%0 Journal Article %A Irene Hunter %A Hannah J. Cobban %A Peter Vandenabeele %A David J. MacEwan %A Graeme F. Nixon %T Tumor Necrosis Factor-α–Induced Activation of RhoA in Airway Smooth Muscle Cells: Role in the Ca2+ Sensitization of Myosin Light Chain20 Phosphorylation %D 2003 %R 10.1124/mol.63.3.714 %J Molecular Pharmacology %P 714-721 %V 63 %N 3 %X Tumor necrosis factor-α (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 MLC20phosphorylation 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. %U https://molpharm.aspetjournals.org/content/molpharm/63/3/714.full.pdf