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. 2006 Mar 22;7(1):42.
doi: 10.1186/1465-9921-7-42.

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

Saula Ravasi  1 , Simona Citro, Barbara Viviani, Valérie Capra, G Enrico Rovati
Affiliations

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

Saula Ravasi et al. Respir Res. .

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 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.

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Figures

Figure 1
Figure 1
RT-PCR, [3H]ICI198,615 binding and LTD4-induced [3H]thymidine incorporation. (A) Final RT-PCR products obtained using inner primers for human CysLT1-R (Upper Panel) and CysLT2-R (Lower Panel). The PCR mediated amplification of cDNA produced the expected 948 bp (CysLT1) and 1117 bp fragments (CysLT2), which were visualized upon agarose gel, by UV irradiation. Lane 1, purchased HASMC and lane 2, HASMC isolated in our lab; STD, standard. (B) Equilibrium binding curve of [3H]ICI198,615 in membranes from HASMC. Mixed type binding curves were performed using 0.1–1 nM [3H]ICI198,615 (saturation part of the curve) and 1 nM – 1 μM of unlabeled ICI198.615 (competition part of the curve). Binding is expressed as the ratio of bound ligand concentration over total ligand concentration, (B/T, dimensionless), vs. the logarithm of total ligand concentration. B (in M) is the sum of "hot", "cold", and non-specific binding; T (in M) is the sum of "hot" and "cold" ligand incubated. Data shown are representative of two independent experiments simultaneously analyzed with LIGAND. (C-D) Effect of LTD4, EGF, and CysLT1-R antagonists on [3H]thymidine incorporation in HASMC. Increase of [3H]thymidine incorporation induced by 1 μM LTD4 and 20 ng/ml EGF alone or in combination, in the absence and presence of 1 μM of the antagonists pranlukast (C) and zafirlukast (D) (30 min pretreatment). Control is represented by MEM additioned with 1% FBS. The results are presented as mean ± S.E.M. of at least three experiments performed in triplicate on two different cell lines. **P < 0.01 (one-way ANOVA).
Figure 2
Figure 2
LTD4-induced EGF-R phosphorylation. (A) Increase of [3H]thymidine incorporation induced by 1 μM LTD4 and 20 ng/ml EGF alone and in combination, in the absence and presence of 250 nM AG1478 (1 h pretreatment). Control is represented by MEM additioned with 1% FBS. The results are presented as mean ± S.E.M. of three experiments performed in triplicate on two different cell lines. (B) Concentration-response curve of EGF-R phosphorylation induced by 3 minutes treatment with the indicated concentrations of LTD4. (C) Time-course of EGF-R phosphorylation induced by 1 μM LTD4. For B and C 1 ng/ml EGF was used as an internal control and the experiments were repeated twice.
Figure 3
Figure 3
Effect of AG1478 and CysLT1-R antagonists on LTD4-induced EGF-R phosphorylation. (A) EGF-R phosphorylation induced by 1 μM LTD4 (n = 20) and 0.1 ng/ml EGF (3 minutes) alone and in combination (n = 4). The results are presented as mean ± S.E.M. (B) Representative experiment. (C) EGF-R phosphorylation induced by 1 μM LTD4, 1 ng/ml EGF (3 minutes), in the absence and presence of 250 nM AG1478 (1 h preincubation). D) EGF-R phosphorylation induced by 1 μM LTD4 in the absence and presence of 1 μM of the antagonists zafirlukast and pranlukast (30 minutes pretreatment). 1 ng/ml EGF was used as an internal control. (C-D) The results presented are representative of at least three experiments performed on different cell lines.
Figure 4
Figure 4
LTD4-induced ERK1/2 phosphorylation. (A) Increase of [3H]thymidine incorporation induced by 1 μM LTD4 and 20 ng/ml EGF alone and in combination in the absence and presence of 20 μM PD98059 (1 h preincubation). Control is represented by MEM additioned with 1% FBS. The results are presented as mean ± S.E.M. of three experiments performed in triplicate on two different cell lines. **P < 0.01 (one-way ANOVA). (B) ERK1/2 phosphorylation induced by 10 nM LTD4 (n = 12) and 0.01 ng/ml EGF (5 minutes) alone and in combination (n = 4). The results are presented as mean ± S.E.M. (C) ERK1/2 phosphorylation induced by 10 nM LTD4 and 0.01 ng/ml EGF (5 minutes) alone and in combination in the absence and presence of 20 μM PD98059 (1 h preincubation). (D) Effect of 1 μM zafirlukast and pranlukast (30 minutes pretreatment) on ERK1/2 phosphorylation induced by 10 nM LTD4 (5 min). 0.1 ng/ml EGF was used as an internal control. (E) ERK1/2 phosphorylation induced by 10 nM LTD4, 0.1 ng/ml EGF (5 minutes), alone or in combination, in the absence and presence of 250 nM AG1478 (1 h preincubation). (D-E) The results presented are representative of at least three experiments performed on two different cell lines.
Figure 5
Figure 5
Effect of PTX on [3H]thymidine incorporation, EGF-R and ERK1/2 phosphorylation induced by LTD4 or EGF. (A) Increase of [3H]thymidine incorporation induced by 1 μM LTD4 and 20 ng/ml EGF alone and in combination in the absence and presence of 100 ng/ml PTX (20 hours pretreatment). Control is represented by MEM additioned with 1% FBS. The results are presented as mean ± S.E.M. of three experiments performed in triplicate on two different cell lines. **P < 0.01 (one-way ANOVA). (B) EGF-R phosphorylation induced by 1 μM LTD4 (3 minutes), in the absence and presence of 100 or 300 ng/ml PTX (20 hours pretreatment). 1 ng/ml EGF was used as an internal control. (C) ERK1/2 phosphorylation induced by 10 nM LTD4 (5 minutes) in the absence and presence of 100 or 300 ng/ml PTX (20 hours pretreatment). The results presented are representative of at least three experiments performed on two different cell lines.
Figure 6
Figure 6
Effect of genistein and PP1 on [3H]thymidine incorporation, EGF-R and ERK1/2 phosphorylation, and Ras activation induced by LTD4 or EGF. (A-B) Increase of [3H]thymidine incorporation induced by 1 μM LTD4 and 20 ng/ml EGF alone and in combination in the absence and presence of (A) 50 μM genistein or (B) 1 μM PP1 (30 minutes pretreatment). Control is represented by MEM additioned with 1% FBS. The results are presented as mean ± S.E.M. of at least three experiments performed in triplicate on at least two different cell lines. **P < 0.01 (one-way ANOVA). (C) EGF-R phosphorylation induced by 1 μM LTD4 (3 minutes), in the absence and presence of 50 μM genistein or 1 μM PP1 (30 minutes pretreatment). 1 ng/ml EGF was used as an internal control. (D) ERK1/2 phosphorylation induced by 10 nM LTD4 and 0.1 ng/ml EGF (5 minutes) in the absence and presence of 50 μM genistein or 1 μM PP1 (30 minutes pretreatment). E) Increase of Ras-GTP levels induced by 10 nM LTD4 and 10 ng/ml EGF alone and in combination (5 minutes). Activated Ras (p21 Ras-GTP) was co-immunoprecipitated and detected by immunoblotting the same amount of proteins for each sample with a pan-Ras antibody. The results presented are representative of at least three experiments performed on two different cell lines.
Figure 7
Figure 7
Effect of LY294002 and wortmannin on [3H]thymidine incorporation, EGF-R and ERK1/2 phosphorylation induced by LTD4 or EGF. (A-B) Increase of [3H]thymidine incorporation induced by 1 μM LTD4 and 20 ng/ml EGF alone and in combination in the absence and presence of (A) 50 μM LY294002 or (B) 200 nM wortmannin (30 minutes pretreatment). Control is represented by MEM additioned with 1% FBS. The results are presented as mean ± S.E.M. of at least three experiments performed in triplicate on at least two different cell lines. **P < 0.01 (one-way ANOVA). (C) EGF-R phosphorylation induced by 1 μM LTD4 (3 minutes), in the absence and presence 50 μM LY294002 or 200 nM wortmannin (30 minutes pretreatment). 1 ng/ml EGF was used as an internal control. (D) ERK1/2 phosphorylation induced by 10 nM LTD4 and 0.1 ng/ml EGF (5 minutes) in the absence and presence of 50 μM LY294002 or 200 nM wortmannin (30 minutes pretreatment). The results presented are representative of at least three experiments performed on two different cell lines.
Figure 8
Figure 8
LTD4-induced ROS generation and effect of NAC on [3H]thymidine incorporation, EGF-R and ERK1/2 phosphorylation. (A) Increase of [3H]thymidine incorporation induced by 1 μM LTD4 and 20 ng/ml EGF alone and in combination in the absence and presence of 10 mM NAC (1 h pretreatment). Control is represented by MEM additioned with 1% FBS. The results are presented as mean ± S.E.M. of four experiments performed in triplicate on two different cell lines. (B) Increase in ROS generation induced by 1 μM LTD4 (5 min) in the absence and presence of 1 μM of the CysLT1-R antagonist montelukast (30 min pretreatment). Control is represented by MEM additioned with 1% FBS. The results are presented as mean ± S.E.M. of four experiments performed in triplicate on two different cell lines. (C) EGF-R phosphorylation induced by 1 μM LTD4 (3 minutes), in the absence and presence of 10 mM NAC (1 h pretreatment). 1 ng/ml EGF was used as an internal control. (D) ERK1/2 phosphorylation induced by 10 nM LTD4 and 0.1 ng/ml EGF (5 minutes) in the absence and presence of 10 mM NAC (1 h pretreatment). The results presented are representative of at least three experiments performed on two different cell lines.
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