Regulation of Hypoxia-inducible Factor-1α Protein Level during Hypoxic Conditions by the Phosphatidylinositol 3-Kinase/Akt/Glycogen Synthase Kinase 3β Pathway in HepG2 Cells

Abstract

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...

Hypoxia promotes glycogen synthesis and accumulation in human ovarian clear cell carcinoma

“…Additionally, it is well known that AKT regulates glycogen synthesis through phosphorylation at Ser9 followed by inactivating the GSK3β. When the cells are exposed to hypoxic conditions, time-dependent biphasic HIF1α stabilization and activation by PI3K/AKT/GSK3β signaling pathway has been proposed but this explanation remains controversial (50). Our present results supported part of the initial response reported in previously findings (50), although the hypoxic treatment of the cells with a different type of PIK3CA mutation was for a longer time compared with that in the previous report (50).…”

“…5B). Since PIK3CA gene mutation causes the continuous activation of AKT, it was not surprising in HAC2 cells that the phosphorylated AKT at Ser473 was elevated under hypoxic culture conditions as well as DFO treatment, moreover, the stabilized HIF1 (48,49) was further activated by PI3K/AKT pathway, one of the main mechanisms of HIF1 activation, as reported previously (50). Additionally, it is well known that AKT regulates glycogen synthesis through phosphorylation at Ser9 followed by inactivating the GSK3β.…”

The TWIST1 oncogene is a direct target of hypoxia-inducible factor-2α

“…We identified three novel genes previously unknown to play a role in the C. elegans HIF pathway. Two of these, spe-8 and gska-3, are predicted homologues of the human tyrosine kinase FER and the serine/threonine glycogen synthase kinase GSK-3 respectively, which have previously been reported to play a role in HIF regulation in mammalian cells (Mottet et al, 2003;Salem et al, 2005). Using gska-3-deficient animals, we confirmed increased hypoxia sensitivity observed with gska-3 knockdown.…”

Loss of expression and nuclear/cytoplasmic localization of the FOXP1 forkhead transcription factor are common events in early endometrial cancer: relationship with estrogen receptors and HIF-1α expression

“…Several studies also suggested that Act significantly contributes to HIF-1a stabilization. 31,32 A direct role of FOXP1 in HIF regulation cannot however, be excluded as a recent study indicated that the nuclear FOXO4 protein downregulates HIF-1a through prolyl hydroxylase/pVHL-independent transcriptional pathway. 33 The biological consequences of the cytoplasmic localization of FOXP1 or its overall loss of expression are obscure.…”