Accumulation and nuclear import of HIF1 alpha during high and low oxygen concentration in skeletal muscle cells in primary culture

Abstract

The hypoxia-inducible-factor-1 (HIF1) mediates the transcriptional upregulation of several target genes during hypoxia. HIF1 itself is known to be regulated essentially by ubiquitinylation and proteolytic degradation of the subunit HIF1α of the dimeric transcription factor HIF1. In contrast to other tissues, skeletal muscle expresses high amounts of HIF1α in normoxia as well as in hypoxia. In view of this, we aimed to investigate HIF1α accumulation and subcellular localization as well as the transcriptional activity of the HIF1α-regulated gene of glyceraldehyde dehydrogenase (GAPDH) in skeletal muscle cells exposed to low oxygen concentration (3% O 2), normoxia (20% O 2) or high oxygen concentration (42% O 2). Immunofluorescence analysis reveals that under normoxic and high oxygen conditions, significant amounts of HIF1α can be found exclusively in the cytoplasm of the myotubes. Muscle cells treated with CoCl 2, a known inhibitor of HIF1α degradation, show even higher levels of HIF1α, again exclusively in the cytoplasm. Under conditions of low oxygen, HIF1α in controls as well as in CoCl 2-treated cells is found in the nuclei. CdCl 2 inhibits nuclear import of HIF1α at low oxygen concentration and leads to a transcriptional downregulation of the marker enzyme of anaerobic glycolysis GAPDH. Immunoprecipitation with anti-HIF1α antibody co-precipitates HSP90 in an oxygen-dependent manner, more at high pO 2 than at low pO 2. Cadmium-treated samples also show high amounts of co-immunoprecipitated HSP90, independent of oxygen concentration. We conclude that in skeletal muscle cells, HIF1α, in contrast to other tissues, may, in addition to its regulation by degradation, also be regulated by binding to HSP90 and subsequent inhibition of its import into the nuclei.