2-2--(hydroxynitrosohydrazono)bis-ethanamine and benzyloxycarbonylleucyl-leucyl-leucine-aldehyde

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.

Topics: Amino Acids, Dicarboxylic; Caspase 3; Caspases; Cell Hypoxia; Cell Line; Cysteine Proteinase Inhibitors; Deferoxamine; Enzyme Inhibitors; Ferric Compounds; Gene Expression; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Iron; Leupeptins; Nitric Oxide; Nitric Oxide Donors; Procollagen-Proline Dioxygenase; Proteasome Endopeptidase Complex; Proteasome Inhibitors; S-Nitrosoglutathione; Transcription Factors; Triazenes; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases; Von Hippel-Lindau Tumor Suppressor Protein

Cells exposed to low oxygen concentrations respond by initiating defense mechanisms, including the stabilization of hypoxia-inducible factor (HIF) 1alpha, a transcription factor that upregulates genes such as those involved in glycolysis and angiogenesis. Nitric oxide and other inhibitors of mitochondrial respiration prevent the stabilization of HIF1alpha during hypoxia. In studies of cultured cells, we show that this effect is a result of an increase in prolyl hydroxylase-dependent degradation of HIF1alpha. With the use of Renilla luciferase to detect intracellular oxygen concentrations, we also demonstrate that, upon inhibition of mitochondrial respiration in hypoxia, oxygen is redistributed toward nonrespiratory oxygen-dependent targets such as prolyl hydroxylases so that they do not register hypoxia. Thus, the signaling consequences of hypoxia may be profoundly modified by nitric oxide.

Topics: Antioxidants; Cell Hypoxia; Cell Line; Cell Respiration; Cycloheximide; Cysteine Proteinase Inhibitors; HeLa Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Leupeptins; Luciferases; Methacrylates; Mitochondria; Nitric Oxide; Nitric Oxide Donors; Oxygen; Procollagen-Proline Dioxygenase; Protein Synthesis Inhibitors; Reactive Oxygen Species; Recombinant Fusion Proteins; Signal Transduction; Thiazoles; Transcription Factors; Transfection; Triazenes