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NFκB and HIF display synergistic behaviour during hypoxic inflammation

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Abstract

The oxygen-sensitive transcription factor hypoxia inducible factor (HIF) is a key regulator of gene expression during adaptation to hypoxia. Crucially, inflamed tissue often displays regions of prominent hypoxia. Recent studies have shown HIF signalling is intricately linked to that of the pro-inflammatory transcription factor nuclear factor kappa B (NFκB) during hypoxic inflammation. We describe the relative temporal contributions of each to hypoxia-induced inflammatory gene expression and investigate the level of crosstalk between the two pathways using a novel Gaussia princeps luciferase (Gluc) reporter system. Under the control of an active promoter, Gluc is expressed and secreted into the cell culture media, where it can be sampled and measured over time. Thus, Gluc constructs under the control of either HIF or NFκB were used to resolve their temporal transcriptional dynamics in response to hypoxia and to cytokine stimuli, respectively. We also investigated the interactions between HIF and NFκB activities using a construct containing the sequence from the promoter of the inflammatory gene cyclooxygenase 2 (COX-2), which includes functionally active binding sites for both HIF and NFκB. Finally, based on our experimental data, we constructed a mathematical model of the binding affinities of HIF and NFκB to their respective response elements to analyse transcriptional crosstalk. Taken together, these data reveal distinct temporal HIF and NFκB transcriptional activities in response to hypoxic inflammation. Furthermore, we demonstrate synergistic activity between these two transcription factors on the regulation of the COX-2 promoter, implicating a co-ordinated role for both HIF and NFκB in the expression of COX-2 in hypoxic inflammation.

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Abbreviations

COX-2:

Cyclooxygenase2

DMOG:

Dimethyl-oxaloylglycine

DMSO:

Dimethyl-sulfoxide

Gluc:

Gaussia Luciferase

HIF:

Hypoxia inducible factor

HRE:

Hypoxia response element

NFκB:

Nuclear factor kappa B

NRE:

NFκB response element

pGluc:

Plasmid encoding Gluc

PHD:

Prolyl hydroxylase

References

  1. Semenza GL (2006) Regulation of physiological responses to continuous and intermittent hypoxia by hypoxia-inducible factor 1. Exp Physiol 91:803–806

    Article  PubMed  CAS  Google Scholar 

  2. Semenza GL (2009) Regulation of cancer cell metabolism by hypoxia-inducible factor 1. Semin Cancer Biol 19:12–16

    Article  PubMed  CAS  Google Scholar 

  3. Eltzschig HK, Carmeliet P (2011) Hypoxia and inflammation. N Engl J Med 364:656–665

    Article  PubMed  CAS  Google Scholar 

  4. Schmedtje JF Jr, Ji YS, Liu WL, DuBois RN, Runge MS (1997) Hypoxia induces cyclooxygenase-2 via the NF-kappaB p65 transcription factor in human vascular endothelial cells. J Biol Chem 272:601–608

    Article  PubMed  CAS  Google Scholar 

  5. Cummins EP, Berra E, Comerford KM, Ginouves A, Fitzgerald KT et al (2006) Prolyl hydroxylase-1 negatively regulates IkappaB kinase-beta, giving insight into hypoxia-induced NFkappaB activity. Proc Natl Acad Sci USA 103:18154–18159

    Article  PubMed  CAS  Google Scholar 

  6. Culver C, Sundqvist A, Mudie S, Melvin A, Xirodimas D et al (2010) Mechanism of hypoxia-induced NF-kappaB. Mol Cell Biol 30:4901–4921

    Article  PubMed  CAS  Google Scholar 

  7. Koong AC, Chen EY, Giaccia AJ (1994) Hypoxia causes the activation of nuclear factor kappa B through the phosphorylation of I kappa B alpha on tyrosine residues. Cancer Res 54:1425–1430

    PubMed  CAS  Google Scholar 

  8. Fitzpatrick SF, Tambuwala MM, Bruning U, Schaible B, Scholz CC et al (2011) An intact canonical NF-kappaB pathway is required for inflammatory gene expression in response to hypoxia. J Immunol 186:1091–1096

    Article  PubMed  CAS  Google Scholar 

  9. Eliasson P, Jonsson JI (2010) The hematopoietic stem cell niche: low in oxygen but a nice place to be. J Cell Physiol 222:17–22

    Article  PubMed  CAS  Google Scholar 

  10. Hayden MS, Ghosh S (2004) Signaling to NF-kappaB. Genes Dev 18:2195–2224

    Article  PubMed  CAS  Google Scholar 

  11. Karin M (2006) Nuclear factor-kappaB in cancer development and progression. Nature 441:431–436

    Article  PubMed  CAS  Google Scholar 

  12. Hoffmann A, Natoli G, Ghosh G (2006) Transcriptional regulation via the NF-kappaB signaling module. Oncogene 25:6706–6716

    Article  PubMed  CAS  Google Scholar 

  13. Simakajornboon N, Gozal E, Gozal D (2001) Developmental patterns of NF-kappaB activation during acute hypoxia in the caudal brainstem of the rat. Brain Res Dev Brain Res 127:175–183

    Article  PubMed  CAS  Google Scholar 

  14. Cockman ME, Lancaster DE, Stolze IP, Hewitson KS, McDonough MA et al (2006) Posttranslational hydroxylation of ankyrin repeats in IkappaB proteins by the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor inhibiting HIF (FIH). Proc Natl Acad Sci USA 103:14767–14772

    Article  PubMed  CAS  Google Scholar 

  15. Belaiba RS, Bonello S, Zahringer C, Schmidt S, Hess J et al (2007) Hypoxia up-regulates hypoxia-inducible factor-1alpha transcription by involving phosphatidylinositol 3-kinase and nuclear factor kappaB in pulmonary artery smooth muscle cells. Mol Biol Cell 18:4691–4697

    Article  PubMed  CAS  Google Scholar 

  16. Bonello S, Zahringer C, BelAiba RS, Djordjevic T, Hess J et al (2007) Reactive oxygen species activate the HIF-1alpha promoter via a functional NFkappaB site. Arterioscler Thromb Vasc Biol 27:755–761

    Article  PubMed  CAS  Google Scholar 

  17. van Uden P, Kenneth NS, Rocha S (2008) Regulation of hypoxia-inducible factor-1alpha by NF-kappaB. Biochem J 412:477–484

    Article  PubMed  Google Scholar 

  18. Rius J, Guma M, Schachtrup C, Akassoglou K, Zinkernagel AS et al (2008) NF-kappaB links innate immunity to the hypoxic response through transcriptional regulation of HIF-1alpha. Nature 453:807–811

    Article  PubMed  CAS  Google Scholar 

  19. Bracken CP, Whitelaw ML, Peet DJ (2005) Activity of hypoxia-inducible factor 2alpha is regulated by association with the NF-kappaB essential modulator. J Biol Chem 280:14240–14251

    Article  PubMed  CAS  Google Scholar 

  20. Walmsley SR, Print C, Farahi N, Peyssonnaux C, Johnson RS et al (2005) Hypoxia-induced neutrophil survival is mediated by HIF-1alpha-dependent NF-kappaB activity. J Exp Med 201:105–115

    Article  PubMed  CAS  Google Scholar 

  21. Tannous BA, Kim DE, Fernandez JL, Weissleder R, Breakefield XO (2005) Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo. Mol Ther 11:435–443

    Article  PubMed  CAS  Google Scholar 

  22. Ruecker O, Zillner K, Groebner-Ferreira R, Heitzer M (2008) Gaussia-luciferase as a sensitive reporter gene for monitoring promoter activity in the nucleus of the green alga Chlamydomonas reinhardtii. Mol Genet Genomics 280:153–162

    Article  PubMed  CAS  Google Scholar 

  23. Chung E, Yamashita H, Au P, Tannous BA, Fukumura D et al (2009) Secreted Gaussia luciferase as a biomarker for monitoring tumor progression and treatment response of systemic metastases. PLoS One 4:e8316

    Article  PubMed  Google Scholar 

  24. Badr CE, Niers JM, Morse D, Koelen JA, Vandertop P et al (2011) Suicidal gene therapy in an NF-kappaB-controlled tumor environment as monitored by a secreted blood reporter. Gene Ther 18:445–451

    Article  PubMed  CAS  Google Scholar 

  25. Badr CE, Niers JM, Tjon-Kon-Fat LA, Noske DP, Wurdinger T et al (2009) Real-time monitoring of nuclear factor kappaB activity in cultured cells and in animal models. Mol Imaging 8:278–290

    PubMed  CAS  Google Scholar 

  26. Bruning U, Cerone L, Neufeld Z, Fitzpatrick SF, Cheong A et al (2011) MicroRNA-155 promotes resolution of hypoxia-inducible factor 1alpha activity during prolonged hypoxia. Mol Cell Biol 31:4087–4096

    Article  PubMed  CAS  Google Scholar 

  27. Appleby SB, Ristimaki A, Neilson K, Narko K, Hla T (1994) Structure of the human cyclo-oxygenase-2 gene. Biochem J 302:723–727

    PubMed  CAS  Google Scholar 

  28. Kaidi A, Qualtrough D, Williams AC, Paraskeva C (2006) Direct transcriptional up-regulation of cyclooxygenase-2 by hypoxia-inducible factor (HIF)-1 promotes colorectal tumor cell survival and enhances HIF-1 transcriptional activity during hypoxia. Cancer Res 66:6683–6691

    Article  PubMed  CAS  Google Scholar 

  29. Higgins CF (1992) ABC transporters: from microorganisms to man. Annu Rev Cell Biol 8:67–113

    Article  PubMed  CAS  Google Scholar 

  30. Cummins EP, Oliver KM, Lenihan CR, Fitzpatrick SF, Bruning U et al (2010) NF-kappaB links CO2 sensing to innate immunity and inflammation in mammalian cells. J Immunol 185:4439–4445

    Article  PubMed  CAS  Google Scholar 

  31. Agbor TA, Cheong A, Comerford KM, Scholz CC, Bruning U et al (2011) Small ubiquitin-related modifier (SUMO)-1 promotes glycolysis in hypoxia. J Biol Chem 286:4718–4726

    Article  PubMed  CAS  Google Scholar 

  32. Bintu L, Buchler NE, Garcia HG, Gerland U, Hwa T et al (2005) Transcriptional regulation by the numbers: models. Curr Opin Genet Dev 15:116–124

    Article  PubMed  CAS  Google Scholar 

  33. Bintu L, Buchler NE, Garcia HG, Gerland U, Hwa T et al (2005) Transcriptional regulation by the numbers: applications. Curr Opin Genet Dev 15:125–135

    Article  PubMed  CAS  Google Scholar 

  34. Shea MA, Ackers GK (1985) The OR control system of bacteriophage lambda. A physical-chemical model for gene regulation. J Mol Biol 181:211–230

    Article  PubMed  CAS  Google Scholar 

  35. Jaakkola P, Mole DR, Tian YM, Wilson MI, Gielbert J et al (2001) Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292:468–472

    Article  PubMed  CAS  Google Scholar 

  36. Koong AC, Chen EY, Lee AS, Brown JM, Giaccia AJ (1994) Increased cytotoxicity of chronic hypoxic cells by molecular inhibition of GRP78 induction. Int J Radiat Oncol Biol Phys 28:661–666

    Article  PubMed  CAS  Google Scholar 

  37. Pouyssegur J, Dayan F, Mazure NM (2006) Hypoxia signalling in cancer and approaches to enforce tumour regression. Nature 441:437–443

    Article  PubMed  CAS  Google Scholar 

  38. Koong AC, Chen EY, Kim CY, Giaccia AJ (1994) Activators of protein kinase C selectively mediate cellular cytotoxicity to hypoxic cells and not aerobic cells. Int J Radiat Oncol Biol Phys 29:259–265

    Article  PubMed  CAS  Google Scholar 

  39. Semenza GL (2009) Regulation of vascularization by hypoxia-inducible factor 1. Ann NY Acad Sci 1177:2–8

    Article  PubMed  CAS  Google Scholar 

  40. Nelson DE, Ihekwaba AE, Elliott M, Johnson JR, Gibney CA et al (2004) Oscillations in NF-kappaB signaling control the dynamics of gene expression. Science 306:704–708

    Article  PubMed  CAS  Google Scholar 

  41. Carey M (1998) The enhanceosome and transcriptional synergy. Cell 92:5–8

    Article  PubMed  CAS  Google Scholar 

  42. Paradisi A, Maisse C, Bernet A, Coissieux MM, Maccarrone M et al (2008) NF-kappaB regulates netrin-1 expression and affects the conditional tumor suppressive activity of the netrin-1 receptors. Gastroenterology 135:1248–1257

    Article  PubMed  CAS  Google Scholar 

  43. Rosenberger P, Schwab JM, Mirakaj V, Masekowsky E, Mager A et al (2009) Hypoxia-inducible factor-dependent induction of netrin-1 dampens inflammation caused by hypoxia. Nature Immunol 10:195–202

    Article  CAS  Google Scholar 

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Acknowledgments

We thank Drs. Catrióna Johnston and Jens Rauch (Systems Biology Ireland) for discussion. This work was supported by Science Foundation Ireland (Grant No. 06/CE/B1129).

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None.

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Authors and Affiliations

  1. Conway Institute, University College Dublin, Dublin, 4, Ireland

    Ulrike Bruning, Susan F. Fitzpatrick & Cormac T. Taylor

  2. Systems Biology Ireland, University College Dublin, Dublin, 4, Ireland

    Susan F. Fitzpatrick, Till Frank, Marc Birtwistle, Cormac T. Taylor & Alex Cheong

  3. School of Physics, University College Dublin, Dublin, 4, Ireland

    Till Frank

Authors
  1. Ulrike Bruning
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  2. Susan F. Fitzpatrick
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  3. Till Frank
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  4. Marc Birtwistle
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  5. Cormac T. Taylor
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  6. Alex Cheong
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Corresponding author

Correspondence to Alex Cheong.

Additional information

U. Bruning, S. F. Fitzpatrick, C. T. Taylor and A. Cheong contributed equally to this work.

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Bruning, U., Fitzpatrick, S.F., Frank, T. et al. NFκB and HIF display synergistic behaviour during hypoxic inflammation. Cell. Mol. Life Sci. 69, 1319–1329 (2012). https://doi.org/10.1007/s00018-011-0876-2

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  • DOI: https://doi.org/10.1007/s00018-011-0876-2

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