Hypoxic induction of the hypoxia-inducible factor is mediated via the adaptor protein Shc in endothelial cells

Circ Res. 2002 Jul 12;91(1):38-45. doi: 10.1161/01.res.0000024412.24491.ca.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing*
  • Adaptor Proteins, Vesicular Transport*
  • Blotting, Western
  • Cell Hypoxia / physiology
  • Cell Line
  • Cell Movement / drug effects
  • Cobalt / pharmacology
  • Cycloheximide / pharmacology
  • DNA-Binding Proteins / metabolism*
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / drug effects
  • Endothelium, Vascular / metabolism*
  • Enzyme Inhibitors / pharmacology
  • Humans
  • Hypoxia-Inducible Factor 1
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Mutation
  • Nuclear Proteins / metabolism*
  • Plasmids / genetics
  • Proteins / genetics
  • Proteins / metabolism*
  • Proto-Oncogene Proteins c-raf / antagonists & inhibitors
  • Proto-Oncogene Proteins c-raf / metabolism
  • Pyrazoles / pharmacology
  • Pyrimidines / pharmacology
  • Shc Signaling Adaptor Proteins
  • Src Homology 2 Domain-Containing, Transforming Protein 1
  • Transcription Factors*
  • Transfection
  • Tyrphostins / pharmacology
  • Ubiquitin / metabolism
  • ras Proteins / genetics
  • ras Proteins / metabolism
  • src-Family Kinases / antagonists & inhibitors
  • src-Family Kinases / metabolism

Substances

  • 4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine
  • Adaptor Proteins, Signal Transducing
  • Adaptor Proteins, Vesicular Transport
  • DNA-Binding Proteins
  • Enzyme Inhibitors
  • HIF1A protein, human
  • Hypoxia-Inducible Factor 1
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Nuclear Proteins
  • Proteins
  • Pyrazoles
  • Pyrimidines
  • SHC1 protein, human
  • Shc Signaling Adaptor Proteins
  • Src Homology 2 Domain-Containing, Transforming Protein 1
  • Transcription Factors
  • Tyrphostins
  • Ubiquitin
  • 6,7-dimethoxy-3-phenylquinoxaline
  • Cobalt
  • Cycloheximide
  • src-Family Kinases
  • Proto-Oncogene Proteins c-raf
  • ras Proteins
  • cobaltous chloride