Published online before print June 6, 2002, doi: 10.1161/01.RES.0000024412.24491.CA
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Submitted on January 9, 2002
Revised on May 21, 2002
Accepted on May 22, 2002
Hypoxic Induction of the Hypoxia-Inducible Factor Is Mediated via the Adaptor Protein Shc in Endothelial Cells
Frank Jung ;
From Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Frankfurt, Germany.
* To whom correspondence should be addressed. E-mail: Dimmeler{at}em.uni-frankfurt.de.
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-1
protein stabilization and endothelial cell migration.
Key words: hypoxia • hypoxia-inducible factor-1 • Shc • Ras • endothelial cells
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