Activation of HIF‐1α in exponentially growing cells via hypoxic stimulation is independent of the Akt/mTOR pathway

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Abstract
Accumulation of HIF‐1α during normoxic conditions at high cell density has previously been shown to occur and can be used to stabilize HIF‐1α protein in the absence of a specific anaerobic chamber. However, the impact and origin of this pool of HIF‐1α, obtained under normoxia, has been underestimated. In this study, we have systematically compared the related pools of HIF‐1α stabilized in normoxia by high cell density to those obtained at low density in hypoxia. At first glance, these two stimuli appear to have similar outcomes: HIF‐1α stabilization and induction of HIF‐1‐dependent genes. However, upon careful analysis, we observed that molecular mechanisms involved are different. We clearly demonstrate that density‐dependant HIF‐1α accumulation during normoxia is due to the cells high consumption of oxygen, as demonstrated by using a respiration inhibitor (oligomycin) and respiratory‐defective mutant cells (GSK3). Finally and most importantly, our data indicate that a decrease in AKT activity followed by a total decrease in p70S6K phosphorylation reflecting a decrease in mTOR activity occurs during high oxygen consumption, resulting from high cell density. In contrast, hypoxia, even at severe low O2 levels, only slightly impacts upon the mTOR pathway under low cell density conditions. Thus, activation of HIF‐1α in exponentially growing cells via hypoxic stimulation is independent of the Akt/mTOR pathway whereas HIF‐1α activation obtained in high confluency is totally dependent on mTOR pathway as rapamycin totally impaired (i) HIF‐1α stabilization and (ii) mRNA levels of CA9 and BNIP3, two HIF‐target genes. J. Cell. Physiol. 218: 167–174, 2009.