Activation of HIF-1 alpha in Exponentially Growing Cells Via Hypoxic Stimulation Is Independent of the Akt/mTOR Pathway

Accumulation of HIF-1 alpha during normoxic conditions at high cell density has previously been shown to occur and can be used to stabilize HIF-1 alpha protein in the absence of a specific anaerobic chamber. However, the impact and origin of this pool of HIF-1 alpha, obtained under normoxia, has been under-estimated. In this study, we have systematically compared the related pools of HIF-1 alpha 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-1 alpha 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-1 alpha 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 chat a decrease in AKT activity followed by a total decrease in p70(S6K) phosphorylation reflecting a decrease in mTOR activity occurs during high oxygen consumption, resulting from high cell density. In contrast, hypoxia, even at severe low O-2 levels, only slightly impacts upon the mTOR pathway under low cell density conditions. Thus, activation of HIF-1 alpha in exponentially growing cells via hypoxic stimulation is independent of the Akt/mTOR pathway whereas HIF-1 alpha activation obtained in high confluency is totally dependent on mTOR pathway as rapamycin totally impaired (i) HIF-1 alpha stabilization and (ii) mRNA levels of CA9 and BNIP3, two HIF-target genes.