mechanisms of gene regulation and signal transduction in...
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Mechanisms of Gene Regulation and Signal!Transduction in Hypoxia!
Lorenz Poellinger!Dept. of Cell and Molecular Biology!
Karolinska Institutet, Stockholm, Sweden!
Normoxia - O2 availability is in balance with! the demand (21% O2)!
Hypoxia - unbalance between oxygen supply! and demand (1% O2)!
O2 supply
O2 consumption
O2 supply O2 consumption Oxygen homeostasis
Hypoxia
Physiological conditions: developing embryo (normal fetal development and stem cell differentiation)
Hypoxia Inducible Factors (HIFs) Mediate Adaptive Responses to Hypoxia!
HIFs
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
Hypoxia
Adaptive responses to hypoxia
Red blood cell !production
Formation and dynamic!regulation of !
blood vessels!
Glucose and!energy metabolism!
Cell Differentiation,!Growth and apoptosis
Cell migration
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
Brain
Heart and Lungs
Liver
Hypoxia and pathologyH!
Defective vascularisation leading to low!pO2 is a characteristic of a number of !diseases - local hypoxia!
Anemia-systemic hypoxia!
Hypoxia and tumor development!
Biochem. Pharm. 73, 450-7, 2007!
Hypoxia-inducible factors!
HIF-1α protein stability is regulated by oxygen levels!
HIF-1α!
HIF-1α N C A B bHLH
A B bHLH
HIF-1β/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, 12, 1191-1197!
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
P P
VHL Degradation
1 822 772 584 531 N C A B bHLH
331 91
PHDs
OH OH
Superfamily of iron II and 2-oxoglutarate!dependent oxygenases.!
402 563!
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���
HIF-1α 1% O2
HIF-2α 1% O2
HIF-2α 5% O2
72
HIF
act
ivity
4 Time (h)
Differential Utilization of HIFs in Response to! Oxygen Shortage!
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!
1 822 772 584 531 N C
P P
PHDs
OH OH
VHL
A B bHLH
Degradation
331 91
Normoxia
N OH
FIH!
FEBS letters, 581, 3582-3591, 2007!
Inhibition of HIF-1α function at normoxia!
FEBS letters, 581, 3582-3591, 2007!Curr. Opinion Cell Biol. 19, 223-9, 2007!
Cellular adaptation to hypoxia!
Cellular adaptation to hypoxia!
Curr. Opinion Cell Biol. 19, 223-9, 2007!
MCT4-H+/lactate !monocarboxylate transporter!
NHE-1-Na+/H+ exchanger!
AE- anion exchanger!
CA IX- carbonic anhydrases!
Intracellular pH= 7.2-7.7 !
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)!
tumour
Role of hypoxia in cancer
Angiogenesis!and vascular tone!
Glucose and!energy metabolism!
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
Taguchi et al., Genes to Cell 16, 123, 2011 ���
Stabilization of Nrf2���
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-oxoglutarate-��� dependent 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 ���