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.
Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Frankfurt, Germany.
Frank
F
Jung
Judith
J
Haendeler
Jörg
J
Hoffmann
Agnes
A
Reissner
Elisabeth
E
Dernbach
Andreas M
AM
Zeiher
Stefanie
S
Dimmeler
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D015153:Blotting, Western; D015687:Cell Hypoxia / Q000502:physiology; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D003035:Cobalt / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014157:Transcription Factors; D014162:Transfection; D020032:Tyrphostins / Q000494:pharmacology; D025801:Ubiquitin / Q000378:metabolism; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C497442:HIF1A protein, human; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D011506:Proteins; D011720:Pyrazoles; D011743:Pyrimidines; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014157:Transcription Factors; D020032:Tyrphostins; D025801:Ubiquitin; C106250:6,7-dimethoxy-3-phenylquinoxaline; D003035:Cobalt; D003513:Cycloheximide; D019061:src-Family Kinases; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins; C018021:cobaltous chloride