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(Circulation Research. 2002;91:38.)
© 2002 American Heart Association, Inc.

Molecular Medicine

Hypoxic Induction of the Hypoxia-Inducible Factor Is Mediated via the Adaptor Protein Shc in Endothelial Cells

Frank Jung^*, Judith Haendeler^*, Jörg Hoffmann, Agnes Reissner, Elisabeth Dernbach, Andreas M. Zeiher, Stefanie Dimmeler

From Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Frankfurt, Germany.

Correspondence to Stefanie Dimmeler, PhD, Department of Internal Medicine IV, Molecular Cardiology, University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany. 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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