1. HIF-1: Hypoxia Inducible Factor-1 Lynn Harrison, Ph.D.

2. HYPOXIA A state where O 2 availability/ delivery is below the level necessary to maintain physiological O 2 tensions for a particular tissue. When tissue demand exceeds its O 2 supply. Different tissues have different oxygenation levels –Po 2 = 18 mm Hg at 1mm depth in cerebral cortex (2.5%) –Po 2 = 20-30 mm Hg in renal cortex (3-4%)

3. 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

4. HIF Is a heterodimer formed from an  and a  subunit History 1.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)

5. HIF-1 bHLH - basic Helix-loop-helix domain required for dimerization PAS - domain identified to be required for dimerization ODD – O 2 -dependent degradation domain TAD – transactivation domain NLS – nuclear localization signal J.Cell. Mol. Med. 8, 423-431

6.  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.

7. 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% O 2 ) to induce HIF-1  than HIF-2  Hypoxia: Through the Lifecycle, Ch. 7, ed. Roach, 2003, Kluwer Academic/Plenum Publishers, NY

8. 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.

9. HIF-1  protein level is regulated by O 2 HIF-1  mRNA and protein are constantly being produced In the presence of O 2, Fe 2+ and 2-oxoglutarate, prolyl hydroxylase hydroxylates prolines 402 and 564 There are 4 prolyl hydroxylases (PHD1- 4) HLH PASODD 401608 Pro 402 Pro 564 TAD 786826 Asn 803

10. 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

11. 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 PASODD 401608 Pro 402 Pro 564 TAD 786826 Asn 803 OH Ubi pVHL ELB ELC CUL2 RXB1

12. 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 O 2, Fe 2+ and 2-oxoglutarate HLH PASODD 401608 Pro 402 Pro 564 TAD 786826 Asn 803 OH

13. 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 PASODD 401608 Pro 402 Pro 564 TAD 786826 Asn 803 Lys 532 AC CONTROVERSIAL

14. Normoxia AC Prolyl hydroxylases Asparagine hydroxylase ARD1? J.Cell. Mol. Med. 8, 423-431 Can overexpression of HIF-1  increase transcription activity? Not necessarily due to the hydroxylation of the asparagine under normoxia

15. 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% O 2 -Brain triggered at 18% O 2 Mechanisms to stabilize HIF without hypoxia: 1.Anything that will inhibit PHD 2.Inhibition of pVHL binding with HIF-1 

16. 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

17. 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

18. Phosphofructokinase is the rate-limiting step Glycolysis Hypoxia: Through the Lifecycle, Ch. 7, ed. Roach, 2003, Kluwer Academic/Plenum Publishers, NY

19. Group 3 – Cell Proliferation / viability -Insulin-like growth factor 2 (IGF-2) -IGF binding protein 1 -IGF binding protein 3 -p21 -endothelin 1

20. 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?

21. Cancer letters 2006, 237,10-21

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

23. 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% O 2 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.

24. 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

25. p53 promotes HIF-1  destruction independent of pVHL

26. Regulation of HIF-1  by phosphorylation Phosphorylation pathways induce increased HIF-1  expression and activity Ribosomal protein P-inactive Increases translation Ribosomal protein kinase Inhibits Akt activation Oncogenes can activate HIF-1: Ras Src kinase Hypoxia: Through the Lifecycle, Ch. 7, ed. Roach, 2003, Kluwer Academic/Plenum Publishers, NY

27. ROS activates survival/death pathways SRC HIF-1  ↑ Dr. Jin’s lecture

28. 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 H 2 O 2 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

29. 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 PASODD Cys 25 TAD HIF-2  JBC 275, 4618

30. Reducing environment promotes binding of HIF-2  -  to HRE but not HIF1 Nucleus Final execution of signaling: Relatively reducing environment HIF2 ++S-++ +ve charge stabilizes de-protonated cysteine ----- GSH GSSG +/- Gene expression + 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

31. 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

32. 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 accumulation Seen as high NO situations

33. RNI interaction with redox environment –low NO concentrations RNI effect stopped by H 2 O 2 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 

34. GSNO attenuates PHD activity Possible that RNI compete with O 2 for the Fe 2+ in the active site Other studies have suggested increased synthesis of HIF-1  rather than decreased degradation Toxicology 208, 223-233

35. 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 PASODD 401608 Pro 402 Pro 564 TAD 786826 Asn 803 cys 533 S-nitrosylation stabilizes protein S-nitrosylation stimulates transcription activity cys 800

36. Radiation may aid vascularization of the tumor Mol. Cell 26,63-74

37. Angiogenesis Cancer Metastasis Rev. 26,281-290

38. 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