* 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
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ABSTRACT |
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Key Words: epithelial lung cells; fluoride; proliferation; apoptosis; MAP kinases.
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INTRODUCTION |
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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 (AlF4-) 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 G-protein 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 MEK-independent 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 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.
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MATERIALS AND METHODS |
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Animals.
Male rats (WKY/NHsd), 180230 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 µg/ml), hydrocortisone (5 µg/ml), ampicillin (100 µg/ml), streptomycin (100 µg/ml), fungizone (0.25 µg/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-cm2 wells, 28-cm2 dishes or 75-cm2 flasks (0.1 ml medium/cm2) in Hams medium, supplemented with ampicillin (100 µg/ml), streptomycin (100 µg/ml), fungizone (0.25 µg/ml) and 10% inactivated FBS. The cells (passage numbers 76100) 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 G2-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 µM or 50 µM) or SB202190 (540 µM), 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 µg/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 G1 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 x 106) were washed in PBS (phosphate-buffered saline without Ca2+ and Mg2+), 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 µl 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 MAP-kinase 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 µg/ml), and aprotinine (10 µg/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 TRI-CARB, 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 ß-glycerophosphate, 1 mM benzamidine, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 1 mM Na3VO4, 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 x g) for 10 min and JNK and p38 were immunoprecipitated from 200 µl 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 ß-glycerophosphate, 22.5 mM MgCl2, 0.05 mM Na3VO4, 1.1 mM DTT, 0.5 mM ATP (including 250 µCi/ml of 32P-ATP), and with 50 µg/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 SDSpolyacrylamide 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.
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RESULTS |
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DISCUSSION |
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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 fluoride-induced apoptosis in A549 cells. In addition, since the inhibition of p38 resulted in only a partial reduction in fluoride-induced apoptosis, and JNK in most systems has been 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 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.
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ACKNOWLEDGMENTS |
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NOTES |
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