Teresa PereiraCMB, Karolinska Institutet

2010-06-15

Reactive oxygen species and theHypoxia-inducible Factor

signaling pathway

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)

Heart and Lungs Brain

Cornea

Cartilage

Physiological oxygen levels

Avascular Tissues

5%O2 14%O2

21%O2

Hypoxia

Adaptive responses to hypoxia

Red blood cell production

Formation and dynamicregulation of blood vessels

Glucose andenergy metabolism

Autophagy

Cell migration

pH regulation

Erythropoietin

VEGF-A

GLUT1,3

CA9

BNIP3

E-cadherin

Hypoxia and pathology

Defective vascularisation leading to lowpO2 is a characteristic of a number of diseases - local hypoxia

Anemia-systemic hypoxia

Hypoxia and tumor development

Carmeliet, P. 2005, Oncology, 69

Hypoxia-inducible factor-1aHIF-1a protein stability is regulated by oxygen levels

HIF-1a

HIF-1aN CA BbHLH

A BbHLH

HIF-1b/Arnt

Von Hippel-Lindau Tumor Suppressor Gene

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.

Hershko, Cell Death Differ., 2005

Degradation of HIF-1a by pVHL is associated with the tumor supressor function of pVHL

Tanimoto et al., EMBO J, 2000,

Degradation of HIF-1a is regulated by two specific proline residues

Normoxia

Superfamily of iron II and 2-oxoglutaratedependent oxygenases.

1 822772584531N CPP

VHL

A BbHLH

Degradation

33191

OH OH

402 563

PHDs

NCB 2007, 3, 144-153

Normoxia

Iron II and 2-oxoglutaratedependent oxygenase

1 822772584531N CPP

VHL

A BbHLH

Degradation

33191

OH OH

402 563

PHDs

NOH

FIH

Hydroxylation of an asparagine residue in HIF-1a inhibits interaction with CBP at normoxia

HIF-a

O2 PHDsFe2+2-oxoglutarate

HIF-aP

P

OHOH

VHL

VHL

HIF-aP

P

OHOH

Proteasome

Oxygen-dependent Regulation of HIF-a Expression

HIF-a

O2 PHDsFe2+2-oxoglutarate

VHL

VHL

HRE

HIF-a

ARNT

Oxygen-dependent Regulation of HIF-a Expression

Coactivators

HIF-a

O2 PHDsFe2+2-oxoglutarate

VHLVHL

HRE

HIF-a

Oxygen-dependent Regulation of HIF-a Activity

HIF-aP

P

OHOH

HIF-aP

P

OHOH

HIF-a

ARNT

OH

N

HIF-aP

P

OHOH

Proteasome

FIH-1

O2

2-oxoglutarateFe2+

Coactivators

HIF-a

ARNT

OHN

Km- 100 mM

Prolyl hydroxylase activity decreases progressively withreduction of O2 levels HIF stabilization begins at 5% O2 and increases exponentially up to 0.5% O2

JBC, 2006, 281, 28712-20

Biochem. J. 2007, 405, 1-9

Increase in ROS production at hypoxia is paradoxical:concentration of O2 decrease at hypoxia O2 is a substrate for ROS production

PNAS, 1998, 95, 11715-720

Ebselen- glutathione peroxidase mimeticPDTC- thiol reductive agent pyrrolidine dithiocarbamater0 -cells lacking mitochondrial DNA-derived proteins

DCFH- 2’-7’-dichlorofluoresceinoxidized by H2O2 but not O2

.–

Generation of ROS in response to hypoxia

wikipedia

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 intensityratiometric

Allows measuring cytosolic thiol redox In live cells

Assessment of cytosolic ROS using a FRET sensor

Assessment of cytosolic ROS using a FRET sensor

Cell Metab, 2005, 1, 401-408

Circ Res, 2010, 106, 526-535

Assessment of the effect of hypoxia on redox signaling using a redox-sensitive ratiometric fluorescent

protein sensor RoGFP

GFP with two engineered cysteine thiolsexcitation maxima- 400 nm oxidized - 484 nm reduced

Cyto-RoGFP

Circ Res, 2010, 106, 526-535

mitochondrialintermembrane space

mitochondrialmatrix

Measuring ROS in hypoxia using RoGFP

JBC, 2000, 275, 25130-38

DFO-deferoxamineiron quelator

Stabilization of HIF-1a in response to hypoxia isdependent on ROS

Cell Death Differ, 2008, 15, 660-666

Electron transport chain

Cell Metab, 2005, 1, 393-399Cell Metab, 2005, 1, 401-408

Rotenone-complex I inhibitorMyxothiazol- complex III inhibitorStigmatelin- complex III inhibitor

Role of complex III on HIF-a stabilization

Exp Physiol 2006, 91, 807-819

Generation of ROS by complex III

Partial pressure of oxygen is reduced – mitochondrial electron transferfrom ubiquinol to cyt c1 by the Reiske iron-sulfur protein is delayedallowing electrons to bind to molecular oxygen forming O2

-

Qo

Qi

Cell Metab, 2005, 1, 393-399

HIF-a stabilization at hypoxia is dependent on Cyt C

HIF-a stabilization in hypoxia is dependent on Rieske iron-sulfur protein of complex III

Cell Metab, 2005, 1, 401-408

Measuring FeII and FeIII by EPR spectroscopy

Cell, 2004, 118, 781-794

How is prolyl hydroxylase activity affected by ROS?

-ROS may trigger signal transduction cascate-change PHDs disulfite bond-oxydize enzyme-bound iron

g=6 hemoproteinsg=4.3 free iron

g= 2.24, 2.01 and 1.93dioxygenases

Why do cancer cells use glycolysis instead of oxidative phophorylation to produce ATP?

Warburg effect

Clin Cancer Res 2007, 13, 789-794

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

Hypoxia and tumor development

Pyruvate dehydrogenase kinase 1 is a HIF-1 target gene

Pyruvate acetyl-CoAPDH

PDK1

Cell Metab 2006, 3, 177-185

HIF-1-induced PDK1 activity inhibits PDH resulting in decreased flux through the TCA cycle

Cell Metab 2006, 3, 177-185

72h hypoxia

Cell Metab 2006, 3, 177-185

Effect of PDK1 on hypoxia-induced ROS production

DCF fluorescence

FEBS letters, 581, 3582-3591, 2007Curr. Opinion Cell Biol. 19, 223-9, 2007

Cellular adaptation to hypoxia

Biochem. J. 2007, 405, 1-9

Cytochrome C oxidase subunit composition is regulated by O2 in yeast and human cells

COX5a high levels of O2COX5b low levels of O2

COX5b increases rate of electron transfer

COX4-1 expression increases production ofROS at hypoxia

siRNA COX4-2 leads to increases levels of ROS at hypoxia

To maintain the efficiency of respiration under conditions of low O2

Regulation of ROS production by the Hypoxia-induciblepathway

-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)

Cardiov Res 2008, 77, 463-470

Role of HIF-1a in the acute phase of ischaemic preconditioning: production of ROS

Thank you for your attention

naked mole rat

Model to cancer research

subterranean mole rat