S-Glutathionylation of LMW-PTP regulates VEGF-mediated FAK activation and endothelial cell migration

Abstract

Although promising, the ability to regulate angiogenesis via delivery of VEGF remains unrealized goal. We have shown previously that physiological levels of peroxynitrite (PN,1 µM) are required for VEGF-mediated angiogenic response, yet the redox-regulated mechanisms governing VEGF signal remain unexplored. We assessed the impact of VEGF and peroxynitrite on modifying redox-state, reduced-glutathion (GSH) and S-glutathionylation on regulation of the low molecular weight protein tyrosine phosphatase (LMW-PTP) and focal adhesion kinase (FAK), key mediators of VEGF-mediated cell migration. Stimulation of human microvascular endothelial (HME) with VEGF (20 ng/ml) or PN (1 µM) caused immediate and reversible negative-shift in cellular redox-state and thiol oxidation of LMW-PTP that culminated in cell migration. VEGF causes reversible S-glutathionylation of LMW-PTP that inhibited its phosphorylation and activity and caused transient FAK activation. Modulating redox-state by decomposing peroxynitrite (FeTPPS, 2.5 µM) or GSH-precursor (NAC, 1 mM) caused positive-shift of redox-state and prevented VEGF-mediated S-glutathionylation and oxidative inhibition of LMW-PTP. NAC and FeTPPS prevented FAK activation, its association with LMW-PTP and cell migration. Inhibiting LMW-PTP expression markedly enhanced FAK activation and cell migration. While mild oxidative stress achieved by combining VEGF with 0.1–0.2 mM PN augmented cell migration, acute shift to oxidative stress achieved by combining VEGF with 0.5 mM PN induced and sustained FAK activation, LMW-PTP S-glutathionylation resulting in LMW-PTP inactivation and inhibited cell migration. In conclusion, our findings demonstrate that balanced redox-state is required for VEGF to facilitate reversible S-glutathionylation of LMW-PTP, FAK activation and endothelial cell migration. Shifting redox-state to reductive stress or oxidative stress blunted VEGF-mediated angiogenic response.