INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Bronchial asthma is a disease that is characterized by episodic reversible airway obstruction, airway hyperresponsiveness, and allergic inflammation in the airway (1). The pathogenesis of allergic inflammation is complex and involves multiple inflammatory cells, cytokines, and mediators (2, 3). A variety of cells produce cytokines that participate in the production of airway inflammation of asthmatic airways (2, 3). Airway smooth muscle (ASM) cells, which have been regarded as having contractile properties in response to contractile inflammatory mediators (4), may also participate in airway inflammatory response by expressing various cytokines (5).

Many extracellular stimuli elicit specific biologic responses through activation of mitogen-activated protein (MAP) kinase cascades (8). The mammalian MAP kinase superfamily has been molecularly characterized: extracellular signal-regulated kinase (Erk), p38 MAP kinase, and c-Jun-NH₂-terminal kinase (JNK). p38 MAP kinase is activated by environmental stresses such as hyperosmotic shock, heat shock, cold shock, UV irradiation, and inflammatory cytokines, and it plays an important role in apoptosis and cytokine expression (9). Erk is activated by mitogenic stimuli and plays a central role in cell proliferation and differentiation (15, 16). In addition, recent studies have suggested that Erk also plays an important role in the signal cascade of induction of various inflammatory mediators, including cytokines and chemical mediators (17).

Phosphorylation and catalytic activation of Erk has implicated the signal-promoting proliferation of ASM cells (21, 22), and it has been proposed that ASM cells act as effector cells in the production of airway inflammation of asthmatics by expressing cytokines (5). However, the intracellular signal that regulates cytokine expression in ASM cells has not been determined.

We used platelet-activating factor (PAF) and tumor necrosis factor (TNF)- as inducers for cytokine production by ASM cells. PAF causes ASM contraction (23); however, it has not been examined whether PAF could stimulate ASM cells to produce cytokines. TNF- has been reported to stimulate ASM cells to produce cytokines (5, 7). In the allergic inflammation of asthmatic airway, eosinophils play a pivotal role in the production of allergic inflammation (2, 3). RANTES is a member of the C-C chemokine family and has been shown to be involved in the production of allergic inflammation through its chemotactic activity for eosinophils (2, 3). RANTES is known to be produced by a variety of cells, including ASM cells (5, 6). Consequently, we measured the concentrations of RANTES.

In the present study, therefore, we attempted to examine the role of p38 MAP kinase and Erk in RANTES production by PAF-stimulated and TNF--stimulated ASM cells. To this end, we examined phosphorylation and activation of these kinase, the effect of SB 203580 as the specific inhibitor for p38 MAP kinase activity (24) and p38 MAP kinase activity and RANTES production, and the effect of PD 98059 as the specific inhibitor for MAP kinase-1 (MEK-1) on Erk activity (25) and RANTES production in PAF- and TNF--stimulated human ASM cells.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Reagents

PAF and platelet-derived growth factor-BB (PDGF) were obtained from Sigma Chemical Co. (St. Louis, MO) and R&D Systems (Minneapolis, MN), respectively. TNF- was kindly provided by Dainippon Pharmaceutical Ltd. (Osaka, Japan). SB 203580 as the specific inhibitor for p38 MAP kinase activity was kindly provided by SmithKline Beecham (Brockham Park, Betchworth, Surrey, UK). PD 98059 as the specific inhibitor for MEK-1 was obtained from New England BioLabs Inc. (Beverly, MA). SB 203580 and PD 98059 were dissolved in dimethyl sulfoxide.

Cell Culture

Human bronchial smooth muscle (BSM) cells derived from normal healthy subjects used as ASM cells in this study were obtained from Clonetics (San Diego, CA). The cells (1 × 10⁴ cells/ml) were placed onto 24-well flat-bottomed tissue culture plate (Corning, Corning, NY) for cytokine production, tissue culture plate (Falcon 1007, Oxnard, CA) for western blot and in vitro kinase assay, and 96-well flat-bottomed tissue culture plate (Corning) for [³H]thymidine incorporation using BSM cell growth medium (SmBM; Clonetics) containing 5% fetal bovine serum (FBS), gentamycin-amphotericin B, epidermal growth factor (EGF), fibroblast growth factor (FGF) and dexamethasone (DEX). In order to examine cytokine production, phosphorylation and activation of p38 MAP and Erk, and [³H]thymidine incorporation in ASM cells, ASM cells were allowed to reach subconfluence, the culture medium was replaced with SmBM without FBS, EGF, FGF, or DEX (growth-factor-free SmBM) and the cells were cultured for 16 h. In order to examine the effect of SB 203580 and PD 98059 on p38 MAP kinase and Erk activity, and cytokine production, the cells that had been preincubated with SB 203580 (10 µM), PD98059 (50 µM), or a combination of SB 203580 and PD 98059 for 1 h were stimulated and cultured in growth-factor-free SmBM for the desired times as indicated. ASM cells at passage numbers 3 to 5 were used for experiments. At confluence of each passage, using immunofluorescence technique for both smooth muscle actin and myosin, more than 95% of the cells displayed the characteristics of smooth muscle cells in culture.

Western Blot Analysis of p38 MAP Kinase, Erk, and JNK

The threonine and tyrosine phosphorylation of p38 MAP kinase was analyzed by commercially available kits (PhosphoPlus p38 MAPK Antibody Kit; New England BioLabs). Analysis of threonine and tyrosine phosphorylation of p38 MAP kinase was performed using an antiphosphorylated threonine and tyrosine of p38 MAP kinase antibody, which is specific for active p38 MAP kinase and does not cross-react with Erk and JNK. Analysis of threonine and tyrosine phosphorylation of Erk was performed using an antiphosphorylated threonine and tyrosine of p42/p44 MAP kinase antibody (anti-phospho-specific p42/p44 MAP kinase antibody; New England BioLabs), which is specific for active p42/p44 MAP kinase and does not cross-react with p38 MAP kinase and JNK. Analysis of threonine and tyrosine phosphorylation of JNK was performed using an antiphosphorylated threonine and tyrosine of JNK antibody (anti-phospho-specific JNK antibody; New England BioLabs), which is specific for active JNK and does not cross-react with p38 MAP kinase and Erk. Analysis of p38 MAP kinase, Erk, and JNK was performed according to manufacturer's instructions as described previously (13). Briefly, after separating proteins from cell lysate by 15% SDS-PAGE, the cell lysate containing 10 µg of protein was electrophoretically transferred to membrane and the membrane was incubated with specific antibody to phosphorylated threonine and tyrosine of p38 MAP kinase (affinity-purified rabbit polyclonal IgG), specific antibody to phosphorylated threonine and tyrosine of Erk (affinity-purified rabbit polyclonal IgG), or specific antibody to phosphorylated threonine and tyrosine of JNK (affinity-purified rabbit polyclonal IgG) for analysis of JNK, and then it was incubated with the horseradish peroxidase (HRP)-conjugated antirabbit antibody (1:2,000) and horseradish-peroxidase-conjugated antibiotin antibody (1:1,000) to detect biotinylated protein markers. Blots were incubated with enhanced chemiluminescence solution (LumiGLO) for 1 min and exposed on Kodak XAR film. In order to show the amounts of p38 MAP kinase, Erk, and JNK precipitated, blots were stripped and reprobed using phosphorylation-state independent p38 MAP kinase-specific antibody (affinity-purified rabbit polyclonal IgG) to determine total p38 MAP kinase levels, phosphorylation-state independent p42/p44 MAP kinase-specific antibody (affinity purified rabbit polyclonal IgG) to determine total p42/p44 MAP kinase levels or phosphorylation-state independent JNK-specific antibody (affinity-purified rabbit polyclonal IgG) to determine total JNK levels, respectively.

p38 MAP Kinase and Erk Kinase Assay

The activity of p38 MAP kinase was analyzed by commercially available kits (p38 MAP Kinase Assay Kit; New England BioLabs). The kit employs two different antibodies, anti-p38 MAP kinase antibody, which is specific for p38 MAP kinase and does not cross-react with ERK1/2 or JNK, and anti-phospho-specific ATF-2 antibody to detect p38 MAP kinase-induced phosphorylation of ATF-2. p38 MAP kinase activity was analyzed by a specific immunoprecipitation with anti-phospho-specific p38 MAP kinase antibody followed by an in vitro kinase assay of its substrate, ATF-2, according to the manufacturer's instruction as described previously (13). The activity of Erk was analyzed by commercially available kits (MAP Kinase Assay Kit; New England BioLabs). The kit employs two different antibodies, anti-phospho-specific p42/p44 MAP kinase antibody, which is specific for active p42/ p44 MAP kinase and does not cross-react with p38 MAP kinase or JNK, and anti-phospho-specific Elk-1 antibody to detect p42/p44-induced phosphorylation of Elk-1. Erk activity was analyzed by a specific immunoprecipitation with anti-phospho-specific p42/p44 kinase antibody followed by an in vitro kinase assay of its substrate, Elk-1, according to manufacturer's instructions as described previously (26). Briefly, the cell lysate containing 200 µg of protein was incubated with anti-p38 MAP kinase antibody to selectively immunoprecipitate p38 MAP kinase or anti-phospho-specific p42/p44 MAP kinase antibody to selectively immunoprecipitate active p42/p44 MAP kinase from cell lysates, and the immunoprecipitates were incubated with ATF-2 fusion protein or Elk-1 fusion protein in the presence of ATP, a process that allowed immunoprecipitated active p38 MAP kinase to phosphorylate its substrate, ATF-2, and immunoprecipitated active p42/p44 to phosphorylate its substrate, Elk-1. The samples were separated by a 15% SDS-PAGE, transferred to membranes, and blotted with anti-phospho-specific ATF-2 antibody or anti-phospho-specific Elk-1 antibody. The membrane was incubated with HRP-conjugated antirabbit antibody (1:2,000) and HRP-conjugated antibiotin antibody (1:2,000), and then the membrane was incubated with 10 ml of Enhanced Chemiluminescence (ECL) solution and exposed on Kodak XAR film for 1 min.

Measurement of RANTES

The concentrations of RANTES in the culture supernatants from ASM cells were measured by commercially available ELISA kits (Amersham International, Aylesbury, UK). ELISA was performed according to the manufacturer's instructions. All samples were assayed in duplicate.

DNA Synthesis

DNA synthesis of ASM cells was measured by incorporation of [³H]thymidine. ASM cells that had been preincubated with or without PD 98059 (50 µM) for 1 h were stimulated with PAF or PDGF and cultured in growth factor free medium at 37° C in humidified 5% CO₂ atmosphere for 24 h. [³H]thymidine was added to each well for the last 4 h of the incubation period. After incubation, the cells were harvested onto glass fiber strips with a cell harvester and retained radioactivity was counted in a scintillation counter.

Statistical Analysis

Statistical significance was analyzed by using analysis of variance (ANOVA). A p value less than 0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

PAF Induces RANTES Production

First, we examined a dose-dependent induction of RANTES production by ASM cells. To this end, the culture supernatants from ASM cells stimulated with various concentrations of PAF were harvested at 24 h after cultivation. The concentrations of RANTES in the culture supernatants from PAF-stimulated culture increased in a dose-dependent manner (Figure 1a). The concentrations of RANTES in the culture supernatants from ASM cells stimulated with 1 µM of PAF at 12, 24, and 48 h after cultivation were determined. The concentrations of RANTES in the culture supernatants from PAF-stimulated culture were maximal at 24 h after cultivation (Figure 1b).

View larger version (14K): [in this window] [in a new window]   Figure 1.   PAF induces RANTES production. ASM cells were culture either with medium or with various concentrations of PAF (0.01, 0.1, and 1.0 µM) and the concentrations of RANTES in the culture supernatants were determined at 24 h after cultivation (a). ASM cells were cultured either with medium (closed circles) or PAF (1 µM) (closed squares) and the concentrations of RANTES in the culture supernatants were determined at 12, 24, and 48 h after cultivation (b). The results are expressed as the mean ± SD of five different experiments. *1: p < 0.01 compared with RANTES concentrations in ASM cells cultured with medium. *2: p < 0.01 compared with RANTES concentration in ASM cells at 12 h.

PAF Induces Phosphorylation of p38 MAP Kinase and Erk

To determine whether PAF could induce the threonine and tyrosine phosphorylation of p38 MAP kinase and Erk, ASM cells were stimulated with PAF for the desired times, and p38 MAP kinase and Erk were immunoblotted. Amounts of phosphorylated threonine and tyrosine of p38 MAP kinase in PAF-stimulated cells increased at 5 min, sustained between 10 and 30 min, and then returned to near basal levels at 60 min (Figure 2a, upper panel). Amounts of phosphorylated threonine and tyrosine of Erk in PAF-stimulated cells increased at 10 min, sustained between 15 and 30 min, and then returned to near basal levels at 60 min (Figure 2b, upper panel). Lower panel of Figure 2a shows that equal amounts of p38 MAP kinase protein were immunoblotted with p38 MAP kinase-specific antibody regardless of time of culture periods, indicating that PAF-stimulation-induced increases in the threonine and tyrosine phosphorylation of p38 MAP kinase occurred in the absence of changes in p38 MAP kinase protein levels. The lower panel of Figure 2b shows that equal amounts of Erk protein were immunoblotted with Erk kinase-specific antibody regardless of time of culture periods, indicating that PAF-stimulation-induced increases in the threonine and tyrosine phosphorylation of Erk occurred in the absence of changes in Erk protein levels.

View larger version (45K): [in this window] [in a new window]   Figure 2.   PAF induces the threonine and tyrosine phosphorylation of p38 MAP kinase and Erk. ASM cells were stimulated with PAF (1 µM) for the desired times as indicated. The lysates from ASM cells were separated by a 15% SDS-PAGE, transferred to membranes, and blotted either with a specific antibody to phosphorylated threonine and tyrosine of p38 MAP kinase (phospho-p38 MAPK; upper panel of [a]) or a specific antibody to phosphorylated threonine and tyrosine of Erk (phospho-Erk; upper panel of [b]). Blots shown in the upper panel of (a) were stripped and reprobed using a phosphorylation state-independent p38 MAP kinase-specific antibody to show the amounts of p38 MAP kinase blotted (p38 MAPK; lower panel of [a]). Lane P of (a) represents positive protein prepared from C-6 glioma cells stimulated with anisomycin for phosphorylated threonine and tyrosine of p38 MAP kinase. Lane N of (a) represents negative protein prepared from C-6 glioma cells unstimulated with anisomycin. Blots shown in the upper panel of (b) were stripped and reprobed using a phosphorylation state-independent Erk-specific antibody to show the amounts of Erk blotted (Erk: lower panel of [b]). Lane P of (b) represents phosphorylated Erk control protein for positive control (New England BioLabs, Inc.). Lane N of (b) represents nonphosphorylated Erk control protein for negative control (New England BioLabs, Inc.). Blots are representatives of three identical experiments independently performed. The amounts of phosphorylated p38 MAP kinase and Erk proteins were quantitated by NIH image analyzer and are presented as the amounts of phosphorylated p38 MAP kinase and Erk proteins relative to control cells treated without agonist (1.0). Fold increase in amounts of phosphorylated p38 MAP kinase and Erk proteins as indicated below are expressed as the mean ± SD in three different experiments.

PAF Induces p38 MAP Kinase and Erk Activity

Activation of p38 MAP kinase and Erk is mediated by dual phosphorylation of the threonine and tyrosine residues of p38 MAP kinase and Erk, respectively (8, 12). Increases in the threonine and tyrosine phosphorylation of p38 MAP kinase and Erk in PAF-stimulated ASM cells shown in Figure 2 reflect activation of p38 MAP kinase and Erk. In addition to analysis of the threonine and tyrosine phosphorylation of p38 MAP kinase and Erk, we next examined whether PAF could induce p38 MAP kinase activity and Erk activity, and the effect of SB 20380 and p38 MAP kinase activity and the effect of PD 98059 on Erk activity in PAF-stimulated ASM cells. p38 MAP kinase activity was analyzed by a specific immunoprecipitation with anti-p38 MAP kinase antibody followed by an in vitro kinase assay of its substrate, ATF-2. PAF induced p38 MAP kinase activity as demonstrated by the increased phosphorylation of its substrate, ATF-2, and pretreatment of the cells with SB 203580 attenuated PAF-induced increases in p38 MAP kinase activity (Figure 3a). Erk activity was analyzed by a specific immunoprecipitation with anti-phospho-specific p42/ p44 MAP kinase antibody followed by an in vitro kinase assay of its substrate, Elk-1. PAF induced Erk activity as demonstrated by the increased phosphorylation of its substrate, Elk-1. Pretreatment of the cells with PD 98059 attenuated PAF-induced increases in Erk activity (Figure 3b).

View larger version (34K): [in this window] [in a new window]   Figure 3.   PAF induces p38 MAP kinase and Erk activity. ASM cells that had been preincubated either with medium or with SB 203580 (10 µM) as the specific inhibitor for p38 MAP kinase activity for 1 h were stimulated with PAF (1 µM) for 10 min. p38 MAP kinase activity was analyzed by a specific immunoprecipitation with anti-p38 MAP kinase antibody followed by an in vitro kinase assay of its substrate, ATF-2 (a). The cells were cultured with medium (lane 1), SB 203580 (lane 2), PAF (lane 3), and PAF and SB 203580 (lane 4). Lane P of (a) represents positive protein prepared from C-6 glioma cells stimulated with anisomycin for phosphorylated threonine and tyrosine of p38 MAP kinase. ASM cells that had been preincubated either with medium or with PD 98059 (50 µM) as the specific inhibitor for MEK-1 for 1 h were stimulated with PAF for 10 min. Erk activity was analyzed by a specific immunoprecipitation with anti-phospho-specific p42/p44 MAP kinase antibody followed by an in vitro kinase assay of its substrate, Elk-1 (b). The cells were cultured with medium (lane 1), PD 98059 (lane 2), PAF (lane 3), and PAF and PD 98059 (lane 4). Lane P represents positive protein prepared from E. coli expressing active p42/p44 MAP kinase by coexpression with a constitutively active form of its activator, MEK-2. The concentrations of dimethyl sulfoxide used in this study were 0.01%, which had no effect. Blots are representatives of three identical experiments independently performed. The amounts of phosphorylated ATF-2 and Elk-1 proteins were quantitated by NIH image analyzer and are presented as the amounts of phosphorylated p38 MAP kinase and Erk proteins relative to control cells treated without agonist (1.0). Fold increase in amounts of phosphorylated ATF-2 and Elk-1 proteins as indicated below are expressed as the mean ± SD in three different experiments.

SB 203580 and PD 98059 Partially Inhibit PAF-induced RANTES and IL-8 Production

PAF induced RANTES production and p38 MAP kinase and Erk activity in ASM cells. These results suggested that PAF-stimulation-induced RANTES production might be mediated through p38 MAP kinase- and Erk-dependent pathways. To test this possibility, ASM cells that had been preincubated with SB 203580, PD 98059, or a combination of both were stimulated with PAF, and the concentrations of RANTES in the culture supernatants were determined at 24 h after cultivation. Preincubation of the cells with SB 203580 (1 to 10 µM) for 1 h demonstrated a concentration-dependent inhibition of RANTES production in response to stimulation with PAF, although a complete inhibition was not seen in any experiment (Figure 4a). PD 98059 (5 to 50 µM) also inhibited PAF-induced RANTES in a concentration-dependent manner, although a compete inhibition was not seen in any experiments (Figure 4b). ASM cells were preincubated with a combination of SB 203580 (10 µM) and PD 98059 (50 µM). SB 203580 and PD 98059 acted additively to inhibit RANTES and IL-8 production (Figure 5), but the inhibition of RANTES and IL-8 production by these inhibitors was not complete.

View larger version (12K): [in this window] [in a new window]   Figure 4.   SB 203580 and PD 98059 partially inhibit PAF-induced RANTES production. ASM cells that had been preincubated with either medium or various concentrations of SB 203580 for 1 h were cultured either with medium (closed circles) or with PAF (1 µM) (closed squares) (a). ASM cells that had been preincubated with either medium or various concentrations of PD 98059 for 1 h were cultured either with medium (open circles) or PAF (1 µM) (open squares) (b). The concentrations of RANTES in the culture supernatants were determined at 24 h after cultivation as described in METHODS. 1 µM, 5 µM, and 10 µM of SB 203580 contained 0.001%, 0.005%, and 0.01% of dimethyl sulfoxide, respectively. 1 µM, 10 µM, and 50 µM of PD 98059 contained 0.0002%, 0.002%, and 0.01% of dimethyl sulfoxide, respectively. These concentrations of dimethyl sulfoxide had no effect on PAF-induced RANTES production. The results are expressed as the mean ± SD in five different experiments. *1: p < 0.05 compared with RANTES concentrations in ASM cells cultured without an inhibitor. *2: p < 0.01 compared with RANTES concentrations in ASM cells cultured without an inhibitor.

View larger version (11K): [in this window] [in a new window]   Figure 5.   SB 203580 and PD 98059 act additively to inhibit PAF-induced RANTES and IL-8 production. ASM cells that had been preincubated with medium, SB 203580 (SB, 10 µM), PD 98059 (PD, 50 µM), or a combination of them (SB + PD) for 1 h were cultured either with medium or with PAF (1 µM), and the concentrations of RANTES in the culture supernatants were determined at 24 h after cultivation as described in METHODS. The concentrations of dimethyl sulfoxide used in this study were 0.01%, which had no effect. The results are expressed as the mean ± SD in five different experiments. *1: p < 0.01 compared with RANTES concentrations in ASM cells cultured without inhibitor. *2: p < 0.01 compared with RANTES concentrations in ASM cells cultured without inhibitor and those in ASM cells cultured with either SB 203580 or PD 98059.

PAF Induces JNK Phosphorylation

Because SB 203580, p38 MAP kinase activity inhibitor, and MEK-1 inhibitor, PD 98059, partially inhibited RANTES production by PAF-stimulated ASM cells, other intracellular signal(s) might involve the regulation of RANTES production by PAF-stimulated BEC. To test this hypothesis, we examined the threonine and tyrosine phosphorylation of JNK in PAF-stimulated ASM cells. Amounts of phosphorylated threonine and tyrosine of JNK in PAF-stimulated cells increased at 15 min and sustained between 30 and 60 min (Figure 6, upper panel). Lower panel of Figure 6 shows that equal amounts of JNK protein were immunoblotted with JNK-specific antibody regardless of time of culture periods, indicating that PAF-stimulation-induced increases in threonine and tyrosine phosphorylation of JNK occurred in the absence of changes in JNK protein levels.

View larger version (26K): [in this window] [in a new window]   Figure 6.   PAF induces the threonine and tyrosine phosphorylation of JNK. ASM cells were stimulated with PAF (1 µM) for the desired times as indicated. The lysates from ASM cells were separated by a 15% SDS-PAGE, transferred to membranes, and blotted with a specific antibody to JNK (phospho-JNK) (upper panel ). Blots shown in the upper panel were stripped and reprobed using a phosphorylation state-independent JNK specific antibody to show JNK blotted (JNK) (lower panel ). Lane P represents positive protein prepared from 293 cells treated with UV light. Lane N represents negative protein prepared from 293 cells treated without UV light. Blots are representatives of three identical experiments independently performed. The amounts of phosphorylated JNK proteins were quantitated by NIH image analyzer and are presented as the amounts of phosphorylated JNK proteins relative to control cells treated without agonist (1.0). Fold increase in amounts of phosphorylated JNK proteins as indicated below are expressed as the mean ± SD in three different experiments.

PAF Does not Promote DNA Synthesis in ASM Cells

DNA synthesis was measured by [³H]thymidine incorporation on Day 1. [³H]thymidine incorporation in the cells cultured with medium, PD 98050 (50 µM), PAF (1 µM), and PD 98059 and PAF were 221 ± 38, 209 ± 23, 222 ± 38, and 218 ± 18 cpm, respectively (mean ± SD in three different experiments), showing that [³H]thymidine incorporation in PAF-stimulated cells was comparable to that in PAF-unstimulated cells and preincubation with PD 98059 did not affect [³H]thymidine incorporation. These results indicated that PAF did not promote DNA synthesis and PD 98059 did not affect [³H]thymidine incorporation in PAF-stimulated cells. Because PDGF is known to promote DNA synthesis in ASM cells (21, 22), we also examined the effect of PD 98059 on PDGF-induced [³H]thymidine incorporation on Day 1 in order to verify our experimental conditions. [³H]thymidine incorporation in the cells cultured with medium, PD 98050 (50 µM), PDGF (30 ng/ml), and PD 98059 and PAF were 221 ± 38, 209 ± 23, 1,235 ± 208, and 322 ± 36 cpm (mean ± SD in three different experiments), respectively, showing that PDGF promoted [³H]thymidine incorporation and PD 98059 inhibited [³H]thymidine incorporation in PDGF-stimulated cells. These results indicated that ASM cells incorporated [³H]thymidine in response to PDGF, but not PAF, under our experimental conditions.

TNF- Induces Phosphorylation of p38 MAP Kinase, Erk, and JNK, and SB 203580 and PD 98059 Do not Inhibit TNF--induced RANTES Production

Finally, we analyzed the threonine and tyrosine phosphorylation of p38 MAP kinase, Erk, and JNK, reflecting activation state of these kinases in TNF--stimulated ASM cells, and examined the effect of SB 203580 and PD 98059 on TNF-- induced RANTES production to clarify a role of p38 MAP kinase and Erk in TNF--induced RANTES production. TNF- induced the threonine and tyrosine phosphorylation of p38 MAP kinase, Erk, and JNK (Figures 7a-7c), whereas neither SB 203580 nor PD 98059 had inhibitory effect on TNF-- induced RANTES production (Figure 8). These results indicated that p38 MAP kinase and Erk were not involved in TNF--induced RANTES production by ASM cells.

View larger version (57K): [in this window] [in a new window]   Figure 7.   TNF- induces the threonine and tyrosine phosphorylation of p38 MAP kinase, Erk, and JNK. ASM cells were stimulated with PAF (1 µM) for the desired times as indicated. The lysates from ASM cells were separated by a 15% SDS-PAGE, transferred to membranes, and blotted with a specific antibody to phosphorylated threonine and tyrosine of p38 MAP kinase (phospho-p38 MAPK) (upper panel of [a]), a specific antibody to phosphorylated threonine and tyrosine of Erk (phospho-Erk) (upper panel of [b]), or a specific antibody to JNK (phospho-JNK) (upper panel of [c]). Blots shown in the upper panel were stripped and reprobed using a phosphorylation state-independent p38 MAP kinase specific antibody, a phosphorylation state-independent p38 MAP kinase specific antibody, a phosphorylation state-independent Erk-specific antibody or a phosphorylation state-independent JNK specific antibody to show the amounts of p38 MAP kinase (p38 MAPK) (lower panel of [a]), Erk (Erk) (lower panel of [b]) and JNK blotted (JNK) (lower panel of [c]), respectively. Blots are representative of three identical experiments independently performed. The amounts of phosphorylated p38 MAP kinase, Erk, and JNK proteins were quantitated by NIH image analyzer and are presented as the amounts of phosphorylated p38 MAP kinase, Erk, and JNK proteins relative to control cells treated without agonist (1.0). Fold increase in amounts of phosphorylated p38 MAP kinase, Erk, and JNK proteins as indicated below are expressed as the mean ± SD in three different experiments.

View larger version (12K): [in this window] [in a new window]   Figure 8.   SB 203580 and PD 98059 do not inhibit TNF--induced RANTES production. ASM cells that had been preincubated with medium, SB 203580 (SB, 10 µM), PD 98059 (PD, 50 µM), or a combination of them (SB + PD) for 1 h were cultured either with medium or with TNF- (100 ng/ml), and the concentrations of RANTES in the culture supernatants were determined at 24 h after cultivation as described in METHODS. The concentrations of dimethyl sulfoxide used in this study were 0.01%, which had no effect. The results are expressed as the mean ± SD in five different experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In the present study, we examined the signal transduction pathway in PAF-induced RANTES production by human ASM cells. The results showed that PAF induced RANTES production. Analysis of signal transduction pathway regulating PAF-induced RANTES production revealed that PAF induced the threonine and tyrosine phosphorylation of p38 MAP kinase and Erk, and p38 MAP kinase and Erk activity in human ASM cells. Although a partial inhibition either by SB 203580 or PD 98050 of RANTES production was observed, SB 203580 and PD 98059 acted additively to inhibit RANTES production by PAF-stimulated ASM cells. These results indicate that parallel pathways that are p38 MAP-kinase-dependent and Erk-dependent regulate PAF-induced RANTES production by human ASM cells, and that other signal(s) might involve PAF-induced RANTES production. In contrast, p38 MAP kinase and Erk are not involved in TNF--induced RANTES production.

The specific inhibitors of p38 MAP kinase and Erk signaling pathways have been identified, providing effective tools for investigating the role of p38 MAP kinase and Erk in cellular signaling. In this study, SB 203580 and PD 98059 were used as the specific inhibitors for p38 MAP kinase activity and MEK-1 activity, respectively, in order to elucidate the biologic functions of different p38 MAP kinase and/or Erk. In PAF-stimulated ASM cells, 10 µM of SB 203580 caused almost complete inhibition of p38 MAP kinase activity. SB 203580 at that concentration caused a 48% decrease in RANTES production, indicating a partial inhibition, 50 µM of PD 98059 caused almost complete inhibition of Erk activity, and PD 98059 at that concentration caused a 40% decrease in RANTES production, indicating a partial inhibition. Also, 10 µM of SB 203580 and 50 µM of PD 98059 were used in this study to examine the inhibitory effect of these inhibitors on cytokine production since the previous studies with analysis of the role of p38 MAP kinase and Erk in eliciting various biologic responses, including cytokine expression, showed that these concentrations of inhibitors almost completely inhibited cell growth and cytokine expression (25, 27). Consequently, 10 µM of SB 203580 and 50 µM of PD 98050 employed in this study were sufficient concentrations to examine the signal transduction pathway. From the results with a partial inhibition of RANTES production by each inhibitor, we next examined the effect of a combination of SB 203580 (10 µM) and PD 98059 (50 µM) on RANTES production by PAF-stimulated ASM cells. SB 203580 and PD 98059 acted additively to inhibit RANTES production by 85%, but the inhibition was not complete. These results indicate that although p38 MAP kinase and Erk, at least in part, regulate PAF-stimulated RANTES production by ASM cells, other signal(s) might be involved in the regulation of RANTES production by PAF-stimulated ASM cells. One possible signal that may regulate PAF-induced RANTES production by ASM cells is JNK. The present study showed that PAF induced the threonine and tyrosine phosphorylation of JNK. The specific inhibitor for JNK has been reported (28), but it is not available yet. Because JNK pathway has been reported to be involved in cytokine expression (29), JNK pathway may participate in the regulation of RANTES production by PAF-stimulated ASM cells; however, further study is needed to clarify this point.

It has been shown that Erk is an important signal in regulating DNA and protein synthesis and cell proliferation in ASM cells as well as other type of cells (21, 22). The present results with Erk-mediated RANTES production by PAF-stimulated ASM cells might result from an increased DNA synthesis and proliferation of cells. To test this possibility, DNA synthesis in ASM cells on Day 1 was measured by [³H]thymidine incorporation. As a result, DNA synthesis in PAF-stimulated ASM cells was comparable to that in unstimulated ASM cells. In addition, PD 98059 did not affect DNA synthesis in PAF-stimulated ASM cells and unstimulated ASM cells. These results indicated that PAF-induced RANTES production was not associated with DNA synthesis.

p38 MAP kinase and Erk activation, and RANTES production were also induced by TNF- stimulation, indicating a possible involvement of p38 MAP kinase and Erk in RANTES production. However, neither SB 203580 nor PD 98059 inhibit RANTES production. These results indicate that PAF and TNF- utilize distinct signal transduction pathway to produce RANTES, and that the role of p38 MAP kinase and Erk in Rantes production by ASM cells appears to be stimulus- dependent.

The mechanism of activation and the function of MAP kinases have been extensively studied. A variety of extracellular stimuli activate p38 MAP kinase and elicit a variety of cellular functions. p38 MAP-kinase-mediated cytokine expression has been well documented (13, 14, 27). Recent studies have indicated the involvement of Erk and the coordinate regulation by p38 MAP kinase and Erk in cytokine expression in a variety of cell types (17); however, the role of Erk in cytokine expression in ASM cells has not been clarified. Phosphorylation and catalytic activation of Erk promoting DNA synthesis and cell proliferation of ASM cells in response to proinflammatory substances and growth factors have been reported (21, 22). The activation of Erk in ASM cells has implicated the intracellular signal promoting DNA synthesis and proliferation of ASM cells. Our results with Erk-mediated cytokine production indicate a new role for Erk in ASM cells, which is the regulation of cytokine production. Thus, the activation of Erk implicates the intracellular signal inducing cytokine expression as well as promoting cell proliferation.

PAF elicits various cell functions utilized by various intracellular signals (30). PAF-activated p38 MAP kinase cascade has been shown to participate in IL-8 expression in human bronchial epithelial cells (27) and neutrophil adhesion (33). Although the activation of Erk by PAF has been demonstrated in a variety of cell types, few studies have demonstrated the role of Erk in cellular responses (32, 34). Erk is involved in the mitogenic signaling of PAF in endometrial adenocarcinoma cell line HEC-1A (32) and may be involved in PAF-induced phospholipase A₂ activation (34). In the present study, we have shown that in ASM cells, PAF induced RANTES production by the simultaneous activation of two distinct pathways, p38 MAP kinase and Erk, indicating that PAF-activated Erk and p38 MAP kinase are important intracellular signals inducing cellular functions, at least in ASM cells.

PAF released in the airway of asthmatics causes airway smooth muscle contraction (23). In addition, our results showed that PAF induced RANTES production. RANTES, which exhibits a chemotactic activity for eosinophils, has been shown to play an important role in the production of airway inflammation of asthmatics through the recruitment of eosinophils into the site of airway inflammation (2, 3). Catalytic activation of Erk has implicated the signal promoting ASM cell proliferation (21, 22). The modulation of Erk pathway in ASM cells indicates a strategy for controlling ASM cell proliferation seen in airway remodeling of asthmatic airway (21, 22, 35). In the present study, we showed that SB 203580 as the specific inhibitor for p38 MAP kinase and PD 98059 as the specific inhibitor for MEK-1, which is an upstream regulator of Erk, inhibited RANTES production by ASM cells. It is not known, at this time, whether SB 203580 and PD 98059 are capable of producing beneficial effect on controlling the production of allergic inflammation. However, understanding and the regulation of signal cascades leading to RANTES production by ASM cells indicate a strategy for the therapy of allergic inflammation of asthmatic airways.

From the data presented here, we conclude that p38 MAP kinase and Erk are involved in regulating RANTES production by ASM cells stimulated with PAF but not with TNF-. These results indicate that the role of p38 MAP kinase and Erk in RANTES production by ASM cells appears to be stimulus-dependent and that multiple intracellular signaling pathways are involved inthe regulation of RANTES production by ASM cells.