61, 83–91 (2001)
Copyright © 2001 by the Society of Toxicology
TOXICOLOGICAL SCIENCES
Fluoride-Induced Apoptosis in Epithelial Lung Cells Involves Activation
of MAP Kinases p38 and Possibly JNK
E. V. Thrane,* M. Refsnes,* G. H. Thoresen,† M. Låg,* and P. E. Schwarze* ,1
*Department of Environmental Medicine, National Institute of Public Health, Oslo, Norway; and †Department of Pharmacology,
Faculty of Medicine, University of Oslo, Oslo, Norway
Received October 25, 2000; accepted December 12, 2000
The mechanisms by which fluoride exerts its diverse effects
have been investigated to some extent. Several studies have
indicated the formation of an aluminum-fluoride complex
(AlF 4-) and a subsequent activation of GTP-binding G-proteins
in eliciting the fluoride-induced effects (Refsnes et al., 1999a;
Sternweis et al., 1982). The further propagation of the Gprotein activating signal has only been elucidated to a minor
extent, but some studies indicate that the G-protein regulator
RGS3, protein kinase C (PKC), the kinase p38, and a serum
response factor may each play a role in the signal transduction
(Dulin et al., 1999; Heidenreich et al., 1999; Schwarze et al.,
2000). Fluoride may also elicit some of its effects through
inhibition of phosphatases, thus, permanently activating protein kinases (Heidenreich et al., 1999; Wergedal et al., 1992).
However, it has been reported that fluoride did not inhibit
tyrosine phosphatases at mitogenic concentrations in bone
cells, indicating that phosphatases are not involved in all of the
fluoride-induced effects (Caverzasio et al., 1998).
Mitogen-activated protein (MAP) kinases (ERK, p38, JNK)
participate in the transmission of signals to the cell nucleus.
The activity of these kinases has been associated with the
initiation of diverse processes in different cells. ERK 1 and 2
are part of the ras/raf/MEK pathway often associated with
proliferation and survival (Lewis et al., 1998). ERK-regulating
kinase (MEK) has been shown to activate ERKs, but MEKindependent activation has also been reported (Band and Posner, 1997; Grammer and Blenis, 1997). Activation of ERKs
plays an important role in alveolar lung cell and bone cell
proliferation (Thrane et al., 1999, Wu et al., 1997). In contrast,
in a nerve cell-derived cell line (PC 12) ERK plays an important role in differentiation (Traverse et al., 1992). ERK activation seems to promote survival in most cell types (Wang et
al., 1998), but it has also been reported that induction of
apoptosis may be mediated via ERK (Ishikawa and Kitamura,
1999). The other MAP kinases (JNK and p38) have been
implicated primarily in the induction of apoptosis and inflammation after exposure to different agents (Lewis et al., 1998;
MacFarlane et al., 2000; Obata et al., 2000). However, several
isoforms of JNK and p38 have been identified that may have
different functions and localization (Gupta et al., 1996; Jiang et
Exposure to fluorides can induce inflammatory reactions, cell
cycle arrest, and apoptosis in different experimental systems. Fluorides are known G-protein activators, but less is known about
fluoride effects downstream of G-protein activation. The aim of
this study was to elucidate whether the induction of apoptosis by
fluorides and inhibition of proliferation is mediated by MAP kinases in primary rat lung, alveolar type 2 cells and the human
epithelial lung cell line A549. Sodium fluoride (NaF) induced
apoptosis in both cell types but at different concentrations, with
the primary cells being more sensitive to NaF. Proliferation of the
type 2 cells and A549 cells was inhibited in the presence of NaF.
NaF induced a prolonged activation of MAP kinase ERK. NaF
also activated p38 and JNK in A549 cells for several hours (maximally 6-fold and 3-fold increase, respectively). Inhibition of ERK
with the MEK1,2 inhibitor PD98059 increased apoptosis 2-fold,
whereas the inhibitor of p38, SB202190, decreased the level of
apoptotic cells by approximately 40%. SB202190 also inhibited
apoptosis by almost 40% when ERK activity was reduced in the
presence of PD98059. Neither PD98059 nor SB202190 did affect
the NaF-induced inhibition of proliferation. These observations
indicate that activation of MAP kinases p38 and possibly JNK are
involved in NaF-induced apoptosis of epithelial lung cells,
whereas ERK activation seems to counteract apoptosis in epithelial lung cells. In contrast, activation of ERK and p38 are not
involved in NaF-induced inhibition of cell proliferation.
Key Words: epithelial lung cells, fluoride, proliferation, apoptosis, MAP kinases.
Exposure to fluorides has been shown to elicit inflammatory
responses in human volunteers (Lund, K. et al., 1999). Epithelial lung cells release increased amounts of inflammatory cytokines in response to fluoride exposure (Refsnes et al., 1999a).
At high concentrations, fluoride has also been found to inhibit
protein synthesis and cell cycle progression (Aardema et al.,
1989; Holland, 1979). It has also been reported that epithelial
lung cells and alveolar macrophages undergo apoptosis after
fluoride exposure (Hirano et al., 1996; Refsnes et al., 1999b).
1
To whom correspondence should be addressed at the Department of
Environmental Health, NIPH, 4404 Torshov, 0403-Oslo, Norway. Fax: ⫹ 47
22 04 22 43. E-mail: [email protected].
83
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THRANE ET AL.
al., 1997). The expression of the different isoforms of p38 or
JNK in lung cells is not known. The published data indicate
that the role of JNK and p38 in apoptosis seems to differ
between cell types (Kang et al., 2000). Thus, it has been
reported that p38 and JNK activation may protect against the
induction of apoptosis (Minamino et al., 1999). However, in
general, ERK and p38/JNK seem predominantly to support the
activation of a survival and a death pathway, respectively.
Furthermore, opposing activities of ERK in relation to JNK/
p38 have been reported (Liu and Holbrook, 1998); Wang et al.,
1995).
A marked increase in the rate of proliferation in adult lung
tissue occurs when lost cells need to be substituted, e.g., after
injury by toxic agents. Epithelial, alveolar type 2 cells have
been shown to proliferate in vivo and in vitro (Kauffman, 1980;
Lag et al., 1996). Many agents, that also stimulate proliferation
of type 2 cells, are known, at least in part, to elicit their effects
through the ras/MAP kinase ERK pathway in other cell types
(Meloche et al., 1992; Thoresen et al., 1998). In contrast,
agents that inhibit this signal pathway are often found to inhibit
proliferation.
Apoptosis is often viewed as a tissue’s possibility to get rid
of damaged cells without inducing an inflammatory reaction,
which might harm more cells. Epithelial lung cells can also be
induced to undergo apoptosis. The agent paraquat has been
shown to elicit apoptosis in these cells (Cappelletti et al.,
1998). However, the precise mechanisms by which these processes are induced and the roles of different MAP kinases in
these processes are still obscure. In this study we have examined the importance of different MAP kinases in apoptosis and
proliferation of primary type 2 cells and a human alveolar
epithelial lung cell line following exposure to NaF.
MATERIALS AND METHODS
Chemicals. Insulin, hydrocortisone, transferrin, epidermal growth factor
(EGF), protease (type 1: crude), DNAse I (type III), N-(2-hydroxyethyl)piperazine-N⬘-(2-ethanesulfonic acid) (HEPES), phenylmethylsulfonyl fluoride
(PMSF), Tris [hydroxymethyl] amino methane (TRIS), ethylene glycol bis(␤aminoethyl ether)-N,N,N⬘,N⬘-tetraacetic acid (EGTA), orthovanadate, dithiothreitol (DTT) and Hams F12 medium with glutamine were obtained from
Sigma Chemical Company (St.Louis, MO). Williams’s E medium without
glutamine was from Bio Whittaker (Walkersville, MD), fetal bovine serum
(FBS) from Gibco BRL (Paisly, Scotland), Hoechst 33258, 2(2-(4-hydroxyphenyl)-6-benzimidazole-6-(1-methyl-4-piperazyl) benzimidazole hydrochloride), from Calbiochem-Boehringer (La Jolla, CA). Leupeptin and aprotinine
were from Amersham, Life Science (Buckinghamshire, U.K.). 2⬘-Amino-3⬘methoxyflavone (PD98059) and 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5(4-pyridyl)1H-imidazole (SB 202190) were obtained from Calbiochem (Nottingham, U.K.). Sodium fluoride (NaF) (pa) was from Riedel DeHaen AG,
(Hannover, Germany). All other chemicals were of analytical grade.
Animals. Male rats (WKY/NHsd), 180 –230 g body weight, were purchased from Harlan, Bicester, U.K. The animals were given Ewos R3 standard
pelleted laboratory chow from Astra Ewos AB, Sweden, and water ad libitum.
Lung cell cultures. Primary type 2 cells were isolated by centrifugal
elutriation of a cell suspension prepared from perfused lungs enzymatically
digested with protease, as previously described Lag et al., 1996). The cell
population was purified by differential attachment. Cell viability, as judged by
trypan blue exclusion, exceeded 90%. The cells were cultured in Williams’s E
medium supplemented with insulin (5 ␮g/ml), hydrocortisone (5 ␮g/ml),
ampicillin (100 ␮g/ml), streptomycin (100 ␮g/ml), fungizone (0.25 ␮g/ml),
HEPES (15 mM), EGF (10 ng/ml) and 5% inactivated FBS. The cultures were
serum-starved for 1 or 2 days before exposure to fluoride.
The human epithelial lung cell line, A549, from the American Type Culture
Collection (ATCC Rockville, MD, USA) was cultured in Costar 10-cm 2 wells,
28-cm 2 dishes or 75-cm 2 flasks (0.1 ml medium/cm 2) in Hams medium,
supplemented with ampicillin (100 ␮g/ml), streptomycin (100 ␮g/ml), fungizone (0.25 ␮g/ml) and 10% inactivated FBS. The cells (passage numbers
76 –100) were seeded at low density and grown for 5 days, before they were
serum-starved for 2 days prior to exposure to NaF.
Flow cytometry. The percentage of apoptotic cells and cells in the S- and
G 2-phases of the cell cycle was determined by flow cytometry. Type 2 and
A549 cells were exposed for various time periods to 1 or 5 mM NaF,
respectively, or to different concentrations of NaF for up to 30 h. A549 cells
were pretreated for 1 h with PD98059 (25 ␮M or 50 ␮M) or SB202190 (5– 40
␮M), and subsequently exposed for 20 h to 5 mM NaF. After exposure, the
cells were prepared for flow cytometry with Triton X-100 (0.1%) and Hoechst
33258 (1 ␮g/ml) for staining of cellular DNA. The histograms were recorded
on a Skatron Argus flow cytometer and analyzed using the Multiplus Program
(Phoenix Flow Systems, San Diego, CA). The apoptotic index was determined
as the percentage of signals between the G 1 peak and the channel positioned at
20% of that peak.
DNA electrophoresis. DNA fragmentation was performed according to the
method of Gorczyca et al., (1993). Briefly, harvested cells (2.5 ⫻ 10 6) were
washed in PBS (phosphate-buffered saline without Ca2⫹ and Mg 2⫹), resuspended
in 0.25 ml of TBE (45 mM Tris borate buffer, 1 mM EDTA, pH 8.0) containing
0.25% Nonidet P-40 and 0.1 mg/ml RNase A, and incubated at 37°C for 30 min.
After treatment with proteinase K (1 mg/ml final concentration) for 30 min, the
samples were subjected to ultrasonic treatment (Transsonic 460 bath) for 1 min.
Fifty ␮l loading buffer (0.01 ml 1 M Tris, pH 7.5; 0.04 ml 0.5 M EDTA, pH 7.5;
0.5 ml glycerol (85%); 0.8 mg bromophenol blue; H2O to 1 ml) was added. The
samples were incubated at 65°C for 10 min just prior to application to the gel.
Horizontal agarose gels (1.5%) were run for 3 h at room temperature at 2V/cm.
The DNA bands were visualized under UV light in gels run with GelStar, as
described in the manufacturer’s instructions (FMC).
MAP kinase ERK activity. A549 cell cultures and type 2 cells were
exposed to 5 mM or 1 mM NaF, respectively. For determination of MAPkinase ERK activity, the cell cultures were lysed and homogenized in a buffer
containing TRIS (10 mM), NaCl (150 mM), EGTA (2 mM), DTT (2 mM),
orthovanadate (1 mM), PMSF (1 mM), leupeptin (10 ␮g/ml), and aprotinine
(10 ␮g/ml). Activity was assessed using the Biotrak p42/p44 MAP kinase
enzyme assay (Amersham, Buckinghamshire, UK). The extent of phosphorylation was analyzed on a liquid scintillation analyzer (Packard 1500 TRICARB, Packard Instrument Co., Inc. Downers Grove, IL).
Immunoprecipitation and JNK/p38 kinase assays. JNK and p38 kinase
activities were measured as described by Dixon et al. (1999) with minor
modifications. Briefly, A549 cell cultures were exposed to NaF for the indicated time period, and the cells were rinsed twice in saline and scraped into a
buffer containing 20 mM TRIS pH 7.4, 137 mM NaCl, 2 mM EDTA, 25 mM
␤-glycerophosphate, 1 mM benzamidine, 10 ␮g/ml leupeptin, 10 ␮g/ml pepstatin A, 1 mM Na 3VO 4, 2 mM pyrophosphate, 10% (v/v) glycerol, and 1%
(v/v) Triton X-100. The cells were disrupted by passage through a syringe. The
lysate was centrifuged (15,000 ⫻ g) for 10 min and JNK and p38 were
immunoprecipitated from 200 ␮l of supernatant with specific polyclonal antibodies (Santa Cruz Biotechnology, Inc.) prebound to protein A-sepharose
CL-4. Kinase assays of the immunocomplexes were performed in a buffer
containing 12.5 mM HEPES pH 7.4, 12.5 mM ␤-glycerophosphate, 22.5 mM
MgCl 2, 0.05 mM Na 3VO 4, 1.1 mM DTT, 0.5 mM ATP (including 250 ␮Ci/ml
of ␥ 32P-ATP), and with 50 ␮g/ml of the substrates MAPKAPK2-GST (kindly
supplied by Dr. P. Sugden, Imperial College, London, U.K.) or c-jun-GST
(Santa Cruz Biotechnology, Inc.). After electrophoresis on SDS–polyacrylam-
MECHANISM OF FLUORIDE-INDUCED APOPTOSIS IN LUNG CELLS
85
cells after readdition of serum to the medium. Whereas the
number of cells in S- and G 2-phase of the cell cycle significantly (p ⫽ ⬍0.001) increased from approximately 27% to
more than 40% by 20 h after serum addition, in the absence of
NaF, the percentage of cells with more than G 1-phase DNA
content remained unaltered in the presence of NaF (5 mM).
The inhibition of proliferation was dose-dependent as shown in
Figure 1B. Maximal inhibition was achieved at 3.75 (p ⫽
0.013, compared to control) to 5 mM NaF. A similar inhibition
of proliferation was observed in type 2 cells at lower concentrations (data not shown). As measured by flow cytometry, an
increase in the percentage of apoptotic A549 cells was observed (Fig. 2) concomitant with an inhibition of proliferation
(Fig. 1). NaF (5 mM) induced a significant increase in apoptotic cells at 10 h after the start of exposure, and this number
increased up to almost 13% at 30 h (p ⫽ 0.021). A roughly
similar time relationship between fluoride exposure and induction of apoptotic cells was obtained by microscopic analysis
(Refsnes et al., 1999b). Qualitatively similar results were observed with type 2 cells (Figs. 3A–3C). However, the fluorideinduced percentage of apoptotic cells was much higher in type
2 cell cultures (48% at 20 h; Fig. 3A) than in A549 cells (Fig.
2). An induction of apoptosis was noted by 2 to 5 h after the
start of NaF exposure and significantly increased after 8 h (p ⫽
0.021). Furthermore, significant increase in apoptosis was
found at 0.5 mM NaF (p ⫽ ⬍0.001). A maximal increase in
apoptosis was induced in type 2 cells at 1 mM NaF compared
to 3.75 to 5 mM in A549 cells (Fig. 3B). The analysis of DNA
fragmentation confirmed the flow cytometry results in type 2
cells with 1 mM NaF (Fig. 3C).
FIG. 1. Inhibition of proliferation of A549 cells by NaF. (A) Timedependent effect of NaF (NaF 5 mM, filled circle; control, open circle); *p ⫽
⬍ 0.001. (B) Concentration-dependent effects of NaF 20 h after start of
exposure. The percentage of cells in the S- and G 2-phases of the cell cycle was
determined by flow cytometry. Results are presented as the mean ⫾ SE of 3
experiments; *p ⫽ 0.003.
ide gels, the phosphorylation of substrate proteins was detected by autoradiography. Optical density of autoradiograms was measured by an image analysis
system.
Statistical analysis. The data were analyzed using the Student’s t-test to
assess the difference between the control and treated groups. A one-way
analysis of variance (ANOVA) was performed on the results in Figure 8,
followed by a pairwise multiple comparison (Tukey test) between the groups
to determine the significance of mean differences among treatments.
RESULTS
Inhibited entry into S- and G 2-phases and induction of
apoptosis in A549 and type 2 cells by fluoride. Figure 1A
shows the time-dependent stimulation of proliferation of A549
FIG. 2. Induction of apoptosis in A549 cells by NaF. Time-dependent
increase in the percentage of apoptotic cells, as determined by flow cytometry
during exposure to NaF (NaF 5 mM, filled circle; control, open circle). Results
are presented as one typical experiment out of 3, with mean ⫾ SE of 3
parallels; *p ⫽ 0.021.
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THRANE ET AL.
and 245% of the control value. In the presence of the MEK1,2
inhibitor PD98059, the NaF-induced activity of ERK was
inhibited approximately to control levels or somewhat below,
except at the 4-h time point, when the activity was comparable
to NaF-induced levels without inhibitor (Fig. 4B). The effect of
NaF on ERK in type 2 cells is displayed in Fig. 4C. Apparently
exposure to NaF resulted in a biphasic activation of ERK. NaF
induced a small, significant increase (50%) in ERK activity at
10 min (p ⫽ 0.016), which declined to low levels by 30 min.
Two h after addition, a maximal activity of 200% compared to
FIG. 3. Induction of apoptosis in type 2 cells by NaF. (A) Time-dependent
increase in the percentage of apoptotic cells exposed to NaF (NaF 1 mM, filled
circle; control, open circle) as determined by flow cytometry; *p ⫽ 0.021. (B)
Concentration-dependent increase of apoptotic cells after 20 h of exposure to
NaF; *p ⫽ 0.001. Results in A and B are presented as the mean ⫾ SE of 4 and
5 experiments, respectively. (C) DNA fragmentation of type 2 cells exposed to
NaF (1 mM) for 20 h. Results of one experiment are shown.
MAP kinase ERK activity in type 2 and A549 cells. Both
the inhibitory effects on proliferation and the induction of
apoptosis may be mediated by different MAP kinases. In A549
cells, NaF induced a dose-dependent stimulation in ERK activity (Fig. 4A) with a steady increase from 2.5 mM NaF to 7.5
mM. At 5 mM NaF, the activity increased approximately
4-fold. In time-dependent activity measurements, there was a
significant increase (p ⬍ 0.05) in ERK activity (40%) during
the first 30 min after start of exposure to NaF (Fig. 4B). During
the next 3.5 h, the activity varied between approximately 210
FIG. 4. ERK activity in A549 cells and type 2 cells during exposure to
NaF. (A) and (B) are A549 cells; (C) is type 2 cells. (A) Concentrationdependent activity in A549 cells; *p ⫽ 0.003. (B) Time-dependent alterations
in activity in A549 cells in the absence (open square) and presence (filled
square) of PD98059 (25 ␮M); *p ⫽ 0.014), **p ⫽ 0.006. (C) Time-dependent
alterations in activity in type 2 cells; *p ⫽ 0.016, **p ⫽ 0.036). Results are
presented as a percentage of control of the mean ⫾ SE of 3, 3–5, and 5
experiments for (A), (B), and (C), respectively.
MECHANISM OF FLUORIDE-INDUCED APOPTOSIS IN LUNG CELLS
87
inhibitor PD98059 (25 and 50 ␮M) resulted in an approximate
doubling of the percentage of apoptotic cells (p ⫽ 0.009),
whereas there was no significant effect on the NaF-induced
inhibition of proliferation (Figs. 6A and 6B). Basal apoptotic
numbers were not affected, even at higher inhibitor concentrations. Inhibition of p38 with 5 ␮M SB202190 did not affect the
NaF-induced inhibition of proliferation (Fig. 7B). In contrast, 5
␮M SB202190 reduced the number of NaF-induced apoptotic
cells by approximately 40% (p ⫽ ⬍0.001), whereas there were
no changes in the basal levels (Fig. 7A). Higher concentrations
of SB202190 did not exert an additional effect. The increased
apoptosis by NaF and PD98059, calculated as a percentage of
the NaF-induced apoptosis, was significantly inhibited by approximately 30% (p ⫽ 0.045) in the presence of the p38
inhibitor SB 202190 (Fig. 8).
FIG. 5. Time-dependent changes in activity of p38 and JNK in A549 cells
after exposure to NaF (5 mM). (A) Blots of phosphorylated p38 and JNK
substrates (see Materials and Methods). (B) Densitometric measurements of
blots, p38 (triangle) and JNK (circle). Results are presented as percent of
control. A typical experiment out of 3 is shown.
control was observed. The activity had not returned to basal
levels by 4 h.
P38 and JNK activities in A549 cells. P38 and JNK have
been implicated in stress-related responses and the induction of
apoptosis. Figure 5A shows blots of p38 and JNK activity after
exposure of A549 cells to NaF. Marked increases in activity
were observed 1 to 4 h after the start of exposure, with roughly
similar time courses for both kinases. The P38 activity was
augmented approximately 6-fold at 2 h, whereas the JNK
activity was almost 3-fold higher than control (Fig. 5B). The
elevated activities were sustained for at least 4 h after addition
of NaF.
Effects of inhibition of ERK and p38 MAP kinases on proliferation and apoptosis of A549 cells. In the presence of 5
mM NaF, the inhibition of ERK activity with the MEK1,2
FIG. 6. Inhibition of proliferation and induction of apoptosis in A549 cells
exposed to NaF (20 h) after pretreatment with the MEK inhibitor, PD98059.
Open bars, no NaF; filled bars, NaF (5 mM) with different concentrations of
PD98059. (A) Percentage of apoptotic cells, *p ⫽ 0.009. (B) Percentage of
cells in S-and G 2-phases of the cell cycle. Results of flow cytometric analysis
are presented as the mean ⫾ SE of 4 experiments.
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THRANE ET AL.
FIG. 7. Inhibition of proliferation and induction of apoptosis in A549 cells
exposed to NaF after pretreatment with the p38 inhibitor SB202190. Open
bars, no NaF; filled bars, NaF (5 mM) with different concentrations of
SB202190. (A) Percentage of apoptotic cells, *p ⫽ ⬍0.001. (B) Percentage of
cells in S- and G 2-phases of the cell cycle. Results of flow cytometric analysis
are presented as the mean ⫾ SE of 4 experiments.
cells from rats and humans. The increased apoptosis seemed to
be related to an activation of MAP kinase p38 and possibly
JNK.
Several studies have indicated that the stress-related kinase
p38 is mainly associated with the induction of responses such
as cytokine release and apoptosis (Lewis et al., 1998). Various
chemically and biologically different agents have been found
to mediate the induction of apoptosis through the activation of
p38 (Chuang et al., 2000; Lewis et al., 1998). The activation of
p38 induced by NaF in epithelial lung cells was associated with
an increase in the number of apoptotic cells. Inhibition of p38
by SB202190 decreased the number of apoptotic cells. However, the reduction in apoptosis was only partial, suggesting
that NaF may also induce apoptosis by other pathways than
p38. Alternatively, the inhibitor SB202190 may not have inhibited p38 activity completely, even when high concentrations
were used. It has been shown that SB202190 is not equally
effective against all p38 isoforms (Lee et al., 1999; Nemoto et
al., 1998). Since it is not known which forms of p38 are
expressed in epithelial lung cells, the possibility cannot be
excluded that p38 isoforms that are less sensitive to SB202190
also exist in these cells.
Also JNK has been predominantly positively associated with
stress responses that include cytokine release and apoptosis
(Lewis et al., 1998). However, alternative roles of JNK have
been reported. Similar to other kinases, JNK occurs in different
isoforms that may have different functions (Bost et al., 1999;
Butterfield et al., 1999). Thus, the possibility cannot be excluded that the activation of JNK by fluoride might have an
inhibiting effect on apoptosis. However, in the present study
we find a correlation between activation of JNK and increased
apoptosis, suggesting a possible role for JNK in fluorideinduced apoptosis in A549 cells. In addition, since the inhibition of p38 resulted in only a partial reduction in fluorideinduced apoptosis, and JNK in most systems has been
DISCUSSION
In experimental systems, fluorides are known to exert a
variety of effects in different cell types. In bone cells, fluorides
have elicited potentially beneficial effects by stimulating
growth of bone cells (Caverzasio et al., 1998). In other cell
cultures, inhibition of protein synthesis and cell-cycle progression (Aardema et al., 1989; Holland, 1979), alterations in
cellular metabolism (Curnutte et al., 1979), induction of inflammatory cytokine release (Refsnes et al., 1999a), and apoptosis (Hirano and Ando (1996) have been observed. The
mechanisms by which fluorides may exert their effects have
been studied to some extent. The involvement of G-protein
activation in fluoride action has been reported (Sternweis and
Gilman, 1982). In this study we demonstrate that fluorides
induced apoptosis and inhibited proliferation in epithelial lung
FIG. 8. Effect of pretreatment with PD98059 and SB202190 together, on
apoptosis in A549 cells exposed to NaF. Apoptosis is calculated as a percentage of apoptotic cells treated with NaF only. Results of flow cytometric
analysis are presented as the mean ⫾ SE of 3– 4 experiments. One-way
ANOVA, significant difference between all groups (p ⫽ ⬍ 0.001); Tukey test,
significant difference between the 2 groups NaF ⫹ PD98059 and NaF ⫹
PD98059 ⫹ SB202190 (p ⫽ 0.045).
MECHANISM OF FLUORIDE-INDUCED APOPTOSIS IN LUNG CELLS
positively associated with apoptosis, it is tempting to speculate
that JNK in A549 cells functions in parallel with p38.
The involvement of ERK in the induction of apoptosis has
been reported (Wang et al., 1998). In our study, NaF-activated
ERK apparently stimulated survival, as judged by the increased
percentage of apoptotic cells after inhibition of MEK1,2 by
PD98059. This indicates that ERK activation in epithelial lung
cells opposes the induction of apoptosis and thus sustains
survival and growth. Furthermore, type 2 cell proliferation in
vitro has been found to partially depend on the activation of
ERK (Thrane et al., manuscript submitted). Several other studies have also indicated that activation of ERK is associated
with the survival and growth of cells (Lewis et al., 1998).
The ERK-induced survival or growth stimulatory effect,
reported in most cell types (Cook and McCormick, 1996;
Ishikawa and Kitamura, 1999; Langan et al., 1994; Meloche et
al., 1992) seems to depend on a prolonged/persistent signal,
although, it has been reported that a short-term signal (⬃5 min)
is sufficient to elicit a positive response (Gotoh et al., 1990;
Sarbassov et al., 1997). The activation of ERK by fluoride in
the epithelial lung cells also showed kinetics of a relatively
prolonged activation. However, rather than increasing the rate
of cell proliferation, NaF inhibited cell growth both in A549
cells and type 2 cells. The inhibition of proliferation seemed to
be mediated neither by ERK nor p38, since the inhibition of
these kinases did not affect the NaF effect on proliferation. In
contrast, fluoride has been found to stimulate proliferation of
bone cells (Caverzasio et al., 1998). NaF might also exert a
similar stimulatory proliferative signal in the lung cells via
prolonged ERK activation. However, another, more dominant
NaF-induced growth inhibitory signal or the induction of
apoptosis in the same concentration range may prevent the
proliferative response.
The activation of JNK/p38 also lasted for hours, indicating
that a prolonged activation may be critical for inducing
apoptosis. These prolonged signals seemed to be stronger than
the prolonged activation of ERK. The combined effect of
inhibition of ERK-dependent survival and NaF-induced apoptosis was still inhibited by SB202190 to a similar extent as the
NAF-induced apoptosis alone. This may indicate that the signal NaF elicited through p38 was of greater importance for the
outcome than the signal induced through ERK.
Both primary type 2 cells from rat and A549 cells, which are
derived from human type 2 cells, undergo apoptosis when
exposed to fluorides. Concomitantly, proliferation was inhibited. However, the human cell line was more resistant to the
fluoride effect than the primary rat cells, for both induction of
apoptosis and inhibition of proliferation. These differences
could be species-specific, because human cells may have different properties than rat cells that render the latter more
susceptible to toxicant effects. However, the A549 cells may
have been selected for properties that make them more resistant
to environmental changes. We show that the mechanistic data
89
FIG. 9. Suggested model for the effect of NaF on apoptosis via different
MAP kinases.
from the human cell line essentially are applicable to primary
epithelial lung cells from rats.
Figure 9 illustrates the summary of the presented findings
put into the context of current knowledge. Thus, the balance
tilts in favor of apoptosis after NaF exposure. However, the
scheme is likely to be oversimplified since other pathways
probably contribute to the result. The fluoride activation of
MAP kinases p38 and possibly JNK in epithelial lung cells is
associated with increased apoptosis, while fluoride-induced
ERK activation counteracted the effect. A prolonged activation
of ERK by fluoride was not sufficient to completely inhibit
apoptosis, possibly because the sustained, strong activation of
p38 and JNK prevailed.
ACKNOWLEDGMENTS
The excellent technical assistance of Nuong Dinh, Tonje Skuland, Edel
Lilleås, and Hans Jørgen Dahlman is gratefully acknowledged.
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