2-2--(hydroxynitrosohydrazono)bis-ethanamine and oxalylglycine

Hypoxia-inducible factor 1 (HIF-1) is controlled through stability regulation of its alpha subunit, which is expressed under hypoxia but degraded under normoxia. Degradation of HIF-1alpha requires association of the von Hippel Lindau protein (pVHL) to provoke ubiquitination followed by proteasomal digestion. Besides hypoxia, nitric oxide (NO) stabilizes HIF-1alpha under normoxia but destabilizes the protein under hypoxia. To understand the role of NO under hypoxia we made use of pVHL-deficient renal carcinoma cells (RCC4) that show a high steady state HIF-1alpha expression under normoxia. Exposing RCC4 cells to hypoxia in combination with the NO donor DETA-NO (2,2'-(hydroxynitrosohydrazono) bis-ethanimine), but not hypoxia or DETA-NO alone, decreased HIF-1alpha protein and attenuated HIF-1 transactivation. Mechanistically, we noticed a role of calpain because calpain inhibitors reversed HIF-1alpha degradation. Furthermore, chelating intracellular calcium attenuated HIF-1alpha destruction by hypoxia/DETA-NO, whereas a calcium increase was sufficient to lower the amount of HIF-1alpha even under normoxia. An active role of calpain in lowering HIF-1alpha amount was also evident in pVHL-containing human embryonic kidney cells when the calcium pump inhibitor thapsigargin reduced HIF-1alpha that was stabilized by the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG). We conclude that calcium contributes to HIF-1alpha destruction involving the calpain system.

Topics: Amino Acid Sequence; Amino Acids, Dicarboxylic; Anaerobiosis; Calcium; Calcium Channel Blockers; Calcium Channels; Calpain; Cells, Cultured; Cysteine Proteinase Inhibitors; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Molecular Sequence Data; Nitric Oxide; Nitric Oxide Donors; Triazenes; Von Hippel-Lindau Tumor Suppressor Protein

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