CysLT1 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 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.


CONCLUSIONS
Collectively, our data demonstrate that in HASMC LTD4 stimulation of a Gi/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.
Ravasi
Saula
S
Laboratory of Molecular Pharmacology, Section of Eicosanoid Pharmacology, Department of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy. Saula.Ravasi@unimi.it
Citro
Simona
S
Viviani
Barbara
B
Capra
Valérie
V
Rovati
G Enrico
GE
D000111:Acetylcysteine / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D001980:Bronchi / Q000166:cytology*; D049109:Cell Proliferation; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066246:ErbB Receptors / Q000235:genetics*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D016166:Free Radical Scavengers / Q000494:pharmacology; D006801:Humans; D017998:Leukotriene D4 / Q000494:pharmacology; D008565:Membrane Proteins / Q000502:physiology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D032389:Myocytes, Smooth Muscle / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D037342:Pertussis Toxin / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D018077:Receptors, Leukotriene / Q000502:physiology*; D015533:Transcriptional Activation / Q000502:physiology*; D000077191:Wortmannin; D018631:ras Proteins / Q000502:physiology
D000730:Androstadienes; D002867:Chromones; D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D008565:Membrane Proteins; D009025:Morpholines; D047428:Protein Kinase Inhibitors; D017382:Reactive Oxygen Species; D018077:Receptors, Leukotriene; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D017998:Leukotriene D4; D004247:DNA; D037342:Pertussis Toxin; D066246:ErbB Receptors; D048049:Extracellular Signal-Regulated MAP Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D018631:ras Proteins; C083169:leukotriene D4 receptor; D000111:Acetylcysteine; D000077191:Wortmannin