Abstract
ADP plays critical signaling roles in the vascular endothelium. ADP receptors are targeted by several cardiovascular drugs, yet the intracellular pathways modulated by ADP are incompletely understood. These studies have identified important roles for the phosphatase PTEN in ADP-dependent modulation of the endothelial isoform of nitric oxide synthase (eNOS) as well as of lipid and protein kinase pathways in endothelial cells. We find that ADP-promoted eNOS activation as well as phosphorylation of p38 MAPK are enhanced by siRNA-mediated PTEN knockdown. However, the increase in ADP-dependent eNOS activation promoted by PTEN knockdown is abrogated by siRNA-mediated knockdown of p38 MAPK. These findings indicate that PTEN tonically suppresses both p38 phosphorylation as well as ADP-stimulated eNOS activity. A key enzymatic activity of PTEN is its role as a lipid phosphatase, catalyzing the dephosphorylation of phosphoinositol-3,4,5-trisphosphate (PIP3) to phosphoinositol-4,5-bisphosphate (PIP2). We performed biochemical analyses of cellular phospholipids in endothelial cells to show that siRNA-mediated PTEN knockdown leads to a marked increase in PIP3. Because these complex lipids activate the small GTPase Rac1, we explored the role of PTEN in ADP-modulated Rac1 activation. We used a FRET biosensor for Rac1 to show that ADP-dependent Rac1 activation is blocked by siRNA-mediated PTEN knockdown. We then exploited a FRET biosensor for PIP3 to show that the striking ADP-dependent increase in intracellular PIP3 is entirely blocked by PTEN knockdown. These studies identify a key role for PTEN in the modulation of lipid mediators involved in ADP receptor-regulated endothelial signaling pathways involving eNOS activation in vascular endothelial cells.