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
Top Related