FGF signaling is required for pulmonary homeostasis following hyperoxia
Isamu Hokuto,
Anne-Karina T. Perl, and
Jeffrey A. Whitsett
Divisions of Neonatology and Pulmonary Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039
Submitted 15 August 2003
; accepted in final form 13 November 2003
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ABSTRACT
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To assess the role of fibroblast growth factor (FGF) signaling in pulmonary function in the postnatal period, we generated transgenic mice in which a soluble FGF receptor (FGFR-HFc) was conditionally expressed in respiratory epithelial cells of the mouse lung, thereby inhibiting FGF activity. Although FGFR-HFc did not alter postnatal lung morphogenesis, male FGFR-HFc transgenic mice were more susceptible to hyperoxia and failed to recover when ambient oxygen concentrations were normalized. Inflammation, alveolar-capillary leak, and mortality were increased following exposure to 95% FIO2. Expression of surfactant protein (SP)-A and SP-B were significantly decreased in association with decreased immunostaining for thyroid transcription factor-1. FGF signaling is required for maintenance of surfactant homeostasis and lung function during hyperoxia in vivo, mediated, at least in part, by its role in the maintenance of SP-B expression.
fibroblast growth factor; lung injury; surfactant protein; thyroid transcription factor-1
INHALED OXYGEN AND MECHANICAL ventilation remain the mainstay of respiratory therapy for acute respiratory distress. Despite the utility of oxygen in the maintenance of arterial oxygenation, oxygen is toxic, causing injury to both epithelial and vascular compartments of the lung. Exposure of adult mammals to 95-100% oxygen generally results in respiratory compromise and death within several days of continuous exposure (34). The lung responds to hyperoxia by enhancing the expression of cytoprotective proteins including antioxidants, surfactant proteins, and DNA repair enzymes (24, 25, 45). Although endotoxin, various cytokines, and polypeptide hormones, including IL-11, IL-6, EGF, and FGF-7 enhance cytoprotection of the lung during hyperoxia (4, 5, 30, 37, 42-44), endogenous factors critical for survival and repair of the lung during oxygen exposure are not known.
Intratracheal FGF-7 decreased lung injury and enhanced survival during acute lung injury caused by oxygen, acid instillation, and infection, presumably by interacting with FGF receptors on target cells. The protective effects of FGF required pretreatment of the animals before exposure to oxygen (26, 46). It is less clear whether oxygen injury stimulates endogenous FGF receptor signaling to maintain pulmonary homeostasis during oxygen injury. In previous studies, transgenic mice were generated in which FGF receptor signaling was inhibited by expression of a dominant-negative soluble FGF receptor (FGFR-HFc) that was secreted by respiratory epithelial cells of the peripheral lung. Although expression of FGFR-HFc in epithelial cells of the embryonic lung blocked peripheral lung formation, its expression in the postnatal period did not alter lung morphogenesis or postnatal survival (16), thereby generating a model in which FGF signaling could be inhibited in the adult. To determine whether FGF signaling influences physiological processes in the postnatal lung, we assessed the effects of FGF receptor inhibition on pulmonary responses to hyperoxia in adult mice.
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MATERIALS AND METHODS
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Gene construction and PCR. We generated surfactant protein C-reverse tetracycline transactivator (SP-C/rtTA) and (tetO)7-cytomegalovirus (CMV)-FGFR-HFc mice as previously described (16). The human SP-C promoter is selectively expressed in peripheral bronchiolar epithelial cells and in type II alveolar cells in transgenic mice. SP-C/rtTA and (tetO)7-CMV-FGFR-HFc mice were mated to produce transgenic mice bearing both genes. For genotyping, DNA was extracted from tails, and PCR was performed for SP-C/rtTA and (tetO)7-CMV-FGFR-HFc genes using primers 5'-AAA ATC TTG CCA GCT TTC CCC-3' and 5'-GAC ACA TAT AAG ACC CTG GTC A-3' for SP-C/rtTA and 5'-CAG GCC AAC CAG TCT GCC TGG C-3' and 5'-CGT CTG AGC TGT GTG CAC CTC C-3' for (tetO)7-CMV-FGFR-HFc. The conditioned PCR was 30 cycles at 95°C for 30 s, 58°C for 30 s, 72°C for 30 s, followed by a 10-min extension at 72°C. Double transgenic mice and littermates were used for the experiments.
Animal use and doxycycline administration. Transgenic and control mice were kept in a pathogen-free vivarium in accordance with institutional guidelines under Institutional Animal Care and Use Committee-approved protocols. Doxycycline was administered to the mice in the chow at a concentration of 625 mg/kg (Harlan Teklad, Madison, WI) from 4 wk of age to the end of the experiment. Experimental mice were exposed either to 95% oxygen or to room air for 72 h at 9-11 wk of age. Oxygen-exposed mice were weaned for 24 h in 60% and then placed in room air for the duration of the experiment. If found in respiratory distress, mice were was recorded.
Tissue preparation, histology, and immunohistochemistry. Mice were killed by lethal dose injection of ketamine, xylazine, and acepromazine. Lungs were inflation fixed with 4% paraformaldehyde in PBS at 25 cmH2O and immersed in same fixative. Tissue was fixed overnight, dehydrated in the series of alcohol embedded in paraffin. Tissue sections were stained with hematoxylin and eosin. Immunohistochemistry for SP-B, pro-SP-C, platelet endothelial cell adhesion molecule 1 (PECAM-1), and thyroid transcription factor (TTF)-1 was performed on tissue sections as previously described (49). Anti-SP-B (AB3426), pro-SP-C (AB3428), and rat TTF-1 antibodies were generated in our laboratory and are available at Chemicon (Temecula, CA). Anti-PECAM-1 antibody was purchased from Pharmingen (San Diego, CA).
Isolation of RNA and RNA analysis. Lung tissues were excised and homogenized with TRIzol reagent (Invitrogen), and the RNA was extracted as directed by the manufacturer. RNA concentration was measured by spectrophotometer, and RNA (3 µg) was used for S1 nuclease protection assays; 10 µg were used for RNase protection assays. S1 nuclease protection assay for SP-A, SP-B, SP-C, and L32, and RNase protection assay for TTF-1 was performed as previously described (2, 14) and quantified by the ImageQuant. For RT-PCR, RNA was treated with DNase, and cDNA was synthesized by using Superscript II (Invitrogen). FGFR-HFc mRNA was detected with primers 5'-CAG GCC AAC CAG TCT GCC TGG C-3' and 5'-CGT CTG AGG TGT GTG CAC CTC C-3' and the following conditions: 30 cycles of 95°C for 30 s, 58°C for 30 s, 72°C for 30 s, and 72°C for 7 min.
Cell counts in bronchoalveolar lavage fluid. Mice were killed by injection of ketamine, xylazine, and acepromazine and exsanguination. PBS (1 ml) was infused into the trachea and withdrawn by syringe three times for each aliquot. This procedure was repeated three times, and the samples were pooled. The bronchoalveolar lavage (BAL) fluid was centrifuged at 1,500 rpm for 5 min to collect cells. Supernatant was stored at -80°C for protein analysis. Cells were suspended in 3 ml of red blood cell lysis buffer (140 mM NH4Cl, 20 mM Tris·Cl, pH 7.6) and recentrifuged. Cell pellets were resuspended in 0.5 ml of PBS, and cell number was counted with a hemacytometer. Differential cell counts were performed on the cytospin preparations after Giemsa staining.
Protein and cytokine analysis of BAL fluid and lung tissue. Total protein in BAL fluid was measured by the method of Lowry et al. (19). SP-B in BAL fluid was analyzed by enzyme-linked immunosorbent assay (ELISA) as previously described (32). For tissue analysis, lungs were homogenized in 1 ml of PBS and centrifuged at 3,000 rpm for 10 min. The supernatant was stored at -80°C. IL-1
and IL-6 were quantitated in aliquots using sandwich ELISA kits (R&D Systems, Minneapolis, MN).
Statistical analysis. Analysis of difference among the groups was carried out by ANOVA for multiple groups and Student's t-test for two groups. Results were expressed as means ± SE. Significance level was taken as P < 0.05.
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RESULTS
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The constructs used to express the FGFR-HFc placed the mutant receptor under conditional control of SP-C/rtTA and doxycycline (Fig. 1A). FGFR-HFc mRNA was assessed in adult transgenic mice in the presence and absence of doxycycline, demonstrating that the receptor was expressed only in double transgenic mice during exposure to doxycycline (Fig. 1B). Under vivarium conditions, survival and lung morphology in the FGFR-HFc transgenic mice were unaltered when doxycycline was administered after birth (data not shown).

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Fig. 1. Gene construction and conditional expression of a soluble FGF receptor (FGFR-HFc). A: surfactant protein C-reverse tetracycline transactivator (SP-C/rtTA) produces the rtTA under control of SP-C promoter. Doxycycline binds to the tetracycline transactivator, and this complex binds to (tetO)7 promoter and activates FGFR-HFc expression. CMV, cytomegalovirus. B: mice were treated with or without doxycycline (Dox) from 4 wk of age. RNA was extracted, and cDNA was synthesized. -Actin and FGFR-HFc was identified by gel electrophoresis. FGFR-HFc was expressed only with doxycycline treatment (lane 2). This figure is representative of n = 10.
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FGFR-HFc enhances susceptibility to oxygen injury. To assess the role of FGF signaling in pulmonary homeostasis during lung injury and repair, we exposed the FGFR-HFc mice to 95% oxygen for 3 days, followed by a 1-day period in 60% oxygen, and then placed them in room air for recovery. Although most control littermates survived after 9 days, the majority of FGFR-HFc transgenic mice developed severe respiratory distress and were killed or died (Fig. 2). Mortality following the initial period of hyperoxia was higher in male than female mice (Fig. 2). Thereafter, experiments were conducted with male mice for consistency. Survival of the non-transgenic and male FGFR-HFc mice that were not treated with doxycycline was similar. Lung histology was assessed before and after oxygen exposure and during recovery. Mild air space enlargement and inflammatory cell infiltrates were observed in both control and transgenic mice, and there were no discernable differences in lung histology (Fig. 3), indicating similarity in extent of initial oxygen injury. Proliferation index was assessed after bromodeoxyuridine (BrdU) labeling, consistent with the lack of differences in lung histology; cell labeling indexes were not altered by expression of FGFR-HFc (data not shown).

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Fig. 2. Survival rate following 95% oxygen exposure. Double transgenic mice and control male or female mice were treated with doxycycline from 4 wk of age and at 8-10 wk of age placed in 95% O2 for 3 days, 60% O2 for 1 day, followed by room air for 5 days. Male double transgenic mice treated with doxycycline were more susceptible to hyperoxia than controls (P < 0.05). Most FGFR-HFc-expressing males were killed or died on day 4 or 5 of the experiment. In female mice, statistically significant differences in mortality were not observed between the genotypes at these times. Statistical differences were assessed by Log rank test, n 12 per group.
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Fig. 3. Effects of FGFR-HFc on lung morphology. Lung sections from double transgenic mice and littermates were obtained after exposure to 95% O2 and stained with hematoxylin and eosin. Lung morphology (before and after exposure to 95% O2) was similar in both control and double transgenic mice. Mild air space enlargement was observed on day 5 of the experiment (C and F) in both control and FGFR-HFc-expressing mice. Photomicrographs are representative of n = 3. Bar = 100 µm.
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Increased alveolar capillary leak in FGFR-HFc-expressing mice. During recovery, lung weight and lung weight-to-body weight ratio were significantly increased in the FGFR-HFc transgenic mice on day 4 (Fig. 4A). Likewise, the protein content and BAL fluid were significantly increased, consistent with alveolar-capillary leak (Fig. 4B). We performed PECAM-1 staining in lung tissue on days 0, 3, and 5 to assess the alveolar capillary bed. The intensity and distribution of PECAM-1 staining were not altered by the FGFR-HFc during oxygen exposure (Fig. 4C).

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Fig. 4. Increased alveolar-capillary leak following hyperoxia. A: lung weight/body weight (%). On days 0, 3, and 4 of the experiment, body weight and lung weight were measured. There were no differences in lung/body wt between control and FGFR-HFc (Fc) mice on days 0 and 3. Lung/body wt (%) were significantly different on day 4 of the experiment. Statistical differences were assessed by ANOVA, n = 3 per group. B: total protein in bronchoalveolar lavage fluid (BALF). The lungs were excised at days 0, 3, and 4 of the experiment and lavaged. Total protein was measured in the pooled BALFs and expressed as µg/ml. On day 0, there were no differences in BALF protein in control and FGFR-HFc mice. On day 4, BALF protein was significantly increased in FGFR-HFc mice. Statistical differences were assessed by ANOVA, n = 3 per group. C: inhibition of FGF signaling did not alter platelet endothelial cell adhesion molecule (PECAM) staining of the endothelium. Lung sections were stained with PECAM-1. No differences in distribution and intensity of staining were observed in control and FGFR-HFc-expressing mice. Photomicrographs are representative of n = 3; bar = 100 µm.
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Effects of FGFR-HFc on lung inflammation. Total and differential cell counts were performed on days 0 and 4 of the experiment. Total cell count and the percentage of neutrophils were increased on day 4 (Fig. 5, A and B). Inflammatory cytokines were assessed in whole lung homogenates. In FGFR-HFc mice, IL-1
and IL-6 levels were significantly increased on day 4 (Fig. 5C).

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Fig. 5. Increased inflammation during recovery from hyperoxia in FGFR-HFc transgenic mice. A: cell counts were performed on BALF on days 0, 3, and 4 of the experiment. Before oxygen exposure and on day 3, there were no differences in the cell count between control and FGFR-HFc mice. On day 4, total cell count in lungs of control mice remained normal, whereas that of FGFR-HFc mice was significantly increased. Statistical differences were assessed by ANOVA, n = 3-4 per genotype. B: cells were collected from BALF and stained with Giemsa. On day 4 of the experiment, the percentage of neutrophils was significantly increased in FGFR-HFc mice compared with that of controls. Statistical differences were assessed by ANOVA, n = 3-4 per group. C: IL-1 and IL-6 were measured in whole lung homogenates on days 0, 3, and 4 by ELISA. IL-1 and IL-6 were significantly increased on day 4 in the FGFR-HFc mice. Differences were analyzed by ANOVA, n = 3-4 per genotype.
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Decreased expression of surfactant proteins in FGFR-HFc transgenic mice. Because pulmonary surfactant is required for maintenance of pulmonary function during hyperoxia, we assessed tissue RNA and BAL protein concentrations for SP-A, SP-B, and SP-C. Before exposure to oxygen, SP-A, SP-B, and SP-C expression was similar in FGFR-HFc and control mice. In both control and FGFR-HFc-expressing mice, surfactant protein (SP-A, SP-B, and SP-C) mRNAs were decreased on days 3 and 4 of hyperoxia; SP-A and SP-B mRNAs were significantly reduced in the FGFR-HFc compared with control mice on day 4 of oxygen exposure (Fig. 6A). Because SP-B activity is critical for survival in hyperoxia, we assessed its content in BAL fluid. Alveolar SP-B was significantly reduced 3 and 4 days after oxygen exposure in the FGFR-HFc compared with control mice (Fig. 6B). Because reduction of SP-B to <20-30% of normal causes alveolar capillary leak and respiratory failure even in room air, the marked reduction in SP-B content is consistent with the increase in respiratory distress and death in the FGFR-HFc mice. Immunohistochemistry for SP-B confirmed the decrease in expression of SP-B in bronchiolar and alveolar regions of the lung (Fig. 7A). In control mice, SP-B staining decreased during 3 days of oxygen exposure but was rapidly restored in the alveolar epithelium during recovery on day 5. In contrast, SP-B staining remained decreased in the FGFR-HFc mice.

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Fig. 6. Effects of FGFR-HFc on surfactant protein expression. A: SP-A, -B, and -C mRNAs were measured by S1 nuclease protection assays on days 0, 3, and 4 of the experiment. Before exposure to 95% oxygen, SP-A, -B, and -C mRNAs were similar in both genotypes. On day 4 of the experiment, SP-A and -B mRNAs were significantly decreased, n = 3-6 per group. Differences were assessed by ANOVA; values are means ± SE. B: on days 0, 3, and 4, BALF SP-B was measured by ELISA. Data were standardized to total protein in BALF and normalized to control on day 0. On days 3 and 4, SP-B content was significantly decreased. Differences were assessed by Student's t-test, n = 3-4 per group.
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Fig. 7. A: decreased SP-B staining in FGFR-HFc mice following hyperoxia. SP-B staining was performed on tissue sections of days 0, 3, and 5 of the experiment. Before oxygen exposure, SP-B staining was similar in control and FGFR-HFc mice. SP-B staining was decreased in FGFR-HFc mice, particularly in bronchiolar epithelium on days 3 and 5 (arrows indicate positive staining; arrowheads indicate negative staining). Photomicrographs are representative of n = 3 per each group (bar = 100 µm). B: decreased thyroid transcription factor (TTF)-1 immunostaining in FGFR-HFc transgenic mice. Lung sections (days 0, 3, and 5) were stained TTF-1. TTF-1 staining was decreased in epithelial cells lining airways of FGFR-HFc compared with control mice. TTF-1 staining was decreased following hyperoxia and did not recover on day 5 in FGFR-HFc mice (arrow indicates positive staining; arrowheads indicate negative staining). Photomicrographs are representative of n = 3 per each group (bar = 100 µm).
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Decreased TTF-1 during recovery from hyperoxia. Because TTF-1 is known to regulate surfactant protein gene transcription, we assessed TTF-1 immunostaining during hyperoxia and recovery. TTF-1 staining was similar in the lungs of FGFR-HFc and control mice in room air. TTF-1 staining was decreased during exposure to 95% oxygen on day 3 but increased during the recovery period, being detected in the nuclei of alveolar type II cells in control mice. In contrast, staining for TTF-1 remained decreased during the recovery period in the FGFR-HFc transgenic mice (as seen on day 5, Fig. 7B). Consistent with this observation, TTF-1 mRNA was significantly decreased in the FGFR-HFc mice on day 4 (Fig. 8). Thus FGF signaling was required for maintenance of both TTF-1 and its transcriptional target, SP-B, during recovery from hyperoxia.

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Fig. 8. Effect of FGFR-HFc on TTF-1 expression. TTF-1 mRNA was determined by RNase protection assay on days 0, 3, and 4 of the experiment. Results were standardized by L32 and normalized to control on day 0. On day 0, expression of TTF-1 was similar in control and FGFR-HFc-expressing mice. On day 3, TTF-1 mRNA was significantly decreased in both control and FGFR-HFc mice. While TTF-1 recovered in control mice on day 4, TTF-1 expression remain decreased in the FGFR-HFc mice. Differences were assessed by ANOVA, n = 3-6 per group.
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DISCUSSION
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Selective inhibition of FGFR signaling in the postnatal mouse lung did not alter lung morphogenesis or function under physiological conditions but rendered male mice susceptible to oxygen-induced injury, limiting their survival during the recovery period. Increased alveolar-capillary leak, inflammation, and mortality were observed in FGFR-HFc-expressing mice. Expression of surfactant proteins SP-A and SP-B were decreased in the lungs of FGFR-HFc transgenic mice following hyperoxia. During recovery in room air, the expression of SP-B staining and mRNA was maintained in control but failed to recover in the FGFR-HFc transgenic mice. Restoration of TTF-1, an FGF-sensitive transcription factor regulating surfactant protein gene expression, was dependent on FGF signaling. Together, the FGF receptor pathway was critical for maintenance of surfactant homeostasis and lung function following hyperoxia.
FGF signaling plays an essential role during lung morphogenesis. Targeted deletion of FGF-10, the FGFR2IIIb receptor, expression of a dominant-negative FGF receptor or Sprouty, an intracellular FGF antagonist, blocked lung morphogenesis during the early embryonic period (13, 16, 20, 23, 27). Although inhibition of FGF signaling until embryonic day 12 caused severe lung hypoplasia, the effects were less extensive when FGF inhibition was induced later in gestation. In previous studies from our laboratory, the ability of the FGFR-HFc to inhibit effects of FGF-7 on lung branching was shown in vitro and in vivo (16). No effects of Sprouty-4 or FGFR-HFc receptors were observed in the postnatal period under physiological conditions (16, 27). Although FGF ligands are present in the adult lung, it is unclear whether FGF signaling plays a role in postnatal lung homeostasis. On the other hand, FGF signaling can be readily activated in the adult lung by exposure to exogenous FGF polypeptides. Intratracheal administration or genetic expression of FGF-7, FGF-10, or FGF-3 caused marked proliferative effects in the respiratory epithelium in vivo (3, 33, 48, 50) and induced the expression of enzymes involved in surfactant homeostasis (21). Physiological roles for endogenous FGF signaling during injury and repair of the lung, however, have not been demonstrated.
The FGFR-HFc peptide expressed in the present study is synthesized in type II epithelial cells and in proliferative bronchiolar epithelial cells under control of the SP-C promoter. Previous studies from this laboratory and others have demonstrated its inhibitory activity on FGF signaling in vitro and on FGF-dependent activities in vivo (9, 16). Because the FGFR-HFc binds to various FGF polypeptides, presumably restricting them from their active sites, it is unclear which FGF ligands mediated the effects of the FGFR-HFc during hyperoxia in the present study. Intratracheal FGFs have marked cytoprotective effects on the lung following various pulmonary injuries. Ulich et al. (40) first demonstrated the marked effect of FGF-7 on lung cell proliferation. Pretreatment of mice with FGF-7 before oxidant injury enhanced epithelial cell proliferation, protecting the lung from hyperoxia (26). Subsequent studies demonstrated the effects of exogenous FGF-7 on lung injury, caused by acid aspiration (46). In a recent study, conditional expression of FGF-7 protected the respiratory epithelial cells during hyperoxia (33). It has been less clear whether endogenous FGFs or other polypeptides mediate endogenous cell responses during lung injury. The present study provides direct evidence for a role of endogenous FGF receptor signaling in the protection of lung function following injury.
Because FGFs have marked and pleotropic effects on cell signaling, proliferation, and gene expression, the mechanisms underlying the cytoprotective effects of FGF on lung injury are likely to be complex. Likewise, the lung responds in complex ways to hyperoxia with induction of cytoprotective responses, enhanced expression of antioxidants, DNA repair enzymes, surfactant proteins, and inflammatory mediators (24, 25, 45). In the present study, a short period of oxygen was administered followed by a weaning into room air for recovery. In control mice, pulmonary homeostasis was maintained and surfactant protein gene expression was rapidly restored during the recovery period. Proliferative effects in this model were modest, as the cytotoxic injury was short and not sufficient to cause death in the FVB/N mouse. BrdU staining was decreased by hyperoxia and was not different in FGFR-HFc and control mice. Likewise, lung histology was not substantially perturbed during oxygen exposure (day 3), and differences in control and FGFR-HFc mice were not noted, indicating that the severity of initial lung injury was similar in both groups of mice. In contrast to wild-type mice, male FGFR-HFc-expressing mice failed to recover following oxygen injury. SP-B and TTF-1 remained at low levels, and alveolar capillary leak, inflammation, and respiratory failure were observed in the FGFR-HFc mice.
SP-B is critical for lung function. Expression of SP-B is required for lung function at birth in mice and in humans. Reduction of SP-B to 20-30% of wild-type levels causes acute respiratory failure and death in mice (12, 22). Reduction of SP-B to 50% of wild-type levels in the Sftpb+/- mice renders them susceptible to hyperoxia, with resultant alveolar capillary leak, inflammation, and death (39). Thus the decrease in SP-B in the FGFR-HFc-expressing mice is sufficient to cause respiratory failure. SP-B is required for the reduction of surface tension and the maintenance of alveolar capillary integrity. Surfactant proteins are expressed in alveolar type II epithelial cells, and their expression is under strict control of a lung selective transcription factor, TTF-1 (6, 8, 17). TTF-1-/- mice lack peripheral lung tissue and fail to express SP-A, -B, and -C (18). Likewise, FGF signaling is required for the formation of peripheral lung during embryogenesis and influences the expression of TTF-1. FGF-7 or FGF-10 stimulated TTF-1 and SP-B and -C expression in vivo and in vitro (11, 38, 47). In the present study, nuclear TTF-1 was markedly decreased during acute hyperoxia and increased during the recovery period in wild-type, but not FGFR-HFc, mice. Likewise, restoration of SP-B, a transcriptional target of TTF-1, was observed during recovery of wild-type but not FGFR-HFc mice. Together, inhibition of FGFR signaling is required for recovery of TTF-1 and SP-B following hyperoxia. Deterioration of lung function in the FGFR-HFc mice is therefore related, at least in part, to the lack of pulmonary SP-B.
In the present study, male mice were more sensitive to oxygen than females. Of interest, survival of control mice was associated with maintenance of TTF-1 and SP-B expression at 5 days (in the recovery period). Because the time of oxygen exposure was limited to 3 days to permit analysis of lung histology and gene expression, the present study prioritized analysis in male mice. We did further study of the effects of a more prolonged period of oxygen exposure in females. It is unclear whether effects of FGF inhibition will be similar in female mice. The mechanisms underlying this sex-related difference in sensitivity to hyperoxia are unclear at present.
Proposed model underlying the protective effects of FGF signaling during hyperoxia. The present study supports a model in which FGF signaling maintains lung function during hyperoxia by influencing SP-B expression and surfactant activity. Oxygen is known to induce pulmonary FGF production in vivo (10, 29). Because FGF signaling is also known to activate several intracellular signaling pathways that influence TTF-1 activity, including Ras and p38 MAP kinases (7), the activity of TTF-1 on target genes (including SP-A, SP-B, and SP-C) may be influenced by maintenance of TTF-1 activity during hyperoxia. Because TTF-1 activity is also regulated by oxidation-reduction, its decline during acute hyperoxia and subsequent fall in SP-B may be mediated by modification of TTF-1 or its transcriptional partners during oxidant injury (1, 36). SP-A, SP-B, and phospholipid synthesis are enhanced by FGF-dependent pathways (11, 21). Thus a model by which TTF-1 activity is influenced by FGF signaling to maintain SP-B production and surfactant homeostasis is consistent with the present observations. It is also likely that other genes and pathways mediating oxidant protection are also influenced by FGFs during repair of the lung following hyperoxia.
FGF signaling influences pulmonary inflammation. Severe inflammation and increased expression of proinflammatory cytokines were noted in the lungs of FGFR-HFc transgenic mice following oxygen exposure, suggesting that FGF signaling may inhibit pulmonary inflammation. An anti-inflammatory role for FGFs has been supported by several studies. FGF-7 decreased concentrations of inflammatory cytokines in the lungs of mice following bone marrow transplantation (15). Likewise, FGF-7 inhibited the migration of inflammatory cells into lung following Pseudomonas infection (41) and decreased the expression of IFN-
-induced genes in vitro (31). Thus the protective effects of endogenous FGFs on lung injury during hyperoxia may be influenced, at least in part, by its effects on inflammation.
Oxygen is widely used for therapy of both acute and chronic lung disorders. Exposure to high ambient concentrations of oxygen is toxic to lung cells, causing epithelial and endothelial cell injury. Previous studies in humans demonstrated the loss of TTF-1 and decreased surfactant protein in epithelial cells in acute lung injury associated with hyaline membrane disease, pulmonary edema, and infection (35). Therefore, strategies to maintain FGF signaling during hyperoxia may enhance recovery of the lung following injury.
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ACKNOWLEDGMENTS
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We thank William Hull and Dr. Cindy Bachurski for technical assistance with surfactant protein and mRNA measurements.
GRANTS
This work was funded by National Heart, Lung, and Blood Institute Grants HL-56387 and HL-38859 (J. A. Whitsett).
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FOOTNOTES
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Address for reprint requests and other correspondence: J. A. Whitsett, Cincinnati Children's Hospital Medical Center, Div. of Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (E-mail: jeff.whitsett{at}cchmc.org).
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. Section 1734 solely to indicate this fact.
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