Hypoxia-inducible factor 1 alpha (HIF-1 alpha) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions - Its stabilization by hypoxia depends on redox-induced changes

The hypoxia-inducible factor 1 transcriptional activator complex (HIF-1) is involved in the activation of the erythropoietin and several other hypoxia-responsive genes, The HIF-1 complex is composed of two protein subunits: HIF-1 beta/ARNT (aryl hydrocarbon receptor nuclear translocator), which is constitutively expressed, and HIF-1 alpha, which is not present in normal cells but induced under hypoxic conditions. The HIF-1 alpha subunit is continuously synthesized and degraded under normoxic conditions, while it accumulates rapidly following exposure to low oxygen tensions. The involvement of the ubiquitin-proteasome system in the proteolytic destruction of HIF-1 in normoxia was studied by the use of specific inhibitors of the proteasome system. Lactacystin and MG-132 were found to protect the degradation of the HIF-1 complex in cells transferred from hypoxia to normoxia. The same inhibitors were able to induce HIF-1 complex formation when added to normoxic cells. Final confirmation of the involvement of the ubiquitin-proteasome system in the regulated degradation of HIF-1 alpha was obtained by the use of ts20TG(R) cells, which contain a temperature-sensitive mutant of E1, the ubiquitin-activating enzyme, Exposure of ts20 cells, under normoxic conditions, to the non-permissive temperature induced a rapid and progressive accumulation of HIF-1. The effect of proteasome inhibitors on the normoxic induction of HIF-1 binding activity was mimicked by the thiol reducing agent N-(2-mercaptopropionyl)glycine and by the oxygen radical scavenger 2-acetamidoacrylic acid. Furthermore, N-(2-mercaptopropionyl)-glycine induced gene expression as measured by the stimulation of a HIF-1-luciferase expression vector and by the induction of erythropoietin mRNA in normoxic Hep 3B cells. These last findings strongly suggest that the hypoxia induced changes in HIF-1 alpha stability and subsequent gene activation are mediated by redox-induced changes.