The Phosphatase SHP-2 Activates HIF-1α in Wounds In Vivo by Inhibition of 26S Proteasome Activity.

Yvonn Heun,Bjoern F Kraemer,Katharina Grundler Groterhorst,Alexander Pfeifer,Ulrich Pohl,Kristin Pogoda,Hanna Mannell

doi:10.3390/ijms20184404

Abstract

Vascular remodeling and angiogenesis are required to improve the perfusion of ischemic tissues. The hypoxic environment, induced by ischemia, is a potent stimulus for hypoxia inducible factor 1α (HIF-1α) upregulation and activation, which induce pro-angiogenic gene expression. We previously showed that the tyrosine phosphatase SHP-2 drives hypoxia mediated HIF-1α upregulation via inhibition of the proteasomal pathway, resulting in revascularization of wounds in vivo. However, it is still unknown if SHP-2 mediates HIF-1α upregulation by affecting 26S proteasome activity and how the proteasome is regulated upon hypoxia. Using a reporter construct containing the oxygen-dependent degradation (ODD) domain of HIF-1α and a fluorogenic proteasome substrate in combination with SHP-2 mutant constructs, we show that SHP-2 inhibits the 26S proteasome activity in endothelial cells under hypoxic conditions in vitro via Src kinase/p38 mitogen-activated protein kinase (MAPK) signalling. Moreover, the simultaneous expression of constitutively active SHP-2 (E76A) and inactive SHP-2 (CS) in separate hypoxic wounds in the mice dorsal skin fold chamber by localized magnetic nanoparticle-assisted lentiviral transduction showed specific regulation of proteasome activity in vivo. Thus, we identified a new additional mechanism of SHP-2 mediated HIF-1α upregulation and proteasome activity, being functionally important for revascularization of wounds in vivo. SHP-2 may therefore constitute a potential novel therapeutic target for the induction of angiogenesis in ischemic vascular disease.

Highlights

The transcription factor hypoxia inducible factor 1α (HIF-1α) is involved in vascular remodeling and angiogenesis [1]
As HIF-1α has been shown to be degraded by the proteasome as well as calpain [10], we first investigated whether this is true in endothelial cells upon hypoxia
We induced the lentiviral expression of a construct containing the oxygen-dependent degradation (ODD) domain of HIF-1α [16], which guides its proteasomal degradation upon ubiquitinylation [3], fused to a luciferase gene (HIF1-ODD-Luc) with simultaneous expression of mCherry, in endothelial cells

Summary

Introduction

The transcription factor hypoxia inducible factor 1α (HIF-1α) is involved in vascular remodeling and angiogenesis [1]. HIF-1α induces the expression of several angiogenic genes, such as vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF) or matrix metalloprotease 2 (MMP-2), which are potent inducers of angiogenesis and arteriogenesis [1,3]. It has been the target of therapeutic strategies to increase tissue perfusion in ischemic limbs [4,5] and to improve wound healing [6,7]. It has been shown that HIF-1α may be degraded by the protease calpain [10]

Methods

Results

Conclusion