title: Central role for hydrogen peroxide in P2Y1 ADP receptor-mediated cellular responses in vascular endothelium.
abstract: 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.
authors.affiliation: Cardiovascular Medicine Division, Department of Medicine, Division of Vascular and Endovascular Surgery, Department of Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115.
authors.forename: Hermann
authors.initials: H
authors.lastname: Kalwa
authors.forename: Juliano L
authors.initials: JL
authors.lastname: Sartoretto
authors.forename: Roberta
authors.initials: R
authors.lastname: Martinelli
authors.forename: Natalia
authors.initials: N
authors.lastname: Romero
authors.forename: Benjamin S
authors.initials: BS
authors.lastname: Steinhorn
authors.forename: Ming
authors.initials: M
authors.lastname: Tao
authors.forename: C Keith
authors.initials: CK
authors.lastname: Ozaki
authors.forename: Christopher V
authors.initials: CV
authors.lastname: Carman
authors.forename: Thomas
authors.initials: T
authors.lastname: Michel
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002460:Cell Line; D017097:Electric Impedance; D042783:Endothelial Cells / Q000378:metabolism* / Q000502:physiology; D004789:Enzyme Activation / Q000235:genetics / Q000502:physiology*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D058472:Receptors, Purinergic P2Y1 / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D051941:fms-Like Tyrosine Kinase 3 / Q000378:metabolism*
chemical_list: D058472:Receptors, Purinergic P2Y1; D000244:Adenosine Diphosphate; D006861:Hydrogen Peroxide; C497970:FLT3 protein, human; D051941:fms-Like Tyrosine Kinase 3
keywords: FRET; purinergic signaling; signal transduction