METOXIA CourseHIF in cell biology and physiology Professor Patrick H. Maxwell 11th October 2012 This course is funded with the support of the METOXIA project under the FP7 Programme.
Learning objectives• Consider how oxygen varies in cell culture and in vivo• Understand the molecular basis of the HIF pathway• Understand the tools available for dissecting the role of HIF• Consider inputs contributing to HIF activation• Understand outputs resulting from HIF activation This course is funded with the support of the METOXIA project under the FP7 Programme.
1660: Robert Boyle - effect of air pumpMammals and candles both need oxygen
Why do we need oxygen? RESPIRATIONglucose + 6 O2 6H2O + 6CO2 DG°’ = -686 kcal 36 ATP GLYCOLYSIS glucose 2 x lactate DG°’ = -47 kcal 2 ATP
Oxygen in vivo Breathing-airways-Cytochome oxidase lungs-heart-bloodHeme based enzyme 140-100 mmHgKm for oxygen < 1 mmHg Blood vessels, tissue 100-30 mmHg Cell- mitochondrion 30-1 mmHg
Metabolism is coordinated with oxygenation This course is funded with the support of the METOXIA project under the FP7 Programme.
Tissue culture20% oxygen: 5% CO2 at sea level = 152 mmHgDiffusion (with some convection) - Fick’s lawConsumption by cells - mitochondria and other processesDo not need mitochondria!
Metzen et al, Respiration Physiology , 1995 24 hours, unstirred, 52 mm height
Many other reactions use are more sensitive to oxygen than cytochrome c oxidase! Km, µMCytochrome c oxidase <0.5Xanthine oxidase 50-240Collagen prolyl hydroxylase 30-72Lysine hydroxylaseHeme oxygenasesNitric oxide synthasesLipoxygenases 40Cholesterol 7 -monooxygenase 20Calcidiol 1-monoxygenaseDopamine hydroxylase 550Tissue oxygenation 10-60 mmHg ~ 13-78 µM
Learning objectives 1• how oxygen varies in cell culture and in vivo – Not simple to relate in cell culture to in vivo setting – In vivo oxygen concentration is very variable – Oxygen is necessary for other reactions besides respiration – Many aspects of anatomy and physiology are consistent with matching oxygen supply and demand This course is funded with the support of the METOXIA project under the FP7 Programme.
RED BLOOD CELLS20,000,000,000,000 red blood cellsVolume about 2 litresCarries 99% of the oxygen in the bloodbound to haemoglobin.~65% of iron in the bodyNo nucleus or genetic materialProduction is tightly physiologically regulated via EPO
The different HIF subunitsHIF-1 : the prototypical HIF subunit – although HIF-2 more important for EPO!HIF-2 : more restricted range of targets, more cell-type specific, not completely essential for mouse developmentHIF-3 : multiple isoforms, some of which have a dominant negative effect
The different HIF prolyl hydroxylasesPHD2: main PHD activity in most cells required for development in mice controls EPO production in humansPHD3: highly inducible by hypoxia
Learning objectives 2• The HIF pathway – Key steps are • Prolyl hydroxylation • Capture by VHL • Ubiquitylation • Proteasomal destruction This course is funded with the support of the METOXIA project under the FP7 Programme.
Tools for analysing HIF pathwayAltering oxygen decreases PHD, FIH activityPHD inhibitors iron chelators (eg desferrioxamine, 100µM) cobalt (eg CoCl2 100µM) 2OG analogues (eg DMOG, 1 mM)PHD inhibitors are HIF activators but are not hypoxia mimetics!
Tools for analysing HIF pathwayGenetic approaches in cell cultureRNAi stable knockdown may be difficult to achieve ?minimal levels of VHL requiredCells with deletion of a component VHL defective human cells CHO cells lacking VHL, lacking HIF mouse embryonic fibroblasts stable expression
Tools for analysing HIF pathwayGenetic approaches in whole organisms mice: knockouts, inducible knockouts, hypomorphs humans: vhl, phd2 worms fish fliesPower of comparison across organisms eg Trichoplax adherens
Inputs to the HIF signalHIF- mRNATranslation efficiencyLevel of PHD enzyme expressionAmount of oxygen ? iron, ascorbate, 2OG ? fumarate, succinateOther post translational modifications of HIF-
Outputs from the HIF signalDirect transcription effects of HIF on HRE’sHighly influenced by cell type and contextInteracts with other pathwaysInfluences epigenetic control
Cellular adaptation and HIF HIF-1GLUCOSE GLUCOSE HIF-1OXYGEN PYRUVATE Mitochondrial respiration LACTATE ATP
Angiogenes Energy metabolism is Glucose transporters VEGF Blood oxygen Glycolytic enzymes PLGF Erythropoietin Mitochondrial respiration PDGF HIF-1 coactivators Transcription HIF-1 HREVasomotor CellNOS isoforms proliferation/survivalEndothelins IGF/IGF-BPsAdrenoreceptors Cyclin G2Tyrosine Nip/Nixhydroxylase Metal Matrix metabolism transport Collagens/prolyl Transferrin hydroxylases Caeruloplasmi Transglutaminase
Effect of HIF may be “good” or “bad”……..Cell and tissue survival Cancer progression
VHL restores the primary cilium in RCC cells Esteban et al JASN 2006
E-Cadherin• Epithelial cell adhesion molecule LOSS OF• Maintains tissue integrity CELL-CELL ADHESION and architecture• Interacts with the beta catenin-Wnt signalling pathway• Reduced expression is PROMOTE TUMOUR seen in many carcinomas GROWTH AND INVASION
Very large numbers of foci of HIF activationMandriota et al Cancer Cell 2002
Loss of VHL reduces E Cadherin expressionCAIX ECAD CAIX ECAD Normal kidney TumourCAIX ECAD Esteban et al, Cancer Res 2006
Re-expression of VHL restores E Cadherin expression VHL RCC - RCC + Esteban et al, Cancer Res 2006
siRNA for HIF-1 and HIF-2 rescues E-cadherin Esteban et al, Cancer Res 2006
In renal epitheliumIndependent effects of VHL loss on tight junction, adherens junction, ciliumEffects substantially mediated via HIFEffects are indirect, involve several mediators, balance of HIF-1 and HIF-2 is variableInteracts with other pathways
Second hit is very commonMinimal effect on proliferation
Progression – a progressive switch from HIF-1 to HIF-2? Normal epithelium Cyst / adenoma / tumour Metastasis HIF-1 HIF-1 + HIF-2 HIF-2
HIF1A inactivated in some CCRCCDalgliesh et al, 2010 3/407 mutations in HIF1AMorris et al 2009 1/40 mutations in HIF1A
HIF will be activated in many settings- but it is not necessarily beneficial
Selection and reproduction POTOSI 4,100m17th Century one of the largest cities in the world53 years before the first child of Spanish parentage survived in PotosiColorado – birthweight falls 100g per 1,000 mWorldwide decline in birthweights with altitude is lowest in longest resident groups