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Published ahead of print on December 30, 2004, doi:10.1165/rcmb.2004-0356OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 32, pp. 326-333, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2004-0356OC

Thromboxane Prostanoid Receptor Signals Through Gi Protein to Rapidly Activate Extracellular Signal–Regulated Kinase in Human Airways

Simona Citro, Saula Ravasi, G. Enrico Rovati and Valérie Capra

Laboratory of Molecular Pharmacology, Section of Eicosanoid Pharmacology, Department of Pharmacological Sciences, University of Milan, Milan, Italy

Correspondence and requests for reprints should be addressed to Valérie Capra, Dept. of Pharmacological Sciences, University of Milan, Via Balzaretti, 9, 20133 Milan, Italy. E-mail: Valerie.Capra{at}unimi.it


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
We showed previously that activation of the thromboxane prostanoid (TP) receptor causes human airway smooth muscle (HASM) cells to proliferate, suggesting a role in airway remodeling. This study aimed at determining the molecular mechanisms underlying this mitogenic action. We found that the MEK inhibitor PD98059 significantly affected agonist-induced DNA synthesis of HASM cells, which suggests that extracellular signal–regulated kinases (ERK) are involved. ERK activation by the agonist U46619 was rapid, sensitive to pertussis toxin, and significantly abrogated by the tyrosine kinase inhibitors genistein and PP1. Stimulation of the TP receptor was also found to translocate phosphorylated ERK into the nucleus. TP receptor was found to activate Ras, as demonstrated by inhibition of ERK activation and DNA synthesis by Clostridium sordellii lethal toxin, and by the ability of U46619 to increase RasGTP. Finally, [3H]thymidine incorporation and ERK phosphorylation were also affected by prior treatment with protein kinase C inhibitor GF109203X, although to different extents. In conclusion, in HASM cells TP receptor, predominantly coupled to Gi/o proteins, activates the Ras/ERK pathway to induce mitogenesis, probably with the involvement of nonreceptor tyrosine kinases and protein kinase C.

Key Words: cell proliferation • human airway smooth muscle • MAP kinase • Ras • thromboxane A2 receptor


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The smooth muscle cells of the airways are believed to play a fundamental role in the inflammatory events leading to the structural changes of the airway wall, or remodeling, which is one of the main characteristics of chronic asthma (1). Today, treatment of airway smooth muscle hyperplasia is considered a major issue in asthma management, especially as it has been shown that airway smooth muscle cells from individuals with asthma proliferate faster than that of subjects without asthma (2), possibly because of the persistent vascular leakage.

In addition to growth factors, many other stimuli such as plasma- or inflammatory cell–derived mediators, contractile agonists, cytokines, and extracellular matrix proteins induce proliferation of human airway smooth muscle (HASM) cells (3). In HASM cells these mitogens act via their respective receptors to stimulate two parallel signaling pathways, namely the extracellular signal–regulated kinase (ERK) or the phosphatidylinositol 3-kinase (PI3K) pathways that regulate the expression of cell cycle proteins and thus modulate cell cycle progression (4).

The prostanoid thromboxane A2 (TXA2) mediates a number of cellular responses through the interaction with a seven transmembrane domains receptor that is termed thromboxane prostanoid or TP (5), whose major signaling pathway involves the activation of the Gq/11 family of G proteins (6). TP receptor can be activated by its natural agonist as well as by high levels of other eicosanoids such as PGH2, PGF2{alpha}, and isoprostanes, all of which may have a role in asthma (7, 8). In humans, a single gene located on chromosome 19p13.3 is alternatively spliced in the carboxyl terminus, giving rise to two isoforms of the receptor that share the first 328 amino acids, and that are termed TP{alpha} and TPß (9). We have previously shown that both mRNAs are expressed in HASM cells (10).

Mitogen-activated protein kinases (MAPK) are ubiquitous protein kinases with a multiplicity of signal-transducing functions activated by neurotransmitters, cytokines, and growth factors, as well as chemical and mechanical stressors (11). The p44/p42 MAPK ERK1 and ERK2 were the first Ras-effector pathway to be identified and are believed to have overlapping, if not redundant, signaling capabilities (they are therefore referred to as ERK1/2). Downstream from Ras the pathway consists of a three-kinase module cascade, Raf/MEK/ERK. The nuclear translocation of ERK1/2 in response to mitogens is considered a strong requirement to initiate gene transcription, cell proliferation, or differentiation, because it has been demonstrated in PC12 cells (12), fibroblasts (13), and HASM cells (14). In the airways, mitogens produce acute responses that can modify smooth muscle contraction and may induce chronic responses that progressively modify the general architecture of this tissue (15).

The involvement of G protein–coupled receptors (GPCRs) in cell cycle control is now widely recognized and can be obtained through the activation of different signaling pathways involving Ras-ERK, PI3K, and protein kinase C (PKC) (16). In particular, the TP receptor has been shown to be involved in different aspects of the remodeling of vascular smooth muscle cells (17, 18), in the activation of MAPK/ERK in smooth muscle from uterus and aorta (19, 20), and of ERK1/2 and PI3K in heterologous systems overexpressing the TP receptor (21).

Our previous results showed that stimulation of TP receptor induced HASM cell proliferation per se, besides potentiating the epidermal growth factor (EGF) mitogenic response independently of the transactivation of EGF receptor (EGFR) (10). On the basis of these initial studies, we have investigated further the signaling pathway linking TP receptor to HASM cell proliferation and found that TP receptor predominantly couples to Gi/o proteins to activate the Ras/ERK cascade, with the involvement of nonreceptor protein tyrosine kinases (PTKs) and a minor contribution of PKC, which could be downstream of Gq and/or Gi. We also provide evidence for the U46619-induced increase of phosphorylated ERK in the nucleus.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Materials
Cell culture supplies (media, serum, trypsin, aminoacids, and antibiotics) and buffers were from Invitrogen Life Technologies (Carlsbad, CA) and Sigma RBI (St. Louis, MO). 9,11-Dideoxy-9{alpha},11{alpha}-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U46619), and [1S-[1{alpha},2{alpha}(Z),3{alpha},4{alpha}]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (SQ29,548) were obtained from Cayman Chemical Co. (Ann Arbor, MI). Stock solutions of these compounds were stored at –20°C. 4-(3-Chloroanilino)-6,7-dimethoxyquinazoline (AG1478), 4',5,7-trihydroxyisoflavone (Genistein), 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF 109203X), 2'-amino-3'-methoxyflavone (PD98059), and 4-amino-1-tert-butyl-3-(1'-naphthyl)pyrazolo[3,4-d]pyrimidine (PP1) were from Calbiochem (San Diego, CA). Pertussis toxin (PTx) and all other chemicals were of analytical grade and purchased from Sigma RBI. Clostridium sordellii lethal toxin was kindly provided by Prof. M.Y. Popoff (Institut Pasteur, Paris, France). The protease inhibitor complex Complete was from Roche Applied Sciences (Basel, Switzerland). Anti-ERK1 antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), anti-phosphorylated ERK1/2 antibody was from Cell Signaling Technology (Beverly, MA), and Alexa-488 goat anti-rabbit antibody was from Molecular Probes (Eugene, OR). Ultima Gold scintillation liquid was from Perkin Elmer life sciences (Boston, MA). All reagents and supplies for electrophoresis and DCProtein assay were obtained from Bio-Rad Laboratories (Richmond, CA). Reagents and films for chemoluminescence and [6-3H]thymidine were from Amersham Biosciences (Piscataway, NJ).

Culture of HASM Cells
Smooth muscle cells from human bronchi were purchased from Cambrex Bio Science (Baltimore, MD) or isolated in our laboratory as previously described (10). Cells were routinely grown in monolayers in modified Eagle's medium (MEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin, passaged at a 1:3 ratio in 75 cm2 culture flasks, and used between the 3rd and 8th passages (our isolates) or between the 3rd and the 12th passages (purchased cells). For this investigation we established one line and purchased three, and cell lines from these two fonts were used to address each topic so that the data shown are the means ± SEM, indicating variability between cell lines. Cells were grown at 37°C in a humidified atmosphere of 5% CO2/95% air and were regularly tested to ensure the absence of mycoplasma.

Proliferation Assay
Proliferation assays were performed essentially as previously described (10). Briefly, cells were synchronized in serum-free MEM for 48 h, then placed in "serum-deficient" MEM containing 1% FBS to provide cells with basic supplements to exit from G0 phase, and representing the control. Monolayers were subjected to 48 h stimulation with 1 µM of the TP receptor agonist U46619 with or without prior incubation with the MEK1 inhibitor PD98059, the Src inhibitor PP1, the broad spectrum PTKs inhibitor genistein, the PKC inhibitor GF109203X, or the Ras inhibitor Clostridium sordellii lethal toxin (L.Tox), for the indicated periods of time and concentrations. The [3H]thymidine pulse lasted the final 4 h (1 µCi/ml). At the end of the experiment cells were washed twice with ice-cold PBS and acid-soluble radioactivity was removed by 20-min treatment with 5% TCA at 4°C followed by a two-step wash with 95% ethanol. The acid-insoluble portion was recovered by 60-min solubilization with 2% Na2CO3 in 0.1 M NaOH and radioactivity was measured by liquid scintillation counting.

Analysis of ERK1/2 Phosphorylation on HASM Cell Extracts
Analysis of ERK1/2 phosphorylation was performed essentially as previously described (22). Subconfluent cells in 35-mm dishes were serum-starved for 48 h, and 2 h before challenge cells were placed in media supplemented with 0.1% FBS. Cells were then preincubated with the inhibitors for the indicated periods of time, and stimulations at 37°C were terminated on ice by addition of ice-cold lysis buffer (20 mM TrisHCl pH 7.5, 1 mM dithiotreitol, 2 mM EGTA, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and the protease inhibitor complex Complete). Thereafter, the whole cell lysates were sonicated four times on ice for 15 s with glass/glass homogenizer, and the protein content was measured and compensated for before SDS-PAGE (15 µg/sample). Cell lysates were solubilized by heating at 95°C for 5 min in sample buffer (200 mM Tris pH 6.5, 6% SDS, 2-ß mercaptoethanol, 24% glycerol, bromophenol blue), subjected to electrophoresis on 15% polyacrylamide gel, and the separated proteins were transferred to a nitrocellulose membrane. Membranes were then blocked for 60 min with 5% nonfat dried milk at room temperature and then incubated overnight at 4°C with an anti–phospho ERK1/2 monoclonal antibody (Cell Signaling Technology) at a concentration of 1 ng/ml. After extensive washing, membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG as a secondary antibody for 60 min at room temperature. After another wash step, the phosphorylated immunoreactive proteins were visualized by chemiluminescence (Amersham Biosciences). Where appropriate, membranes were stripped and re-probed with anti-ERK1 antibody to detect total ERK protein (to further control protein loading). Signals were quantified by scanning densitometry. Where specified the analysis of ERK1/2 was also run on cytosolic and nuclear fractions, which were obtained as follows. Subconfluent cells in 100-mm dishes were growth-arrested by incubating for 48 h in serum-free medium, equilibrated for 2 h in MEM containing 0.1% FBS, and then stimulated with mitogenic compounds. Monolayers were lysed (20 mM Hepes, 25 mM KCl, 1.5 mM MgCl2, 1 mM dithiotreitol, 1 mM phenylmethylsulfonyl fluoride, 20 mM NaF, 0.2% Triton, and Complete) and homogenized in a glass/glass potter. After centrifugation at 1,000 x g for 10 min at 4°C, the supernatant was collected as cytosolic fraction. The remaining pellet was resuspended in lysis buffer and held on ice for 10 min. This lysate was used as the nuclear fraction.

Indirect Immunofluorescence
HASM cells were grown on glass coverslips and serum-starved for 48 h. After treatment with the specified stimuli for different times, monolayers were washed with PBS, fixed with 3.7% paraformaldehyde, and permeabilized with 0.1% Triton for 5 min at room temperature. Samples were incubated with 2% bovine serum albumin as blocking solution and with anti-ERK1 (1:500) or anti–phosphorylated ERK1/2 (1:500) antibodies for 1 h at 37°C. After a washing step, coverslips were treated with Alexa-488 goat anti-rabbit antibody (1:500) for 45 min at room temperature. Confocal laser scanning microscopy was performed using a Bio-Rad confocal microscope (Radiance 2,100) with a x60 lens.

Ras Activation Assay (Pull-Down Assay)
Ras activation assay was essentially as previously described (22), following the affinity precipitation protocol provided by the manufacturer (Ras pull-down assay kit; Upstate Biotechnology, Lake Placid, NY), which uses the GST fusion protein of human Ras binding domain of Raf-1 to specifically bind and precipitate RasGTP from cell lysates. Briefly, cells were serum-starved overnight, treated with appropriate stimuli, and then lysed as above. Lysates (1 mg/ml of total cell protein for each sample) were incubated with 10 µg of Raf-1 RBD for 45 min at 4°C and then centrifuged for 15 s at 14,000 x g. The pellets were resuspended in 2x Laemmli sample buffer, boiled for 5 min, and finally centrifuged for 15 s at 14,000 x g. The supernatant was collected, cellular proteins were resolved by SDS-PAGE using 11% (wt/vol) acrylamide, and analyzed by Western blotting. Signals were quantified by scanning densitometry.

Experimental Design and Statistical Analysis
Having established one line and purchased three, we used cell lines from the two fonts to address each topic in at least three separate experiments, so that in the data shown as means ± SEM, the latter indicates the degree of variability between experiments and cell lines. When indicated, Student's t test or ANOVA followed by post hoc test for multiple comparison was performed. P < 0.05 was accepted as statistically significant.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Stimulation of DNA Synthesis
Effect of PD98059. Agonist stimulation of TP receptor by the stable analog of TXA2, U46619, at a concentration of 1 µM increased DNA synthesis by 110% ± 15 SEM over control (Figure 1A). This confirmed our previous observations in HASM cells (10). To test the involvement of the MAPK/ERK pathway in the TP receptor mediation of HASM cell proliferation, we analyzed the effect of pretreatment for 30 min with 20 µM PD98059, a compound known to block MAPK cascade and thus prevent the activation of MEK1 by upstream activators such as Raf-1 (23). Figure 1 shows that PD98059 inhibited U46619-induced DNA synthesis by 73% ± 7.7 SEM (Figure 1B).



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  		Figure 1. Effect of inhibitors on the mitogenic response exerted by exogenously added U46619, measured as [3H]thymidine incorporation in HASM cells. (A) Synchronized cells were pretreated with the MEK1 inhibitor PD98059 (PD98, 20 µM for 60 min), the PTK inhibitors PP1 (1 µM for 30 min) or genistein (Gen, 50 µM for 30 min), or the protein kinase C inhibitor GF109203X (GFX, 500 nM for 60 min) and then stimulated with 1 µM U46619 for 48 h. Absolute counts for basal incorporation are equal to 2,391 dpm/well ± 292 SEM. Means ± SEM of five experiments, each performed in triplicate. *P < 0.05 versus U46619-stimulated cells (t test). (B) The same results as above are presented as % inhibition versus U46619-induced [3H]thymidine incorporation ± SEM.

 
Effect of PP1, genistein, and GF109203X. A series of experiments investigated the involvement of PTKs and PKC, which are generally believed to be involved in the signaling pathway linking GPCRs to cell proliferation. The effects of 30 min pretreatment before agonist stimulation with 1 µM PP1, 50 µM genistein, and 500 nM GF109203X are summarized in Figure 1B, and indicate that both PTKs and PKC inhibit the TP-mediated DNA synthesis of HASM cells (> 75% inhibition versus U46619-induced [3H]thymidine incorporation). We did not observe any significant detrimental effect of the inhibitors on cell viability at the concentrations and times tested (trypan blue cell exclusion method).

Activation of ERK
Time- and dose-dependent activation. Exposure of HASM cells to increasing concentrations of U46619 resulted in a dose-dependent phosphorylation of ERK1/2, with maximal activation between 100 and 300 nM (Figure 2A). The response to U46619 was rapid, reaching a peak at 5 min and returning to the basal level after 30 min (Figure 2B). As a result, all subsequent ERK1/2 phosphorylations were performed with 300 nM U46619 for 5 min, a condition that generated an average increase in ERK phosphorylation over control equal to 206% ± 51 SEM. As expected, 30 min preincubation with 20 µM PD98059 completely abolished U46619-induced ERK1/2 phosphorylation (data not shown). EGF at a concentration of 0.1 ng/ml was always added as an internal positive control.



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  		Figure 2. Effect of U46619 on ERK1/2 activation and intracellular localization in HASM cells. (A) Concentration-dependent activation of ERK1/2. Cells were stimulated for 5 min with the indicated concentrations of the agonist, and cell lysates were resolved by SDS-PAGE and immunoblotted with anti-ERK antibodies to detect the phosphorylated (pp, top) and the total (bottom) ERK immunoreactive proteins. EGF, 0.1 ng/ml, was added as an internal positive control. (B) Time-dependent activation of ERK1/2. Cells were stimulated with 300 nM U46619 for the indicated times; lysates were resolved by SDS-PAGE and immunoblotted with anti-ERK antibodies to detect the phosphorylated (pp, top) and the total (bottom) ERK immunoreactive proteins. EGF, 0.1 ng/ml, was included as an internal positive control. (C) Detection of phosphorylated ERK in the cytosolic and nuclear fractions of HASM cells. Cells were stimulated with 300 nM U46619 (U46) for 5 min, 0.1 ng/ml EGF being included as an internal positive control. After separation of the cytosolic and nuclear fractions, samples were resolved by SDS-PAGE and immunoblots screened with anti-ERK antibody to detect the phosphorylated (pp) ERK immunoreactive protein. Results shown in all panels are representative of three separate experiments.

 
Localization of phosphorylated ERK. Because MAPK nuclear translocation is thought to represent a crucial step in the modulation of gene expression and cell cycle, we considered whether activation of ERK1/2 in response to stimulation with U46619 resulted in the presence of phosphorylated ERK1/2 in the nucleus. The subcellular localization of ERK during U46619 stimulation in HASM cells is presented in Figures 2C and 3. Figure 2C shows that 5 min challenge with 300 nM of U46619 resulted in negligible signals for activated ERK in the cytosol, whereas phosphorylated ERK were clearly observed in the nuclear fraction. Confocal image presented in Figure 3Aa shows that in serum-deprived cells the ERK pool was primarily located in the cytoplasm, where it was inactive (Figure 3Ba). However, within 2 min activated ERK was present both in the cytoplasm and in the nucleus (Figure 3Bb), and between 5 and 10 min most of the ERK translocated to the nucleus (Figures 3Ac–3Ad), where it accumulated in its active form (Figures 3Bc–3Bd); at 30 min after stimulation there was a reduction in the signal for phosphorylated ERK (Figure 3Be), whereas a part of the cellular ERK pool was still present in the nucleus at this time point (Figure 3Ae). EGF at a concentration of 0.1 ng/ml was always added as an internal positive control (Figures 3Af and 3Bf).



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  		Figure 3. ERK1/2 localization following U46619 stimulation in HASM cells. Cells were serum-starved for 48 h before stimulation with 300 nM U46619 for the indicated times, and processed for indirect immunofluorescence with anti-ERK antibodies to detect the total (A, panels a–e) and the phosphorylated (B, panels a–e) ERK immunoreactive proteins. EGF at a concentration of 0.1 ng/ml was included as an internal positive control (panels f in both A and B).

 
Effect of SQ29,548 and PTx. To verify the specificity of ERK1/2 activation, we pretreated HASM cells with 1 µM SQ29,548 for 30 min, before agonist stimulation. Figures 4A and 4C show that preincubation with the specific TP receptor antagonist completely abolished U46619-induced ERK1/2 phosphorylation. We have indeed tested the effect of SQ29,548 on EGF-induced ERK activation and, as expected, found no inhibitory activity (data not shown).



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  		Figure 4. Effect of SQ29,548 (SQ) and pertussis toxin (PTx) on ERK1/2 activation induced by U46619 in HASM cells. (A) Cells pretreated with 1 µM of the specific receptor antagonists SQ29,548 for 30 min were then stimulated for 5 min with 300 nM U46619; 0.1 ng/ml EGF was added as an internal positive control. Cell lysates were resolved by SDS-PAGE and immunoblotted as in Figure 2. Results are representative of three experiments. (B) As in A, but cells preincubated with 300 ng/ml PTx for 18 h. Results shown are representative of five experiments. (C) Percentage of ERK phosphorylation as means ± SEM. **P < 0.01 versus U46619-stimulated cells (one-way ANOVA).

 
Furthermore, we examined whether a PTx-sensitive G protein mediated U46619-induced ERK1/2 phosphorylation. We found that preincubation with 300 ng/ml PTx for 18 h completely inhibited the TP receptor–mediated ERK1/2 activation (Figures 4B and 4C).

Effect of AG1478. Figures 5A and 5B show that 250 nM AG1478, a receptor tyrosine kinase inhibitor specific for EGFR, had no effect on U46619-induced ERK activation, whereas, as expected, it completely abolished the effect of EGF alone. Moreover, when tested on the combination of U46619 and EGF, AG1478 reduced ERK1/2 phosphorylation back to the level induced by U46619 alone, indicating that ERK activation by TP receptor stimulation does not involve EGFR transactivation.



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  		Figure 5. Effect of AG1478 (AG) on ERK activation induced by U46619 (U46), alone or in combination with EGF, in HASM cells. (A) As in Figure 4, but cells pretreated with 250 nM AG1478 for 30 min; 0.1 ng/ml EGF was added as an internal positive control. Cell lysates were resolved by SDS-PAGE and immunoblotted as in Figure 2. Results are representative of three experiments. (B) Percentage of ERK phosphorylation as means ± SEM. *P < 0.05 (one-way ANOVA).

 
Effect of PP1, genistein, and GF109203X. To identify the signal pathways involved in TP receptor–induced ERK1/2 activation and to compare them with the results obtained in proliferation studies, we investigated the role of PTKs and PKC. After pretreatment of HASM cells for 30 min with 1 µM PP1 or 50 µM genistein, TP-dependent activation of ERK1/2 was decreased by 67% ± 18 SEM and > 90%, respectively (Figures 6A and 6B). When HASM cells were pretreated with 500 nM of the specific PKC inhibitor GF109203X (Figures 6C and 6D), TP-dependent activation of ERK1/2 was only partially inhibited (45% ± 8.4 SEM), which suggests that the role for PKC in mediating ERK activation in these cells is limited.



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  		Figure 6. Effect of PTK inhibitors, namely PP1 and genistein (Gen), and of the PKC inhibitor GF109203X (GFX) on ERK activation induced by U46619 (U46) in HASM cells. (A) As in Figure 4, but cells pretreated with 1 µM PP1 or 50 µM genistein for 30 min; 0.1 ng/ml EGF was added as an internal positive control. Cell lysates were resolved by SDS-PAGE and immunoblotted as in Figure 2. Results are representative of three experiments. (B) Percentage of ERK phosphorylation as means ± SEM. *P < 0.05 versus U46619-stimulated cells (one-way ANOVA). (C) As in Figure 4, but cells pretreated with 500 nM GF109203X for 60 min; 0.1 ng/ml EGF was added as an internal positive control. Cell lysates were resolved by SDS-PAGE and immunoblotted as in Figure 2. Results are representative of four experiments. (D) Percentage of ERK phosphorylation as means ± SEM.

 
Activation of Ras
The postulated mechanism underlying GPCR-mediated proliferation and ERK1/2 activation frequently entails the participation of Ras. We found that 10 ng/ml of Clostridium sordellii lethal toxin (L.Tox), a specific inhibitor of the small GTPases Ras, Rap, and Rac (24), had a significant inhibitory effect on TP receptor–mediated proliferation of HASM cells (Figure 7A). To confirm this observation, we measured Ras activation by performing a RBD pull-down assay, and found that 5 min stimulation of HASM cells with 300 nM U46619 efficiently induced the small G protein activation, an effect specifically inhibited by 30 min pretreatment with 1 µM SQ29,548 (Figure 7B). Finally, Figure 7C shows that, besides being able to abolish the EGF-induced signal, L.Tox almost completely inhibited ERK phosphorylation induced by U46619, a finding that substantiates the role of Ras as an upstream modulator of ERK1/2 in HASM proliferation induced by TP receptor activation.



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  		Figure 7. Ras involvement following U466619 stimulation of HASM cells, by [3H]thymidine incorporation, Ras pull-down, and ERK activation assays. (A) Percentage of [3H]thymidine incorporation induced by 1 µM U46619 with and without pretreatment with 10 ng/ml of L.Tox for 18 h. Means ± SEM of three experiments, each performed in triplicate. *P < 0.05 versus U46619-stimulated cells (Student's t test). (B) Cells were pretreated with 1 µM SQ29,548 for 30 min and then stimulated with 300 nM U46619 for 5 min, with 0.1 ng/ml EGF included as an internal positive control. Activated Ras (p21ras GTP) was co-immunoprecipitated with Raf-1 RBD from cell lysates and detected by immunoblotting with a pan-Ras antibody. (C) Cells were pretreated with 10 ng/ml L.Tox for 18 h and then stimulated with 300 nM U46619 for 5 min; 0.1 ng/ml EGF, with and without L.Tox pretreatment, was included as an internal positive control. Cell lysates were resolved by SDS-PAGE and immunoblotted as in Figure 2. Results are representative of three experiments.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
The signaling pathways evoked by TP receptor stimulation have been well documented in the vascular smooth muscle and platelets because of their great importance in the cardiovascular system in health and disease (6, 25), but only few data have been reported on signal transduction by the TP receptor in HASM cells (10). TP receptor modulators have recently received attention for asthma therapy (8), and we and others have recently shown that stimulation of the TP receptor induces DNA synthesis in these cells (10, 26). This proliferative effect might be important in a number of other pathologic conditions, such as chronic bronchitis, bronchiolitis obliterans, and chronic obstructive pulmonary disease, where an essential step in the disease progression is airway remodeling.

Herein we show that the TP receptor, independently of EGFR phosphorylation, requires the Gi/o protein to activate the Src-Ras-ERK1/2 cascade, providing the first evidence for a rapid nuclear translocation of activated ERK1/2. We also demonstrate that PKC is involved in DNA synthesis despite its limited role as an activator of ERK1/2.

PD98059, a specific MEK1 inhibitor, almost completely inhibited U46619-induced thymidine incorporation in a manner consistent with its ability to block the activation of p42/p44 MAPK. The same results have been obtained with a structurally different MEK1 inhibitor, U0126 (data not shown). This suggests that ERK1/2 promote the mitogenesis of HASM cells. Accordingly, the stable analog of TXA2, U46619, induced ERK1/2 phosphorylation in a rapid and dose-dependent manner.

There is disagreement in the literature on the necessity for prolonged activation of the MAPK cascade to produce a significant mitogenic effect (14, 27). Here we demonstrate that despite the rapid and transient activation of ERK following TP receptor stimulation, U46619 is able to translocate ERK into the nucleus as early as 2 min, where it accumulates in its active form up to 10 min. Given that it is known that MAPK nuclear translocation represents a critical step in signal transduction (13, 14), our observations suggest that stimulation of the TP receptor in HASM cells 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. However, it is also possible that the cytoplasmic pool of active MAPK could phosphorylate transcription factors in the cytoplasm when they are released from the synthetic machinery. The identification of the possible cytoplasmic and/or nuclear targets for ERK activated in response to TP receptor stimulation deserves further investigation.

TP receptor activation is known to potentiate the mitogenic effects of RTK signaling either through RTK transactivation (20) or not (10). Here, we observed that stimulation of the TP receptor specifically potentiated EGF-induced ERK1/2 activation, in contrast with data present in the literature for other contractile or inflammatory stimuli, which suggest an absence of synergy at the ERK1/2 level between GPCRs and EGFR in HASM cells (28).

Pathways for activation of nuclear transcription in response to stimulation of GPCRs have been delineated in recombinant systems (16, 29). In fact, distinct GPCRs use different but interconnected pathways to signal to the nucleus, and the signaling events that mediate such augmented growth vary with cell line and tissue, and remain to be fully characterized, particularly in natural systems (30). Several lines of evidence suggest that an essential step for the mitogenic stimulus induced by a number of GPCRs is the transactivation of a growth factor receptor, such as the EGFR and its tyrosine kinase activity (31), as has been shown for TP receptor in heterologous or native systems (19–21, 32). However, our previous observations on DNA synthesis and EGFR phosphorylation (10) already suggested the independence of a TP-mediated effect from its transactivation. Here, we observed that blocking the EGFR tyrosine kinase activity only inhibited EGF-induced ERK1/2 phosphorylation without affecting U46619-induced signal, even when both agonists were used in combination. These data ultimately demonstrate that in HASM cells TP receptor itself triggers mitogenic downstream signaling.

In HASM cells many GPCRs induce mitogenic effects through mechanisms different from transactivation (33), although these signaling events are still unclear or not fully established for both Gq- and Gi-coupled receptors. Because it is known that TP receptors have the ability to couple to more than one heterotrimeric G protein (e.g., Gq/11 and Gi [6]), and its coupling specificity has been poorly characterized in HASM cells, we questioned which pathways were involved in the mitogenic response after TP receptor activation. The capacity of Gq signaling to stimulate ERK through PKC-mediated phosphorylation of Raf-1 has been well documented (34), as well as the capacity of Gi to directly activate Ras and then ERK1/2 (35). Thus, theoretically, ERK1/2 activation may be achieved by promiscuous coupling of TP receptor with both PTx-sensitive and -insensitive G proteins.

Our data with the PKC inhibitor GF109203X, as well as with the structurally different inhibitor H7 (data not shown), support a role for PKC in TP-dependent proliferation. In fact, although PKC inhibition substantially reduced thymidine incorporation, only partial inhibition of ERK activation was observed, suggesting a minor involvement of the Gq-PLC-PKC-ERK1/2 pathway and, conversely, a possible involvement of PKC in a different pathway(s) linking TP receptor to the nucleus.

In the light of previous observations by us and others (10, 20) that PTx was able to inhibit U46619-induced mitogenic response, we investigated the role of Gi/0 proteins in TP receptor–mediated ERK1/2 activation and found a total inhibition by PTx, PP1/genistein and L.Tox, indicating a predominant involvement of a Gi/o protein that signals through Src/Ras to activate p42/p44 MAPK. Accordingly, inhibition of PTK and Ras resulted in a substantial reduction of DNA synthesis. Furthermore, to our knowledge this is the first time that the TXA2 mimetics have been demonstrated to increase the amount of GTP-bound p21ras in membranes from HASM cells, clearly indicating the involvement of the small G protein Ras in TP-mediated HASM cell proliferation. These results are in agreement with previous observations that Gi but not Gq is linked to activation of p21ras in HASM cells (36).

In conclusion, this study shows the existence of at least two pathways through which TP receptor can induce G1 progression of HASM cells: (1) PTx-sensitive G proteins such as Gi/o, where both {alpha} and ß{gamma} could potentially activate Src/Ras/ERK and possibly PKC; and (2) PTx-insensitive G proteins such as Gq/11 that can trigger PKC to induce proliferation either directly or through Raf/MEK/ERK and/or other PKC-dependent pathways. Thus, the observations that PKC inhibition produced a substantial reduction in thymidine incorporation and to a lesser extent in ERK1/2 phosphorylation suggest that TP-mediated effects only partially converge at the level of MAPK cascade, hence indicating the contribution of pathways other than MAPKs downstream of TP receptor. The detailed understanding of the contribution of all these interconnected pathways is the object of ongoing investigation in our laboratory.

Collectively, these data improve our understanding of the transduction pathways involved in the proliferative response of HASM cells after TP receptor activation and the general comprehension of the mediators and the mechanisms that regulate the proliferation of airway myocytes; such proliferation can result from recurrent stimulation by contractile, inflammatory mediators, and by growth factors. Because TP receptor may be activated by many inflammatory mediators, these results may suggest new therapeutic approaches to alter the airway remodeling observed in patients with chronic airflow obstruction and airway inflammation, for example asthma, chronic bronchitis, bronchiolitis obliterans, and chronic obstructive pulmonary disease.


   Acknowledgments
 
The authors thank Prof. M.Y. Popoff (Institut Pasteur, Paris, France) for kindly providing the Clostridium sordellii lethal toxin.


   Footnotes
 
This research was partially supported by PRIN 2003 from the Italian Ministry of University and Research, MIUR (to G.E.R.).

Conflict of Interest Statement: S.C. has no declared conflicts of interest; S.R. has no declared conflicts of interest; G.E.R. has no declared conflicts of interest; and V.C. has no declared conflicts of interest.

Received in original form November 15, 2004

Received in final form December 16, 2004


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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