Role of CPI-17 in the regulation of endothelial cytoskeleton.
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-alpha abolished the effect of CPI-17 on actin cytoskeleton, suggesting that the PKC-alpha 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.
Kolosova
Irina A
IA
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, USA.
Ma
Shwu-Fan
SF
Adyshev
Djanybek M
DM
Wang
Peyi
P
Ohba
Motoi
M
Natarajan
Viswanathan
V
Garcia
Joe G N
JG
Verin
Alexander D
AD
D008841:Actin Cytoskeleton / Q000378:metabolism*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D022001:Focal Adhesions / Q000378:metabolism; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008168:Lung / Q000098:blood supply*; D008833:Microcirculation / Q000502:physiology; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism; D010749:Phosphoprotein Phosphatases; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011651:Pulmonary Artery / Q000166:cytology; D034741:RNA, Small Interfering; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology
D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; D018994:Myosin Light Chains; C107507:PPP1R14A protein, human; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D006632:Histamine; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases