p38 and Src-ERK1/2 Pathways Regulate Crystalline Silica-Induced Chemokine Release in Pulmonary Epithelial Cells

Johan Øvrevik1, Marit Låg, Per Schwarze and Magne Refsnes

Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, N-0403 Oslo, Norway

Received April 19, 2004; accepted July 4, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Crystalline silica has been shown to trigger pulmonary inflammation both in vivo and in vitro, but the underlying molecular mechanisms remain unclear. In the present study we focus on the intracellular signaling pathways regulating chemokine release from lung epithelial cells after crystalline silica exposure. Our results show that silica particles induced a concentration- and time-dependent increase in interleukin (IL)-8 release from the human epithelial lung cell line A549. The IL-8 induction was significantly attenuated by inhibitors of the mitogen-activated protein kinases (MAPKs), p38 (SB202190) and extracellular signal-regulated kinase (ERK)-1 and -2 (PD98059), as well as a general protein tyrosine kinase (PTK) inhibitor (genistein). However, IL-8 induction was most efficiently inhibited by the Src family kinase (SFK) inhibitor, PP2, suggesting a crucial role of SFKs in regulating silica-induced IL-8 release from A549 cells. Silica exposure induced phosphorylation of the MAPKs p38 and ERK1/2, but not JNK or ERK5. Silica also induced a significant phosphorylation of SFKs. Moreover, PP2 inhibited silica-induced phospho-ERK1/2 to near-control levels, whereas phospho-p38 was not significantly reduced by the SFK inhibitor. Our results suggest the presence of two separate signaling pathways which are important in the regulation of silica-induced IL-8 release from A549 cells; one involving SFK-dependent activation of ERK1/2, and the other activation of p38, at least partly independent of SFKs. Experiments with primary type 2 (T2) cells from rat lungs suggest that crystalline silica-induced release of macrophage inflammatory protein (MIP)-2 is regulated through similar mechanisms.

Key Words: quartz; mitogen activated protein kinases; protein tyrosine kinases; interleukin-8; macrophage inflammatory protein-2.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The harmful effects of crystalline silica (SiO2) are well documented. Epidemiological, clinical, and pathological studies have associated inhalation of crystalline silica with pulmonary diseases such as silicosis, chronic bronchitis, and lung cancer (American Thoracic Society, 1997Go). Inflammatory responses appear to be important to the development of these diseases, and recent investigations have provided substantial evidence for the potential of crystalline silica to induce pulmonary inflammation (Becher et al., 2001Go; Olbruck et al., 1998Go; Vallyathan et al., 1995Go). Of special significance is the ability of silica to induce production and release of CXC-chemokines such as interleukin (IL)-8 (Hetland et al., 2001Go; Schins et al., 2000Go; Stringer et al., 1996Go; Stringer and Kobzik, 1998Go). IL-8 is a potent neutrophil chemoattractant and plays a key role in the development of acute inflammation (Harada et al., 1996Go). Recent studies on IL-8 have revealed a variety of biological functions in various cell types, suggesting a pivotal role in several pathological conditions including chronic inflammation, fibrosis, and cancer (Mukaida, 2003Go). Thus, understanding the specific mechanisms of silica-induced generation of IL-8 may be important to the understanding of crystalline silica pathology in general.

Presently, numerous environmental stimuli are known to activate intracellular signaling cascades in the bronchial epithelium, triggering the release of pro-inflammatory mediators. Among the key enzymes in these events are the mitogen-activated protein kinase (MAPK) family of serine/threonine kinases (Puddicombe and Davies, 2000Go). MAPKs are activated in response to a range of extracellular stimuli (growth factors, cytokines, hormones, oxidants, toxins, physical stress) and regulate a variety of cellular responses including apoptosis, immune activation, and inflammation (Puddicombe and Davies, 2000Go). Currently, the best described MAPK members are the extracellular signal-regulated kinase-1 and -2 (ERK1/2), the c-Jun-N-terminal kinases (JNKs), and the p38 MAPKs. Activation of ERK1/2, JNK, and p38 appears to be crucial for optimal IL-8 gene expression, as they contribute through both activation of transcription factors and posttranscriptional mechanisms such as IL-8 mRNA stabilization (Hoffmann et al., 2002Go). Crystalline silica may activate all the three MAPK cascades. This activation seems to control silica-induced activation of activator protein (AP)-1 (Ding et al., 1999Go, 2001Go; Shukla et al., 2001Go), which is an important transcription factor in the regulation of basal and induced cytokine expression (Hoffmann et al., 2002Go). Taken together, this suggests the involvement of MAPKs in silica-induced cytokine release.

Another group of signaling molecules potentially important in silica-induced inflammation are the protein tyrosine kinases (PTKs). PTKs are roughly divided into the receptor PTKs and the non-receptor PTKs, which both are involved in the early stages of intracellular signaling. Silica particles have been shown to induce NF-{kappa}B activation through tyrosine phosphorylation of I{kappa}B-{alpha} and this phosphorylation was attenuated by PTK inhibitors (Kang et al., 2000Go). It has also recently been shown that PTK inhibitors may block silica-induced generation of reactive oxygen species (ROS) in fibroblasts (Kim et al., 2002Go). However, the participation of specific PTKs in silica-induced signaling cascades has yet to be established. Of special interest are the Src family kinases (SFKs), which belong to the non-receptor PTKs. SFKs are activated following engagement of several different classes of cell surface receptors, including immuno-receptors, cytokine receptors, G protein-coupled receptors (GPCRs), and receptor PTKs (Thomas and Brugge, 1997Go). Moreover, SFKs have been shown to mediate MAPK activation in response to various extracellular stimuli, including ROS, UV irradiation, monosodium urate (MSU) crystals, and ligands of scavenger receptors (Hsu et al., 2001Go; Kitagawa et al., 2002Go; Liu et al., 2001Go; Nishida et al., 2000Go).

The aim of this study was to investigate intracellular signaling pathways regulating crystalline silica-induced IL-8 release from a human lung epithelial cell line (A549), and to compare this with the mechanisms regulating chemokine release from primary rat type 2 (T2) cells. We have focused on determining whether the different MAPK cascades are involved in silica-induced chemokine release, and by what mechanisms MAPK activation is regulated. On the basis of the results presented below, we suggest the presence of two separate signaling pathways, which are important in the regulation of crystalline silica-induced IL-8 release from A549 cells; one involving SFK-dependent activation of ERK1/2, and the other activation of p38, at least partly independent of SFKs. We further suggest that similar mechanisms may be involved in silica-induced macrophage inflammatory protein (MIP)-2 release from primary rat T2 cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents. Culture medium, Nutrition Mixture F12 HAM Kaigin's modification (F12K) was obtained from Sigma-Aldrich (St. Louis, MO), whereas William's medium E was from Bio Whittaker Europe (Verviers, Belgium). Fetal bovine serum (FBS) was from EuroClone (Pero, Italy). Ampicillin and fungizone were from Bristol-Myer Squibb (Bromma, Sweden) and penicillin/streptomycin was from Bio Whittaker (Walkersville, MD). Phenylmethylsulfonyl fluoride (PMSF) and propidium iodide (PI) were from Sigma-Aldrich. Cytokine ELISA assays for IL-8 and MIP-2 were purchased from Biosource International (Camarillo, CA). The inhibitors PD98059 (2'-amino-3'-methoxyflavone), SB202190 (4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl) 1H-imidazole), genistein (4',5,7-trihydroxyisoflavone), and PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) were purchased from Calbiochem-Novabiochem Corporation (La Jolla, CA). Specific antibodies against phospho-p38, p38, phospho-JNK, JNK, phospho-ERK5, ERK5, and phospho-Src family kinases (Tyr416) were obtained from Cell Signaling Technology Inc. (Beverly, MA). Antibodies against phospho-ERK1/2, ERK2, and Src were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). All other chemicals used were purchased from commercial sources at the highest purity available.

Crystalline silica particles. The commercially available crystalline silica particle MIN-U-SIL 5 Ground Silica (U.S. Silica Company, Berkley Springs, WV) was kindly provided by Dr. Paul Borm, Medical Institute of Environmental Hygiene, Düsseldorf, Germany. MIN-U-SIL 5 is a high purity, natural crystalline silica. According to the manufacturer, this ground silica is at least 98% SiO2 and a size distribution with typically 96% passing 5 µm and a median diameter of 1.6 µm. The crystalline silica particles were prepared and exposed to the cell cultures as previously described by Hetland et al. (2000b)Go.

Cell cultures. The human epithelial lung cell line A549 from American Tissue Type Culture Collection (ATCC, Rockville, MD) was cultured in F12K medium, supplemented with ampicillin (100 µg/ml), penicillin/streptomycin (100 µg/ml), fungizone (0.25 µg/ml) and 10% heat-inactivated FBS. The A549 cells were plated in 35 mm six-well culture dishes (2 x 104 cells/well) or 90 mm dishes (2 x 105 cells/dish) and grown to confluence at 37°C in a humidified atmosphere of 5% CO2 in air (prior to exposure). T2 cells were isolated from rat lungs by the method described by Lag et al. (1996)Go. In short, lungs were removed from six anaesthetized male, inbred Wistar Kyoto (Wky/NHds) rats between six and eight weeks old. Cells were isolated by sequential use of enzymatic digestion, centrifugal elutriation, and differential attachment. The isolated T2 cells were cultured in William's medium E supplemented with insulin (5 µg/ml), hydrocortisone (0.087 µg/ml), transferrin (5 µg/ml), EGF (10 ng/ml), sodium selenite (6.2 ng/ml), ascorbic acid (5 µg/ml), gluthatione (5 µg/ml), ampicillin (100 µg/ml), penicillin/streptomycin (100 µg/ml), fungizone (0.25 µg/ml), Hepes (15 mM), and 5% heat-inactivated FBS. The T2 cells were plated at a density of 4 x 105/cm2 and grown for two days at 37°C in a humidified atmosphere of 5% CO2 in air, prior to exposure.

IL-8 and MIP-2 assays. Cells were grown in 35 mm, six-well culture dishes, exposed to crystalline silica and incubated at 37°C. When inhibitors of MAPKs or PTKs were used, the cells were pre-incubated with the inhibitor for 1 h prior to crystalline silica exposure. All inhibitors were kept in the culture throughout the particle exposure period. After particle exposure supernatants were removed and centrifuged in two steps, first at 250 x g to remove cells, then at 2500 x g to remove the remaining silica particles. The final supernatants were stored at –70°C. IL-8 and MIP-2 protein levels were determined by enzyme-linked immunosorbant assay (ELISA) according to the manufacturer's guideline. Absorbance was measured and quantified using a plate reader (TECAN Sunrise, Phenix Research Products, Hayward, CA) complete with software (Magellan V 1.10).

Cell viability assay. Cell suspensions were stained with PI (5 µg/ml). Uptake of PI by damaged cells was analyzed using fluorescence microscopy, and 300–400 cells from each sample were counted.

Immunoblotting. Cells were grown to confluence in 90 mm culture dishes and serum starved for 24 h prior to crystalline silica exposure. Inhibitors were applied in the same manner as for the cytokine assay. Cells were harvested in ice-cold PBS containing PMSF (1 mM) and resuspended in sample-buffer (3.1 ml destilled H2O, 1.0 ml 0.5 M Tris-HCl pH 6.8, 0.8 ml glycerol and 2.5 ml 10% SDS) prior to protein determination by the BioRad DC Protein Assay (BioRad Life Science, Camarillo, CA). Proteins (12.5 µg/well for all analyses except total and phosphorylated p38 with 25 µg/well) from whole-cell lysates were separated by 10% SDS-PAGE and blotted onto nitrocellulose membranes. To ensure that the protein levels of each well were equal, Ponceau-staining was used for loading control. The membranes were then probed for phosphorylated ERK1/2, p38, JNK, and ERK5 as well as phosphorylated SFKs (Tyr416), prior to incubation with horseradish peroxidase-conjugated secondary antibodies. The blots were developed using the SuperSignal West Dura chemoluminescence system (Pierce, Perbio Science, Sweden) according to the manufacturer's instructions. Finally the membranes were stripped by incubation for 1 h at 60°C in stripping-buffer (62.5 mM Tris-HCl, 100 mM 2-mercaptoethanol and 2% SDS) and re-probed for total ERK2, p38, JNK, ERK5, and Src. Quantification of optical densities and estimation of molecular weights of the protein bands, were performed by the use of KODAK 1D Image Analysis Software.

Statistical analysis. Statistical significance of treatments where evaluated using one-way ANOVA with Dunnett's post-test for multiple comparisons (Figs. 1A, 1B, 2A, 2B, 3, 4, 5, and 6), or two-way ANOVA with Bonferroni post-test (Figs. 1C and 2C). Values are presented as means ± SEM.



View larger version (12K):
[in this window]
[in a new window]
 
FIG. 1. Induction of IL-8 release by crystalline silica in human lung epithelial cells (A549). A549 cells were exposed to different concentrations of crystalline silica and incubated for 24 h, prior to analysis of IL-8 release (A) and viability (B). A549 cells were also exposed to crystalline silica (40 µg/cm2) and incubated for increasing periods of time (C). IL-8 release was measured by ELISA as described under Materials and Methods. Each point represents mean ± SEM of independent experiments (n ≥ 3). —•—Silica-exposed cells, ·····{odot}····control cells, *significantly different from unexposed controls (p < 0.05).

 


View larger version (15K):
[in this window]
[in a new window]
 
FIG. 2. Involvement of MAPKs in crystalline silica-induced IL-8 release from human lung epithelial cells (A549). A549 cells were pre-treated with different concentrations of the p38 inhibitor SB202190 (A), the ERK1/2 inhibitor PD98059 (B), and a combination of both inhibitors (C), for 1 h prior to crystalline silica (40 µg/cm2) exposure and incubated for 24 h. IL-8 release was measured by ELISA as described under Materials and Methods. The results are expressed as relative IL-8 induction compared to crystalline silica exposed cells in absence of inhibitor (100% response). Each value represents mean ± SEM of independent experiments (n ≥ 3). —•—Silica-exposed cells, ·····{odot}····control cells, *significantly different from unexposed controls (p < 0.03), {dagger}significantly different from silica-exposed cells in absence of inhibitor (p < 0.05).

 


View larger version (12K):
[in this window]
[in a new window]
 
FIG. 3. Involvement of PTKs in crystalline silica-induced IL-8 release from human lung epithelial cells (A549). A549 cells were pre-treated with the general PTK inhibitor genistein (A), and the SFK inhibitor PP2 (B) for 1 h prior to crystalline silica (40 µg/cm2) exposure and incubated for 24 h. IL-8 release was measured by ELISA as described under Materials and Methods. The results are expressed as relative IL-8 induction compared to crystalline silica-exposed cells in absence of inhibitor (100% response). Each value represents mean ± SEM of independent experiments (n = 3–4). —•—Silica-exposed cells, ·····{odot}····control cells, {dagger}significantly different from silica-exposed cells in absence of inhibitor (p < 0.05).

 


View larger version (32K):
[in this window]
[in a new window]
 
FIG. 4. Activation of MAPKs and SFKs by crystalline silica in human lung epithelial cells (A549). A549 cells were exposed to crystalline silica (60 µg/cm2) and incubated for the indicated periods of time. Total and phosphorylated p38 (A) and total ERK2 and phosphorylated ERK1/2 (B) as well as total c-Src and phosphorylated SFKs (C) were detected by Western blotting as described under Materials and Methods. The figure displays optical quantification of protein bands as well as typical blots. Values are presented as relative phosphorylation compared to total levels of the respective protein (i.e., phospho-p38/p38) ± SEM (max ratio = 1.0). The results are representative of independent experiments (n = 3–6). *Significantly different from unexposed controls (p < 0.05).

 


View larger version (27K):
[in this window]
[in a new window]
 
FIG. 5. Involvement of SFKs in crystalline silica-induced MAPK activation in human lung epithelial cells (A549). A549 cells were pretreated with PP2 for 1 h prior to exposure to crystalline silica (60 µg/cm2) for 30 min. Total and phosphorylated p38 (A) and total ERK2 and phosphorylated ERK1/2 (B) were detected by Western blotting as described under Materials and Methods. The figure displays optical quantification of protein bands as well as typical blots. Values are presented as relative phosphorylation compared to total levels of the respective protein (i.e., phospho-p38/p38) ± SEM (max ratio = 1.0). The results are representative of independent experiments (n = 4). *Significantly different from unexposed controls (p < 0.05), {dagger}significantly different from silica-exposed cells in absence of inhibitor (p < 0.05).

 


View larger version (20K):
[in this window]
[in a new window]
 
FIG. 6. Induction of MIP-2 release by crystalline silica from primary type 2 (T2) cells from rat lungs. T2 cells were exposed to increasing concentrations of crystalline silica for 24 h (A). T2 cells were pre-treated with SB202190, PD98059, and PP2 1 h prior to crystalline silica exposure and incubated for 24 h (B). MIP-2 release was measured by ELISA as described under Materials and Methods. The results are expressed as relative MIP-2 induction compared to crystalline silica exposed cells in absence of inhibitor (100% response). Each value represents mean ± SEM of independent experiments (n = 4). *Significantly different from unexposed controls (p < 0.05), {dagger}significantly different from silica exposed cells in absence of inhibitor (p < 0.05).

 

    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IL-8 Release by Crystalline Silica-Exposed A549 Cells
To explore the effects of crystalline silica on IL-8 release, A549 cells were exposed to various silica concentrations for 24 h. Silica caused a significant concentration-dependent induction of IL-8 from A549 cells (Fig. 1A). The exposure also caused a reduction in cell viability, which was significantly different from unexposed cells at the two highest concentrations tested (Fig. 1B). To minimize the impact of silica-induced cell death, 40 µg/cm2 was chosen as the concentration to be used in the following time-course and inhibitor studies of IL-8 release. A549 cells produced a time-dependent increase in IL-8 release, which was significant after 24 h of silica exposure (Fig. 1C).

Involvement of MAPKs in Crystalline Silica-Induced IL-8 Release from A549 Cells
MAPK signaling is important in various cellular stress responses including cytokine expression (Puddicombe and Davies, 2000Go). To investigate the involvement of MAPKs in crystalline silica-induced IL-8 release, A549 cell cultures were pre-incubated with the p38 inhibitor SB202190 or the MAPK/ERK kinase (MEK) 1 inhibitor PD98059, for 1 h prior to particle exposure. The presence of SB202190 significantly attenuated the silica-induced IL-8 release from A549 cells at the tested concentrations (Fig. 2A). Inhibition of MEK1, an activator protein located upstream of ERK1/2 (Puddicombe and Davies, 2000Go), also significantly inhibited the chemokine induction (Fig. 2B). To investigate the p38 and ERK1/2 pathways, SB202190 is recommended to be used at 10 µM and PD98059 at 50 µM (Cuenda and Alessi, 2000Go). At these concentrations SB202190 and PD98059 inhibited silica-induced IL-8 release by approximately 65 and 60%, respectively, when corrected for basal IL-8 levels. In the presence of both SB202190 (10 µM) and PD98059 (50 µM) crystalline silica-induced chemokine release was reduced to near-control levels (Fig. 2C). Basal IL-8 levels in controls were not significantly affected by inhibitor treatments (p > 0.05). We did not observe any obvious changes in cell viability due to the presence of the inhibitors (not shown).

Involvement of PTKs in Crystalline Silica-Induced IL-8 Release from A549 Cells
Having established that chemical inhibitors of the p38 and ERK1/2 pathways attenuate crystalline silica induced IL-8 release, we investigated the involvement of signaling events potentially located upstream of MAPK activation. In the presence of a general PTK inhibitor, genistein (50–200 µM), silica-induced IL-8 release from A549 cells was significantly attenuated (Fig. 3A). Since SFKs may be important in MAPK activation and cytokine release (Hsu et al., 2001Go; Kitagawa et al., 2002Go; Liu et al., 2001Go), we also examined whether SFKs were involved in silica-induced IL-8 release. Pre-incubation with the SFK inhibitor, PP2 (1–100 µM), led to a concentration-dependent reduction of crystalline silica-induced IL-8 release from A549 cells (Fig. 3B). At 10 µM, PP2 reduced silica-induced IL-8 release by approximately 80%, when corrected for basal IL-8 levels. Basal IL-8 levels in controls where not significantly affected by inhibitor treatments (p > 0.05). We did not observe any obvious changes in cell viability due to the presence of the inhibitors (not shown).

Activation of MAPKs and PTKs by Crystalline Silica in A549 Cells
To further explore the involvement of MAPKs and SFKs in crystalline silica-induced IL-8 release, we examined the effect of the particles on phosphorylation of MAPKs and SFKs. Compared to the IL-8 ELISA assay, detection of protein phosphorylation by Western blotting was less sensitive to silica exposure. This might be partly because IL-8 accumulates in the growth medium throughout the exposure time, whereas protein phosphorylations do not, but also because of the less quantitative nature of the Western technique. Therefore, the silica concentration was increased to 60 µg/cm2 in these experiments. As assessed with specific antibodies, silica exposure induced phosphorylation of p38 and ERK1/2 from 15 min after the particles were added to the cell cultures (Figs. 4A and 4B). We did not, however, observe phosphorylation of neither JNK nor ERK5 (also known as big mitogen-activated protein kinase [BMK]-1) in response to the silica concentrations tested (not shown). Activation of the SFKs was examined by using a phospho-specific (Tyr416) antibody which cross-reacts with several SFK members (c-Src, Lyn, Fyn, Lck, Yes, and Hck). Crystalline silica induced a significant phosphorylation of a protein estimated to approximately 61 kDa, corresponding to c-Src (60 kDa), visible from the earliest time-point tested (Fig. 4C). The antibody also revealed induction of additional phospho-proteins, mainly a 53 kDa protein, but also a band at approximately 57 kDa (only visible in some blots—not shown), possibly corresponding to Lyn (53 and 56 kDa isoforms). Indeed, recent work has shown that c-Src and Lyn, but not Lck and Fyn, were abundantly expressed in A549 cells (Huang et al., 2003Go). The phosphorylation levels of the 53 and 57 kDa bands were, however, considerably weaker than the 61 kDa band and were not quantified. As seen from the figure, the antibody against total c-Src also seemed to cross-react with other SFK-members, giving rise to protein bands at approximately 61, 57, and 53 kDa (Fig. 2C).

Optical band quantification showed that the kinetics of phospho-p38 followed a biphasic pattern with a short transient peak at 15 min followed by a longer sustained phase, with a second peak at 2 h (Fig. 4A). In comparison, phosphorylation of ERK1/2 increased more gradually, peaked at 30 min and then decreased until 2 h of silica exposure, where it stabilized at approximately twice the basal phospho-ERK1/2 level (Fig. 4B). The kinetics of silica-induced phospho-c-Src had more resemblance with ERK1/2 than with p38. Src phosphorylation peaked after 1 h of silica exposure before it gradually decreased until 4 h (Fig. 4C). Initial screenings suggested that ERK1/2 and SFKs were phosphorylated for up to 16 h, whereas p38 phosphorylation disappeared between 8 and 16 h (not shown).

Involvement of SFKs in Crystalline Silica-Induced MAPK Activation in A549 Cells
SFKs have been shown to activate MAPKs in response to various stimuli (Kitagawa et al., 2002Go; Liu et al., 2001Go; Nishida et al., 2000Go), and it was therefore of interest to determine whether SFK activation was located upstream of MAPKs in the signaling pathway leading to IL-8 release. Thus, we examined the effect of PP2 on crystalline silica-induced MAPK activation in A549 cells. Pre-incubation with PP2 (10 µM) did not affect silica-induced p38 phosphorylation significantly (Fig. 5A). ERK1/2-phosphorylation was, however, inhibited to near-control levels in the presence of PP2 (Fig. 5B). PP2 did not affect basal phosphorylation levels of ERK1/2 and p38 (not shown).

MIP-2 Release by Crystalline Silica-Exposed Primary Rat T2 Cells
We examined if crystalline silica exposure of primary epithelial rat T2 cells would elicit a similar response as observed with the A549 cells. The release of MIP-2, a chemokine which in rats has an analogous function to IL-8 in humans (Driscoll, 2000Go; Harada et al., 1996Go), was thus measured. T2 cells exposed to silica particles produced a concentration-dependent increase in MIP-2 release, with a significant increase at a particle concentration of 20 µg/cm2 and peaking at 30 µg/cm2 (Fig. 6A). We also examined whether signaling pathways similar to the above described for A549 cells might be involved in the observed MIP-2 release from crystalline silica-exposed T2 cells. The inhibitors SB202190 (10 µM) and PD98059 (50 µM) almost completely prevented silica-induced MIP-2 release from T2 cells (Fig. 6B). Furthermore, PP2 (10 µM) strongly attenuated silica-induced MIP-2 (Fig. 6B). Basal MIP-2 levels in controls were, however, not significantly affected by inhibitor treatments (p > 0.05). Western blotting revealed that crystalline silica induced phosphorylation of the MAPKs p38 and ERK1/2, as well as SFKs (Fig. 7). However, use of the phospho-Src family (Tyr416) antibody only revealed phosphorylation of a 52 and a 57 kDa band, but no band corresponding to the 60 kDa c-Src as observed in the A549 cells.



View larger version (38K):
[in this window]
[in a new window]
 
FIG. 7. Activation of MAPKs and SFKs by crystalline silica in primary T2 cells from rat lungs. T2 cells were incubated for 30 min, with or without crystalline silica (30 µg/cm2). Total and phosphorylated p38 (A) and total ERK2 and phosphorylated ERK1/2 (B) as well as ß-actin and phosphorylated SFKs (C) were detected by Western blotting as described under Materials and Methods. The results are representative of independent experiments (n = 3).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous studies have shown that crystalline silica may induce the release of pro-inflammatory cytokines and chemokines (Barrett et al., 1999Go; Desaki et al., 2000Go; Hetland et al., 2001Go; Olbruck et al., 1998Go; Schins et al., 2000Go; Stringer et al., 1996Go), and activate signaling cascades involving MAPKs or PTKs (Ding et al., 1999Go, 2001Go; Kang et al., 2000Go; Kim et al., 2002Go; Shukla et al., 2001Go). How these events are connected has, however, remained unclear. Our present results extend and complement the knowledge on crystalline silica-induced signaling cascades by (1) providing a connection between silica-induced phosphorylation of SFKs and MAPKs and silica-induced chemokine release, and (2) by showing that SFKs regulate silica-induced ERK1/2 activation.

In accordance with previous findings (Desaki et al., 2000Go; Hetland et al., 2001Go; Schins et al., 2000Go; Stringer et al., 1996Go), our results show that crystalline silica induced a significant increase in IL-8 release from A549 cells. Our results further show that the IL-8 induction was significantly attenuated by the p38 inhibitor SB202190 and by the MEK inhibitor PD98059. Although PD98059 has been considered a relatively specific inhibitor of the ERK1/2 pathway, recent studies have shown that it may also inhibit other pathways such as JNK and in particular the MEK5-ERK5 pathway (Kamakura et al., 1999Go; Mody et al., 2001Go; Salh et al., 2000Go). However, since crystalline silica did only induce phosphorylation of p38 and ERK1/2 but not JNK or ERK5, the observed effect of PD98059 was presumably due to inhibition of the ERK1/2 pathway. Thus, our findings suggest that crystalline silica induces IL-8 release through activation of the MAPKs p38 and ERK1/2, but not JNK or ERK5.

The role of MAPK pathways in IL-8 regulation seems to be highly cell type and stimulus specific. Previous studies have shown that IL-8 may be regulated by p38, ERK1/2, or JNK, alone (Jung et al., 2002Go; Kawaguchi et al., 2002Go; Li et al., 2003Go) or in different combinations (Furuichi et al., 2002Go; Kumar et al., 2003Go; Li et al., 2002Go; Wu et al., 2002Go). However, it appears that a maximal production of IL-8 and other cytokines may require a combined activation of all the three major MAPK cascades (Li et al., 2002Go; Zhu et al., 2000Go). Therefore, the potential to induce IL-8 of a given substance may partly depend on the ability to induce multiple MAPK-pathways. The IL-8 promoter region, which has been extensively studied, contains binding sites for NF-{kappa}B, CAAT/enhancer-binding protein (C/EBP), and AP-1 (see Hoffmann et al., 2002Go). Whereas NF-{kappa}B binding seems to be pivotal to the IL-8 promoter activity for most cell types studied, the AP-1 and C/EBP (also called NF-IL-6) sites are not essential for induction but required for maximal gene expression (Hoffmann et al., 2002Go). It appears from the literature that MAPKs primarily regulate IL-8 through activation of AP-1 and C/EBP (Jung et al., 2002Go; Kumar et al., 2003Go; Wu et al., 2002Go). However, MAPKs may also regulate IL-8 through activation of NF-{kappa}B and through posttranscriptional mechanisms such as mRNA stabilization (Jijon et al., 2002Go; Li et al., 2002Go).

The knowledge on silica-induced MAPK activation and the intracellular processes it affects is limited. Studies by Ding et al. (1999Go, 2001Go) have shown that crystalline silica induced phosphorylation of ERK1/2 and p38, but not JNK, and that both the ERK1/2 and the p38 pathways are involved in AP-1 induction in a mouse epidermal cell line (JB6). In a non-transformed alveolar T2 epithelial cell line (C10) crystalline silica-induced phosphorylation ERK1/2 and JNK, but not p38, were reported to be associated with AP-1 activation (Shukla et al., 2001Go). Whether this slightly contradicting MAPK activation pattern may be a result of different cell systems or silica qualities remains unclear. However, more importantly these findings show that crystalline silica have the potential to activate all three MAPK cascades, and that these may regulate silica-induced AP-1 activity.

In our study we observed that crystalline silica induced a biphasic phosphorylation of p38. Also, the silica-induced kinetics of phospho-ERK1/2 displayed resemblance of a biphasic activation with a rapid increasing peak followed by a second sustained phase. Information on the role of transient versus sustained MAPK activation is mostly limited to the role of ERK1/2 in cell growth and proliferation (see Marshall, 1995Go, for review). Initially, it was believed that a sustained ERK1/2 activation was required for translocation of ERK1/2 to the nucleus, a process that is necessary for MAPKs to activate transcription factors, thus suggesting that transient and sustained MAPK activation would have different effects on gene expression (Marshall, 1995Go). However, recent studies have also shown that a transient activation is sufficient both to translocate ERK1/2 to the nucleus and to induce gene expression (Chen et al., 2003Go; Horgan and Stork, 2003Go). Thus, it is uncertain what roles the different phases of MAPK activation may play in the regulation of cytokine production. However, silica appears to induce AP-1 activity in a slow and sustained manner, similar to the IL-8 release observed in our study (Ding et al., 1999Go; Shukla et al., 2001Go). Also, others have shown that silica-induced IL-8 mRNA levels peak at 3 h, and that IL-8 protein release appears later, at 24 h (Desaki et al., 2000Go). Therefore, it seems likely that the late, sustained phases of silica-induced MAPK activity are more closely linked to regulation of the IL-8 induction, than the early peaks.

Genistein attenuated IL-8 release after crystalline silica exposure, suggesting the involvement of PTKs. More interestingly, the SFK inhibitor PP2, strongly attenuated silica-induced IL-8 release. Indeed, PP2 was a more efficient inhibitor of IL-8 induction than both the MAPK inhibitors tested, suggesting a crucial role of SFKs in silica-induced chemokine release. Although previous studies have implicated the involvement of PTKs in silica-induced signaling pathways such as NF-{kappa}B activation (Kang et al., 2000Go) and intracellular ROS generation (Kim et al., 2002Go), the involvement of specific PTKs have not previously been described. SFKs are known to regulate MAPK activation, for example through phosphorylation of Shc which stimulates the Ras-MAPK pathway (see Thomas and Brugge, 1997Go, for review). Here, we observed that although the overall silica-induced kinetics of phospho-ERK1/2 and phospho-SFKs differs, the early phases seem to parallel. Since PP2 strongly attenuated ERK1/2 phosphorylation, it is likely that SFK activation precedes and regulates ERK1/2 activity in response to silica exposure. In contrast, PP2 exposure did not affect silica-induced p38 phosphorylation significantly, indicating that p38 was mainly activated independently of SFKs, through a signaling pathway separate from the one activating ERK1/2. Differences in phosphorylation kinetics, between p38 and ERK1/2, support the idea that the two MAPK cascades are regulated through different mechanisms in response to silica exposure. Although our results suggest that SFKs regulate ERK1/2 activity in response to silica exposure, inhibition of silica-induced IL-8 release by the SFK inhibitor PP2 appears to be much more complete than with the ERK1/2-pathway inhibitor PD98059. This apparent discrepancy may be explained by the ability of SFKs to regulate a variety of signaling pathways involved in IL-8 induction. Indeed, we did observe a partial but nonsignificant effect of PP2 on phospho-p38, which could contribute to the observed effect on IL-8. Furthermore, SFKs have been shown to regulate NF-{kappa}B activity (Huang et al., 2003Go; Liu et al., 2001Go). Thus, the potent effect of PP2 compared to PD98059 suggests that silica-induced SFK activation may regulate other signaling pathways in addition to the ERK1/2 cascade, which contribute to the IL-8 release (Fig. 8).



View larger version (37K):
[in this window]
[in a new window]
 
FIG. 8. Proposed model for crystalline silica-induced IL-8 release. Solid arrows represent silica-induced signaling pathways shown in this study. Dotted arrows represent silica-induced signaling pathways shown by other authors or hypothetical pathways discussed in the text.

 
Obviously, the pitfalls of generalizing results obtained from immortalized carcinoma cell lines are many. Therefore, we compared our findings from the A549 cells with primary T2 cells from rat lungs. Previous work from our group has shown that particle-exposed T2 cells released increased levels of MIP-2 (Becher et al., 2001Go), the rat analogue to human IL-8 (Driscoll 2000Go; Harada et al., 1996Go). Our present results confirmed that crystalline silica induces MIP-2 release from the T2 cells. Although the T2 cells were more sensitive to crystalline silica exposure than the A549 cells, similar signaling pathways seemed to be involved in silica-induced chemokine release, that is activation of ERK1/2, p38, and SFKs. However, the phospho-SFK antibody only revealed induction of two phospho-protein bands estimated to 52 and a 57 kDa, and no band corresponding to the 60 kDa c-Src as in the A549 cells. A recent screen of protein kinase expression in the T2 cells, show expression of the SFK members c-Src and Lyn, but not Fyn, or Lck (Låg et al., unpublished results). Thus, it is tempting to speculate that the only SFK-members phosphorylated in silica-exposed T2 cells are the two isoforms of Lyn, which are known to be 53 and 56 kDa, respectively. In contrast, our results suggest that silica-exposure mainly induced phosphorylation c-Src, and to a lesser extent Lyn, in the A549 cells. The significance of this discrepancy is not clear, but it could point to differences in how silica particles initiate signaling cascades in A549 and T2 cells.

The origin of silica-induced signal transduction remains uncertain. Several reports have suggested that the observed effects of crystalline silica are largely due to the generation of ROS (Ding et al., 2001Go; Kang et al., 2000Go; Shukla et al., 2001Go), while others have focused on interactions between silica particles and cell surface receptors such as scavenger receptors or Fc-receptors (Hamilton et al., 2000Go; Hetland et al., 2000aGo; Stringer et al., 1996Go). Interestingly, both of these scenarios may in theory lead to activation of SFKs. Both GPCRs (Luttrell et al., 1997Go) and scavenger receptors (Hsu et al., 2001Go) may signal through SFK activation, but evidence also indicates that SFKs can be activated by ROS, independent of receptor activation. H2O2 has been shown to activate {alpha}-subunits of small G-proteins directly, leading to dissociation of the ß{gamma}-subunits (Gß{gamma}) and activation of ERK1/2 (Nishida et al., 2000Go). This Gß{gamma}-responsive ERK1/2 activation by H2O2 was independent of ligands binding to Gi-coupled receptors, but required Src activity (Nishida et al., 2000Go). A future challenge will be to clarify whether silica-induced SFK activation is due to ROS generation or receptor interactions. To characterize the initiating mechanism(s) of the signaling pathways described in this work will be important for the understanding of crystalline silica-induced inflammation, and this is thus a prioritized focus in our current studies.

In summary, our results suggest the presence of two separate signaling pathways which are important in the regulation of silica-induced IL-8 release from A549 cells. One pathway involves SFK-dependent activation of ERK1/2, and the other activation of p38, at least partly independent of SFKs. The results further suggest that similar mechanisms may be involved in MIP-2 release from primary rat T2 cells.


    ACKNOWLEDGMENTS
 
We thank E. Lilleaas, H. S. Hopen, H. J. Dahlman, and T. Skuland for expert assistance throughout the study, and J. A. Holme for valuable discussions. Financial support was provided by the Research Council of Norway.


    NOTES
 

1 To whom correspondence should be addressed at Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo, Norway. Fax: +47 22 04 26 86. E-mail: johan.ovrevik{at}fhi.no.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
American Thoracic Society (1997). Adverse effects of crystalline silica exposure. American Thoracic Society Committee of the Scientific Assembly on Environmental and Occupational Health. Am. J. Respir. Crit. Care Med. 155, 761–768.[ISI][Medline]

Barrett, E. G., Johnston, C., Oberdorster, G., and Finkelstein, J. N. (1999). Silica-induced chemokine expression in alveolar type II cells is mediated by TNF-alpha-induced oxidant stress. Am. J. Physiol. Lung Cell. Mol. Physiol. 276, L979–L988.[Abstract/Free Full Text]

Becher, R., Hetland, R. B., Refsnes, M., Dahl, J. E., Dahlman, H. J., and Schwarze, P. E. (2001). Rat lung inflammatory responses after in vivo and in vitro exposure to various stone particles. Inhal. Toxicol. 13, 789–805.[CrossRef][ISI][Medline]

Chen, J. X., Berry, L. C., Christman, B. W., and Meyrick, B. (2003). Glutathione mediates LPS-stimulated COX-2 expression via early transient p42/44 MAPK activation. J. Cell Physiol. 197, 86–93.[CrossRef][ISI][Medline]

Cuenda, A., and Alessi, D. R. (2000). Use of kinase inhibitors to dissect signaling pathways. Methods Mol. Biol. 99, 161–175.[Medline]

Desaki, M., Takizawa, H., Kasama, T., Kobayashi, K., Morita, Y., and Yamamoto, K. (2000). Nuclear factor-kappa B activation in silica-induced interleukin-8 production by human bronchial epithelial cells. Cytokine 12, 1257–1260.[CrossRef][ISI][Medline]

Ding, M., Shi, X. L., Dong, Z. G., Chen, F., Lu, Y. J., Castranova, V., and Vallyathan, V. (1999). Freshly fractured crystalline silica induces activator protein-1 activation through ERKs and p38 MAPK. J. Biol. Chem. 274, 30611–30616.[Abstract/Free Full Text]

Ding, M., Shi, X. L., Lu, Y. J., Huang, C. S., Leonard, S., Roberts, J., Antonini, J., Castranova, V., and Vallyathan, V. (2001). Induction of activator protein-1 through reactive oxygen species by crystalline silica in JB6 cells. J. Biol. Chem. 276, 9108–9114.[Abstract/Free Full Text]

Driscoll, K. E. (2000). TNF[alpha] and MIP-2: Role in particle-induced inflammation and regulation by oxidative stress. Toxicol. Lett. 112–113, 177–183.[CrossRef]

Furuichi, S., Hashimoto, S., Gon, Y., Matsumoto, K., and Horie, T. (2002). p38 mitogen-activated protein kinase and c-Jun-NH2-terminal kinase regulate interleukin-8 and RANTES production in hyperosmolarity stimulated human bronchial epithelial cells. Respirology 7, 193–200.[CrossRef][ISI][Medline]

Hamilton, R. F., deVilliers, W. J. S., and Holian, A. (2000). Class A type II scavenger receptor mediates silica-induced apoptosis in Chinese hamster ovary cell line. Toxicol. Appl. Pharmacol. 162, 100–106.[CrossRef][ISI][Medline]

Harada, A., Mukaida, N., and Matsushima, K. (1996). Interleukin 8 as a novel target for intervention therapy in acute inflammatory diseases. Mol. Med. Today 2, 482–489.[CrossRef][ISI][Medline]

Hetland, G., Namork, E., Schwarze, P. E., and Aase, A. (2000a). Mechanism for uptake of silica particles by monocytic U937 cells. Hum. Exp. Toxicol. 19, 412–419.[CrossRef][ISI][Medline]

Hetland, R. B., Refsnes, M., Myran, T., Johansen, B. V., Uthus, N., and Schwarze, P. E. (2000b). Mineral and/or metal content as critical determinants of particle-induced release of IL-6 and IL-8 from A549 cells. J. Toxicol. Env. Health - Part A 60, 47–65.[CrossRef][ISI]

Hetland, R. B., Schwarze, P. E., Johansen, B. V., Myran, T., Uthus, N., and Refsnes, M. (2001). Silica-induced cytokine release from A549 cells: Importance of surface area versus size. Hum. Exp. Toxicol. 20, 46–55.[CrossRef][ISI][Medline]

Hoffmann, E., Dittrich-Breiholz, O., Holtmann, H., and Kracht, M. (2002). Multiple control of interleukin-8 gene expression. J. Leukoc. Biol. 72, 847–855.[Abstract/Free Full Text]

Horgan, A. M., and Stork, P. J. S. (2003). Examining the mechanism of Erk nuclear translocation using green fluorescent protein. Exp. Cell Res. 285, 208–220.[CrossRef][ISI][Medline]

Hsu, H. Y., Chiu, S. L., Wen, M. H., Chen, K. Y., and Hua, K. F. (2001). Ligands of macrophage scavenger receptor induce cytokine expression via differential modulation of protein kinase signaling pathways. J. Biol. Chem. 276, 28719–28730.[Abstract/Free Full Text]

Huang, W. C., Chen, J. J., and Chen, C. C. (2003). c-Src-dependent tyrosine phosphorylation of IKKbeta is involved in tumor necrosis factor-alpha-induced intercellular adhesion molecule-1 expression. J. Biol. Chem. 278, 9944–9952.[Abstract/Free Full Text]

Jijon, H. B., Panenka, W. J., Madsen, K. L., and Parsons, H. G. (2002). MAP kinases contribute to IL-8 secretion by intestinal epithelial cells via a posttranscriptional mechanism. Am. J. Physiol. Cell Physiol. 283, C31–C41.[Abstract/Free Full Text]

Jung, Y. D., Fan, F., McConkey, D. J., Jean, M. E., Liu, W., Reinmuth, N., Stoeltzing, O., Ahmad, S. A., Parikh, A. A., Mukaida, N., and Ellis, L. M. (2002). Role of p38 MAPK, AP-1, and NF-[kappa]B in interleukin-1[beta]-induced IL-8 expression in human vascular smooth muscle cells. Cytokine 18, 206–213.[CrossRef][ISI][Medline]

Kamakura, S., Moriguchi, T., and Nishida, E. (1999). Activation of the protein kinase ERK5/BMK1 by receptor tyrosine kinases. Identification and characterization of a signaling pathway to the nucleus. J. Biol. Chem. 274, 26563–26571.[Abstract/Free Full Text]

Kang, J. L., Pack, I. S., Hong, S. M., Lee, H. S., and Castranova, V. (2000). Silica induces nuclear factor-kappa B activation through tyrosine phosphorylation of I kappa B-alpha in RAW264.7 macrophages. Toxicol. Appl. Pharmacol. 169, 59–65.[CrossRef][ISI][Medline]

Kawaguchi, M., Onuchic, L. F., and Huang, S. K. (2002). Activation of extracellular signal-regulated kinase (ERK)1/2, but not p38 and c-Jun N-terminal kinase, is involved in signaling of a novel cytokine, ML-1. J. Biol. Chem. 277, 15229–15232.[Abstract/Free Full Text]

Kim, K. A., Kim, Y. H., Seok Seo, M., Kyu Lee, W., Won Kim, S., Kim, H., Lee, K. H., Shin, I. C., Han, J. S., Joong Kim, H., and Lim, Y. (2002). Mechanism of silica-induced ROS generation in Rat2 fibroblast cells. Toxicol. Lett. 135, 185–191.[CrossRef][ISI][Medline]

Kitagawa, D., Tanemura, S., Ohata, S., Shimizu, N., Seo, J., Nishitai, G., Watanabe, T., Nakagawa, K., Kishimoto, H., Wada, T., Tezuka, T., Yamamoto, T., Nishina, H., and Katada, T. (2002). Activation of extracellular signal-regulated kinase by ultraviolet is mediated through Src-dependent epidereal growth factor receptor phosphorylation. Its implication in an anti-apoptotic function. J. Biol. Chem. 277, 366–371.[Abstract/Free Full Text]

Kumar, A., Knox, A. J., and Boriek, A. M. (2003). CCAAT/Enhancer-binding protein and activator protein-1 transcription factors regulate the expression of interleukin-8 through the mitogen-activated protein kinase pathways in response to mechanical stretch of human airway smooth muscle cells. J. Biol. Chem. 278, 18868–18876.[Abstract/Free Full Text]

Lag, M., Becher, R., Samuelsen, J. T., Wiger, R., Refsnes, M., Huitfeldt, H. S., and Schwarze, P. E. (1996). Expression of CYP2B1 in freshly isolated and proliferating cultures of epithelial rat lung cells. Exp. Lung Res. 22, 627–649.[ISI][Medline]

Li, J., Kartha, S., Iasvovskaia, S., Tan, A., Bhat, R. K., Manaligod, J. M., Page, K., Brasier, A. R., and Hershenson, M. B. (2002). Regulation of human airway epithelial cell IL-8 expression by MAP kinases. Am. J. Physiol. Lung Cell. Mol. Physiol. 283, L690–L699.[Abstract/Free Full Text]

Li, L. F., Ouyang, B., Choukroun, G., Matyal, R., Mascarenhas, M., Jafari, B., Bonventre, J. V., Force, T., and Quinn, D. A. (2003). Stretch-induced IL-8 depends on c-Jun NH2-terminal and nuclear factor-{kappa}B-inducing kinases. Am. J. Physiol. Lung Cell. Mol. Physiol. 285, L464–L475.[Abstract/Free Full Text]

Liu, R., Aupperle, K., and Terkeltaub, R. (2001). Src family protein tyrosine kinase signaling mediates monosodium urate crystal-induced IL-8 expression by monocytic THP-1 cells. J. Leukoc. Biol. 70, 961–968.[Abstract/Free Full Text]

Luttrell, L. M., Della Rocca, G. J., van Biesen, T., Luttrell, D. K., and Lefkowitz, R. J. (1997). Gbeta gamma subunits mediate Src-dependent phosphorylation of the eidermal growth factor receptor. A scaffold for G protein-coupled receptor-mediated Ras activation. J. Biol. Chem. 272, 4637–4644.[Abstract/Free Full Text]

Marshall, C. J. (1995). Specificity of receptor tyrosine kinase signaling: Transient versus sustained extracellular signal-regulated kinase activation. Cell 80, 179–185.[ISI][Medline]

Mody, N., Leitch, J., Armstrong, C., Dixon, J., and Cohen, P. (2001). Effects of MAP kinase cascade inhibitors on the MKK5/ERK5 pathway. FEBS Lett. 502, 21–24.[CrossRef][ISI][Medline]

Mukaida, N. (2003). Pathophysiological roles of interleukin-8/CXCL8 in pulmonary diseases. Am. J. Physiol. Lung Cell. Mol. Physiol. 284, L566–L577.[Abstract/Free Full Text]

Nishida, M., Maruyama, Y., Tanaka, R., Kontani, K., Nagao, T., and Kurose, H. (2000). G alpha(i) and G alpha(o) are target proteins of reactive oxygen species. Nature 408, 492–495.[CrossRef][ISI][Medline]

Olbruck, H., Seemayer, N. H., Voss, B., and Wilhelm, M. (1998). Supernatants from quartz dust treated human macrophages stimulate cell proliferation of different human lung cells as well as collagen-synthesis of human diploid lung fibroblasts in vitro. Toxicol. Lett. 96–7, 85–95.

Puddicombe, S. M., and Davies, D. E. (2000). The role of MAP kinases in intracellular signal transduction in bronchial epithelium. Clin. Exp. Allergy 30, 7–11.[CrossRef][ISI][Medline]

Salh, B. S., Martens, J., Hundal, R. S., Yoganathan, N., Charest, D., Mui, A., and Gomez-Munoz, A. (2000). PD98059 attenuates hydrogen peroxide-induced cell death through inhibition of Jun N-terminal kinase in HT29 cells*1, *2. Mol. Cell Biol. Res. Com. 4, 158–165.[CrossRef][Medline]

Schins, R. P. F., McAlinden, A., MacNee, W., Jimenez, L. A., Ross, J. A., Guy, K., Faux, S. P., and Donaldson, K. (2000). Persistent depletion of I kappa B alpha and interleukin-8 expression in human pulmonary epithelial cells exposed to quartz particles. Toxicol. Appl. Pharmacol. 167, 107–117.[CrossRef][ISI][Medline]

Shukla, A., Timblin, C. R., Hubbard, A. K., Bravman, J., and Mossman, B. T. (2001). Silica-induced activation of c-Jun-NH2-terminal amino kinases, protracted expression of the activator protein-1 proto-oncogene, fra-1, and S-phase alterations are mediated via oxidative stress. Cancer Res. 61, 1791–1795.[Abstract/Free Full Text]

Stringer, B., Imrich, A., and Kobzik, L. (1996). Lung epithelial cell (A549) interaction with unopsonized environmental particulates: Quantitation of particle-specific binding and IL-8 production. Exp. Lung Res. 22, 495–508.[ISI][Medline]

Stringer, B., and Kobzik, L. (1998). Environmental particulate-mediated cytokine production in lung epithelial cells (A549): Role of preexisting inflammation and oxidant stress. J. Toxicol. Env. Health -Part A 55, 31–44.[CrossRef][ISI]

Thomas, S. M., and Brugge, J. S. (1997). Cellular functions regulated by Src family kinases. Annu. Rev. Cell Dev. Biol. 13, 513–609.[CrossRef][ISI][Medline]

Vallyathan, V., Castranova, V., Pack, D., Leonard, S., Shumaker, J., Hubbs, A. F., Shoemaker, D. A., Ramsey, D. M., Pretty, J. R., and McLaurin, J. L. (1995). Freshly fractured quartz inhalation leads to enhanced lung injury and inflammation. Potential role of free radicals. Am. J. Respir. Crit. Care Med. 152, 1003–1009.[Abstract]

Wu, H. M., Wen, H. C., and Lin, W. W. (2002). Proteasome inhibitors stimulate interleukin-8 expression via Ras and apoptosis signal-regulating kinase-dependent extracellular signal-related kinase and c-Jun N-terminal kinase activation. Am. J. Respir. Cell Mol. Biol. 27, 234–243.[Abstract/Free Full Text]

Zhu, W., Downey, J. S., Gu, J., Di Padova, F., Gram, H., and Han, J. (2000). Regulation of TNF expression by multiple mitogen-activated protein kinase pathways. J. Immunol. 164, 6349–6358.[Abstract/Free Full Text]