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Originally published In Press as doi:10.1074/jbc.M407670200 on October 21, 2004

J. Biol. Chem., Vol. 279, Issue 53, 55946-55957, December 31, 2004
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Increased Phosphatidylcholine Production but Disrupted Glycogen Metabolism in Fetal Type II Cells of Mice That Overexpress CTP:Phosphocholine Cytidylyltransferase*

Ross Ridsdale, Irene Tseu, Matthias Roth-Kleiner, Jinxia Wang, and Martin Post{ddagger}

From the Canadian Institutes of Health Research Group in Lung Development, Hospital for Sick Children Research Institute and Institute of Medical Sciences, University of Toronto, Toronto, M5G 1X8 Canada

Received for publication, July 8, 2004 , and in revised form, October 1, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
CTP:phosphocholine cytidylyltransferase (CCT) is a rate-determining enzyme in the de novo synthesis of phosphatidylcholine (PtdCho). Alveolar type II cells synthesize large quantities of disaturated PtdCho, the surface-active agent of pulmonary surfactant, particularly at late gestation when the lung prepares itself for postnatal air breathing. To clarify the role of CCT{alpha} in lung surfactant maturation, we overexpressed CCT{alpha}1-367 using the surfactant protein-C promoter. Lungs of transgenic mice were analyzed at day 18 of gestation (term = 19 days). Overexpression of CCT{alpha}1-367 increased the synthesis and content of PtdCho in fetal type II cells isolated from the transgenic mice. Also, PtdCho content of fetal lung fluid was increased. No changes in surfactant protein content were detected. Interestingly, fetal type II cells of transgenic mice contained more glycogen than control cells. Incorporation studies with [U-14C]glucose demonstrated that overexpression of CCT{alpha}1-367 in fetal type II cells increased glycogen synthesis without affecting glycogen breakdown. To determine which domain contributes to this glycogen phenotype, two additional transgenes were created overexpressing either CCT{alpha}1-239 or CCT{alpha}239-367. Glycogen synthesis and content were increased in fetal type II cells expressing CCT{alpha}239-367 but not CCT{alpha}1-239. We conclude that overexpression of CCT{alpha} increases surfactant PtdCho synthesis without affecting surfactant protein levels but that it disrupts glycogen metabolism in differentiating type II cells via its regulatory domain.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In dividing cells, large quantities of phosphatidylcholine (PtdCho)1 are required for membrane synthesis. The lung also requires a steady synthesis of PtdCho for pulmonary surfactant, a lipoprotein that is synthesized and secreted by the alveolar type II epithelial cell into the thin liquid layer that lines the epithelium (1). One of the functions of pulmonary surfactant is to reduce surface tension at the air-liquid interface of the alveoli during expiration. This surface-active function requires sufficient amounts of dipalmitoyl-PtdCho (1). Deficiencies of surfactant have been associated with a variety of lung diseases, including respiratory distress syndrome in premature neonates, which is the most common respiratory disorder of premature infants. The production of surfactant is set into gear during the latter part of gestation (2). How maturing type II cells are able to increase and direct their PtdCho production toward pulmonary surfactant at late gestation without compromising PtdCho demand for membrane biogenesis and homeostasis remains unknown. The major pathway for de novo synthesis of PtdCho in most mammalian cells is the CDP-choline pathway. Output from this pathway is determined by the activity of the rate-limiting enzyme CTP:phosphocholine cytidylyltransferase (CCT), which catalyzes the formation of CDP-choline from phosphocholine (3). The mammalian genome contains two CCT genes that encode four isoforms (4-6). The {alpha} isoform (CCT{alpha}) is ubiquitously expressed, whereas the three {beta} isoforms (CCT{beta}1, -2, and -3) are more restricted in distribution (4-6). Most tissues that express a CCT{beta} also express CCT{alpha}. Although the particular role of CCT{beta} is not understood, the overlapping expression suggests that CCT{beta} acts to augment PtdCho production in certain cell types. CCT{alpha} is the predominant isoform in the fetal and adult lung, and its expression and activity increases during the latter part of gestation (7-9). Structurally, CCT{alpha} can be divided into four domains (Fig. 1a). The N terminus contains a well characterized nuclear localization signal (10) and may play a role in dimerization (11). The highly conserved central domain is the catalytic domain (12, 13). This region shares high identity between isoforms and widely divergent species (12, 14). The catalytic domain is flanked on the C terminus by membrane (M) binding and phosphorylation (P) domains. The M region is an extended amphipathic {alpha}-helix that promotes reversible interaction with membranes (15). Membrane association of CCT{alpha} has been shown to increase catalysis (13, 14, 16). The P domain of the C terminus contains 16 serine and 2 tyrosine residues that are subject to multiple phosphorylations (17, 18). Increased phosphorylation is associated with decreased membrane binding and, therefore, decreased activity (17, 19, 20). Little is known about which kinases and phosphatases target CCT{alpha} and what role they play in modulating CCT{alpha} activity. The unusual requirements of the developing lung for PtdCho synthesis and, therefore, CCT activity suggest a unique mechanism of regulation of CCT{alpha} in differentiating type II cells.



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  		FIG. 1.
Confirmation of CCT{alpha}1-367 overexpression in mice. a, schematic diagram of rat CCT{alpha} protein sequence and three constructs that were generated based on the rat sequence. NLS, nuclear localization signal; MBD, membrane binding. b, upper panel, PCR genotyping for SPC-CCT{alpha}1-367 using genomic DNA from mouse tails. The negative control sample was wild type C57BL/6 genomic DNA. The positive control was a C57BL/6 genomic sample spiked with 20 pM SPC-CCT{alpha}1-367 plasmid. Middle panel, immunoblot of E18 murine lungs using the anti-FLAG antibody. Lower panel, immunofluorescence image of E18 lung with anti-FLAG antibody and a corresponding differential interference contrast (DIC) image from the same region. Immunolabeling is restricted to alveolar type II cells. kb, kilobases.

 
In the present study we hypothesized that an increased CCT{alpha} activity in fetal type II cells would accelerate the maturation of the surfactant system in the developing lung. We overexpressed full-length and truncated CCT{alpha} domains in maturing type II cells using the surfactant protein (SP)-C promoter. There was an enhanced surfactant PtdCho production without a change in surfactant protein levels. However, the most striking feature of these mice was that CCT{alpha} overexpression led to increased glycogen content in the maturing type II cells. This contrasts the normal decline in glycogen content with increased surfactant synthesis at late gestation (21, 22). The increased glycogen content was dependent on the presence of the regulatory (M and P) domain of CCT{alpha} and occurred due to an increase in glycogen synthesis. No change in surfactant proteins was noted. Although no overall acceleration in the maturation of the surfactant system was noted, the results corroborate recent findings (29) linking glycogen and surfactant PtdCho metabolism in maturing type II cells via CCT{alpha}.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—C57BL/6 and SJL mice were obtained from Charles River (St. Constant, Quebec, Canada). Anti-SPA and SPB antibodies were obtained from Chemicon (Temecula, CA). Anti-pro-N-SPC antiserum was generously provided by Dr. M. Beers (University of Pennsylvania, Philadelphia, PA). Epon was purchased from Marivac (Montreal, Quebec, Canada). All culture medium was obtained from Invitrogen. Osmium tetroxide and anti-FLAG M2 antibodies were obtained from Sigma). Deuterated choline and phosphocholine were obtained from CDN Isotopes Inc. (Pointe-Claire, Quebec, Canada), whereas deuterated dipalmitoyl-PtdCho (deuterated in choline head group) was obtained from Avanti%20Polar%20Lipids">Avanti Polar Lipids (Alabaster, AL). [Methyl-14C]choline and [U-14C]glucose were purchased from Amersham Biosciences.

Transgene Construction—Rat CCT{alpha} (9) was used as the basis to generate three constructs, each augmented with a FLAG sequence (DYKD-DDDK) at the C terminus. The first construct encompassed the full translated sequence for CCT{alpha} (CCT{alpha}1-367), the second truncated construct contained the N terminus and the catalytic domain (CCT{alpha}1-239), and the third construct was composed of the membrane (M) and phosphorylation (P) domains (CCT{alpha}203-367) (Fig. 1a). The resultant 1.2-, 0.8-, and 0.5-kilobase FLAG-tagged CCT{alpha} cDNAs were subcloned 3' of the 3.7-kilobase human SPC promoter (23) and 5' of the SV40 small T intron and polyadenylation sequences. The SPC-CCT{alpha}-FLAG expression cassettes were excised with NdeI and NotI, purified using Glass Milk (Gene Clean Kit Bio101, BioCan, Mississauga, Ontario, Canada) and Elutip-D columns (Schleier and Schuell, NY, NY, USA), and ethanol-precipitated.

Production of Transgenic Mice—Transgenic embryos were generated according to Hogan et al. (24). DNA injections into the pronuclei of (C57BL/6 x SJL) F2 embryos were carried out at a concentration of 3 ng/µl. The genotype was established by PCR analysis of genomic DNA extracted from the embryonic tail (Fig. 1b) and confirmed by Southern blot analysis. The primers used were 5'-TCACCTCTGTCCCCTCTCCCTACG-3' (SPC primer for 5') and either 5'-TGCCTGCTTCCTTGATGTGCTTAT-3' (CCT{alpha}1-367 primer for 3'), 5'-GCCGCCGTCCCCTTCTCCAT-3' (CCT{alpha}1-239 primer for 3'), or 5'-TGAACAGACTGTGAGTGAG-3' (CCT{alpha}203-367 primer for 3'). The annealing temperature was 60 °C for CCT{alpha}1-367, 58 °C for CCT{alpha}203-367, and 53 °C for CCT{alpha}1-239. A total of 35 cycles were used for amplification. Mice were bred to homozygosity. To allow for comparison of expression between transgenes carrying different constructs, exogenous CCT{alpha} protein expression was determined by immunoblotting using anti-FLAG antibodies. Only mice showing comparable expression levels of protein in the lung were used in the analysis.

Choline and Glucose Incorporation—Timed pregnant litters were obtained by breeding either negative male and female C57BL/6 mice or homozygous transgenic male and negative C57BL/6 female mice. Detection of coitus was determined by the appearance of a vaginal plug that was designated day 0 of gestation (E0), and fetuses were harvested at E18 of gestation (term = day 19). Lungs were dissected from the fetuses, and distal epithelial type II cells were isolated (25). Purity of the epithelial cell cultures as determined by proSPC staining was >70%. After a 48-h culture, cells were rinsed with serum-free minimal essential medium (MEM) and incubated with choline-free MEM media containing 1 µCi/ml [methyl-3H]choline. After 4 h of incubation, the medium was removed, and the cells were washed with Hanks' balanced medium, trypsinized, and counted, and cellular lipids were extracted according to the method of Bligh and Dyer (26). Lipid samples were dried under nitrogen, and radioactivity was quantified using a scintillation counter. To investigate the synthesis of glycogen, cells were incubated in glucose-free MEM containing 1 µCi/ml [U-14C]glucose. After a 2-24-h incubation period, medium was removed, and cells were washed with Hanks' balanced medium, trypsinized, and counted. To determine glycogen degradation, cells were incubated for 2 h with 5 µCi/ml [U-14C]glucose in glucose-free MEM. After this pulse period, cells were extensively washed with normal MEM and then incubated for 0-48 h in normal MEM medium. At timed intervals cellular reactions were stopped by washing and trypsinizing the cells. Glycogen was isolated as previously described (27), and radioactivity was quantified using a scintillation counter. Glycogen synthase activity in whole lung homogenates or isolated fetal type II cells was assayed in the forward direction by measuring the rate of incorporation of UDP-D-[U-14C]glucose into glycogen (28).

Electron Microscopy—Lung tissue removed from fetuses was rinsed in 1 unit/ml heparin in PBS to remove blood and minced in ~1-mm pieces. For routine electron microscopy the tissue was fixed for 1 h in 4% (w/v) paraformaldehyde and 1% (w/v) glutaraldehyde in PBS. Tissues were then rinsed 3 times in PBS and exposed to 1% (w/v) osmium tetroxide for 1 h followed by another three rinses with PBS. The samples were then dehydrated through an ascending alcohol series ending in propylene oxide. Propylene oxide was then exchanged with an increasing concentration of Epon (Marivac, St. Laurent, Quebec, Canada) until the samples were fully infiltrated with 100% Epon. Samples were placed in molds, and the Epon was polymerized at 70 °C overnight. For immunogold electron microscopy the tissue was processed as described previously (29). Ultrathin sections of the resulting blocks were cut using a diamond knife on a Reichert Ultracut microtome to gold thickness and placed onto 400-mesh copper grids or nickel-plated grids for immunogold. Immunogold labeling was performed as previously (29) using a 1:200 diluted anti-FLAG antibody (Sigma) followed by a 1:300 diluted 10-nm gold-conjugated goat anti-mouse IgG (Nanoprobes, Yaphank, NY). All samples were stained for 10 min in 3% (w/v) uranyl acetate in double-distilled water and 5 min in 1% (w/v) lead citrate followed by double wash distilled water to remove the excess stain. Samples were examined on a Philips 430 electron microscope.

Glycogen Content—Glycogen content was determined according to Seifter et al. (30). Briefly, fetal lungs dissected from genotyped fetuses were individually boiled in 100 µl of 30% KOH for 30 min. The resulting slurry was diluted with 1.5 ml of distilled water. A 0.4-µl aliquot of each sample was added to 800 µl of 0.2% (w/v) anthrone reagent in 95% sulfuric acid. Samples were boiled for 10 min and cooled, and absorbance was read at 620 nm using a Beckman UV spectrophotometer.

Immunoblot Analysis—Three CCT{alpha} positive and three negative E18 lungs were rinsed with PBS and lysed in homogenization buffer using a Dounce homogenizer. Homogenates were diluted in Laemmli loading buffer to a final concentration of 25 µg/µl protein and then boiled for 5 min. Samples (60 µg) were subjected to SDS-PAGE on a 10% (w/v) polyacrylamide gel and then electrophoretically transferred to a nitrocellulose membrane. Nonspecific binding was blocked by incubating the nitrocellulose membrane with 3% (w/v) dry skim milk in Tris-buffered saline at 4 °C for 60 min, and the membrane was then treated with the specified primary antibody (see "Results"). After overnight incubation at 4 °C, the nitrocellulose membrane was washed 3 times with Tris-buffered saline plus % Tween 20) followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:20,000) for 1 h at room temperature. The membranes were then washed thoroughly with cold Tris-buffered saline plus Tween 20 (5 x 5 min), and bands were visualized using an enhanced chemiluminescence detection kit (Amersham Biosciences).

Cytidylyltransferase Assay—Lungs were collected and homogenized in homogenization buffer of 145 mM NaCl, 50 mM Tris-HCl (pH 7.4), 50 mM NaF, and 2.5 mM EDTA. Postmitochondrial supernatant and membrane-rich and cytosolic fractions were obtained as previously described (31, 32).

Measurement of Choline-containing Metabolites—The aqueous phase remaining after lipid extraction from fetal lung tissue was used to determine the choline metabolites (33). Briefly, a 200-µl aliquot of the aqueous layer was spiked with 5 nM deuterated choline and phosphocholine and subjected to liquid chromatography on a Altech Absorbo-sphere silica column fitted with a Altech Solvent Miser Silica guard column (Deerfield, IL), and peaks were analyzed by an API4000 triple-quadrupole mass spectrometer (MDS SCIEX, Concord, On, Canada) using multiple reaction monitoring and quantified by Analyst 1.2 software (MDS SCIEX).

Fetal Lung Liquid Lavage—E18 pregnant mice were euthanized with diethyl ether, and fetuses were extracted by caesarian section. With the aid of a dissecting microscope, a thoracotomy was performed to expose the lungs, and a 30-gauge needle (with a blunted tip) was inserted through a tracheostomy. The fluid-filled lungs were lavaged with 50 µl of PBS containing 0.05 mg/ml 70-kDa dextran-FITC (PBS/dextran-FITC) (Molecular Probes, Eugene, OR). The recovered lung liquid was diluted with PBS/dextran-FITC to a final volume of 200 µl. A standard curve was generated using PBS/dextran-FITC and PBS alone and compared with lung liquid samples to correlate loss of FITC signal with a volume of diluted PBS/dextran-FITC. Samples were measured fluorometrically using a Molecular Devices SpectraMax Gemini electron microscope (Sunnyvale, CA). This provided an accurate measurement of the volume of fetal lung liquid obtained by the lavage method.

Laser Capture Microscopy (LCM)—Optimal cutting temperature compound frozen lung sections from control and transgenic mice were fixed with 75% (v/v) ethanol, rehydrated, stained with rabbit pro-SPC antibody diluted in normal goat serum followed by FITC-anti rabbit IgG, and then dehydrated. Alveolar type II cells identified by pro-SPC immunofluorescence were dissected using a PixCell II System (Arcturus Engineering, Mountain View, CA), and the RNA was extracted. For mass spectral analysis of PtdCho, sections were fixed with 3% (w/v) paraformaldehyde in PBS for 5 min. These sections were then rinsed in distilled water for 5 min. Excess water was removed, and the tissue was rapidly frozen on dry ice. Tissues were thawed in distilled water and stained with rabbit pro-SPC antibody diluted in 5% (w/v) bovine serum albumin followed by FITC-anti rabbit IgG. The tissue was washed, rapidly frozen on dry ice, and immediately freeze-dried for 1 h. Alveolar type II cells were captured as described above.

Real-time Reverse Transcription-PCR—Total RNA was extracted from the LCM-captured type II cells using the PicoPure RNA isolation kit (Arcturus Engineering). After DNase I treatment, total RNA was reverse-transcribed using random hexamers (Applied Biosystems, Foster City, CA). The resulting templates (20 ng of cDNA for our target genes and 2 ng for 18 S) were quantified by real-time PCR (ABI Prism 7700, Foster City, CA). Primers and TaqMan probes for CCT{alpha} were purchased from Applied Biosystems as Assays-on-DemandTM (Foster City, CA). A dilution series determined the efficiency of amplification, allowing the relative quantification method to be employed (34). For relative quantitation, PCR signals were compared between groups after normalization using 18 S as an internal reference. -Fold change was calculated according to Livak and Schmittgen (34).

Mass Spectral Analysis of PtdCho—Homogenized tissues, fetal lung liquid material, or microdissected type II cells were spiked with 2.5 nM deuterated dipalmitoyl-PtdCho as an internal standard and then extracted by a two-step Bligh and Dyer (26). For lipid analysis the chloroform layers were removed and dried under nitrogen gas. Samples were then reconstituted in 200 µl of 3:1 chloroform/methanol, and 30 µl was injected by auto-sampler into an API4000 triple-quadrupole mass spectrometer (MDS SCIEX). Individual PtdCho species were detected at M+1 and M+Na in positive mode and quantified from the common phosphocholine daughter ion at 184 m/z (193 m/z for the internal standard).

Statistics—All values are shown as the means ± S.E. Statistical analysis was done by Student's t test or, for comparison of more than two groups, by one-way analysis of variance followed by Duncan's multiple range comparison test, with significance defined as p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
CCT{alpha} Expression in Transgenic Mice Lungs—To establish a role for CCT{alpha} in regulating pulmonary surfactant formation during late fetal development, we expressed FLAG-tagged full-length CCT{alpha}1-367 in lung epithelial type II cells using the SPC enhancer/promoter. Western blot analysis using monoclonal FLAG M2 antibodies demonstrated exogenous CCT{alpha}-FLAG expression in the E18 transgenic lungs, whereas no expression was noted in the lungs of littermate wild type controls (Fig. 1b). As anticipated, CCT{alpha}-FLAG protein expression at E18 was restricted to the distal lung epithelial cells of the transgene (Fig. 1b). Overexpression of full-length CCT{alpha} in distal lung epithelial cells caused no embryonic or postnatal lethality. The transgenic mice were in no obvious distress. No differences were observed in body or lung weight between wild type and transgenic mice (results not shown).

CCT Activity in CCT{alpha}1-367 Transgenic Lungs—E18 fetal lung were fractionated by centrifugation, and CCT{alpha} activity was measured. The postmitochondrial (3,000 x g) and membrane-enriched (300,000 x g) pellet of transgenic lungs displayed a significantly greater CCT activity than those of littermate control lungs (Fig. 2a). Examination of the soluble choline metabolites from the fetal lung tissue indicated that there was a significant decrease in phosphocholine content of E18 transgenic lungs compared with littermate controls with no significant changes in the amount of choline and CDP-choline (Fig. 2b). This increased consumption of phosphocholine supports the idea of an increased flux through the reaction catalyzed by CCT{alpha}. The amount of glycerophosphocholine, a breakdown product of PtdCho, was not different between transgenic and control lungs (Fig. 2b).



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  		FIG. 2.
CCT activity and choline metabolites in lungs of SPC-CCT{alpha}1-367 mice. a, CCT activity in lung tissue of E18 CCT{alpha}1-367 transgenic mice and control liter mates. Tissue samples were subfractionated by differential centrifugation, and activity was measured. b, micromolar content of choline metabolites, including glycerophosphocholine, of E18 lung tissue. Open bars are control mice, and closed bars are CCT{alpha}1-367 transgenic mice. n = 4; *, p < 0.05; **, p < 0.01.

 
Choline Incorporation and PtdCho Content in Type II Cells of CCT{alpha}1-367 Transgenic Lungs—Because the SPC promoter restricts CCT{alpha} protein expression to the distal epithelial cells of the lung, we measured radioactive choline incorporation into PtdCho and PtdCho content in E18 epithelial type II cells (Fig. 3). First, we found using laser capture microscopy (Fig. 3a) and real-time PCR (Fig. 3b) that E18 type II cells of transgenic mice had a 6-fold greater CCT mRNA expression than E18 type II cells from control mice. For [methyl-3H]choline incorporation studies, multiple litters had to be pooled to provide sufficient cell numbers. Progeny of CCT{alpha} negative parents produced wild type (control) cell cultures, whereas progeny of homozygous transgenic males and negative females were used to obtain enriched fetal epithelial type II cell cultures in which 100% of the epithelial cells overexpressed CCT{alpha}. In agreement with the aforementioned increased mRNA expression and CCT activity, choline incorporation into total lipids of E18 type II cells isolated from the transgenic mice was significantly (>6-fold) greater than that of E18 control type II cell cultures (Fig. 3c). In previous studies we have found that >90% of the radioactive choline incorporated in total lipids is incorporated into PtdCho (36). To determine whether this increase in PtdCho synthesis led to increased PtdCho content, we quantitatively assessed PtdCho in LCM-dissected E18 type II cells using tandem mass spectrometry. In line with the increased PtdCho synthesis, E18 type II cells of transgenic mice had an ~2-fold greater dipalmitoyl-PtdCho content compared with cells of control mice (Fig. 3d).



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  		FIG. 3.
CCT{alpha} mRNA and PtdCho analysis of type II cells of SPC-CCT{alpha}1-367 mice. a, an example of laser captured E18 type II cells identified with pro-N-SPC antibody. Top panels from right to left, a rhodamine-positive E18 type II cell before capture and after capture. Bottom panels from right to left, tissue remaining on the slide after removal of the LCM cap and captured E18 type II cell on the cap. b, relative abundance of CCT{alpha} transcript in LCM captured type II cells from E18 transgenic and control mice as determined by real-time PCR. c, incorporation of [methyl-3H]choline into PtdCho by type II cells isolated from E18 transgenic and control mouse lungs. d, dipalmitoyl-PtdCho content in LCM captured type II cells from E18 CCT{alpha} overexpressors and negative littermates. Open bars are control mice, and closed bars are CCT{alpha}1-367 transgenic mice. n = 3; **, p < 0.01.

 
PtdCho Content in CCT{alpha}1-367 Transgenic Lungs—To determine whether the increase in CCT{alpha} expression and activity of E18 type II cells also resulted in an increase in surfactant PtdCho content, we isolated fetal lung liquid fluid from E18 mice and with the aid of tandem mass spectrometry quantitatively analyzed the PtdCho molecular species of both fetal lung liquid and residual lung tissue (Fig. 4). Although a moderate increase in dipalmitoyl-PtdCho content was observed in whole fetal lung tissue of mice overexpressing CCT{alpha} (Fig. 4b), no significant difference in total PtdCho content was noted between the CCT{alpha} transgenes and their wild type siblings (Fig. 4a). Conversely, lung liquid fluid of E18 transgenes displayed a significant increase in the amount of all major PtdCho species when compared with wild type (control) siblings (Fig. 4, a and b). However, the PtdCho species composition in the fetal lung fluid of CCT{alpha} transgenes was not different from that of control siblings (Fig. 4c). Fetal lung fluid of control and transgenic mice alike contained larger amounts of palmitoylmyristoyl (16:0/14:0)-PtdCho and palmitoylpalmitoleoyl (16:0/16:1)-PtdCho than expected, based on published adult rat surfactant PtdCho values (37). Fetal lung tissue and lung liquid were also examined for phosphatidylglycerol and phosphatidylinositol content and composition, but no significant differences were observed between the transgenic and control mice (not shown).



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  		FIG. 4.
Phospatidylcholine content of lung liquid and post-lavaged lung tissue of E18 SPC-CCT{alpha}1-367 mice. a, total PtdCho content. b, content of PtdCho species. c, percentage distribution of each PtdCho species. Open bars are control mice, and closed bars are CCT{alpha}1-367 transgenic mice. n = 6; *, p < 0.05; **, p < 0.01.

 
Surfactant Proteins in CCT{alpha} Transgenic lungs—To examine if CCT{alpha} overexpression in lung epithelial type II cells had any impact on SP production, we measured SPA, SPB, and pro-SPC content by immunoblotting (Fig. 5). Because the surfactant proteins are mainly expressed by distal lung epithelial cells (38), surfactant proteins were only assessed in E18 whole lung tissue. No significant difference in protein content was observed for any of the three surfactant proteins between the transgenic mice and littermate controls.



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  		FIG. 5.
Surfactant protein content in fetal lung tissue of SPC-CCT{alpha}1-367 mice. Shown are Western blotting for surfactant proteins A, B, and C in E18 lung tissue and densitometric analysis of blots. Open bars are control mice, and closed bars are CCT{alpha}1-367 transgenic mice. n = 3.

 
Type II Cell Development in CCT{alpha}1-367 Transgenic Lungs—The most striking morphological feature of differentiating type II cells at late fetal gestation is the decrease in glycogen content and a concomitant increase in the number and size of lamellar bodies, the intracellular storage organelles of surfactant (2, 27). To investigate whether overexpression of CCT{alpha}1-367 had any ultrastructural effects on differentiating type II cells, E18 mouse lungs were fixed, processed, and examined by transmission electron microscopy. Contrary to the normal decline in glycogen content at late gestation, CCT{alpha}1-367 overexpression led to increased glycogen content in differentiating type II cells (Fig. 6b). However, secreted surfactant material (lamellar bodies and tubular myelin) was clearly visible in the fluid-filled alveolar spaces of the CCT{alpha}1-367 transgene (Fig. 6b), suggesting that surfactant maturation was not delayed. Biochemical assessment of glycogen content confirmed that E18 CCT{alpha}1-367 transgenic lungs contained significantly more glycogen than E18 wild type control lungs (Fig. 6c). Additionally, labeling studies showed that [U-14C]glucose incorporation into glycogen of E18 epithelial type II cells isolated from SPC-CCT{alpha}1-367 transgenic lungs was significantly greater than that of wild type control cells (Fig. 7a).



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  		FIG. 6.
Glycogen and PtdCho content of SPC-CCT{alpha}1-367, SPC-CCT{alpha}1-239 and SPC-CCT{alpha}203-367 mice. a, left panel, immunoblot of lungs from E18 CCT{alpha}1-239 (28 kDa) and CCT{alpha}203-367 (15 kDa) transgenic mice and littermate controls using the anti-FLAG antibody. n = 3 separate mice. Right panel, CCT{alpha} activity in lung tissue of E18 CCT{alpha}1-367, CCT{alpha}1-239, and CCT{alpha}203-367 transgenic mice and littermate controls. b, representative ultrastructural electron micrographs of E18 lung tissue. Glycogen stores are marked by an asterisk. Scale bar = 200 nm c, total PtdCho content of fetal lung fluid. Open bars are control mice, and closed bars are CCT{alpha} transgenic mice. n = 4; **, p < 0.01.

 



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  		FIG. 7.
Glycogen metabolism of type II cells isolated from SPC-CCT{alpha}1-367 mice, SPC-CCT{alpha}1-239, and SPC-CCT{alpha}203-367 mice. a, incorporation of [U-14C]glucose into glycogen over a 24-h period in isolated type II cells from E18 transgenic and control mice. Open bars are control mice, and closed bars are either SPC-CCT{alpha}1-367, SPC-CCT{alpha}1-239, or SPC-CCT{alpha}203-367 transgenic mice. b, immunogold localization of the exogenously expressed FLAG-tagged CCT{alpha} in a type II cell from an E18 SPC-CCT{alpha}1-367 transgenic mouse lung using a monoclonal FLAG antibody. Circles indicate gold particles. The scale bar equals 100 nm. c, incorporation of [U-14C]glucose into glycogen over 2-4 h in isolated type II cells from E18 CCT{alpha}1-367 (black circles) and control mice (black squares). d, pulse-chase study monitoring loss of 14C-labeled glycogen in isolated type II cells from E18 CCT{alpha}1-367 (black circles) and control mice (black squares). Cells were labeled for 2 h with 5 µCi/ml of [U-14C]glucose, washed, and incubated with fresh medium. At the indicated time intervals, incubations were terminated, and the disappearance of 14C-labeled glycogen was measured. e, glycogen synthase activity in whole lung tissue and isolated type II cells from E18 CCT{alpha}1-367 (black bar) and control C57Bl/6 mice (white bar). n = 4-8; *, p < 0.05.

 
PtdCho and Glycogen Metabolism in CCT{alpha}1-239 and CCT{alpha}203-367 Mutant Lungs—The glycogen accumulation in differentiating type II cells of SPC-CCT{alpha}1-367 transgenes could be a direct result of increased CCT{alpha} activity. Alternatively, it could be the result of an excess of CCT{alpha} protein and, therefore, CCT{alpha} phosphorylation sites, which interfere with the phosphorylation/dephosphorylation regulation of glycogen metabolism. Interestingly, exogenously expressed CCT{alpha} localized predominantly to the glycogen stores in E18 fetal type II cells (Fig. 7b). The glycogen stores of fetal type II cells are also important cellular sites for endogenous CCT{alpha} (29), implicating a direct linkage of CCT{alpha} and glycogen. To test the aforementioned possibilities, two additional transgenes were created overexpressing truncated CCT{alpha} proteins in the differentiating type II cells. One protein (CCT{alpha}1-239) consisted of the N terminus and catalytic domain but lacked the regulatory M and P domains. This truncated CCT{alpha} protein is thought to be constitutively active (39). The second construct (CCT{alpha}203-367) contained the regulatory M and P domains but lacked the catalytic domain. Both constructs included a FLAG epitope, and the expression of the truncated proteins in the E18 transgenic lungs was confirmed by immunoblotting using an anti-FLAG antibody (Fig. 6a). Both CCT{alpha} mutants were analyzed at 18 days of gestation for CCT activity and PtdCho content in whole lung tissue and bronchiolar alveolar fluid. No increase in CCT activity compared with control siblings was noted (Fig. 6a). Also, no significant difference in PtdCho content of fetal lung liquid was observed between CCT{alpha}1-239 and CCT{alpha}203-367 mutant mice and their littermate controls (Fig. 6d). E18 lungs of CCT{alpha}1-239 transgenes had a similar ultrastructural appearance as their control siblings. In contrast, CCT{alpha}203-367 transgenes showed an increased glycogen deposition in E18 type II cells (Fig. 6b), and biochemically, the lungs of these mice showed a modest but significant increase in glycogen content (Fig. 6c). In addition, E18 type II cells isolated from CCT{alpha}203-367 transgenes incorporated significantly greater amounts of [U-14C]glucose into glycogen relative to epithelial type II cells of CCT{alpha}1-239 transgenic lungs or nontransgenic lungs (Fig. 7c).

Glycogen Metabolism in CCT{alpha}1-367 Mutant Lungs—Initially, we assessed the incorporation of [U-14C]glucose into glycogen over a 24-h period (Fig. 7a). Such a long labeling period does not differentiate between glycogen synthesis and degradation. To determine whether CCT{alpha} overexpression increased the rate of glycogen synthesis, we pulse-labeled type II cells isolated from E18 control and SPC-CCT{alpha}1-367 mice for 2-4 h with radioactive glucose. As shown in Fig. 7c, the rate of [U-14C]glucose incorporation into glycogen was significantly increased in the cells overexpressing CCT{alpha}1-367 when compared with wild type control cells. In contrast, no significant differences in glycogen degradation were observed between wild type control and CCT{alpha}-overexpressing cells (Fig. 7d). Glycogen synthase activity was determined in whole lung homogenates and type II cells isolated from E18 control and SPC-CCT{alpha}1-367 mice. Both whole lung homogenate and isolated type II cells of the CCT{alpha}1-367 transgene had significantly greater GS activity relative to littermate controls (Fig. 7e). Together, the data suggest that overexpression of CCT{alpha} affects the glycogenic pathway, thereby increasing the glycogen content of the fetal type II cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Herein, we demonstrate that overexpression of CCT{alpha} in fetal distal epithelial cells results in an increased surfactant PtdCho formation without affecting surfactant protein levels. Most interestingly, CCT{alpha} overexpression led to increased glycogen content in the maturing type II cells, which contrasts with the normal decline in glycogen content and increased surfactant PtdCho synthesis at late gestation. The increased glycogen deposition appeared to be dependent on the presence of the regulatory domain of CCT{alpha}. The increased glycogen content was due to an augmented glycogen synthesis. Earlier studies have suggested a precursor-product relationship between glycogen and surfactant production during development (27, 40, 41), and recently we have shown that endogenous CCT{alpha} localizes within the glycogen pools of maturing type II cells (29). The data herein strengthen the idea that CCT{alpha} provides a link between glycogen and surfactant PtdCho metabolism in differentiating type II cells.

Although transgenic mice overexpressing CCT{alpha} in epithelial type II cells using the SPC promoter have been created, those mice were only investigated for their effect on the adult surfactant system (35). In agreement with our findings, the lungs of the transgenic mice exhibited a 6-7-fold greater expression of CCT{alpha} relative to wild type controls (35). The authors reported that the rate of disaturated PtdCho synthesis was significantly increased in adult type II cells isolated from the transgenic mice but that the disaturated PtdCho content of alveolar lavage and lung tissue did not differ between transgenic and control mice. This suggests that the increase in surfactant PtdCho synthesis may be counterbalanced by an increase in surfactant PtdCho degradation. In contrast to adult lung, we found that the increase in the rate of PtdCho synthesis in fetal type II cells from transgenic mice was accompanied by an increase in (disaturated) PtdCho content in whole lung tissue, dissected type II cells, and lung liquid fluid. Given that macrophages are involved in surfactant clearance (42), it is plausible that the excess PtdCho content generated in the postpartum lungs was consumed by resident macrophages that are not present in the fetal lung. In the present study, we observed that the content of glycerophosphocholine, a PtdCho degradation product, did not significantly differ between transgenic and control mice, suggesting that overexpression of CCT{alpha} in fetal type II cells does not increase PtdCho degradation.

Mass spectral analysis showed a significant increase in PtdCho content in the fetal lung liquid of the transgenic mice; however, no significant change in species distribution was observed. Therefore, although PtdCho synthesis was increased, the mechanisms involved in generating the PtdCho species profile remained unaffected. Independent of CCT{alpha} overexpression, the concentrations of palmitoylmyristoyl-PtdCho and palmitoylpalmitoleoyl-PtdCho were much higher in the fetal mouse lung liquid than the reported values for adult rat lung lavage fluid (10% 16:0/14:0-PtdCho and 30% 16:0/16:1-PtdCho versus 4% 16:0/14:0-PtdCho and 10% 16:0/16:1-PtdCho) (37). A postpartum decrease of both PtdCho species has been reported for rodent and pig surfactants (43). The content of both PtdCho species was also significantly higher in the fetal murine lung liquid than that of newborn rat (43), suggesting that they may play an important role at term when the air/fluid interface is first established.

The increase in PtdCho content in the lung liquid fluid was significantly greater than that of type II cells or lung tissue, suggesting an activation of the apical secretory pathway. How the type II cell senses the excess of PtdCho and stimulates its apical efflux remains to be investigated.

No significant change in content and composition of fetal lung liquid was observed for phosphatidylglycerol or phosphatidylinositol, indicating that other lipid synthetic pathways were unaffected by the increase in surfactant PtdCho production. Interestingly, the large increase in pulmonary surfactant PtdCho content within and outside (fetal lung liquid) type II cells also did not affect the expression levels of pulmonary surfactant proteins A, B, or C. Similar observations have been made in transgenic mice, where surfactant protein levels were altered without affecting surfactant PtdCho levels (44-48). Thus, although both surfactant lipid and protein components are closely regulated during development (49), there appears to be no cross-regulation between the surfactant protein and lipid biosynthetic pathways.

Our findings of high glycogen content occurring concurrent with high PtdCho production were unexpected since previous morphological studies had suggested an opposite correlation, i.e. glycogen depletion with increased surfactant production (21, 22). We suspected that the increase in glycogen content was not the direct result of increases in CDP-choline production and speculated that the excess of phosphorylation targets in the CCT{alpha} transgenes (14-fold increase in CCT{alpha} expression relative to littermate controls) interfered with the coordinated phosphorylation-dephosphorylation control of glycogen metabolism in the fetal lung. To test this possibility we generated two transgenic mice, CCT{alpha}1-239 and CCT{alpha}203-367. Neither transgene showed a change in CCT activity and PtdCho content. Wang and Kent (39) have reported that CCT{alpha} constructs composed of the first 236 amino acids are constitutively active in CHO cells. In contrast, Yang et al. (16) showed that CCT{alpha}1-231 has low activity in vitro and in CHO cells. Our observation of CCT{alpha}1-239 not being active in vivo is consistent with the idea that the M domain is required for its activity (16). Overexpression of CCT{alpha}203-367 was not expected to result in any change in PtdCho content because it lacks the catalytic domain. Like full-length CCT{alpha}1-367, CCT{alpha}203-367 had higher glycogen content in the E18 lung tissue compared with littermate controls, and this glycogen was found in the alveolar type II cells by transmission electron microscopy. There was no significant accumulation of glycogen in the E18 lungs of CCT{alpha}1-239 transgenic mice. Clearly, the amino acids contributing to the glycogen phenotype are those unique to the C terminus of CCT{alpha}. Both the M and P domain are encompassed by these amino acids. Of both domains, the P domain is the most likely candidate to contribute to the phenotype since it contains consensus phosphorylation sites for glycogen synthase kinase-3{beta}, an enzyme implicated in controlling glycogen synthase. The increased rate of incorporation of [U-14C]glucose into glycogen and the unchanged rate of glycogen degradation are consistent with an increase in glycogen synthase activity in type II cells of the transgene. Subsequent enzyme activity measurements revealed that the activity of glycogen synthase was indeed increased in the type II cells of CCT{alpha}1-239 transgenic mice. Although the exact mechanism responsible for glycogen synthase activation remains to be investigated, one possibility is that the P-domain of CCT{alpha} acts as a competitive inhibitor of glycogen synthase phosphorylation by glycogen synthase kinase-3{beta}, thereby keeping glycogen synthase in its active state and stimulating glycogen production. The localization of exogenously expressed CCT{alpha} to the glycogen stores in the fetal type II cells agrees with an interaction of CCT{alpha} with glycogen-metabolizing enzymes. Recent immunogold data have revealed a similar cellular localization of endogenous CCT{alpha} to the glycogen deposits in fetal type II cells (29), supporting the idea of a physiologic linkage between glycogen and surfactant PtdCho via CCT{alpha} during gestation. The current data indicate that this relationship is more than glycogen utilization. It would appear that CCT{alpha} may also play a role in regulating glycogen production in the type II cells.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Division of Lung Biology, Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-6772; Fax: 416-813-5002; E-mail: martin.post{at}sickkids.ca.

1 The abbreviations used are: PtdCho, phosphatidylcholine; CCT{alpha}, CTP:phosphocholine cytidylyltransferase; SPA, -B, and -C, surfactant protein A, B, and C; M, membrane domain; P, phosphorylation; MEM, minimal essential medium; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; LCM, laser capture microscopy. Back



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 ABSTRACT
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 DISCUSSION
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