Hypoxia initiates an intracellular signaling pathway leading to the activation of the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1 activity is regulated through different mechanisms involving stabilization of HIF-1␣, phosphorylations, modifications of redox conditions, and interactions with coactivators. However, it appears that some of these steps can be cell type-specific. Among them, the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in the regulation of HIF-1 by hypoxia remains controversial. Here, we investigated the activation state of PI3K/Akt/glycogen synthase kinase 3␤ (GSK3␤) in HepG2 cells. Increasing incubation times in hypoxia dramatically decreased both the phosphorylation of Akt and the inhibiting phosphorylation of GSK3␤. The PI3K/Akt pathway was necessary for HIF-1␣ stabilization early during hypoxia. Indeed, its inhibition was sufficient to decrease HIF-1␣ protein level after 5-h incubation in hypoxia. However, longer exposure (16 h) in hypoxia resulted in a decreased HIF-1␣ protein level compared with early exposure (5 h). At that time, Akt was no longer present or active, which resulted in a decrease in the inhibiting phosphorylation of GSK3␤ on Ser-9 and hence in an increased GSK3␤ activity. GSK3 inhibition reverted the effect of prolonged hypoxia on HIF-1␣ protein level; more stabilized HIF-1␣ was observed as well as increased HIF-1 transcriptional activity. Thus, a prolonged hypoxia activates GSK3␤, which results in decreased HIF-1␣ accumulation. In conclusion, hypoxia induced a biphasic effect on HIF-1␣ stabilization with accumulation in early hypoxia, which depends on an active PI3K/Akt pathway and an inactive GSK3␤, whereas prolonged hypoxia results in the inactivation of Akt and activation of GSK3␤, which then down-regulates the HIF-1 activity through down-regulation of HIF-1␣ accumulation.Mammalian cells require a constant supply of oxygen to maintain adequate energy production, which is essential for maintaining normal function and for ensuring cell survival. The cellular response to decreased oxygen levels is regulated by a hypoxia-inducible factor-1 (HIF-1) 1 heterodimeric complex composed of two subunits, HIF-1␣ and arylhydrocarbon receptor nuclear translocator (1).This transcription factor binds to conserved regulatory sequences known as hypoxia-responsive element (HRE) found in the promoter of several target genes such as vascular endothelial growth factor (VEGF), erythropoietin, or glycolytic enzymes (aldolase A, enolase-␣, etc.) and controls their expression in response to hypoxia, leading to the adaptation of cells to decreased oxygen level (2).Only the HIF-1␣ subunit is regulated by a reduced oxygen level, and the regulation occurs in large part at post-translational modifications, resulting in its stabilization, nuclear translocation, DNA binding activity, and proper transcriptional activity. Under normal conditions, HIF-1␣ is hydroxylated at the prolines 564 and 462 residues in the oxygen-dependent degradation domain and, hence, inter...

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Hypoxic environment in solid tumor is known to favor cell survival and to initiate the formation of new capillaries. In this work, we identified by 2D gel analysis 94-kDa glucose-regulated protein (GRP94) as being upregulated in human endothelial cells in response to hypoxia. Three putative hypoxia responsive elements (HRE) were found in the GRP94 promoter. Competition experiments of HIF-1 DNA binding using specific probes containing each HRE sequence of the GRP94 promoter clearly evidenced that HIF-1 binds these sequences with high affinity. The human GRP94 promoter was then cloned upstream of the luciferase gene and showed enhanced activity in hypoxic conditions. Mutation of two of the three HREs present in this promoter completely inhibited the hypoxia-induced increase in luciferase activity.