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

Abstract

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. Key Words: hypoxia Ⅲ hypoxia-inducible factor-1 Ⅲ Shc Ⅲ Ras Ⅲ endothelial cells H ypoxia is an important regulatory stimulus for a variety of different biological processes, such as vascular tone and structure, angiogenesis, and erythropoiesis. These events are mediated by a variety of hypoxia-sensitive cellular proteins, which modulate cell-cell interaction and cell proliferation and differentiation. Many of these proteins, such as erythropoietin, vascular endothelial growth factor, the inducible NO synthase gene, or glycolytic enzymes, 1-4 are under the control of a master regulator of O 2 homeostasis, hypoxia-inducible factor (HIF)-1, a transcription factor. This DNA binding protein is a heterodimer composed of 2 subunits, HIF-1␣ and HIF-1␤. HIF-1␤ can dimerize with several different basic helix-loop-helix-PAS proteins, whereas HIF-1␣ is the O 2 -regulated subunit that determines its biological activity. 5,6 The regulation of HIF is complex and involves not only changes on the transcriptional level but also posttranscriptional and posttranslational alterations in response to hypoxia. 6 -9 Perhaps most striking is HIF protein stabilization under hypoxic conditions and its rapid degradation on reoxygenation. Under normoxic conditions, HIF is subject to ubiquitination and proteasomal degradation. 6,9,10 However, the regulatory step in hypoxia-mediated HIF regulation has not been determined. The von Hippel-Lindau tumor suppressor protein, a putative ubiquitin-protein ligase, 11 appears to play a role in HIF protein stabilization, inasmuch as loss of the von Hippel-Lindau protein results in constitutive expression of the HIF protein. 12 In addition, there is evidence that multiple domains in the HIF protein regulate its expression and that they do so by altering the ubiquitination of HIF-1␣ under nonhypoxic conditions. 13 In addition to changes in the redox state affecting hypoxic signaling, changes in phosphorylation may influence HIF-1 expression and, eventually, the transactivation of genes. Indeed, hypoxic induction of HIF-1 is inhibited by pretreating cells with the tyrosine kinase inhibitor genistein or the serine/threonine phosphatase inhibitor sodium fluoride. 14 The adaptor protein Shc is an immediate substrate of tyrosine kinase and may play an important role in linking activated tyrosine kinase receptors to downstream hypoxic signaling, involving HIF protein stabilization. 15,16 The Shc family of adaptor proteins consists of multiple protein-protein interaction domains: an amino-terminal phosphotyrosine binding domain, a central collagen homology domain, and...

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“…Thus, it is possible that the hypoxic effects are mediated by the recruitment of specific signaling relay proteins and nonreceptor tyrosine kinases. For example, the adaptor protein shc, an immediate substrate of tyrosine kinases, may play an important role in linking hypoxic conditions to downstream signaling events, such as induction of hypoxic inducible factor (37). The IC 50 for inhibiting EGFR phosphorylation by PKI 166 is reported to be 10 nM (28), but in our experiment, PKI 166 inhibited PI3K/Akt/ NF-B and MEK/MAPK(Erk) at a micromolar concentration.…”

The adaptor protein Ruk/CIN85 activates plasminogen activator inhibitor-1 (PAI-1) expression via hypoxia-inducible factor-1α

“…HIF-mediated reporter gene activity was shown to be decreased by Morg1 while suppression of Morg1 led to a marked increase of HIF-1 activity. There are also data showing that the adaptor protein Shc which possess two distinct domains that bind phosphotyrosine containing sequences (PTB and SH2) and a cen- tral region that contains critical tyrosine phosphorylation sites (47) and its downstream effectors, Ras and Raf-1, are involved in hypoxia-induced HIF-1 stabilisation in human umbilical vein endothelial cells (48). In these cells overexpression of a dominantnegative Shc mutant resulted in significantly reduced HIF-1α protein levels as compared with control.…”

Hypoxia and HIF-1α Stability

“…Jung et al 4 report that a Shc-Ras signaling pathway regulates endothelial cell migration, probably as a result of HIF-1␣ protein stabilization. Previously, Gu et al 21 reported that Shc and FAK regulated cell migration through two different mechanisms: one is a pathway from Shc through the MAP kinase pathway leading to the stimulation of random cell motility, and the other is from FAK through p130Cas leading to stimulation of directionally persistent cell migration.…”

“…Among these pathways, the hypoxia-inducible transcription factor-1 (HIF-1␣) plays an essential role by transactivating the VEGF gene and activating a pattern of gene expression associated with mobilization, migration, and recruitment of endothelial cells and smooth muscle cells to form new blood vessels. [1][2][3] The report by Jung et al 4 in this issue of Circulation Research investigates the signaling pathway responsible for hypoxia-induced HIF-1␣ protein stabilization and concludes that a mechanism involving Src, Shc, Ras, and Raf-1 is critical in endothelial cells.…”

“…In this editorial, we discuss 5 key issues raised by the work of Jung et al 4 (1) Signal transduction pathways that regulate protein stabilization are important pathophysiological mechanisms that may provide novel therapeutic approaches. (2) Shc isoforms represent a family of stress-regulated proteins that act as sensors for redox, oxygen species (ROS), and other genotoxic stresses.…”