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Fibroblast Growth Factor-10 Attenuates H₂O₂-Induced Alveolar Epithelial Cell DNA Damage

Role of MAPK Activation and DNA Repair

Division of Pulmonary and Critical Care Medicine, Northwestern University Feinberg School of Medicine, Chicago; Veterans Administration Chicago Health Care System, Lakeside Division, Chicago, Illinois; and Stanford University Medical Center, Stanford, California

Address correspondence to: David Kamp, Pulmonary and Critical Care Medicine, Northwestern University Feinberg School of Medicine, 303 East Chicago Ave., Tarry Bldg, 14-707, Chicago, IL 60611. E-mail: d-kamp{at}northwestern.edu

	Abstract

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Fibroblast growth factor-10 (FGF-10), an alveolar epithelial cell (AEC) mitogen that is critical for lung development, may promote AEC repair. We determined whether FGF-10 attenuates H₂O₂-induced, A549 and rat alveolar type II cell DNA damage. We show that FGF-10 prevents H₂O₂-induced DNA damage assessed by an alkaline elution, ethidium bromide fluorescence as well as by a comet assay. Mitogen-activated protein kinase inhibitors abolished the protective effect of FGF-10 against H₂O₂-induced DNA damage yet had no effect on H₂O₂-induced DNA damage. A Grb2-SOS inhibitor (SH3 binding peptide), an Ras inhibitor (farnesyl transferase inhibitor 277), and an Raf-1 inhibitor (forskolin) each prevented FGF-10- and H₂O₂-induced A549 cell ERK1/2 phosphorylation. Also, FGF-10 and H₂O₂ each induced negligible ERK1/2 phosphorylation in Ras dominant-negative (N17) cells. Inhibitors of Ras and Raf-1 blocked the protective effect of FGF-10 against H₂O₂-induced DNA damage but had no effect on H₂O₂-induced DNA damage. Furthermore, cold conditions and aphidicolin, an inhibitor of DNA polymerase-, -, and -, each blocked the protective effects of FGF-10, suggesting a role for DNA repair. We conclude that FGF-10 attenuates H₂O₂-induced AEC DNA damage by mechanisms that involve activation of Grb2-SOS/Ras/RAF-1/ERK1/2 pathway and DNA repair.

Abbreviations: alveolar epithelial cells, AEC • DNA strand breaks, DNA-SB • extracellular signal-regulated kinases, ERKs • fibroblast growth factor-10, FGF-10 • forskolin, FSK • farnesyl transferase inhibitor, FTI • keratinocyte growth factor, KGF • mitogen-activated protein kinase, MAPK • SH3 binding peptide, SH3b-p

	Introduction

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Alveolar epithelial cell (AEC) injury and repair are important in the pathogenesis of oxidant-induced lung damage and fibrosis caused by various agents (1–4). Generation of reactive oxygen species (ROS) in response to oxidative stress induces lung injury in part by causing damage to DNA, lipids, and proteins (5, 6). DNA strand break (DNA-SB) formation is among the earliest abnormalities that occur within cells exposed to an oxidative stress (7). Although ROS-induced DNA damage can be readily repaired, extensive DNA derangement can alter the physiologic stability of the cell and thereby promote cell death by apoptosis and/or necrosis (1, 8, 9).

Accumulating evidence suggests that growth factors, particularly keratinocyte growth factor (KGF; also known as fibroblast growth factor 7), are important in preventing lung injury from various causes including H₂O₂, radiation, and bleomycin (2–4, 10). Although, the mechanisms involved in mediating the protective effects of KGF are incompletely defined, some of the implicated mechanisms include stimulating AEC proliferation, augmenting surfactant protein synthesis, limiting oxidant-induced increases in lung epithelial cell permeability, and promoting AEC DNA repair (2, 4, 10–12). Finally, growth factors may decrease oxidant-induced lung damage by inhibiting apoptosis, a central pathway regulating cellular homeostasis (10).

Fibroblast growth factor-10 (FGF-10) is a recently described 13.9-kD heparin-binding protein that is structurally and functionally similar to KGF (10, 13). FGF-10 is a potent alveolar type II cell (AT2 cell) mitogen that is predominantly expressed by lung mesenchymal cells and is required for lung development (10, 13–15). FGF-10 promotes epithelial cell motility, differentiation, migration and wound healing. KGF and FGF-10 are expressed in the lung and are unique among the over 20 FGF family members because they bind with high affinity to a spliced variant of fibroblast growth factor receptor 2-IIIb (FGFR-2IIIb) located on epithelial cells (10, 13). However, unlike KGF, FGF-10 has high affinity to FGFR1-IIIb receptors expressed on epithelial cells (16).

The above data suggest an important role of FGF-10 in preventing AEC injury but there are few studies that have directly assessed this possibility. We recently showed that FGF-10 attenuates cyclic-stretch induced DNA damage by mechanisms involving mitogen-activated protein kinase (MAPK) activation via the Grb2-SOS/Ras/Raf-1/ERK1/2 pathway (17). Additionally, recent studies demonstrate that H₂O₂-induced MAPK activation may play a key role in cell survival (18, 19). In this study, we have explored the role of FGF-10 against oxidant-induced AEC DNA damage. We reasoned that FGF-10 would attenuate H₂O₂-induced AEC DNA damage by mechanisms involving MAPK activation. We report that FGF-10 attenuates H₂O₂-induced, A549, and rat AT2 cell DNA-SB as assessed by alkaline-unwinding, ethidium bromide fluorescence, and comet assays. In addition, inhibitor studies suggest that FGF-10 prevents AEC DNA damage by mechanisms involving the Grb2-SOS/Ras/Raf-1/ERK1/2 pathways. Finally, we show that the protective effects of FGF-10 are blocked by cold conditions and by an inhibitor of DNA polymerase, which suggests a role for DNA repair.

	Materials and Methods

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FGF-10 was purchased from R&D Systems (Minneapolis, MN). U0126, PD98059, and polyclonal anti-ERK1/2 antibodies p44/p42 were purchased from Promega (Madison, WI). We purchased monoclonal anti-phosphorylated ERK1/2 antibodies and polyclonal ERK-2 antibodies from Biolabs (Beverly, MA) and Cell Signaling Technologies (Beverly, MA), Farnesyl transferase inhibitor (FTI 277) and SH3 binding peptide (SH3b-p) from Calbiochem-Novabiochem (La Jolla, CA), and aphidicolin and all other chemicals from Sigma Chemicals (St. Louis, MO).

Cell Culture
A549 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with L-glutamine (0.3 µg/ml), nonessential amino acids, penicillin (100 U/ml), streptomycin (200 µg/ml), and 10% fetal bovine serum (FBS; GIBCO, Grand Island, NY). A549 cells were plated at a density of 3.0 x 10⁵ cells/well in a six-well plate and were grown to confluency in a humidified 95% air–5% CO₂ incubator at 37°C over 24 h. Rat AT2 cells were isolated from specific pathogen-free adult Harlan Sprague-Dawley rats (200–250 g) using technique previously described (1). Cells were plated at a density of 1 x 10⁶ cells/well in a six-well plate and were grown to confluency in a humidified 95% air–5% CO₂ incubator at 37°C over 24 h.

DNA-SB Assay
A549 cells as well as rat AT2 cells were treated with FGF-10 (10 ng/ml) for 1 h followed by H₂O₂ (50 µM) for 30 min. DNA-SB formation was assessed by alkaline unwinding and ethidium bromide fluorescence technique, exactly as previously described (1, 2, 4). In some experiments, cells were treated with selective MAPK inhibitors, U0126 (10 µM) or PD98059 (100 µM), for 15 min before FGF-10 (10 ng/ml) was added for 1 h. Because ethidium bromide preferentially binds to double-stranded DNA (ds-DNA) in alkali, the relative amounts of nonbroken ds-DNA and broken single-stranded DNA can be assessed. Fluorescence was determined with a model 450 Sequoia Turner fluorometer (Mountain View, CA) with excitation at 520 nm and emission at 585 nm. The results were expressed as previously described (1, 2) in which the percentage of total double stranded DNA is defined as (F – Fmin)/(Fmax – Fmin) x 100, where F is the fluorescence in the experimental condition, Fmin is the background ethidium bromide fluorescence determined after converting all the DNA into single-strand form, and Fmax is the fluorescence determined from cells not exposed to alkaline unwinding conditions. The reductions in ds-DNA in this assay are due to increased DNA-SB formation.

Comet Assay
The Comet assay was performed as previously described by our laboratory using a Comet Assay kit (Trevigen Inc) according to the manufacturer's instructions (17). A549 cells were treated with FGF-10 (10 ng/ml) for 1 h followed by H₂O₂ (50 µM) for 30 min. DNA-SB formation was assessed by single cell gel electrophoresis as described previously (16, 19). The cell suspension was mixed with liquefied agarose at a 1:10 (vol/vol) ratio. A small aliquot of this mixture was immediately transferred onto the slide provided. After cell-lysis at 4°C, slides were treated with alkali solution (0.3 M NaOH, 1 mM EDTA) for 60 min to unwind the double-stranded DNA. Slides were electrophoresed at 1 V/cm for 10 min. After staining with SYBR green dye, samples were visualized and photographed by fluorescent microscope. Tail length was defined as the distance between the leading edge of the nucleus and the end of the tail. NIH image software was used for image analysis.

Effect of Cold Conditions and a DNA Polymerase Inhibitor on FGF-10's Protective Effect against H₂O₂-Induced AEC DNA Damage
To determine whether FGF-10 protects A549 cell from H₂O₂-induced DNA-SB formation by augmenting DNA repair, A549 cells were exposed either to cold conditions (0°C) or aphidicolin (Sigma), a DNA polymerase -, -, and - inhibitor. For the cold conditions, cells were pretreated with FGF-10 (10 ng/ml) for 1 h, then placed on ice during H₂O₂ exposure. For the aphidicolin treatment, the cells were exposed to aphidicolin (1 µM) for 1 h after FGF-10 (10 ng/ml) pretreatment. The cells were then washed in PBS and exposed to H₂O₂ (50 µM) for 30 min. After treatment, the cells were washed and then DNA damage was assessed using the Comet assay described above.

MAPK Assay
A549 cell MAPK activation was assessed using techniques previously described by our laboratory (17) and briefly summarized below.

Preparation of lysates and total protein isolation. A549 and rat AT2 cells were treated with FGF-10 (200 ng/ml) for 10 min. In some of the ERK1/2 Western blot experiments; cells were treated with U0126 (10 µM) for 1.5 h before exposure to FGF-10 (200 ng/ml) for 10 min. The treated cells were washed with ice cold PBS, lysed with 0.5 ml of ice-cold cell lysis buffer containing PMSF (1 mM), incubated on ice for 5 min, sonicated four times for 5 s each, microcentrifuged at 15,000 rpm (Beckman centrifuge) for 5 min, and then the supernatant was collected. Protein content was determined by the Bradford technique using a Bio-Rad protein assay system (Bio-Rad, Hercules, CA).

Immunoprecipitation. Cell lysates (200 µg total protein in 200 µl) were mixed with a 15 µl of suspension of immobilized phospho-p44/42 MAP kinase monoclonal antibody and incubated for 4 h at 4°C with continuous gentle rocking. After microcentrifugation for 30 s at 4°C, the pellet was washed twice with 500 µl of 1x lysis buffer and kept on ice until use in the Western analysis.

Western blot analysis. Thirty-five-microgram samples of protein were size fractionated by 1% SDS/12% polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Optitran; Schleider and Schuell, Keene, NH) using a semidry transfer apparatus (Bio-Rad). Incubation of blots with a monoclonal antibody that specifically recognizes the dually phosphorylated active form of ERK1 and ERK2 was performed overnight at 4°C. Blots were developed with an enhanced chemiluninescence detection kit (ECL+; Amersham, Buckinghamshire, UK) used as recommended by the manufacturer. The bands were quantified by densitometric scan (Eagle Eye II; Stratagene, La Jolla, CA).

ERK assay. The ERK activity was determined exactly as described in a commercially available p44/p42 assay kits. Total cell protein of 45 µg from cell lysates were immunoprecipitated with an immobilized by crosslinking to agarose hydrazide beads phosphospecific p44/p42 MAP kinase (Thr202/Tyr204) monoclonal antibody by overnight incubation at 4°C with gentle rocking. The beads were washed twice with lysis buffer and then twice with kinase reaction buffer (KRB) (25 mM Tris, pH 7.5; 5 mM ß-glycerolphosphate, 2 mM DTT, 0.1 mM Na₃VO₄, 10 mM MgCl₂). The beads were then resuspended in 50 µl of KRB supplemented with the 200 µM ATP and 2 µg Elk-1 fusion protein. The reaction was performed at 30°C for 30 min and terminated with 25 µl of 3x SDS loading buffer (187 mM Tris-HCL, pH 6.8, 10% glycerol, 2% SDS, 5% ß-mercaptoethanol; 1.25 x 10^–3% bromophenol blue. Samples were fractionated in 12.5% SDS-PAGE and analyzed by immunoblotting using a phosphospecific Elk-1 antibody (Biolabs and Cell Signaling Technologies) or phosphorylated ERK1/2 antibody (p42/p44 MAPK antibody) (Promega, Madison, WI) as a probe. The blots were developed. The densitometric values of phosphorylated ERK1/2 and ELK1 were and quantified as described. The band densitometry values from individual bands were quantified using a densitometry scanner (Eagle Eye II; Stratagene, La Jolla, CA). Densitometries of individual p42/p44 bands were added up together. Total ERK bands were measured similarly. The densitometry data were normalized for protein loading by dividing the p-ERK densitometry reading with total-ERK densitometry reading.

Statistics
All data are expressed as the mean ± SEM. An unpaired Student's t test was used to assess the difference between two groups. One-way analysis of variance (ANOVA) was performed when more than two groups were compared with a single control and then differences between individual groups within the set were assessed by a multiple comparison test (Tukey) when the F statistic was < 0.05. A P value of < 0.05 was considered significant.

	Results

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FGF-10 Attenuates H₂O₂-Induced AEC DNA-SB Formation
To determine whether FGF-10 prevents H₂O₂-induced AEC DNA damage, A549 cells were exposed to FGF-10 (10 ng/ml) for various times (0, 1, 4, or 24 h) and then H₂O₂ (50 µM)-induced DNA-SB were assessed after 30 min. As shown in Figure 1A, FGF-10 exposure for as little as 1 h completely blocked H₂O₂-induced DNA-SB formation as assessed by an alkaline elution, ethidium bromide fluorescence technique. Notably, FGF-10 exposure at the same time of H₂O₂ (0 h) was not protective, suggesting that the protective effects of FGF-10 are not due to an antioxidant mechanism. We also assessed DNA-SB formation by single cell gel electrophoresis. As shown in Figure 1B, H₂O₂ (50 µM) induced A459 cell DNA-SB formation as shown by an increased comet tail length and this effect was abolished by FGF-10 (10 ng/ml). The protective effect of FGF-10 peaked at a dose 10 ng/ml, and a dose-dependent protective effect was seen between the dose of 1 and 10 ng/ml (P < 0.05 FGF-10 1 versus 10 ng/ml) (Figure 1C). Because the maximum protective effect of FGF-10 was noted after 1 h treatment, this time point was chosen for all subsequent experiments.

View larger version (70K): [in this window] [in a new window]   Figure 1. (A) Pretreatment of A549 cells with FGF-10 for as little as 1 h blocks the DNA damaging effect of H2O2. A549 cells were pretreated with FGF-10 (10 ng/ml) at 0, 1, 4, and 24 h periods then exposed to H2O2 (50 µM) for 30 min and DNA damage was assessed by the alkaline elution technique. The protective effect of FGF-10 was seen in as little as 1-h treatment with FGF-10. *P < 0.05 FGF-10 + H2O2 at 0 h versus 1, 4, and 24 h, n = 3. Control, open bars; H2O2, dashed bars; FGF-10 + H2O2, filled bars. (B) FGF-10 attenuates H2O2-induced AEC DNA-SB formation as assessed by Comet assay. A549 cells were pretreated with FGF-10 (10 ng/ml) for 1 h, followed by H2O2 (50 µM) for 30 min. DNA damage was determined by single cell gel electrophoresis (Comet assay; Trevigen Inc) exactly as described by manufacturer's protocol. As shown in the panel, FGF-10 prevented H2O2-induced A549 cell DNA damage. (C) FGF-10 attenuates H2O2-induced AEC DNA-SB formation. A549 cells were pretreated with FGF-10 at variable dose (1, 10, and 100 ng/ml) followed by H2O2 (50 µM) for 30 min. DNA-SB formation was assessed by the alkaline elution technique. The protective effect of FGF-10 against H2O2-induced A549 cell DNA-SB formation was seen with as little as 1 ng/ml dose. *P < 0.05 H2O2 versus FGF-10 (1, 10, and 100 ng/ml) + H2O2, n = 3. A dose-dependent protective effect of FGF-10 was seen only between the dose of 1 and 10 ng/ml (P < 0.05 FGF-10 1 versus 10 ng/ml). (D) FGF-10 and KGF attenuate H2O2-induced AEC DNA damage. A549 cells were treated with FGF-10 (10 ng/ml) or KGF (100 ng/ml), followed by H2O2 (50 µM) for 30 min, and DNA damage was assessed by DNA-SB assay. H2O2 caused 80% reduction in double-stranded DNA. FGF-10 and KGF both attenuated H2O2-induced A549 cell DNA damage. *P < 0.05 control versus H2O2, P < 0.05 H2O2 versus KGF (100 ng/ml)/FGF-10 (10 ng/ml) + H2O2, n = 3.

MAPK-Dependent Pathways Mediate the Protective Effects of FGF-10
We recently showed that FGF-10 attenuates cyclic stretch-induced DNA damage by mechanisms involving MAPK activation (17). To explore whether MAPK activation also mediates the protective effect of FGF-10 against H₂O₂-induced AEC DNA-SB formation, A459 and rat AT2 cells were pretreated with specific MAPK-inhibitors (U0126: 10 µM or PD98059: 100 µM) for 15 min followed by FGF-10 (10 ng/ml) for 1 h, and then H₂O₂ for 30 min. U0126 and PD98059 both blocked the protective effect of FGF-10 against H₂O₂-induced DNA-SB formation in A549 cells and rat AT2 cells (Figure 2). PD98059 did not alter the DNA damaging effect of H₂O₂ (Figure 2A), which suggests that H₂O₂-induced MAPK activation does not mediate H₂O₂-induced DNA damage. Collectively, these data suggest that MAPK-dependent pathways are important in mediating the protective effect of FGF-10 against H₂O₂-induced AEC DNA damage.

View larger version (34K): [in this window] [in a new window]   Figure 2. (A–C) FGF-10 prevents H2O2-induced AEC DNA-SB formation via MAPK-dependent pathways. A549 as well as rat AT II cells were pretreated with MAPK-inhibitors U0126 or PD98059 before FGF-10 (10 ng/ml), followed by H2O2 (50 µM) for 30 min. Both inhibitors blocked the protective effects of FGF-10 against H2O2-induced A549 and ATII cell DNA-SB formation. Notably, PD98059 did not block H2O2-induced A549 cell DNA-SB formation (A) as well as U0126 did not block H2O2-induced DNA-SB formation in rat AT2 cells (C). *P < 0.005 control versus H2O2, P < 0.005 H2O2 versus FGF-10 + H2O2, P < 0.005 FGF-10 + H2O2 versus U0126 + FGF-10 + H2O2 and P < 0.005 FGF-10 + H2O2 versus PD98059 + FGF-10 + H2O2, n = 3. (A) PD98059 (–), filled bars; PD98059 (+), open bars.

View larger version (160K): [in this window] [in a new window]   Figure 3. FGF-10 and H2O2 both activate MAPK in AEC that is dependent on Ras/Raf-1 pathway activation. (A) Subconfluent A549 cells were serum-starved overnight and then pretreated with FGF-10 (200 ng/ml) or H2O2. Cells were washed, lysed with lysis buffer, and MAPK activation was assessed by Western immunoblotting by using ERK1/2 antibodies. Both FGF-10 and H2O2 induced ERK1/2 activation in A549 cells. Pretreatment of A549 cells with MAPK inhibitor, U0126, blocked FGF-10 as well as H2O2-induced A549 cell MAPK activation. When added together, FGF-10 and H2O2 induced a significant increase in MAPK activation as compared with FGF-10 or H2O2 alone. *P < 0.005 versus control. (B) Pretreatment of A549 cells with SH3b-p, an agent that blocks Ras/Raf-1 signaling via Grb2-SOS, blocked FGF-10 (200 ng/ml for 10 min) as well as H2O2-induced A549 cell ERK1/2 phosphorylation. *P < 0.05 Control versus FGF-10, or H2O2, or FGF-10 + H2O2; P < 0.05 FGF-10 versus U0126 + FGF-10, or SH3b-p + FGF-10, or FTI + FGF-10; P < 0.05 H2O2 versus U0126 + H2O2, or SH3b-p + H2O2, FTI + H2O2, or FSK + H2O2; P < 0.005 FGF-10 + H2O2 versus either U0126, SHJ3b-p, FTI or FSK plus FGF-10 + H2O2; n = 3. (C) Pretreatment of A549 cells with a Ras inhibitor, FTI 277 (10 µM), blocked FGF-10 (200 ng/ml for 10 min) as well as H2O2-induced A549 cell ERK1/2 phosphorylation. (D) Pretreatment of A549 cells with a Raf-1 inhibitor, FSK (50 µM) for 15 min, blocked FGF-10 (200 ng/ml for 10 min) as well as H2O2-induced A549 cell ERK1/2 phosphorylation. These data suggest that both FGF-10 and H2O2-induced ERK1/2 phosphorylation is dependent of Ras/Raf-1 pathway. Mean ± SEM, n = 3. *P < 0.05 Control versus FGF-10, or H2O2, or FGF-10 + H2O2; P < 0.05 FGF-10 versus U0126 + FGF-10, or SH3b-p + FGF-10, or FTI + FGF-10; P < 0.05 H2O2 versus U0126 + H2O2, or SH3b-p + H2O2, FTI + H2O2, or FSK + H2O2; P < 0.005 FGF-10 + H2O2 versus either U0126, SH3b-p, FTI or FSK plus FGF-10 + H2O2; n = 3. (E) Both FGF-10 and H2O2 failed to induce ERK1/2 phosphorylation in Ras dominant-negative N17 A549 cells, suggesting that both FGF-10 and H2O2 activate ERK/MAPK via Ras/Raf-1-dependent pathway.

View larger version (61K): [in this window] [in a new window]   Figure 4. The Ras/Raf-1 pathways mediate the protective effects of FGF-10 against H2O2-induced DNA damage. An inhibitor of Ras, FTI 277 (10 µM) (A), as well as a Raf-1, FSK (50 µM) (B), both blocked the protective effect of FGF-10 against H2O2-induced A549 cells DNA strand-break formation as assessed by comet assay. (A) Control, open bars; FTI, filled bars; FGT-10, dotted bars; FTI/FGF-10/H2O2, hatched bars. (B) No Forskolin, filled bars; Forskolin, hatched bars. Mean ± SEM, n = 3, *P < 0.05 versus control, P < 0.05 H2O2 versus FGF-10 + H2O2, P < 0.05 FGF-10 + H2O2 versus FTI or Forskolin + FGF-10 + H2O2. Similarly, the Ras dominant-negative N17 cell line also demonstrated H2O2-induced DNA strand-break formation as assessed by comet assay (C). Control, filled bars; FGT-10, hatched bars; H2O2, open bars; FGF-10 + H2O2, checkered bars. Mean ± SEM, n = 3, *P < 0.05 versus control, P < 0.05 H2O2 versus FGF10 + H2O2 (A549 cells), P < 0.05 H2O2 versus FGF10 + H2O2 (N17 cells). However, FGF-10 did not show any protective effects against H2O2-induced AEC DNA-SB formation in N17 cells.

View larger version (37K): [in this window] [in a new window]   Figure 5. Protective effect of FGF-10 against H2O2-induced DNA damage occurs in part by augmentation of AEC DNA repair via DNA polymerase. A549 cells were pretreated with FGF-10 (100 ng/ml) followed by H2O2 (50 µM) for 30 min. As assessed by comet assay, FGF-10 augmented A549 cell DNA repair against H2O2-induced DNA damage. This protective effect of FGF-10 was abolished by aphidicolin, a DNA polymerase inhibitor-, -, and -, and cold conditions, suggesting that the protective effects of FGF-10, in part, are mediated by DNA polymerase. (Left) Filled bars, 37°C; hatched bars, 0°C. (Right) Filled bars, control; hatched bars, aphidicolin.

	Discussion

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DNA damage surveillance mechanisms are crucial for maintaining genome integrity and cell survival (9). DNA-SB formation is among the earliest abnormalities that occur in cells exposed to oxidative stress such as H₂O₂, asbestos, radiation, and mechanical stretch (1, 2, 4, 7). The alkaline unwinding, ethidium bromide fluorescent assay for measuring DNA-SB is one of the most sensitive assays for detecting DNA damage, with a detection threshold of one break per chromosome (1). The Comet assay enables individual cells to be analyzed for DNA-SB formation (20). Using both assays of DNA-SB formation, we noted that FGF-10 nearly blocked H₂O₂-induced DNA damage. Moreover, the protective effects observed with FGF-10 were greater than that noted with KGF (Figure 1D).

Our understanding of MAPK activation in response to DNA damage is incomplete. Recent studies suggest that MAPK signaling is important for cell survival after exposure to oxidative stress, including H₂O₂ (17, 18, 21). Further, MAPK activation by DNA damaging agents, including H₂O₂, is directly correlated to the levels of DNA damage (19). Similar to these studies, we noted that H₂O₂ stimulated A549 cell MAPK activation. Several lines of evidence presented in this study demonstrate that the protective effects of FGF-10 against H₂O₂-induced A549 cell and rat AT2 cell DNA-SB formation are dependent upon an intact MAPK signaling pathway. First, MAPK inhibitors blocked the protective effects of FGF-10 against H₂O₂-induced A549 and AT2 cell DNA-SB formation (Figure 2). Notably, we observed that MAPK inhibitors did not augment H₂O₂-induced A549 cell and rat AT2 cell DNA-SB formation (Figures 2A and 2C) suggesting that MAPK activation is critical in mediating the protective effects of FGF-10 rather than inducing DNA-SB. Second, inhibitor studies focusing on various aspects of the Grb2-SOS/Ras/Raf-1 pathway implicate a role for the Grb2-SOS, Ras, and Raf-1 in mediating FGF-10–induced ERK1/2 phosphorylation (Figures 3B–3D). We also used Ras dominant-negative N17 A549 cells to show the importance of Ras in FGF-10-mediated MAPK activation (Figure 3E). The inhibitors of Grb2-SOS, Ras, and Raf-1 inhibitors as well as N17 cells also blocked H₂O₂-induced MAPK activation, suggesting that these pathways also mediate H₂O₂-induced MAPK activation. However, H₂O₂-induced A549 cell DNA-SB damage was independent of H₂O₂-induced A549 cell MAPK activation because MAPK inhibitors did not augment H₂O₂-induced A549 cell DNA-SB formation (Figures 2A and 2C). In addition, inhibitors of Ras and Raf-1 blocked the protective effect of FGF-10 against H₂O₂-induced DNA damage in A549 cells, however, had no effect on H₂O₂-induced DNA damage (Figures 4A and 4B). Similarly, the Ras dominant-negative N17 cell line also demonstrated that FGF-10 did not show any protective effects against H₂O₂-induced DNA damage (Figure 4C). The above data firmly support the conclusion that Ras-mediated MAPK activation is important in exerting the protective effects of FGF-10, but not in triggering H₂O₂-induced DNA damage (22). These findings parallel our recent study showing that FGF-10 attenuates cyclic stretch induced DNA-SB (17). Our findings also concur with a recent report demonstrating that MAPK signaling pathways are critical in fetal rat lung branching morphogenesis, a key role shared by FGF-10 (23). Collectively, these data firmly implicate the Grb2-SOS/Ras/Raf-1/MAPK pathway in mediating the protective effects of FGF-10.

The precise molecular mechanism by which MAPK activation by FGF-10 affords protection in our model requires further study. However, at least three possible mechanisms were considered. First, FGF-10 may augment antioxidant defenses. This possibility seems unlikely based upon previous studies, including one from our group, showing that KGF does not increase the activity of two antioxidants involved in H₂O₂ clearance from AEC, catalase, and GSH (2, 24). In contrast, Frank and coworkers (25) used differential display RT-PCR to show that KGF augments keratinocyte expression of a nonselenium glutathione peroxidase (GPX) gene, an enzyme that utilizes GSH to decrease the toxic effects of H₂O₂ and organic peroxides. Although additional studies exploring the effects of FGF-10 on AEC antioxidant levels may yield interesting results, the protective effects observed in this study after as little as a 1 h treatment period suggests that signaling mechanisms rather than transcriptional or translational changes in proteins are important.

Second, because FGF-10 is a potent AT2 cell mitogen (14), we questioned whether FGF-10's protective effects were due to increased AEC proliferation. This possibility seems unlikely for several reasons. First, using flow cytometry, we previously showed that a high percentage of A549 cells are in the proliferative stages of the cell cycle (S phase: 34% and G2/M phase: 10%) and that KGF (100 ng/ml) did not alter these percentages (4). Second, the protective effects observed in this study occurred after incubation with FGF-10 for as little as 1 h, which is not associated with cell doubling (data not shown). Finally, we previously noted that KGF did not increase A549 cell DNA synthesis as assessed by bromodeoxyuridine labeling over 24 h (2). Collectively, these data suggest that FGF-10 attenuates H₂O₂-induced AEC DNA damage through mechanisms that are independent of cell proliferation.

Third, growth factors may decrease oxidant-induced cellular damage by inhibiting apoptosis, a central pathway regulating cellular homeostasis (21). FGF family members bind specific tyrosine kinase receptors (FGFR) that are coupled to multiple signaling pathways. Signaling via the phosphatidyl 3-kinase (PI3K)/protein kinase B pathway is known to mediate survival signals from growth factors, including KGF (10, 17, 21). However, unlike the findings from the current investigation, MAPK inhibitors fail to block the protective effects of growth factors in studies implicating the PI3K pathway. We have also observed that, similar to other growth factors (2, 12, 21), FGF-10 may exert its protective effect in part via other signaling pathways involving protein kinase C (PKC) and tyrosine protein kinases (PTK) (data not shown). In this study, we focused on FGF-10-induced MAPK activation because previous studies have shown that signaling through this pathway provides a mechanism by which some growth factors, including FGF-10, prevent DNA damage and apoptosis (17, 21, 26, 27). There are at least three implicated mechanisms by which MAPK activation prevents apoptosis including: (i) the phosphorylation of pro-apoptotic Bcl-2 family members (e.g., Bax) that renders them inactive, (ii) the transcriptional increase of anti-apoptotic Bcl-2 family members (e.g., Bcl-2 or Bcl-X_L), and (iii) the translational upregulation of Bcl-2 and Bcl- X_L (21). In this study, the latter two possibilities seem unlikely to account for the protective effects of FGF-10 because we noted protection after as little as a 1-h treatment period (Figure 1A). Although further studies are necessary to address the first possibility, our data suggest an important role for MAPK activation for transmitting FGF-10-induced survival signals by markedly reducing H₂O₂-induced DNA damage.

In this study we provide some evidence that FGF-10 protects A549 cell by augmenting DNA repair (Figure 5). Both cold conditions and a DNA polymerase inhibitor blocked the protective effects of FGF-10. These findings with FGF-10 parallel our prior studies demonstrating that the protective effects of KGF against both H₂O₂- and radiation-induced AEC DNA damage are due in part to DNA repair mechanisms (2, 4). Because FGF-10, similar to KGF, binds the FGFR2iiib receptor located exclusively on epithelial cells, it seems highly likely that FGF-10 may augment DNA repair in manner similar to KGF. The mechanism by which eukaryotic cells repair H₂O₂-induced DNA damage is complex and not well established (5, 9). Our current findings suggest that detailed mechanistic studies exploring the role of MAPK in FGF-10-induced AEC DNA repair should prove interesting.

In summary, we demonstrate that FGF-10 attenuates H₂O₂-induced DNA-SB formation via the Grb2-SOS/Ras/Raf-1/MAPK pathway. Furthermore, the protective effects of FGF-10 occur in part by augmentation of AEC DNA repair. These findings add to the accumulating body of evidence implicating the MAPK signaling pathway in DNA repair and cell survival. Future investigations will be necessary to determine the downstream molecular mechanisms mediating protective effects of FGF-10 as well as the in vivo relevance of our findings. Our data suggest that FGF-10 may have an important role in preventing oxidant-induced lung injury.

	Acknowledgments

 
This work was supported by a National Research Science Award (D.U.) and a Merit Review grant from the Department of Veterans Affairs (D.W.K.). The authors are grateful to Dr. J. I. Sznajder for providing N17 A549 cells.

Received in original form February 26, 2003

Received in final form February 13, 2004

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