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...

Premature senescence of human diploid fibroblasts (HDFs) can be induced by exposures to a variety of oxidative stress and DNA damaging agents. In this study we developed a robust model of UVB-induced premature senescence of skin HDFs. After a series of 10 subcytotoxic (nonproapoptotic) exposures to UVB at 250 mJ/cm 2 , the socalled biomarkers of senescence were markedly expressed: growth arrest, senescence-associated β-galactosidase activity, senescence-associated gene overexpression, deletion in mitochondrial DNA. A set of 44 stress-and senescence-associated genes were found to be differentially expressed in this model, among which clusterin/ apolipoprotein J (apo J) and transforming growth factor-β1 (TGF-β1). Transfection of apo J cDNA provided protection against premature senescence-inducing doses of UVB and other stressful agents. Neutralizing antibodies against TGF-β1 or its receptor II (TβRII) sharply attenuated the senescence-associated features, suggesting a role for TGF-β1 in UVB-induced premature senescence. Both the latent and active forms of TGF-β1 were increased with time after the last UVB stress. Proteasome inhibition was ruled out as a potential mechanism of UVB-induced stress-induced premature senescence (SIPS). This model represents an alternative in vitro model in photoaging research for screening potential anti-photoaging compounds.Supplementary material available online at

As a result of oxygen deprivation (hypoxia), cells undergo different transcriptional adaptations that are in majority dependent on one key transcription factor, hypoxia-inducible factor-1 (HIF-1)

Oxygen-dependent regulation of HIF-1 activity occurs at multiple levels in vivo. The mechanisms regulating HIF-1alpha protein expression have been most extensively analyzed but the ones modulating HIF-1 transcriptional activity remain unclear. Changes in the phosphorylation and/or redox status of HIF-1alpha certainly play a role. Here, we show that ionomycin could activate HIF-1 transcriptional activity in a way that was additive to the effect of hypoxia without affecting HIF-1alpha protein level. In addition, a calmodulin dominant negative mutant and W7, a calmodulin antagonist, as well as BAPTA, an intracellular calcium chelator, inhibited the hypoxia-induced HIF-1 activation. These results indicate that elevated calcium in hypoxia could participate in HIF-1 activation. Furthermore, ERK but not JNK phosphorylation was evidenced in both conditions, ionomycin and hypoxia. PD98059, an inhibitor of the ERK pathway as well as a ERK1 dominant negative mutant also blocked HIF-1 activation by hypoxia and by ionomycin. A MEKK1 (a kinase upstream of JNK) dominant negative mutant had no effect. In addition, BAPTA, calmidazolium, a calmodulin antagonist and PD98059 inhibited VEGF secretion by hypoxic HepG2. All together, these results suggest that calcium and calmodulin would act upstream of ERK in the hypoxia signal transduction pathway.

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