HIF-1:Hypoxia Inducible Factor-1

• Lynn Harrison, Ph.D.

HYPOXIA• A state where O2 availability/ delivery is

below the level necessary to maintain physiological O2 tensions for a particular tissue.

• When tissue demand exceeds its O2 supply.

• Different tissues have different oxygenation levels– Po2 = 18 mm Hg at 1mm depth in cerebral cortex

(2.5%)

– Po2 = 20-30 mm Hg in renal cortex (3-4%)

• Hypoxia can be local or systemic

• E.g. systemic = high altitude– Organism response mediated by the chemoreceptors

– Cellular response mediated by HIFs

• E.g. local = solid tumor, stroke, MI

• Hypoxia in one cell type maybe normal in another

HIF• Is a heterodimer formed from an and a

subunit

• History1. Semenza and Wang 1992 identified a 50 nt

enhancer element from the 3’ flanking sequence of the Human EPO gene. Used EMSA to identify nuclear factor that bound the region during hypoxia (Mol. Cell. Biol. 12, 5447)

2. Wang et al in 1995 identified a heterodimer HIF-1 (PNAS USA 92, 5510)

HIF-1

• bHLH - basic Helix-loop-helix domain required for dimerization

• PAS - domain identified to be required for dimerization • ODD – O2-dependent degradation domain

• TAD – transactivation domain

• NLS – nuclear localization signal

J.Cell. Mol. Med. 8, 423-431

subunit of HIF• There are three subunits:

• HIF-1 – ubiquitously expressed, baseline protein

• HIF-2 – endothelial and macrophage specific, baseline protein is not detectable until induced by hypoxia for other tissues/cells

• HIF-3 – lacks the C-term transactivation domain and is thought to be a negative regulator of HIF. Found in distal tubules of the kidney and corneal epithelial cells.

HIF-1 and 2

• HIF-1 and 2 are 48% identical in amino acid sequence.• Highest identity in the bHLH domain (85%) and PAS A (68%) and

B (73%) domains• Both bind to the subunit and bind to a core DNA sequence called

the Hypoxia Response Element (HRE) to increase transcription• HIF-2 has a stronger transactivation of the VEGF promoter,

showing the importance in the vasculature• HIF-1 null mice die at embryonic day 10, HIF-2 null mice die

at mid-gestation days 12.5-16. They have different roles in development.

• In liver and kidney need more severe hypoxia (6% O2) to induce HIF-1 than HIF-2

Hypoxia: Through the Lifecycle, Ch. 7, ed. Roach, 2003, Kluwer Academic/Plenum Publishers, NY

HIF subunit• Is a nuclear localized protein, also known as ARNT (aryl

hydrocarbon receptor nuclear translocator)

• Aryl hydrocarbon receptor is translocated to the nucleus after binding with an exogenous inducer. It binds to the ARNT and the dimer binds to the Xenobiotic Response Element.

• Constitutively expressed.

• Three types 1, 2 and 3

• 1 and 2 can bind the subunit. The third not involved in hypoxic response, has weak HIF dimerization.

• 2 is found primarily in the brain and kidney, 1 is ubiquitous.

HIF-1 protein level is regulated by O2

• HIF-1 mRNA and protein are constantly being produced

• In the presence of O2, Fe2+ and 2-oxoglutarate, prolyl hydroxylase hydroxylates prolines 402 and 564

• There are 4 prolyl hydroxylases (PHD1- 4)

HLH PAS ODD

401 608

Pro402

Pro564

TAD TAD

786 826

Asn803

PHD enzymes

• 2-oxoglutarate (or -ketoglutarate) is generated from the TCA cycle which occurs under aerobic conditions

• Pyruvate → acetyl CoA → 2-oxoglutarate

FASEB J. Warnecke et al

2-oxoglutarate

TCA

Ubi

Hydroxylated HIF-1 is ubiquitinated by E3-ubiquitin ligase

• The E3-ubiquitin ligase complex contains the– von Hippel Lindau protein (pVHL)– Elongin B (ELB) and elongin C (ELC)– CUL2– RXB1

• HIF-1 is then degraded by the 26S proteasomal complex

HLH PAS ODD

401 608

Pro402

Pro564

TAD TAD

786 826

Asn803

OH OH

UbiUbi

Ubi

pVHLELBELCCUL2RXB1

HIF-1 DNA binding and transcription factor activity

• The C-term transactivation domain binds to the p300/CBP

• This is critical for the activation of transcription through the HRE element (core sequence is 5’-ACGTG-3’)

• Hydroxylation of Asn803 blocks this interaction

• Hydroxylation is by an asparagine hydroxylase called inhibiting HIF-1 or FIH-1

• This also requires O2, Fe2+ and 2-oxoglutarate

HLH PAS ODD

401 608

Pro402

Pro564

TAD TAD

786 826

Asn803

OH

Acetylation may also be involved in proteasome degradation

• ARD1 is an acetyl transferase that may acetylate Lysine 532. This may not function alone, point in question at the moment. Acetylation is believed to increase the interaction with pVHL.

• This inhibits transcriptional activation, protein stability and degradation

• ARD1 seems to act in cytoplasm not nucleus

• ARD1 expression decreases during hypoxia

HLH PAS ODD

401 608

Pro402

Pro564

TAD TAD

786 826

Asn803

Lys532

AC

CONTROVERSIAL

Normoxia

AC

AC

Prolyl hydroxylases

Asparagine hydroxylase

ARD1?

J.Cell. Mol. Med. 8, 423-431

Can overexpression of HIF-1increase transcription activity? Not necessarily due to the hydroxylation of the asparagine under normoxia

Hypoxia

J.Cell. Mol. Med. 8, 423-431

AC

ARD1 is decreased?Loss of acetylation?

Severity of hypoxic exposure to achieve response varies from tissue to tissue:-Kidney and liver need systemic exposure of 6% O2

-Brain triggered at 18% O2

Mechanisms to stabilize HIF without hypoxia:1. Anything that will inhibit PHD2. Inhibition of pVHL binding with HIF-1

Genes regulated by HIF-1

• Group 1 – Oxygen delivery-Erythropoeitin (EPO)

-Nitric oxide synthase 2 (NOS2)

-Transferrin

-Transferrin receptor

-Vascular endothelial growth factor (VEGF)

-VEGF receptor FLT-1

• Group 2 – Glucose/ Energy metabolism-Aldolase A-Aldolase C-Enolase 1 (ENO1) -Glucose transporter 1-Glyceraldehyde phosphate dehydrogenase -Hexokinase 1-Hexokinase 2-Lactate dehydrogenase A-Phosphofructokinase L-Phosphoglycerate kinase 1-Pyruvate kinase M

Phosphofructokinaseis the rate-limitingstep

Glycolysis

Hypoxia: Through the Lifecycle, Ch. 7, ed. Roach, 2003, Kluwer Academic/Plenum Publishers, NY

• Group 3 – Cell Proliferation / viability-Insulin-like growth factor 2 (IGF-2)

-IGF binding protein 1

-IGF binding protein 3

-p21

-endothelin 1

• Over 70 genes identified as HIF-1 regulated

• They are pro-survival as well as pro-apoptotic

• So does HIF-1 activate pro-survival or pro-death pathways?

• Is this the length of time of hypoxia or severity or other contributing factors?

Cancer letters 2006, 237,10-21

Interaction with p53

• Under normoxia and no DNA damage, p53 interacts with MDM2 , promoting poly-ubiquitination and degradation. MDM2 required to export p53 where degradation occurs

• Very low in normal, non-stressed cell

E3-ligase

UbiUbi

J. Cell. Mol. Med. 8, 423-431

p53 and hypoxia• Conflicting information relating to whether HIF-1 causes

accumulation of p53• Probably relates to the severity of hypoxia/ other

environmental stresses• Determined by Pan et al (Oncogene 23,4975) that 0.02%

O2 not enough to accumulate p53, also needed acidosis/ nutrient deprivation.

• Adaptation to hypoxia results in a ↓ intracellular pH.• Occurs due to lactic acid production from anaerobic

glycolysis. Also hypoxic cells hydrolyse ATP and produce protons. These protons are not reused for ATP re-synthesis as under aerobic conditions and so cause acidosis.

• Apoptosis correlates with acidosis more so than with the hypoxia.

p53 and HIF-1 use the same co-activator for transcription

• The more p53 accumulates, the lower the capability of HIF-1 regulate gene transcription.

• This then shifts the balance in favor of apoptosis

• The contribution of HIF-1 to apoptosis independent of p53 by expression of apoptotic genes is still unknown

Toxicology 208, 223-233

p53 promotes HIF-1 destruction independent of pVHL

Regulation of HIF-1 by phosphorylation

• Phosphorylation pathways induce increased HIF-1 expression and activity

Ribosomal protein

P-inactiveIncreases translation

Ribosomal protein kinase

Inhibits Akt activation

Oncogenes can activate HIF-1:

RasSrc kinase

Hypoxia: Through the Lifecycle, Ch. 7, ed. Roach, 2003, Kluwer Academic/Plenum Publishers, NY

ROS activates survival/death pathways

SRC

HIF-1 ↑

Dr. Jin’s lecture

Superoxide from mitochondria stabilize HIF-1

• ROS from complex III stabilizes HIF-1• Does not happen in rho zero cells where there is no functional mitochondria• Isolated mitochondria produce ROS under hypoxia• H2O2 stabilizes HIF-1 during normoxia• This is abolished by overexpression of catalase• Chandel et al JBC 275, 25130-25138

Dr. Aw said to remind you of reductive stress caused by hypoxia on mitochondria

JBC 275, 25130

Redox control of HIF through Cysteine

• Critical cys in the HLH only in HIF-2• Fos and Jun have a similar cys and binding is

also regulated by redox control

HIF-2

HLH PAS ODD

Cys25

TAD TADHIF-2

JBC 275, 4618

Reducing environment promotes binding of HIF-2- to HRE but not HIF1

Nucleus

Final execution of signaling:Relatively reducing environment

HIF2++S-++

+ve charge stabilizesde-protonated cysteine

-----

GSHGSSG

+/-

Geneexpression

+ +

REF1

Dr Aw’s lecture

1. Ref1 interacts with the N-term of HIF-2

2. In vitro Ref1 enhances binding to the HRE

3. siRNA of REF1 decreases HIF-2 transcriptional activity

Loss of Ref1 decreases transcriptional activity of HIF-1

• Ref 1 does not interact with the N-term of HIF-1 or promote binding to the HRE

• Possible it influences cysteines in the transactivation domains to increase transcriptional activity

Reactive Nitrogen Intermediates• Under normoxia, RNI cause:

– HIF-1 stabilization– HIF1 DNA binding– HIF1 transactivation

• Shown using different NO donors with different half-lives• NO induction of the VEGF promoter was at the HIF1

binding site• GSNO caused HIF-1 accumulation and this can be

reversed by DTT - believed S-nitrosation of HIF-1• Overexpression of iNOS also resulted in HIF-1

accumulation under normoxia• Activated but not resting macrophages could also cause

accumulationSeen as high NO situations

RNI interaction with redox environment –low NO concentrations

• RNI effect stopped by H2O2 and superoxide

• Peroxynitrite formation involved??

• If inhibit mitochondrial respiration then HIF-1 is destabilized, even if from low NO production

• Possible inhibition of ETC, causes more availability of oxygen for the action of PHD enzymes to hydroxylate HIF -1

GSNO attenuates PHD activity

• Possible that RNI compete with O2 for the Fe2+ in the active site

• Other studies have suggested increased synthesis of HIF-1 rather than decreased degradation

Toxicology 208, 223-233

NO mediated S-nitrosylation and stabilization of HIF-1

• Macrophages in tumors have been shown to be involved in the s-nitrosylation of cys533

HLH PAS ODD

401 608

Pro402

Pro564

TAD TAD

786 826

Asn803

cys533

S-nitrosylationstabilizes protein

S-nitrosylationstimulates transcription

activity

cys800

Radiation may aid vascularization of the tumor

Mol. Cell 26,63-74

Angiogenesis

Cancer Metastasis Rev. 26,281-290

Roles in other diseases

• Cancer –solid tumor survival• Cerebral ischemia

– activates HIF1, causes production of VEGF and EPO, implicated in neurogenesis

• Chondrocyte survival– Live deep in matrix, a good distance from vessels

– important for growth plates and cartilaginous tissue

– May play a role in osteoarthritis

• Duodenal ulceration• Ischemia-reperfusion injury