Journal
CIRCULATION RESEARCH
Volume 91, Issue 1, Pages 38-45Publisher
LIPPINCOTT WILLIAMS & WILKINS
DOI: 10.1161/01.RES.0000024412.24491.CA
Keywords
hypoxia; hypoxia-inducible factor-1; Shc; Ras; endothelial cells
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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 She tyrosine phosphorylation, with a maximum at 10 minutes. To test whether She directly mediates hypoxia-induced HIF stabilization, human umbilical vein endothelial cells were transfected with a dominant-negative She 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 She. Hypoxia-induced Ras activity was significantly reduced in cells transfected with dnShc compared with control levels, indicating that Ras indeed acts downstream from She. 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 She 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, She in concert with Ras and Raf-1 contributes to hypoxia-induced HIF-1alpha protein stabilization and endothelial cell migration.
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