Abstract 19886: Delocalization of Endogenous Human Microvascular Smooth Muscle A-kinase Antagonizes the Positive Effects of Rap-Rho Signaling on Cell Integrity and Survival
Background: Vascular stress or injury may trigger an inflammatory response leading to increased release of prostaglandin E2 (PGE2) and prostacyclin (PGI2), activation of Gs-coupled receptors, and increased intracellular cyclic AMP (cAMP). This study elucidated the targets of cAMP signaling in the vessel wall, specifically in human vascular smooth muscle cells explanted from skin punch biopsy arterioles of healthy volunteers (microVSM).
Methods & Results: Intracellular concentrations of cAMP were increased by treating quiescent cells with forskolin (10 μM, an activator of adenylyl cyclase). Global protein expression was determined by Differential-in-Gel electrophoresis. Protein profiles showed detectable changes in proteins associated with the cytoskeleton, gene expression, protein -synthesis, -trafficking, and extracellular matrix. Notably, actin modulating proteins caldesmon, ezrin-moesin, LASP-1, zyxin and α-adducin, as well as cytoskeletal proteins, tubulin and syncoilin-1 were differentially expressed. In support of these results, cAMP increased phosphorylation of A-kinase substrate CREB at Ser133 (4.2 ± 0.7 fold at 10min, n=4, P<0.01), activated the integrin-coupled small GTPase Rap1, and Rho (2.6± 0.6 fold and 4.2± 0.9 fold at 5 min, respectively, n=3, P<0.05, assessed by pull-down assays measuring the activated GTP-bound forms), increased actin stress fibers and the focal adhesion rich protein vinculin, and increased cell attachment to fibronectin substrate (∼3-fold at 30 min versus control). The selective A-kinase inhibitor H-89 (2 μM) inhibited CREB Ser133 phosphorylation (1.3±0.21 fold, n=4, P=NS), but did not diminish Rap1-Rho activation coupled stress-fiber formation, suggesting compartmentalized A-kinase local signaling facilitated by A-kinase anchoring proteins (AKAPs). Global disruption of this interaction by the anchoring inhibitor decoy peptide Ht31, but not control peptide Ht31P, abolished Rap-Rho mediated effects.
Conclusions: Delocalized A-kinase potentially disrupts inside-out integrin-matrix contacts leading to anoikis. Together, compartmentalized cAMP signaling elicits a protective cellular response to maintain structure and vessel wall integrity in response to stress or injury.
- © 2010 by American Heart Association, Inc.