Tumor necrosis factor-a-induced activation of RhoA in airway smooth muscle cells: Role in the Ca2+ sensitization of myosin light chain20 phosphorylation

Irene Hunter, H. J. Cobban, P. Vandenabeele, David Joseph MacEwan, Graeme Fleming Nixon

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Abstract

Tumor necrosis factor-alpha (TNF), an inflammatory cytokine, has a potentially important role in the pathogenesis of bronchial asthma and may contribute to airway hyper-responsiveness. Recent evidence has revealed that TNF can increase the Ca2+ sensitivity of agonist-stimulated myosin light chain(20) (MLC20) phosphorylation and contractility in guinea pig airway smooth muscle (ASM). In the present study, the potential intracellular pathways responsible for this TNF-induced Ca2+ sensitization were investigated. In permeabilized cultured guinea pig ASM cells, recombinant human TNF stimulated an increase in Ca2+ activated MLC20 phosphorylation under Ca2+ "clamp" conditions. This increased MLC20 phosphorylation was inhibited by preincubation with the Rho-kinase inhibitor Y27632. TNF also increased the proportion of GTP-bound RhoA, as measured using rhotekin Rho-binding domain, in a time course compatible with a role in the TNF-induced Ca2+ sensitization. In cultured human ASM cells, recombinant human TNF also activated RhoA with a similar time course. In addition, TNF stimulated phosphorylation of the regulatory subunit of the myosin phosphatase, which was inhibited by Y27632. Although human ASM cells expressed both receptor subtypes, TNF-R1 and TNF-R2, the activation of RhoA was predominantly via stimulation of the TNF-R1, although RhoA did not immunoprecipitate with the TNF-R1. In conclusion, the TNF-induced increase in the Ca2+ sensitivity of MLC20 phosphorylation is through stimulation of the TNF-R1 receptor and via a RhoA/Rho-kinase pathway leading to inhibition of the myosin light chain phosphatase. This intracellular mechanism may contribute to TNF-induced airway hyper-responsiveness.

Original languageEnglish
Pages (from-to)714-721
Number of pages7
JournalMolecular Pharmacology
Volume63
Issue number3
DOIs
Publication statusPublished - Mar 2003

Keywords

  • BINDING PROTEIN-RHO
  • TNF-ALPHA
  • KINASE INHIBITOR
  • P115 RHOGEF
  • RECEPTOR
  • PHOSPHATASE
  • GTPASE
  • CYTOSKELETON
  • STIMULATION
  • G-ALPHA(13)

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