Regulation of hypoxia-inducible factor-1alpha protein level during hypoxic conditions by the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase 3beta pathway in HepG2 cells.
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-1alpha, 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 3beta (GSK3beta) in HepG2 cells. Increasing incubation times in hypoxia dramatically decreased both the phosphorylation of Akt and the inhibiting phosphorylation of GSK3beta. The PI3K/Akt pathway was necessary for HIF-1alpha stabilization early during hypoxia. Indeed, its inhibition was sufficient to decrease HIF-1alpha protein level after 5-h incubation in hypoxia. However, longer exposure (16 h) in hypoxia resulted in a decreased HIF-1alpha 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 GSK3beta on Ser-9 and hence in an increased GSK3beta activity. GSK3 inhibition reverted the effect of prolonged hypoxia on HIF-1alpha protein level; more stabilized HIF-1alpha was observed as well as increased HIF-1 transcriptional activity. Thus, a prolonged hypoxia activates GSK3beta, which results in decreased HIF-1alpha accumulation. In conclusion, hypoxia induced a biphasic effect on HIF-1alpha stabilization with accumulation in early hypoxia, which depends on an active PI3K/Akt pathway and an inactive GSK3beta, whereas prolonged hypoxia results in the inactivation of Akt and activation of GSK3beta, which then down-regulates the HIF-1 activity through down-regulation of HIF-1alpha accumulation.
Mottet
Denis
D
Laboratory of Biochemistry and Cellular Biology, University of Namur, 61 Rue de Bruxelles, 5000 Namur, Belgium.
Dumont
Valéry
V
Deccache
Yann
Y
Demazy
Catherine
C
Ninane
Noelle
N
Raes
Martine
M
Michiels
Carine
C
D000276:Adjuvants, Immunologic / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular; D002867:Chromones / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D018021:Lithium Chloride / Q000494:pharmacology; D008113:Liver Neoplasms; D009025:Morpholines / Q000494:pharmacology; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D014157:Transcription Factors; D014407:Tumor Cells, Cultured
D000276:Adjuvants, Immunologic; D002867:Chromones; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D018021:Lithium Chloride; D009025:Morpholines; D009687:Nuclear Proteins; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D014157:Transcription Factors; C497442:HIF1A protein, human; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C494918:AKT1 protein, human; C000605506:GSK3B protein, human