Introduction

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Mitogen-activated protein kinases (MAPKs) are important regulatory proteins that include the extracellular signal- regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs) or stress-activated protein kinases, and p38 kinases. Activation of MAPK pathways is under intense investigation because of its association with phosphorylation of a number of proteins and transcription factors. Moreover, MAPK signaling is causally linked to transcriptional activation of genes intrinsic to cell proliferation, cell survival, and apoptosis (reviewed in 1). Western blot analyses and in vitro kinase activity assays have been used extensively to investigate the phosphorylation and activity of MAPK proteins in cell and tissue homogenates. However, more sensitive cell imaging methods are necessary to complement these techniques as well as to illustrate cellular patterns of MAPK activation and phenotypic endpoints that may be unique to certain stimuli.

The ERK pathway is stimulated by a number of growth factors as well as various environmental and oxidative stresses and is causally linked to the development of proliferation or apoptosis in a variety of cell types (2). Epidermal growth factor (EGF) is a cytokine studied widely as an activator of ERK via phosphorylation of the EGF receptor (EGFR) (5), whereas hydrogen peroxide (H₂O₂) may activate ERK through EGFR-dependent or -independent pathways (6, 7). The interrelationships between EGF and H₂O₂ are complex, as EGF causes formation of H₂O₂ in cells (8). Moreover, H₂O₂ is required for ultraviolet B (UVB)- induced EGFR and ERK activation in keratinocytes (9).

Parallels also exist between UVB- and asbestos-associated cell signaling events, in that asbestos fibers induce oxidative stress by generating extracellular and intracellular reactive oxygen species, including H₂O₂ (reviewed in 10). Moreover, activation of ERKs by asbestos in mesothelial cells is also mediated through phosphorylation of the EGFR (11), an event triggering increased expression of c-fos and the development of apoptosis (12). In mesothelial cells, ERK activation by asbestos or H₂O₂ can be blocked by antioxidants, suggesting an oxidant-dependent mechanism (14). Interestingly, H₂O₂ and other reactive oxygen and nitrogen species are proposed mediators of asbestos-induced proliferation and toxicity in a variety of cell types (reviewed in 10 and 15). Moreover, these oxidants may activate or modify the EGFR (6, 9, 16). Because of the multiple interrelationships between EGF, H₂O₂, and asbestos described earlier and their implications in the development of a number of fibroproliferative diseases, a goal of the present studies was to determine whether patterns of ERK phosphorylation and nuclear translocation differed in pulmonary epithelial cells in response to these agents. Moreover, we were interested in whether patterns of ERK activation were cell-cycle specific and causally related to cell-cycle changes and functional outcomes, i.e., proliferation and apoptosis. We show here that patterns of ERK activation and phenotypic outcomes are different in response to EGF and the oxidative stresses, H₂O₂ and asbestos. ERK activation governs cell-cycle alterations by these agents, reflected by increased proportions of cells in the G₂/M and S phases, and suggesting a common mechanism to explain the development of apoptosis or proliferation by various ERK stimuli.

	Materials and Methods

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Pulmonary Epithelial Cell Cultures and Exposure to EGF, H₂O₂, and Asbestos

A contact-inhibited, nontransformed murine alveolar type II epithelial cell line (C10) (17) was propagated in CMRL-1066 medium containing penicillin, streptomycin, L-glutamine, and 10% fetal bovine serum (FBS) (GIBCO BRL, Grand Island, NY). For all experiments, cells were grown to near confluency, complete medium was removed, and serumless medium was added 24 h before exposure to agents. Mouse EGF (Upstate Biotechnology, Lake Placid, NY) was added directly to the medium for a concentration of 5 ng/ml, previously shown to cause proliferation and ERK activation in pleural mesothelial cells (11, 12). H₂O₂ (Sigma, St. Louis, MO) was added directly to the medium for concentrations from 100 to 300 µM. Crocidolite asbestos fibers (NIEHS reference sample) were suspended in Hanks' balanced salt solution (HBSS) (1 mg/ml), triturated 10× through a 22-gauge needle to obtain a homogeneous suspension, and added directly to the medium for a concentration of 5 µg/cm² culture dish, previously shown to induce ERK activity and apoptosis in pleural mesothelial cells (11, 18, 19). Sham control cultures received medium without agents and were treated identically. Groups in all experiments consisted of two or three determinations per time point, and all experiments were performed in duplicate.

Immunohistochemistry for Localization of Phosphorylated ERKs

Cell monolayers grown on glass coverslips were exposed to agents as described earlier and examined at various time points from 15 min to 24 h. At each time point, culture dishes were placed on ice, the medium was aspirated, and the cells were washed twice with phosphate-buffered saline (PBS). Cells were fixed in 4% paraformaldehyde in PBS for 30 min at room temperature (RT), then washed in PBS and permeabilized in 100% methanol for 10 min at 20°C followed by 0.1% Triton X-100/PBS for 15 min at RT. Endogenous peroxidase activity was quenched by incubation with 3% H₂O₂ in methanol for 10 min at RT. Cells were then washed in PBS and incubated in blocking buffer (2% goat serum in 10 ml of 0.1% Triton X-100/PBS) for 1 h at RT. For detection of activated/phosphorylated ERKs (pERKs), cells were incubated with an anti-pERK antibody (New England Biolabs, Beverly, MA) at 1:250 dilution in blocking buffer overnight at 4°C. This antibody detects both p44 (ERK1) and p42 (ERK2) phosphorylated proteins. Cells were then washed twice for 20 min in blocking buffer and incubated with a biotinylated secondary antibody (goat antirabbit immunoglobulin [Ig] G; Vectastain ABC Elite kit; Vector Laboratories, Burlingame, CA) for 1 h at RT. After washing in PBS for 5 min, cells were incubated with the ABC reagent for 60 min according to the manufacturer's protocol, then washed twice for 5 min in PBS before incubation with the chromagen 3,3'-diaminobenzidine (DAB) according to the manufacturer's protocol (Vector Laboratories). Cells were washed in H₂O, counterstained with hematoxylin, and mounted on slides using 90% glycerol in H₂O for subsequent examination using light microscopy. Controls consisted of cells stained in the absence of primary antibody.

Localization of pERKs by Immunofluorescence and Confocal Scanning Laser Microscopy

For detection of pERKs by immunofluorescence, cell monolayers grown on glass coverslips were fixed and permeabilized as described earlier, then washed in PBS and incubated in 0.1% Triton X-100/PBS containing 2% nonfat milk for 30 min at RT. Cells were washed in PBS and then in 1% bovine serum albumin (BSA)/PBS before incubation with the anti-pERK antibody (1:250 dilution in 1% BSA/PBS) overnight at 4°C. After washing in 1% BSA/PBS twice for 20 min, cells were incubated with an Alexa 488-conjugated secondary antibody (goat antirabbit IgG; Molecular Probes, Eugene, OR) diluted 1:200 in 1% BSA/PBS for 1 h at RT. After washing in PBS, cells were incubated in propidium iodide (PI) solution (0.1% Triton X-100, 20 µg/ml PI, 0.2 mg/ml ribonuclease [RNase] A, and 0.5 mM ethylenediaminetetraacetic acid [pH 8.0]) for 25 min at RT, then washed and mounted on slides using antifade medium (KPL mounting medium; Kirkegaard and Perry Laboratories, Gaithersburg, MD). Slides were examined using a Bio-Rad MRC1024ES confocal scanning laser microscope (Bio-Rad, Hercules, CA). For each sample, confocal images were collected in the fluorescence modes, followed by electronic merging of the images.

Western Blot Analysis for pERKs

Cells grown in 100-mm culture dishes were washed three times with ice-cold PBS and collected in lysis buffer (20 mM Tris [pH 8.0]), 150 mM NaCl, 1% Triton X-100, 10% glycerin, 1 mM Na₃O₄V, 10 mM NaF, 10 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride), then vortexed for 1 min at 4°C and centrifuged at 14,000 rpm for 20 min. Protein concentrations were determined using the Bradford assay (Bio-Rad), and the soluble cell extracts were diluted in 2× Laemmli sample buffer (125 mM Tris [pH 6.8], 20% glycerol, 4% sodium dodecyl sulfate [SDS], 0.01% bromphenolblue, and 0.4 M -mercaptoethanol). Samples (40 µg protein/lane) were electrophoresed in 12.5% SDS-polyacrylamide gels and electroblotted (Ellard Instrumentation, Seattle, WA) onto nitrocellulose membranes according to standard procedures. Membranes were washed in Tris-buffered saline (TBS) and blocked overnight at 4°C in TBS containing 5% nonfat milk, then incubated with the anti-pERK antibody at a dilution of 1:1,000 in TBS containing 0.1% Tween-20 (TTBS) and 5% BSA overnight at 4°C. Membranes were then washed three times with TTBS and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (antirabbit IgG; Vector Laboratories) diluted 1:2,000 in TTBS containing 5% nonfat milk for 1 h at RT. Membranes were washed three times with PBS, and antibody binding was visualized by enhanced chemiluminescence according to the manufacturer's protocol (Kirkegaard and Perry). Scanning densitometry (Bio-Rad GS700 and Multi-Analyst 1.1 software) was used for quantitation of p44 and p42 in each lane.

Detection and Quantitation of Apoptosis

For detection of apoptosis, cell monolayers grown on glass coverslips were treated with agents as described earlier, then fixed in 100% methanol at 20°C for 24 h. To induce DNA denaturation in situ, cells were heated to 100°C in PBS containing 5 mM MgCl₂ for 5 min, then immersed in ice-cold water for 10 min. After incubation with 40% FBS in PBS on ice for 15 min, cells were incubated with a monoclonal antibody to single-stranded DNA (10 µg/ml, Apostain [F7-26]; Alexis, San Diego, CA) for 30 min at RT, then washed twice in PBS and incubated with a HRP-conjugated secondary antibody (15 µg/ml, goat antimouse IgM; Jackson Laboratories, West Grove, PA) for 30 min at RT. To visualize secondary antibody binding, the peroxidase substrate DAB (Sigma) was used. Cells were washed and mounted on slides in 90% glycerol in PBS for subsequent examination using brightfield light microscopy. To determine numbers of apoptotic cells and total cell numbers per field, four fields were evaluated at ×400 magnification on duplicate coverslips. Apoptosis was confirmed by transmission electron microscopy (TEM). In brief, C10 cells on Thermanox coverslips (13 mm diameter; NUNC, Naperville, IL) were fixed for 30 min in 2% glutaraldehyde/1% formaldehyde in Mellonig's phosphate buffer, postfixed for 30 min in 1% O_sO₄ on ice, dehydrated in a graded series of alcohol, and embedded in Spurr's epoxy resin. Ultrathin sections (~ 60 nm thick) were cut with a diamond knife, retrieved onto copper grids, and contrasted with uranyl acetate and lead citrate. Finally, the sections were examined with a JEOL 1210 transmission electron microscope operating at 60 kV.

Immunofluorescence Technique for Detection of Proliferating Cell Nuclear Antigen

To verify whether agents induced DNA synthesis, an antibody against proliferating cell nuclear antigen (PCNA) was used. PCNA is synthesized in the early G₁ and S phases of the cell cycle and is used as a marker of cell proliferation (20). Cell monolayers grown on glass coverslips were fixed in 100% methanol for 30 min at 20°C, washed in PBS, then washed in PBS containing 1% Triton X-100 (PBST). After incubation in PBS containing 2% nonfat milk and 0.1% Triton X-100 for 1 h at RT, cells were incubated with a biotinylated antibody to PCNA (1:1,000; Pharmingen, San Diego, CA) overnight at 4°C, then washed twice for 20 min in PBST. Primary antibody binding was detected using a 1:200 dilution of streptavidin-conjugated Alexa 568 (Molecular Probes). After washing with PBS, cells were incubated in PI solution for 25 min at RT, then washed with PBS and mounted on slides using antifade medium. Slides were examined using confocal scanning laser microscopy (CSLM) as described earlier. For quantitation of PCNA-positive cells and total cell numbers per field, four fields were evaluated at ×400 magnification on duplicate coverslips.

Cell-Cycle Distribution of Cells Expressing pERKs Using Laser Scanning Cytometry

Because patterns of ERK phosphorylation were most striking and protracted after exposure to asbestos, the cell-cycle distribution of pERKs was determined in sham and asbestos-exposed C10 cells at 2 and 8 h after exposure to fibers (5 µg/cm² dish). Cell monolayers grown on glass coverslips were immunostained using the anti-pERK antibody and the Alexa 488-conjugated secondary antibody, followed by nuclear DNA counterstaining with PI, as described earlier for CSLM. Laser scanning cytometry (LSC) analysis was performed using an instrument manufactured by CompuCyte (Cambridge, MA) with WinCyte 3.3 data analysis software (CompuCyte). Detailed descriptions of LSC methodology have been published previously (21). To perform LSC, scan areas were set to include at least 5,000 cells for analysis on each coverslip. Slides were scanned under a 20× objective using the instrument's 488-nm wavelength argon ion laser with red and green fluorescence detectors. The primary contouring parameter, used to detect and quantify cells, was set on red fluorescence of PI-stained nuclei. Detector gain voltages were set so that a maximum of 75% saturation was achieved for the brightest maximum pixel event scanned. Using the instrument's scan data display, the threshold contour was placed within the nucleus at a distance one-third of the way from the nuclear border; and the integration contour, used to compute nuclear integrated fluorescence, was located 4 pixels outside the threshold contour. Peripheral contours were set to measure integrated fluorescence of the cytoplasm by specifying an inner and outer boundary 10 pixels wide that included the majority of the cytoplasmic rim outside the measured nuclear area. Background was measured outside the cell and automatically subtracted from the nuclear and cytoplasmic integrated fluorescence values. Cell-cycle analyses were performed using ModFit LT data analysis software (Verity Software House, Inc., Portland, ME) with subsequent quantification of percentages of cells expressing pERKs in various phases of the cell cycle.

Cell-Cycle Alterations by Agents with and without Addition of the MAPK/ERK Kinase 1 Inhibitor PD98059 Using Fluorescence-Activated Cell Sorting

Cells were exposed to EGF (5 ng/ml), H₂O₂ (200 µM), or asbestos (5 µg/cm²) in the absence (0.1% dimethyl sulfoxide [DMSO] in medium) or presence of PD98059 (30 µM) (New England Biolabs) diluted in DMSO (final concentration, 0.1% in medium) as described previously (14). In vitro kinase activity assays showed that this concentration of PD98059 inhibited H₂O₂-induced ERK, but not JNK activity (data not shown). At 24, 48, and 72 h, medium and cells trypsinized from plates were centrifuged at 1,200 rpm for 10 min, and cell suspensions were pelleted and filtered through a 53-µM nylon mesh in HBSS (19). Approximately 10⁶ cells were added to 1 ml containing PI (50 µg/ml), Triton X-100 (1%), sodium citrate (4 mM), and RNase (0.5 mg). After a 1-h incubation at 4°C, preparations were analyzed on an Epics Elite Cytometer (Coulter Corp., Miami, FL). A total of 20,000 gated events were analyzed and recorded on a 1,024-linear-channel histogram with Elite software, and the percentages of cells in G₀/G₁, S, G₂M, and subG₀/G₁ were determined.

Statistical Analysis

Results were evaluated by one-way analysis of variance using the Student-Newman-Keuls procedure for adjustment of multiple comparisons. Differences with P values  0.05 were considered statistically significant. All experiments were performed in duplicate with n = 2-3/group/time period.

	Results

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Localization of pERKs in Pulmonary Epithelial Cells (C10)

The goal of initial studies, using the immunoperoxidase technique, was to determine changes in cellular distributions of ERKs after exposure to different stimuli. In control C10 cells, we observed little immunoreactivity for phosphorylated ERKs (Figure 1A). When exposed to EGF, C10 cells showed a generally diffuse activation of ERKs as indicated by increases in immunoreactivity (Figure 1B). The cellular distributions of pERKs ranged from occasional punctate granules at the plasma membrane (Figure 1B, arrowhead) and perinuclear localization (Figure 1B, arrow) to homogeneous cytoplasmic accumulation observed in most cells. Cells exposed to H₂O₂ showed more heterogeneous patterns of pERK immunoreactivity (Figures 1C-1F) with less intense cytoplasmic distribution of pERKs, accompanied by a more common pattern of vesicular accumulation of pERKs and nuclear condensation at 15 and 30 min (Figure 1C, arrow). After 1 h exposure to H₂O₂, cells with pERK-positive vesicles were fewer in number, and striking cytoplasmic and nuclear localization of phosphorylated ERKs were observed (Figures 1D-1F). In contrast to sham controls (Figure 2A), increased intensity and distribution of pERKs were also observed in cells exposed to asbestos (Figure 2B). Membrane vesicles of pERKs were noted in cells phagocytizing long asbestos fibers (Figure 2B, arrow).

View larger version (162K): [in this window] [in a new window]   Figure 1.   Distribution of pERKs in pulmonary epithelial cells using the immunoperoxidase technique. (A) Sham control cells. (B) Exposure to EGF (5 ng/ml) for 1 h. Arrowhead shows occasional punctate granules. Arrow shows perinuclear localization. (C) Exposure to H2O2 (300 µM) for 15 min. Arrow shows prominent vesicular and nuclear accumulation in cell with condensed nucleus. (D) Exposure to H2O2 (300 µM) for 1 h. (E) Exposure to H2O2 (300 µM) for 1 h. (F) Exposure to H2O2 (300 µM) for 2 h. Original magnifications: A, ×500; B, C, E, and F, ×1,000; D, ×25.

View larger version (142K): [in this window] [in a new window]   Figure 2.   Immunoperoxidase methods showing pERKs (A and B) and apoptosis (C-F) in asbestos-exposed pulmonary epithelial cells. (A) Sham control cells. (B) Cells exposed to crocidolite asbestos (5 µg/cm2) for 1 h. Arrow indicates punctate accumulation of pERKs in cell phagocytizing a long asbestos fiber. (C) Apostain technique in sham controls at 24 h. Arrow shows localization in cell exhibiting apoptotic bodies. (D) Cells exposed to H2O2 (300 µM) for 24 h. (E) Field showing distribution of apoptotic cells in areas of deposition of long fibers at 24 h. (F) Cluster of apoptotic cells in area of accumulation of asbestos fibers (5 µg/cm2) at 24 h. Original magnifications: A-D, ×1,000; E, ×25; F, ×2,500.

The time frame of pERK distribution after exposure to different agents was examined over a 24-h period using CSLM (Figure 3). In comparison with sham controls, a rapid, diffuse increase in ERK immunoreactivity was observed within the cytoplasm (Figure 3, green) of EGF- exposed cells within 15 min. Some nuclear localization (Figure 3, yellow) was also noted. Both cytoplasmic and nuclear pERKs were decreased after 1 h exposure to EGF and not apparent at 24 h. Exposure to H₂O₂ also resulted in rapid (15 min) increases in both nuclear and cytoplasmic pERKs, which, like EGF, were markedly decreased after 1 h. In contrast, exposure to asbestos resulted in protracted phosphorylation of ERKs with intense nuclear localization (Figure 3, yellow) at 4 h. pERK-positive cells were still apparent at 24 h. Patterns of ERK1 (p44) and ERK2 (p42) phosphorylation by Western blot analyses were similar to those indicated by immunocytochemistry (Figure 4). Significant increases (P  0.05) in phosphorylation of ERKs, peaking at 15 and 30 min, were seen after addition of EGF or H₂O₂. After exposure to asbestos, significant elevations (P  0.05) in pERKs were observed at 1 h and increased over a 24-h period.

View larger version (60K): [in this window] [in a new window]   Figure 3.   Time frame of pERK accumulation as documented by CSLM using immunofluorescence technique. In comparison with sham control cells, exposure to EGF (5 ng/ml), H2O2 (200 µM), or asbestos (5 µg/cm2) caused increases in both cytoplasmic (green) and nuclear localization (yellow reflecting merged image with PI). PI-stained nuclei appear red. Original magnification: all panels, ×1,000.

View larger version (29K): [in this window] [in a new window]   View larger version (26K): [in this window] [in a new window]   Figure 4.   Patterns of ERK1 (p44) and ERK2 (p42) phosphorylation in pulmonary epithelial cells exposed to agents as shown by Western blot analysis. (A) Western blot; (B) quantitation of results using phosphoimaging (means ± standard error of the mean [SEM]). Open bars, p44; shaded bars, p42. *P  0.05 in comparison with sham control.

Apoptosis and Proliferation in Pulmonary Epithelial Cells Exposed to ERK-Inducing Stimuli

Because transfection and inhibitor studies have linked ERK activation to the development of apoptosis or proliferation in various cell types (2, 7, 14), we examined these phenotypic endpoints in C10 cells exposed to EGF, H₂O₂, or asbestos. For detection of apoptosis, we first used a commercially available antibody to single-stranded DNA (Apostain). As illustrated in Figure 2C, only an occasional apoptotic cell was noted in sham cultures. Increased apoptosis was observed after exposure to H₂O₂ (Figure 2D) as well as asbestos (Figures 2E and 2F), with increased numbers of apoptotic cells generally occurring in areas of accumulation of long asbestos fibers.

Exposure to H₂O₂ caused a dose-dependent increase in apoptosis. Apoptosis was not observed with 100 µM H₂O₂ (data not shown) but was increased in a time-dependent fashion after addition of 200 µM H₂O₂ without significant decreases in total cell numbers (Figure 5C). Proportions of cells exhibiting apoptosis were also increased after exposure to 300 µM H₂O₂ (Figure 5A), but total cell numbers on dishes were reduced significantly (P  0.05) (data not shown). In contrast, exposure to EGF did not induce apoptosis at any time point (Figure 5A), and significant increases (P  0.05) in numbers of S-phase cells, as detected with the PCNA technique, as well as elevations in total cell numbers were seen (Figure 5C). Exposure to asbestos showed striking increases in apoptosis at all time periods (Figure 5A) and increases in PCNA-positive cells at 72 h in the absence of changes in total cell numbers (Figures 5B and 5C). Apoptosis by asbestos was confirmed by TEM (Figure 6), which occurred simultaneously with lytic cell death in these cultures.

View larger version (22K): [in this window] [in a new window]   Figure 5.   Determination of phenotypic endpoints of exposures to EGF (5 ng/ml), H2O2 (200 µM), or asbestos (5 µg/cm2). (A) Quantitation of numbers of Apostain-positive cells; (B) numbers of cells undergoing DNA synthesis as detected using an antibody to PCNA; (C) total cell numbers per field. Data are expressed as means ± SEM. *P  0.05 in comparison with sham group at each time point.

View larger version (223K): [in this window] [in a new window]   Figure 6.   TEM photograph showing apoptosis in epithelial cells exposed to asbestos (5 µg/cm2) for 24 h. Note the condensation of chromatin in the nucleus (N) and apoptotic bodies (*). Original magnification: ×8,000.

Changes in Cell-Cycle Distribution and Expression of pERKs by LSC

Because exposure to asbestos induced a unique pattern of protracted activation and nuclear translocation of ERKs, we focused on whether expression of pERKs was cell cycle-related in epithelial cells with and without addition of asbestos fibers (5 µg/cm² dish) at 2, 4, and 8 h, i.e., time points showing significant increases in activated ERKs as indicated by Western blot analyses. A representative display of LSC measurement of cell-cycle distribution and pERK expression in sham control and asbestos-exposed (4 h) cells is shown in Figure 7. At all time points, over 90% of sham control cells were in G₀/G₁, with only 1% in S phase, 5% in G₂/M, and 1 to 2% in the subdiploid fraction. Although similar cell-cycle distributions were observed in asbestos-exposed cells, the S phase and G₂/M fractions appeared to expand with longer exposure times. A significant increase (P  0.05) in the proportion of cells in G₂/M occurred at 8 h (data not shown).

View larger version (27K): [in this window] [in a new window]   Figure 7.   Representative displays of LSC measurements of cell-cycle distribution (DNA content) and pERK expression in pulmonary epithelial cells. DNA histograms show the subdiploid (sub), G0/G1, S phase (S), and G2/M fractions in sham control cells (A) and in cells exposed to asbestos (5 µg/cm2) for 4 h (B). Beneath each histogram is the corresponding scatter plot showing pERK expression in relation to the cell cycle in sham control cells (C) and in cells exposed to asbestos (5 µg/cm2) for 4 h (D). pERK was detected immunocytochemically using an Alexa 488-conjugated secondary antibody. Nuclear DNA was counterstained with PI. At least 5,000 cells were analyzed for each sample. Gated region in scatter plots (C and D) indicates the population of pERK-positive cells.

Table 1 shows the percentages of pERK-positive cells per total cell number in sham control versus asbestos- exposed groups at 2 and 8 h (Table 1, third column). At both time points, groups exposed to asbestos exhibited approximately 3-fold increases in the percentage of cells expressing pERKs in comparison with sham controls. Subsequently, the percentage of pERK-positive cells in each compartment of the cell cycle (Table 1, right columns) was assessed individually. At 2 and 8 h, ERK immunoreactivity was expressed in less than 10% of sham control cells in the subdiploid and G₀/G₁ fractions, and 27 to 35% of cells in the G₂/M and S fractions. In asbestos-exposed groups, patterns of cell-cycle distribution of ERKs were similar to sham controlsless than 10% of cells with a subdiploid DNA content expressed pERKs, and the majority of cells exhibiting immunoreactivity (40 to 57%) were in G₂/M or S. 

In comparison with sham controls or groups with addition of PD98059 alone, ERK stimuli induced increases in the percentage of cells in G₂/M at 24 h that were significantly inhibited (P  0.05) by PD98059 (Figure 8). This compound also abrogated more persistent increases in G₂/M cells occurring at 48 h after exposure to H₂O₂ or asbestos. S-phase elevations by EGF and H₂O₂ at 24 h and by asbestos at 72 h, a time point corresponding to increases in PCNA-positive cells by asbestos (Figure 5), were also diminished by blocking ERK activation.

View larger version (39K): [in this window] [in a new window]   Figure 8.   Effects of the MEK1 inhibitor PD98059 (30 µM) on proportions of cells in G2/M or S phase after exposure to EGF (5 ng/ml), H2O2 (200 µM), or asbestos (5 µg/cm2) using fluorescence-activated cell sorter. Data are expressed as means ± SEM of duplicate or triplicate determinations. *Significantly increased (P  0.05) from sham control. + Significantly different (P  0.05) from groups without PD98059.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We show here that cell-imaging techniques can detect different patterns of cytoplasmic and nuclear localization of pERKs in pulmonary epithelial cells exposed to diverse stimuli. Patterns of pERKs by immunocytochemical approaches using CSLM and LSC correlated directly with profiles of kinase activation using Western blot analyses. Moreover, cell-imaging techniques were vital to establishing subsequent end points of proliferation and apoptosis in response to agents as well as determining the cell-cycle kinetics and proportions of pERK-positive cells in different phases of the cell cycle.

Biochemical and cell fractionation studies have indicated that ERKs translocate from the cytoplasm to the nucleus where they phosphorylate transcription factors and are required for growth factor-induced gene expression and cell-cycle entry (22). Phosphorylation of ERK2 promotes its homodimerization and active transport to the nucleus, but passive diffusion of ERK2 monomers may be a more predominant route for entering the nucleus (25). Our studies using immunocytochemistry revealed that cellular patterns of cytoplasmic ERK phosphorylation and nuclear translocation differ in response to disparate ERK stimuli. With EGF, a diffuse cytoplasmic localization predominated followed by rapid nuclear accumulation of pERKs within a 30-min period. EGF-induced ERK phosphorylation preceded increased numbers of cells in S phase and cell proliferation as documented by elevations in total cell numbers. Although Western blot analyses showed that the time frames of increased ERK activity by EGF and H₂O₂ were similar, a unique pattern of granular or vesicular-like accumulations of ERK predominated in H₂O₂-exposed cells as early as 15 min after exposure. Subsequently, these vesicles became consolidated in condensed cells exhibiting apoptosis. Because similar-appearing vesicles are associated with early depolymerization of cytoskeletal proteins and cell contraction observed after addition of 200 µM H₂O₂ to human umbilical cord vein endothelial cells (26), and activated focal adhesion kinase (FAK) is seen in vascular endothelial cells at higher concentrations of H₂O₂ (27), we used an antibody to FAK to determine whether it exhibited similar patterns of accumulation and colocalization with pERKs. These results show that pERKs did not colocalize with FAK (data not shown). The differences in patterns of cytoplasmic phosphorylation of ERKs observed with EGF versus H₂O₂ are intriguing and may be related to the development of different outcomes, i.e., proliferation versus apoptosis, after exposures to these agents.

In contrast to EGF and H₂O₂, asbestos is an insoluble fiber causing delayed and persistent activation of ERKs, i.e., from 1 to 24 h. Patterns of cytoplasmic ERK accumulation most resembled those seen with H₂O₂, but were restricted to areas of fiber deposition and accumulation. Inasmuch as fibers persist and are encompassed or phagocytized by cells, these phenomena might account for the prolonged increases in nuclear localization and ERK activity as documented here using CSLM and Western blot analyses. Subsequent phenotypic responses to asbestos included both striking and early apoptosis and delayed but significant increases in S phase and PCNA-positive cells in the absence of increases in total cell numbers. These results suggest that apoptosis may trigger compensatory cell proliferation which may be dynamic processes occurring in preneoplasia (28).

LSC was used as a novel tool to determine whether expression of pERKs occurred in cells in specific phases of the cell cycle. This technique revealed that in both sham control and asbestos-exposed groups, accumulation of pERK protein predominated in cells in the S and G₂/M phases of the cell cycle. More importantly, inhibition of ERK phosphorylation by the MAPK/ERK kinase (MEK) 1 inhibitor PD98059 inhibited both S- and G₂/M-phase alterations by ERK stimuli. In the case of the mitogenic agent EGF, progression from S phase into G₂/M resulted in cell proliferation. In contrast, after exposure to toxic agents such as asbestos and H₂O₂, cells entered the S phase, often as a result of unscheduled DNA synthesis, and could be arrested in G₂/M preceding apoptosis. Thus, our findings are consistent with the hypothesis that early expression of pERKs in S and G₂/M by these oxidative stresses precedes the development of apoptosis (12, 14). A causal relationship between ERK activation and apoptosis has been demonstrated in asbestos-exposed pleural mesothelial cells after pretreatment with PD98059 (14). Moreover, hyperoxia-induced apoptosis in macrophages is inhibited after addition of PD98059 and with use of dominant negative mutants of ERK (3). The unique and protracted increases in S-phase cells by asbestos, as documented by increases in PCNA-positive cells at 72 h, may reflect a dual role of ERK activation in compensatory hyperplasia or cell survival after early cell injury by asbestos.

Our results here, suggesting a role of ERK in cell-cycle progression by mitogenic stimuli, provide insight into the limited observations on ERK activation in epithelial cells and tumors using in vivo models. In an experimental model of colon carcinoma, tumor growth is diminished by a small molecule MEK inhibitor, thus indicating involvement of the ERK pathway in propagation and progression of epithelial tumors (29). Moreover, increases in pERKs have been found using immunocytochemistry in low-grade and malignant gliomas, suggesting that activation of ERKs may be critical in instigation of cell proliferation or malignant progression (30). After injection of the lung carcinogen urethane, increased immunoreactivity of pERKs occurs in alveolar epithelial cells (31), presumably an early response to pulmonary carcinogens.

Several reports also suggest a role of ERK in epithelial cell repair. For example, repair of ischemic injury in intestinal epithelium is triggered by binding of transforming growth factor- to the EGFR and activation of ERKs (32). During healing of experimental gastric ulcers, nuclear translocation of ERKs is a prominent response that accompanies increased c-fos expression (33). ERKs also may be important in proliferation or repair in lung fibrogenesis, inasmuch as pERKs localize in epithelial cells at sites of developing lesions after inhalation of asbestos fibers (34). The causal relationship between ERK activation, apoptosis, and epithelial cell proliferation by asbestos is currently being evaluated using transgenic approaches to create dominant negative MEK mutants with lung epithelial cell-specific promoters.