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Iron-regulatory proteins limit hypoxia-inducible factor-2α expression in iron deficiency

Nature Structural & Molecular Biology volume 14, pages 420–426 (2007)Cite this article

Abstract

Hypoxia stimulates erythropoiesis, the major iron-utilization pathway. We report the discovery of a conserved, functional iron-responsive element (IRE) in the 5′ untranslated region of the messenger RNA encoding endothelial PAS domain protein-1, EPAS1 (also called hypoxia-inducible factor-2α, HIF2α). Via this IRE, iron regulatory protein binding controls EPAS1 mRNA translation in response to cellular iron availability. Our results uncover a regulatory link that permits feedback control between iron availability and the expression of a key transcription factor promoting iron utilization. They also show that an IRE that is structurally distinct from, for example, the ferritin mRNA IRE and that has been missed by in silico approaches, can mediate mechanistically similar responses.

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Figure 1: Identification of a novel IRE in the human EPAS1 mRNA.
Figure 2: The EPAS1 IRE binds IRP1 and IRP2.
Figure 3: The EPAS1 IRE mediates iron-dependent post-transcriptional regulation.
Figure 4: Post-transcriptional regulation of EPAS1 expression by iron.
Figure 5: Polysome analysis of EPAS1 and ACTB mRNA translation in response to iron treatment.
Figure 6: Proposed model for the feedback regulation between iron and oxygen metabolism via the IRE-IRP regulatory system and the 5′ UTR IRE of EPAS1 mRNA.

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References

  1. Eckardt, K.U. & Kurtz, A. Regulation of erythropoietin production. Eur. J. Clin. Invest. 35 Suppl. 3, 13–19 (2005).

    Article  CAS  Google Scholar 

  2. Leung, P.S., Srai, S.K., Mascarenhas, M., Churchill, L.J. & Debnam, E.S. Increased duodenal iron uptake and transfer in a rat model of chronic hypoxia is accompanied by reduced hepcidin expression. Gut 54, 1391–1395 (2005).

    Article  CAS  Google Scholar 

  3. Wang, G.L., Jiang, B.H., Rue, E.A. & Semenza, G.L. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc. Natl. Acad. Sci. USA 92, 5510–5514 (1995).

    Article  CAS  Google Scholar 

  4. Ema, M. et al. A novel bHLH-PAS factor with close sequence similarity to hypoxia-inducible factor 1alpha regulates the VEGF expression and is potentially involved in lung and vascular development. Proc. Natl. Acad. Sci. USA 94, 4273–4278 (1997).

    Article  CAS  Google Scholar 

  5. Flamme, I. et al. HRF, a putative basic helix-loop-helix-PAS-domain transcription factor is closely related to hypoxia-inducible factor-1 alpha and developmentally expressed in blood vessels. Mech. Dev. 63, 51–60 (1997).

    Article  CAS  Google Scholar 

  6. Hogenesch, J.B. et al. Characterization of a subset of the basic-helix-loop-helix-PAS superfamily that interacts with components of the dioxin signaling pathway. J. Biol. Chem. 272, 8581–8593 (1997).

    Article  CAS  Google Scholar 

  7. Tian, H., McKnight, S.L. & Russell, D.W. Endothelial PAS domain protein 1 (EPAS1), a transcription factor selectively expressed in endothelial cells. Genes Dev. 11, 72–82 (1997).

    Article  CAS  Google Scholar 

  8. Wenger, R.H. & Gassmann, M. Oxygen(es) and the hypoxia-inducible factor-1. Biol. Chem. 378, 609–616 (1997).

    CAS  PubMed  Google Scholar 

  9. Schofield, C.J. & Ratcliffe, P.J. Oxygen sensing by HIF hydroxylases. Nat. Rev. Mol. Cell Biol. 5, 343–354 (2004).

    Article  CAS  Google Scholar 

  10. Kallio, P.J., Wilson, W.J., O'Brien, S., Makino, Y. & Poellinger, L. Regulation of the hypoxia-inducible transcription factor 1alpha by the ubiquitin-proteasome pathway. J. Biol. Chem. 274, 6519–6525 (1999).

    Article  CAS  Google Scholar 

  11. Lando, D., Peet, D.J., Whelan, D.A., Gorman, J.J. & Whitelaw, M.L. Asparagine hydroxylation of the HIF transactivation domain a hypoxic switch. Science 295, 858–861 (2002).

    Article  CAS  Google Scholar 

  12. Semenza, G.L. Hypoxia-inducible factor 1: oxygen homeostasis and disease pathophysiology. Trends Mol. Med. 7, 345–350 (2001).

    Article  CAS  Google Scholar 

  13. Hu, C.J., Wang, L.Y., Chodosh, L.A., Keith, B. & Simon, M.C. Differential roles of hypoxia-inducible factor 1alpha (HIF-1alpha) and HIF-2alpha in hypoxic gene regulation. Mol. Cell. Biol. 23, 9361–9374 (2003).

    Article  CAS  Google Scholar 

  14. Warnecke, C. et al. Differentiating the functional role of hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha (EPAS-1) by the use of RNA interference: erythropoietin is a HIF-2alpha target gene in Hep3B and Kelly cells. FASEB J. 18, 1462–1464 (2004).

    Article  CAS  Google Scholar 

  15. Smith, K. et al. Silencing of epidermal growth factor receptor suppresses hypoxia-inducible factor-2-driven VHL−/− renal cancer. Cancer Res. 65, 5221–5230 (2005).

    Article  CAS  Google Scholar 

  16. Hentze, M.W., Muckenthaler, M.U. & Andrews, N.C. Balancing acts: molecular control of mammalian iron metabolism. Cell 117, 285–297 (2004).

    Article  CAS  Google Scholar 

  17. Wallander, M.L., Leibold, E.A. & Eisenstein, R.S. Molecular control of vertebrate iron homeostasis by iron regulatory proteins. Biochim. Biophys. Acta 1763, 668–689 (2006).

    Article  CAS  Google Scholar 

  18. Sanchez, M. et al. Iron regulation and the cell cycle: identification of an iron-responsive element in the 3′-untranslated region of human cell division cycle 14A mRNA by a refined microarray-based screening strategy. J. Biol. Chem. 281, 22865–22874 (2006).

    Article  CAS  Google Scholar 

  19. Muckenthaler, M., Gray, N.K. & Hentze, M.W. IRP-1 binding to ferritin mRNA prevents the recruitment of the small ribosomal subunit by the cap-binding complex eIF4F. Mol. Cell 2, 383–388 (1998).

    Article  CAS  Google Scholar 

  20. Gray, N.K., Pantopoulos, K., Dandekar, T., Ackrell, B.A. & Hentze, M.W. Translational regulation of mammalian and Drosophila citric acid cycle enzymes via iron-responsive elements. Proc. Natl. Acad. Sci. USA 93, 4925–4930 (1996).

    Article  CAS  Google Scholar 

  21. Liu, X.B., Hill, P. & Haile, D.J. Role of the ferroportin iron-responsive element in iron and nitric oxide dependent gene regulation. Blood Cells Mol. Dis. 29, 315–326 (2002).

    Article  CAS  Google Scholar 

  22. Binder, R. et al. Evidence that the pathway of transferrin receptor mRNA degradation involves an endonucleolytic cleavage within the 3′ UTR and does not involve poly(A) tail shortening. EMBO J. 13, 1969–1980 (1994).

    Article  CAS  Google Scholar 

  23. Smith, S.R., Ghosh, M.C., Ollivierre-Wilson, H., Hang Tong, W. & Rouault, T.A. Complete loss of iron regulatory proteins 1 and 2 prevents viability of murine zygotes beyond the blastocyst stage of embryonic development. Blood Cells Mol. Dis. 36, 283–287 (2006).

    Article  CAS  Google Scholar 

  24. Galy, B., Ferring, D. & Hentze, M.W. Generation of conditional alleles of the murine Iron Regulatory Protein (IRP)-1 and -2 genes. Genesis 43, 181–188 (2005).

    Article  CAS  Google Scholar 

  25. Hentze, M.W. et al. A model for the structure and functions of iron-responsive elements. Gene 72, 201–208 (1988).

    Article  CAS  Google Scholar 

  26. Gunshin, H. et al. Iron-dependent regulation of the divalent metal ion transporter. FEBS Lett. 509, 309–316 (2001).

    Article  CAS  Google Scholar 

  27. Goossen, B. & Hentze, M.W. Position is the critical determinant for function of iron-responsive elements as translational regulators. Mol. Cell. Biol. 12, 1959–1966 (1992).

    Article  CAS  Google Scholar 

  28. Paraskeva, E., Gray, N.K., Schlager, B., Wehr, K. & Hentze, M.W. Ribosomal pausing and scanning arrest as mechanisms of translational regulation from cap-distal iron-responsive elements. Mol. Cell. Biol. 19, 807–816 (1999).

    Article  CAS  Google Scholar 

  29. Hentze, M.W. et al. Identification of the iron-responsive element for the translational regulation of human ferritin mRNA. Science 238, 1570–1573 (1987).

    Article  CAS  Google Scholar 

  30. Maxwell, P.H. et al. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399, 271–275 (1999).

    Article  CAS  Google Scholar 

  31. Bernardi, R. et al. PML inhibits HIF-1alpha translation and neoangiogenesis through repression of mTOR. Nature 442, 779–785 (2006).

    Article  CAS  Google Scholar 

  32. Lang, K.J., Kappel, A. & Goodall, G.J. Hypoxia-inducible factor-1alpha mRNA contains an internal ribosome entry site that allows efficient translation during normoxia and hypoxia. Mol. Biol. Cell 13, 1792–1801 (2002).

    Article  CAS  Google Scholar 

  33. Kallio, P.J. et al. Signal transduction in hypoxic cells: inducible nuclear translocation and recruitment of the CBP/p300 coactivator by the hypoxia-inducible factor-1alpha. EMBO J. 17, 6573–6586 (1998).

    Article  CAS  Google Scholar 

  34. Semenza, G.L. et al. Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1. J. Biol. Chem. 271, 32529–32537 (1996).

    Article  CAS  Google Scholar 

  35. Wiesener, M.S. et al. Widespread hypoxia-inducible expression of HIF-2alpha in distinct cell populations of different organs. FASEB J. 17, 271–273 (2003).

    Article  CAS  Google Scholar 

  36. Ryan, H.E., Lo, J. & Johnson, R.S. HIF-1 alpha is required for solid tumor formation and embryonic vascularization. EMBO J. 17, 3005–3015 (1998).

    Article  CAS  Google Scholar 

  37. Peng, J., Zhang, L., Drysdale, L. & Fong, G.H. The transcription factor EPAS-1/hypoxia-inducible factor 2alpha plays an important role in vascular remodeling. Proc. Natl. Acad. Sci. USA 97, 8386–8391 (2000).

    Article  CAS  Google Scholar 

  38. Tian, H., Hammer, R.E., Matsumoto, A.M., Russell, D.W. & McKnight, S.L. The hypoxia-responsive transcription factor EPAS1 is essential for catecholamine homeostasis and protection against heart failure during embryonic development. Genes Dev. 12, 3320–3324 (1998).

    Article  CAS  Google Scholar 

  39. Compernolle, V. et al. Loss of HIF-2alpha and inhibition of VEGF impair fetal lung maturation, whereas treatment with VEGF prevents fatal respiratory distress in premature mice. Nat. Med. 8, 702–710 (2002).

    Article  CAS  Google Scholar 

  40. Scortegagna, M., Morris, M.A., Oktay, Y., Bennett, M. & Garcia, J.A. The HIF family member EPAS1/HIF-2alpha is required for normal hematopoiesis in mice. Blood 102, 1634–1640 (2003).

    Article  CAS  Google Scholar 

  41. Scortegagna, M. et al. HIF-2alpha regulates murine hematopoietic development in an erythropoietin-dependent manner. Blood 105, 3133–3140 (2005).

    Article  CAS  Google Scholar 

  42. Morita, M. et al. HLF/HIF-2alpha is a key factor in retinopathy of prematurity in association with erythropoietin. EMBO J. 22, 1134–1146 (2003).

    Article  CAS  Google Scholar 

  43. Ding, K., Scortegagna, M., Seaman, R., Birch, D.G. & Garcia, J.A. Retinal disease in mice lacking hypoxia-inducible transcription factor-2alpha. Invest. Ophthalmol. Vis. Sci. 46, 1010–1016 (2005).

    Article  Google Scholar 

  44. Rosenberger, C. et al. Expression of hypoxia-inducible factor-1alpha and -2alpha in hypoxic and ischemic rat kidneys. J. Am. Soc. Nephrol. 13, 1721–1732 (2002).

    Article  CAS  Google Scholar 

  45. Gray, N.K. et al. Recombinant iron-regulatory factor functions as an iron-responsive-element-binding protein, a translational repressor and an aconitase. A functional assay for translational repression and direct demonstration of the iron switch. Eur. J. Biochem. 218, 657–667 (1993).

    Article  CAS  Google Scholar 

  46. Korner, C.G. et al. The deadenylating nuclease (DAN) is involved in poly(A) tail removal during the meiotic maturation of Xenopus oocytes. EMBO J. 17, 5427–5437 (1998).

    Article  CAS  Google Scholar 

  47. Benes, V. & Muckenthaler, M. Standardization of protocols in cDNA microarray analysis. Trends Biochem. Sci. 28, 244–249 (2003).

    Article  CAS  Google Scholar 

  48. Hanson, E.S., Foot, L.M. & Leibold, E.A. Hypoxia post-translationally activates iron-regulatory protein 2. J. Biol. Chem. 274, 5047–5052 (1999).

    Article  CAS  Google Scholar 

  49. Toth, I., Yuan, L., Rogers, J.T., Boyce, H. & Bridges, K.R. Hypoxia alters iron-regulatory protein-1 binding capacity and modulates cellular iron homeostasis in human hepatoma and erythroleukemia cells. J. Biol. Chem. 274, 4467–4473 (1999).

    Article  CAS  Google Scholar 

  50. Christova, T. & Templeton, D.M. Effect of hypoxia on the binding and subcellular distribution of iron regulatory proteins. Mol. Cell. Biochem., published online 3 January 2007.

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Acknowledgements

We thank Y. Vainshtein for support with microarray data analysis, J. Stolte for maintenance of the IronChip platform, V. Benes and members of the European Molecular Biology Laboratory Genomics Core Facility for help with qPCR and sequencing, and D. Shouval (Hadassah University Hospital) for kindly providing the RC1 renal carcinoma cell line. This work was supported by grants from the Marie Curie Quality of Life Programme, CORDIS FP5 (QLGA-CT-2001-52011) and the Young Investigator Award of the Medical Faculty, University of Heidelberg to M.S., by grants from the Forschungsschwerpunktprogramm des Landes Baden-Württemberg (RNA and disease) to M.U.M. and M.W.H. and by funds from the Gottfried Wilhem Leibniz Prize to M.W.H.

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Author notes

  1. Martina U Muckenthaler and Matthias W Hentze: These authors contributed equally to this work.

Authors and Affiliations

  1. Molecular Medicine Partnership Unit, Im Neuenheimer Feld 153, Heidelberg, 69120, Germany

    Mayka Sanchez, Martina U Muckenthaler & Matthias W Hentze

  2. European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, 69117, Germany

    Mayka Sanchez, Bruno Galy, Martina U Muckenthaler & Matthias W Hentze

  3. Department of Pediatric Oncology, Hematology and Immunology, University of Heidelberg, Im Neuenheimer Feld 153, Heidelberg, 69120, Germany

    Mayka Sanchez & Martina U Muckenthaler

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Contributions

M.S., B.G., M.U.M. and M.W.H designed and analyzed the experiments and wrote the paper. M.S. and B.G. performed the experiments.

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Correspondence to Martina U Muckenthaler or Matthias W Hentze.

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Supplementary information

Supplementary Fig. 1

Phylogenetic conservation of the EPAS1 5′ untranslated region iron-responsive element. (PDF 341 kb)

Supplementary Fig. 2

Iron regulation of the EPAS1 iron-responsive element in HeLa cells. (PDF 323 kb)

Supplementary Fig. 3

EPAS1 and HIF1α expression in RC1. (PDF 1478 kb)

Supplementary Table 1

Oligonucleotides used in this study. (PDF 21 kb)

Supplementary Methods

5′ rapid amplification of cDNA ends. (PDF 16 kb)

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Sanchez, M., Galy, B., Muckenthaler, M. et al. Iron-regulatory proteins limit hypoxia-inducible factor-2α expression in iron deficiency. Nat Struct Mol Biol 14, 420–426 (2007). https://doi.org/10.1038/nsmb1222

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