The fully linked HTML version of this article has now been published.
JCS ePress
online publication date 12 Feb 2003
doi: 10.1242/jcs.00318
Research Article
Intracellular localisation of human HIF-1
hydroxylases: implications for oxygen sensing
Eric Metzen*,
Utta Berchner-Pfannschmidt,
Petra Stengel,
Jan H. Marxsen,
Ineke Stolze,
Matthias Klinger,
Wei Qi Huang,
Christoph Wotzlaw,
Thomas Hellwig-Bürgel,
Wolfgang Jelkmann,
Helmut Acker,
and
Joachim Fandrey
* Author for correspondence (e-mail: metzen{at}physio.uni-luebeck.de)
Hypoxia-inducible factor1 (HIF-1) is an essential transcription factor for cellular adaptation to decreased oxygen availability. In normoxia the oxygen-sensitive 
-subunit of HIF-1 is hydroxylated on Pro564 and Pro402 and thus targeted for proteasomal degradation. Three human oxygen-dependent HIF-1 prolyl hydroxylases (PHD1, PHD2, and PHD3) function as oxygen sensors in vivo. Furthermore, the asparagine hydroxylase FIH-1 (factor inhibiting HIF) has been found to hydroxylate Asp803 of the HIF-1 C-terminal transactivation domain, which results in the decreased ability of HIF-1 to bind to the transcriptional coactivator p300/CBP. We have fused these enzymes to the N-terminus of fluorescent proteins and transiently transfected the fusion proteins into human osteosarcoma cells (U2OS). Three-dimensional 2-photon confocal fluorescence microscopy showed that PHD1 was exclusively present in the nucleus, PHD2 and FIH-1 were mainly located in the cytoplasm and PHD3 was homogeneously distributed in cytoplasm and nucleus. Hypoxia did not influence the localisation of any enzyme under investigation. In contrast to FIH-1, each PHD inhibited nuclear HIF-1 accumulation in hypoxia. All hydroxylases suppressed activation of a cotransfected hypoxia-responsive luciferase reporter gene. Endogenous PHD2mRNA and PHD3mRNA were hypoxia-inducible, whereas expression of PHD1mRNA and FIH-1mRNA was oxygen independent. We propose that PHDs and FIH-1 form an oxygen sensor cascade of distinct subcellular localisation.
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U. Berchner-Pfannschmidt, F. Petrat, K. Doege, B. Trinidad, P. Freitag, E. Metzen, H. de Groot, and J. Fandrey
Chelation of Cellular Calcium Modulates Hypoxia-inducible Gene Expression through Activation of Hypoxia-inducible Factor-1{alpha}
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T. Acker and H. Acker
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G. L. Semenza
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Physiology,
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[Abstract]
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H. B. Suliman, M. Ali, and C. A. Piantadosi
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Blood,
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J. Fandrey
Oxygen-dependent and tissue-specific regulation of erythropoietin gene expression
Am J Physiol Regulatory Integrative Comp Physiol,
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[Abstract]
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S. Linke, C. Stojkoski, R. J. Kewley, G. W. Booker, M. L. Whitelaw, and D. J. Peet
Substrate Requirements of the Oxygen-sensing Asparaginyl Hydroxylase Factor-inhibiting Hypoxia-inducible Factor
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G. Hopfl, O. Ogunshola, and M. Gassmann
HIFs and tumors--causes and consequences
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P. H. Maxwell
Regulation of HIF-1; enzymatic hydroxylation, the role of VHL and activation in renal cell carcinoma.
AACR Meeting Abstracts,
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N. Erez, M. Milyavsky, R. Eilam, I. Shats, N. Goldfinger, and V. Rotter
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R. K. Bruick
Oxygen sensing in the hypoxic response pathway: regulation of the hypoxia-inducible transcription factor
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N. Masson and P. J. Ratcliffe
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© The Company of Biologists Ltd 2003
