title: S-glutathionylation of LMW-PTP regulates VEGF-mediated FAK activation and endothelial cell migration.
abstract: Although promising, the ability to regulate angiogenesis through delivery of VEGF remains an unrealized goal. We have shown previously that physiological levels of peroxynitrite (1 µM) are required for a VEGF-mediated angiogenic response, yet the redox-regulated mechanisms that govern the VEGF signal remain unexplored. We assessed the impact of VEGF and peroxynitrite on modifying redox-state, the level of reduced-glutathione (GSH) and S-glutathionylation on regulation of the low molecular weight protein tyrosine phosphatase (LMW-PTP) and focal adhesion kinase (FAK), which are key mediators of VEGF-mediated cell migration. Stimulation of human microvascular endothelial (HME) cells with VEGF (20 ng/ml) or peroxynitrite (1 µM) caused an immediate and reversible negative-shift in the cellular redox-state and thiol oxidation of LMW-PTP, which culminated in cell migration. VEGF causes reversible S-glutathionylation of LMW-PTP, which inhibits its phosphorylation and activity, and causes the transient activation of FAK. Modulating the redox-state using decomposing peroxynitrite (FeTPPS, 2.5 µM) or the GSH-precursor [N-acetylcysteine (NAC), 1 mM] caused a positive-shift of the redox-state and prevented VEGF-mediated S-glutathionylation and oxidative inhibition of LMW-PTP. NAC and FeTPPS prevented the activation of FAK, its association with LMW-PTP and cell migration. Inhibiting LMW-PTP expression markedly enhanced FAK activation and cell migration. Although mild oxidative stress achieved by combining VEGF with 0.1-0.2 mM peroxynitrite augmented cell migration, an acute shift to oxidative stress achieved by combining VEGF with 0.5 mM peroxynitrite induced and sustained FAK activation, and LMW-PTP S-glutathionylation, resulting in LMW-PTP inactivation and inhibited cell migration. In conclusion, our findings demonstrate that a balanced redox-state is required for VEGF to facilitate reversible S-glutathionylation of LMW-PTP, FAK activation and endothelial cell migration. Shifting the redox-state to reductive stress or oxidative stress inhibited the VEGF-mediated angiogenic response.
authors.affiliation: Program in Clinical and Experimental Therapeutics, University of Georgia, USA.
authors.forename: Mohammed A
authors.initials: MA
authors.lastname: Abdelsaid
authors.forename: Azza B
authors.initials: AB
authors.lastname: El-Remessy
mesh_terms: D002465:Cell Movement / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051417:Focal Adhesion Kinase 1 / Q000235:genetics / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D018919:Neovascularization, Physiologic / Q000235:genetics; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress; D030421:Peroxynitrous Acid / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*
chemical_list: C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D030421:Peroxynitrous Acid; D051417:Focal Adhesion Kinase 1; C496050:PTK2 protein, human; D017027:Protein Tyrosine Phosphatases; D005978:Glutathione