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1 Department of Medicine, Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA
2 Institute of Molecular Oncology, Showa University, Tokyo, Japan
* To whom correspondence should be addressed. E-mail: averin{at}jhmi.edu.
We have previously shown that myosin light chain phosphatase (MLCP) is critically involved in the regulation of agonist-mediated endothelial permeability and cytoskeletal organization (Verin et al., Am J 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 non-stimulating 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 to 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 effect of CPI-17 on actin cytoskeleton, suggesting that PKC isoform is most likely responsible for CPI-17 activation in endothelium. Depletion of endogenous CPI-17 in lung microvascular EC significantly attenuated histamine-induced increase in endothelial permeability. Taken together these data suggest potential importance of PKC/CPI-17-mediated pathway in histamine- triggered cytoskeletal rearrangements leading to lung microvascular barrier compromise.
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