1. Reactive oxygen species and the Hypoxia-inducible Factor
signaling pathway
Teresa Pereira
CMB, Karolinska Institutet

2. Normoxia - O2 available is in balance with
the demand (21% O2 in the lab)
Hypoxia - unbalance between oxygen supply
and demand (1% O2 in the lab)

3. Physiological oxygen levels
Heart and Lungs
Brain
Avascular Tissues
Cornea
5%O2
14%O2
21%O2
Cartilage

4. Adaptive responses to hypoxia
Glucose and
energy metabolism
GLUT1,3
Hypoxia
Red blood cell
production
Cell migration
E-cadherin
Erythropoietin
Formation and dynamic
regulation of
blood vessels
Autophagy
BNIP3
VEGF-A
pH regulation
CA9

5. Hypoxia and pathology Defective vascularisation leading to low
pO2 is a characteristic of a number of
diseases - local hypoxia
Anemia-systemic hypoxia

6. Hypoxia and tumor development
Carmeliet, P. 2005, Oncology, 69

7. Hypoxia-inducible factor-1a
HIF-1a protein stability is regulated by oxygen levels
HIF-1a N C A B
bHLH
HIF-1b/Arnt
HIF-1a

8. Hershko, Cell Death Differ., 2005
Von Hippel-Lindau Tumor Suppressor Gene
Hershko, Cell Death Differ., 2005
Mutated in VHL disease- hereditary cancer syndrome:
retinal and CNS hemangioblastomas
renal cell carcinomas and
pheochromocytomas.
Mutated in sporadic renal cell carcinomas and hemangioblastomas.
Hypervascularized tumors.
Constitutive expression of VEGF in VHL inactivated cells.

9. Degradation of HIF-1a by pVHL is associated with the tumor supressor function of pVHL
Tanimoto et al., EMBO J, 2000,

10. Degradation of HIF-1a is regulated by two specific proline residues
Normoxia
PHDs
VHL
Degradation OH OH P P
bHLH A B N C 1 91
331
531
584
772
822
Superfamily of iron II and 2-oxoglutarate
dependent oxygenases.
NCB 2007, 3,

11. Hydroxylation of an asparagine residue in HIF-1a inhibits
interaction with CBP at normoxia
Normoxia
PHDs
FIH
VHL
Degradation OH OH N OH P P
bHLH A B N C 1 91
331
531
584
772
822
Iron II and 2-oxoglutarate
dependent oxygenase

12. Oxygen-dependent Regulation of HIF-a Expression
PHDs
Fe2+
2-oxoglutarate
HIF-a P OH
HIF-a P OH
HIF-a
VHL
VHL
Proteasome

13. Oxygen-dependent Regulation of HIF-a Expression
PHDs
Fe2+
2-oxoglutarate
HIF-a
HIF-a
VHL
ARNT
VHL
Coactivators
HRE

14. Oxygen-dependent Regulation of HIF-a Activity
PHDs
Fe2+
2-oxoglutarate
FIH-1 O2
2-oxoglutarate
Fe2+
HIF-a P OH
HIF-a P OH
HIF-a
HIF-a
Coactivators
HIF-a P OH
VHL
VHL
HIF-a
ARNT OH N
HIF-a
ARNT OH N
Proteasome
HRE

15. Prolyl hydroxylase activity decreases progressively with
reduction of O2 levels
HIF stabilization begins at 5% O2 and increases
exponentially up to 0.5% O2
Km- 100 mM
JBC, 2006, 281,

16. Increase in ROS production at hypoxia is paradoxical:
concentration of O2 decrease at hypoxia
O2 is a substrate for ROS production
Biochem. J. 2007, 405, 1-9

17. Generation of ROS in response to hypoxia
DCFH- 2’-7’-dichlorofluorescein
oxidized by H2O2 but not O2.–
Ebselen- glutathione peroxidase mimetic
PDTC- thiol reductive agent pyrrolidine dithiocarbamate
r0 -cells lacking mitochondrial DNA-derived proteins
PNAS, 1998, 95,

18. Assessment of cytosolic ROS using a FRET sensor
CFP-69 aa cysteine-containing from the
redox-regulated HSP-33- YFP
Oxidation of cysteine thiols causes
separation of the CFP and YFP – increase in
CFP intensity and decrease in YFP intensity
ratiometric
Allows measuring cytosolic thiol redox
In live cells
wikipedia

19. Assessment of cytosolic ROS using a FRET sensor
Cell Metab, 2005, 1,

20. Assessment of the effect of hypoxia on redox signaling
using a redox-sensitive ratiometric fluorescent
protein sensor RoGFP
Cyto-RoGFP
GFP with two engineered cysteine thiols
excitation maxima- 400 nm oxidized
- 484 nm reduced
Circ Res, 2010, 106,

21. Measuring ROS in hypoxia using RoGFP
mitochondrial
intermembrane
space
mitochondrial
matrix
Circ Res, 2010, 106,

22. Stabilization of HIF-1a in response to hypoxia is
dependent on ROS
DFO-deferoxamine
iron quelator
JBC, 2000, 275,

23. Electron transport chain
Cell Death Differ, 2008, 15,

24. Role of complex III on HIF-a stabilization
Rotenone-complex I inhibitor
Myxothiazol- complex III inhibitor
Stigmatelin- complex III inhibitor
Cell Metab, 2005, 1,
Cell Metab, 2005, 1,

25. Generation of ROS by complex IIIQo Qi
Partial pressure of oxygen is reduced – mitochondrial electron transfer
from ubiquinol to cyt c1 by the Reiske iron-sulfur protein is delayed
allowing electrons to bind to molecular oxygen forming O2-
Exp Physiol 2006, 91,

26. HIF-a stabilization at hypoxia is dependent on Cyt C
Cell Metab, 2005, 1,

27. HIF-a stabilization in hypoxia is dependent on
Rieske iron-sulfur protein of complex III
Cell Metab, 2005, 1,

28. How is prolyl hydroxylase activity affected by ROS?
-ROS may trigger signal transduction cascate
-change PHDs disulfite bond
-oxydize enzyme-bound iron
g=6 hemoproteins
g=4.3 free iron
g= 2.24, 2.01 and 1.93
dioxygenases
Measuring FeII and FeIII by EPR spectroscopy
Cell, 2004, 118,

29. Why do cancer cells use glycolysis instead of
oxidative phophorylation to produce ATP?
Warburg effect
2 ATP versus 38 ATPs
cytochrome oxidase activity is only limited by O2 availability
when O2 is lower than 1 mM (0.1% O2)
support cell growth- pyruvate used in lipid synthesis for membrane assembly
Clin Cancer Res 2007, 13,

30. Hypoxia and tumor development

31. Pyruvate dehydrogenase kinase 1 is a HIF-1 target gene
acetyl-CoA
PDH
PDK1
Cell Metab 2006, 3,

32. HIF-1-induced PDK1 activity inhibits PDH resulting in
decreased flux through the TCA cycle
Cell Metab 2006, 3,

33. Effect of PDK1 on hypoxia-induced ROS production
72h hypoxia
DCF fluorescence
Cell Metab 2006, 3,

34. Cellular adaptation to hypoxia
FEBS letters, 581, , 2007
Curr. Opinion Cell Biol. 19, 223-9, 2007

35. Cytochrome C oxidase subunit composition is
regulated by O2 in yeast and human cells
To maintain the efficiency of respiration under conditions of low O2
COX5a high levels of O2
COX5b low levels of O2
COX5b increases rate of electron transfer
COX4-1 expression increases production of
ROS at hypoxia
siRNA COX4-2 leads to increases levels of ROS
at hypoxia
Biochem. J. 2007, 405, 1-9

36. Regulation of ROS production by the Hypoxia-inducible
pathway
-induction of glycolytic enzymes and LDH
-induction of PDK1 –reducing flux through the TCA cycle
-induction of COX4-2 and inhibition of COX4-1 –efficient respiration
-inhibition of genes involved in mitochondria biogenesis (PGC1a)

37. Role of HIF-1a in the acute phase of ischaemic
preconditioning: production of ROS
Cardiov Res 2008, 77,

38. Thank you for your attention
naked mole rat
subterranean mole rat
Model to cancer research