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 ���