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J Cell Biochem. 2005 Feb 1;94(2):351-64.

Endothelial cell myosin light chain kinase (MLCK) regulates TNFalpha-induced NFkappaB activity.

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1
Division of Pulmonary and Critical Care Medicine, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, USA.

Abstract

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.

PMID:
15526279
DOI:
10.1002/jcb.20250
[Indexed for MEDLINE]
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