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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 620-626, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.4912

Maturational Factors Modulate Transcription Factors CCAAT/Enhancer-Binding Proteins {alpha}, ß, {delta}, and Peroxisome Proliferator–Activated Receptor-{gamma} in Fetal Rat Lung Epithelial Cells

Anne-Marie Barlier-Mur, Bernadette Chailley-Heu, Claudie Pinteur, Alexandra Henrion-Caude, Christophe Delacourt and Jacques R. Bourbon

Inserm U 492, Faculté de Médecine, Université Paris XII, Créteil; and Inserm E 213, Hôpital Trousseau Assistance Publique-Hopitaux de Paris, Paris, France

Address correspondence to: J. R. Bourbon, Inserm U 492, Faculté de Médecine, 8 rue du Général Sarrail, 94010 Créteil cedex, France. E-mail: Jacques.Bourbon{at}creteil.inserm.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous investigations have evidenced the importance of CCAAT/enhancer-binding proteins (C/EBPs) and peroxisome proliferator–activated receptor (PPAR){gamma} for lung development, especially for alveolar type II cells (ATII). This prompted us to explore whether ATII maturation-promoting mediators controlled their expression in isolated ATII. In whole rat lung, C/EBP{alpha}, ß, {delta}, and PPAR{gamma} mRNAs increased 3–5 times between gestational day 18 and term (Day 22), dropped around birth, then reincreased. C/EBPß and {delta}, but not PPAR{gamma}, displayed similar profile in isolated ATII; C/EBP{alpha} transcript disappeared and the protein became hardly detectable in isolated cells. In cultured ATII, dexamethasone increased C/EBPß and PPAR{gamma} mRNAs 2–4 times, and cyclic AMP increased C/EBPß and {delta} mRNAs ~ 1.5 times. Whereas retinoic acid increased C/EBPß and PPAR{gamma} mRNAs 1.5 times in ATII in vitro, vitamin-A deficiency strongly decreased fetal lung C/EBP{alpha}, ß, and PPAR{gamma} transcripts in vivo. C/EBPß, {delta}, and PPAR{gamma} mRNAs were also increased in vitro by epidermal growth factor and keratinocyte growth factor, whereas they were unchanged by the maturation inhibitor transforming growth factor-ß. C/EBP{alpha} expression was not reinduced by any mediator. Changes in transcripts were reflected in protein levels analyzed through Western blotting. These results argue for a role of these factors in ATII functional maturation, and indicate a multifactorial control of their ontogeny.

Abbreviations: alveolar type II cells, ATII • CCAAT/enhancer-binding protein, C/EBP • dexamethasone, Dex • Dulbecco's modified Eagle's medium, DMEM • epidermal growth factor, EGF • Engelbreth-Holm-Swarm, EHS • fetal bovine serum, FBS • keratinocyte growth factor, KGF • peroxisome proliferator–activated receptor, PPAR • retinoic acid (all trans), RA • surfactant protein, SP • transforming growth factor, TGF • vitamin A–deficient, VAD


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CCAAT/enhancer-binding proteins (C/EBPs), a family of basic leucine-zipper transcription factors control a wide array of genes and have been postulated to serve a crucial role in normal tissue development and regulation of cell proliferation or differentiation in various organs (1). With regard to lung development and functions, various studies have shown the involvement of these factors. Thus, the homozygous C/EBP{alpha} gene null mutation in the mouse resulted in animal death shortly after birth, with abnormal lung histology including interstitial thickening (2) and hyperproliferation of alveolar epithelial type II cells (ATII) (3). C/EBP{alpha} expression level is the highest in the lung after adipose tissue and liver, and it correlates with expression of surfactant proteins (SPs) in cultured ATII (4). C/EBP{alpha} also transactivates the bronchiolar Clara cell secretory protein (CCSP) gene (5), and its expression correlates with that of CCSP in cultured Clara cells (6). Mice lacking both C/EBPß and {delta} also die during early postnatal development, but contrary to C/EBP{alpha}-/- mice, they manifest no histological lung abnormality (7). A developmental increase of C/EBP{delta} has nevertheless been reported in the rabbit, and cAMP and dexamethasone, two enhancing mediators of lung maturation, enhanced the expression level of this factor in human lung explants (8). C/EBP{delta} has been shown to reverse the action of a transcriptional silencer just upstream of the SP-A promoter (9), whereas an antisense oligonucleotide overlapping the translational start site of C/EBP{delta} simultaneously reduced C/EBP{delta} mRNA and protein and SP-A expression (10). Moreover, a double-stranded oligonucleotide matching the consensus C/EBP binding site reduced SP-A mRNA as much as 75% in the human cell line NCI-H441 (9). C/EBPs have also been shown to control the expression of SP-D, a secreted protein from both ATII and Clara cells (11). Last, both C/EBPß and {delta} were increased in rat lung after exposure to hyperoxia (12), a cause of lung epithelial injury.

C/EBPß and {delta} are also known to act synergistically with the nuclear hormone receptor superfamily member, peroxisome proliferator–activated receptor (PPAR) {gamma}, for triggering the adipocyte differentiation program (13). As regards lung development and similar to C/EBP{alpha}, PPAR{gamma} was induced during in vitro maturation of ATII and was enhanced by cAMP (14). The precocious death of mice with homozygous null mutation for PPAR{gamma} (15) prevents, however, evaluation of the role of this transcription factor in lung development.

C/EBPs and PPAR{gamma} are therefore involved in lung organogenesis and in the expression control of several lung-specific proteins. The correlation with SPs raises the question of the expression control of these transcriptional regulators during development, especially during the process of ATII maturation that precedes birth. This maturation, which consists in the rise of synthesis and storage of the various surfactant components, is known to be submitted to a multifactorial control that includes glucocorticoids (16, 17), cAMP (17), retinoids (18, 19) and several growth factors acting as paracrine mediators (16, 17, 20). We therefore analyzed whether those mediators that control ATII maturation controlled the expression of C/EBPs and PPAR{gamma} in developing lung cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Virgin Wistar female rats (Charles River, Saint-Aubin-Lès-Elbeuf, France) were mated overnight in the animal care facilities of the laboratory. The following morning was designated Day 0 of pregnancy. Pregnancy was checked 14 d later by palpation. Term is 22 d. Lungs were collected from fetuses under pentobarbital anesthesia of the pregnant rats and from anesthetized young rats aged 0–25 d.

Lung Cell Isolation and Primary Culture
ATII were isolated from the lung of rat fetuses and newborns ranging from gestational day 18 to postnatal day 1. Fetal lung cells were enzymatically dispersed. ATII were separated from other cell types by serial differential adhesions to plastic and low speed centrifugations as described previously (20, 21). Immunolabeling for cytokeratins and vimentin has shown epithelial cells obtained through this procedure to be over 95% pure (21). Purified ATII cells were either used immediately for RNA or protein extraction (thereafter designated freshly isolated ATII), or seeded on multiwell plastic culture plates coated with Engelbreth-Holm-Swarm (EHS) tumor basement membrane matrix prepared in the lab (20), and allowed to adhere overnight under air–CO2 (95%–5%) at 37°C in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). The following morning, FBS-containing medium was removed, cells were rinsed twice with DMEM, and experimental media were introduced. For studying changes with culture time and the effects of various mediators on ATII, experiments were conducted in a defined culture medium based on DMEM enriched with various additives including penicillin, streptomycin, bovine transferrin, biotin, sodium selenite, and metal trace elements (20). Cultures were run with immature cells from 18-d-old fetuses. Media, serum and antibiotics were from Gibco BRL (Grand Island, NY); additives were from Sigma (L'Isle d'Abeau, France) and were cell-culture tested.

Cell Treatments
Cells were treated for 24 or 48 h in defined medium either with dexamethasone (Dex; Sigma), dibutyryl-cAMP (thereafter simply designated cAMP; Sigma), all-trans retinoic acid (RA; Sigma), human platelet transforming growth factor ß1 (TGF-ß; BD Biosciences, San Diego, CA), mouse submaxillary-gland epidermal growth factor (EGF; Sigma), or human recombinant keratinocyte growth factor (KGF; R&D Systems Europe, Abingdon, UK). Control medium was defined medium without added mediator, except for cAMP, for which it was added with Na butyrate. Concentrations that were used for each mediator are indicated in RESULTS.

Vitamin A–Deficient Rat Fetuses
Partial vitamin A deficiency was established by submitting virgin rats to a vitamin A–deprived diet around 5 wk before mating and for the whole duration of pregnancy as described previously (18). This treatment has been reported to decrease fetal plasma retinol about half as compared with fetuses from rats receiving the standard chow. Fetal blood and lung tissue were collected at term. Samples were from the same litters as those used for previous evaluation of lung maturation, and plasma retinol was similarly determined by high-performance liquid chromatography (18).

RNA Isolation and Northern Blot Analysis
Total RNAs were isolated from homogenized tissues and cells using Trizol reagent (Gibco BRL) as specified by the manufacturer. The precipitated RNA was resuspended in sterile water and quantified by absorbance at 260 nm. Twenty-five micrograms of RNAs were fractionated by electrophoresis through 1.2% agarose–2.2M formaldehyde gels and blotted onto nylon membranes (GeneScreen; NEN-Perkin Elmer, Courtaboeuf, France). The integrity of RNAs was assessed by visual examination of the ethidium bromide-stained gel. C/EBP{alpha} and {delta} cDNA probes were gifts from Prof. S. L. McKnight (Dallas, TX). C/EBPß and PPAR{gamma} cDNA probes were gifts from Dr. A. Balsalobre (Geneva, Switzerland) and Dr. F. Djouadi (Paris, France), respectively. Probes were labeled with [{alpha}-32P]-dCTP (NEN-Perkin Elmer) using Rediprime DNA labeling system (Amersham Pharmacia Biotech, Saclay, France) and recovered with probe purification MicroSpin G25 columns (Amersham Pharmacia Biotech). Conditions for prehybridization, hybridization, and membrane stripping were as described previously (21). Autoradiograms of blots were obtained with Kodak X-OMAT AR films at -70°C. The relative intensity of bands was quantified by scanning densitometry using the NIH Image program. Results were normalized for variations in gel loading by 18 S rRNA blotting.

Protein Electrophoresis and Immunoblotting
Tissue and cells were lysed in buffer as previously described (22) and protein extracts were obtained after centrifugation at 10,000 x g for 10 min. Protein content of the supernatant was determined by the Bradford method. Crude protein extracts from the various samples were either dropped directly onto nitrocellulose membrane (Amersham Pharmacia Biotech) for dot-blot analysis, or mixed with Laemmli sample buffer to be submitted to electrophoresis for Western blot analysis. Equal amounts of proteins were subjected to 12% SDS-PAGE, then blotted to nitrocellulose membranes. After blocking in 5% nonfat milk in phosphate-buffered saline for 1 h at room temperature, dot- and Western-blot membranes were incubated overnight at 4°C with an antibody directed against either C/EBP{alpha}, C/EBPß, C/EBP{delta}, or PPAR{gamma} (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1/250. To monitor equal loading of proteins, membranes were incubated with an antibody directed against ß-actin (Sigma). Immunoreactive proteins were revealed using a chemiluminescent substrate (ECL kit, Amersham Pharmacia Biotech).

Statistical Analysis
Results are reported as mean ± SEM. Data were analyzed using either the nonparametric U test, the two-tailed t test, or one-way ANOVA according to applicability. Significance level is assigned for P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Developmental Expression Profile of C/EBPs and PPAR{gamma}
Messenger RNAs (mRNAs) of C/EBP{alpha}, ß, {delta}, and PPAR{gamma}1 (thereafter simply designated PPAR{gamma}) were readily detected in the 18 d fetal lung (Figure 1, upper panel). PPAR{gamma}2 transcript was not found, consistently with previous observations in whole lung (23) or in ATII (14). All four factors displayed a 3- to 5-fold increase from Day 18 of gestation, peaking either at 1 or 2 d before birth (Figure 1, lower panel), a transient decline during the first postnatal day, and a return to prenatal level on postnatal Day 5 (Figure 1, lower panel). In freshly isolated ATII, C/EBP{alpha} transcripts were not detected whatever the developmental stage; C/EBPß and {delta} mRNAs exhibited, as in whole lung, a prenatal rise, whereas PPAR{gamma} did not display any developmental increase (Figure 2).



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  		Figure 1. Relative changes in steady-state levels of mRNAs for C/EBP{alpha} (squares), ß (circles), {delta} (diamonds), and PPAR{gamma} (triangles) in the developing fetal rat lung. Upper panel: representative Northern blots obtained with specific cDNA probes; lower panel: densitometric analysis of Northern blots (mean ± SEM on three individuals for each stage). Day 0 is birth time. Average steady-state level at a given developmental stage is expressed as a percentage of the level at -4 d (= gestational day 18) taken as a reference. All four transcription factors displayed a prenatal peak of expression (peak value significantly different from that at -4 d for P < 0.05 by the nonparametric U test), tended to decrease around birth, then regained the prenatal level.

 


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  		Figure 2. Relative changes in steady-state levels of mRNAs for C/EBPß (circles), {delta} (diamonds), and PPAR{gamma} (triangles) in alveolar epithelial type II cells (ATII) immediately after isolation and purification from fetal and newborn rat lung. Upper panel: representative Northern blots obtained with specific cDNA probes; lower panel: densitometric analysis of Northern blots (mean ± SEM on three or four different cell isolates for each stage; absence of error bar means that it is too low to be represented at this scale). Average steady-state level is expressed as a percentage of the level at -4 d taken as a reference. C/EBP{alpha} was not detected. A prenatal peak of expression was observed for both C/EBPs, followed by a drop around birth. No developmental change was observed for PPAR{gamma}.

 
Changes in Expression of C/EBPs and PPAR{gamma} in Cultured Cells and Effects of Various Mediators Changes with Time in Basal Medium
ATII isolated at 18 d were cultured for 12 h in MEM containing 10% FBS to allow cells to adhere to the substratum, then this medium was replaced by defined medium for a further 72-h period. After an initial decline in expression for the first 12 h, the level of transcripts of C/EBPß, {delta}, and PPAR{gamma} reincreased to regain the initial level of freshly isolated cells after 84 h of culture (Figure 3). C/EBP{alpha} transcripts remained undetectable in cultured cells. Using dot blot analysis, a strong signal was obtained with both antibodies against C/EBP{alpha} and C/EBPß proteins in whole tissue; a signal for C/EBP{alpha} was also found in freshly isolated ATII, although already less abundant than that for C/EBPß, but it became extremely faint in cultured cells whereas C/EBPß remained easily detectable (Figure 4). Although labeling may be partly unspecific, this approach therefore evidenced progressive disappearance of C/EBP{alpha} protein from isolated ATII, consistent with the absence of its transcript.



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  		Figure 3. Changes as a function of culture time in the steady-state levels of mRNAs for C/EBPß (circles), {delta} (diamonds), and PPAR{gamma} (triangles) in isolated fetal rat lung ATII. Cells were seeded onto EHS matrix and cultured for 12 h in the presence of 10% FBS (adhesion period to the substratum), then for the following 72 h in defined medium as described in MATERIALS AND METHODS. Densitometric analysis of Northern blots; average steady-state level is expressed as a percentage of the initial level at the time of culture initiation. Expression level of the three transcription factors fell down about one half during the first 12 h, then reincreased to recover progressively the initial level. Mean ± SEM on six to eight independent culture experiments; significant difference with initial level (ANOVA) for: *P < 0.05; **P < 0.01.

 


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  		Figure 4. Dot blot analysis of C/EBP{alpha} and ß in whole fetal rat lung tissue (1) and in ATII immediately after cell isolation (2) and after a 24 h-culture (3). Same amounts of the same samples were spotted for both proteins. The top and bottom lines were probed with specific anti-C/EBP{alpha} and anti-C/EBPß antibodies, respectively, at the same dilution. C/EBP{alpha} progressively disappeared from isolated and cultured ATII.

 
Effects of Mediators
The effects of various mediators with known effects upon alveolar cell maturation were tested in cultured ATII at the pre- and post-translational levels for C/EBPß, {delta}, and PPAR{gamma}. Mediators were used at a single concentration elicited as optimal for stimulation or inhibition of ATII maturation on the basis of data in the literature. Results for transcripts are shown in Table 1, and most prominent induced changes are depicted in Figure 5. Dex 10-7 M enhanced C/EBPß (Figure 5) and PPAR{gamma} transcripts 3.6 times and 1.9 times, respectively, at time 48 h after a transient decrease for C/EBPß at time 24 h. C/EBP{delta} expression, by contrast, was slightly diminished at both times. Cyclic AMP 10-4 M increased C/EBPß and {delta} expression 40–70% at both times, whereas PPAR{gamma} expression was unchanged. Similar to Dex, RA 10-6 M increased C/EBPß and PPAR{gamma} expression only after 48 h, although the increase was ~ 50% only, and it slightly decreased C/EBP{delta} expression. Both EGF (20 ng/ml) and KGF (50 ng/ml) increased C/EBPß and PPAR{gamma} (Figure 5) transcripts ~ 70–80%, and led C/EBP{delta} expression unchanged. TGF-ß (10 ng/ml) had no significant effect on any of the three transcription factors. None of the tested mediators reinduced C/EBP{alpha} expression in cultured cells.


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  		TABLE 1 Effects of various mediators on the steady-state levels of mRNAs for C/EBPß, {delta}, and PPAR{gamma} in cultured fetal rat lung ATII expressed as percent of control values

 


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  		Figure 5. Effects of mediators in cultured fetal rat lung ATII. Northern blots representative of the most prominent effects observed (see Table 1), i.e., effects of Dex and KGF on the steady-state levels of mRNAs for C/EBPß and PPAR{gamma}, respectively. Cells were seeded onto EHS matrix and cultured in MEM with 10% FBS for 12 h, then for 48 h in defined medium with or without (control) the mediators, and collected for RNA extraction and probing.

 
Western blot analysis of cell protein contents (Figure 6) indicated that C/EBPß protein was increased the most by EGF, KGF, and Dex, but unchanged by TGF-ß. Densitometric analysis of two repeated experiments indicated a 30% increase with KGF and a 50% increase with EGF and Dex after correction for loading. Changes in C/EBP{delta} protein were limited to a slight decrease with RA. PPAR{gamma} protein was enhanced 50% by KGF, but not by EGF or Dex.



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  		Figure 6. Effects of mediators in cultured fetal rat lung ATII. Western blot analysis of C/EBPß, C/EBP{delta}, and PPAR{gamma} proteins. Cells were cultured and treated as in Figure 5. C: control medium; mediators: EGF 20 ng/ml, KGF 50 ng/ml, Dex 10-7 M, RA 10-6 M, or TGF-ß 10 ng/ml. Thirty and 70 µg of cell proteins were loaded for the detection of C/EBPß and of the two other transcription factors, respectively. Representative experiment is shown. The antibody against C/EBPß labeled a major band at 38 kD and a minor band at 20 kD, whereas the antibodies against C/EBP{delta} and PPAR{gamma} labeled bands at 32 kD and 48 kD, respectively, consistently with previous observations (14, 24). Each experimental membrane was subsequently labeled with an anti–ß-actin antibody to monitor equal loading. Increased amount of C/EBPß protein was manifest in the presence of EGF, KGF, and Dex. No significant changes were observed for C/EBP{delta}, except a slight decrease with RA. PPAR{gamma} protein was enhanced only by KGF.

 
Effects of Vitamin A Deficiency on C/EBP and PPAR{gamma} Expressions
Lungs from vitamin A–deficient (VAD) fetuses were collected at term and compared with those of age-matched control fetuses. Plasma retinol concentration was 13.0 ± 1.9. mg/dl in VAD fetuses versus 22.9 ± 1.4 mg/dl in controls (n = 7, P < 0.001). Messenger RNAs of C/EBP{alpha}, ß, and PPAR{gamma} were reduced by 60, 65, and 25%, respectively, in the lung of VAD fetuses, whereas C/EBP{delta} mRNA level was unchanged (Figure 7).



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  		Figure 7. Steady-state levels of mRNAs for C/EBP{alpha}, ß, {delta}, and PPAR{gamma} in the lung of VAD fetal rats at term. Rats were submitted to VAD diet around 5 wk before mating and for the whole duration of pregnancy. Control rats received the standard chow. Fetal lungs were collected at term and kept frozen until RNA extraction. Plasma retinol concentration was 22.9 ± 1.4 and 13.0 ± 1.9 mg/dl in control (open bars) and VAD (shaded bars) fetuses, respectively (n = 7; P < 0.001). Results are expressed as the percentage of average steady-state level in control lungs. Mean ± SEM on seven individuals; significant difference with control lungs for: **P < 0.01; ***P < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The developing lung stores surfactant in ATII in preparation for birth. Because previous data indicated that C/EBP and PPAR {gamma} expressions seemed to correlate with ATII marker proteins and that they controlled their expression, we sought to discover whether this maturational event is associated with changes in the expression of these transcription factors and whether this expression is submitted to a regulation by the various mediators involved in lung maturation. Our results indicate that these factors are developmentally regulated at the pretranslational level by mediators that control lung maturation.

Because no comprehensive study of their ontogeny has been performed formerly, a preliminary step was to determine changes in the expression of C/EBP{alpha}, ß, {delta} and PPAR{gamma} in the lung and in ATII during the perinatal period. The expression of the four studied factors indeed displayed a prenatal increase, peaking up at end of gestation. This coordinate rise that correlates temporally with surfactant storage (25, 26) suggests a common integrated developmental control. Indeed, previous studies have pointed out spatial-temporal correlations between some C/EBPs or PPAR{gamma} and surfactant proteins (4, 9, 14). Taking into account the essential role of these transcription factors for adipocyte differentiation that is controlled through their coordinate regulation (13, 29), an involvement in the synthesis of lipid precursors of surfactant phospholipids through lipogenesis (30) also appears likely. A prenatal increase of C/EBPß and {delta} was consistently found in isolated ATII; the absence of change for PPAR{gamma} transcripts in ATII suggest the possibility that changes in whole lung for this factor reflect those in other cell types, presumably lipofibroblasts (27).

C/EBP{alpha} mRNA was not detected in freshly isolated or cultured cells, and C/EBP{alpha} protein previously shown to be present in the nucleus of ATII (4, 28), progressively disappeared from cultured cells. An almost complete loss of the transcript was also reported in adult rat ATII over a 48–h culture (4). It is therefore likely that both the protein and its transcript are quite labile, that the transcript was lost during the isolation procedure of ATII, and that transcription did not resume in our culture conditions leading to decay of the protein. This prevented us from studying the developmental profile and the control of C/EBP{alpha} by various mediators in isolated cells.

Cultured ATII initially displayed a decrease of about half of their C/EBPß, {delta}, and PPAR{gamma} transcripts during the phase of adhesion onto the matrix, possibly due to the presence of serum, because the initial level was recovered during the further 72 h of culture in defined medium. No increase mimicking the in vivo rise of expression occurred, however. This suggests that the prenatal expression rise of C/EBPs and PPAR{gamma} is not due to an endogenous developmental program of ATII, but results from the inductive action of mediators.

Dexamethasone was the most potent stimulator of expression in cultured cells. This is in keeping with the prominent role of glucocorticoids in the maturation of ATII and the ontogenetic control of all surfactant components (16, 17). Particularly, our findings may be in connection with the demonstrated increasing effect of dexamethasone on de novo fatty acid synthesis and on fatty acid synthase in fetal lung explants (31, 32), strengthening the assumption of an involvement in the control of lipogenesis in ATII. Cyclic AMP, which is also a stimulus of both surfactant phospholipid and protein syntheses (16, 17) and of fatty acid synthase expression (31), also stimulated C/EBPß/{delta} expression, although to a lesser extent than Dex for C/EBPß. It is worth underlining that Dex and cAMP have previously been reported to synergistically stimulate both C/EBP{delta} expression (8) and fatty acid synthase expression (31) in cultured human fetal lung explants.

Retinoids were reported previously to increase surfactant phospholipids in fetal rat lung and cultured ATII, and to enhance the synthesis of phospholipids and neutral lipids from acetate in fetal ATII (19, 33). Whereas RA enhanced C/EBPß and PPAR{gamma} mRNA steady state levels in ATII, vitamin A deficiency that becomes limiting for surfactant synthesis below a certain threshold of fetal blood retinol (18) reciprocally led to a strongly decreased expression of C/EBPß and PPAR{gamma}. C/EBP{delta} that was little changed by RA in vitro was unchanged in VAD fetuses. The slight C/EBP{delta} decrease observed in cultured ATII might be related to the proliferation-inhibiting activity evidenced for RA in these cells (33), because proliferation is known to be associated with C/EBP{delta} induction (34). In addition, C/EBP{alpha} expression was considerably decreased in VAD fetuses, indicating that C/EBP{alpha} expression is probably regulated by retinoids in a similar way as that of C/EBPß.

A large number of growth factors have been shown to influence maturation of ATII and the synthesis of surfactant components in the mature cell. Among these, we studied two potent stimulating factors, EGF (33, 35) and KGF (20, 36), and one inhibiting factor, TGF-ß (37, 38). Both EGF and KGF stimulated the expression of C/EBPß and PPAR{gamma} to a similar extent, but let unchanged C/EBP{delta} expression. TGF-ß did not significantly change the expression of C/EBPs and PPAR{gamma}, indicating that its impairment of ATII maturation involves other mechanism(s).

Some mediators exerted in ATII opposite effects to those reported in other cell types. For instance, ATII appeared to respond to retinoids similarly to hepatocytes (39), but differently from adipocytes in which RA inhibited the expression of all C/EBPs (40). This is consistent with the opposite biological effects of retinoids in ATII and adipocytes, i.e., enhanced lipogenesis in ATII (18) and inhibited adipocyte differentiation (41, 42). Similarly, EGF and TGF-ß exerted opposite effects on the expression of these factors in ATII and adipocytes (43), which is in keeping with the fact that EGF and TGF-ß are stimulatory and inhibitory for ATII differentiation and inhibitory and stimulatory for adipocyte differentiation, respectively. This suggests that a factor that promotes differentiation in a given cell type invariably exerts a stimulation on expression of C/EBPß and PPAR{gamma}.

When comparing the effects of the various mediators, C/EBPß and PPAR{gamma} on the one hand and C/EBP{delta} on the other hand appear to be submitted to a differential control: except for cAMP, the various mediators enhanced the expression of the two formers whereas they either let unchanged or tended to diminish the latter. This suggests that mediators that induce cAMP synthesis are involved in triggering the prenatal rise of C/EBP{delta} expression.

Comparison of data from mRNA and protein analyses showed that although there was no strict proportionality between transcript and protein changes, most of changes induced by mediators at the pretranslational level were reflected at the post-translational level. Thus, various factors that stimulated C/EBPß expression also enhanced the protein. Little changes were induced for both C/EBP{delta} transcript and protein. The only apparent discrepancy was seen for PPAR{gamma}, the transcript of which was enhanced by Dex, EGF, and KGF, whereas the protein appeared to be enhanced by KGF only. Taken together, the transcript and protein analyses nevertheless indicate that the ontogeny of C/EBPs and PPAR{gamma} is actually controlled by those mediators that control epithelial lung maturation.

In conclusion, the coordinated developmental profile of C/EBPs and PPAR{gamma}, their simultaneous presence in ATII, and the effects of lung maturation-promoting mediators on their expression altogether indicate an involvement of these transcription factors in lung development, more especially in prenatal ATII maturation. The fact that immature lung epithelial cells responded in vitro to various mediators that enhance lung maturation by an increased expression of these transcription factors indicates that the ontogeny of the latter is submitted to a multifactorial developmental control.


   Acknowledgments
 
This work was supported in part by the Fondation pour la Recherche Médicale and by a Legs Poix grant from the Chancellerie des Universités de Paris.

Received in original form June 17, 2002

Received in final form March 14, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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P. C. Martis, J. A. Whitsett, Y. Xu, A.-K. T. Perl, H. Wan, and M. Ikegami
C/EBP{alpha} is required for lung maturation at birth
Development, March 15, 2006; 133(6): 1155 - 1164.
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