Intracellular localisation of human HIF-1 alpha hydroxylases: implications for oxygen sensing.
Hypoxia-inducible factor1 (HIF-1) is an essential transcription factor for cellular adaptation to decreased oxygen availability. In normoxia the oxygen-sensitive alpha-subunit of HIF-1 is hydroxylated on Pro564 and Pro402 and thus targeted for proteasomal degradation. Three human oxygen-dependent HIF-1 alpha 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 alpha 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.
Metzen
Eric
E
Institute of Physiology, University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany. metzen@physio.uni-luebeck.de
Berchner-Pfannschmidt
Utta
U
Stengel
Petra
P
Marxsen
Jan H
JH
Stolze
Ineke
I
Klinger
Matthias
M
Huang
Wei Qi
WQ
Wotzlaw
Christoph
C
Hellwig-Bürgel
Thomas
T
Jelkmann
Wolfgang
W
Acker
Helmut
H
Fandrey
Joachim
J
D002451:Cell Compartmentation / Q000502:physiology; D015687:Cell Hypoxia / Q000502:physiology*; D002467:Cell Nucleus / Q000201:enzymology*; D003593:Cytoplasm / Q000201:enzymology*; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008164:Luminescent Proteins; D006899:Mixed Function Oxygenases; D012516:Osteosarcoma / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D011393:Procollagen-Proline Dioxygenase / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000235:genetics; D014407:Tumor Cells, Cultured
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008164:Luminescent Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D006899:Mixed Function Oxygenases; C439790:HIF1AN protein, human; D011393:Procollagen-Proline Dioxygenase; D010100:Oxygen