Oxygen Biology and Hypoxia (eBook)

Cover 1
Contents 6
Contributors 14
Preface 22
Volumes in Series 24
Section I: Hypoxia-Inducible Factor 50
Chapter 1: Hypoxia-Inducible Factors Per/ARNT/Sim Domains: Structure and Function 52
1. Introduction 53
2. Delineation of the HIF PAS Domains 54
3. Expression and Characterization of HIF PAS Domains 56
3.1. Recombinant protein expression 56
3.2. Limited proteolysis 57
3.3. Solution NMR spectroscopy 59
4. Assessing PAS Domain Protein-Protein Interactions 60
4.1. NMR spectroscopy 60
4.2. Reporter genes of HIF function 62
4.3. Preparation of nuclear lysates 64
4.4. Coimmunoprecipitation of HIF-alpha and HIF-beta 64
4.5. Electrophoretic mobility shift assay 66
5. Discussion 67
Acknowledgments 69
References 70
Chapter 2: Hypoxia-Inducible Factor Prolyl-Hydroxylase: Purification and Assays of PHD2 74
1. Introduction 75
2. Preparation of Purified PHD2 from a Bacterial Source 77
3. Assaying of PHD2 Activity 78
4. Indirect Measurements of PHD2 Activity 78
4.1. 1-[14C]-CO2 capture assay 79
4.2. Fluorescence derivatization of 2OG 81
4.3. Oxygen consumption assay 82
4.4. 1-[14C]succinate quantification 84
5. Direct Measurements of PHD2 Hydroxylation Activity 84
5.1. LC/MS identification of hydroxylated HIF-1alpha 556 to 574 84
5.2. pVHL capture assay 85
6. Binding Assays 86
7. Comparison of Assay Formats 87
References 74
Chapter 3: Determination and Modulation of Prolyl-4-Hydroxylase Domain Oxygen Sensor Activity 92
1. Introduction 93
2. Production of Functionally Active PHDs 97
3. Determination of PHD Activity by VHL Binding to Peptides Derived from the HIF-1alpha ODD Domain 97
4. Determination of Prolyl-4-Hydroxylation by Oxidative Decarboxylation of 2-Oxoglutarate 100
5. Crude Tissue Extracts are not a Suitable Source of PHD Activity for the 2-Oxoglutarate Conversion Assay 102
6. Thin Layer Chromatography to Assess the Purity of [5-14C] 2-Oxoglutarate 102
7. Application of the 2-Oxoglutarate Conversion Assay to Protein Targets 104
8. Conclusions 104
Acknowledgments 74
References 106
Chapter 4: Characterization of Ankyrin Repeat-Containing Proteins as Substrates of the Asparaginyl Hydroxylase Factor Inhibiting Hypoxia-Inducible Transcription Factor 110
1. Introduction 111
2. Experimental Techniques 114
2.1. Production of FIH-1 114
2.2. Protein quantitation 117
2.3. Production of substrates 118
2.4. CO2 capture assays 120
2.5. Interaction assays 125
2.6. In vitro pull-down assay 125
2.7. Co-immunoprecipitation assay 128
3. Discussion/Conclusion 131
Acknowledgments 74
References 132
Chapter 5: Transgenic Models to Understand Hypoxia-Inducible Factor Function 136
1. Introduction 137
2. Hypoxia Response Pathway Genes and Development 139
2.1. HIF-alpha subunits 139
2.2. HIF-beta/ARNT subunits 140
2.3. pVHL 141
2.4. PHDs 141
2.5. VEGF 142
3. HIF in Physiology 142
3.1. HIF-1alpha heterozygotes 142
3.2. Conditional knockouts using the loxP/cre recombinase system 143
3.3. Liver 143
3.4. Myeloid lineage 143
3.5. Lymphocytes 144
3.6. Hematopoiesis 144
3.7. Skeletal muscle 144
3.8. Heart and cardiovascular system 145
3.9. Mammary gland 145
3.10. Colon 145
3.11. Chondrocytes 145
3.12. Skin 146
3.13. Motor neurons and brain 146
4. HIF Function in Tumor Biology 147
4.1. First insights-xenografts 147
4.2. Astrocytoma 148
4.3. Mammary carcinogenesis 148
4.4. pVHL and VHL disease 148
5. Summary 149
References 74
Chapter 6: The Silencing Approach of the Hypoxia-Signaling Pathway 156
1. A Brief History of RNAi 157
2. The Hypoxia-Signaling Pathway 158
3. HIF-alpha Stability 159
3.1. The family of the HIF prolyl-hydroxylases 159
3.2. Acetylation/deacetylation (ARD1/HDAC1) 163
4. HIF Activity 163
4.1. The factor-inhibiting HIF 163
5. HIF-1/HIF-2 Target Gene Specificity 165
6. RNAi as a New Potential Therapeutic Strategy 165
Acknowledgments 167
References 167
Chapter 7: Cellular and Developmental Adaptations to Hypoxia: A Drosophila Perspective 172
1. Introduction 173
2. Drosophila melanogaster as a Model System to Study Physiological Responses to Hypoxia 173
3. Experimental Advantages of the Model System 174
4. The Drosophila Respiratory System 175
5. Occurrence of a Drosophila System Homologous to Mammalian HIF 177
6. Regulation of Sima by Oxygen Levels 180
7. Role of Sima and Fatiga in Drosophila Development 181
8. Hypoxia-Inducible Genes and the Adaptation of Drosophila to Oxygen Starvation 183
9. Regulation of Sima by the PI3K and TOR Pathways 183
10. Role of the HIF System in Growth Control and Cell Size Determination 185
11. Concluding Remarks 187
Acknowledgments 188
References 188
Section II: Erythropoietin 194
Chapter 8: Constitutively Overexpressed Erythropoietin Reduces Infarct Size in a Mouse Model of Permanent Coronary Artery Ligation 196
1. Introduction 197
2. Material and Methods 198
2.1. Animals and surgery 198
2.2. Determination of infarct size and immunohistochemical analysis 199
3. Results 200
3.1. EPO plasma levels and EPO-R expression in the myocardium 200
3.2. Cardiac protection by EPO 202
3.3. Infarct size correlates with WT1 staining 202
4. Discussion 202
Acknowledgments 74
References 74
Chapter 9: Use of Gene-Manipulated Mice in the Study of Erythropoietin Gene Expression 206
1. Introduction 207
2. Materials 210
2.1. Genetic manipulation of mouse lines 210
2.2. Hypoxic chamber 210
2.3. BAC clones 211
2.4. Plasmids and bacterial strains for recombination of BAC clones 211
2.5. Antibodies and immunohistochemistry 211
3. Methods and Results 212
3.1. Real-time and noninvasive monitoring of EPO activity in vivo 212
3.2. Analysis of erythropoietic and non-erythropoietic function of EPO-EPOR pathway in vivo 214
3.3. Transgenic mouse-expressing GFP under the control of Epo gene regulatory region 215
3.4. Regulatory region sufficient for in vivo Epo gene expression 217
3.5. Identification of the REP cell 218
3.6. Essential cis-elements for cell type-specific and inducible Epo gene expression in vivo 220
4. Conclusion 222
Acknowledgments 74
References 74
Chapter 10: Control of Erythropoietin Gene Expression and its Use in Medicine 228
1. Introduction 229
2. Native EPO Gene Expression and its Pharmacologic Stimulation 1
3. EPO Gene Transfer 1
4. Recombinant EPOS 1
5. Conclusions 239
References 74
Chapter 11: Role of Hypoxia-Inducible Factor-2alpha in Endothelial Development and Hematopoiesis 248
1. Introduction 249
2. Vasculogenesis/Angiogenesis and HIFS 249
2.1. HIF-2a null mice 250
2.2. HIF-2alpha knockdown mice 250
2.3. HIF-1alpha null mice 252
2.4. HIF-1alpha null EC 253
3. HIF-1beta/ARNT Null Mice 253
4. Neovascularization and HIFS 254
4.1. HIF-2alpha knockdown mice 254
4.2. HIF-1alpha null EC 259
5. Hematopoiesis and HIFS 260
5.1. HIF-2alpha null mice 260
5.2. HIF-2alpha knockdown mice 261
5.3. HIF-1a null mice 262
5.4. HIF-1beta/ARNT null mice 262
6. Conclusion 263
References 74
Section III: Hypoxia and Adaptation 268
Chapter 12: Organ Protection by Hypoxia and Hypoxia-Inducible Factors 270
1. Introduction 271
2. From Ischemic to Hypoxic Preconditioning 1
3. Hypoxia-Inducible Transcription Factors 1
4. Strategies to Activate HIF and HIF Target Genes 1
4.1. Hypoxic hypoxia and carbon monoxide 1
4.2. Inhibition of HIF prolyl hydroxylases 278
4.3. Additional strategies to activate the HIF pathway 279
5. Hypoxic Preconditioning and HIF 280
5.1. Protective role of HIF target genes 280
5.2. HIF activation and organ protection 281
6. HIF in Chronic Hypoxic/Ischemic Diseases 1
7. Conclusions and Perspectives 1
References 74
Chapter 13: Hypoxia and Regulation of Messenger RNA Translation 296
1. Introduction 297
2. Changes in Global mRNA Translation During Hypoxia 1
2.1. Kinetics and oxygen dependency 299
2.2. Influence of genetic background 299
3. Molecular Mechanisms that Regulate mRNA Translation During Hypoxia 1
3.1. Translational regulation during hypoxia by eIF2alpha phosphorylation 301
3.2. Translational regulation during hypoxia by eIF4F complex availability 303
3.3. Translational regulation by eEF2 phosphorylation 304
4. Methods Employed to Study mRNA Translation During Hypoxia 305
4.1. Protein synthesis 305
4.2. The polysome assay 305
4.3. Enzymatic activity of reporter constructs 316
5. Protocols 317
5.1. 35S methionine labeling 317
5.2. Polysome fractionation 318
References 74
Chapter 14: Hypoxia and the Unfolded Protein Response 324
1. Introduction 325
1.1. Mechanisms of cellular adaptation to low oxygen environment 325
1.2. Causes of ER stress and UPR 326
1.3. The PERK-eIF2alpha-ATF4 arm of the UPR 327
1.4. The IRE1-XBP1 arm of the UPR 328
1.5. The ATF6 pathway 329
1.6. Consequences of aberrant UPR induction for tumor formation 330
2. Methods Employed in Detecting Hypoxic Induction of ER Stress 331
2.1. Events proximal to ER stress 331
2.2. Events distal to ER stress 335
Acknowledgments 74
References 74
Section IV: Hypoxia and Tumor Biology 344
Chapter 15: Tumor Hypoxia in Cancer Therapy 346
1. Hypoxia in Human Tumors 347
2. The Dynamic Nature of Hypoxia in Tumors 349
3. Consequences of Tumor Hypoxia for Cancer Treatment 349
4. Size of the Oxygen Effect with Radiation 351
5. The Influence of Tumor Hypoxia on Cancer Treatment by Radiotherapy 352
6. Influence of Tumor Hypoxia on Response to Chemotherapy 356
7. Exploiting Hypoxia in Cancer Treatment 357
7.1. Hypoxic cytotoxins 357
7.2. Hypoxia-selective gene therapy 360
7.3. Targeting HIF-1 361
7.4. Exploiting tumor necrosis with obligate anaerobes 363
References 74
Chapter 16: HIF Gene Expression in Cancer Therapy 372
1. Introduction 373
2. Experimental Procedures 375
2.1. Induction by hypoxia or chemical mimetics 375
2.2. Genetic mutations 376
2.3. HIF hydroxylation and other posttranslational modifications 377
2.4. HIF inhibitors 378
2.5. RNA interference 380
2.6. HIF knockout mice 381
2.7. Somatic cell HIF knockout 381
2.8. HIF activity (reporter assays) 382
2.9. HIF responsive elements 382
2.10. RNA analysis 382
2.11. Quantitative RT-PCR 383
2.12. Western blot analysis 383
2.13. Chromatin immunoprecipitation 383
2.14. HEEBO microarray analysis 384
3. Conclusions 386
Acknowledgments 74
References 74
Chapter 17: Analysis of Hypoxia-Inducible Factor 1alpha Expression and its Effects on Invasion and Metastasis 396
1. Introduction 396
2. Protocol 1: HIF-1alpha Immunohistochemistry 398
3. Protocol 2: Invasion Assay 399
4. Protocol 3: Transepithelial Resistance Measurement of Cell-Cell Adhesion 400
5. Protocol 4: Analysis of MRNA Expression by QRT-PCR 400
References 74
Chapter 18: Macrophage Migration Inhibitory Factor Manipulation and Evaluation in Tumoral Hypoxic Adaptation 404
1. Introduction 405
2. Modulation of MIF Levels by Targeted shRNAs and Assessment of Knockdown Efficiency 406
2.1. MIF-specific shRNA transfection 406
2.2. Assessment of MIF knockdown and associated phenotypes by RT-PCR 408
2.3. Assessment of MIF knockdown and associated loss of HIF-alpha stability by Western blotting 409
2.4. Enzymatic analyses 410
3. Analysis of MIF-Dependent CSN5 and COP9 Signalosome Function 411
3.1. CSN5 co-immunoprecipitations 412
3.2. CSN-dependent deneddylation 412
3.3. Determination of CSN-associated versus-disassociated CSN5 413
4. Determination of Tumor-Associated MIF Expression and MIF Polymorphic Disparity 413
4.1. Immunohistochemistry of MIF and CSN5 tumor expression levels and correlation to hypoxic adaptation 414
4.2. MIF plasma analysis and genomic DNA extraction 414
4.3. Genotyping of MIF -173 G/C and MIF5-8 CATT repeats from human samples 415
5. Conclusions 416
References 416
Chapter 19: The von Hippel-Lindau Tumor Suppressor Protein: An Update 420
1. Introduction 421
2. Regulation of Epithelial Differentiation by pVHL 422
2.1. E-cadherin 422
2.2. The primary cilium 422
3. Crosstalk between c-Met and VHL 423
4. Regulation of Neuronal Apoptosis by pVHL 424
5. Possible Links Between p53 and pVHL 425
6. Regulation of pVHL by Phosphorylation 425
7. Polyubiquitylation of pVHL 426
8. Mouse Models for Studying pVHL Function 426
References 74
Chapter 20: Hypoxia-Inducible Factor 1 Inhibitors 434
1. Introduction 435
2. Cell-Based High Throughput Screens 436
2.1. Cell-based HTS protocol 438
2.2. Validation of active "hits" from HTS 439
2.3. Small molecule inhibitors of HIF-1 identified in cell-based assays 441
3. Cell-Free Assays 442
3.1. Inhibition of HIF-1 DNA binding 442
3.2. Inhibition of protein-protein interaction 444
3.3. Inhibition of HIF-1 transcriptional activity 446
4. Bioassay-Directed Isolation of Natural Product HIF-1 Inhibitors 447
5. Conclusions 448
Acknowledgments 74
References 449
Section V: Hypoxia and Inflammatory Mediators 452
Chapter 21: Regulation of Hypoxia-Inducible Factors During Inflammation 454
1. Introduction 455
2. Regulation of HIF at the Transcriptional Level 457
3. Regulation of HIF at the Translational Level 459
4. Regulation of HIF-1alpha at the Posttranslational Level 460
5. Regulation of HIF-1 Activity 462
6. Perspectives 463
7. Conclusions 463
Acknowledments 74
References 74
Chapter 22: Superoxide and Derived Reactive Oxygen Species in the Regulation of Hypoxia-Inducible Factors 470
1. Introduction 471
2. Reactive Oxygen Species Act as Signaling Molecules 472
3. HIFs are Sensitive to Oxygen 473
4. Reactive Oxygen Species Modulate HIF 474
5. How are HIFs Regulated by Reactive Oxygen Species? 476
5.1. Regulation of HIF-alpha synthesis by reactive oxygen species? 476
5.2. Direct regulation of HIF-a by reactive oxygen species 477
5.3. Regulation of HIF by reactive oxygen species via interference with a regulatory signaling pathway 477
6. Summary 480
7. Methods 480
8. The Cytochrome C Reduction Assay for Detection of Extracellular Reactive Oxygen Species 480
9. Chemiluminescence Assay for Detection of Extracellular Reactive Oxygen Species 481
10. Measuring Intracellular Production of Reactive Oxygen Species using Fluorescent Dyes 482
10.1. DCF fluorescence 483
10.2. Hydroethidine fluorescence 483
10.3. Dihydrorhodamine fluorescence 484
10.4. ROS measurements in tissues 485
11. Detection of Reactive Oxygen Species by Electron Paramagnetic Resonance 485
Acknowledgments 74
References 487
Chapter 23: Genetics of Mitochondrial Electron Transport Chain in Regulating Oxygen Sensing 496
1. Introduction 497
2. Detecting HIF-1alpha Protein Levels 498
2.1. Background 498
2.2. Materials 499
2.3. Equipment 500
2.4. Methods 500
3. Detecting Intracellular ROS Levels 501
3.1. Background 501
3.2. Materials 502
3.3. Equipment 502
3.4. Methods 502
4. Method 1: Examining Hypoxic Stabilization of HIF-1alpha Protein in Cells Containing RNAI against the Rieske Fe-S Protein 503
4.1. Background 503
4.2. Materials 503
4.3. Equipment 504
4.4. Methods 504
5. Method 2: Examining the Role of ROS Generated from Mitochondrial Electron Transport in Hypoxic Stabilization of HIF-1alpha Protein 507
5.1. Background 507
5.2. Materials 507
5.3. Methods 507
6. Concluding Remarks 508
Acknowledgments 74
References 74
Chapter 24: Hypoxia-Inducible Factor-1alpha Under the Control of Nitric Oxide 512
1. HIF-1 and Oxygen Sensing 513
2. Nitric Oxide: A Multifunctional Messenger 514
3. Accumulation of HIF-1alpha and Activation of HIF-1 by NO 516
4. Superoxide Stabilizes HIF-1alpha but Antagonizes NO Actions 519
5. Hypoxic Signal Transmission is Antagonized by NO 521
6. Summary and Conclusions 522
Acknowledgments 74
References 524
Chapter 25: Hypoxic Regulation of NF-kappaB Signaling 528
1. Background 529
2. Treatment Protocols for Cellular Hypoxia Studies 530
2.1. Exposure of cells to ambient atmospheric hypoxia 531
2.2. Inhibition of hydroxylases in cultured cells 531
2.3. Manipulation of NF-kappaB signaling in cultured cells 532
3. Measurement of NF-kappaB Activity in Cultured Cells 533
3.1. NF-kappaB-luciferase promoter-reporter assay 533
3.2. NF-kappaB DNA-binding assay 534
3.3. Immunoblotting analysis 537
3.4. Peptide pull-down assay 539
4. Summary/Conclusions 540
References 74
Author Index 542
Subject Index 584