Research

CysLT₁ receptor-induced human airway smooth muscle cells proliferation requires ROS generation, EGF receptor transactivation and ERK1/2 phosphorylation

Saula Ravasi¹ , Simona Citro¹ , Barbara Viviani² , Valérie Capra¹ and G Enrico Rovati¹

¹  Laboratory of Molecular Pharmacology, Section of Eicosanoid Pharmacology, Department of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy

²  Laboratory of Toxicology, Department of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy

author email corresponding author email

Respiratory Research 2006, 7:42doi:10.1186/1465-9921-7-42

Published:	22 March 2006

Abstract

Background

Cysteine-containing leukotrienes (cysteinyl-LTs) are pivotal inflammatory mediators that play important roles in the pathophysiology of asthma, allergic rhinitis, and other inflammatory conditions. In particular, cysteinyl-LTs exert a variety of effects with relevance to the aetiology of asthma such as smooth muscle contraction, eosinophil recruitment, increased microvascular permeability, enhanced mucus secretion and decreased mucus transport and, finally, airway smooth muscle cells (ASMC) proliferation. We used human ASMC (HASMC) to identify the signal transduction pathway(s) of the leukotriene D₄ (LTD₄)-induced DNA synthesis.

Methods

Proliferation of primary HASMC was measured by [³H]thymidine incorporation. Phosphorylation of EGF receptor (EGF-R) and ERK1/2 was assessed with a polyclonal anti-EGF-R or anti-phosphoERKl/2 monoclonal antibody. A Ras pull-down assay kit was used to evaluate Ras activation. The production of reactive oxygen species (ROS) was estimated by measuring dichlorodihydrofluorescein (DCF) oxidation.

Results

We demonstrate that in HASMC LTD₄-stimulated thymidine incorporation and potentiation of EGF-induced mitogenic signaling mostly depends upon EGF-R transactivation through the stimulation of CysLT₁-R. Accordingly, we found that LTD₄ stimulation was able to trigger the increase of Ras-GTP and, in turn, to activate ERK1/2. We show here that EGF-R transactivation was sensitive to pertussis toxin (PTX) and phosphoinositide 3-kinase (PI3K) inhibitors and that it occurred independently from Src activity, despite the observation of a strong impairment of LTD₄-induced DNA synthesis following Src inhibition. More interestingly, CysLT₁-R stimulation increased the production of ROS and N-acetylcysteine (NAC) abolished LTD₄-induced EGF-R phosphorylation and thymidine incorporation.

Conclusion

Collectively, our data demonstrate that in HASMC LTD₄ stimulation of a G_i/o coupled CysLT₁-R triggers the transactivation of the EGF-R through the intervention of PI3K and ROS. While PI3K and ROS involvement is an early event, the activation of Src occurs downstream of EGF-R activation and is followed by the classical Ras-ERK1/2 signaling pathway to control G1 progression and cell proliferation.