Mechanisms of Gene Regulation and Signal! Transduction in Hypoxia! Lorenz Poellinger! Dept. of Cell and Molecular Biology! Karolinska Institutet, Stockholm, Sweden!
Normoxia - O 2 availability is in balance with! the demand (21% O 2 )! Hypoxia - unbalance between oxygen supply! and demand (1% O 2 )!
Hypoxia Inducible Factors (HIFs) Mediate Adaptive Responses to Hypoxia! Oxygen homeostasis O 2 supply O 2 consumption Hypoxia HIFs O 2 supply O 2 consumption Pathological conditions: Ischemia (myocardial infarction or stroke) cancer pulmonary and hematological diseases kidney diseases (acute kidney injury, chronic kidney disease and diabetic kidney) wound healing, inflammation and infection Physiological conditions: developing embryo (normal fetal development and stem cell differentiation)
Adaptive responses to hypoxia Glucose and! energy metabolism! Red blood cell! production Hypoxia Cell migration Formation and dynamic! regulation of! blood vessels! Cell Differentiation,! Growth and apoptosis Matrix metabolism
HIF-1 target genes Red blood cell production! Erythropoietin Iron metabolism! Transferrin (transport),! Transferrin receptor (uptake)! Cerulopasmin Formation and dynamic regulation! of blood vessels! VEGF, Flt1 (VEGF receptor 1),! Plasminogen activator inhibitor-1,! Nitric oxide synthase-2 (NO production)! Glucose and energy metabolism! Glucose transporter 1 and 3,! Hexokinase 1 and 2,! Aldolase A and C,! Phosphoglycerate kinase 1, Enolase 1,! Lactate dehydrogenase A,! Cellular differentiation,! proliferation and viability! TGF-β3, p21, Nip3, Cyclin G2! Differentiated embryo chondrocyte 1 ph regulation! Carbonic anhydrase 9 and 12 Matrix metabolism! Prolyl-4-hydroxylase-α1,! Collagen type V-α1
Hypoxia and pathologyh! Brain Heart and Lungs Defective vascularisation leading to low! po2 is a characteristic of a number of! diseases - local hypoxia! Liver Anemia-systemic hypoxia!
Hypoxia and tumor development!
Hypoxia-inducible factors! Biochem. Pharm. 73, 450-7, 2007!
HIF-1α protein stability is regulated by oxygen levels! bhlh A B HIF-1α N C bhlh A B HIF-1β/Arnt HIF-1α!
Von Hippel-Lindau Tumor Suppressor Gene Hershko, Cell Death Differ., 2005, 12, 1191-1197! 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-1α by pvhl is associated with! the tumor supressor function of pvhl
Degradation of HIF-1α is regulated by two specific proline! residues! Normoxia Degradation OH P PHDs VHL bhlh A B N C 1 91 331 531 584 772 822 402 563! Superfamily of iron II and 2-oxoglutarate! dependent oxygenases.! OH P
Hershko, Cell Death Differ., 2005, 12, 1191-1197!
Cellular Adaptation to Hypoxia -mediated via hypoxia-inducible factors (HIFs)! -Three known members, HIF-1α, HIF-2α, and HIF-3α! -bhlh /PAS transcription factors! -Dimerization partner Arnt! -Complexes bind to hypoxia responsive elements (HREs)! -HIF protein stabilization at low oxygen concentrations (HIF-1α, HIF-2α, and HIF-3α)! -Derepression of HIF function at low oxygen concentrations (HIF-1α, and HIF-2α, but not HIF-3α)!
Different HIF Functions in Tumor Cells (neuroblastoma, breast cancer cells): - HIF-1: mediator of acute responses to hypoxia - HIF-2: mediator of long-term (chronic) responses to hypoxia
Differential Utilization of HIFs in Response to! Oxygen Shortage! HIF-1α 1% O 2 HIF-2α 1% O 2 HIF-2α 5% O 2 HIF activity 4 Time (h) 72
Signal transduction via Nrf2 Taguchi et al., Genes to Cell 16, 123, 2011
Degradation of Nrf2 versus HIF1-alpha! Taguchi et al., Genes to Cell 16, 123, 2011
Hypoxia and ROS?? Studies in cardiomyocytes: - Hypoxia -> mitochondrial redox potential reduced -> low electron flow is accompanied by increased ROS generation - Stress, increased cardiac work load -> mitochondrial redox potential highly oxidized -> increased ROS levels
Hydroxylation of an asparagine residue in HIF-1α inhibits! interaction with CBP at normoxia! Normoxia Degradation PHDs FIH! VHL OH OH OH P P N bhlh A B N C 1 91 331 531 584 772 822
Inhibition of HIF-1α function at normoxia! FEBS letters, 581, 3582-3591, 2007!
Cellular adaptation to hypoxia! FEBS letters, 581, 3582-3591, 2007! Curr. Opinion Cell Biol. 19, 223-9, 2007!
Cellular adaptation to hypoxia! MCT4-H + /lactate! monocarboxylate transporter! NHE-1-Na + /H + exchanger! AE- anion exchanger! CA IX- carbonic anhydrases! Intracellular ph= 7.2-7.7! Curr. Opinion Cell Biol. 19, 223-9, 2007!
Local adaptation to hypoxia! Angiogenesis! VEGF! VEGFR1! VEGFR2! PDGF-β Angiopoietin-2! Tie-2! Vascular tone and blood flow! Endothelin-1! Plaminogen activator inhibitor-1 (PAI-1)! inos2 Adrenomedullin (ADM)!
Role of hypoxia in cancer Glucose and! energy metabolism! Angiogenesis! and vascular tone! tumour Cell survival! IGF-2! TGF-a! TGF-b Cell migration and metastasis Matrix metalloproteinase-2 (MMP-2 )- matrix metabolism! E-cadherin (negative regulation)- adhesion molecule (EMT)! Lysyl oxidase- promotes metastasis
Stabilization of Nrf2 Taguchi et al., Genes to Cell 16, 123, 2011
Conclusions - Under hypoxic conditions, HIFs mediate adaptive physiological responses - The mechanism of activation of HIFs is protein stabilization - The mechanism of activation of Nrf2 is protein stabilization - Oxygen sensing depends on Fe-containing, 2-oxoglutaratedependent oxygenases (prolyl and aspariginyl hydroxylases) - In cardiomyocytes there is increased ROS production under hypoxic conditions - Mutations in the VHL/HIF or Keap1/Nrf2 genes correlate with tumorigenesis