Endothelial cell myosin light chain kinase (MLCK) regulates TNFα-induced NFκB activity

Raj Wadgaonkar, Laura Linz-McGillem, Ari L. Zaiman, Joe G.N. Garcia

Research output: Contribution to journalArticle

39 Scopus citations

Abstract

Tumor necrosis factor (TNFα-) generates both apoptotic and survival signals with endothelial cell (EC) survival dependent on nuclear factor kappa-B (NFκB) 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 TNFα-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 NFκB activation is unknown, we studied the role of MLCK in the regulation of NFκB-dependent transactivation in bovine pulmonary artery EC. Both TNFα-induced increase in NFκB dependent transactivation measured by NFκB luciferase reporter assay (∼ fivefold) and nuclear translocation of NFκB 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 TNFα-induced IκB phosphorylation, translocation of p65, NFκB-DNA binding, and NFκB 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 TNFα-induced NFκB response. In contrast, a constitutively active MLCK mutant (EC MLCK1745) enhanced TNFα-induced luciferase activity. Together, these novel observations indicate that TNFα-induced cytoskeletal rearrangement driven by MLCK activity is necessary for TNFα-dependent NFκB activation and amplification of pro-survival signals.

Original languageEnglish (US)
Pages (from-to)351-364
Number of pages14
JournalJournal of Cellular Biochemistry
Volume94
Issue number2
DOIs
StatePublished - Feb 1 2005
Externally publishedYes

Keywords

  • Actin
  • Apoptosis
  • Cytoskeleton
  • E-selectin
  • Survival

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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