Central role for hydrogen peroxide in P2Y1 ADP receptor-mediated cellular responses in vascular endothelium

ADP activates a family of cell surface receptors that modulate signaling pathways in a broad range of cells. ADP receptor antagonists are widely used to treat cardiovascular disease states. These studies identify a critical role for the stable reactive oxygen species hydrogen peroxide (H2O2) in medi...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2014-03, Vol.111 (9), p.3383-3388
Main Authors: Kalwa, Hermann, Sartoretto, Juliano L., Martinelli, Roberta, Romero, Natalia, Steinhorn, Benjamin S., Tao, Ming, Ozaki, C. Keith, Carman, Christopher V., Michel, Thomas
Format: Article
Language:English
Subjects:
Adenosine Diphosphate - metabolism
Animals
Biological Sciences
Biosensing techniques
Cancer
Cardiovascular disease
Cattle
Cell Line
Chemotherapy
Cultured cells
Cytoskeleton
Electric Impedance
Endothelial cells
Endothelial Cells - metabolism
Endothelial Cells - physiology
Enzyme Activation - genetics
Enzyme Activation - physiology
Fluorescence
Fluorescence Resonance Energy Transfer
fms-Like Tyrosine Kinase 3 - metabolism
Humans
Hydrogen peroxide
Hydrogen Peroxide - metabolism
Imaging
Immunoblotting
Microscopy, Fluorescence
Modulated signal processing
Oxidases
Phosphorylation
Physiological regulation
Proteins
Receptors
Receptors, Purinergic P2Y1 - metabolism
Signal Transduction - physiology
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Summary:ADP activates a family of cell surface receptors that modulate signaling pathways in a broad range of cells. ADP receptor antagonists are widely used to treat cardiovascular disease states. These studies identify a critical role for the stable reactive oxygen species hydrogen peroxide (H2O2) in mediating cellular responses activated by the G protein-coupled P2Y1 receptor for ADP. We found that ADP-dependent phosphorylation of key endothelial signaling proteins—including endothelial nitric oxide synthase, AMP-activated protein kinase, and the actin-binding MARCKS protein— was blocked by preincubation with PEG-catalase, which degrades H2O2. ADP treatment promoted the H2O2-dependent phosphorylation of c-Abl, a nonreceptor tyrosine kinase that modulates the actin cytoskeleton. Cellular imaging experiments using fluorescence resonance energy transfer-based biosensors revealed that ADP-stimulated activation of the cytoskeleton-associated small GTPase Rac1 was independent of H2O2. However, Rac1-dependent activation of AMP-activated protein kinase, the signaling phospholipid phosphatidylinositol-(4, 5)-bisphosphate, and the c-Abl–interacting protein CrkII are mediated by H2O2. We transfected endothelial cells with differentially targeted HyPer2 H2O2 biosensors and found that ADP promoted a marked increase in H2O2 levels in the cytosol and caveolae, and a smaller increase in mitochondria. We performed a screen for P2Y1 receptor-mediated receptor tyrosine kinase transactivation and discovered that ADP transactivates Fms-like tyrosine kinase 3 (Flt3), a receptor tyrosine kinase expressed in these cells. Our observation that P2Y1 receptor-mediated responses involve Flt3 transactivation may identify a unique mechanism whereby cancer chemotherapy with receptor tyrosine kinase inhibitors promotes vascular dysfunction. Taken together, these findings establish a critical role for endogenous H2O2 in control of ADP-mediated signaling responses in the vascular wall.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.1320854111