Abstract 13255: 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. Angiotensin-II (Ang-II) is implicated both in physiological responses and in vascular disease states. The role of MARCKS in Ang-II signaling is largely unknown, and the pathways connecting Ang-II and endothelial cytoskeletal remodeling are incompletely understood. These studies used molecular imaging methods, powerful biosensors, and small interfering RNA approaches to explore the role of MARCKS in Ang-II signal transduction in vascular tissues and endothelial cells. Immunofluorescence studies in intact mouse arterial preparations revealed robust MARCKS expression in vascular endothelium. Ang-II treatment of arterial preparations markedly increased MARCKS phosphorylation in the vascular endothelium. In bovine aortic endothelial cells, Ang-II treatment promoted a 1.9+/-0.2-fold increase in MARCKS phosphorylation (p<0.05, n=4) with an EC50 of 1 μM. The effects of Ang-II on MARCKS phosphorylation were abrogated by PEG-catalase, implicating endogenous H2O2 in the Ang-II response. Ang-II promoted the rapid and reversible translocation of MARCKS from the cytoskeleton to the cytoplasm. Studies using the H2O2 biosensor HyPer2 revealed that Ang-II promotes increases in intracellular H2O2 (p<0.05, n=4). We used a Rac1 FRET biosensor to show that angiotensin-II promotes Rac1 activation that is significantly attenuated by PEG-catalase (p<0.05, n=4). siRNA-mediated Rac1 knockdown blocked angiotensin-II-stimulated MARCKS phosphorylation. Cell imaging studies using a phosphoinositide 4,5-bisphosphate (PIP2) biosensor revealed that angiotensin-II regulation of PIP2 depends on MARCKS and H2O2. siRNA-mediated knockdown of either MARCKS or Rac1 attenuates Ang-II-mediated activation of the tyrosine kinase c-Abl and disrupts actin fiber formation. Imaging studies using vinculin and phalloidin showed that Ang-II promotes rearrangements in the actin cytoskeleton and leads to increases in focal adhesion complexes that are entirely dependent on MARCKS. These studies establish a critical role for MARCKS in angiotensin-II signaling to the endothelial cytoskeleton in a novel pathway that is dependent on H2O2, Rac1, and c-Abl.
- © 2012 by American Heart Association, Inc.