Reactive oxygen species and the Hypoxia-inducible Factor signaling pathway Teresa Pereira CMB, Karolinska Institutet 2010-06-15

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)

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

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

Hypoxia and pathology Defective vascularisation leading to low pO2 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-1a HIF-1a protein stability is regulated by oxygen levels HIF-1a N C A B bHLH HIF-1b/Arnt HIF-1a

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.

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 PHDs VHL Degradation OH OH P P bHLH A B N C 1 91 331 531 584 772 822 402 563 Superfamily of iron II and 2-oxoglutarate dependent oxygenases. NCB 2007, 3, 144-153

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 91 331 531 584 772 822 402 563 Iron II and 2-oxoglutarate dependent oxygenase

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

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

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

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, 28712-20

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

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, 11715-720

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

Assessment of cytosolic ROS using a FRET sensor Cell Metab, 2005, 1, 401-408

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, 526-535

Measuring ROS in hypoxia using RoGFP mitochondrial intermembrane space mitochondrial matrix Circ Res, 2010, 106, 526-535

Stabilization of HIF-1a in response to hypoxia is dependent on ROS DFO-deferoxamine iron quelator JBC, 2000, 275, 25130-38

Electron transport chain Cell Death Differ, 2008, 15, 660-666

Role of complex III on HIF-a stabilization Rotenone-complex I inhibitor Myxothiazol- complex III inhibitor Stigmatelin- complex III inhibitor Cell Metab, 2005, 1, 393-399 Cell Metab, 2005, 1, 401-408

Generation of ROS by complex III Qo 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, 807-819

HIF-a stabilization at hypoxia is dependent on Cyt C Cell Metab, 2005, 1, 393-399

HIF-a stabilization in hypoxia is dependent on Rieske iron-sulfur protein of complex III Cell Metab, 2005, 1, 401-408

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, 781-794

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, 789-794

Hypoxia and tumor development

Pyruvate dehydrogenase kinase 1 is a HIF-1 target gene acetyl-CoA PDH 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

Effect of PDK1 on hypoxia-induced ROS production 72h hypoxia DCF fluorescence Cell Metab 2006, 3, 177-185

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

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

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)

Role of HIF-1a in the acute phase of ischaemic preconditioning: production of ROS Cardiov Res 2008, 77, 463-470

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