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Am J Physiol Lung Cell Mol Physiol 288: L43-L51, 2005. First published September 24, 2004; doi:10.1152/ajplung.00096.2004
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FGF-18 is upregulated in the postnatal rat lung and enhances elastogenesis in myofibroblasts

Bernadette Chailley-Heu, Olivier Boucherat, Anne-Marie Barlier-Mur, and Jacques R. Bourbon

Physiopathologie et Thérapeutique Respiratoires, Institut National de la Santé et de la Recherche Médicale U492, Faculté de Médecine, Université Paris XII, Créteil, France

Submitted 17 March 2004 ; accepted in final form 31 August 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The fibroblast growth factors (FGFs) are key players in fetal lung development, but little is known about their status in postnatal lung. Here, we investigated the expression pattern of FGF-18 transcripts through the perinatal period and evidenced a sevenfold increase after birth that paralleled changes in elastin expression. In vitro, recombinant human (rh)FGF-18 had a mitogenic activity on day 21 fetal rat lung fibroblasts and stimulated its own expression in the latter, whereas FGF-2 inhibited it. At 50 or 100 ng/ml, rhFGF-18 increased the expression of {alpha}-smooth muscle actin ({alpha}-SMA; 2.5-fold), a characteristic marker of myofibroblasts, of tropoelastin (6.5-fold), of lysyl oxidase (2-fold), and of fibulins 1 and 5 (8- and 2.2-fold) in confluent fibroblasts isolated from fetal day 21 lung; similar results were obtained with fibroblasts from day 3 postnatal lungs. Elastin protein expression was also slightly increased in fetal fibroblasts. Lung analysis on day 4 in rat pups that had received rhFGF-18 (3 µg) on days 0 and 1 showed a 1.7-fold increase of tropoelastin transcripts, whereas {alpha}-SMA transcripts were unchanged. In contrast, rhFGF-2 markedly decreased expression of elastin in vitro and in vivo and of fibulin 5 in vitro. In addition, vitamin A, which is known to enhance alveolar development, elevated FGF-18 and elastin expressions in day 2 lungs, thus advancing the biological increase. We postulate that FGF-18 is involved in postnatal lung development through stimulating myofibroblast proliferation and differentiation.

lung development; elastin; lysyl oxidase; fibulin; {alpha}-smooth muscle actin; fibroblast growth factor-18

LUNG DEVELOPMENT PROCEEDS via a series of epithelial-mesenchymal cell interactions, regulated by growth factors and components of the extracellular matrix. Among growth factors, several fibroblast growth factors (FGFs) are expressed in embryonic and adult lung tissues and are essential to lung morphogenesis (25).

At early stages, the lung branching process requires FGF-10 (1), the gene null mutation of which induced absence of bronchial branching from trachea (33). The same consequence was noted in the transgenesis of a dominant negative receptor FGFR-2IIIb (26). Other FGFs are implicated later and more specifically in cell proliferation and differentiation. FGF-1 stimulates lung epithelial proliferation and airway bud formation (4), and both FGF-1 and FGF-7 have been shown to be required for alveolar epithelial type II cell proliferation and differentiation in vitro (34). Moreover, FGF-7 also enhanced maturation of cultured fetal rat type II cells by increasing the expression of surfactant components, including phospholipids and surfactant protein (SP)-A, SP-B, SP-C (5), and SP-D (38). The null mutation of FGF-7 had no effect on lung morphogenesis, however (9).

By contrast, little is known about the possible role of FGFs during the terminal phase of lung development, although alveologenesis necessitates cell proliferation, differentiation, and morphogenetic changes to increase the exchange surface area of the lung through the formation of secondary septa, vascular growth, and remodeling. Although a previous report evidenced the requirement of FGF signaling since the null mutation of both Fgfr3 and Fgfr4 genes led to failure of septation and alveolarization (35), uncertainty remains as to which members of the FGF family are actually involved. Both receptors are not expressed similarly in mice. FGFR-3 is increased during the 2-wk postnatal period, whereas FGFR-4 is present more abundantly later (29). Among FGFs present in the developing lung, FGF-9 and FGF-18 display high affinity for these receptors (6, 24). FGF-9 regulates lung size by stimulating mesenchymal proliferation, and although the null mutation of its gene did not alter epithelial differentiation, it did not allow mouse postnatal survival (6). In addition, FGF-9 has been shown in vitro to activate FGFR-3 and FGFR-4 and could therefore play a role in alveologenesis, although its expression in visceral pleura argues for an involvement mostly in peripheral alveologenesis, which is a later process in final lung growth. FGF-18 expression, which is preferentially detected in the lung among various adult rat tissues, is also present in rat embryos (11, 23), and its role in embryogenesis and fetal lung development has been well demonstrated. Thus FGF-18 gene loss of function leads to lethality just before or at birth (17, 24). Transgenic overexpression of FGF-18 targeted on lung epithelium suggested a possible influence in enhancing proximal program during lung morphogenesis (37). FGF-18 has so far not been investigated postnatally.

In the present study, we examined the expression pattern of FGF-18 during lung perinatal development, which led us to hypothesize that it could play a role in the postnatal period. We therefore characterized in vitro and in vivo biological effects of FGF-18 on growth and expression of markers of myofibroblasts that are cells implicated at this developmental stage. The effects of FGF-2, a downregulator of elastogenesis (3, 31), were analyzed in parallel.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and lung tissue sampling. Dated pregnant Sprague-Dawley rats were purchased from Charles River (Saint-Germain sur l’Arbresle, France). Procedures involving animals were in accordance with the rules of the Guide for Care and Use of Laboratory Animals, and under the authority of the French Ministry of Agriculture. The day after mating was designated day 0 of gestation. Term is 22 days. Lungs were collected between fetal day 17 and postnatal day 21. Fetuses were retrieved by cesarean section from pentobarbital-anesthetized pregnant females. Lungs from fetuses and pups were either immediately frozen in liquid nitrogen and kept at –80°C until RNA extraction or biochemical analyses or used for cell isolation and culture.

In vivo treatments of rat pups. Neonates were treated once daily by subcutaneous dorsal injections of 3 µg of recombinant human (rh) FGF-18 or FGF-2 (PeproTech, Rocky Hill, NJ) in 0.9% NaCl in a total volume of 50 µl on day 0 and day 1, and the lungs were collected on day 4. Other pups received on day 0 a single injection of all trans-retinol palmitate (Sigma, L’Isle d’Abeau, France) at various concentrations (1,800, 3,000, and 5,000 IU, 4 pups each), and their lungs were collected 2 days later. Control pups received 50 µl of vehicle (0.9% NaCl) only.

Lung cell culture. Enzymatically dispersed epithelial cells and fibroblasts were isolated under aseptic conditions from the lungs of 21-day-old fetuses or 3-day-old pups through differential adhesions to plastic and low-speed centrifugation as previously described (5). Fibroblasts were collected directly on plastic by short-lasting adhesion and grown in DMEM containing 10% FBS until confluence. Epithelial cells were allowed to adhere overnight under air-CO2 (95%-5%) at 37°C in MEM containing 10% FBS, either on plastic or on the basement membrane-like matrix from Engelbreth-Holm-Swarm (EHS) murine tumor, and used immediately. Cells were treated with rhFGF-18 (10, 25, 50, and 100 ng/ml) or rhFGF-2 (50 and 100 ng/ml) for 48 h in defined culture medium.

[3H]thymidine incorporation. [3H]thymidine (1.89 TBq/mmol; Amersham, Orsay, France) was added to culture medium at a final volumetric activity of 7.4 kBq/ml (0.2 µCi/ml). After a 48-h incorporation, epithelial cells and fibroblasts were rinsed twice with cold PBS, and DNA was precipitated with 5% TCA, dissolved in 200 µl of 1 N sodium hydroxide, and neutralized by 200 µl of 1 N acetic acid. Material was scraped with the aid of a rubber policeman and homogenized by repeated pipetting, and 200-µl aliquots were used to determine incorporated radioactivity in OptiPhase scintillation cocktail (Wallac Scintillation Products, PerkinElmer, Courtaboeuf, France). Incorporated radioactivity was normalized for cell protein amount determined by the Bradford method.

RNA extraction and Northern blot analysis. Total RNAs were isolated from lung tissue or cultured cells using TRIzol reagent (Invitrogen, Cergy-Pontoise, France). The pelleted RNA was dissolved in sterile water and quantified by absorbance at 260 nm. Twenty-five micrograms of RNA were fractionated by electrophoresis through 1% agarose and 2.2 M formaldehyde gels and were then blotted onto nylon membranes (Gene Screen, PerkinElmer). The membranes were preincubated and successively probed for FGF-18, {alpha}-smooth muscle actin ({alpha}-SMA), elastin, lysyl oxidase, fibulin-1, or fibulin-5 in a hybridization buffer containing 50% formamide, 50 mM Tris·HCl (pH 7.5), 0.8 M NaCl, 10% dextran sulfate, 0.1% sodium pyrophosphate, 5x Denhardt’s solution, 0.1% SDS, and 75 µg/ml denatured salmon sperm DNA. The rat cDNA probes consisted, respectively, of a 904-bp sequence for FGF-18 (gift from Dr. N. Itoh, Kyoto, Japan), a 187-bp sequence for {alpha}-SMA (gift from Dr. K. M. McHugh, Philadelphia, PA), a 1,100-bp sequence for elastin (gift from Dr. C. Rich, Boston, MA), a 700-bp sequence for lysyl oxidase (gift from Dr. P. C. Trackman, Boston, MA), a 501-bp sequence for fibulin-1, and a 451-bp sequence for fibulin-5 designed from Primer Express software (Applied Biosystems). Probes were labeled with [{alpha}-32P]dCTP (NEN, PerkinElmer) using the Rediprime DNA labeling system (Amersham) and purified on G-50 probe purification columns (Amersham). The blots were exposed to X-Omat Kodak scientific imaging films for a suitable exposure time at –80°C. Autoradiographic signals were quantified by densitometry using image analysis software (NIH Image, Bethesda, MD) and normalized to the relative amount of 18S ribosomal RNA.

Western blot analysis of proteins. Lung sample homogenates or cultured fibroblasts were lysed (50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 5 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM leupeptin, 25 mM NaF, 0.3% Nonidet P-40, and 0.2% Triton X-100). Protein concentration was determined by the Bradford method. Lysates were suspended in reducing Laemmli sample buffer, boiled for 5 min, and separated by 12% SDS-PAGE. After proteins were transferred onto Immobilon membranes (Millipore, Bedford, MA), nonspecific binding was blocked with 10 mM Tris-buffered saline (TBS) containing 10% nonfat dry milk for 1 h. {alpha}-SMA was detected with a monoclonal antibody (Cymbus Biotechnology, Chandlers Ford, UK) and elastin with a rabbit polyclonal antibody (Chemicon International, Temecula, CA). Membranes were incubated overnight at 4°C. After being washing three times with TBS plus 0.05% Tween 20, secondary anti-mouse or anti-rabbit IgG antibody conjugated with horseradish peroxidase was applied for 1 h at room temperature, followed by addition of chemiluminescent substrate (ECL Western Blotting Analysis System, Amersham). The blots were exposed to Hyperfilm ECL (Amersham). To control for variations in protein loading, each membrane was labeled with a monoclonal anti-{beta}-actin antibody (Sigma). Band densitometry analysis was performed with the NIH Image analysis software.

Histological analysis of lung tissue. For morphometric histological studies, postnatal pup lungs were inflated at 20 cmH2O pressure with fixative (4% paraformaldehyde in PBS) via the trachea and immersed in fixative overnight. They were washed in PBS, dehydrated, and embedded in paraffin. Frontal sections of the lungs were stained with hematoxylin and eosin.

Statistical analysis. Data are presented as means ± SE. Multiple comparisons of mean values were made by ANOVA (Fisher’s protected least significant differences test), and two-group comparisons were made by two-tailed unpaired Student’s t-test, with P = 0.05 considered the limit of statistical significance.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Developmental expression of FGF-18 and elastin mRNA in rat lung. To examine the expression of FGF-18 gene during rat lung development and maturation, Northern blot analyses were performed on lung mRNAs during fetal and postnatal periods until the third postnatal week, corresponding to the completion of lung organogenesis with end of alveolarization. As shown in Fig. 1A, FGF-18 mRNA level was rather weakly expressed during the fetal period between day 14 and day 18, increased approximately twice in late gestation between day 19 and day 21, and then transiently fell on the day of birth before increasing sharply from postnatal day 3. A high expression level approximately sevenfold over day 0 was maintained from day 3 to day 10, and then it progressively decreased. The level of expression in adults (day 28) was as low as in fetal day 14.



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  		Fig. 1. Fibroblast growth factor (FGF)-18 mRNA expression during rat lung development and in isolated pulmonary cells. A: expression pattern from fetus to adult; steady-state level of FGF-18 mRNA was obviously increased in the postnatal period between days 3 and 15. Data are means ± SE (n = 4). Significant difference for: P < 0.01 vs. day 0 (a); P < 0.001 vs. day 0 and fetal day 14 (b); P < 0.01 vs. day 0 and postnatal day 10 (c) (ANOVA). B: representative Northern blots of the comparative patterns of FGF-18 and elastin mRNAs showing that both increased in the same postnatal period corresponding to alveolar septation in the rat. C: detection of FGF-18 mRNA in 2 separate primary cultures of isolated lung fetal epithelial cells or fibroblasts (day 21). Lanes 1 and 2, epithelial cells cultured on Engelbreth-Holm-Swarm (EHS) matrix; lanes 3 and 4, epithelial cells cultured on plastic; lanes 5 and 6, fibroblasts cultured on plastic. Only fibroblasts expressed the FGF-18 gene.

 
This expression pattern was strikingly similar to the one of elastin in the same postnatal rat lungs, i.e., both were elevated along the same period between postnatal days 3 and 16 (Fig. 1B).

In primary cultures of isolated fetal rat pulmonary cells, FGF-18 was found to be expressed by fibroblasts but not by epithelial cells grown on either plastic or EHS matrix (Fig. 1C).

In vitro effect of FGF-18 on cell proliferation. In dose-response analysis of [3H]thymidine incorporation by cells isolated from 21-day-old fetal lungs and cultured for 48 h, we tested for FGF-18 concentrations ranging from 10 to 100 ng/ml (Fig. 2). No concentration of FGF-18 induced a significant change for epithelial cells compared with control conditions (Fig. 2). FGF-7 (50 ng/ml) tested in parallel as positive control increased thymidine incorporation 4.5-fold in epithelial cells (P < 0.001; data not shown). By contrast, a substantial increase of incorporation was induced in fibroblasts in a dose-dependent manner; the maximal increase (~2.3-fold) occurred from 50 ng/ml of FGF-18 (Fig. 2).



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  		Fig. 2. Effect of exogenous FGF-18 on proliferation of fetal lung cells. Epithelial cells and fibroblasts isolated from day 21 fetal lung were exposed in culture to 0–100 ng/ml of recombinant human (rh)FGF-18 for 48 h. Cell proliferation was evaluated by [3H]thymidine incorporation into DNA. Data are means ± SE (n = 6). Significant difference vs. control for *P < 0.05 and ***P < 0.001 (ANOVA). Absence of error bars indicates that SE was too low to be represented at this scale. FGF-18 did not induce any significant change in thymidine incorporation in epithelial cells, whereas it enhanced the incorporation in fibroblasts in a dose-dependent manner.

 
Effect of FGF-18 and FGF-2 on FGF-18 expression in cultured fetal lung fibroblasts. Northern blot analysis showed that fibroblasts cultured in the presence of FGF-18 increased their expression of endogenous FGF-18 in a dose-dependent manner (Fig. 3). The increase was fourfold the basal level with 100 ng/ml of FGF-18. Conversely, FGF-2 at same concentration inhibited FGF-18 expression in cultured fibroblasts (Fig. 3).



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  		Fig. 3. Effects of exogenous FGF-18 and FGF-2 on FGF-18 expression in fetal rat lung fibroblasts. Twenty-one-day-old fibroblasts were primary cultured with and without rhFGF-18 (25, 50, and 100 ng/ml) or rhFGF-2 (50 and 100 ng/ml) for 48 h. FGF-18 mRNA expression was increased linearly by exogenous FGF-18. Conversely, rhFGF-2 inhibited FGF-18 expression. Data are means ± SE (n = 5). Significant difference for *P < 0.05 vs. control and FGF-18 (25 ng/ml), **P < 0.01 vs. control, ***P < 0.001 vs. control (ANOVA).

 
Effects of FGF-18 on expression of elastin, lysyl oxidase, fibulins, and {alpha}-SMA mRNAs and proteins in isolated fibroblasts. Northern blot analysis showed that in fibroblasts from 21-day-old fetuses cultured in the presence of FGF-18, a 3.9-kb signal was detected for elastin mRNA, and its expression was upregulated at 25, 50, and 100 ng/ml (3-, 6-, and 5-fold, respectively; Fig. 4, A and D). Conversely, elastin mRNA expression was inhibited in the presence of FGF-2 (Fig. 4, A and D). Elastin is synthesized as the soluble monomer tropoelastin (TE), and extracellularly, TE molecules are cross-linked by lysyl oxidase to form insoluble elastin. Lysyl oxidase mRNAs were detected at 5.6 kb and were increased by approximately twofold when fibroblasts were exposed to 50 and 100 ng/ml of FGF-18 (Fig. 4, B and D). {alpha}-SMA mRNA, which characterizes the myofibroblast phenotype, was detected at 1.4 kb and increased 2.5-fold in cultured fibroblasts with 100 ng/ml of FGF-18. FGF-2 had no effect on {alpha}-SMA expression (Fig. 4, C and D). Figure 4E shows Western blot protein analysis indicating that TE protein expression detected at 67 kDa was slightly increased by FGF-18 and markedly inhibited by FGF-2. {alpha}-SMA detected at 45 kDa was expressed in cultured fibroblasts with a scarcely discernible increase at 100 ng/ml of FGF-18 and a slight decrease at 50 and 100 ng/ml of FGF-2. As shown in Fig. 5, the transcripts of fibulin-1 and fibulin-5 that are extracellular matrix proteins that have overlapping binding sites for TE were increased 4- and 8-fold and 1.8- and 2.2-fold with 50 and 100 ng/ml of FGF-18, respectively.



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  		Fig. 4. In vitro effects of exogenous FGF-18 and FGF-2 on tropoelastin, lysyl oxidase, and {alpha}-smooth muscle actin ({alpha}-SMA) mRNA and protein expressions in fetal lung fibroblasts. Primary cultures of 21-day-old fetal lung fibroblasts were maintained for 48 h in the absence (control, C) or presence of rhFGF-18 or rhFGF-2. Densitometric Northern blot analysis: A, the level of tropoelastin mRNA was increased 3-, 6.5-, and 5-fold by rhFGF-18 (25, 50, and 100 ng/ml), respectively, and strongly inhibited by rhFGF-2 (100 ng/ml); B, the level of lysyl oxidase mRNA was increased ~2-fold in parallel of tropoelastin expressions; C, the level of {alpha}-SMA mRNA was increased 2.5-fold by rhFGF-18 (100 ng/ml); data are means ± SE (n = 7). Significant difference vs. control for *P < 0.05, **P < 0.01, and ***P < 0.001 (ANOVA). D: representative Northern blots of the 3 mRNA patterns and subsequent hybridization with an 18S ribosomal RNA as an RNA loader control. E: Western blot analysis of tropoelastin and {alpha}-SMA proteins in fetal lung fibroblasts. Cell lysates (25 µg) were subjected to 12% SDS-PAGE and immunoblotted with antibodies against {alpha}-SMA and elastin. Membranes were reprobed with a monoclonal antibody for {beta}-actin as a protein loader control. Tropoelastin expression was slightly increased by rhFGF-18 (100 ng/ml) and obviously inhibited by rhFGF-2. Similar results were obtained in 2 separate culture experiments.

 


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  		Fig. 5. In vitro effects of exogenous FGF-18 on fibulin-1 and fibulin-5 mRNA expression in fetal lung fibroblasts. Primary cultures of 21-day-old fetal lung fibroblasts were maintained for 48 h in the absence (control, C) or presence of rhFGF-18. Densitometric Northern blot analysis: A, the level of fibulin-1 mRNA was increased 4- and 9-fold by rhFGF-18 (50 and 100 ng/ml), respectively; B, the level of fibulin-5 mRNA was increased approximately twice at both concentrations. Data are means ± SE (n = 4). Significant difference vs. control for *P < 0.05, **P < 0.01 (ANOVA). C: representative Northern blots of the 2 mRNA patterns and subsequent hybridization with an 18S ribosomal RNA as an RNA loader control.

 
Similar effects were obtained with fibroblasts isolated from 3-day-old pups and cultured in the presence of FGF-18 or FGF-2 (Fig. 6). TE mRNA expression was upregulated at 50 and 100 ng/ml of FGF-18 (~5-fold). Conversely, TE mRNA expression was inhibited in the presence of FGF-2 (Fig. 6). Lysyl oxidase mRNAs were increased 2.7- and 4.6-fold with 50 and 100 ng/ml of FGF-18, respectively. {alpha}-SMA mRNA was increased 3- and 2.5-fold with 50 and 100 ng/ml of FGF-18, respectively. FGF-2 had also a slight enhancing effect on {alpha}-SMA expression (Fig. 6). Fibulin-1 and fibulin-5 transcripts were increased 2- and 3-fold and 1.6- and 2.8-fold, respectively, with 50 and 100 ng/ml of FGF-18. FGF-2 had a marked inhibitor effect on fibulin-5 expression as well as on TE expression (30% of control level in both instances).



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  		Fig. 6. In vitro effects of exogenous FGF-18 and FGF-2 on tropoelastin, lysyl oxidase, {alpha}-SMA, fibulin-1, and fibulin-5 mRNA expressions in postnatal lung fibroblasts. Two separate primary cultures of 3-day-old pup lung fibroblasts were maintained for 48 h in the absence (control, C) or presence of rhFGF-18 or rhFGF-2. Northern blot analysis shows the expression of all 5 transcripts was increased similarly as observed for 21-day-old fetal fibroblasts. Tropoelastin mRNA was increased ~5-fold, lysyl oxidase mRNA 2.7- and 4.6-fold, fibulin-1 and fibulin-5 ~2- and 3-fold, and {alpha}-SMA 3- and 2.5-fold by rhFGF-18 (50 and 100 ng/ml), respectively. Tropoelastin and fibulin-5 transcripts were reduced approximately one-third of control level by rhFGF-2 (100 ng/ml).

 
Effect of FGF-18 on pulmonary expression of TE and {alpha}-SMA mRNAs and proteins in vivo. Lungs of 4-day-old pups treated with two injections of rhFGF-18 (3 µg) displayed a 1.7-fold increase of TE mRNA, whereas the {alpha}-SMA mRNA level was not significantly changed (Fig. 7). Lysyl oxidase mRNA was also not significantly changed on the average, although it was enhanced in one-half of the treated pups (data not shown). No significant change in TE and {alpha}-SMA mRNAs was observed following FGF-2 treatment (Fig. 7). Histological observation of the lungs of FGF-18-treated pups showed a morphology similar to control lungs.



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  		Fig. 7. In vivo effects of FGF-18 on tropoelastin and {alpha}-SMA expressions in postnatal lung. Rat pups were treated by a dorsal subcutaneous injection of rhFGF-18 (3 µg) or rhFGF-2 (3 µg) on postnatal days 0 and 1 and killed on day 3. A: Northern blot analysis showing tropoelastin mRNA expression was increased 1.7-fold by rhFGF-18, whereas {alpha}-SMA mRNA expression was unchanged. Both expressions were not significantly changed by rhFGF-2. Data are means ± SE (n = 4). Significant difference vs. control for *P < 0.05 (Student’s t-test). B: representative Northern blots of mRNA patterns and subsequent hybridization with an 18S ribosomal RNA as an RNA loader control.

 
Effect of vitamin A on pulmonary expression of FGF-18 mRNA in vivo. Because vitamin A is known to play an important role in postnatal lung development (19), we have searched for a possible effect on FGF-18 gene expression. Newborn rats were submitted to a single injection of retinol palmitate at various concentrations on day 0, and their lungs were retrieved 2 days later. The expression of FGF-18 mRNA was increased in all treated fetuses for all the tested concentrations, and the stimulation was proportional to the vitamin A dosage. FGF-18 mRNA level was increased 4.5-fold at the highest vitamin A dose (5,000 IU; Fig. 8A). TE expression examined in parallel to FGF-18 was also increased in the same samples (Fig. 8B). No histological difference was observed between the lungs of retinol palmitate-treated pups and control lungs.



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  		Fig. 8. Effect of retinol palmitate on FGF-18 mRNA expression in neonatal lung. Rat pups were treated by a single dorsal subcutaneous injection of retinol palmitate (1,800, 3,000, or 5,000 IU) on the day of birth. Lungs were collected on postnatal day 2. A: FGF-18 expression was significantly increased by vitamin A in a dose-dependent manner as shown by Northern blot analysis. Data are means ± SE (n = 6 in each group). *P < 0.05 vs. control (ANOVA). B: Northern blot of FGF-18 and tropoelastin mRNA expressions displayed that they were both enhanced by 5,000 IU vitamin A.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present report, we provide evidence implicating the growth factor FGF-18 in the postnatal development of rat lung. The burst in expression level of this factor, which occurs during the first two postnatal weeks, as well as the in vivo and in vitro effects of exogenous FGF-18, are highly suggestive of its involvement in processes taking place during this period.

Previous observations that reported the high expression level of FGF-18 in the embryonic lung relative to other organs (11) and the suppressed alveolarization in fgfr3–/–fgfr4–/– double-null mutant mice (35) prompted us to investigate a possible involvement of FGF-18 in postnatal developmental process. In rodents as in humans, the terminal phase of pulmonary maturation during which secondary alveolar septa form is a postnatal event. This includes proliferation of interstitial fibroblasts, alveolar budding, and septation, which requires coordinated outgrowth of epithelial cells, increase of capillary network, and the presence of myofibroblasts in interstitium. The latter cells produce elastin that make deposits at alveolar septal tips (26). We show herein a striking similarity between the postnatal pattern of FGF-18 and elastin expression profiles, highly suggestive of a possible involvement of this factor in lung elastogenesis.

Among rat lung cells, we found that isolated fibroblasts, but not epithelial cells, expressed FGF-18. This is in agreement with previous data about mouse fetal tissue from embryonic days 12.5 to 19.5 in which FGF-18 transcripts were localized by in situ hybridization in areas of lung mesenchymal cells adjacent to airways (11, 23, 37) surrounding the cartilage rings and in peripheral lung mesenchyme (37).

FGFs are known to play diverse roles in regulating cell proliferation, migration, and differentiation. The present thymidine incorporation data indicated that exogenous rhFGF-18 protein stimulated proliferation of primary cultured fetal rat lung fibroblasts in a dose-dependent manner. A proliferative effect has similarly been shown previously in the fibroblast cell line NIH/3T3 in a heparan sulfate-dependent manner using rh (12) and murine (11) FGF-18 proteins. By contrast, FGF-18 displayed no proliferative effect for alveolar epithelial cells. Although this does not rule out the possibility that it may exert paracrine effects on other cell types, our results indicate that FGF-18 is an autocrine mitogen for fibroblasts and therefore appears as at least one of the signal molecules involved in the previously reported autocrine growth regulation in fibroblasts (30). Moreover, our data indicate that FGF-18 positively regulates its own expression in cultured fibroblasts; this may, therefore, represent a self-maintained regulatory mechanism in these cells.

Fibroblasts cultured in the conditions used in the present study display a myofibroblast phenotype, otherwise known as smooth muscle-like cell phenotype, characterized by the expression of {alpha}-SMA and TE (28, 39). Enhancing effects of FGF-18 were also demonstrated for these markers as well as for the cross-linking enzyme of TE, lysyl oxidase, and for elastin ligands such as fibulin-1 and fibulin-5. These two latter proteins have been found to be expressed in the lung (32) and more especially in myofibroblasts isolated from postnatal lung (13) in which they were associated to elastin. The null mutation of fibulin-5 demonstrated its essential role in elastic fiber organization (21). Messenger RNA expression of all five was upregulated dose dependently by FGF-18. The latter, therefore, coordinately enhanced a set of genes involved in the elastogenesis process. These effects of FGF-18 were similarly obtained in fibroblasts isolated from 3-day-old newborn lungs, i.e., at the time of alveolar elastogenesis initiation and in "immature" 21-day-old fetal fibroblasts. The responsiveness of fetal fibroblasts indicates not only that FGF-18 would represent a maturational factor for these cells toward the myofibroblast phenotype and a stimulus of elastogenesis but that lung fibroblasts are able to undergo elastin accumulation early, which suggests that the increase of FGF-18 may represent the signal that initiates the process.

To determine whether rhFGF-18 was also biologically active in vivo and in an attempt to define its functional role in postnatal period, rhFGF-18 was given twice to normal rat pups just after birth in the goal of inducing a more precocious raising of FGF-18. Whereas elastin was increased significantly, although at a lesser extent than in cultured fibroblasts, {alpha}-SMA expression was unchanged. Change of elastin expression without change of {alpha}-SMA expression was also reported previously by immunohistochemical observations in mice lacking elastin (36). In the FGF-18-treated pups, we have not observed any significant morphological change in lungs and distinguishable difference in {alpha}-SMA immunolabeling (data not shown). The less marked effects of FGF-18 in vivo than in vitro may be accounted for by the fact that the markers under study were not restricted to myofibroblasts but were abundantly expressed in blood vessel smooth muscle cells. Whether or not myofibroblasts were preferential target cells for FGF-18, in vivo effects on {alpha}-SMA, TE, and lysyl oxidase of these cells could be hidden when whole tissue was studied by in vivo approach. Alternatively, it could be assumed that too low amounts of rhFGF-18 reached the lung after subcutaneous injection. However, the fact that FGF-2 given at similar dosage the same way markedly decreased elastin expression, indicating that significant amounts of injected factors reached the lung, argues against this assumption.

An involvement of FGF-18 in myofibroblast growth and maturation raises the question of the interrelationships of this factor with other mediators known to be implicated in differentiation of the same cells. The differentiation and maturation mechanisms of myofibroblasts appear to be submitted to a complex control. Depletion of myofibroblasts from alveolar walls in the platelet-derived growth factor-deficient mice results in an absence of elastic fibers in alveolar walls and incomplete alveolarization (2, 15). Similarly, mice bearing deletions of retinoic acid receptors exhibit reduced lung elastin and alveolar numbers (20). Transforming growth factor-{beta}1 has been recognized for a long time as a differentiation factor of myofibroblasts upregulating {alpha}-SMA (7) and elastin (39) expressions, but its effects on elastin expression appear to result essentially from stabilization of elastin mRNA (14). Another paracrine regulatory factor, insulin-like growth factor-I, stimulates TE synthesis in neonatal rat pulmonary fibroblasts (22), as it does in aortic smooth muscle cells. FGF-2, a well-known mitogenic agent for pulmonary capillary endothelial cells, has also been reported to have proliferative effects for fibroblasts, even stronger than those of FGF-18 (11, 12). By contrast, FGF-2 is known to downregulate elastin (3, 31) and lysyl oxidase (8); both these effects are opposite to those of FGF-18 evidenced in the present work. In addition, we show also that FGF-2 downregulates the elastin-binding protein fibulin-5, which had never been observed, and inhibits FGF-18 expression in fibroblasts. The biological effects of FGF-18 could therefore be complementary to those of the different other maturational factors and would necessitate a precise balance with FGF-2 in a complex, multifactorial control of myofibroblast growth, differentiation, and elastogenesis.

Retinoids promote alveolarization (10, 19). Although the liver is the major site of retinoid storage, the perinatal lung also stores significant amounts in the form of retinol esters (18). These have been observed in the lipid droplets of fibroblasts located at the base of new septa and designated lipofibroblasts, whereas those at the tips of newly formed septa, engaged in synthesis and secretion of TE, exhibit the features of myofibroblasts (27, 28). Moreover, retinoic acid was shown to increase transcription of the elastin gene in cultured developing rat lung fibroblasts (16). Because retinoids are among the primary morphogens that regulate the spatio-temporal expression of numerous growth factors in both mesenchyme and epithelium, we assumed possible relationships between retinoids and FGF-18 expression. Indeed, a dose-dependent increase of the latter was observed in 2-day-old rat newborns that received retinol on the first postnatal day. In parallel, elastin expression was stimulated, displaying an accelerated rise compared with normal timing, i.e., postnatal day 4 (20). This FGF-18 upregulation was without effect on lung morphology at 2 or 4 days old, as observed in FGF-18-treated pup lungs. This may result from stages too early to show modifications.

Together, the temporal coincidence of the postnatal peak of FGF-18 expression with the period of alveologenesis, the effects of exogenous FGF-18 on fibroblast proliferation and on elastin anabolism, and the vitamin A-enhanced expression of FGF-18 in rat newborns strongly argue for an involvement of FGF-18 in the control of myofibroblast development in connection with alveolar morphogenetic mechanisms. Our results extend the field of FGF-18 activities to the last part of pulmonary development and come in addition to its implication at earlier stages of embryo-fetal lung development reported previously. Determining whether FGF-18 expression is altered in pathophysiological processes in which alveolarization is impaired, such as in bronchopulmonary dysplasia and emphysema, would be of particular interest.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. Chailley-Heu, Institut National de la Santé et de la Recherche Médicale U492, Faculté de Médecine, 8 rue du Général Sarrail, 94010 Créteil cedex, France (E-mail: bernadette.chailley-heu{at}creteil.inserm.fr)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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