help button home button
AJRCCM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on May 25, 2006, doi:10.1164/rccm.200511-1718OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
200511-1718OCv1
174/5/581    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yi, M.
Right arrow Articles by Tanswell, A. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yi, M.
Right arrow Articles by Tanswell, A. K.
American Journal of Respiratory and Critical Care Medicine Vol 174. pp. 581-589, (2006)
© 2006 American Thoracic Society
doi: 10.1164/rccm.200511-1718OC

Original Article

Fibroblast Growth Factor-2 and Receptor-1{alpha}(IIIc) Regulate Postnatal Rat Lung Cell Apoptosis

Man Yi, Rosetta Belcastro, Samuel Shek, Daochun Luo, Martin Post and A. Keith Tanswell

Canadian Institutes of Health Research Group in Lung Development, Lung Biology Programme, Hospital for Sick Children Research Institute; and the Departments of Paediatrics and Physiology, University of Toronto, Toronto, Ontario, Canada

Correspondence and requests for reprints should be addressed to A. Keith Tanswell, M.B., Division of Neonatology, The Hospital for Sick Children, 555 University Avenue, Toronto, ON, M5G 1X8 Canada. E-mail: keith.tanswell{at}sickkids.ca


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Fibroblast growth factor receptor-1{alpha}(IIIc) [FGF-R1{alpha}(IIIc)] regulates recovery of neonatal rat lung growth, after 95% oxygen–mediated growth arrest. Its role in normal postnatal alveologenesis is unknown.

Objective: To determine if FGF-R1{alpha}(IIIc) regulates normal postnatal alveologenesis.

Methods: Truncated soluble FGF-R1{alpha}(IIIc) or neutralizing antibodies to FGF-1 or FGF-2 were injected intraperitoneally into 3-d-old rats. The pups were killed at Day 7 for studies of alveolar development.

Measurements and Main Results: Injected, truncated soluble FGF-R1{alpha}(IIIc) inhibited phosphorylation of the endogenous FGF-R1, and downstream pathway, and paradoxically increased lung DNA content and tissue fraction while inhibiting lung cell DNA synthesis. The increase in tissue thickness was due to reduced apoptosis, as indicated by reductions in cleaved effector caspases 3 and 7. Inhibition of the intrinsic apoptosis pathway was suggested by decreases in the proapoptotic protein Bax and mitochondrial cytochrome c release, and an increase in the antiapoptotic protein Bcl-xL. Injected antibodies to FGF-1 and FGF-2 had no effect on DNA synthesis, but both increased Bcl-xL content and decreased cytochrome c release and cleaved caspase-7 protein expression. However, only injection of the antibody to FGF-2 replicated the increased tissue fraction and inhibited apoptosis observed with the injection of truncated soluble FGF-R1{alpha}(IIIc).

Conclusions: Inhibition of ligand binding, most likely of FGF-2, to the FGF-R1{alpha}(IIIc) inhibits normal postnatal lung cell apoptosis.

Key Words: fibroblast growth factors • lung development • lung growth • truncated soluble receptors

Fibroblast growth factors (FGFs) are a large family of low-molecular-weight polypeptides that regulate morphogenesis of many organs, including the lung (1, 2). Several of the FGFs, including FGF-1 (acidic FGF), FGF-2 (basic FGF), FGF-7, FGF-9, FGF-10, and FGF-18 are expressed in the developing lung (3–10). The FGF family acts through five high-affinity receptors (FGF-R1 [flg], FGF-R2 [bek], FGF-R3, FGF-R4, and FGF-R5), all of which are expressed in the lung (11, 12). With the exception of FGF-R5, the FGF receptors all have an intracellular tyrosine kinase domain, a single membrane-spanning domain, and an extracellular region consisting of three immunoglobulin-like domains (D1, D2, D3). The C-terminus portion of the D3 domain mediates ligand binding, and classification of the FGF receptors is based on three discrete subgroups of D3 domains, which represent variably spliced isoforms (13) with unique FGF-binding specificities (14).

FGF-R1 RNA is detected in the fetal rat from Day 18 of gestation and its expression remains relatively constant until 28 d after birth (15), covering the period of postnatal alveologenesis. FGF-R1 binds FGF-1, FGF-2, FGF-3, FGF-4, and FGF-8, but not FGF-7 (16). FGF-1 binds with equivalent affinity to FGF-R1{alpha}(IIIc) and FGF-R1{alpha}(IIIb), but FGF-2 binds with higher affinity to FGF-R1{alpha}(IIIc) (17). Also, a naturally occurring secreted form of FGF-R1 has a greater affinity for FGF-2 than for FGF-1 (18).

Alveoli initially form from prealveolar saccules by a process of secondary septation, which involves the in-growth of secondary crests from the walls of the saccule, thus subdividing it into smaller gas exchange units, the alveoli (19). Approximately 85% of alveoli form after birth in the human, whereas alveologenesis is a completely postnatal event in the rat (20). In the neonatal rat, the most rapid increase in alveolar density occurs between Days 3 and 8 of life (21), and alveolar formation is essentially complete by Day 21 of life (22). In addition to cell proliferation, apoptosis of alveolar epithelial cells and interstitial fibroblasts is an important component of the lung remodeling that occurs during alveologenesis in both the rat and human (23–25).

The growth factors, and growth factor receptors, that regulate the process of alveologenesis have not been defined. With respect to the FGF family, there is evidence to suggest that FGF-R3 and FGF-R4 interact to play a critical role in the postnatal formation of alveoli (11), whereas epithelial cell FGF-R2 apparently does not play a role (26). FGF-R1 is essential for murine embryonic development, with embryos carrying homozygous deletions of FGF-R1 dying in early gestation (27). The contribution, if any, of the FGF-R1 to normal postnatal formation of alveoli has not, to our knowledge, been reported. On the basis of our previous observation that inhibition of ligand binding to the FGF-R1, using a truncated soluble FGF-R1{alpha}(IIIc)/Fc chimeric protein (sFGF-R1), blocked restoration of alveologenesis in rats recovering from lung growth arrest induced with 95% oxygen (28), we hypothesized that the FGF-R1{alpha}(IIIc) would also play a critical role in physiologic postnatal alveologenesis.

Some of the results of these studies have been previously reported in the form of an abstract (29).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
In Vivo Interventions
Animal studies were conducted according to criteria established by the Canadian Council on Animal Care and were approved by the Hospital for Sick Children Research Institute Animal Care Review Committee. Nine pups from each of three equal-sized litters received either phosphate-buffered saline (PBS), sFGF-R1, or a truncated soluble p75 neurotrophin receptor chimeric protein (sNGF-R; R&D Systems, Minneapolis, MN) in 100 µl PBS (10 µg/g body weight). Time points were selected to capture the rapid increase in alveolar density that occurs between Days 3 and 8 of life in the rat (21). Intraperitoneal injections were given once, on Day 3 of life, via a 30-gauge needle into the right iliac fossa. For neutralizing antibody studies, 24 pups from four equal-sized litters were injected with either rabbit anti-human FGF-1/acidic FGF neutralizing antibody (R&D Systems), neutralizing mouse anti-human FGF-2/basic FGF (clone bFM-1; Upstate, Lake Placid, NY), or isotype IgG on Days 3, 4, 5, and 6 of life (10 µg/g body weight). Animals were killed on Day 7 of life.

Lung DNA Content and Synthesis
DNA content and synthesis ([3H]thymidine incorporation into DNA) were measured as previously described (28, 30). Intraperitoneal [3H]thymidine (1 µCi/g) was injected 2 h before removal of lung tissue. Lungs were weighed and then homogenized for DNA isolation (31).

TUNEL Assay
Details of the terminal deoxynucleotide nick end labeling (TUNEL) assay are provided in the online supplement.

Western Blot Analyses
Western blots of lysates from perfused lung tissue were performed as previously described (32). Protein content was measured as described by Bradford (33). Details of antibody sources, dilutions, and immunoprecipitation are provided in the online supplement. Blots were imaged and bands quantified by enhanced chemiluminescence detection as previously described (32). The software used calculated integrated band densities after subtraction of background values (FluorChem 8000, Ver. 3.04A; Alpha Innotech Corp., San Leandro, CA) (32).

Immunohistochemistry
Lungs were perfusion-fixed at a constant inflation pressure of 20 cm water. Details are provided in the online supplement. Intraperitoneal injection (20 µg/g) of bromodeoxyuridine (BrdU) was performed 2 h before animals were killed. Immunohistochemical staining of paraformaldehyde-fixed paraffin-embedded lung tissue was with a BrdU In-Situ Detection Kit (BD Biosciences Pharmingen, San Diego, CA). Frozen sections were used to stain for cleaved caspase 3. Details are provided in the online supplement.

Morphometric Analyses
Morphometric assessments were performed on coded images to mask the treatment category. Images were captured with OpenLab, version 3.5.2 (Improvision, Guelph, ON, Canada), using a Leica camera and microscope (Leica Microsystems GmbH, Wetzlar, Germany) attached to a Macintosh computer (Mac 10.3.5 operating system; Apple Computer, Cupertino, CA). Because of regional variations in lung maturation (34), all assessments were performed on the right middle lobe. Mean linear intercept, tissue fraction, secondary crest density, and alveolar density were calculated as previously described (35).

Data Presentation
All values are presented as mean ± SEM. Parametric data were subjected to one-way analysis of variance (ANOVA), followed by Student-Newman-Keuls post hoc test for between-group differences (36). For categorical data, a Kruskal-Wallis ANOVA was used to detect differences across groups, followed by the Mann-Whitney test for between-group differences. A p value < 0.05 was regarded as statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Reduced FGF-R1 Phosphorylation after Injections of sFGF-R1 or Neutralizing Antibody to FGF-2
FGF-R1 was immunoprecipitated with an antibody against the C-terminal domain of the FGF-R1 and was analyzed by immunoblotting with either anti–FGF-R1 (N-terminal domain) or an antiphosphotyrosine antibody. Injection of sFGF-R1 or FGF-2 neutralizing antibody, but not FGF-1 neutralizing antibody, reduced phosphorylation of the endogenous FGF-R1 (Figure 1).


Figure 1
View larger version (36K):
[in this window]
[in a new window]
  		Figure 1. Analysis of fibroblast growth factor receptor-1 (FGF-R1) phosphorylation. (A) FGF-R1 was immunoprecipitated with an antibody against the C-terminal intracellular domain of the FGF-R1 and was analyzed by immunoblotting with either an antibody against the N-terminal extracellular domain of FGF-R1 or an antiphosphotyrosine antibody (anti-pTyr). (B) By densitometry, phosphorylation of FGF-R1 in the lung was significantly reduced in those rat pups injected with soluble FGF-R1 (sFGF-R1), when compared with pups injected with vehicle alone. (C) Injection of anti–FGF-2 neutralizing antibody, but not anti–FGF-1 neutralizing antibody, also significantly reduced FGF-R1 phosphorylation. *p < 0.05 by one-way analysis of variance (ANOVA) compared with values for animals that had received vehicle alone. Values are means ± SEM for three to four animals in each group. Ab = antibody; term = terminal.

 
Increased Lung Tissue Fraction in sFGF-R1–treated Pups
Day 7 pups that had been injected with the control sNGF-R had lungs with a similar histologic appearance to those that had been injected with the vehicle alone. However, pups that had been injected with the truncated sFGF-R1 had lungs with an obvious increase in interstitial thickness between Days 3 and 7, when compared with those that had been injected with the vehicle alone (Figure 2). Morphometric analysis of lung tissue, after intervention studies with the sFGF-R1, confirmed a significant increase in tissue fraction (Table 1).


Figure 2
View larger version (96K):
[in this window]
[in a new window]
  		Figure 2. Grayscale images from hematoxylin-and-eosin–stained postnatal rat lungs at Days 3 or 7 of life. Day 3 and Day 7 pups had received intraperitoneal injections of phosphate-buffered saline (PBS; Day 3 and Day 7 vehicle). Other Day 7 pups had received sFGF-R1{alpha}(IIIc)/Fc chimera (Day 7 sFGF-R1) or, as a control for the chimeric protein, soluble p75 neurotrophin receptor chimeric protein (sNGF-R/Fc; Day 7 sNGF-R). Lung tissue from pups that had received sNGF-R appeared no different than tissue from pups injected with vehicle. In contrast, a marked increase in interstitial thickness was evident in the lungs from pups that had been injected with the sFGF-R1. Bar length = 200 µm.

 

View this table:
[in this window]
[in a new window]
  		TABLE 1. MORPHOMETRIC ANALYSES OF LUNGS FROM DAY 7 PUPS THAT HAD RECEIVED INJECTIONS OF SOLUBLE RECEPTOR OR NEUTRALIZING ANTIBODY

 
Paradoxical Changes in Lung DNA Content and Synthesis in sFGF-R1–treated Pups
Likely explanations for an intervention causing an increase in cellularity are stimulation of lung cell proliferation or inhibition of cell death. Our initial step, therefore, was to measure lung weight, lung DNA content, and lung DNA synthesis, as assessed by [3H]thymidine incorporation into DNA. For these particular measurements, we did not include the use of the control sNGF-R, because we have previously demonstrated its lack of effect on these parameters (28, 37). Injection of the truncated sFGF-R1 had no significant effect on body weight, lung weight, or total lung protein content. Total lung DNA content was significantly increased in sFGF-R1–treated animals, but was accompanied by a marked inhibition of DNA synthesis, as assessed by [3H]thymidine incorporation into DNA (Table 2). To confirm the inhibitory effect on DNA synthesis, in the absence of an effect on total DNA content, we used BrdU incorporation into lung tissue as an additional marker for cell proliferation (38). Lungs from 7-d-old rats injected with PBS or sNGF-R had large numbers of BrdU-positive cells, but there was an obvious reduction in the number of BrdU-positive cells in lung tissue from the Day 7 rat pups that had received the sFGF-R1 (Figure 3). This was confirmed by counting the number of BrdU-positive cells per unit area (Figure 4). That this inhibition of lung cell DNA synthesis translated into an impairment of alveolar formation was confirmed by morphometric analysis. Injection of the sFGF-R1 resulted in a significant increase in mean linear intercept and a significant decrease in the number of secondary crests (Table 1).


Figure 3
View larger version (84K):
[in this window]
[in a new window]
  		Figure 3. Bromodeoxyuridine (BrdU) incorporation in neonatal rat lung as a marker of cell proliferation. Small intestine, from a mouse injected with BrdU to show positively stained nuclei (brown), was used as a positive control. Lung tissue from 7-d-old rats that had been injected with vehicle (Day 7 vehicle) or sNGF-R/Fc (Day 7 sNGF-R) on Day 3 of life had large numbers of BrdU-positive cells. In contrast, lung tissue from rat pups that had been injected with the sFGF-R1 (Day 7 sFGF-R1) had an apparent reduction in BrdU-positive cells. Arrows identify actively proliferating cells. Bar length = 50 µm.

 

Figure 4
View larger version (16K):
[in this window]
[in a new window]
  		Figure 4. Quantification of proliferating cells in Day 7 rat lung tissue by BrdU incorporation. After a single intraperitoneal injection of sFGF-R1 on Day 3 of life, the number of BrdU-positive cells in lung tissue from 7-d-old pups was significantly reduced, compared with the 7-d-old pups that had received either PBS alone (vehicle) or the sNGF-R. *p < 0.05 by one-way ANOVA compared with values for animals that had received vehicle alone. Values are means ± SEM for four animals in each group.

 

View this table:
[in this window]
[in a new window]
  		TABLE 2. BODY AND LUNG WEIGHTS, LUNG PROTEIN AND DNA CONTENTS, AND DNA SYNTHESIS IN DAY 7 PUPS THAT HAD RECEIVED INJECTIONS OF SOLUBLE RECEPTOR OR VEHICLE

 
Inhibition of Apoptosis by sFGF-R1
The reduction in both [3H]thymidine incorporation into DNA and BrdU-positive cells, after treatment with sFGF-R1, in the presence of an increased tissue fraction, suggested that the intervention had inhibited the apoptosis-mediated thinning of the interstitium, which is normally seen after birth. Attempts to assess apoptosis in the lung interstitium using the TUNEL assay were unsuccessful, due to labeling of neonatal erythrocytes (Figure E1 of the online supplement), because it is very difficult to completely remove erythrocytes from the neonatal lung. It is not clear if this labeling was an artifact or if it represented detection of the reticulocytes normally present in the neonatal circulation. Immunohistochemistry to identify cells containing cleaved caspase 3 was therefore used as the initial screen for detection of apoptosis. Seven-day-old pups that had been injected with vehicle on Day 3 of life had easily identified cleaved caspase-3–positive cells (Figure 5A). The number of cleaved caspase-3–positive cells appeared to be reduced in pups that had received sFGF-R1 (Figure 5B), which was confirmed by quantifying the number of caspase-3–positive cells per unit area (Figure 5C). We then went on to examine a panel of anti- and proapoptotic proteins by Western blot, as shown in Figure 6A. Pups that had received sFGF-R1 had an unchanged expression of Fas, but did have a reduction in the expression of the proapoptotic protein Bax, and an increase in the antiapoptotic protein Bcl-xL. The expression of mitochondria-derived cytoplasmic cytochrome c was inhibited by the injection of sFGF-R1. The contents of cleaved caspases 3 and 7 were also suppressed by the injection of sFGF-R1. Densitometric analysis was used to confirm the statistical significance of these changes (Figure 6B).


Figure 5
View larger version (47K):
[in this window]
[in a new window]
  		Figure 5. Detection of cells undergoing apoptosis in neonatal rat lung tissue by immunohistochemistry for an apoptosis marker protein, cleaved caspase 3. Apoptotic cells were labeled with a fluorescent antibody to cleaved caspase 3 (green), and cell nuclei were visualized with 4',6-diamidino-2-phenylindole (blue). (A) Lung tissue from 7-d-old pups that had received an intraperitoneal injection of PBS on Day 3 of life had numerous cleaved caspase-3–positive cells. The white box identifies the area shown under higher power as an inset. (B) Lung tissue from 7-d-old pups that had received an intraperitoneal injection of sFGF-R1 chimera on Day 3 of life had a reduced number of cleaved caspase-3–positive cells (arrow). Bar length = 100 µm. (C) This apparent reduction in cleaved caspase-3–positive cells was statistically significant. *p < 0.05 by one-way ANOVA compared with values for animals that had received vehicle alone. Values are means ± SEM for four animals in each group.

 

Figure 6
View larger version (48K):
[in this window]
[in a new window]
  		Figure 6. (A) Western blots of neonatal rat lung tissue for the proapoptotic proteins Bax and cytochrome c, the antiapoptotic protein Bcl-xL, and apoptosis regulators cleaved caspases 3 and 7, and Fas. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a control for protein loading of gels. Protein sizes are shown in kilodaltons (kDa). Gels were loaded with samples from individual Day 7 pups that had received PBS (vehicle) or sFGF-R1 on Day 3 of life. (B) As assessed by densitometry, pups that had received the sFGF-R1 had significantly reduced lung contents of Bax, cleaved caspase 3, cleaved caspase 7, and cytoplasmic cytochrome c, and a significantly increased lung content of Bcl-xL. *p < 0.05 by one-way ANOVA compared with values for animals that had received vehicle alone. Values are means ± SEM for four animals in each group.

 
Differing Effects of Anti–FGF-1 and Anti–FGF-2
FGF receptors are generally promiscuous, binding multiple FGF family members. Both FGF-1 and FGF-2 bind with high affinity to the FGF-R1. We used intraperitoneal injections of neutralizing antibodies to explore their possible contribution to the effects mediated by the FGF-R1. Anti–FGF-2, but not anti–FGF-1, significantly inhibited phosphorylation of the FGF-R1 (Figure 1) and a downstream mediator, Erk1/2 (Figure E2). Interestingly, neither the anti–FGF-1 nor the anti–FGF-2 antibodies had any apparent effect on DNA synthesis, as assessed by BrdU immunoreactivity (Figure E3), which was confirmed by counting labeled cells (Figure E4), suggesting that inhibition of binding of another FGF family member accounted for the inhibition of DNA synthesis observed with the sFGF-R1. There was no obvious effect of the neutralizing antibody to FGF-1 on either the tissue fraction (Table 1) or the apparent thickness of the lung interstitium (Figure 7). In contrast, neutralizing antibodies to FGF-2 did increase the tissue fraction (Table 1) and caused an obvious thickening of the lung interstitium (Figure 7), comparable to that observed after injection of the sFGF-R1. Neither of the antibodies to FGF-1 or FGF-2 had any significant effect on mean linear intercept, but, as observed with the sFGF-R1, both significantly decreased the number of secondary crests. All three interventions reduced the alveolar density, but this effect was only statistically significant for the FGF-2 antibody (Table 1). By Western blot, neither the FGF-1 nor the FGF-2 antibodies had any effect on Fas expression (Figure 8A), Bax, or cleaved caspase-3 expression (data not shown). However, both antibodies caused an increase in the antiapoptotic protein Bcl-xL, and a decrease of cytoplasmic cytochrome c and cleaved caspase 7 (Figure 8A), which were statistically significant (Figure 8B).


Figure 7
View larger version (106K):
[in this window]
[in a new window]
  		Figure 7. Hematoxylin-and-eosin–stained sections of postnatal rat lungs at Day 7 of life from rat pups that had received intraperitoneal injections of neutralizing antibody to FGF-1 or FGF-2, or their respective isotype IgGs. There was no obvious effect of the anti–FGF-1 antibody on interstitial thickness, but the anti–FGF-2 antibody produced an interstitial thickening similar to that which had been observed with the sFGF-R1. Bar length = 200 µm.

 

Figure 8
View larger version (38K):
[in this window]
[in a new window]
  		Figure 8. (A) Western blots of neonatal rat lung tissue for the apoptosis-related proteins cleaved caspase 7, cytoplasmic cytochrome c, Fas, and Bcl-xL. GAPDH served as a control for protein loading of gels. Protein sizes are shown in kilodaltons (kDa). Gels were loaded with samples from individual Day 7 pups that had received daily intraperitoneal injections of PBS (vehicle), neutralizing antibody to FGF-1 (FGF-1 Ab), or neutralizing antibody to FGF-2 (FGF-2 Ab) from Day 3 until Day 6 of life. (B) Densitometric analyses revealed a significant increase in the content of the antiapoptotic protein Bcl-xL, and a decrease in the contents of cleaved caspase 7 and cytosolic cytochrome c, with no effect on Fas content, after injection of neutralizing antibodies to FGF-1 and FGF-2. *p < 0.05 by one-way ANOVA compared with values for animals that had received vehicle alone. #p < 0.05 by one-way ANOVA compared with values for animals that had received the neutralizing antibody to FGF-1. Values are means ± SEM for three animals in each group.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The primary intervention used in this study was the injection of a recombinant human sFGF-R1{alpha}(IIIc)/Fc chimera. This protein is initially derived from a DNA sequence that encodes the entire extracellular domain of the human FGF-R1{alpha}(IIIc), fused to the carboxy-terminal Fc region of human IgG1 via a polypeptide linker. There is a very high degree of homology between the amino acid sequences for the FGF-R1 proteins from different species (14), making it likely that the recombinant human sFGF-R1{alpha}(IIIc)/Fc chimera used in our studies has a similar affinity for members of the FGF family as does the endogenous receptor, which includes binding of both FGF-1 and FGF-2 (18). The desired effect of injecting the sFGF-R1 was for it to act as a decoy to prevent binding of those ligands that would normally have bound to the endogenous FGF-R1{alpha}(IIIc). To confirm that there were no nonspecific effects of the fused Fc portion of the chimera, we used an sNGF-R/Fc chimera as a control, which we have previously found to have no effect on postnatal lung growth (28, 37).

On the basis of our previous observations, with a model of lung growth arrest followed by recovery (28), we were not surprised that the truncated sFGF-R1 caused inhibition of DNA synthesis, as assessed by both [3H]thymidine and BrdU incorporation, and impairment of alveolarization, as assessed by morphometry. What we had not anticipated, however, was that this would occur in the absence of any change in lung weight or protein content. It became apparent from lung morphology and morphometry that this was due to an increase in the tissue fraction, secondary to an increased cellularity of the lung interstitium, as reflected by an increase in lung DNA content. That this increase in DNA content was not accompanied by an increase in lung weight or protein content is consistent with the observed reduction in protein-to-DNA ratio. The most obvious explanation was that apoptosis, induced by the binding of one or more FGF family members to the endogenous FGF-R1, was inhibited by the binding of these ligands to the decoy truncated sFGF-R1{alpha}(IIIc)/Fc chimera. Apoptosis is involved in the remodeling of both rat and human lung during both normal prenatal and postnatal development (24, 25). Postnatally, the role of apoptosis appears to be in ridding the lung of excess fibroblasts and epithelial cells so as to increase the gas exchange surface area (23, 25). Support for the inhibition of apoptosis by the sFGF-R1 came from the measurement of proapoptotic and antiapoptotic proteins. Treatment with the sFGF-R1 caused a reduction in the concentration of the proapoptotic protein Bax, an increased concentration of the antiapoptotic protein Bcl-xL, and an inhibition of the downstream factors cytoplasmic cytochrome c, cleaved caspase 3, and cleaved caspase 7, without affecting Fas. Fas had been implicated in the postnatal apoptosis of murine lung alveolar epithelial cells, and this pathway was inhibited in vitro by FGF-7 (39, 40).

FGF-R1{alpha}(IIIc) binds several members of the FGF family, including high-affinity binding to FGF-1 and FGF-2. We specifically addressed the possible impact of the decoy sFGF-R1 binding to these two ligands by the use of neutralizing antibodies to FGF-1 and FGF-2. The neutralizing antibody to FGF-2, but not the antibody to FGF-1, increased interstitial thickness and the tissue fraction, to a degree comparable to that observed with the decoy sFGF-R1. The observed effect on lung histology by the decoy sFGF-R1 could, therefore, be completely accounted for by its binding to FGF-2. There are precedents for FGF-2 serving such a function, in that it has been reported to promote apoptosis and suppress granulation tissue formation in acute incision wounds (41), and induce cell death in Ewing's sarcoma (42) and various tumor cell lines (43–45).

The two major routes to apoptosis are through the intrinsic and extrinsic pathways. Both pathways ultimately lead to the cleavage of inactive procaspase molecules into active caspase proteins (46). Caspases are a family of aspartate-specific cysteine proteases that cleave and degrade a specific subset of cellular proteins in cells undergoing apoptosis (47). Initiator caspases, including caspases 8 and 9, are responsible for either directly or indirectly activating various effector caspases, including caspases 3, 6, and 7. We used caspases 3 and 7 as markers for this final component of the apoptotic pathway, which was inhibited by the decoy sFGF-R1. Initiation of this terminal cascade may be through the extrinsic pathway, which is induced by binding of death-signal ligands, such as FasL, to integral membrane proteins, such as Fas, with subsequent caspase-8 activation (48). We found no evidence for an effect of the sFGF-R1 on Fas expression. Alternatively, apoptosis can be initiated through an intrinsic pathway, mediated by the mitochondria. The Bcl-2 family consists of several proapoptotic and antiapoptotic proteins that determine cell death and survival by controlling mitochondrial membrane ion permeability, cytochrome c release, and the subsequent activation of caspase 9 (49, 50). Injection of the sFGF-R1 inhibited expression of the proapoptotic protein Bax and stimulated expression of the antiapoptotic protein Bcl-xL. There was a corresponding downstream inhibition of mitochondrial release of cytochrome c and a decrease in the expression of cleaved caspases 3 and 7. These findings suggest that the decoy sFGF-R1 acts through binding an FGF family member, or members, to prevent apoptosis initiated through the intrinsic pathway, and that the FGF-R1{alpha}(IIIc) can act as a death receptor.

To determine if FGF-1 or FGF-2 were FGF family members mediating this process, we selectively inhibited their binding to FGF receptors by the use of neutralizing antibodies. The antibody to FGF-2, but not the antibody to FGF-1, replicated the interstitial thickening and increased tissue fraction observed with the sFGF-R1. This may have resulted from the anti–FGF-2 antibody inhibiting FGF-R1 and downstream Erk1/2 phosphorylation. Anti–FGF-1 had no effect on this transduction pathway. Interestingly, of the apoptosis-related proteins studied, the neutralizing antibody to FGF-2 affected the antiapoptotic protein Bcl-xL, cytochrome c release, and cleaved caspase-7 expression, in a similar fashion to the decoy sFGF-R1. Bcl-xL was also increased to a lesser degree after treatment with the anti–FGF-1 antibody, but this treatment did not affect the tissue fraction. It seems reasonable to assume that the increase in Bcl-xL observed after treatment with the decoy sFGF-R1 can be attributed to its binding of FGF-1 and FGF-2. Because neither the anti–FGF-1 antibody nor the anti–FGF-2 antibody affected Bax expression, the increase in the expression of this protein after treatment with the sFGF-R1 must be accounted for by the binding of ligands other than FGF-1 or FGF-2. However, the contribution of these ligands, and the effect on Bax, may not be essential because the neutralizing antibody to FGF-2 alone was sufficient to increase the tissue fraction. FGF-R1 activation leads to receptor tyrosine phosphorylation and increased phosphorylation of three major intracellular pathways. These are the Ras–mitogen-activated protein (MAP) kinase (Erk1/2, p38, c-Jun N-terminal kinases [JNKs]) pathway, the phosphoinositide-3 kinase (PI-3) kinase–Akt pathway, and the phospholipase C (PLC{gamma}) pathway (51). Our data are consistent with the effect of inhibiting FGF-2 binding to the FGF-R1 being mediated, at least in part, through the MAP kinase (Erk1/2) pathway. Inhibition of FGF-1 binding to its receptors did not affect FGF-R1 tyrosine phosphorylation, suggesting that its effects in the neonatal lung, if any, are mediated through binding to alternative FGF receptors.

In summary, ligand binding to the FGF-R1{alpha}(IIIc) is an effector of postnatal lung growth. In addition, both FGF-2 and the FGF-R1{alpha}(IIIc) appear to regulate the physiologic lung cell apoptosis normally seen shortly after birth.


   FOOTNOTES
 
Supported by a group grant from the Canadian Institutes of Health Research (M.P., A.K.T.).

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200511-1718OC on May 25, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form November 7, 2005; accepted in final form May 22, 2006


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Yamaguchi TP, Rossant J. Fibroblast growth factors in mammalian development. Curr Opin Genet Dev 1995;5:485–491.[CrossRef][Medline]
  2. Goldfarb M. Functions of fibroblast growth factors in vertebrate development. Cytokine Growth Factor Rev 1996;56:14–27.
  3. Bellusci S, Grindley J, Emoto H, Itoh N, Hogan BL. Fibroblast growth factor 10 (FGF10) and branching morphogenesis in the embryonic mouse lung. Development 1997;124:4867–4878.[Abstract]
  4. Han RNN, Liu J, Tanswell AK, Post M. Expression of basic fibroblast growth factor and receptor: immunolocalization studies in developing rat fetal lung. Pediatr Res 1992;31:435–440.[Medline]
  5. Hebert JM, Basilico C, Goldfarb M, Haub O, Martin GR. Isolation of cDNAs encoding four mouse FGF family members and characterization of their expression patterns during embryogenesis. Dev Biol 1990;138:454–463.[CrossRef][Medline]
  6. Colvin JS, Feldman B, Nadeau JH, Goldfarb M, Ornitz DM. Genomic organization and embryonic expression of the mouse fibroblast growth factor 9 gene. Dev Dyn 1999;216:72–88.[CrossRef][Medline]
  7. Fu YM, Spirito P, Yu ZX, Biro S, Sasse J, Lei J, Ferrans VJ, Epstein SE, Casscells W. Acidic fibroblast growth factor in the developing rat embryo. J Cell Biol 1991;114:1261–1273.[Abstract/Free Full Text]
  8. Chailley-Heu B, Boucherat O, Barlier-Mur AM, Bourbon JR. 2005. FGF-18 is upregulated in the postnatal rat lung and enhances elastogenesis in myofibroblasts. Am J Physiol Lung Cell Mol Physiol 2005;288:L43–L51.[Abstract/Free Full Text]
  9. Mason IJ, Fuller-Pace F, Smith R, Dickson C. FGF-7 (keratinocyte growth factor) expression during mouse development suggests roles in myogenesis, forebrain regionalisation and epithelial-mesenchymal interactions. Mech Dev 1994;45:15–30.[CrossRef][Medline]
  10. Sannes PL, Burch KK, Khosla J. Immunohistochemical localization of epidermal growth factor and acidic and basic fibroblast growth factors in postnatal developing and adult rat lungs. Am J Respir Cell Mol Biol 1992;7:230–237.[Medline]
  11. Weinstein M, Xu X, Ohyama K, Deng CX. FGFR-3 and FGFR-4 function cooperatively to direct alveologenesis in the murine lung. Development 1998;125:3615–3623.[Abstract]
  12. Sleeman M, Fraser J, McDonald M, Yuan S, White D, Grandison P, Kumble K, Watson JD, Murison JG. Identification of a new fibroblast growth factor receptor, FGFR5. Gene 2001;271:171–182.[CrossRef][Medline]
  13. Goldfarb M. Functions of fibroblast growth factors in vertebrate development. Cytokine Growth Factor Rev 1996;7:311–325.[CrossRef][Medline]
  14. Johnson DE, Williams LT. Structural and functional diversity in the FGF receptor multigene family. Adv Cancer Res 1993;60:1–41.[Medline]
  15. Powell PP, Wang CC, Horinouchi H, Shepherd K, Jacobson M, Lipson M, Jones R. Differential expression of fibroblast growth factor receptors 1 to 4 and ligand genes in late fetal and early postnatal rat lung. Am J Respir Cell Mol Biol 1998;19:563–572.[Abstract/Free Full Text]
  16. De Paepe ME, Sardesai MP, Johnson BD, Lesieur-Brooks AM, Papadakis K, Luks FI. The role of apoptosis in normal and accelerated lung development in fetal rabbits. J Pediatr Surg 1999;34:863–870.[CrossRef][Medline]
  17. Werner S, Duan DS, de Vries C, Peters KG, Johnson DE, Williams LT. Differential splicing in the extracellular region of fibroblast growth factor receptor 1 generates receptor variants with different ligand-binding specificities. Mol Cell Biol 1992;12:82–88.[Abstract/Free Full Text]
  18. Duan DS, Werner S, Williams LT. A naturally occurring secreted form of fibroblast growth factor (FGF) receptor 1 binds basic FGF in preference over acidic FGF. J Biol Chem 1992;267:16076–16080.[Abstract/Free Full Text]
  19. Burri PH, Moschopulos M. Structural analysis of fetal rat lung development. Anat Rec 1992;234:399–418.[CrossRef][Medline]
  20. Burri PH. Structural aspects of prenatal and postnatal development and growth of the lung. In: McDonald JA, editor. Lung growth and development. New York: Marcel Dekker; 1997. pp. 1–35.
  21. Meyrick B, Reid L. Pulmonary arterial and alveolar development in normal postnatal rat lung. Am Rev Respir Dis 1982;125:468–473.[Medline]
  22. Randell SH, Mercer RR, Young SL. Postnatal growth of pulmonary acini and alveoli in normal and oxygen exposed rats studied by serial section reconstructions. Am J Anat 1989;186:55–68.[CrossRef][Medline]
  23. Kresch MJ, Christian C, Wu F, Hussain N. Ontogeny of apoptosis during lung development. Pediatr Res 1998;43:426–431.[Medline]
  24. Scavo LM, Ertsey R, Chapin CJ, Allen L, Kitterman JA. Apoptosis in the development of rat and human fetal lungs. Am J Respir Cell Mol Biol 1998;18:21–31.[Abstract/Free Full Text]
  25. Schittny JC, Djonov V, Fine A, Burri PH. Programmed cell death contributes to postnatal lung development. Am J Respir Cell Mol Biol 1998;18: 786–793.[Abstract/Free Full Text]
  26. Hokuto I, Perl A-KT, Whitsett J. Prenatal, but not postnatal, inhibition of fibroblast growth factor receptor signaling causes emphysema. J Biol Chem 2003;278:415–421.[Abstract/Free Full Text]
  27. Deng C, Bedford M, Li C, Xu X, Yang X, Dunmore J, Leder P. Fibroblast growth factor receptor-1 (FGFR-1) is essential for normal neural tube and limb development. Dev Biol 1997;185:42–54.[CrossRef][Medline]
  28. Jankov RP, Luo X, Campbell A, Belcastro R, Cabacungan J, Johnstone L, Frndova H, Lye SJ, Tanswell AK. Fibroblast growth factor receptor-1 and neonatal compensatory lung growth after exposure to 95% oxygen. Am J Respir Crit Care Med 2003;167:1554–1561.[Abstract/Free Full Text]
  29. Yi M, Belcastro R, Cabacungan J, Tanswell K. Soluble fibroblast growth factor receptor-I(IIIc) reduced postnatal rats lung proliferation and apoptosis [abstract]. Proc Am Thorac Soc 2005;2:A465.
  30. West DC, Sattar A, Kumar S. A simplified in situ solubilization procedure for the determination of DNA and cell number in tissue cultured mammalian cells. Anal Biochem 1985;147:289–295.[CrossRef][Medline]
  31. Buch S, Han RNN, Cabacungan J, Wang JX, Yuan SZ, Belcastro R, Deimling J, Jankov R, Luo XP, Lye SJ, et al. Changes in expression of platelet-derived growth factor and its receptors in the lungs of newborn rats exposed to air or 60% O2. Pediatr Res 2000;48:423–433.[Medline]
  32. Jankov RP, Luo X, Belcastro R, Copland I, Frndova H, Lye SJ, Hoidal JR, Post M, Tanswell AK. Gadolinium chloride inhibits pulmonary macrophage influx and prevents O2-induced pulmonary hypertension in the neonatal rat. Pediatr Res 2001;50:172–183.[Medline]
  33. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–254.[CrossRef][Medline]
  34. Brumley GW, Chernick V, Hodson WA, Normand C, Fenner A, Avery ME. Correlations of mechanical stability, morphology, pulmonary surfactant, and phospholipid content in the developing lamb lung. J Clin Invest 1967;46:863–873.[Medline]
  35. Yi M, Jankov RP, Belcastro R, Humes D, Copland I, Shek S, Sweezey NB, Post M, Albertine KH, Auten RL, et al. Opposing effects of 60% oxygen and neutrophil influx on alveologenesis in the neonatal rat. Am J Respir Crit Care Med 2004;170:1188–1196.[Abstract/Free Full Text]
  36. Snedecor G, Cochran WG. Statistical methods. Ames, IA: Iowa State University Press; 1980. pp. 215–237.
  37. Yuan S, Hannam V, Belcastro R, Cartel N, Cabacungan J, Wang J, Diambomba Y, Johnstone L, Post M, Tanswell AK. A role for platelet-derived growth factor-BB in rat postpneumonectomy compensatory lung growth. Pediatr Res 2002;52:25–33.[Medline]
  38. Gardner SY, Brody AR. Incorporation of bromodeoxyuridine as a method to quantify cell proliferation in bronchiolar-alveolar duct regions of asbestos-exposed mice. Inhal Toxicol 1995;7:215–224.
  39. De Paepe ME, Mao QF, Embree-Ku M, Rubin LP, Fas LF. FasL-mediated apoptosis in perinatal murine lungs. Am J Physiol Lung Cell Mol Physiol 2004;287:L730–L742.[Abstract/Free Full Text]
  40. Bao SY, Wang YJ, Sweeney P, Chaudhuri A, Doseff AI, Marsh CB, Knoell DL. Keratinocyte growth factor induces Akt kinase activity and inhibits Fas-mediated apoptosis in A549 epithelial cells. Am J Physiol Lung Cell Mol Physiol 2005;288:L36–L42.[Abstract/Free Full Text]
  41. Akasaka Y, Ono I, Yamashita T, Jimbow K, Ishii T. Basic fibroblast growth factor promotes apoptosis and suppresses granulation tissue formation in acute incisional wounds. J Pathol 2004;203:710–720.[CrossRef][Medline]
  42. Sturla LM, Westwood G, Selby PJ, Lewis IJ, Burchill SA. Induction of cell death by basic fibroblast growth factor in Ewing's sarcoma. Cancer Res 2000;60:6160–6170.[Abstract/Free Full Text]
  43. Russo VC, Andaloro E, Fornaro SA, Najdovska S, Newgreen DF, Bach LA, Werther GA. Fibroblast growth factor-2 over-rides insulin-like growth factor-1 induced proliferation and cell survival in human neuroblastoma cells. J Cell Physiol 2004;199:371–380.[CrossRef][Medline]
  44. Maloof P, Wang Q, Wang H, Stein D, Denny TN, Yahalom J, Fenig E, Wieder R. Overexpression of basic fibroblast growth factor (FGF-2) downregulates Bcl-2 and promotes apoptosis in MCF-7 human breast cancer cells. Breast Cancer Res Treat 1999;56:153–167.[CrossRef][Medline]
  45. Duplan SM, Theoret Y, Kenigsberg RL. Antitumor activity of fibroblast growth factors (FGFs) for medulloblastoma may correlate with FGF receptor expression and tumor variant. Clin Cancer Res 2002;8:246– 257.[Abstract/Free Full Text]
  46. El-Deiry JZ. Overview of cell death signaling pathways. Cancer Biol Cell 2005;4:139–163.
  47. Earnshaw WC, Martins LM, Kaufmann SH. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem 1999;68:383–424.[CrossRef][Medline]
  48. Ferri KF, Kroemer G. Organelle-specific initiation of cell death pathways. Nat Cell Biol 2001;3:E255–E263.[CrossRef][Medline]
  49. Allen RT, Cluck MW, Agrawal DK. Mechanisms controlling cellular suicide: role of Bcl-2 and caspases. Cell Mol Life Sci 1998;54:427– 445.[CrossRef][Medline]
  50. Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell survival. Science 1998;281:1322–1326.[Abstract/Free Full Text]
  51. Schlessinger J. Cell signaling by receptor tyrosine kinase. Cell 2000; 103:211–225.[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
200511-1718OCv1
174/5/581    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yi, M.
Right arrow Articles by Tanswell, A. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yi, M.
Right arrow Articles by Tanswell, A. K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Cell Mol. Biol.
Copyright © 2006 American Thoracic Society