Thrombin Activates the Hypoxia-Inducible Factor-1 Signaling Pathway in Vascular Smooth Muscle Cells

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230

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...

Section: Fig 7 Effect Of Pi3k Inhibition On Hif-1␣ Expressionmentioning

confidence: 99%

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

Mottet¹,

Dumont

Deccache

et al. 2003

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230

“…This may occur through direct phosphorylation because these kinases have been shown to phosphorylate HIF-1␣ in vitro (28,29). In some cases, p44/p42 MAPK and p38 can also influence HIF-1␣ protein induction (8,30). The stress-activated protein kinases/c-Jun N-terminal kinases (SAPKs/JNKs) do not phosphorylate HIF-1␣ in vitro, but may indirectly regulate HIF-1-mediated transcription of VEGF under hypoxia through phosphorylating the transcription factor c-Jun (18,23).…”

Section: Vasular Endothelial Growth Factor (Vegf)mentioning

confidence: 99%

Evidence for a Role of p38 Kinase in Hypoxia-inducible Factor 1-independent Induction of Vascular Endothelial Growth Factor Expression by Sodium Arsenite

Duyndam

Hulscher

Wall

et al. 2003

Recently we have demonstrated that sodium arsenite induces the expression of hypoxia-inducible factor 1␣ (HIF-1␣) protein and vascular endothelial growth factor (VEGF) in OVCAR-3 human ovarian cancer cells. We now show that arsenic trioxide, an experimental anticancer drug, exerts the same effects. The involvement of phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK) pathways in the effects of sodium arsenite was investigated. By using kinase inhibitors in OVCAR-3 cells, both effects of sodium arsenite were found to be independent of phosphatidylinositol 3-kinase and p44/p42 MAPKS but were attenuated by inhibition of p38 MAPK. A role for p38 in the regulation of HIF-1␣ and VEGF expression was supported further by analysis of activation kinetics. Experiments in mouse fibroblast cell lines, lacking expression of c-Jun N-terminal kinases 1 and 2, suggested that these kinases are not required for induction of HIF-1␣ protein and VEGF mRNA. Unexpectedly, sodium arsenite did not activate a HIF-1-dependent reporter gene in OVCAR-3 cells, indicating that functional HIF-1 was not induced. In agreement with this hypothesis, up-regulation of VEGF mRNA was not reduced in HIF-1␣ ؊/؊ mouse fibroblast cell lines. Altogether, these data suggest that not HIF-1, but rather p38, mediates induction of VEGF mRNA expression by sodium arsenite.

“…Recent reports show the induction of HIF under normoxic conditions by certain stimuli such as cytokines (25,26), hormones (27,28), growth factors (17, 29 -31), and viral-encoded receptors (32). The activation of HIF is dependent on the PI3K/ Akt pathway in some cases, such as EGF (17,31) and insulinlike growth factor 1 (IGF-1) (30) stimulation, but not others.…”

mentioning

confidence: 99%

Lack of Evidence for the Involvement of the Phosphoinositide 3-Kinase/Akt Pathway in the Activation of Hypoxia-inducible Factors by Low Oxygen Tension

Álvarez-Tejado

Alfranca

Aragonés

et al. 2002

107

Hypoxia-inducible factors (HIF) belong to an evolutionary conserved family of transcription factors, the activity of which is tightly regulated by oxygen levels. We have recently demonstrated that hypoxia activates the phosphoinositide 3-kinase (PI3K)/Akt pathway in some cell types, and other works have suggested that this pathway is involved in the activation of HIF. In the present work we studied the role of this pathway in the induction of HIF by hypoxia. Under hypoxic conditions the PI3K/Akt pathway was activated in some (PC12 and HeLa) but not all cell types (HepG2) tested, whereas the HIF protein was induced by hypoxia in all cases. Kinetics analysis showed that, when observed, the activation of PI3K/Akt occurred after HIF induction. In addition, the chemical inhibition of PI3K had no significant effect on the induction of the HIF protein or its transcriptional activity but prevented Akt activation. Accordingly, transient overexpression of a dominant negative form of the regulatory subunit of PI3K in HEK293T cells did not interfere with the induction of the HIF-␣ protein by hypoxia or affect HIF-mediated transcription in any of the cell types tested. Moreover, forced activation of the PI3K/Akt pathway did not affect the transcriptional activity of HIF under normoxic or hypoxic conditions. Thus, our data suggest that the activation of PI3K/Akt by hypoxia is cell type-specific and, when observed, lies downstream of HIF activation or in a parallel pathway. Furthermore, the activity of the PI3K/Akt is not sufficient for the activation of HIF nor is it essential for its induction by hypoxia.The reduction in oxygen levels in aerobic organisms triggers specific cellular and systemic adaptive responses. Among the molecular responses to hypoxia found in multicellular organisms, the activation of HIF 1 transcription factors is the best characterized and the one responsible for the activation of genes involved in energy metabolism, angiogenesis, and apoptosis. HIF proteins belong to the basic helix-loop-helix-Per/ ARNT/Sim (bHLH-PAS) family of transcription factors. The functional HIF unit is a heterodimer composed of an HIF-␣ and a HIF-␤ (ARNT) subunit. The ␤ subunit is common to all HIF complexes, whereas there are three different HIF-␣ subunits that can participate in the complex: HIF-1␣, HIF-2␣ (EPAS), and HIF-3␣. Both ␣ and ␤ mRNAs as well as the HIF-␤ protein are constitutively expressed; however, HIF-␣ protein levels are tightly regulated by the level of oxygen. Under normoxic conditions HIF proteins show a remarkably short half-life (Ͻ5 min), whereas a decline in oxygen tension results on its accumulation (1). Further analysis demonstrated that a specific region of HIF, the oxygen-dependent degradation domain, mediates its targeting for ubiquitination and proteasomal degradation under normoxic conditions (1, 2). Importantly, the von Hippel-Lindau tumor suppressor protein (pVHL) binds to HIF and functions as part of an E3 ubiquitin ligase complex that mediates its degradation (3). Recently, several groups (4 -...