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INSERM Unit 319, Développement Normal et Pathologique des Fonctions Epitheliales, Université Paris 7-Denis Diderot, 75251 Paris, France
Address all correspondence and requests for reprints to: Jacques R. Bourbon, INSERM U319, Université Paris 7-Denis Diderot, Tour 33-43, Case courrier 7126, 2 Place Jussieu, 75251 Paris Cedex 05, France. E-mail: bourbon{at}paris7.jussieu.fr
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Keratinocyte growth factor (KGF), otherwise designated fibroblast growth factor 7 (FGF-7), was originally purified and cloned as a mitogen with predominant activity for human keratinocytes (6). KGF, which is a 28-kDa protein, is produced by stromal mesenchyme-derived cells, including lung fibroblasts (6). KGF effects were shown on lung epithelial cell proliferation and growth (7, 8). Lately, the role of KGF during lung development was mainly studied at the morphogenesis level. Besides FGF-10, which seems to represent the major mediator in bronchial branching (9), KGF was also found to modulate branching morphogenesis in an in vitro model (10, 11). However, although the use of a KGF antibody reduced branching in this model, the branching pattern in the presence of exogenous KGF was abnormal (10, 11). Yet, we have demonstrated that KGF also acts as an enhancer of fetal type II cell maturation (12). We and others reported that KGF strongly stimulates the synthesis of all surfactant components, including DSPC (12, 13) and the various surfactant-specific proteins (12). We have shown that KGF stimulation of DSPC synthesis specifically concerns the fraction of DSPC that is incorporated in surfactant, and correlates with increased activities of choline phosphate cytidylyltransferase and fatty acid synthase (12). Recently, dexamethasone was found to enhance KGF gene expression in the cultured whole embryonic lung (14). Based on these findings, we hypothesized that KGF controls alveolar maturation and participates in mediating the so-called FPF effect.
The current study was designed to examine the relative part of KGF in the control of surfactant synthesis by alveolar type II cells. The hypothesis, that KGF-mediated stimulation of DSPC synthesis is regulated by glucocorticoids, is tested. KGF-neutralizing antibody was used to abrogate the stimulating effect of fetal lung FCM. Surfactant synthesis was monitored in isolated type II cells by measuring the rate of choline incorporation into DSPC, i.e. using the same experimental conditions that led to FPF original description. Previously, we have shown that such a rate directly reflects actual changes in DSPC accumulation (12). In addition, we examined whether glucocorticoids control KGF production from isolated lung fibroblasts.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preparation of conditioned media
Fibroblasts from first-step differential adhesion
(15) were grown to confluence in MEM containing 10% FBS.
After rinsing twice with PBS, they were maintained for 8 h in
serum-free DMEM. Then, cells were cultured in fresh serum-free DMEM for
24 h. This medium designated FCM was collected, filtered at 0.22
µm, and kept at -20 C until use.
To compare the stimulating effect of FCM prepared in these basal conditions to that of medium conditioned by fibroblasts submitted to previous glucocorticoid stimulation, other fibroblasts were exposed for 16 h to dexamethasone 10-7 M in serum-free DMEM, rinsed, and cultured in fresh DMEM to condition for 12 h. Medium conditioned by dexamethasone-treated fibroblasts is designated FCM-dex.
[3H]-choline incorporation into DSPC and
determination of radiolabeled DSPC
Incorporation of tritiated choline into DSPC was evaluated from
type II cells that were cultured in the presence of a 1:1 mixture,
vol/vol, of FCM medium and fresh DM, i.e. FCM/DM containing
0.5 µCi/ml (18.5 kilobecquerels/ml) of [methyl
3H]-choline [80.7 Ci/mmol (3.03
terabecquerels/mmol), Amersham Pharmacia Biotech, Les
Ulis, France] as previously described (12). The
consequence of diluting DM components by addition of another medium was
checked. Cultures of cells in either fresh complete DM, or DM/DMEM
(1:1), or a mixture of DM/DMEM previously conditioned by type II cells
for 24 h, all led to the same level of choline incorporation into
DSPC synthesis (data not shown). Thus, to assess FCM effects on type II
cells, we compared choline incorporation in type II cells that were
either exposed to a DM/DMEM mixture (vol/vol; designated as the control
medium), or to a FCM/DM mixture.
After incorporation, radioactive medium was removed, and cells were PBS-rinsed twice. Cells were then incubated with Dispase (Sigma) 10 mg/ml in PBS at 37 C until complete digestion of the EHS matrix, and pelleted (4000 x g, 10 min). Radioactive DSPC was essentially determined directly on cell pellets. Alternatively, to specifically address the effects of dexamethasone on surfactant DSPC synthesis, surfactant material was extracted from cells before determination of DSPC radioactivity. Hence, in FCM-dex studies, DSPC was measured after surfactant extraction through a density-gradient fractionation technique adapted for cultured cells and quantitative analysis (16). Before lipid extraction, a trace amount of [14C]-dipalmitoylphosphatidylcholine [113 mCi/mmol (4.2 gigabecquerels/mmol), Amersham Pharmacia Biotech] was added to cell pellets or to surfactant fractions, for determination of recovery.
Lipids were extracted by chloroform/methanol/water, 1:2:0.8 (vol/vol/vol). DSPC was further extracted from lipid extracts by the osmium tetroxide method and TLC separation on silica gel 60 chromatography plates (Merck & Co., Inc., Darmstadt, Germany) in chloroform/methanol/water, 65:25:4 (vol/vol/vol) (17). DSPC was identified by comparison with a standard run in parallel, eluted from gel by chloroform/methanol/water, 1:2:0.8 (vol/vol/vol), dried, and redissolved in chloroform/methanol, 2:1 (vol/vol), to count activities in Optiscint scintillation cocktail (EEG Instrument, Evry, France) using a double-channel dpm program.
Neutralization of KGF
Polyclonal antihuman KGF and antihuman epidermal growth factor
(EGF) antibodies, produced in goats immunized with the respective human
recombinant proteins, were purchased from R&D Systems Europe Ltd.
(Abingdon, UK) and used for neutralization in FCM. These two antibodies
were selected for their ability to neutralize the biological activities
of KGF and EGF, respectively.
RNA extraction and Northern blot analysis
Total RNA was isolated from cultured fibroblasts using the TRI
REAGENT kit from Euromedex (Souffel-Weyersheim, France). Twenty
micrograms of RNA were fractionated by electrophoresis through 1%
agarose, 2.2 M formaldehyde gels, and blotted onto nylon
membranes (Gene Screen, NEN Life Science Products, Boston,
MA). The KGF probe was amplified by RT-PCR from RNA extracted from
adult rat lung. The primers used were: sense, 5'ATC CTG CCG ACT CCG CTC
TAC-3'; antisense, 5'-CCC TCC GCT GTG TGT CCA TTT A-3', encoding a
459-bp fragment between positions 132 and 590 of rat KGF complementary
DNA (11). Probes were radiolabeled with
[
-32P] deoxycytidine triphosphate
(ICN Biomedicals, Inc., Irvine, CA) using a
Rediprime DNA labeling system from Amersham Pharmacia Biotech, and purified on NucTrap probe purification columns
(Stratagene, Cambridge, UK). Prehybridization with salmon
sperm DNA was performed for 3 h, and hybridization was carried out
overnight at 42 C. To allow correction for variations in loaded amounts
of RNA, blots were stripped and hybridized with an 18S ribosomal RNA
(rRNA) probe. Autoradiographs were made by exposing blots at -80 C to
x-ray film (Reflection, NEN Life Science Products).
Quantification of signal intensity was performed by densitometric
analysis of autoradiograms using the NIH Image analysis program.
Statistical analysis
All data were obtained from a minimum of three different
experiments and are presented as mean ± SEM.
Statistical analyses were performed using ANOVA
(Fisher\\'s PLSD) or, when applicable, using
Student’s t test for unpaired values. In both instances, a
P value < 0.05 was considered statistically
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Addition of the antibody to DM alone
did not change incorporation, showing its inocuity for this pathway
(Table 1). Consistent with previous findings (12), a
stimulating effect of rh-KGF on DSPC synthesis was observed (Table 1).
Sufficient concentration of the antibody to abolish stimulating effects
of rh-KGF was chosen at 5 µg/ml, after testing various concentrations
ranging from 0–20 µg/ml (Table 1, and data not shown).
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. On the
average, choline incorporation in DSPC was increased by 52.8% in type
II cells exposed to FCM, compared with control medium (Fig. 1).
Addition of the KGF-neutralizing antibody to suppress KGF endogenous
activity from the FCM led to suppress about half of the FCM-stimulating
effect on the incorporation of tritiated choline, which was this time
increased by 25.2%, compared with control medium (Fig. 1). Raising
antibody concentration to 10 µg/ml did not lead to further reduction
of the FCM-stimulating effect (not shown). Moreover, we tested the
possible effect of another growth factor, EGF, which is known to be
produced in the developing lung mesenchyme (18). The use
of a goat anti-EGF antibody, used at a final concentration of 10
µg/ml, showed no effect on the stimulating activity of FCM (Fig. 1).
This result indicates the absence of any nonspecific influence of added
goat IgGs on choline incorporation and suggests the specific relevance
of KGF among other mesenchymal-produced factors on surfactant
phospholipid synthesis. From these results, we conclude that about half
of the stimulating effect of FCM can be accounted for by KGF
activity.
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). Then, dose response of dexamethasone
was thus assessed at 24 h and revealed no stimulating effect of a
10-9 M and
10-8 M
concentration, whereas
10-7 M and
10-6 M
dexamethasone led to, respectively, 50 and 60% of stimulation of KGF
mRNA (Fig. 3).
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, FCM-dex
further enhanced choline incorporation into surfactant DSPC by 38%,
compared with FCM alone. To evaluate KGF implication in mediating the
dexamethasone effect, anti-KGF antibody was added to FCM-dex.
Importantly, addition of anti-KGF neutralizing antibody totally
suppressed the effect of dexamethasone treatment (Fig. 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In an effort to define the contribution of fibroblasts to the stimulation of phospholipid surfactant synthesis from type II cells and, in the light of our findings presenting KGF as a stimulating factor, we chose to impair KGF function ex vivo through the use of a neutralizing antibody. This approach allowed us to evaluate, to about 50%, the relative part of KGF in the stimulating activity of medium conditioned by fetal lung fibroblasts in the absence of any stimulus. The possibility that neutralization was partial and that the actual part of KGF was therefore underestimated seems unlikely. Indeed, we found that the same antibody concentration was sufficient to totally inhibit the effect of exogenous rh-KGF (10 ng/ml). Because such concentration was found to actively increase choline incorporation at a higher level than the one obtained with FCM (see Results and 12), this finding argues for an efficient inhibition of the KGF present in FCM. It is highly likely that the antibody was equally potent in neutralizing the activity of rat KGF present in FCM as that of rh-KGF, because rat and human KGFs are highly conserved [91% ungapped amino acid identity (19, 20)] and because rh-KGF displays biological activity in the rat (7, 8, 12, 21), which indicates close 3-dimensional structure.
Fibroblast-pneumonocyte factor was defined as a lung mesenchymal cell product, (2) presumably of peptidic nature (2, 5), that stimulates surfactant phosphatidylcholine production in activating choline phosphate cytidylyltransferase activity (22) through paracrine effect on the alveolar type II cell (2, 22). FPF production was proposed to be stage-specific and induced by glucocorticoids (2) at a pretranslational level (23). Though the mediating substance(s) has (have) yet to be identified, data reported herein strongly argue for an important role of KGF and suggest its involvement in mediating the FPF effect. Indeed, in the current study, the most significant observation is the ability of an anti-KGF neutralizing antibody to reproducibly suppress about half of the stimulation of DSPC synthesis observed when type II cells are exposed to FCM, and to completely abolish the further enhancement of DSPC synthesis observed after previous exposure of fibroblasts to dexamethasone treatment. In addition, we previously reported KGF-induced stimulation of choline phosphate cytidylyltransferase activity in isolated type II cells (12). Also, our present findings reveal that KGF expression is enhanced at the pretranslational level in fibroblasts treated by dexamethasone, using the same experimental conditions as the ones that led to evidence the effect of FPF. Previously, KGF expression was shown to be stimulated by glucocorticoids, though at a much earlier stage of development and in a whole-lung explant culture model (14). At last, KGF expression is regulated in the course of development and shows a marked increase in the fetal rat lung between gestational days 18 and 20 (11), i.e. at the time when surfactant starts accumulating. Altogether, these data suggest that KGF is responsible for most effects attributed to the FPF. However, because mice homozygous for the KGF null-mutation present no obvious respiratory failure (24) and our results impairing KGF reveal some remaining stimulating activity, we propose that the FPF effect is mediated through a combination of factors. Among these factors, the drastic consequences of impairing KGF from the FCM reveals that KGF plays a major contribution. Simultaneous action of other growth factors is likely to account for some redundancy and/or compensation explaining the absence of lung phenotype in the KGF null-mutation.
Although lung epithelial development is delayed in mice lacking EGFR
(25), our findings with anti-EGF antibody argue against a
role of EGF. Instead, transforming-growth factor (TGF), which is
produced by interstitial lung tissue (26, 27) and exerts
its activity through the same receptor, is likely to be involved.
Another mediator, namely TGFß3, could have been a good candidate
because: 1) it was up-regulated by corticosteroid in fetal lung
fibroblasts (28); and 2) its inactivation, in mice, by
gene targeting led to delayed lung maturation (29).
However, the observation that such mutation did not suppress the
corticosteroid-promoted maturation (30) stands sufficient
to question the contribution of TGFß3 to FPF. Other members of the
FGF family, such as FGF-1 or FGF-10, are also very likely to be
involved. Among FGFs, FGF-10 is the most closely related to KGF (47%
of amino acid identity in human), and both factors bind the same
receptor FGF-R2 IIIb with high affinity (31). The earlier
onset of FGF-10 expression, as compared with KGF, in the course of
embryonic lung development (9, 10, 11), however, argues for
different functions of these factors and for an implication of KGF in
later developmental events such as alveolar cell maturation. Whereas
FGF-10 is sufficient to elicit branching in the isolated,
mesenchyme-free embryonic lung epithelium (9, 32), KGF
elicits an abnormal cystic-like growth in this model, as well as in the
isolated whole lung (9, 11, 32). Moreover, FGF-10 gene
targeting in the mouse totally prevents lung organogenesis
(33), which is, by contrast, normal in the presence of KGF
null-mutation (24). Thus, despite their similarities,
their differential effects on lung morphogenesis demonstrate that these
factors have distinct roles in lung development. As for FGF-1, the
simultaneous presence of both KGF and FGF-1 in a complex medium has
been found to be necessary for eliciting the expression of the alveolar
type II cell phenotype from cultured embryonic tracheal epithelial
cells in mesenchyme-free culture (34). FGF-1 has also been
reported to enhance surfactant protein expression in isolated adult rat
type II cells, although to a lesser extent than KGF (21).
As a whole, although the implication of FGF-10 in the control of
surfactant synthesis cannot be ruled out, a predominant role of KGF in
this process seems therefore likely, probably in association with
FGF-1. Having clearly defined the conditions of stimulation by the FCM
herein, implication of these various candidates, discussed here, to
mediate the FPF effect, is worth testing in future studies.
In conclusion, the present results bring new insights into the nature of diffusible mediators from mesenchyme that control surfactant synthesis and reveal the importance of KGF among these factors. Importantly, the current study also establishes that glucocorticoid maturational effects on fetal lung are mediated, at least in part, through the production of KGF by lung mesenchymal cells.
Received March 1, 2000.
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