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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 759-765, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.2002-0070OC

p38 Mitogen-Activated Protein Kinase Regulates Growth Factor–Induced Mitogenesis of Rat Pulmonary Myofibroblasts

Annette B. Rice, Jennifer L. Ingram and James C. Bonner

Airway Inflammation Group, Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina

Address correspondence to: James C. Bonner, Ph.D., NIEHS, PO Box 12233, Research Triangle Park, NC 27709. E-mail: bonnerj{at}niehs.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Myofibroblast proliferation is a central feature of pulmonary fibrogenesis. Several growth factors, including platelet-derived growth factor (PDGF) and epidermal growth factor (EGF), stimulate myofibroblast growth by activating extracellular signal regulated kinases 1 and 2 (ERK1/2). In this report, we demonstrate that PDGF-BB and EGF also activate the p38 mitogen-activated protein (MAP) kinase. Inhibition of p38 activity with the pyridinylimidazole compound SB203580 enhanced both PDGF-BB and EGF-stimulated DNA synthesis in rat lung myofibroblasts. ERK1/2 phosphorylation in response to either PDGF-BB or EGF treatment was significantly increased by pretreatment of cells with SB203580. We also demonstrated that ERK1/2-induced phosphorylation of PHAS-1 substrate was enhanced by inhibition of p38 MAP kinase with SB203580. However, SB203580 did not significantly increase growth factor–induced activation of MEK, the upstream kinase that phosphorylates ERK1/2. p38 MAP kinase was co-immunoprecipitated with ERK-1/2 following growth factor stimulation. Collectively, these data demonstrate that p38 MAP kinase activation negatively regulates PDGF- and EGF-mediated growth responses by directly interacting with ERK1/2 and suppressing its phosphorylation.

Abbreviations: Dulbecco's modified Eagle's medium, DMEM • epidermal growth factor, EGF • extracellular signal-regulated kinases 1 and 2, ERK1/2 • fetal bovine serum, FBS • interleukin, IL • jun amino-terminal kinase, JNK • mitogen-activated protein, MAP • MAP kinase kinase, MEK • phosphate-buffered saline, PBS • platelet-derived growth factor, PDGF • phosphorylated heat- and acid stable protein, PHAS • sodium dodecyl sulfate, SDS • serum-free defined medium, SFDM • tumor necrosis factor, TNF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pulmonary fibrosis is characterized by the proliferation of pulmonary myofibroblasts, which are the principal cell type in the lung that deposit collagen to define the fibrotic lesion (1–3). Several polypeptide growth factors, including epidermal growth factor (EGF) ligands and platelet-derived growth factor (PDGF) isoforms, stimulate the proliferation of myofibroblasts in vitro and are elevated during pulmonary fibrogenesis (4–6). The intratracheal instillation of PDGF-BB or overexpression of the PDGF-B gene causes lung fibrosis in vivo (7, 8). Moreover, receptor tyrosine kinase inhibitors selective for either the EGF receptor (EGFR) or PDGF receptor (PDGFR) significantly reduce pulmonary fibrosis in rats in vivo, suggesting that these growth factors are important to the progression of fibroproliferative lung disease (9).

PDGF-BB or EGF stimulate cell growth by binding to the extracellular domains of their respective cell surface receptors. The PDGF receptor system consists of two subunits termed {alpha} and ß which dimerize in the presence of PDGF-BB to form functional {alpha}{alpha}, {alpha}ß, or ßß dimers (10). Four EGF receptors (erbB1, erbB2, erbB3, and erbB4) form homo- or heterodimeric complexes in the presence of EGF or other ligands such as transforming growth factor-{alpha} and HB-EGF (11). The dimerization of these receptors following ligand binding results in the phosphorylation of specific tyrosine residues on the intracellular domain of the receptor, which results in the docking of a variety of signaling proteins. In particular, mitogenesis is mediated by the sequential phosphorylation of Raf and MEK. MEK then phosphorylates mitogen-activated protein (MAP) kinases termed extracellular signal-regulated kinases 1 and 2 (ERK1/2) (12, 13). Activated ERKs translocate to the nucleus, where they phosphorylate a variety of transcription factors involved in cell cycle progression. It is well established that ERK1/2 activation is required for the growth of mesenchymal cells, including myofibroblasts.

Recent studies have demonstrated cross-talk between p38 MAP kinase and ERK (14, 15). Zhang and coworkers demonstrated that phosphorylated p38 MAP kinase is capable of forming a complex with ERK1/2, thereby preventing the phosphorylation of ERK1/2 by MEK (14). p38 MAP kinases are activated by a variety of agents, including environmental stress (e.g., reactive oxygen species, UV radiation), cytokines (e.g., interleukin [IL]-1ß, tumor necrosis factor [TNF]-{alpha}), or growth factors such as EGF and PDGF (16, 17). Several different isozymes of p38 MAP kinase regulate a diversity of functions, including growth inhibition (18), apoptosis (19), cytokine production (20), and growth factor receptor expression (21). A pyridinylimidazole compound, SB203580, is a highly specific inhibitor of p38 MAP kinase (22). In this study, we report that treatment of rat lung myofibroblasts with SB203580 enhanced both PDGF- and EGF-stimulated DNA synthesis. The phosphorylation of ERK1/2, but not MEK, by these growth factors was also significantly enhanced by SB203580. p38 MAP kinase was co-immunoprecipitated with ERK1/2 following growth factor stimulation. These data suggest that p38 MAP kinase negatively regulates PDGF- and EGF-stimulated mitogenesis by a direct physical interaction with ERK1/2.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
SB203580 was purchased from Calbiochem, La Jolla, CA, and PD98059 was purchased from New England Biolabs Inc., Beverly, MA. Recombinant human EGF, and recombinant human PDGF-BB were obtained from Upstate Biotechnologies, Lake Placid, NY. Anti–phospho-p38 MAP kinase and anti-p38 (total) MAP kinase were from Cell Signaling Technologies, Beverly, MA; Anti–phospho-ERK was purchased from New England Biolabs; anti-ERK polyclonal used for detection of total ERK in Western blotting (Santa Cruz Biotechnology, Santa Cruz, CA); anti–ERK-CT used for immunoprecipitation experiments (StressGen Biotechnologies, Victoria, BC, Canada); rabbit anti-goat HRP IgG, and swine anti-rabbit HRP IgG were obtained from Dakopatts, Carpenteria, CA.

Cell Culture
Primary passage rat pulmonary myofibroblasts were isolated from male Sprague-Dawley rats as described previously (23). These cells stain positively for vimentin, desmin, and {alpha}-smooth muscle actin, which indicated a myofibroblast phenotype (3, 23). In addition, examination of glutaraldehyde-fixed cell pellets by transmission electron microscopy showed ultrastructural features consistent with a myofibroblast phenotype (abundant intermediate filaments and rough endoplasmic reticulum, and lack of Weibel-Palade bodies characteristic of endothelial cells). Cells were grown to confluence in 10% fetal bovine serum (FBS)-Dulbecco's modified Eagle's medium (DMEM) before being seeded for the assays described below.

Western Blot Analysis
Cells were grown to a confluent state in 10% FBS-DMEM in 75 cm2 tissue culture dishes, then rendered quiescent for 24 h with serum-free defined medium (SFDM) consisting of Ham's F-12 medium supplemented with 0.25% bovine serum albumin and an insulin/transferrin/selenium mixture (Boehringer Mannheim, Indianapolis, IN). After treating with the agent of interest, the cultures were washed with ice-cold phosphate-buffered saline (PBS), and cell lysates collected by incubation with 250 µl of lysis buffer consisting of 50 mM Tris-HCl (pH 7.4), 1% Triton X-100, 150 mM NaCl, l mM EGTA, 1 mM Na3V04, l mM NaF, l mM PMSF, 0.25% sodium deoxycholate, and 20 µg/ml of each of the following proteinase inhibitors: aprotinin, leupeptin, and pepstatin. Twenty microliters of each sample were mixed with 5 µl sample buffer (0.5 M Tris-HCl pH 6.8, 10% sodium dodecyl sulfate [SDS], 0.1% bromphenol blue, 20% glycerol, and 50 mM 2-mercaptoethanol and separated by SDS-polyacrylamide gel electrophoresis [PAGE] in a 10–20% Tris-glycine gel; Novex, San Diego, CA). The proteins were transferred to Hybond nitrocellulose membrane (Amersham, Arlington Heights, IL). The membrane was blocked for 2 h at room temperature with 5% nonfat milk in TBS-Tween buffer (20 mM Tris, 500 mM NaCl, 0.01% Tween 20). The membranes were incubated with a 1:1,000 dilution of primary phospho-p38 MAP kinase or phospho-ERK antibodies overnight at 4°C. The membranes were washed 3x with PBS-Tween before a 90-min incubation with a 1:1,000 dilution of HRP-swine anti-rabbit IgG or HRP-rabbit anti-goat IgG (Dakopatts) for phospho-p38 and phospho-ERK, respectively. After thoroughly washing in PBS-Tween, the HRP-labeled proteins were visualized with an enhanced chemiluminescence kit (Amersham). Blots were subsequently stripped at 50°C for 30 min in a buffer containing 62.5 mM Tris (pH 6.7), 2% SDS, and 100 mM ß-mercaptomethonal, then reblotted with 1:1,000 dilution of antibodies against total (activated and unactivated) p38 MAP kinase (Cell Signaling) or ERK (Santa Cruz), and developed with a 1:1,000 dilution of anti-rabbit HRP or anti-goat HRP, respectively.

Immunoprecipitation
Cell lysates from growth factor-stimulated myofibroblasts were collected as described above for Western blotting. Lysates were precleared with 20 µl of protein A-agarose beads (Santa Cruz) and then incubated with a 1:25 dilution of anti–ERK-CT antibody (StressGen Biotechnologies) for 2 h at 4°C. Twenty microliters of protein A agarose beads were then added to precipitate immunocomplexes and washed with lysis buffer three times. The immunoprecpitates were analyzed by Western blot analysis with a PhosphoPlus p38 MAP kinase kit (Cell Signaling).

PHAS-1 Kinase Assay
ERK activity in cell lysates was measured as described previously (24) by phosphorylation of PHAS-1, a substrate for ERK1/2 (25). Briefly, confluent rat lung myofibroblasts in 75 cm2 dishes were growth-arrested in SFDM for 24 h, then treated with 10 µg/cm2 of V2O5 or TiO2 for 5 min to 2 h. The cells were placed on ice, then washed twice with PBS, and scraped off with 800 µl of lysate buffer consisting of 50 mM HEPES, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, and 20 µg/ml each of aprotinin, leupeptin, and pepstatin. Lysates were clarified by centrifugation at 13,000 rpm for 10 min, and protein concentrations were determined by Bradford assay. Immunoprecipitation was performed by incubating 200 µl of lysate with 2 µg of anti-ERK antibody for 2 h, then adding 20 µl of protein-A/agarose (Santa Cruz). After an overnight incubation at 0–4°C with end-over-end mixing, the immune complex was recovered by centrifugation, washed three times with lysis buffer and once with 250 mM HEPES (pH 7.4), 10 mM MgCl2, 200 µM Na3VO4. Immune complex kinase assays were performed using a MAP Kinase Assay Kit (Stratagene, La Jolla, CA). The ERK pellets were resuspended in Stratagene reaction buffer containing 120 µg of PHAS-1 substrate along with 3–5 µCi [{gamma}-32P]ATP in a final volume of 180 µl. Kinase reactions took place for 30 min at RT and were stopped by adding 4x SDS-PAGE reducing sample buffer and boiling for 10 min. ERK-PHAS samples were resolved on 4–20% PAGE gels, dried, and autoradiographed.

[3H]Thymidine Incorporation Assay
Cells were grown to confluence with 10% FBS-DMEM in 24-well tissue culture plates (2 cm2 wells) and then rendered quiescent for 24 h with SFDM containing 0.5% FBS. The cells were pretreated with fresh 0.5% FBS-SFDM containing SB203580 (p38 inhibitor) or PD98059 (MEK inhibitor) in DMSO, or DMSO alone (vehicle control) for 1 h at 37°C, then recombinant human PDGF-BB or EGF was spiked into the medium along with 5 µCi/ml [3H]thymidine (Amersham) for 24 h. The cells were washed with Ham's F-12 at 25°C, placed on ice, and incubated with 0.5 ml/well 5% trichloroacetic acid for 10 min. After washing 3x with ice-cold distilled water, solubilization was performed with 0.5 ml/well 0.2 N NaOH containing 0.1% SDS for 30 min on an oscillating platform. Each sample (100 µl) was added to 1 ml of Ecolume (Costa Mesa, CA) and radioactivity measured on a liquid scintillation counter.

Statistical Analysis
Statistical analysis was performed by ANOVA and two-sample t-tests. A P value of < 0.05 was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Inhibition of p38 MAP Kinase Enhances Growth Factor–Induced Mitogenesis of Rat Lung Myofibroblasts
Confluent, quiescent cultures of myofibroblasts were pretreated with inhibitors of the p38 MAP kinase pathway (SB203580) or an inhibitor of the MEK–ERK pathway (PD98059) before measuring PDGF- or EGF-stimulated mitogenesis by [3H]thymidine incorporation. It is well known that activation of the MEK–ERK pathway is required for growth factor–induced mitogenesis (12), and our experiments confirmed that PD98059 blocked either PDGF- or EGF-induced [3H]thymidine uptake (Figure 1). Pretreatment of myofibroblasts with SB203580 enhanced PDGF- or EGF-stimulated mitogenesis, indicating that p38 MAP kinase has an anti-mitogenic effect on growth factor–induced myofibroblast growth (Figure 1). SB203580 treatment in the absence of growth factors also resulted in increased basal [3H]thymidine incorporation in quiescent myofibroblasts. This could be due to the enhancing effect of SB203580 on endogenous growth factors produced in an autocrine manner, or the enhancing effect of SB203580 on low serum (0.5% FBS) that was used to supplement the medium. It is noteworthy that a similar growth–enhancing effect of SB203580 was observed using normal human lung fibroblasts (data not shown), indicating that the p38 MAP kinase regulatory mechanism was not unique to rat mesenchymal cells and that p38-mediated suppression of growth occurred in cells that possessed either a myofibroblast or fibroblast phenotype.



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  		Figure 1. Mitogenesis of rat lung myofibroblasts is enhanced following treatment with p38 MAP kinase inhibitor, SB203580. Confluent, quiescent cell monolayers were pretreated with 10 µM SB203580 for 1 h before the addition of 5 mCi [3H]thymidine and an increasing concentration of PDGF (A) or EGF (B). [3H]thymidine uptake was measured as described in MATERIALS AND METHODS. The data are the mean ± SEM of three separate experiments, each performed in quadruplicate. Circles, control; triangles, SB203580; squares, PD98059.

 
PDGF and EGF Activate p38 MAP Kinase in Rat Lung Myofibroblasts
To determine the time-course of ERK and p38 MAP kinase activation, myofibroblasts were treated with PDGF or EGF and cell lysates collected at various time points after treatment. Western blotting was then performed using antibodies specific for the phosphorylated forms of ERK or p38 MAP kinase. After blotting with phospho-antibodies, the membranes were stripped and reblotted with polyclonal antibodies that recognized total ERK or p38 protein. ERK was activated by either PDGF or EGF within 5 min and remained activated for as long as an hour after treatment (Figure 2). p38 MAP kinase was also activated rapidly (within 5 min) but dephosphorylated within 30 min following treatment with growth factors (Figure 2). In all further experiments, the activation of ERK (or MEK) was measured at 10 and 30 min after treatment with growth factors.



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  		Figure 2. Time course of ERK and p38 MAP kinase activation in rat pulmonary myofibroblasts. Confluent, quiescent cultures of myofibroblasts were treated with 50 ng/ml PDGF or EGF and cell lysates were harvested at the indicated time points for Western blotting using antibodies specific for phosphorylated p38 MAP kinase (phospho-p38) or total p38 MAP kinase protein (p38). The data are from a single experiment typical of three separate experiments with similar results.

 
Inhibition of p38 MAP Kinase Enhances ERK Activation in Rat Lung Myofibroblasts
Next we evaluated the effect of p38 MAP kinase inhibition on growth factor–induced phosphorylation of ERK. Cells were pretreated with 10 µM SB203580 or DMSO vehicle as a control, then stimulated with PDGF or EGF. Pretreatment with SB203580 significantly enhanced PDGF- or EGF-stimulated ERK1/2 phosphorylation (Figure 3). Another p38 MAP kinase inhibitor (SB22025) that is structurally distinct from SB203580 also enhanced PDGF- and EGF-stimulated ERK phosphorylation and growth (data not shown). Pretreatment of cells with 40 µM MEK inhibitor (PD98059) significantly inhibited PDGF- or EGF-stimulated ERK phosphorylation (data not shown). These changes in ERK1/2 phosphorylation mirrored the enhancement of growth factor–induced mitogenesis by SB203580 and inhibition of growth factor–induced mitogenesis by PD98059 (Figure 1). To verify that enhanced phosphorylation of ERK resulted in enhanced kinase activity, we employed a PHAS-1 assay (24). Cells were pretreated with SB203580 or DMSO vehicle, then stimulated with or without PDGF before immunoprecipitation of ERK1/2 using a polyclonal anti-ERK antibody. The immunoprecipitated ERK1/2 was then incubated with PHAS-1 as a substrate in the presence of 32P-ATP. PDGF caused an increase in ERK1/2-induced phosphorylation of PHAS-1, and SB203580 enhanced PDGF-induced PHAS-1 activation several fold (Figure 4).



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  		Figure 3. p38 MAPK inhibitor (SB203580) enhances PDGF- or EGF-stimulated ERK1/2 phosphorylation. Confluent, quiescent myofibroblasts were pretreated with 10 µM SB203580 for 1 h before and then treated with PDGF-BB (50 ng/ml) or EGF (50 ng/ml) for the indicated time points before collecting cell lysates. Western blotting was performed using antibodies against phosphorylated ERK1/2 (p-ERK) or total ERK1/2 protein (ERK) as described in MATERIALS AND METHODS. For each experiment, p-ERK1/2 was measured first, then the blots were stripped and reblotted for total ERK1/2 protein. (A) Representative western blots showing p-ERK (upper panel) or ERK (middle panel) after pretreatment with SB203580 (+SB; striped bars) or with vehicle control (-SB; open bars), then stimulation with PDGF-BB. The lower panel shows the results of scanning densitometry from three separate experiments, including the representative Western blots. Data are expressed as the mean ± SEM. The p-ERK1/2 signal was normalized against the total ERK1/2 signal for each separate experiment. (B) Same as A, except cells were treated with EGF after SB pretreatment.

 


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  		Figure 4. Inhibition of p38 MAP kinase enhances ERK1/2-induced phosphorylation of PHAS-1. Confluent, quiescent rat lung myofibroblasts were pretreated for 1 h with 10 µM SB203580 or DMSO vehicle, then treated with 50 ng/ml PDGF-BB or medium alone as a control for the indicated time points. ERK1/2 was immunoprecipited from cell lysates as described in MATERIALS AND METHODS, and a MAP kinase assay was performed that assayed phosphorylation of the PHAS-1 substrate by activated ERK1/2.

 
Inhibition of p38 MAP Kinase Does Not Affect PDGF- or EGF-Induced Activation of MEK
We next determined whether or not p38 MAP kinase inhibition would affect the phosphorylation of MEK, the upstream kinase that phosphorylates ERK1/2. Pretreatment of myofibroblasts with SB203580 did not significantly affect PDGF- or EGF-induced phosphorylation of MEK (Figure 5).



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  		Figure 5. p38 MAPK inhibitor (SB203580) does not affect PDGF- or EGF-stimulated MEK phosphorylation. Confluent, quiescent myofibroblasts were pretreated with 10 µM SB203580 for 1 h before and then treated with PDGF-BB (50 ng/ml) or EGF (50 ng/ml) for the indicated time points before collecting cell lysates. Western blotting was performed using antibodies against phosphorylated MEK-1 (p-MEK) or total MEK-1 protein (MEK) as described in MATERIALS AND METHODS. For each experiment, p-MEK was measured first, then the blots were stripped and reblotted for MEK. (A) Representative western blots showing p-MEK (upper panel) or MEK (middle panel) after pretreatment with SB203580 (+SB; striped bars) or with vehicle control (-SB; open bars), then stimulation with PDGF-BB. The lower panel shows the results of scanning densitometry from three separate experiments, including the representative Western blots. Data are expressed as the mean ± SEM. The p-MEK signal was normalized against the total MEK signal for each separate experiment. (B) Same as A, except cells were treated with EGF after SB pretreatment.

 
Activated p38 MAP Kinase is Co-immunoprecipitated with ERK
To determine whether p38 MAP kinase was directly or indirectly suppressing ERK1/2 activity, we immunoprecipitated ERK from cell lysates obtained from growth factor–stimulated myofibroblasts using a anti–ERK-CT antibody, then performed Western blot analysis for p38 MAP kinase. p38 MAP kinase was not co-immunoprecipitated with ERK in cell lysates from myofibroblasts that were not treated with PDGF. However, p38 MAP kinase was co-immunoprecipated with ERK after 5 or 10 min of stimulation with growth factor (Figure 6). Activation of p38 MAP kinase by anisomycin also induced co-immunoprecipitation, as visualized by Western blotting with anti-ERK antibody (data not shown).



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  		Figure 6. p38 MAP kinase is co-immunoprecipitated with ERK following growth factor stimulation. Confluent, quiescent cultures of rat lung myofibroblasts were treated with PDGF-BB (50 ng/ml) for the indicated time points and immunoprecipitation of ERK was performed as described in MATERIALS AND METHODS. Western blotting was then performed using antibodies against total (phosphorylated and unphosphorylated) ERK-2 (upper panel) or total p38 MAP kinase (lower panel). p38 MAP kinase was not co-immunoprecipitated with ERK in lysates from untreated cells, but p38 MAP kinase was co-immunoprecipitated in lysates from PDGF-treated cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
ERKs are the principal MAP kinases that mediate cellular growth in response to polypeptide growth factors. In this study, we have shown that p38 MAP kinase and ERK1/2 are co-activated by growth factors (EGF and PDGF-BB), and that inhibition of p38 MAP kinase by the pyridinylimidazole SB203580 enhances growth factor–induced mitogenesis. SB203580 also increased basal [3H]thymidine uptake in quiescent cells that received no exogenous growth factors, and this was presumably due to the production of low levels of endogenous growth factors, such as PDGF-AA (26), which cause some basal stimulation of [3H]thymidine uptake via an autocrine mechanism. We further demonstrated that growth factor–induced ERK phosphorylation and ERK kinase activity were enhanced by SB203580. However, inhibition of p38 MAP kinase did not affect growth factor–induced phosphorylation of MEK, the upstream activator of ERK1/2. Finally, p38 MAP kinase was co-immunoprecpitated with ERK using an anti-ERK antibody in lysates from growth factor–stimulated myofibroblasts. Collectively, these data suggest that phosphorylated p38 MAP kinase interacts with ERK1/2 to suppress its activity. To our knowledge, this is the first report to show enhanced growth factor–induced mitogenesis through inhibition of p38 MAP kinase.

Other investigators have reported interaction (i.e., cross-talk) between p38 MAP kinase and ERK1/2. Singh and coworkers reported that SB202190 (another specific inhibitor of p38 MAP kinase), induced low-density lipoprotein receptor expression by increasing ERK1/2 activation (15). Therefore, they suggested that p38 MAP kinase negatively regulated the ERK1/2 signaling cascade. More recently, Zhang and coworkers demonstrated for the first time a direct physical interaction between p38 MAP kinase and ERK1/2 (14). They reported that phosphorylated p38{alpha} was capable of forming a complex with ERK1/2, and this correlated with inhibition of ERK1/2 phosphotransferase activity. Finally, these investigators concluded that p38{alpha} may sequester ERK1/2 and sterically block ERK1/2 phosphorylation by MEK. Our findings also demonstrated direct cross-talk between ERK1/2 and p38 MAP kinase, because (i) we showed that SB203580 enhanced the activation of ERK1/2, but not MEK, and (ii) we co-immunoprecipitated activated p38 MAP kinase with an antibody raised against total (phosphorylated and unphosphorylated) ERK. In our study, we could not confirm whether one-way cross-talk or two-way cross-talk exists between ERK and p38 MAPK, as PDGF and EGF activated both ERK1/2 and p38 MAPK. However, growth factor–induced p38 MAPK phosphorylation occurred relatively early and was maximal within 5 min of stimulation, compared with ERK activation, which remained sustained for as long as an hour after stimulation with growth factors. These data suggest that growth factor–activated p38 MAPK would be phosphorylated first to bind ERK. Moreover, we observed that a p38 MAP kinase antibody co-immunoprecipitated ERK in cells treated with anisomycin, which is consistent with the findings of Zhang and coworkers, who demonstrated one-way cross-talk between p38 MAPK and ERK (14).

ERK-independent mechanisms could also contribute to suppression of growth factor–induced mitogenesis by p38 MAP kinase. Page and colleagues reported that p38 MAP kinase negatively regulates PDGF-induced transcription from the cyclin D1 promoter in airway smooth muscle cells via an ERK-independent pathway (27). They proposed that negative regulation of cyclin D1 by p38 would attentuate cell cycle traversal, although they did not measure the net effect of p38 activation on airway smooth muscle cell DNA synthesis and proliferation. They also showed that p38 inhibitors (SB203580 and SB202190) increased PDGF-induced cyclin D1 promoter activity and protein abundance. Moreover, p38 inhibitors increased PDGF-induced ERK activation, yet dominant-negative MKK3 (the upstream activator of p38{alpha}), was insufficient to activate ERK (27). Finally, they suggested that pyridinylimidazole compounds (SB203580 and SB202190) might activate ERK via a MEK-dependent pathway. In our study, we clearly demonstrated that MEK was not phosphorylated by the addition of SB203580 in the absence of growth factors (Figure 5). Moreover, MEK phosphorylation after growth factor treatment was not affected by SB203580 (Figure 5), indicating that the enhancement of growth factor–induced ERK activation by SB203580 is MEK-independent.

Several studies have shown that p38 MAP kinase inhibitors block the production of proinflammatory cytokines such as TNF-{alpha}, IL-6, and IL-1ß (28, 29). Therefore, it has been suggested that pyridinylimidazole compounds such as SB203580 could be used for therapeutic intervention of inflammatory lung diseases such as asthma or pulmonary fibrosis (30). Although these compounds clearly have anti-inflammatory properties, our data suggest that they could also enhance the proliferation of myofibroblasts in vivo and could potentially excacerbate a fibrogenic response. Nevertheless, a recent study by Matsuoka and coworkers showed that the p38 MAPK inhibitor, FR-167653, ameliorated bleomycin-induced pulmonary fibrosis in mice and this was likely due to the suppression of p38-induced profibrotic cytokines such as TNF-{alpha} (31). Therefore, in vivo it appears that the beneficial effect of p38 MAPK inhibitors in suppressing profibrotic cytokines could outweigh the detrimental effect of p38 MAPK inhibitors in enhancing growth factor–stimulated myofibroblast proliferation.

In summary, we have shown that the p38 MAP kinase inhibitor, SB203580, increased both PDGF- and EGF-stimulated mitogenesis in rat pulmonary myofibroblasts in vitro. Both PDGF and EGF induced transient phosphorylation of p38 MAP kinase that coincided with ERK1/2 phosphorylation. The enhanced growth factor–induced mitogenic response caused by SB203580 was correlated with increased phosphorylation and activation of ERK1/2, but no increase in upstream MEK activity was observed. Importantly, we demonstrated that phosphorylated p38 MAP kinase was co-immunoprecipitated with ERK in growth factor–stimulated myofibroblasts. Our findings support the hypothesis that cross-talk exists between p38 MAP kinase and ERK, and that p38 MAP kinase activation suppresses growth factor–induced mitogenesis by inhibiting the phosphorylation of ERK1/2.


   Acknowledgments
 
The authors thank Dr. John O'Bryan and Dr. John Roberts at the NIEHS for helpful comments during the preparation of this manuscript.

Received in original form May 17, 2002

Received in final form July 16, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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