Hypoxic induction of the hypoxia-inducible factor is mediated via the adaptor protein Shc in endothelial cells.
Tyrosine kinase cascades may play a role in the hypoxic regulation of hypoxia-inducible factor (HIF)-1. We investigated the role of tyrosine kinase phosphorylation and of the Shc/Ras cascade on hypoxic HIF-1 stabilization. Exposure of human umbilical vein endothelial cells to hypoxia results in HIF protein stabilization as early as 10 ... minutes, with a maximum at 3 hours, and also in Shc tyrosine phosphorylation, with a maximum at 10 minutes. To test whether Shc directly mediates hypoxia-induced HIF stabilization, human umbilical vein endothelial cells were transfected with a dominant-negative Shc mutant (dnShc), resulting in significantly reduced HIF protein levels compared with control. Similar results were obtained with cells transfected with dominant-negative Ras, a known downstream effector of Shc. Hypoxia-induced Ras activity was significantly reduced in cells transfected with dnShc compared with control levels, indicating that Ras indeed acts downstream from Shc. Moreover, cells pretreated with a specific Raf-1 kinase inhibitor, a known downstream effector of Ras, exhibited reduced HIF protein levels. To examine the functional consequences of Shc in hypoxic signaling, HIF-1 ubiquitination, protein stabilization, and endothelial cell migration were assessed. Overexpression of dnShc increased ubiquitination of HIF-1 and reduced the half-life of the protein. Moreover, dnShc, dominant-negative Ras, or the Raf-1 kinase inhibitor significantly inhibited migration under hypoxia. Thus, Shc in concert with Ras and Raf-1 contributes to hypoxia-induced HIF-1alpha protein stabilization and endothelial cell migration.
Mesh Terms:
Adaptor Proteins, Signal Transducing, Adaptor Proteins, Vesicular Transport, Blotting, Western, Cell Hypoxia, Cell Line, Cell Movement, Cobalt, Cycloheximide, DNA-Binding Proteins, Endothelium, Vascular, Enzyme Inhibitors, Humans, Hypoxia-Inducible Factor 1, Hypoxia-Inducible Factor 1, alpha Subunit, Mutation, Nuclear Proteins, Plasmids, Proteins, Proto-Oncogene Proteins c-raf, Pyrazoles, Pyrimidines, Shc Signaling Adaptor Proteins, Transcription Factors, Transfection, Tyrphostins, Ubiquitin, ras Proteins, src-Family Kinases
Adaptor Proteins, Signal Transducing, Adaptor Proteins, Vesicular Transport, Blotting, Western, Cell Hypoxia, Cell Line, Cell Movement, Cobalt, Cycloheximide, DNA-Binding Proteins, Endothelium, Vascular, Enzyme Inhibitors, Humans, Hypoxia-Inducible Factor 1, Hypoxia-Inducible Factor 1, alpha Subunit, Mutation, Nuclear Proteins, Plasmids, Proteins, Proto-Oncogene Proteins c-raf, Pyrazoles, Pyrimidines, Shc Signaling Adaptor Proteins, Transcription Factors, Transfection, Tyrphostins, Ubiquitin, ras Proteins, src-Family Kinases
Circ. Res.
Date: Jul. 12, 2002
PubMed ID: 12114320
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