Differential endothelial signaling responses elicited by chemogenetic HO synthesis.

Hydrogen peroxide (HO) 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 HO 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 HO 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 HO only when its d-amino acid substrate is provided. DAAO was expressed as a fusion protein with the new HO biosensor HyPer7.2, which allowed us to quantitate intracellular HO levels by ratiometric imaging in living endothelial cells following the activation of DAAO by d-alanine. The addition of extracellular HO to the HyPer-DAAO-transfected cells led to increases in HO throughout different regions of the cell, as measured using the differentially-targeted HyPer biosensor for HO. The sensor response to extracellular HO 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 HO observed in response to the addition of extracellular HO vs. intracellular (DAAO-generated) HO were quantitatively similar. Despite these similarities in the measured levels of intracellular HO, we observed a remarkable quantitative difference in the activation of endothelial phosphorylation pathways between chemogenetically-generated intracellular HO and the phosphorylation responses elicited by the addition of extracellular HO to the cells. Addition of extracellular HO had only a nominal effect on phosphorylation of eNOS, kinase Akt or AMP-activated protein kinase (AMPK). By contrast, intracellular HO 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 HO-promoted eNOS phosphorylation. However, Compound C had no effect on eNOS phosphorylation following HO generation from cytosol- or caveolae-targeted DAAO. We conclude that HO generated in the cell nucleus activates AMPK, leading to eNOS phosphorylation; in contrast, AMPK activation by cytosol- or caveolae-derived HO does not promote eNOS phosphorylation via AMPK. These findings indicate that HO 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.