Cyclic AMP blocks bacterial lipopolysaccharide-induced myosin light chain phosphorylation in endothelial cells through inhibition of Rho/Rho kinase signaling.
During Gram-negative sepsis bacterial LPS induces endothelial cell contraction, actin reorganization, and loss of endothelial integrity by an unknown signal mechanism. In this study, we provide evidence that LPS-stimulation of endothelial cells (HUVEC) decreases myosin light chain (MLC) phosphatase, resulting in an increase in MLC phosphorylation followed by cell contraction. All of these LPS effects could be blocked by the Rho-GTPase inhibitor C3 transferase from Clostridium botulinum or the Rho kinase inhibitor Y-27632. These data suggest that LPS induces MLC phosphorylation via Rho/Rho kinase-mediated inhibition of MLC phosphatase in HUVEC. Furthermore, we observed that cAMP-elevating drugs, known to exert a vasoprotective function, mimicked the effects of C3 transferase and Y-27632, i.e., inhibited LPS-induced MLC phosphatase inactivation and MLC phosphorylation. cAMP elevation did not inhibit myosin phosphorylation induced by constitutively active V14Rho or the MLC phosphatase inhibitor calyculin and did not induce phosphorylation of RhoA in HUVEC, indicating inhibition of an upstream regulator of Rho/Rho kinase. Taken together, Rho/Rho kinase appears to be a central target for inflammatory mediators causing endothelial cell contraction such as bacterial toxins, but also for vasoprotective molecules elevating intracellular cAMP.
Essler
M
M
Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten and Max-von-Pettenkofer-Institut für Medizinische Mikrobiologie, Universität München, Germany. messler@klp.med.uni.muenchen.de
Staddon
J M
JM
Weber
P C
PC
Aepfelbacher
M
M
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000096:biosynthesis / Q000502:physiology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology / Q000378:metabolism* / Q000502:physiology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000276:immunology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors* / Q000276:immunology*; D008387:Marine Toxins; D018994:Myosin Light Chains / Q000037:antagonists & inhibitors* / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D014176:Protein Biosynthesis / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000502:physiology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D015398:Signal Transduction / Q000276:immunology*; D014158:Transcription, Genetic / Q000187:drug effects; D014471:Umbilical Veins; D020741:rho GTP-Binding Proteins / Q000037:antagonists & inhibitors* / Q000502:physiology; D054460:rho-Associated Kinases
D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides; D008387:Marine Toxins; D018994:Myosin Light Chains; D010080:Oxazoles; D011500:Protein Synthesis Inhibitors; C059041:calyculin A; D000242:Cyclic AMP; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase; D020741:rho GTP-Binding Proteins; D015056:1-Methyl-3-isobutylxanthine