Role of CPI-17 in the regulation of endothelial cytoskeleton

1 Department of Medicine, Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224; and 2 Institute of Molecular Oncology, Showa University, Tokyo, Japan Submitted 19 November 2003 ; accepted in final form 25 June 2004 We have previously shown that myosin light chain (MLC) phosphatase (MLCP) is critically involved in the regulation of agonist-mediated endothelial permeability and cytoskeletal organization (Verin AD, Patterson CE, Day MA, and Garcia JG. Am J Physiol Lung Cell Mol Physiol 269: L99–L108, 1995). The molecular mechanisms of endothelial MLCP regulation, however, are not completely understood. In this study we found that, similar to smooth muscle, lung microvascular endothelial cells expressed specific endogenous inhibitor of MLCP, CPI-17. To elucidate the role of CPI-17 in the regulation of endothelial cytoskeleton, full-length CPI-17 plasmid was transiently transfected into pulmonary artery endothelial cells, where the background of endogenous protein is low. CPI-17 had no effect on cytoskeleton under nonstimulating conditions. However, stimulation of transfected cells with direct PKC activator PMA caused a dramatic increase in F-actin stress fibers, focal adhesions, and MLC phosphorylation compared with untransfected cells. Inflammatory agonist histamine and, to a much lesser extent, thrombin were capable of activating CPI-17. Histamine caused stronger CPI-17 phosphorylation than thrombin. Inhibitory analysis revealed that PKC more significantly contributes to agonist-induced CPI-17 phosphorylation than Rho-kinase. Dominant-negative PKC- abolished the effect of CPI-17 on actin cytoskeleton, suggesting that the PKC- isoform is most likely responsible for CPI-17 activation in the endothelium. Depletion of endogenous CPI-17 in lung microvascular endothelial cell significantly attenuated histamine-induced increase in endothelial permeability. Together these data suggest the potential importance of PKC/CPI-17-mediated pathway in histamine-triggered cytoskeletal rearrangements leading to lung microvascular barrier compromise. myosin phosphatase; myosin light chain phosphorylation; endothelial barrier Address for reprint requests and other correspondence: A. D. Verin, 5200 Eastern Ave., Center Tower, MFL Bldg., 6th Fl., Baltimore, MD 21224 (E-mail: averin1{at}jhmi.edu )