ERK activation protects against DNA damage and apoptosis in hyperoxic rat AEC2

S. Buckley1,2, B. Driscoll1,2, L. Barsky3, K. Weinberg3, K. Anderson2, and D. Warburton1,2

1 Developmental Biology, 2 Pediatric Surgery, and 3 Bone Marrow Transplant Programs, Childrens Hospital Los Angeles Research Institute, Los Angeles, California 90027


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The survival of type 2 alveolar epithelial cells (AEC2) in the lung after hyperoxic injury is regulated by signals from the cellular environment. Keratinocyte growth factor and Matrigel can ameliorate the hallmarks of apoptosis seen in hyperoxic AEC2 after 24-h culture on plastic [S. Buckley, L. Barsky, B. Driscoll, K. Weinberg, K. D. Anderson, and D. Warburton. Am. J. Physiol. 274 (Lung Cell. Mol. Physiol. 18): L714-L720, 1998]. We used the same model of in vivo short-term hyperoxia to characterize the protective effects of substrate attachment. Culture of hyperoxic AEC2 on various biological adhesion substrates showed reduced DNA end labeling in cells grown on all biological substrates compared with growth on plastic. In contrast, the synthetic substrate poly-D-lysine conferred no protection. Hyperoxic AEC2 cultured on laminin showed an increased ratio of expression of Bcl-2 to interleukin-1beta -converting enzyme compared with culture on plastic. Laminin also partially restored hyperoxia-depleted glutathione levels and conferred improved optimal mitochondrial viability as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Conversely, attachment to the nonphysiological substrate poly-D-lysine afforded no such protection, suggesting that protection against hyperoxia-induced damage may be associated with integrin signaling. Increased activation of extracellular signal-regulated kinase (ERK), as detected by increased ERK tyrosine phosphorylation, was seen in hyperoxic AEC2 as soon as the cells started to attach to laminin and was sustained after 24 h of culture in contrast to that in control AEC2. To confirm that protection against DNA strand breakage and apoptosis was being conferred by ERK activation, the cells were also plated in the presence of 50 µM PD-98059, an inhibitor of the ERK-activating mitogen-activating kinase. Culture for 24 h with PD-98059 abolished the protective effect of laminin. We speculate that after hyperoxic lung injury, signals through the basement membrane confer specific protection against oxygen-induced DNA strand breakage and apoptosis through an ERK activation-dependent pathway.

extracellular signal-regulated kinase; type 2 alveolar epithelial cells; hyperoxia-induced deoxyribonucleic acid damage; tyrosine phosphorylation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TYPE 2 ALVEOLAR EPITHELIAL CELLS (AEC2), which are normally quiescent in the adult lung, respond to lung injury by proliferating and migrating to repair the alveolar epithelium (24). In the rat model of short-term hyperoxic injury, the response of AEC2 to damage is reproducible and transient. Induction of key cell cycle genes and downregulation of autocrine transforming growth factor-beta secretion, followed by proliferation, occur within 24 h postinjury, with complete resolution occurring by 72 h (6, 7).

Apoptosis, a genetically controlled cellular response to developmental or environmental stimuli that culminates in cell death, is an important mechanism of negative selection that removes damaged cells that may be deleterious to the host (31). Apoptosis, as measured by terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick end labeling (TUNEL), has been reported in sections of hyperoxic lungs (15). Apoptosis has also been reported in fibrotic human lungs (25, 26) and after acute lung injury (3), suggesting that apoptosis may play a key role in the resolution of lung injury. In our previous study using a rat model of in vivo short-term hyperoxia, we (5) showed that AEC2 isolated from hyperoxic lungs showed the hallmarks of apoptosis after 24 h of culture on plastic: significant TUNEL labeling and DNA fragmentation together with increased expression of p53, p21, and Bax proteins as well as DNA laddering. Interestingly, the DNA damage could be ameliorated by incubation with 20 ng/ml of keratinocyte growth factor (KGF) or by culture on Matrigel (5).

Although the protective effects of KGF in a variety of damage models are well documented (8, 20, 22, 30, 35) and cell-matrix adhesion is a recognized physiological determinant of cell growth and survival (1, 21, 23), little is known about the protective role of specific basement membrane components during recovery from acute hyperoxic injury. In this study, we again utilized the rat in vivo hyperoxic injury model to generate hyperoxic AEC2 that undergo reproducible DNA damage and apoptosis in culture. We demonstrated a significant inverse correlation between glutathione levels and DNA strand breakage in hyperoxic AEC2 after 24 h of culture on plastic, confirming that glutathione measurement would be useful when protective strategies in our model of hyperoxia were assessed. We compared hyperoxic AEC2 cultured on plastic, biological adhesion substrates, and poly-D-lysine, measuring various parameters associated with DNA damage and apoptosis, including glutathione levels, mitochondrial viability, DNA end labeling, and expression of interleukin-1beta -converting enzyme (ICE) and Bcl-2 proteins. We showed that the significant protection against hyperoxia-induced DNA damage conferred by laminin was associated with increased activation of extracellular signal-related kinase (ERK) during the early period of attachment. Finally, we abolished the protective effect of laminin by blockade of the mitogen-activated protein (MAP) kinase cascade using PD-98059, an inhibitor of ERK-activating MAP kinase, showing that ERK activation protects against hyperoxia-induced DNA damage in AEC2 cultured on laminin. This leads us to speculate that in vivo, laminin in the alveolar basement membrane may contribute to the survival of hyperoxia-damaged AEC2.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Oxygen treatment and recovery. Adult male Sprague-Dawley rats were exposed to short-term hyperoxia exactly as previously described (7). Briefly, the rats were placed in a 90 × 42 × 38-cm Plexiglas chamber, exposed to humidified >90% oxygen for 48 h, a 48-h oxygen exposure time inducing lung damage with minimal mortality. Control rats were kept in room air. At the end of the exposure period, the rats were anesthetized by an intraperitoneal injection of pentobarbital sodium. After complete exsanguination by saline perfusion via the pulmonary artery, the lungs were lavaged to remove macrophages. The lavaged lungs were then used for AEC2 isolation and culture.

Isolation and culture of AEC2. AEC2 were isolated from lavaged lungs by elastase digestion followed by differential adherence on IgG plates as described by Dobbs et al. (9). The cells were either used immediately for glutathione measurement or plated at 2 ×105 cells/cm2 in DMEM with 10% FCS on plastic or on commercially prepared plates coated with biological substrates for 24 h of culture. Collagen 1-, collagen IV-, fibronectin-, laminin-, Matrigel-, and poly-D-lysine-coated plates were all from Becton Dickinson (Franklin Lakes, NJ). Freshly isolated AEC2 attached more efficiently to the biological substrates than to plastic and less efficiently to poly-D-lysine; therefore, for some experiments, parallel wells were set up for cell counts to correct for varying plating efficiencies. After 24 h of culture, the cells were lysed on ice to extract proteins for Western blotting (7) or trypsinized from the plate and fixed in ice-cold 1% paraformaldehyde for analysis of FITC-dUTP staining by fluoresence-activated cell sorting (FACS) analysis. In some experiments with laminin as the substrate, the cells were lysed early after attachment (2-5 h after being plated) or were treated with 50 µM PD-98059 (Calbiochem, La Jolla, CA) from the time of plating. Immunostaining of attached cells after 24 h of culture with a surfactant protein (SP) C antibody confirmed that >95% of the attached cells were SP-C positive. The antibody to SP-C was kindly provided by Jeffrey Whitsett (Children's Hospital Medical Center, Cincinnati, OH).

Flow cytometric method for measuring DNA end labeling in fixed cells. DNA strand breaks were measured in 1% paraformaldehyde-fixed AEC2 by labeling with fluorescent FITC-dUTP with an APO-DIRECT kit (PharMingen, San Diego, CA) according to the manufacturer's instructions. Fixed cells were incubated at 37°C for 1 h with TdT and FITC-labeled dUTP before being counterstained with propidium iodide. The stained cells were analyzed with a Becton Dickinson FACScan equipped with a 488-nm argon laser, with control positive and negative cells end labeled in parallel with the test samples supplied with the kit. The percentage of gated cells that were FITC positive was compared for each treatment group. No FITC fluorescence was seen in either control or oxygen-treated AEC2 that had been incubated in the labeling reaction mixture in the absence of TdT. Cellquest software (Becton Dickinson) was used for the analyses.

Western blotting of proteins. Western analysis was performed on cell lysates as described by Bui et al. (7), with 10-20 µg protein/lane depending on the sensitivity of the antibody used. Equal loading was confirmed by blotting with an antibody to actin. Proteins of interest were detected with horseradish peroxidase-linked secondary antibodies and the enhanced chemiluminescence system following the manufacturer's instructions (Amersham, Arlington Heights, IL). Antibodies to phosphotyrosine, ERK, p-ERK, c-Jun NH2-terminal kinase (JNK), p-JNK, Bcl-2, and ICE were from Santa Cruz Biotechnology (Santa Cruz, CA). The antibody to actin was from ICN (Irvine, CA). The secondary antibodies were from Sigma (St. Louis, MO).

Glutathione measurement. Measurement of total glutathione (GSH plus GSSG) was performed with AEC2 immediately after isolation and after 24 h of culture. Freshly lysed hyperoxic and control AEC2 were assayed immediately with a modification of the recycling method of Owens and Belcher (19), which colorimetrically measures the formation of 2-nitro-5-thiobenzoic acid from 5,5'-dithiobis(2-nitrobenzoic acid) in the presence of glutathione reductase and NADPH (19). Glutathione levels in the AEC2 were calculated relative to 1-4 nmol glutathione standards freshly prepared for each assay.

Assay of mitochondrial viability. Mitochondrial viability was determined by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay exactly as described by Atabay et al. (2). Briefly, control and hyperoxic cells growing on plastic or on various adhesion substrates were incubated with buffered MTT for 30 min at 37°C, and the purple formazan color generated by viable mitochondria was measured colorimetrically. Because the measurements were performed on 24-h cultures, the cells in parallel wells were counted to correct for the differing attachment efficiencies on the various substrates. All chemicals used were from Sigma.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hyperoxia-induced DNA strand breakage in cultured AEC2 is significantly correlated with depletion of cellular glutathione. AEC2 from hyperoxic and control rats were lysed and assayed for total cellular glutathione (GSH plus GSSG) immediately after isolation and after 24 h of culture, by which time DNA strand breakage in the hyperoxic population was apparent. Total glutathione levels were significantly depleted (~70%) in the fresh isolates of hyperoxic AEC2 compared with those in control cells (Fig. 1A). This depletion was sustained after 24 h of culture. There was a significant inverse correlation between DNA damage, as measured by FITC-dUTP DNA end labeling, and glutathione levels, as measured in fresh isolates of AEC2 (Fig. 1B).



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Fig. 1.   A: total cellular glutathione (GSH+GSSG) levels for control and hyperoxic type 2 alveolar epithelial cells (AEC2). Values are means ± SD of total cellular glutathione levels (GSH+GSSG); n = 4-5 animals/group. Both freshly isolated and 24-h cultured AEC2 from hyperoxic rats have significantly lower glutathione levels than control AEC2. ** P < 0.0125. * P < 0.05 (both by Student's t-test). B: correlation between total glutathione levels in freshly isolated AEC2 and DNA damage measured after a 24-h culture on plastic. No damage was detected before culture. DNA strand breakage has a significant inverse correlation with cellular glutathione levels in hyperoxic AEC2, making it a suitable marker to include when protective strategies are assessed.

Attachment to biological substrates reduces hyperoxia-induced DNA strand breaks in 24-h cultured AEC2. Because we (5) have previously shown Matrigel to be protective against hyperoxia-induced DNA damage, some Matrigel components and other adhesion substrates, including laminin, fibronectin, collagen I, collagen IV, and the attachment factor poly-D-lysine, were compared. Hyperoxic AEC2 were plated on plastic or substrate for 24 h, then recovered and fixed for FITC-dUTP DNA end labeling. DNA damage measured in hyperoxic AEC2 cultured on the various substrates was compared with damage on plastic. Figure 2 shows that all biological substrates tested were significantly protective against hyperoxia-induced DNA damage. In contrast, poly-D-lysine provided no protection at all. Control cells, which showed no significant DNA end labeling on plastic (~10% of cells were end labeled) showed no change on any substrate (data not shown).


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Fig. 2.   DNA strand breakage as measured by fluorescence-activated cell sorting (FACS) analysis of FITC-dUTP DNA end labeling in hyperoxic AEC2 plated on various commercially prepared adhesion substrates for 24 h compared with adhesion to plastic. End labeling of hyperoxic AEC2 on plastic was 60 ± 30% of total cells and 10 ± 5% for control AEC2. Data are means ± SD of ratio of matrix to plastic labeling; n = 4-6 animals/group. Hyperoxia-induced DNA strand breakage in AEC2 is decreased by growth on biological substrates. All substrates tested except poly-D-lysine were significantly more protective than plastic, P < 0.05 by Student's t-test.

Attachment to biological adhesion substrates increases mitochondrial viability in 24-h cultured hyperoxic AEC2. Because mitochondrial damage is more extensive and persists longer than nuclear damage in oxidative stress (33), we compared the mitochondrial viability of hyperoxic AEC2 grown for 24 h on plastic and other biological substrates with the MTT assay. Figure 3 shows that all biological substrates tested significantly improved mitochondrial viability compared with that on plastic, whereas poly-D-lysine afforded no protection, in agreement with the DNA end-labeling data.


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Fig. 3.   Mitochondrial activity as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay of hyperoxic AEC2 grown for 24 h on various commercially prepared adhesion substrates. Results are means ± SD; n = 4 animals/group. Mitochondrial viability in hyperoxic AEC2 is increased by growth on biological substrates. All substrates except poly-D-lysine provided significantly enhanced mitochondrial viability compared with that on plastic, P < 0.05 by Student's t-test, in agreement with the data in Fig. 2.

Laminin increases the expression ratio of Bcl-2 to ICE. Expression of the apoptosis-protective protein Bcl-2 and the apoptosis-associated caspase ICE in lysates of hyperoxic AEC2 growing on the various substrates were compared by Western analysis (Fig. 4). The ratio of Bcl-2 to ICE expression was increased by growth on laminin compared with growth on plastic.


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Fig. 4.   Western analysis of apoptosis-associated caspase interleukin-1beta -converting enzyme (ICE) and apoptosis-protective protein Bcl-2 in lysates of AEC2 growing on various adhesion substrates compared with growth on plastic. Culture on laminin increases ratio of Bcl-2 to ICE expression. Note: polyclonal antibody used for this Bcl-2 blot had relatively poor cross-reactivity and high background with rat lysates. A new monoclonal antibody became available after this work was done and was used for the Western analysis seen in Fig. 7B.

Laminin increases total cellular glutathione levels in hyperoxic AEC2. To see whether laminin, which was protective against hyperoxia-induced DNA damage to AEC2, could restore hyperoxia-depleted glutathione levels, we cultured control and hyperoxic AEC2 on laminin and plastic for 24 h and compared total cellular glutathione levels (Fig. 5). A 24-h culture on laminin resulted in a 1.5-fold increase in glutathione levels in hyperoxic AEC2 compared with culture on plastic.


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Fig. 5.   Total glutathione levels of hyperoxic AEC2 were measured after 24 h of culture on plastic or laminin, the most protective substrate tested. Data are means ± SD; n = 4-5 animals/group. The 24-h culture on laminin significantly increased glutathione levels in hyperoxic AEC2 compared with culture on plastic. * P < 0.025 by Student's t-test. Glutathione levels of control AEC2 on laminin did not differ significantly from those on plastic (data not shown)

During early attachment to laminin, increased activation (phosphorylation) of ERK is seen in hyperoxic AEC2 compared with that in control AEC2. Hyperoxic and control AEC2 were plated on laminin and plastic and lysed after 2, 3, 4, 5, and 24 h on laminin and 24 h only on plastic. The cells attached to plastic so slowly that there was insufficient material for analysis at the earlier time points. Western blotting for tyrosine phosphorylation showed increased phosphorylation of a ~45-kDa protein during the early period of attachment in the hyperoxic AEC2 compared with that in control AEC2 (Fig. 6, top). There was also increased expression of a higher-molecular-mass protein, ~120 kDa, after 24 h of culture in both hyperoxic and control cells. The latter ~120-kDa protein was later confirmed by Western blotting to be focal adhesion kinase (FAK; data not shown), but because there was no difference between hyperoxic and control AEC2, we limited our investigations to the lower-molecular-mass protein. Based on the approximate molecular mass, we performed Western analysis with p-ERK and p-JNK and showed that p-ERK had a similar pattern of expression to that seen with the phosphotyrosine antibody (Fig. 6, middle). Although the activity of p-ERK was increased during attachment of the hyperoxic AEC2 to laminin, the expression of ERK was not significantly altered and the expression and activity of JNK did not change significantly (data not shown). Blotting with an anti-actin antibody confirmed equal loading (Fig. 6, bottom).


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Fig. 6.   Extracellular signal-related kinase (ERK) activation during early attachment to laminin is increased in hyperoxic AEC2 compared with control AEC2. Top: Western analysis with a phosphotyrosine (p-Tyr) antibody of control and hyperoxic AEC2 attached to laminin (+) and plastic (-). Increased tyrosine phosphorylation of an ~45-kDa protein is seen as hyperoxic AEC2 attach to laminin compared with control AEC2. In contrast, tyrosine phosphorylation of a higher-molecular-mass protein of >= 100 kDa seen after 24 h, later confirmed to be focal adhesion kinase (molecular mass 125 kDa), did not differ between hyperoxic and control AEC2. The ~45-kDa protein was confirmed to be p-ERK (middle). ERK expression and c-Jun NH2-terminal kinase (JNK) activation and expression did not differ between control and hyperoxic AEC2 (data not shown). Actin was included as a loading control (bottom). Nos. on right, molecular-mass markers.

MAP kinase pathway inhibitor PD-98059 blocks the protective effect of laminin on hyperoxia-mediated DNA damage to AEC2. To confirm the involvement of ERK activation with protection against hyperoxia-induced DNA damage, hyperoxic AEC2 were plated on plastic and laminin for 4 and 24 h in the presence and absence of 50 µM PD-98059, a specific inhibitor of ERK-activating MAP kinase. Four hours was selected as the optimum time to observe differences in p-ERK, whereas all measurements of DNA damage were made at 24 h, by which time DNA damage was apparent. Inhibition of ERK activity by PD-98059 abolished the protective effect of laminin, resulting in increased DNA strand breakage (Fig. 7A). Western analysis of ERK activation as hyperoxic AEC2 attach to laminin confirms successful blockade by PD-98059 (Fig. 7B, top row). Expression of ERK did not change with PD-98059 treatment (Fig. 7B, second row). Bcl-2 levels were slightly offset by 24 h of PD-98059 treatment, in agreement with the end-labeling data (Fig. 7B, third row). Actin served as a loading control (Fig. 7B, bottom row). ERK activation in control AEC2, much reduced in comparison with hyperoxic AEC2, was also inhibited by PD-98059, with no change in Bcl-2 levels (data not shown).



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Fig. 7.   A: DNA strand breakage as measured by FACS analysis of FITC-dUTP end labeling of hyperoxic AEC2 grown for 24 h on plastic and on laminin with (+) and without (-) 50 µM PD-98059, a specific inhibitor of ERK-activating mitogen-activating protein (MAP) kinase. Inhibitor was added at time of plating. Data are means ± SD; n = 4 animals/group. PD-98059 abolished protective effect of laminin against hyperoxia-induced DNA damage in AEC2. * Significant protective effect of laminin compared with that of plastic in absence of PD-98059, P < 0.05 by Student's t-test. B: Western analysis of lysates from hyperoxic AEC2 at 4 and 24 h after attachment to laminin with and without PD-98059. Four hours was the earliest time point at which sufficient cells were attached to enable recovery of 20 µg of lysate protein (enough to run 2 lanes on a minigel). Twenty-four-hour lysates were also used because DNA damage, if present, was apparent by this time. PD-98059 blocks ERK activation and reduces Bcl-2 expression in hyperoxic AEC2 on laminin. Top row: successful and sustained blockade of ERK activity with PD-98059. In contrast, ERK expression is unchanged (second row). Bcl-2 expression is slightly offset by 24-h PD-98059 treatment (third row), in agreement with end-labeling data of Fig. 7A. Actin was used as a loading control (bottom row). ERK activation in laminin-plated control AEC2, much reduced in comparison with hyperoxic AEC2 in Fig. 6, was also inhibited by PD-98059 but with no change in Bcl-2 levels (data not shown).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The alveolar epithelium of the lung is a major target for oxidant injury. The survival of AEC2 is critical to ensure timely and efficient repopulation of the damaged alveolar surface during the repair process after hyperoxic injury. Depending on the net balance of environmental cues, AEC2 can remain quiescent or proceed toward proliferation or apoptosis. In this paper, we used an in vivo model of hyperoxia to examine the balance between DNA damage, which accompanies a depletion of cellular glutathione, and protective signaling in hyperoxic AEC2 grown on laminin, a key component of the alveolar basement membrane.

Glutathione is an efficient antioxidant, maintaining a reduced intracellular state in the face of a highly oxidizing environment. Epithelial lining fluid of the lung contains GSH at an ~100-fold higher concentration than that found in the fluid of many other tissues (27). The observation that certain tumor cells have evolved resistance to oxidant injury suggests that manipulation of the cellular redox status may provide a role in future therapies, a compelling reason to correlate antioxidant levels, apoptosis, and putative protective strategies in normal, untransformed AEC2. AEC2 from rats subjected to short-term hyperoxia were found to have significantly lower total glutathione (GSH plus GSSG) levels than control AEC2 both immediately on isolation and after 24 h of culture. DNA damage as measured by FITC-dUTP DNA end labeling after 24 h of culture had a significant inverse correlation with initial glutathione levels, making glutathione a useful parameter in assessing hyperoxia-induced DNA damage. Low glutathione levels associated with DNA damage and apoptosis have also been reported in AEC2 derived from a more chronic model of hyperoxia involving exposure to 60 or 85% oxygen for 7 days (27).

In a previous study using the same model of hyperoxia, we (5) showed that Matrigel could significantly reduce hyperoxia-induced DNA damage at 24 h, indicating that the initial in vivo damage is not lethal and may be reversed by a favorable environment in vitro. Antiapoptotic signaling through cell-matrix interaction has been reported in a variety of epithelial cells, including bronchial epithelial cells, mammary epithelial cells, and retinal pigment epithelium (1, 18, 23). We therefore decided to focus on some key components of the alveolar basement membrane as a potential source of protective signaling. We plated hyperoxic AEC2 for 24 h on a selection of biological adhesion substrates using commercially prepared, thinly coated plates to ensure reproducibility and measured DNA damage by FACS analysis of FITC-dUTP end-labeled cells. Because end labeling may not always discriminate between apoptosis and necrosis (14), we also examined expression of proteins specifically related to apoptosis, the caspase ICE and the protective protein Bcl-2. Bcl-2 acts upstream of the execution caspases, preventing their proteolytic processing into active mediators of cell death. Bcl-2 also dramatically alters intracellular glutathione compartmentalization, promoting sequestration of glutathione into the nucleus to restore nuclear antioxidant status and block apoptosis (28). Mitochondrial viability was also assessed because the mitochondria have been described as "the central executioner of programmed cell death" and can contribute to apoptotic signaling via the production of reactive oxygen intermediates (28). FACS analysis of FITC-dUTP DNA end labeling showed that significant protection against hyperoxia-induced DNA damage was conferred by all biological substrates tested but not by the synthetic attachment factor poly-D-lysine. MTT data were in agreement with the FACS analysis, showing that mitochondrial viability was significantly increased when the hyperoxic cells were grown for 24 h on biological substrates compared with those cultured on plastic or poly-D-lysine. Laminin was effective in reducing DNA strand breakage while increasing the ratio of Bcl-2 to ICE expression, suggesting an amelioration of apoptosis. The protective effect of laminin was confirmed further by the observation that depleted glutathione levels seen in hyperoxic AEC2 after 24 h of culture were significantly increased by culture on laminin.

Although the protective effects of laminin against hyperoxia-induced DNA damage and apoptosis in cultured AEC2 were apparent, the mechanism of protection was less clear. The fact that the hyperoxic AEC2 survived relatively poorly on poly-D-lysine or plastic compared with natural biological adhesion substrates suggested the involvement of integrins in protective signaling. Interactions of integrins with the extracellular matrix activates FAK and suppresses apoptosis in diverse cell types (29). Cell interaction with adhesive proteins or growth factors in the extracellular matrix is reported to initiate MAP kinase signaling and cell migration (16), an important function of the AEC2 when repopulating the pulmonary epithelium after lung injury. The observation that laminin strongly promotes AEC2 migration (17) suggests a possible further role for MAP kinase signaling through laminin in the hyperoxic lung because protection against oxidant damage combined with increased capacity for migration would ensure a timely and efficient repopulation of the damaged epithelium.

Comparison of phosphotyrosine expression by Western analysis during attachment of hyperoxic and control AEC2 to laminin showed increased activation of ERK in the hyperoxic population during early attachment. In contrast, p-JNK expression did not differ between the control and hyperoxic populations. Tyrosine-phosphorylated FAK expression was increased in both control and hyperoxic cells after 24 h of culture on plastic or laminin, but there was no difference between the groups. Therefore, we concluded that ERK activation played at least a correlative role in protection and used an inhibitory strategy to test whether the protective signaling in hyperoxic AEC2 cultured on laminin was occurring through an MAP kinase pathway. Blockade of the MAP kinase pathway by 50 µM PD-98059 abolished the protective effect of laminin against DNA strand breakage, showing that activation of ERK confers a protective effect against DNA damage. The observation that the cells attach faster to laminin than to plastic may indicate that timely reattachment confers or maintains viability of damaged cells. Alternatively, laminin may specifically select for a population of "survivors," a population in which protective signaling has already been initiated. Thus an important caveat concerning the data presented herein is that the specific protective effects associated with ERK activation may be due to signaling originating from more than one source. The laminin itself may contribute diverse signals because the commercial plates are prepared by extracting laminin from Engelbreth-Holm-Swarm tumors and therefore may contain other factors. Signaling through ERK activation can be antiapoptotic or apoptotic depending on the cell type and the nature of the apoptotic stimulus. Survival through ERK activation against a diverse range of apoptotic signals has been reported, including serum-induced apoptosis of PC12 cells (34), tumor necrosis factor-alpha -induced apoptosis of L929 cells (12), and ultraviolet-induced apoptosis of human primary neutrophils (10). In contrast, apoptotic signaling through ERK activation has also been described: Fas-mediated apoptosis in a neuroblastoma cell line was blocked by interference with either the ERK or JNK pathways (with dominant-interfering mutant proteins), indicating that ERK and JNK cooperate in the induction of apoptosis by Fas (13). AEC2 express Fas receptors and undergo Fas-dependent apoptosis under the appropriate stimuli (11). Therefore, the survival of AEC2 in vivo after hyperoxic injury will be determined by integration of diverse signals, both protective and destructive, from the cellular environment through ERK activation. In our model of short-term hyperoxic injury, the net signal transmitted to the hyperoxic AEC2 cultured on laminin is protective. We therefore speculate that laminin in the alveolar basement membrane may promote survival of AEC2 during lung repair after acute hyperoxic injury.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: D. Warburton, Childrens Hospital Los Angeles Research Institute, 4650 Sunset Blvd. (MS 35), Los Angeles, CA 90027 (E-mail: dwarburton{at}chla.usc.edu).

Received 11 November 1998; accepted in final form 18 March 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 277(1):L159-L166
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