Differential endothelial signaling responses elicited by chemogenetic H₂O₂ synthesis

Hydrogen peroxide (H₂O₂) modulates critical phosphorylation pathways in vascular endothelial cells, many of which affect endothelial nitric oxide synthase (eNOS) signal transduction. Both intracellular and extracellular sources of H₂O₂ have been implicated in eNOS regulation, yet the specific endothelial pathways remain incompletely understood. Here we exploited chemogenetic approaches and live-cell imaging methods to both generate and detect H₂O₂ in different subcellular compartments (cytosol, nucleus, and caveolae) of cultured EA.hy926 human endothelial cells. We developed novel recombinant constructs encoding differentially-targeted yeast d-amino acid oxidase (DAAO), which generates H₂O₂ only when its d-amino acid substrate is provided. DAAO was expressed as a fusion protein with the new H₂O₂ biosensor HyPer7.2, which allowed us to quantitate intracellular H₂O₂ levels by ratiometric imaging in living endothelial cells following the activation of DAAO by d-alanine. The addition of extracellular H₂O₂ to the HyPer-DAAO-transfected cells led to increases in H₂O₂ throughout different regions of the cell, as measured using the differentially-targeted HyPer biosensor for H₂O₂. The sensor response to extracellular H₂O₂ was more rapid than that quantitated following the addition of d-alanine to transfected cells to activate differentially-targeted DAAO. The maximal intracellular levels of H₂O₂ observed in response to the addition of extracellular H₂O₂ vs. intracellular (DAAO-generated) H₂O₂ were quantitatively similar. Despite these similarities in the measured levels of intracellular H₂O₂, we observed a remarkable quantitative difference in the activation of endothelial phosphorylation pathways between chemogenetically-generated intracellular H₂O₂ and the phosphorylation responses elicited by the addition of extracellular H₂O₂ to the cells. Addition of extracellular H₂O₂ had only a nominal effect on phosphorylation of eNOS, kinase Akt or AMP-activated protein kinase (AMPK). By contrast, intracellular H₂O₂ generation by DAAO caused striking increases in the phosphorylation of these same key signaling proteins. We also found that the AMPK inhibitor Compound C completely blocked nuclear H₂O₂-promoted eNOS phosphorylation. However, Compound C had no effect on eNOS phosphorylation following H₂O₂ generation from cytosol- or caveolae-targeted DAAO. We conclude that H₂O₂ generated in the cell nucleus activates AMPK, leading to eNOS phosphorylation; in contrast, AMPK activation by cytosol- or caveolae-derived H₂O₂ does not promote eNOS phosphorylation via AMPK. These findings indicate that H₂O₂ generated in different subcellular compartments differentially modulates endothelial cell phosphorylation pathways, and suggest that dynamic subcellular localization of oxidants may modulate signaling responses in endothelial cells.