Nitric oxide reverses desferrioxamine- and hypoxia-evoked HIF-1alpha accumulation--implications for prolyl hydroxylase activity and iron.
Hypoxia inducible factor 1 (HIF-1) senses and coordinates cellular responses towards hypoxia. HIF-1 activity is primarily determined by stability regulation of its alpha subunit that is degraded by the 26S proteasome under normoxia due to hydroxylation by prolyl hydroxylases (PHDs) but is stabilized under hypoxia. Besides hypoxia, nitric oxide (NO) stabilizes HIF-1alpha and promotes hypoxia-responsive target gene expression under normoxia. However, in hypoxia, NO attenuates HIF-1alpha stabilization and gene activation. It was our intention to explain the contrasting behavior of NO under hypoxia. We used the iron chelator desferrioxamine (DFX) or hypoxia to accumulate HIF-1alpha in HEK293 cells. Once the protein accumulated, we supplied NO donors and followed HIF-1alpha disappearance. NO-evoked HIF-1alpha destabilization was reversed by proteasomal inhibition or by blocking PHD activity. By using the von Hippel Lindau (pVHL)-HIF-1alpha capture assay, we went on to demonstrate binding of pVHL to HIF-1alpha under DFX/NO but not DFX alone. Showing increased intracellular free iron under conditions of hypoxia/NO compared to hypoxia alone, we assume that increased free iron contributes to regain PHD activity. Variables that allow efficient PHD activation such as oxygen availability, iron content, or cofactor accessibility at that end allow NO to modulate HIF-1alpha accumulation.
Callapina
Melvin
M
Faculty of Medicine, Institute of Biochemistry I, Johann Wolfgang Goethe-University, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany.
Zhou
Jie
J
Schnitzer
Steffen
S
Metzen
Eric
E
Lohr
Christian
C
Deitmer
Joachim W
JW
Brüne
Bernhard
B
D000600:Amino Acids, Dicarboxylic / Q000494:pharmacology; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D015687:Cell Hypoxia; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003676:Deferoxamine / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005290:Ferric Compounds / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007501:Iron / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology; D009569:Nitric Oxide / Q000494:pharmacology / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D011393:Procollagen-Proline Dioxygenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D026422:S-Nitrosoglutathione / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014226:Triazenes / Q000494:pharmacology; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D051794:Von Hippel-Lindau Tumor Suppressor Protein
C105351:1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene; D000600:Amino Acids, Dicarboxylic; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D005290:Ferric Compounds; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007976:Leupeptins; D020030:Nitric Oxide Donors; D061988:Proteasome Inhibitors; D014157:Transcription Factors; D014226:Triazenes; D025521:Tumor Suppressor Proteins; D009569:Nitric Oxide; C024823:ferric sulfate; D026422:S-Nitrosoglutathione; D007501:Iron; D011393:Procollagen-Proline Dioxygenase; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D046988:Proteasome Endopeptidase Complex; C497446:VHL protein, human; D003676:Deferoxamine; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; C040947:oxalylglycine