Angiotensin-II and MARCKS A HYDROGEN PEROXIDE- AND RAC1-DEPENDENT SIGNALING PATHWAY IN VASCULAR ENDOTHELIUM

MARCKS is an actin-binding protein that modulates vascular endothelial cell migration and cytoskeleton signaling (Kalwa, H., and Michel, T. (2011) J. Biol. Chem. 286, 2320-2330). Angiotensin-II is a vasoactive peptide implicated in vascular physiology as well as pathophysiology; the pathways connecting angiotensin-II and cytoskeletal remodeling are incompletely understood. Here we show that MARCKS is expressed in intact arterial preparations, with prominent staining of the endothelium. In endothelial cells, angiotensin-II-promoted MARCKS phosphorylation is abrogated by PEG-catalase, implicating endogenous H2O2 in the angiotensin-II response. Studies using the H2O2 biosensor HyPer2 reveal that angiotensin-II promotes increases in intracellular H2O2. We used a Rac1 FRET biosensor to show that angiotensin-II promotes Rac1 activation that is attenuated by PEG-catalase. siRN-mediated Rac1 knockdown blocks angiotensin-II-stimulated MARCKS phosphorylation. Cell imaging studies using a phosphoinositide 4,5-bisphosphate (PIP2) biosensor revealed that angiotensin-II PIP2 regulation depends on MARCKS and H2O2. siRN-mediated knockdown of MARCKS or Rac1 attenuates receptor-mediated activation of the tyrosine kinase c-Abl and disrupts actin fiber formation. These studies establish a critical role for H2O2 in angiotensin-II signaling to the endothelial cytoskeleton in a novel pathway that is critically dependent on MARCKS, Rac1, and c-Abl.