Endothelial cell myosin light chain kinase (MLCK) regulates TNFalpha-induced NFkappaB activity.
Tumor necrosis factor (TNFalpha-) generates both apoptotic and survival signals with endothelial cell (EC) survival dependent on nuclear factor kappa-B (NFkappaB) activation, a regulator of anti-apoptotic genes. We previously demonstrated that increased EC contractility, rearrangement of the actin cytoskeleton, and increased myosin light chain (MLC) phosphorylation occurs as a consequence of TNFalpha-induced activation of EC MLC kinase (EC MLCK) and is required for bovine lung EC apoptosis. As the association between MLCK and pro-survival signals such as NFkappaB activation is unknown, we studied the role of MLCK in the regulation of NFkappaB-dependent transactivation in bovine pulmonary artery EC. Both TNFalpha-induced increase in NFkappaB dependent transactivation measured by NFkappaB luciferase reporter assay (approximately fivefold) and nuclear translocation of NFkappaB were significantly inhibited by MLCK-selective inhibitors, KT5926 (60% inhibition of luciferase activity) and ML7 (50% decrease). Furthermore, our data revealed that inhibition of MLCK attenuated the TNFalpha-induced IkappaB phosphorylation, translocation of p65, NFkappaB-DNA binding, and NFkappaB transcriptional activity. Molecular approaches to either reduce EC MLCK expression (AdV EC MLCK antisense construct) or to reduce kinase activity (kinase-dead EC MLCK ATPdel mutant) produced similar attenuation of the TNFalpha-induced NFkappaB response. In contrast, a constitutively active MLCK mutant (EC MLCK1745) enhanced TNFalpha-induced luciferase activity. Together, these novel observations indicate that TNFalpha-induced cytoskeletal rearrangement driven by MLCK activity is necessary for TNFalpha-dependent NFkappaB activation and amplification of pro-survival signals.
Wadgaonkar
Raj
R
Division of Pulmonary and Critical Care Medicine, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, USA.
Linz-McGillem
Laura
L
Zaiman
Ari L
AL
Garcia
Joe G N
JG
D000818:Animals; D017209:Apoptosis; D001381:Azepines / Q000494:pharmacology; D002227:Carbazoles / Q000494:pharmacology; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation; D020672:I-kappa B Proteins / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D009154:Mutation; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000502:physiology*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D009281:Naphthalenes / Q000494:pharmacology; D010766:Phosphorylation; D021381:Protein Transport; D011651:Pulmonary Artery / Q000166:cytology* / Q000201:enzymology; D017384:Sequence Deletion; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D001381:Azepines; D002227:Carbazoles; D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; D007211:Indoles; D016328:NF-kappa B; D009281:Naphthalenes; D014409:Tumor Necrosis Factor-alpha; C070571:ML 7; C063769:KT 5926; D000072000:NF-KappaB Inhibitor alpha; D008156:Luciferases; D009219:Myosin-Light-Chain Kinase