3 gene and characterized them. Compared with wild-type mice, the IRS-3-null mice showed normal body weight throughout development, normal blood glucose levels in the fed and fasted state and following an oral glucose bolus, and normal fed and fasted plasma insulin levels. IRS-3 is most abundant in adipocytes and is tyrosine-phosphorylated in response to insulin in these cells. Therefore, isolated adipocytes were analyzed for changes in insulin effects. Insulin-stimulated glucose transport in the adipocytes from the IRS-3-null mice was the same as in wild-type cells. The extent of tyrosine phosphorylation of IRS-1/2 following insulin stimulation was similar in adipocytes from IRS-3-null and wild-type mice, and the insulin-induced association of tyrosine-phosphorylated IRS-1/2 with phosphatidylinositol 3-kinase and SHP-2 was not detectably increased by IRS-3 deficiency. Thus, IRS-3 was not essential for normal growth, glucose homeostasis, and glucose transport in adipocytes, and in its absence no significant compensatory augmentation of insulin signaling through IRS-1/2 was evident.
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Affiliation(s)
- S C Liu
- Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA
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