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Published ahead of print on August 1, 2003, doi:10.1165/rcmb.2003-0235OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 174-183, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0235OC

Lipogenesis in Fetal Rat Lung

Importance of C/EBP{alpha}, SREBP-1c, and Stearoyl-CoA Desaturase

Feijie Zhang, Tianli Pan, Larry D. Nielsen and Robert J. Mason

National Jewish Medical and Research Center, Denver, Colorado

Address correspondence to: Robert J. Mason, M.D., National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail: masonb{at}njc.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Alveolar type II cells increase lipogenesis and convert glycogen into the phospholipids of surfactant in the late term fetal lung. Recent studies suggest that CCAAT/enhancing-binding protein (C/EBP) isoforms and sterol regulatory element binding protein (SREBP)-1c regulate fatty acid synthesis in adult type II cells in vitro. To define the temporal relationships and enzymes involved in lipogenesis in fetal rat lung, the mRNA levels of selected transcription factors and enzymes were determined. There was an increase in the mRNA levels of C/EBP{alpha}, C/EBPß, C/EBP{delta}, peroxisomal proliferator–activated receptor {gamma} (PPAR{gamma}), and SREBP-1c, but not SREBP-1a or SREBP-2 from fetal Days 19–21. There was also an increase in the mRNA levels of fatty acid synthase, stearoyl-CoA desaturase 1 (SCD-1), fatty acid translocase, glycerol-3-P acyl transferase, and phosphatidate cytidylyltransferase. By in situ hybridization, there was detectible expression of fatty acid synthase, SCD-1, and C/EBP{alpha} along the alveolar septae with the same distribution pattern as surfactant protein-C, whereas PPAR{gamma} expression appeared to be restricted to macrophages. Regulation of lipogenesis at the mRNA level is predominately on enzymes of fatty acid synthesis and appears to be regulated by C/EBP{alpha} and SREBP-1c. SCD-1 and phosphatidate cytidylyltransferase are important components of the lipogenic response in the fetal lung that have not been recognized previously.

Abbreviations: acetyl-CoA decarboxylase, ACC • adipocyte determination differentiation dependent factor, ADD-1 • CCAAT/enhancer-binding protein, C/EBP • CTP:phosphocholine cytidylyltransferase, CCT • digylceride acyltransferase, DGAT • epidermal fatty acid–binding protein, E-FABP • fatty acid synthase, FAS • fatty acid translocase, FAT • glycerol-3P acyltransferase, GPAT • keratinocyte growth factor, KGF • phosphatidate cytidylyltransferase, PCT • phosphatidylglycerophosphate synthase, PGPS • phosphastidylinositol synthase, PIS • peroxisomal proliferator–activated receptor, PPAR • ribonuclease protection assay, RPA • stearoyl-CoA desaturase, SCD • surfactant protein, SP • sterol regulatory element binding protein, SREBP


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Maturation of the lung is critical at the time of birth. In the last few days of gestation in the rodent lung, the alveolar epithelium undergoes marked differentiation in preparation for air breathing. Alveolar type II cells convert glycogen into surfactant phospholipids, and surfactant appears in the alveolar fluid (1–7). Numerous studies have documented an increase in lipogenesis and an increase in lipogenic enzymes in the late term fetal lung (8–12). The fetal lung appears to prefer de novo synthesized fatty acids for phosphatidylcholine synthesis, and it is de novo fatty acid synthesis that appears to be principally increased at this time (13, 14). Specifically, there is an increase in mRNA for fatty acid synthase (FAS) and ATP citrate-lyase but not acetyl-CoA carboxylase (ACC) or malic enzyme (12, 15). There is also an increase in the incorporation of radiolabeled choline into phosphatidylcholine and an increase in some of the enzymes involved in phospholipid synthesis (16–18). However, fatty acids themselves can also increase CTP: phosphocholine cytidylyltransferase (CCT) activity and thereby phosphatidylcholine synthesis (19). Although type II cells increase fatty acid synthesis and surfactant phospholipid production in the late term fetal lung, little is known about its molecular regulation.

Recent studies on the regulation of fatty acid synthesis in adult rat alveolar type II cells have underscored the importance of two sets of transcription factors, CCAAT-enhancer-binding protein (C/EBP) {alpha} and {delta} and SREBP-1c (20). When type II cells are grown on tissue culture plastic, they rapidly lose their differentiated functions. However, when the cells are cultured on a permissive substrate, they can be induced to differentiate upon the addition of keratinocyte growth factor (KGF; FGF-7). Under these conditions, KGF stimulates fatty acid synthesis and a variety of lipogenic enzymes including FAS, stearoyl-CoA desaturase (SCD)-1, SCD-2, and epidermal fatty acid–binding protein (E-FABP) (21). KGF also increases the expression of C/EBP{alpha}, C/EBP{delta}, and SREBP-1c, but not C/EBPß, SREBP-2, SREBP-1a, or peroxisomal proliferator–activated receptor {gamma} (PPAR{gamma}) (21). Because KGF also stimulates lipogenesis in fetal rat type II cells, the regulation of the pathways in adult type II cells in vitro and in the developing lung in vivo may be similar (22).

In liver and adipocytes, lipogenesis is also induced by C/EBP{alpha} and SREBP-1c, which coordinately induce the expression of enzymes of de novo fatty acid synthesis. In the liver, SREBP-2 predominantly regulates enzymes involved with cholesterol and bile acid synthesis, whereas SREBP-1c predominantly regulates those involved in fatty acid biosynthesis (23). In adipocytes, C/EBP{alpha}, SREBP-1c, and PPAR{gamma} all appear to be important in stimulating fatty acid synthesis, although only C/EBP{alpha} appears to be essential (24). However, additional transcription factors may also be important (25, 26). It is recognized that the regulation of lipogenesis in adipocytes is very complex and involves many signaling molecules and transcription factors (26).

Defining the regulation of fatty acid synthesis in the fetal lung is important and could lead to new forms of therapy targeted toward increasing endogenous surfactant production. The current study was designed to define the temporal relationship of certain lipogenic enzymes and transcription factors based on relative mRNA levels in the developing rat lung. The recent availability of specific primers and probes for individual gene products makes this approach possible. The selected transcription factors and lipogenic enzymes were chosen based on a previous study of lipogenesis in adult rat type II cells in vitro (21). The current study in the fetal lung was designed to extend the observations with adult type II cells by defining their similarities and differences. In addition, in situ hybridizations allowed for a comparison of gene expression in lung to that of nearby adipose tissue and skin, which served as positive controls.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Timed-pregnant Sprague-Dawley rats were obtained from Charles River Laboratories (Raleigh, NC). A sperm-positive vaginal smear confirmed mating and was designated Day 0 of gestation (day of birth, Day 22). The pregnant female rats dated as gestational Days 17, 18, 19, 20, 21, and 22 were killed with an intraperitoneal dose of pentobarbital sodium, and the fetuses were quickly removed by hysterotomy. The fetuses were weighed to confirm their gestational age. The lungs were removed en bloc from the chest cavity. The lobes of the lung were dissected free from the major airways and maintained on ice in sterile saline. Lungs from littermates were pooled for RNA preparation. A slice through the thorax of one fetus from each litter was removed, fixed with 4% paraformaldehyde overnight, and embedded in paraffin for histology and in situ hybridization.

Real-Time Polymerase Chain Reaction Measurement Preparation of RNA
Homogenates of fetal lungs were directly lysed into 4 M guanidinium isothiocyanate, 0.5% N-laurylsarcosine, and 0.1 M ß-mercaptoethanol in 25 mM sodium citrate buffer. Total cellular RNA was isolated by ultracentrifugation for 18 h at 150,000 x g through a 5.7 M CsCl cushion as previously described (27). To remove any genomic DNA contamination, isolated RNA samples were treated with 4 U of RNase-free DNase I (Ambion, Austin, TX) for 30 min at 37°C.

Reverse Transcription Reaction
Total RNA (2 µg) was used in 100 µl of reverse transcription reaction mix to synthesize cDNA by using TaqMan Reverse transcription reagents according to the manufacturer's protocol (Applied Biosystems, Branchburg, NJ). Random hexamers were used as primers. The reactions were incubated at 25°C for 10 min, at 48°C for 30 min, at 95°C for 5 min, and then stored at –20°C until use. For most genes the resultant cDNA was diluted 1:100 for the real-time PCR measurement, but for the less abundant genes, i.e., ACC, SP-D, SCD-1, and PPAR{gamma}, the cDNA was diluted 1:10.

Real-Time Polymerase Chain Reaction
Real-time polymerase chain reaction (PCR) primer and probe sets were designed for each cDNA with PRIMER EXPRESS software (Version 1.5; Applied Biosystems; Table 1). Sequences for cDNAs were obtained from GenBank for use in primer design. Real-time PCR reactions were performed with TaqMan universal PCR master mix (Applied Biosystems) on an ABI Prism 7,700 Sequence Detection System (Applied Biosystems). The universal 18S rRNA primer/probe set used to normalize all assays was also from Applied Biosystems. Samples were run in triplicate. Each 50-µl PCR reaction contained 20 µl of the diluted relevant cDNA, 100 nM of each primer, 200 nM of probe, 200 µM of each dATP, dCTP, and dGTP, 400 µM of dUTP, 0.5 unit of AmpErase UNG, 0.25 U of AmpliTaq Polymerase, 5.5 mM MgCl2, 1x TaqMan Buffer A. The thermal cycling program consisted of 50°C for 2 min for UNG digestion, 95°C for 10 min for AmpliTaq polymerase activation, and then 40 cycles for denaturing (95°C for 15 s) and annealing and extending (60°C for 1 min). The reactions were quantitated by selecting the amplification cycle when the PCR product of interest was first detected (the threshold cycle [CT]). Data were analyzed with the comparative CT method by using arithmetic formulas to achieve the results for relative quantitation, as recommended by the manufacturer. Validation experiments were performed to demonstrate that the efficiency of target and 18S rRNA amplification was approximately equal.


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  		TABLE 1 Probes and primers for real-time PCR

 
RNase Protection Assay for SREBP
Because SREBP-1a and 1c are so similar, their mRNA relative concentrations were determined by a RNase protection assay (RPA) as described previously (28). Plasmid constructs containing cDNA for rat SREBP-1 and SREBP-2 were obtained from Iichiro Shimomura and Joel Goldstein (University of Texas Southwestern Medical Center, Dallas, TX). Radiolabeled antisense riboprobes were prepared, and the RPA was performed as described (21). The protected fragments for the single SREBP-1 riboprobe were resolved into two bands. The protected fragments for SREBP-2, SREBP-1a, and SREBP-1c corresponded to bands of 520 bp, 257 bp, and 160 bp, respectively.

In Situ Hybridization
In situ hybridization was performed as described previously (29). Tissues were fixed with 4% paraformaldehyde and embedded in paraffin. Radiolabeled sense and anti-sense riboprobes were transcribed from cDNAs that had been cloned into plasmid pGEM 4Z (Promega Biotech, Madison, WI). Most of the probes were as reported (21). In addition, for this study, probes for rat PPAR{gamma} were prepared as described previously (21). Briefly, full-length cDNA was prepared from whole rat lung. PCR primers were based on the reported sequence for rat PPAR{gamma} (accession number AB011365). The forward primer (including an added Bam H I restriction site) was 5'CGGATCCTTTCAAGGGTGCCAGTTTCG-3'. The backward primer (including an added Eco R I site) was 5'-GGAATTCCGATAGAAGGAACACTTTGTCAGCG-3'. The resultant probe corresponded to the 631 bases between nucleotides 848 and 1,479. The vectors were linearized with BamH I and transcribed using SP6 polymerase for antisense riboprobe and were linearized with EcoR I and transcribed using T7 polymerase for sense control riboprobes. Riboprobes were transcribed with [33P]-UTP. Hybridization with radiolabeled sense riboprobes was done as a control.

Statistics
A one-way ANOVA was used to determine if gene expression varied with time. The Dunnet's test was used and data were compared with the level of expression on Day 17. A t test was used to compare threshold cycle numbers of Day 17 to Day 21 fetal lung. Statistical significance was determined as P < 0.05 (JMP software; SAS Institute Inc., Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Surfactant Protein Expression
There have been numerous studies detailing the biochemical and morphologic changes in fetal rat lung toward the end of gestation (2–4). At Fetal Day 17, the epithelial tubules appear undifferentiated and are surrounded by mesenchyme. By Day 19, the alveolar epithelial cells proliferate and accumulate an extensive amount of intracellular glycogen. From Day 19 to Day 21 the glycogen deposits decrease, and phospholipid lamellar inclusions appear. There is subsequent secretion of phospholipid into the alveolar fluid and formation of tubular myelin (2). These morphologic changes indicate the development of the surfactant system. As expected in the current study, there was a marked increase in the mRNA values for the surfactant proteins (SPs) SP-A, SP-B, SP-C, and SP-D at the end of gestation (Figure 1). The expression of SP-A was different from the expression of the other SPs in that at the time of birth the mRNA level was significantly lower than in the adult. Most of the increase in mRNA levels for the SPs occurred after Day 19. The changes in mRNA for the SPs confirm observations previously reported by other investigators and document the maturation of the epithelium in the current experiments (30).



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  		Figure 1. Changes in mRNA levels of the surfactant proteins with gestational age. RNA was isolated and mRNA quantified as described in MATERIALS AND METHODS. All values were normalized to 18S, and the relative expression was compared with Day 17, which is given a value of 1. Hence, the y-axis is fold change normalized to the Day 17 level. One of the four litters for Day 22 was born at the time of the tissue collection. However, the results were the same as those of the three unborn litters, and therefore the results were pooled. The mean ± SE of four independent litters or adults is shown. Values that are significantly different from Day 17 (P < 0.05) are designated with an asterisk. Some data appear without SEM bars simply because SEM were so small that they fell within the dot that designates the mean. The abbreviation F indicates fetal day.

 
Transcription Factor Expression
Toward the end of gestation there was a marked increase in the mRNA values for all the C/EBP isoforms measured (Figure 2). The time course appears similar for each isoform. This parallel increase in all C/EBP isoforms is different from that observed during fat cell differentiation, where C/EBPß and C/EBP{delta} expression precedes C/EBP{alpha} expression (31, 32). In contrast, there was little change in the mRNA levels for SREBP-1 and SREBP-2. In other tissues, SREBP-1c increases concomitant with fatty acid de novo biosynthesis (23). However, the real-time PCR primers and probes that were used in these analyses do not differentiate SREBP-1a from SREBP-1c. Because SREBP-1c is expressed in low levels and an increase in SREBP-1c mRNA might be obscured by SREBP-1a, we performed a RNase protection assay to determine the relative expression of these isoforms. As can be seen in Figure 3, there was relatively little change in SREBP-2 or SREBP-1a from Fetal Day 17 to Fetal Day 21, but there was an increase in SREBP-1c. These data suggest that SREBP-1c may be important but will be difficult to evaluate because of the relatively high levels of expression of SREBP-1a and SREBP-2 in the whole lung. The mRNA level of PPAR{gamma} also increased with lung maturation.



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  		Figure 2. Changes in mRNA levels for selected transcription factors with gestational age. Samples were processed and results expressed as stated in Figure 1. The y-axis is fold change relative to the Day 17 level.

 


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  		Figure 3. Changes in SREBP-1a, SREBP-1c, and SREBP-2 mRNA levels with gestational age. The relative level of expression of SREBP la, 1c, and 2 was determined by a RNase protection assay (RPA). In lanes 1–12, 30 µg RNA was used from whole lung, and in lane 13, 15 µg RNA from freshly isolated adult type II cells was used. The RPA was performed as stated in MATERIALS AND METHODS. Lanes 1–4 contained RNA from four separate litters of Day 19 fetal lungs; lanes 5–8 contained RNA from four litters of Day 21 fetal lungs; lanes 9–12 contained RNA from lungs from four adult animals; and lane 13 contains RNA from freshly isolated type II cells.

 
Fatty Acid Synthesis
As shown in Figure 4, there was a marked increase in FAS. There was little change in ACC mRNA levels, which is similar to previous reports (10, 15). Because ACC is regulated predominantly post-transcriptionally, there may be a significant change in activity without a change in mRNA levels. There was a large increase in SCD-1, but interestingly there was no change in SCD-2. There was also no increase in expression of E-FABP. This fatty acid–binding protein increases in adult type II cells stimulated with KGF (21). There was, however, an increase in fatty acid translocase (FAT), which is also stimulated by KGF in type II cells in vitro (21).



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  		Figure 4. Changes in mRNA levels of enzymes involved in fatty acid synthesis and transport proteins with gestational age. Samples were processed and results expressed as stated in Figure 1. The y-axis is fold change relative to the Day 17 level.

 
Phospholipid Synthesis
Some, but not all, of the enzymes involved in phosphatidylcholine synthesis have been cloned and can be evaluated by real time PCR at this time. Glycerol-3P acyl transferase (GPAT) is the gate keeper for glycerolipid synthesis, and its expression is regulated during lipogenesis in many cell types (17, 33–35). The increased expression of GPAT mirrors the increase in phospholipid in developing rat lung (3) (Figure 5). The rapid increase occurred after Fetal Day 19. Diglyceride acyl transferase (DGAT) is the major enzyme for converting diglyceride to triglyceride. The mRNA for this enzyme changed little during fetal development. There was a slight decrease in expression of CCT. This enzyme is thought to be a key regulatory enzyme in phosphatidylcholine synthesis, but its regulation is also predominantly posttranslational (17, 33). There were slight increases in the mRNA levels for phosphatidylglycerophosphate synthase (PGPS), and phosphatidylinositol synthase (PIS). One enzyme whose mRNA increased markedly is phosphatidate cytidylyltransferase (PCT). This enzyme is important for the synthesis of both phosphatidylglycerol and phosphatidylinositol.



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  		Figure 5. Changes in mRNA levels for selected enzymes of phospholipid synthesis with gestational age. Samples were processed and results expressed as stated in Figure 1. The y-axis is fold change relative to the Day 17 level.

 
The level of expression may be as important as a change in expression. The level of mRNA for individual genes was compared with the level on Day 17 to show the change in expression with fetal development in Figures 1, 2, 4, and 5. However, the absolute levels of expression cannot be evaluated in this format. To indicate the relative levels of abundance, the data were also compared with the level of 18S rRNA. For each gene in each analysis, the threshold cycle number was calculated for the gene of interest and 18S rRNA (Table 2). The cycle number difference (threshold cycle number of gene of interest minus threshold cycle of 18S rRNA) reflects the relative abundance of a specific gene. A large cycle number difference (PPAR{gamma}) indicates a low abundance mRNA and a small cycle number difference indicates a highly expressed gene (SP-C). The important points from these analyses are that SREBP-1 and SREBP-2 are relatively highly expressed even though there is no change during development. Because the regulation of these transcription factors is predominantly at the protein level, these transcription factors may be important even though there was no change in mRNA levels. On the other hand, mRNA levels of PPAR{gamma} increased with gestational age, but the level of expression was very low.


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  		TABLE 2 Real-time PCR threshold cycle number difference

 
In Situ Hybridization
The real-time PCR analyses provide data on the changes in the level of expression with time but do not provide information on the localization of gene expression within the lung. In situ hybridization provides information on both the relative abundance of a specific mRNA and its location. Expression can be readily visualized in autoradiograms of whole lung sections after hybridization with 33P-riboprobes (Figure 6). As can be seen from the tissue sections, lung has a relatively high level of expression of FAS, but there is also expression in subcutaneous fat, especially the dorsal fat pad, which is designated by the arrows (Figure 6). Expression of SP-C is restricted to the lung as expected. SCD-1 and SCD-2 are both expressed in the lung and extrapulmonary tissues. SCD-1 is highly expressed in the dorsal fat pad. In the fetal lung, it is difficult to assign expression to individual cell types along the alveolar wall by in situ hybridization (Figure 7). However, expression can be compared with that of SP-C, which is restricted to type II cells and not expressed in pulmonary vessels and the bronchial epithelium. In the lung, SP-C was expressed only in some cells along the alveolar wall and identified the location and relative abundance of type II cells. The pattern of expression of SCD-1 is similar to that of SP-C. Importantly, SCD-1 is also expressed in a similar pattern to SP-C in the adult rat and murine lung (21; R.J. Mason and L.D. Nielsen, unpublished observations). However, SCD-2 was expressed in many additional cell types, including those of pulmonary arteries and bronchi. FAS was heavily expressed in some of the cells along the alveolar wall, presumably type II cells, but also in the bronchial epithelium. C/EBP {alpha}, ß, and {delta} were expressed widely. C/EBPß appeared to be expressed more intensely in the bronchial epithelium. SREBP-2 was also expressed in most cell types. Unfortunately, the expression of SREBP-1 was below the level of detection. PPAR{gamma} was expressed in only a few scattered cells. By in situ hybridization both FAS and SCD-1 increased in alveolar cells from Day 19 to Day 21 (data not shown).



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  		Figure 6. Autoradiograms of tissue sections hybridized with 33P-riboprobes. After the tissue sections were hybridized with 33P-riboprobes, the sections were overlaid with X-ray film to record exposure. All of the sections are Day 21 fetal lungs that are transected across the thorax. As can be with the SCD-2 probe, the section contains skin, thorax, spinal column, heart, and lungs. The arrows indicate the location of the dorsal fat pads. The dorsal fat pads have high levels of expression of FAS and SCD-1.

 


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  		Figure 7. In situ hybridization of selected mRNA in fetal Day 21 lung. To compare the location and relative expression of selected genes, in situ hybridization was performed as stated in MATERIALS AND METHODS on fetal Day 21 lungs. (A) Hematoxylin and eosin–stained lung. (B) SP-C. (C) SCD-1. (D) SCD-1, dark field. (E) FAS. (F) SCD-2. (G) C/EBP{alpha}. (H) C/EBP{alpha}, dark field. (I) PPAR{gamma}. (J) PPAR{gamma}, dark field. All the micrographs are taken at the same magnification (x200).

 
Because of the low level of expression of SREBP-1 and expression of PPAR{gamma} in only a few cells, we wanted positive controls to demonstrate that the probes were valid and to compare the level of expression to that in the lung. Because the Day 21 fetal lung sample was prepared as a tissue block that included the whole thorax, expression levels of lung, skin, and a dorsal fat pad as well in the heart, developing bone, and spinal chord could be compared. As shown in Figure 8, many of the genes of interest were expressed in the dorsal fat pad and in skin as well as in the lung. In developing adipose tissue, there was intense expression of C/EBP{alpha}, PPAR{gamma}, SREBP-1, FAS, and SCD-1. Expression of SREBP-2, SCD-2, and C/EBPß were more widely expressed. There was a marked difference in SCD-1 and SCD-2 expression in skin, developing hair follicles, and fat. SCD-1 expression appeared restricted to adipocytes. The level of expression of FAS and SCD-1 was similar in developing adipocytes and type II cells. PPAR{gamma} was readily detected in developing adipocytes but not type II cells.



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  		Figure 8. In situ hybridization of selected mRNA in Day 21 fetal adipose tissue and skin. In situ hybridization was done as described in MATERIALS AND METHODS. (A) Hematoxylin and eosin. (B) FAS. (C) SCD-1. (D) SCD-2. (E) C/EBP{alpha}. (F) PPAR{gamma}, dark field. (G) SREBP-1, dark field. (H) SREBP-2, dark field. All the micrographs are taken at the same magnification (x100).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
One of the purposes of this study was to determine if the same transcription factors and lipogenic enzymes that were activated in adult alveolar type II cells by KGF in vitro also increase during the lipogenic response at the end of fetal development in vivo. The assumption is that the transcription factors important for endogenous surfactant regulation should be stimulated in both situations. KGF greatly stimulates lipogenesis in adult rat type II cells in vitro, and this is accompanied by increased expression of C/EBP{alpha}, C/EBP{delta}, and SREBP-1c, but not C/EBPß, SREBP-1a, or SREBP-2 (21). In vitro, there is also a marked increase in the mRNA levels of FAS, SCD-1, and SCD-2, but not ACC (21). In type II cells, there is also an increase in mRNA for GPAT, PCT, and PGPS, but little change in other enzymes in phospholipid synthesis.

During the increased lipogenesis in the late term fetal lung, there were many similarities to the results with adult type II cells in vitro. The similarities include an increase in C/EBP{alpha}, C/EBP{delta}, SREBP-1c, FAS, SCD-1, GPAT, and PCT mRNA levels. The increase in C/EBP{alpha} mRNA levels in the fetal lung has been observed previously and has been associated with an increase in SP-A expression (36). Similarly, an increase in C/EBP{delta} has been reported in the developing lung, and this has been associated with increased SP-A and SP-D expression (37). The increase in SREBP-1c could only be shown by an RPA assay, because there is a significant amount of SREBP-1a in the lung and this does not change with differentiation. The level of SREBP-1c expression was below the level of detection in our in situ hybridization studies, so the location of expression could not be defined. Studies on protein expression of SREBP-1 were not done, because immunocytochemistry is difficult for SREBP isoforms and immunoblotting of whole lung extracts would be uninterpretable because of cellular heterogeneity. From our studies, we conclude that C/EBP{alpha}, C/EBP{delta}, and SREBP-1c are likely to be important in lipogenesis in both the adult and fetal type II cells.

The studies in the developing lung also had some important differences from adult type II cells in vitro. One of the major differences was in PPAR{gamma}. The lung expresses PPAR{gamma}1 but not PPAR{gamma}2 (38). In adult type II cells, we were unable to detect any increase in PPAR{gamma} with differentiation (21). The mRNA levels in type II cells were very low, the protein level was below the level of detection by immunoblotting, and there was no increase in acetate incorporation with the PPAR{gamma} agonist 15-deoxy-{Delta}12,14 prostaglandin J2. However, in the fetal lung, there was a significant increase in PPAR{gamma} 1 mRNA level in whole fetal lung during maturation as measured by real-time PCR. However, the absolute level of expression was low. By in situ hybridization, PPAR{gamma} was highly expressed in developing adipose tissue but only in a few lung cells. We think that the cells that express PPAR{gamma} in the fetal lung are macrophages. In the adult lung, PPAR{gamma} is expressed in macrophages but not alveolar type II cells (21, and data not shown). It should be noted that the differences between the previous in vitro study and the current in vivo study were not due to method of measurement, because the same techniques, primers, and probes were used for the real-time PCR and the same probes for in situ hybridization. However, the data for the fetal lung are difficult to interpret in terms of lipid synthesis because of the cellular heterogeneity. All cells synthesize phospholipids, and whole lung mRNA was used in these studies. Proliferating cells, as in the fetal lung, will also increase phospholipid synthesis as they form new membranes. Nevertheless, the data suggest that the C/EBP isoforms and SREBP-1c are important in lipogenesis in the fetal lung. This concept is supported by observations in C/EBP{alpha} gene-targeted mice. C/EBP{alpha} knockout mice die in the newborn period, although their death has been attributed to hypoglycemia (39, 40). However, a more profound respiratory insufficiency may be present. The SREBP-1c knockout is normal, but there may be compensation by overexpression of SREBP-1a (41). Many of the total SREBP-1 null mice die in the neonatal period (42). SREBP-2 may also compensate for the loss of SREBP-1. The actual physiologic role of C/EBP{alpha} and SREBP-1c in regulation of fatty acid synthesis in vivo will require additional studies.

SCD-1 appears to be a critical enzyme in the pulmonary lipogenic response in this as well as in studies with adult rat type II cells. SCD-1 and SCD-2 are known to be expressed in lung, but their importance to the surfactant system are not defined (43, 44). SCD-1 showed a marked increase in expression at the end of gestation, whereas there was no change in SCD-2 mRNA level. SCD-1 was expressed in cells along the alveolar wall in a pattern that was similar to SP-C expression (type II cells), whereas SCD-2 was more widely expressed. SCD-1 is thought to be primarily responsible for the conversion of palmitate (16:0) to palmitoleate (16:1) (45). The phosphatidylcholine in lung surfactant contains a relatively high amount of 16:1 compared with other sources of phosphatidylcholine (46), which suggests that SCD-1 is important in surfactant phospholipid synthesis. One of the major pathways for dipalmitoylphosphatidylcholine synthesis is by deacylation-reacylation for remodeling existing unsaturated phosphatidylcholine (17). The predominant form of unsaturated phosphatidylcholine for remodeling is 16:0, 18:1 phosphatidylcholine (47). SCD-1 is quite low in the undifferentiated type II cells in vitro and increases markedly in response to KGF (21). Similarly, there is a large increase in the fetal lung at the time of surfactant production. By in situ hybridization, SCD-1 is highly expressed in type II cells in the normal adult lung and the expression is largely restricted to this cell type (21). SCD-1 may, therefore, be very useful for defining the molecular regulation of differentiation in type II cells. However, SCD-1 is not critical for surfactant production in that SCD-1 and SCD-2 gene-targeted mice appear normal and do not have an increased perinatal mortality or morbidity. However, it is also possible that SCD-1 and SCD-2 could compensate for the loss of the other. Rodents have two SCD genes, whereas humans have only one (45).

In summary, C/EBP{alpha}, C/EBP{delta}, and SREBP-1c appear to be important in the lipogenic response in the fetal lung and in adult type II cells in vitro. The primary regulation at the mRNA level are in enzymes of fatty acid synthesis and in glycero1–3-P acyltransferase and PCT synthase. SCD-1 is markedly stimulated during type II cell differentiation in vivo and in vitro. In the whole fetal thorax, SCD-1 expression appears to be restricted to the two lipogenic cells, alveolar type II cells and adipocytes.


   Acknowledgments
 
The authors thank Dr. Frank McCormack for suggesting these studies and Dr. John Shannon for his advice on processing fetal lungs for in situ hybridization. Dr. Lening Zhang performed the statistical analyses. These studies were funded by National Institutes of Health grant HL-29891.

Received in original form June 17, 2003

Received in final form July 28, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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