CysLT(1 )receptor-induced human airway smooth muscle cells proliferation requires ROS generation, EGF receptor transactivation and ERK1/2 phosphorylation

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 D(4 )(LTD(4))-induced DNA synthesis. METHODS: Proliferation of primary HASMC was measured by [(3)H]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 LTD(4)-stimulated thymidine incorporation and potentiation of EGF-induced mitogenic signaling mostly depends upon EGF-R transactivation through the stimulation of CysLT(1)-R. Accordingly, we found that LTD(4 )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 LTD(4)-induced DNA synthesis following Src inhibition. More interestingly, CysLT(1)-R stimulation increased the production of ROS and N-acetylcysteine (NAC) abolished LTD(4)-induced EGF-R phosphorylation and thymidine incorporation. CONCLUSION: Collectively, our data demonstrate that in HASMC LTD(4 )stimulation of a G(i/o )coupled CysLT(1)-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.