Hypoxia-inducible factor-1 alpha (HIF-1 alpha) escapes O(2)-driven proteasomal degradation irrespective of its subcellular localization: nucleus or cytoplasm

Abstract

Eukaryotic cells sense oxygen and adapt to hypoxia by regulating a number of genes. Hypoxia-inducible factor 1 (HIF-1) is the 'master' in this pleiotypic response. HIF-1 comprises two members of the basic helix--loop--helix transcription factor family, HIF-1 alpha and HIF-1 beta. The HIF-1 alpha protein is subject to drastic O(2)-dependent proteasomal control. However, the signalling components regulating the 'switch' for 'escaping' proteasomal degradation under hypoxia are still largely unknown. The rapid nuclear translocation of HIF-1 alpha could represent an efficient way to escape from this degradation. We therefore asked, where in the cell is HIF-1 alpha degraded? To address this question, we trapped HIF-1 alpha either in the cytoplasm, by fusing HIF-1 alpha to the cytoplasmic domain of the Na(+)-H(+) exchanger (NHE-1), or in the nucleus, by treatment with leptomycin B. Surprisingly, we found that HIF-1 alpha is stabilized by hypoxia and undergoes O(2)-dependent proteasomal degradation with an identical half-life (5--8 min) in both cellular compartments. Therefore, HIF-1 alpha entry into the nucleus is not, as proposed, a key event that controls its stability. This result markedly contrasts with the mechanism that controls p53 degradation via MDM2.

Fig. 1. Schematic representation of the NHE-1>>HIF-1α chimera. The figure outlines the main features of both the plasma membrane-bound Na⁺-H⁺ exchanger and transcription factor HIF-1α.

Fig. 2. Stable expression of the chimerical NHE-1>>HIF-1α protein. NHE-1 or NHE-1>>HIF-1α cells incubated either in normoxia (20% O₂) (A and C) or hypoxia (1–2% O₂) (B and D) for 3 h were analysed by immunofluorescence using the anti-HIF-1α antiserum. In addition, control and chimerical protein expressing cells were incubated in the absence or in the presence of CoCl₂ (200 µM) for 3 h and whole cellular extracts (50 µg) were separated by SDS–PAGE and revealed by immunoblot analysis using the same anti-HIF-1α antibody (E). Note that the experiment shown in (E) was done with the stable transfected population whereas (D) is representative of a subclone expressing high levels of the chimera. Results are representative of three independent experiments.

Fig. 2. Stable expression of the chimerical NHE-1>>HIF-1α protein. NHE-1 or NHE-1>>HIF-1α cells incubated either in normoxia (20% O₂) (A and C) or hypoxia (1–2% O₂) (B and D) for 3 h were analysed by immunofluorescence using the anti-HIF-1α antiserum. In addition, control and chimerical protein expressing cells were incubated in the absence or in the presence of CoCl₂ (200 µM) for 3 h and whole cellular extracts (50 µg) were separated by SDS–PAGE and revealed by immunoblot analysis using the same anti-HIF-1α antibody (E). Note that the experiment shown in (E) was done with the stable transfected population whereas (D) is representative of a subclone expressing high levels of the chimera. Results are representative of three independent experiments.

Fig. 3. Oxygen-driven proteasome-mediated regulation of cytoplasmic HIF-1α. Control cells (left panel) or cells expressing the chimerical protein NHE-1>> HIF-1α either untreated (middle panel) or pre-treated with 20 µM of the proteasome inhibitor lactacystin for 30 min (right panel), were incubated under normoxia (20% O₂; N) or hypoxia (1–2% O₂; H) for 4 h after which hypoxic cells were returned to 20% O₂ for 0–40 min. Total cell lysates were analysed by immunoblotting with the antibody to HIF-1α. Essentially identical results were obtained in three independent experiments.

Fig. 4. Blocking nuclear export does affect O₂-dependent degradation of HIF-1α. HeLa cells cultured either in the absence or in the presence of leptomycin B (20 nM) for 16 h were incubated under normoxia (20% O₂; N) or hypoxia (1–2% O₂; H) for an additional 4 h after which hypoxic cells were returned to 20% O₂ for 0–20 min. Total cell lysates were analysed by immunoblotting with antibodies to HIF-1α and p53. Results are representative of three independent experiments.