1. METOXIA Course
HIF 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.
2. 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.
3. 1660: Robert Boyle - effect of air pump
Mammals and candles both need oxygen
4. Why do we need oxygen?
RESPIRATION
glucose + 6 O2 6H2O + 6CO2
DG°’ = -686 kcal 36 ATP
GLYCOLYSIS
glucose 2 x lactate
DG°’ = -47 kcal 2 ATP
5. Oxygen in vivo
Breathing-airways-
Cytochome oxidase lungs-heart-blood
Heme based enzyme 140-100 mmHg
Km for oxygen < 1 mmHg
Blood vessels,
tissue
100-30 mmHg
Cell-
mitochondrion
30-1 mmHg
9. Metabolism is coordinated with oxygenation
This course is funded with the support of the METOXIA project under the FP7 Programme.
10. Tissue culture
20% oxygen: 5% CO2
at sea level = 152 mmHg
Diffusion (with some convection) - Fick’s law
Consumption by cells - mitochondria and other
processes
Do not need mitochondria!
11. Metzen et al, Respiration Physiology , 1995
24 hours, unstirred, 52 mm height
12. Many other reactions use are more sensitive
to oxygen than cytochrome c oxidase!
Km, µM
Cytochrome c oxidase <0.5
Xanthine oxidase 50-240
Collagen prolyl hydroxylase 30-72
Lysine hydroxylase
Heme oxygenases
Nitric oxide synthases
Lipoxygenases 40
Cholesterol 7 -monooxygenase 20
Calcidiol 1-monoxygenase
Dopamine hydroxylase 550
Tissue oxygenation 10-60 mmHg ~ 13-78 µM
13. 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.
14. RED BLOOD CELLS
20,000,000,000,000 red blood cells
Volume about 2 litres
Carries 99% of the oxygen in the blood
bound to haemoglobin.
~65% of iron in the body
No nucleus or genetic material
Production is tightly physiologically regulated via EPO
23. The different HIF subunits
HIF-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
development
HIF-3 : multiple isoforms, some of which
have a dominant negative effect
24. The different HIF prolyl
hydroxylases
PHD2: main PHD activity in most cells
required for development in mice
controls EPO production in humans
PHD3: highly inducible by hypoxia
25. 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.
26. Tools for analysing HIF pathway
Altering oxygen
decreases PHD, FIH activity
PHD 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!
27. Tools for analysing HIF pathway
Genetic approaches in cell culture
RNAi
stable knockdown may be difficult to achieve
?minimal levels of VHL required
Cells with deletion of a component
VHL defective human cells
CHO cells lacking VHL, lacking HIF
mouse embryonic fibroblasts
stable expression
28. Tools for analysing HIF pathway
Genetic approaches in whole organisms
mice: knockouts, inducible
knockouts, hypomorphs
humans: vhl, phd2
worms
fish
flies
Power of comparison across organisms
eg Trichoplax adherens
29. Inputs to the HIF signal
HIF- mRNA
Translation efficiency
Level of PHD enzyme expression
Amount of oxygen
? iron, ascorbate, 2OG
? fumarate, succinate
Other post translational modifications of HIF-
30. Outputs from the HIF signal
Direct transcription effects of HIF on HRE’s
Highly influenced by cell type and context
Interacts with other pathways
Influences epigenetic control
31. Cellular adaptation and HIF
HIF-1
GLUCOSE GLUCOSE
HIF-1
OXYGEN PYRUVATE
Mitochondrial respiration
LACTATE
ATP
32. Angiogenes
Energy metabolism
is
Glucose transporters
VEGF
Blood oxygen Glycolytic enzymes
PLGF
Erythropoietin Mitochondrial respiration
PDGF
HIF-1 coactivators
Transcription
HIF-1
HRE
Vasomotor Cell
NOS isoforms proliferation/survival
Endothelins IGF/IGF-BPs
Adrenoreceptors Cyclin G2
Tyrosine Nip/Nix
hydroxylase
Metal Matrix metabolism
transport Collagens/prolyl
Transferrin hydroxylases
Caeruloplasmi Transglutaminase
33. Effect of HIF may be “good” or “bad”……..
Cell and tissue survival Cancer progression
38. VHL restores the primary cilium in RCC cells
Esteban et al JASN 2006
39. 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
41. Very large numbers of foci of HIF activation
Mandriota et al Cancer Cell 2002
42. Loss of VHL reduces E Cadherin expression
CAIX ECAD CAIX ECAD
Normal kidney
Tumour
CAIX ECAD
Esteban et al, Cancer Res 2006
43. Re-expression of VHL restores E Cadherin
expression
VHL
RCC - RCC +
Esteban et al, Cancer Res 2006
44. siRNA for HIF-1 and HIF-2 rescues E-cadherin
Esteban et al, Cancer Res 2006
45. In renal epithelium
Independent effects of VHL loss on tight
junction, adherens junction, cilium
Effects substantially mediated via HIF
Effects are indirect, involve several
mediators, balance of HIF-1 and HIF-2 is
variable
Interacts with other pathways
46. Second hit is very common
Minimal effect on proliferation
47. Progression – a progressive switch from HIF-1 to HIF-2?
Normal epithelium Cyst / adenoma / tumour Metastasis
HIF-1 HIF-1 + HIF-2 HIF-2
48. HIF1A inactivated in some CCRCC
Dalgliesh et al, 2010 3/407 mutations in HIF1A
Morris et al 2009 1/40 mutations in HIF1A
49. HIF will be activated in many settings
- but it is not necessarily beneficial
52. Selection and reproduction
POTOSI
4,100m
17th Century one of the largest cities in the world
53 years before the first child of Spanish parentage survived in Potosi
Colorado – birthweight falls 100g per 1,000 m
Worldwide decline in birthweights with altitude is lowest in longest resident groups
