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Vol. 63, Issue 3, 714-721, March 2003
-Induced Activation of RhoA in Airway
Smooth Muscle Cells: Role in the Ca2+ Sensitization of
Myosin Light Chain20 Phosphorylation
Department of Biomedical Sciences, Institute of Medical Sciences,
University of Aberdeen, Aberdeen, United Kingdom (I.H., H.J.C., D.J.M.,
G.F.N.); and Department of Molecular Biology, Flanders Interuniversity,
Institute for Biotechnology, University of Gent, Ghent, Belgium (P.V.)
Tumor necrosis factor-
(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 chain20 (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.
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