Cell culture supplies, EGF, N-acetylcysteine (NAC), CRM197 and anti-smooth muscle α-actin antibody were purchased from Sigma Chemical Co (St. Louis, MO); anti EGF-R, p-EGF-R (Tyr 1173) and ERK1/2 were from Santa Cruz Biotechnology (Santa Cruz, CA). Ras activation assay kit containing a GST fusion protein corresponding to the human RBD of Raf1 and a pan-Ras mouse monoclonal antibody (clone RAS10) was purchased from Upstate biotechnology (Lake Placid, NY); Anti-p-ERK1/2 (Thr 202 and Tyr 204) monoclonal antibody is from Cell Signaling Technology (Beverly, MA); Fetal bovine serum (FBS), TriZol^® Reagent, CysLT₁ receptor oligonucleotide primers and Platinum Taq DNA Polymerase were from Life Technologies (NY, USA); LTD₄ from Cayman Chemical Co. (Ann Arbor, MI); zafirlukast, pranlukast and montelukast were a gift from Merck & Co. (West Point, PA). AG1478, pertussis toxin, PD98059, U73122 and genistein, were from Calbiochem (La Jolla, CA); PP1 from Biomol (Plymouth Meeting, PA). DCFH-DA (6-carboxy-2',7'-dichloro-dihydrofluorescein diacetate, di(acetoxymethyl ester)) was from Molecular Probes (Eugene, OR). Ultima Gold scintillation liquid and [³H] ICI198,615 were from Perkin Elmer life sciences (Boston, MA); MMLV-Reverse Transcriptase RETROscript™ for RT-PCR was from Ambion (Austin, TX); the protease inhibitor complex Complete™ from Roche Applied Sciences (Basel, Switzerland). Reagents and films for chemiluminescence and [³H]Thymidine were from Amersham Bioscience (Piscataway, NJ). All reagents and supplies for electrophoresis and DC™ Protein assay were from Bio-Rad Laboratories (Richmond, CA).

Smooth muscle cells from human bronchi were purchased from Cambrex (Walkersville, MD) or isolated in our laboratory as previously described 33. Briefly, macroscopically normal lung fragments were obtained at thoracotomy. Third order bronchi were removed under sterile conditions, the connective tissue and the epithelium were removed and the smooth muscle cut into pieces approximately 10 mg each. The explants were grown at 37°C in a humidified atmosphere of 5% CO₂ in Medium 199, additioned with 20% (v/v) FBS, 100 U/ml penicillin and 100 μg/ml streptomycin in 25 cm² culture flask. The primary isolates were positively stained with an anti-smooth muscle α-actin antibody to assess the identity of the cultures. Thereafter, cells were routinely grown in monolayers in MEM supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin, passaged at a 1:3 ratio in 75 cm² culture flask and used between the 3^rd and 8^th passage (our isolates) or between the 3^rd and the 10^th passage (purchased cells).

Total RNA was extracted from HASMC using "TRIZOL^® Reagent", according to the manufacturer's instructions. After denaturation (75°C, 3 minutes), 1–2 μg of total purified RNA was retrotranscribed in the presence of MMLV-Reverse Transcriptase (5 U/μl) in optimized reaction conditions (RT-Buffer: 50 mM Tris-HC1, pH 8.3, 75 mM KC1, 3 mM MgCl₂, 5 mM DTT, 2 mM dNTPs, 0.5 U/μl RNAse inhibitors 42–44°C, 1 h). Specific amino- and carboxyl-terminal primers for hCysLT₁ receptor (N-terminal, 5'-ATGGATGAAACAGGAAATCTGACAG-3'; C-terminal, 3'-CTATACTTTACATATTTCTTCTCC-5') and for CysLT₂ receptor (N-terminal, 5'-ACCTTCAGCAATAACAACAGC-3'; C-terminal, 3'-CTTTATGCAGTCTGTCTTTGC-5') have been selected on the basis of the sequences previously published 3435. The PCR mediated amplification of cDNA employed Taq Platinum Polymerase (0.03 U/μl), in optimized conditions (PCR-Buffer: 20 mM Tris-HCl, pH 8.4, 75 mM KC1,0.2 mM dNTPs, 2 mM MgCl₂, 0.2 μM forward and reverse primers) using a Bio-Rad/I-Cycler PCR system. Specific cDNA fragments of 948 bp and 1117 bp were amplified (25–30 cycles: denaturation: 95°C, 30 sec; annealing 60°C, 20 sec; extension: 68°C, 45 sec) and visualized after electrophoresis in 1% agarose gels by UV irradiation.

Radioligand binding was performed at equilibrium on membranes of HASMC (0.25 mg/samples) prepared as previously described 36, in a final volume of 500 μl of PBS containing 2.5 mM CaCl₂, 2.5 mM MgCl₂, 10 mM glycine and 20 mM penicillamine. Equilibrium binding studies were performed at 25°C for 1 h with a mixed type binding protocol, obtained by combining both saturation (0.1–1 nM of [³H]ICI198,615) and competition (3 nM – 1 μM of ICI198,615) protocols in a single curve 37. Unbound ligand was separated by rapid vacuum filtration using a Brandel cell harvester and radioactivity measured by liquid scintillation counting. Analysis of binding data was performed by means of the computer program LIGAND (see Statistical analysis). The protein content was determined by Lowry quantization assay. Non specific binding ranged between 40 and 50% of the total binding.

[³H]Thymidine incorporation assay was performed as previously described 31. Briefly, cells were subcultured into 24 well plates and subconfluent cells were serum-starved for 48 hours to synchronize the entire population cell cycle. Medium was then replaced with MEM containing 1% FBS and cells stimulated with LTD₄ (two pulses at 4 hrs interval) and EGF (single pulse) at the indicated concentration for 48 hours and incubated with [³H]thymidine for the last 4 hours at a final concentration of 1 μCi ml^-1. Cells were then washed twice with ice-cold phosphate buffered saline to rinse loosely associated radioactive tracer. Acid-soluble radioactivity was removed by 20 minutes treatment with 5% trichloroacetic acid at 4°C followed by a two-step washing with 95% ethanol. The acid-insoluble portion was recovered by 60 minutes digestion with 2% Na₂CO₃ in 0.1 M NaOH. The radioactivity was then measured by liquid scintillation counting.

Phosphorylation of EGF-R was performed as previously described 31. Briefly, subconfluent cells in 60-mm dishes were serum-starved for 48 h, and 2 h before stimulation the medium was replaced by MEM containing 0.1% fetal bovine serum. The cells were then stimulated at 37°C with LTD₄ or EGF at the concentrations indicated for 5 min. AG1478 was preincubated for 1 hour before the addition of EGF. Monolayers were then placed on ice, washed twice with phosphate buffered saline, lysed (20 mM TrisHCl pH 7.5, 1 mM dithiotreitol, 2 mM EGTA, 20 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na₃VO₄ and the protease inhibitor complex Complete™) and sonicated four times for 15 s. The samples were subsequently diluted in Laemmli buffer, resolved by SDS-PAGE (sodium dodecyl sulfate polyacrilamide gel electrophoresis, 20 μg/lane – 6% gel) and transferred to a nitrocellulose membrane. Immunoblotting was performed with a polyclonal anti-EGF-R antibody at a concentration of 0.2 μg/ml for 18 h at 4°C and immune complex was detected by chemiluminescence using horseradish peroxidase-conjugated goat anti-rabbit IgG (immunoglobulin G) as a secondary antibody. The membrane was stripped with stripping buffer and re-blotted with polyclonal anti-EGF-R antibody.

ERK1/2 phosphorylation was performed as previously described 32. Briefly, confluent cells into 35 mm dishes were serum-starved for 48 h, preincubated with the inhibitors for the indicated time and stimulations at 37°C were terminated by addition of ice-cold lysis buffer (see above). Thereafter, the lysates were sonicated four times on ice for 15 sec, the protein content was measured and compensated for prior to SDS-PAGE. Cell lysates were solubilized by boiling at 95°C for 5 min in Laemmli buffer and subjected to electrophoresis on 15% polyacrylamide gel. The separated proteins were transferred to a nitrocellulose membrane. Membranes were then blocked for 1 hour with 5% non-fat dried milk at room temperature and then incubated overnight at 4°C with an anti-ERK2 or anti-phosphoERK1/2 monoclonal antibody at the concentration of 1 ng/ml. Next, membranes were washed extensively and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG as a secondary antibody for 1 hour at room temperature. After three washes, the immunoreactive proteins were visualized by chemiluminescence.

Ras activation assay was performed following the affinity precipitation protocol provided by the manufacturer (Ras pull down assay kit) as previously described 26. Briefly, cells were serum-starved overnight, treated with appropriate stimuli, and then lysed as previously described (see above). Lysates (1 mg/ml of total cell proteins in each sample) were incubated with 10 μg of Raf-1 RBD for 45 minutes at 4°C and then centrifuged for 15 seconds at 14000 × g to pellet the agarose beads. After discarding the supernatant, agarose beads were washed with 1 ml PBS and then the pellets were resuspended in 2X Laemmli sample buffer containing DTT, boiled for 5 minutes, and finally centrifuged for 15 sec at 14000 × g. The supernatant was collected and cellular proteins resolved by SDS-PAGE using 11% (w/v) acrylamide and analyzed by western blotting (see above).

HASMC were loaded with 10 μM DCFH-DA in saline buffer supplemented with 0.1% of bovine serum albumin for 1 hat 37°C (stained). At the end of the incubation, cells were washed in PBS and oxidative activity was assessed as follows. The production of reactive oxygen species (ROS) was measured by the intensity of DCF emission at 525 nm (excitation 503 nm – Perkin-Elmer LS 50B) in both stained and unstained cells. Results are expressed as the difference in fluorescence (in arbitrary units, AU) calculated as AU = [I_t5 - I_t0], where I represents the intensity of fluorescence at the specified time points.

Ligand binding studies were analyzed using LIGAND computer program. Non specific binding was calculated by LIGAND as one of the unknown parameters of the model. Selection of the best fitting model and evaluation of the statistical significance of the parameter difference was based on the F-test for the extra sum of square principle. Parameter errors are always expressed in % coefficient of variation (%CV). The curve shown was computer generated. Statistical comparison of two groups was performed using an independent t test; multiple groups were analyzed using one way ANOVA followed by either Bonferroni or Dunnett post hoc test. Data are expressed as means ± S.E.M. Each experiment was performed at least three times in triplicate (were possible) on at least two different cell lines. Basal condition refers to cells unexposed to antagonists or inhibitors and, where necessary, vehicle-treated.

In order to confirm whether the different HASMC utilized expressed an LTD₄ receptor, we routinely performed a RT-PCR reaction to amplify the human CysLT₁-R specific DNA sequence. Fig. 1A (Upper Panel) shows the expected product of 948 bp. Indeed, HASMC also express the CysLT₂-R (Fig. 1A, Lower Panel). We performed equilibrium binding studies in membranes from HASMC using [³H]ICI198,615 as labeled ligand (Fig. 1B). Computer analysis of the mixed type curve revealed the presence of two classes of binding sites, as previously reported 38. The high affinity binding site, representative of the CysLT₁-R 20, exhibited a K_d = 0.16 nM ± 68 %CV and a B_max = 6.6 fmol/mg/prot ± 82 %CV. Both parameters were as expected for a constitutive CysLT₁-R.

In HASMC 1 μM LTD₄ was able to produce an increase in [³H]thymidine incorporation (53% ± 4.1 S.E.M. increase vs. control; n = 27), and to potentiate EGF-induced proliferation (39% ± 4.7 S.E.M. increase vs. EGF alone; n = 15). Two pulses of 1 μM LTD₄ has been utilized because it is known that LTD₄ is highly unstable and rapidly metabolized to LTE₄, a much weaker partial agonist at the CysLT₁-R. Furthermore, 30 minutes pretreatment with 1 μM of the two CysLT₁-R antagonists pranlukast and zafirlukast strongly prevented LTD₄-induced HASMC [³H]thymidine incorporation (86% ± 15 S.E.M. and 75% ± 12, respectively), as well as its potentiating effect of the EGF-induced DNA synthesis (Fig. 1C–D), demonstrating that LTD₄ is mostly acting through a classical CysLT₁-R. As expected, neither pranlukast nor zafirlukast were able to influence the mitogenic effect of EGF.

To investigate a possible transactivation of the EGF-R by LTD₄ in HASMC, we tested the EGF-R tyrosine kinase inhibitor AG1478. Figure 2A shows that 1 hour pretreatment with 250 nM AG1478 fully inhibited DNA synthesis induced by LTD₄ and EGF alone or by their combination.

Because we were interested in investigating whether the EGF-R can function as a downstream signaling partner of cysteinyl-LTs, therefore acting as a point of convergence for heterogeneous signaling pathways, we used immunoblot analysis to assess the phosphorylation state of this receptor following the treatment of the cells with LTD₄. As shown in Figure 2B and 2C, EGF-R tyrosine phosphorylation was concentration- and time-dependent, starting as early as 2 min and peaking at 3 min at a concentration of 1 μM. Thus, all subsequent experiments were performed stimulating cells with 1 μM LTD₄ for 3 min, which produced an average EGF-R phosphorylation of 80% ± 7.8 S.E.M., (Fig. 3A, n = 20). Furthermore (Fig. 3B), LTD₄ also induced potentiation of EGF-stimulated autophosphorylation (47% ± 4.8 S.E.M. increase vs. 0.1 ng/ml EGF, Fig. 3A, n = 4), a result in agreement with LTD₄ potentiation of EGF-stimulated DNA synthesis. Unsurprisingly, 1 hour pretreatment with 250 nM AG1478 totally inhibited LTD₄-induced EGF-R phosphorylation (Fig. 3C). In addition, LTD₄ effect was significantly prevented by 1 μM zafirlukast and pranlukast (~70% and ~60%, respectively) (Fig. 3D), a result again in good agreement with the CysLT₁-R antagonist inhibition of thymidine incorporation (see above).

Because we and others have already suggested that LTD₄ is able to activate ERK 263940, we tested the effect of a specific MAPK/ERK kinase (MEK1) inhibitor, i.e. PD98059, on the LTD₄-induced [³H]thymidine incorporation. Indeed, 1 hour pretreatment with 20 μM PD98059 fully prevented LTD₄-induced DNA synthesis (Fig. 4A), as well as the basal level of [³H]thymidine incorporation in the presence of 1% FBS. At variance, PD98059 only partially inhibited the effect caused by EGF and by the combined effect of LTD₄ and EGF.

To confirm the involvement of ERK 1/2, we measured the amount of their phosphorylated form by western blotting. Stimulation of HASMC with10 nM LTD₄ for 5 minutes (time course and concentration-response curves not shown) produced a maximal ERK1/2 phosphorylation of 162% ± 16 S.E.M. (Fig. 4B n = 12). This effect was completely inhibited by 1 μM zafirlukast and pranlukast (Fig. 4D). Furthermore (Fig. 4C), LTD₄ also induced potentiation of EGF-stimulated MAPK activation (104% ± 28 increase vs. 0.01 ng/ml EGF; Fig. 4B n = 4).

To test the hypothesis that MAPK activation could be dependent upon EGF-R transactivation, we also tested the effect of AG1478 on LTD₄-induced ERK1/2 phosphorylation and found that 1 hour pretreatment with 250 nM of the EGF-R phosphorylation inhibitor totally prevented ERK1/2 activation by EGF and LTD₄ alone or by their combination (Fig. 4E).

The time frame of EGF-R activation (3 min) is fully compatible with that of MAPK activation (5 min). Despite there is disagreement in the literature on the necessity for prolonged activation of the MAPK cascade to produce a significant mitogenic effect 4142, we recently demonstrated that a rapid and transient activation of ERK following TP receptor stimulation translocates ERK into the nucleus as early as 2 min, where it accumulates in its active form 32. Thus, these data suggest that that stimulation of the CysLT₁-R in HASMCs might be important for the control of transcription factors and cell cycle re-entry, especially with other mitogenic stimuli (e.g., EGF), if efficient cell proliferation is to be achieved.

To identify which class of G protein is involved in LTD₄-induced [³H]thymidine incorporation, we pretreated HASMC with 100 ng/ml PTX for 20 hours. Preincubation of HASMC with PTX fully abolished LTD₄-induced DNA synthesis (Fig. 5A). On the contrary, the toxin failed to affect EGF mitogenic response, while it reverted [³H]thymidine uptake level to the one induced by EGF alone. Furthermore, PTX (either 100 or 300 ng/ml) also fully inhibited LTD₄-induced EGF-R and ERK1/2 phosphorylation, demonstrating that not only EGF-R transactivation, but also ERK1/2 phosphorylation is totally dependent upon a G_i/o protein activation (Fig. 5B and 5C, respectively).

It is known that GPCRs might trigger activation of a protein tyrosine kinase (PTK) such as Src in order to activate MAPKs. Thus, we tested the effect of genistein, a broad spectrum PTK inhibitor, and of PP1, a specific Src kinase inhibitor, on LTD₄-stimulated [³H]thymidine incorporation in HASMC. It is clear from Figure 6A and 6B that 30 minutes pretreatment with 50 μM genistein or 1 μM PP1 strongly inhibited DNA synthesis caused by LTD₄ (> 90%), or by costimulation of EGF and LTD₄ (> 70%). Furthermore, PP1 and to a lesser extent genistein were able to inhibit the effect induced by EGF alone.

It is also known that a possible pathway leading to EGF-R transactivation by G_i coupled receptors requires Src kinase activity. However, Figure 6C shows that neither genistein nor PP1 significantly affected LTD₄-stimulated EGF-R transactivation as well as EGF-induced autophosphorylation (data not shown). At variance, both compounds inhibited LTD₄- (~33% and > 90%, respectively) or EGF-stimulated (~34% and ~86%, respectively) ERK1/2 phosphorylation in HASMC in a similar way (Fig. 6D).

Finally, through a Ras pull-down assay we directly demonstrated that 10 nM LTD₄ was able to increase the amount of Ras-GTP in HASMC (157% ± 37 S.E.M.), confirming that also in these cells LTD₄ is able to activate the small G protein Ras at a concentration relevant for its pathophysiological role in HASMC proliferation (Fig. 6E).

It has been previously suggested that different isoforms of PI3K might be involved in the mitogenic signal induced by G_i-coupled receptors. Thus, we tested two different PI3K inhibitors, i.e. wortmannin and LY29004, and found that 30 minutes pretreatment blunted LTD₄-induced DNA synthesis. On the contrary, both compounds were only partially able to inhibit [³H]thymidine incorporation produced by EGF alone or in combination with LTD₄ (Fig. 7A–B). Surprisingly, a partial inhibition (~65% for both compounds) was observed on EGF-R transactivation (Fig. 7C). Furthermore, both inhibitors totally ablated LTD₄-induced ERK1/2 phosphorylation without having an effect on EGF-stimulated MAPK activation (Fig. 7D).

Production of ROS has been noted upon growth factor stimulation of arterial smooth muscle cells 43 and bovine tracheal myocytes 44. We show here that pretreatment for 1 h with 10 mM of the antioxidant NAC, a ROS scavenger, completely inhibits LTD₄-induced [³H]thymidine incorporation (Fig. 8A). Similarly, NAC was also able to entirely inhibit EGF-induced DNA synthesis (Fig. 8A). Thus, to test whether LTD₄ treatment induces the intracellular generation of ROS, HASMC were loaded with DCFH-DA (10 μM), and stimulated with either LTD₄ or PDGF (as internal control).

LTD₄ at the concentration of 1 μM was observed to induce almost a 4-fold increase of fluorescence over baseline, within 5 min (AU = 5.50 ± 1.0 S.E.M vs. AU = 1.42 ± 0.21 S.E.M, P < 0.01). This effect was specifically inhibited by a 30 min pretreatment with 1 μM of the specific CysLT₁-R antagonist montelukast (Fig. 8B). Finally, we also show that NAC completely ablated LTD₄-induced EGF-R phosphorylation (Fig. 8C) and MAPK activation (Fig. 8D) as well as EGF-R autophosphorylation (data not shown).