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Am J Physiol Lung Cell Mol Physiol 293: L1395-L1405, 2007. First published September 21, 2007; doi:10.1152/ajplung.00275.2007
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Sterol response element binding protein and thyroid transcription factor-1 (Nkx2.1) regulate Abca3 gene expression

Valérie Besnard, Yan Xu, and Jeffrey A. Whitsett

Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center and The University of Cincinnati College of Medicine, Cincinnati, Ohio

Submitted 16 July 2007 ; accepted in final form 19 September 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The ATP-binding cassette (ABC) ABCA3 gene encodes a lipid transporter critical for surfactant function at birth. To identify transcription factors that regulate ABCA3 expression in the lung, we identified by bioinformatic and functional analyses two positive regulatory regions, located between bp –2591 and –1102 and bp –1102 and +11, relative to the exon 1 of the Abca3 gene promoter. The distal cassette contains consensus sequences predicting binding to lung transcription factors including FOXA2, CCAAT/enhancer binding protein-{alpha} (C/EBP{alpha}), GATA-6, thyroid transcription factor-1 (TTF-1 or Nkx2.1), and nuclear factor of activated T cells-c3 (NFATc3). The activity of the distal region from bp –2591 to –1102 was assessed in HeLa and mouse lung epithelial MLE-15 cells. FOXA2, C/EBP{alpha}, GATA-6, TTF-1, and NFATc3 increased the activity of the Abca3 luciferase construct in a dose-dependent manner. The distal cassette conferred activation by FOXA2, C/EBP{alpha}, GATA-6, TTF-1, and NFATc3 in a position- and orientation-independent manner, serving as an enhancer-like regulatory element. The proximal Abca3 promoter region contained multiple sterol responsive element (SRE) binding sites. SRE binding protein (SREBP)-1c significantly increased the activity of the Abca3 luciferase construct in a dose-dependent manner, whereas SREBP-1a and SREBP-2 did not influence the Abca3 promoter activity. Chromatin immunoprecipitation (ChIP) analyses demonstrated the binding of SREBP-1c, C/EBP{alpha}, and TTF-1 to their respective regulatory elements. Conditional deletion of SREBP cleavage-activating protein (Scap) in respiratory epithelial cells in the mouse lung in vivo inhibited the expression of SREBPs in concert with Abca3. Abca3 gene expression is mediated by discrete cis-acting cassettes that mediate pulmonary cell- and lipid-sensitive pathways regulating surfactant homeostasis.

lung; development; Nkx2.1

ABCA3 IS A 1,704 AMINO ACID member of the ATP-binding cassette (ABC)-containing family of transport proteins associated with the translocation of various substrates across cellular membranes (22, 43). ABCA3 was detected at high levels in type II epithelial cells of the lung where it was associated with lamellar bodies (28, 52) but was also noted in other organs including liver, stomach, kidney, adrenal, intestine, trachea, and brain (28, 41). Mutations in the human ABCA3 gene are associated with lethal respiratory distress in newborn infants (19, 40). Abnormalities in lipid content and function were observed in surfactant from patients with ABCA3-related pulmonary disease (19). Inhibition of ABCA3 expression in type II cells impaired lamellar body formation in vitro (10). Recently, Ban et al. (1) and Fitzgerald et al. (17) generated Abca3 null (Abca3–/–) mice. Abca3–/– mice die within an hour after birth due to respiratory failure. Ultrastructural analysis of the Abca3–/– lungs revealed an absence of normal surfactant in the alveolar space and loss of mature lamellar bodies, consistent with findings in human infants with mutations in ABCA3 (19, 40). These results demonstrate that ABCA3 is required for both lamellar body formation and pulmonary surfactant function. Whereas Abca3 mRNA dramatically increases in the developing fetal lung (41), relatively little is known about its regulation.

Maturation of pulmonary surfactant system occurs in the last trimester of gestation, in association with an increased expression of surfactant proteins (A, B, C, and D) and surfactant phospholipids (47). Coordination of the synthesis and packaging of surfactant components is essential for the transition to air breathing at birth. Expression of surfactant proteins is dependent on a number of transcription factors that are expressed in respiratory epithelial cells, including thyroid transcription factor-1 (TTF-1 or Nkx2.1; Refs. 4, 14), forkhead orthologs (FOX; Refs. 3, 44), GATA-6 (23, 24), CCAAT/enhancer binding protein-{alpha} (C/EBP{alpha}; Ref. 25), and nuclear factor of activated T cells-c3 (NFATc3; Refs. 12, 13). Transcriptional mechanisms regulating genes controlling surfactant lipid homeostasis in the respiratory epithelium are less well-understood. In other tissues, C/EBP isoforms, liver X receptor (LXR), peroxisome proliferator-activated receptor isoforms, and sterol regulatory element-binding proteins (SREBPs) play central roles in the regulation of lipid synthesis (16, 18, 31, 32). During lipogenesis in fetal rat lung, mRNA levels of C/EBP isoforms and SREBP-1c, but not SREBP-1a or SREBP-2, increase before birth (55). Recent studies suggest that regulation of lipogenic enzymes and transport proteins in the lung are influenced by SREBP-1c, C/EBP{alpha}, and C/EBP{delta} (8, 9). Several members of the ABC family of lipid transport proteins are regulated by SREBPs either directly or indirectly (20, 21, 46, 50, 54). The mammalian genome encodes three SREBP isoforms, designated SREBP-1a, SREBP-1c, and SREBP-2. Newly synthesized SREBP are synthesized as inactive precursors and are inserted into the membranes of the endoplasmic reticulum (ER), where their COOH-terminal regulatory domain binds to the COOH-terminal domain of SREBP cleavage-activating protein (SCAP; Refs. 5, 6). To be an active transcription factor, the NH2-terminal domain of SREBP must be released from the membrane. SREBP processing requires SCAP to transfer the SREBP from the ER to the Golgi apparatus where they are finally cleaved by site 1 protease (S1P) and S2P and released to translocate to the nucleus. The expression of SREBP mRNA was previously detected in alveolar type II cells in the murine lung, supporting its potential role in the regulation of surfactant homeostasis (5, 26, 49).

In the present study, we identified distinct regulatory cassettes in the 5' region of the Abca3 gene that regulate cell-selective and lipid-sensitive responses. TTF-1, FOXA2, GATA-6, NFATc3, and C/EBP{alpha} influence Abca3 gene expression by interacting with a distal region of the Abca3 gene promoter, whereas SREBP-1c, activated by SCAP, interacts with a distinct region located in the proximal region of the promoter. Deletion of Scap in respiratory epithelial cells in vivo inhibited SREBP and ABCA3 mRNAs. These results demonstrate the regulation of Abca3 by distinct regulatory regions of the Abca3 gene that interact with transcription factors that mediate tissue-selective and lipid-sensitive regulation to influence surfactant homeostasis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Plasmids and constructs. The expression vectors used were the following: rat FOXA2 cDNA cloned into the pRc/cytomegalovirus (CMV) expression vector (pRc/CMV-rFOXA2), kindly provided by Dr. R. Costa (Chicago, IL); pCMV5-C/EBP{alpha} (25); constitutively active (CA) CA-NFATc3 in pAC-CMV pLpA-5+ expression vector and pCMV/GATA-6, kind gifts from Dr. J. Molkentin (Cincinnati, OH); and pRc/CMV containing the full-length rat TTF-1 cDNA, provided by Dr. R. DiLauro (Naples, Italy). pCMV-nSREBP-1c plasmid, encoding amino acids 1-436 of human nuclear (n) SREBP-1c, was purchased from the American Type Culture Collection (ATCC, Rockville, MD). nSREBP-1a and nSREBP-2 cDNAs encoding amino acids 1-460 of human nuclear SREBP-1a and amino acids 1-431 of human nuclear SREBP-2 were cloned by PCR amplification of human SREBP-1a and SREBP-2 into pcDNA3 (Invitrogen) using the primers described in Supplemental Table 1, containing BamHI and XhoI sites at the 5' and 3' ends for hSREBP-1a, NheI and XhoI sites at the 5' and 3' ends for hSREBP-2, followed by digestion of amplified inserts and vector with the corresponding enzyme. All PCR amplifications for subcloning SREBPs were conducted with Pfu Turbo DNA Polymerase (Stratagene). Insertion of SREBP-1a and SREBP-2 into pcDNA3 was confirmed by sequencing.

The –2.591/+0.011-kb mouse Abca3 promoter was generated by PCR and cloned into the KpnI/XhoI sites of the pGL3-basic vector (Promega) as previously described (13). Unidirectional deletions of the Abca3 promoter were produced using enzymatic digestion. The –2.000/+0.011-, –1.400/+0.011-, –1.102/+0.011-, –0.882/+0.011-, –0.340/+0.011-, –0.238/+0.11-, and –0.128/+0.011-kb promoters were generated by using EcoRI, SpeI, MscI, AccI, AfeI, ApaLI, and SmaI, respectively. After cleavage of the original Abca3 (–2.592/+0.11)-pGL3 construct followed by fill-in of 5' overhangs to generate blunt ends, constructs were digested by XhoI and then ligated in pGL3-basic (blunt indicated restriction enzyme/XhoI to SmaI/XhoI).

Heterologous Abca3/simian virus 40 (SV40) promoter constructs were created by ligating the –2591- to –1102-bp region obtained by PCR amplification of mouse Abca3 promoter into pGL3 promoter (Promega) using the following primers: mAbca3-XmaI-forward 5'-cca cta gta acg ccc ggg agt gtgc-3', mAbca3-XmaI-reverse 5'-ctt cag tgc ccg ggg cca cgg acag-3', mAbca3-BamHI-forward 5'-gta ccg agc tcg gat cca cta gta acg-3', mAbca3-BamHI-reverse 5'-ctt cag tgc ccg gat cca cgg aca ggg-3' containing XmaI sites at the 5' and 3' ends for 5' (sense and antisense orientation) and BamHI sites at the 5' and 3' ends for 3' (sense and antisense orientation) of the luciferase gene, respectively. Orientation of the Abca3 fragment was confirmed by DNA sequencing. Underlining represents DNA sequence recognized by the restriction enzyme.

Promoter analysis. Original genomic sequences and gene mapping information were downloaded from Ensemble. The region 3-kb upstream of exon 1 was identified and retrieved from human and mouse genome assemblies and used to form a promoter database. The human and mouse promoter regions were identified and compared with the position of exon 1 localized at position 40001–40021 for the human ABCA3 and position 40001–40124 for the mouse Abca3. Human and mouse genes were searched for overrepresented cis-elements using MatInspector (Genomatix) vertebrate matrix library. Human and mouse promoters (–3 kb relative to exon 1) were compared using CisMols Analyzer, an internally developed program that identifies compositionally similar and phylogenetically conserved cis-element modules from a list of coexpressed genes.

Cell culture and transfection assays. MLE-15, an immortalized mouse lung epithelial cell line that maintains some morphological and functional characteristics of type II epithelial cells, were cultured in HITES medium (48). Cervical cancer cells HeLa (ATCC CCl-2) were grown in DMEM supplemented with 50 units of penicillin per milliliter, 50 µg of streptomycin per milliliter, and 10% fetal calf serum. Cells were transfected with the FuGENE 6 transfection reagent (Roche Applied Science) according to the manufacturer's protocol. The reporter constructs were cotransfected with either empty vector (pcDNA3) or the indicated expression vector. Forty-eight hours after transfection, luciferase activity was assessed and normalized for cotransfection efficiency by β-galactosidase activity. All transfections were performed in triplicate. pcDNA3 and pCMV β-galactosidase (Clontech) vectors were used to normalize DNA and transfection efficiency, respectively.

Chromatin immunoprecipitation assays. nSREBP-1a, nSREBP-1c, or nSREBP2 expression vectors were transfected into 80% confluent MLE-15 cells on two 150-mm dishes using FuGENE 6 (Roche Applied Science). After 24 h, cells were treated with 1% formaldehyde for 10 min at 24°C, cross-linking was terminated by 0.125 M glycine, and cells were then washed in cold PBS and centrifuged to collect pellets that were resuspended in 1-ml hypotonic buffer (10 mM HEPES-KOH, pH 7.8; 10 mM KCl; 1.5 mM MgCl2; and protease inhibitor cocktail from Sigma) and incubated for 10 min at 4°C. The lysates were collected by centrifugation, resuspended in 1-ml lysis buffer (50 mM Tris·HCl, pH 8; 10 mM EDTA; 1% SDS; and protease inhibitors from Sigma), and incubated for 10 min at 4°C. Lysates were sonicated using Bioruptor (Diagenode, Philadelphia, PA) to shear DNA. Cell debris was removed by centrifugation. To reduce nonspecific background, soluble chromatin from the supernatant was precleared with protein A beads (Santa Cruz) at 4°C for 1–2 h. After centrifugation, precleared chromatin is mixed at 4°C overnight with no antibody or anti-C/EBP{alpha} (Santa Cruz), anti-TTF-1 monoclonal antibody (Seven Hills Bioreagents, Cincinnati, OH), anti-SREBP-1 (H160), or anti-SREBP-2 (N19) (Santa Cruz). Chromatin was incubated for 4 h with protein A beads (Santa Cruz). After incubation, the beads were washed with dialysis buffer (50 mM Tris·HCl, pH 8; 2 mM EDTA; 0.2% Sarkosyl; and protease inhibitors from Sigma) and wash buffer (100 mM Tris·HCl, pH 9; 500 mM LiCl; 1% Nonidet P-40; 1% deoxycholic acid; and protease inhibitors from Sigma). Beads were resuspended in 120-µl elution buffer (50 mM NaHCO3 and 1% SDS; and protease inhibitors from Sigma). Chromatin-protein complexes were incubated at 65°C overnight to reverse the cross-links in presence of 0.3 M NaCl. The next day, the DNA was phenol-extracted, precipitated with ethanol, and dissolved in 50 µl of 10 mM Tris·HCl (pH 8.0). PCRs were performed using primers spanning the 5'-regulatory region of the Abca3 and Gapdh genes (Supplemental Table 1). The products were analyzed by agarose gel electrophoresis.

Small interfering RNA transfection assay. MLE-15 cells were seeded at 40% density the day before transfection. Transfection of small interfering RNAs (siRNAs) was carried out with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instruction. siRNAs for TTF-1 (ON-TARGETplus SMARTpool L-041979-01), SREBP-1 (ON-TARGETplus SMARTpool L-040814-01), SREBP-2 (ON-TARGETplus SMARTpool L-050073-01), and ON-TARGETplus siControl nontargeting pool D-001810-10-05 were generated by Dharmacon (Chicago, IL). Per well, 80 pmol of siRNA duplex of TTF-1, SREBP-1, SREBP-2, or nontargeting siRNA formulated into liposomes were added. Seventy-two hours after transfection, luciferase activity was assessed and normalized for cotransfection efficiency by β-galactosidase activity.

Animals and transgene genotype. Scapflox/flox mice bearing loxP-flanked neo cassette located 3-kb 5' of Scap exon 1 and a third loxP site located in intron 1 were purchased from The Jackson Laboratory (Bar Harbor, ME). Homologous recombination between loxP sites was accomplished by expression of Cre recombinase using (tetO)7CMV-Cretg/tg mice. The SP-C-rtTA–/tg transgenic mouse line (29, 30) was used for respiratory epithelium-specific expression of rtTA (reverse tetracycline transactivator) to cause recombination of the floxed allele after exposure of the dam to doxycycline (29, 30). Triple transgenic mice, herein termed Scap{Delta}/{Delta} mice, were generated by mating (tetO)7CMV-Cre–/tg/Scapflox/flox to SP-C-rtTA–/tg/Scapflox/flox. Scapflox/flox littermates lacking either rtTA or Cre genes served as controls. Genotypes were identified by PCR with genomic DNA from the tails of fetal mice using forward primer 5'-GCT CTG CGC ATC CTA TCC AAT TCC C-3', reverse primer 5'-CAG CCG GCA AGT AAC AAG GGA TCC G-3' for Scapflox/flox. Genotyping for SP-C-rtTA and (tetO)7CMV-Cre DNA was performed by PCR as described previously (29).

Animal husbandry and doxycycline administration. Animals were maintained in a pathogen-free environment in accordance with protocols approved by the Institutional Animal Care and Use Committee of the Cincinnati Children's Hospital Research Foundation. All animals were housed in humidity- and temperature-controlled rooms on a 12:12-h light-dark cycle. Mice were allowed food and water ad libitum. There was no serological evidence of pulmonary pathogens or bacterial infections in sentinel mice maintained within the colony. Gestation was dated by detection of the vaginal plug [at embryonic day 0.5 (E0.5)] and correlated with weight of each pup at the time of death. Dams bearing control and Scap{Delta}/{Delta} mice were maintained on doxycycline in food (625 mg/kg; Harlan Teklad, Madison, WI) from E6.5 to E12.5.

RNA isolation and analysis. At E18.5, dams were killed by exsanguination, and lungs were dissected for analysis. Fetuses were removed from the uterine sac and weighed, and crown-to-rump lengths were determined to assess developmental age (33). RNA was isolated from mouse lung using TRIzol reagent (Invitrogen; Carlsbad, CA). RNA was treated with DNase at room temperature for 15 min before cDNA synthesis. RNA (4 µg) was reverse-transcribed and then analyzed by quantitative RT-PCR for Scap, Srebp-1a, Srebp-1c, Srebp-2, Abca3, stearoyl-CoA desaturase 1 (Scd1), fatty acid synthase (Fas), and β-actin mRNAs using Smart Cycler (Cepheid, Sunnyvale, CA). Primers 5'-TGA CCA CAA ACA AGG AGA GC-3' and 5'-CAG GAA CAC CAA ACA GCA AG-3' for Scap, 5'-ATC GGC GCG GAA GCT GTC GGG GTA G-3' and 5'-ACT GTC TTG GTT GTT GAT GAG CTG G-3' for Srebp-1c, 5'-GTC GTT CAA AAC CGC TGT GTC CAG-3' and 5'-AAG CCG GGT GGG CGC CGG CGC CAT-3' for Srebp-1a, 5'-CAT CCA GCA GCC TTT GAT ATA CCA G-3' and 5'-AGG ACC GGG ACC TGC TGC ACC TGT G-3' for Srebp-2, 5'-GCA TTG CCC TCA TTG GAG AGC CTG-3' and 5'-TCC GGC CAT CCT CAG TGG TGG G-3' for Abca3, 5'-GTG AGG CGA GCA ACT GAC TA-3' and 5'-GGG CAC TGT CTT CAC CTT CT-3' for Scd1, 5'-TTA CAC CTT GCT CCT TGC TG-3' and 5'-TTG ATG ATT CAG GGA GTG GA-3' for Fas, and 5'-TGG AAT CCT GTG GCA TCC ATG AAC-3' and 5'-TAA AAC GCA GCT CAG TAA CAG TCC G-3' for β-actin were used. Experiment is representative of findings from at least two independent dams, each generating at least two or three Scap{Delta}/{Delta} mice that were compared with control littermates.

Statistical analysis. Either Mann-Whitney U test or Student's t-test were used to determine the levels of difference between groups. Values for all measurements were expressed as means ± SE, and P values for significance were 0.05 and 0.01.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Potential regulatory elements in the Abca3 promoter. To understand the molecular mechanisms underlying transcriptional activation of the Abca3 gene in the lung, potential regulatory elements were sought upstream of the transcriptional start site (Fig. 1). We identified two distinct regulatory regions that were well-conserved in human and mouse genes. The upstream region near the transcriptional start site lacks a TATA box but has promoter-like elements consisting of several CpG islands containing a number of potential regulatory motifs including binding sites for stimulating protein 1 (SP1) and SREBP. In a more distal 5'-upstream region (bp –2592 to –1102), potential binding sites for FOXA2, C/EBP{alpha}, GATA-6, TTF-1, and NFATc3 were clustered. Comparison of the human and mouse genes encoding ABCA3 demonstrated close sequence similarities that included shared sites for potential transcription factor binding (Supplemental Table 2).


Figure 1
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  		Fig. 1. Sequence of the mouse Abca3 gene promoter is shown. The transcription initiation site and translation start site (ATG) are indicated by the bent arrow and bold underline, respectively. Putative regulatory elements are identified for the transcription factors forkhead ortholog FOXA2 (discontinuous underline), GATA (white boxed), thyroid transcription factor-1 (TTF-1; gray boxed), CCAAT/enhancer binding protein (C/EBP; gray circled), nuclear factor of activated T cells (NFAT; white circled), sterol responsive element binding protein (SREBP; black boxed), and stimulating protein 1 (SP1) (underlined). The numbering of nucleotides starts at the transcription initiation site (+1).

 
To define functional elements in the Abca3 promoter, Abca3 gene promoter luciferase (Abca3-Luc promoter) constructs were transiently transfected into MLE-15 cells (Fig. 2). Reporter activity demonstrated that DNA sequences (bp –2591 to –1102) produced high levels of luciferase activity in MLE-15 cells, suggesting the presence of a positive regulatory element in this distal region. Deletion of the DNA fragment in a more proximal region, located bp –1102 to –238 from the transcriptional start site, also inhibited promoter activity, indicating an additional positive regulatory element within this region of the promoter (Fig. 2B).


Figure 2
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  		Fig. 2. A and B: deletion analysis of the activity of the Abca3 promoter. A: serial deletions were created within the Abca3 promoter region from positions –2592 to +11 bp relative to the transcription initiation site. Abca3 promoter deletions were fused to the luciferase reporter gene in the pGL3 vector. B: luciferase activities of Abca3 gene promoter luciferase (Abca3-Luc) deletion constructs were assessed in mouse lung epithelial MLE-15 cells. Results are expressed as the means ± SE of 3 experiments performed in triplicate. *P < 0.05 vs. Abca3(2592)-Luc; **P < 0.01 vs. Abca3(2592)-Luc.

 
Lung-enriched transcription factors regulate Abca3 gene. The distal promoter region of the Abca3 gene (bp –2591 to –1102) contains a cluster of potential DNA binding sites for transcription factors known to influence lung developmental gene expression, including FOXA2, C/EBP{alpha}, GATA-6, TTF-1, and NFATc3. To determine whether these transcription factors activate the Abca3 promoter, the Abca3 promoter construct was cotransfected with the expression vectors for each of these transcription factors in both MLE-15 (Fig. 3) and HeLa cells (data not shown). In response to increasing amounts of each expression vector, Abca3 promoter activity was significantly increased in a dose-dependant manner. These findings are consistent with recent studies using C/EBP{alpha}, TTF-1, and NFATc3 in HeLa cells (13, 25, 41).


Figure 3
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  		Fig. 3. FOXA2, C/EBP{alpha}, GATA-6, TTF-1, and NFATc3 regulate the Abca3 promoter. Effects of FOXA2, C/EBP{alpha}, GATA-6, TTF-1, and NFATc3 on Abca3 promoter activity were assessed after cotransfection of Abca3(2592)-Luc (0.5 µg) with increasing amounts of expression plasmids pRc/CMV-rFOXA2 (A), pCMV/GATA-6 (B), pRc/CMV-rTTF-1 (C), pAC-CMV-CA-NFATc3 (D), and pCMV5-C/EBP{alpha} (E) at 0 (–), 0.5, 1.0, and 2 µg per well into MLE-15 cells. Results are expressed as the means ± SE of 3 separate experiments performed in triplicate. *P < 0.05 vs. pcDNA3 condition; **P < 0.01 vs. pcDNA3 condition.

 
Analysis of the Abca3 promoter indicated putative DNA binding sites for FOXA2, C/EBP{alpha}, GATA-6, TTF-1, and NFATc3 in the nucleotide sequence located from bp –2591 to –1102. To determine if this upstream regulatory region acts as an enhancer, the 1489-bp fragment (bp –2591 to –1102) was cloned in sense and antisense orientations upstream of a minimal SV40 promoter or 3' of the luciferase reporter gene in the pGL3 promoter vector. The SV40/Abca3 promoter constructs and the control vector (empty pGL3 promoter vector) were transiently transfected into MLE-15 and HeLa cells (Fig. 4B). Presence of the 1489-bp fragment (in sense and antisense orientations) at the 5' of the SV40 minimal promoter or the 3' end of the luciferase gene significantly increased luciferase activity (2- to 4-fold) in both MLE-15 and HeLa cells demonstrating no lung specificity. In addition, cotransfection of the SV40/Abca3 promoter construct in both sense and antisense orientations 5' of the SV40 minimal promoter or the 3' end of the luciferase gene with expression vectors encoding FOXA2, C/EBP{alpha}, GATA-6, TTF-1, and NFATc3 significantly increased luciferase activity (Fig. 4C). The –2591 to –1102 bp region contains important cis-acting elements for FOXA2, C/EBP{alpha}, GATA-6, TTF-1, and NFATc3. To determine whether C/EBP{alpha} and TTF-1 bind to this region, chromatin immunoprecipitation (ChIP) assays were performed using the distal 1489-bp Abca3 gene fragment with a series of oligonucleotides mapping this region. ChIP readily detected C/EBP{alpha} and TTF-1 binding on the Abca3 promoter (Fig. 5).


Figure 4
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  		Fig. 4. A–C: enhancer element in the 5'-upstream region of the Abca3 promoter. A: the 1489-bp fragment of the Abca3 promoter region was cloned into the pGL3 promoter vector. Abca3 promoter –2592- to –1102-bp fragment was fused in both sense (a) and antisense (b) orientations to the 5' region of the minimal simian virus 40 (SV40) promoter or in both sense (c) and antisense (d) orientations at the 3' region of the luciferase reporter gene in the pGL3 promoter vector. B: the activity of the 1489-bp Abca3 fragment-Luc constructs were assessed in HeLa and MLE-15 cells. C: activity of the 1489-bp Abca3 fragment-Luc constructs were determined in MLE-15 cells in response of pcDNA3 (black bar) or expression plasmids (white bar) pRc/CMV-rFOXA2, pCMV5-C/EBP{alpha}, pCMV/GATA-6, pRc/CMV-rTTF-1, and pAC-CMV-CA-NFATc3. Results are expressed as the means ± SE of 3 experiments performed in triplicate. *P < 0.05 vs. pcDNA3 condition; **P < 0.01 vs. pcDNA3 condition.

 

Figure 5
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  		Fig. 5. C/EBP{alpha} and TTF-1 bind to the Abca3 promoter in chromatin immunoprecipitation (ChIP) assays. A: schematic representation of the Abca3 gene promoter showing the –2592 to +1 bp region. The locations of FOXA (white), GATA (horizontal hatched), C/EBP (black), TTF-1 (gray), and NFAT (oblique hatched) sites are illustrated. Localization of the ChIP primers (p1–p3) is shown (arrows). B: ChIP assays on 5'-regulatory regions of Abca3 gene were performed on cross-linked chromatin in MLE-15 cells with either no antibody (–) or antibodies (+) against C/EBP{alpha} and TTF-1 as described in MATERIALS AND METHODS. DNA was analyzed by PCR with the appropriate primers (Supplemental Table 1). The proximal promoter of the mouse Gapdh gene served as a control. Input (Inp) DNA (precleared soluble chromatin) was used as a PCR control.

 
SREBPs regulate Abca3 gene expression. Multiple ABC superfamily members are associated with lipid transport (7, 36), and several are regulated by the SREBPs, a family of transcription factors known to influence lipid homeostasis in various organs (35, 46, 54). In the Abca3 promoter, potential SRE sequences were detected in a proximal (bp –1102 to +11) region of the promoter. To determine whether SREBP regulated Abca3, MLE-15 and HeLa cells were cotransfected with the Abca3-Luc promoter construct and an expression vector encoding a nuclear form of SREBP-1a (nSREBP-1a, amino acids 1-460), SREBP-1c (nSREBP-1c, amino acids 1-436), or SREBP-2 (nSREBP2, amino acids 1-431). nSREBP-1c increased the luciferase activity of the Abca3 promoter construct in a dose-dependant manner (Fig. 6B). In contrast, nSREBP-1a and nSREBP-2 inhibited the Abca3 promoter activity (Fig. 6, A–C).


Figure 6
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  		Fig. 6. SREBPs regulate the Abca3 promoter in vitro. Effects of active forms of SREBP-1a, SREBP-1c, and SREBP-2 on Abca3 promoter activity were assessed after cotransfection of Abca3-Luc (0.5 µg) with increasing amounts of expression plasmid pcDNA3-nSREBP-1a (A), pCMV-nSREBP-1c (B), and pcDNA3-nSREBP-2 (C) at 0, 0.5, 1.0, and 2 µg per well into MLE-15 cells. Results are expressed as the means ± SE of 3 experiments performed in triplicate. *P < 0.05 and **P < 0.01 vs. 0 µg per well condition.

 
To demonstrate direct regulation of the Abca3 gene promoter by TTF-1 and SREBPs, luciferase activity was assessed on MLE-15 cells cotransfected with Abca3-Luc promoter and siRNA specific for TTF-1, SREBP-1, and SREBP-2 (Fig. 7). TTF-1 siRNA significantly inhibited Abca3-Luc promoter activity. Likewise, siRNA specific for SREBP-1 and SREBP-2 significantly inhibited Abca3-Luc promoter activity compared with the nontargeting siRNA (Fig. 7).


Figure 7
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  		Fig. 7. Regulation of Abca3 gene expression by TTF-1 and SREBP small interfering RNAs (siRNAs) is depicted. Effects of siRNA for TTF-1, SREBP-1, and SREBP-2 on Abca3 promoter activity were assessed after cotransfection of Abca3-Luc (0.5 µg) with 80 pmol of siRNA for either TTF-1, SREBP-1, or SREBP-2 per well into MLE-15 cells. Each siRNA selectively inhibited the targeted gene (data not shown). As a negative control, cells were transfected with nontargeting siRNAs (80 pmol). In parallel wells, as a positive control, MLE-15 cells were transfected with pGL3-basic or Abca3-Luc (0.5 µg). Seventy-two hours after transfection, luciferase activity was assessed and normalized by β-galactosidase activity. Results are expressed as the means ± SE of 3 experiments performed in triplicate. *P < 0.05 vs. nontargeting siRNA condition; **P < 0.01 vs. nontargeting siRNA condition as assessed by Student's t-test.

 
To identify potential SRE binding sites, deletion constructs of the Abca3 promoter were cotransfected with the expression vectors encoding nSREBP-1a, -1c, and -2. nSREBP-1c activated constructs containing the Abca3 promoter sequence from bp –2591 to –238. Further deletion of the construct from –238 bp abolished the effect of nSREBP-1c. Cotransfection with nSREBP-1a or nSREBP-2 inhibited luciferase activity of the (bp –2591 to –340) Abca3 promoter construct (Fig. 8, A–C). Further deletion from –340 bp suppressed the inhibitory effects of nSREBP-2 but not SREBP-1a. Thus SREBP-1c activates the Abca3 promoter, and a region located between bp –340 to –238 is differentially regulated by the different SREBP forms.


Figure 8
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  		Fig. 8. A–C: localization of SREBP binding sites on the Abca3 promoter. Effects of active forms of SREBP-1a, SREBP-1c, and SREBP-2 on Abca3 promoter deletion construct activity were assessed after cotransfection of Abca3-Luc (0.5 µg) with 1 µg of pcDNA3 (black bar) or expression plasmids (white bar) pcDNA3-nSREBP-1a (A), pCMV-nSREBP-1c (B), and pcDNA3-nSREBP-2 (C) into MLE-15 cells. Results are expressed as the means ± SE of 3 experiments performed in triplicate. *P < 0.05 vs. pcDNA3 condition; **P < 0.01 vs. pcDNA3 condition.

 
SREBP-1c binds the Abca3 promoter in ChIP assay. To determine whether the putative SREs are able to bind the SREBPs, ChIP experiments using DNA primers spanning the Abca3 5'-regulatory region containing consensus SRE sites were performed. MLE-15 cells were transfected with expression vectors for each SREBP to enrich DNA-protein complexes. Binding of SREBP-1c and -1a were observed in the regions (bp –882 to –340 and bp –340 to –238), supporting findings from the reporter studies in Fig. 9. In contrast, no SREBP-2 DNA-protein complex was observed within the bp –1102 to –340 region. SREBP-1a, SREBP-1c, and SREBP-2 failed to bind the Gapdh proximal promoter. The active forms of SREBP-1a or SREBP-2 were cotransfected with Abca3-Luc and increasing amounts of nSREBP-1c into MLE-15 cells. Abca3 promoter activity increased in a dose-dependant manner in response to nSREBP-1c (Fig. 10), indicating that SREBP-1c activates the Abca3 gene independently of the inhibitory effect of SREBP-1a and SREBP-2.


Figure 9
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  		Fig. 9. SREBP-1 isoforms bind to the Abca3 promoter. A: schematic representation of the Abca3 gene promoter showing the –2592 to +1 bp region. The locations of SREBP (white circle) and SP1 (black circle) sites are illustrated. Localization of the ChIP primers is shown (arrows). B: ChIP assays on 5'-regulatory regions of Abca3 gene were performed on cross-linked chromatin in MLE-15 cells with either no antibody (–) or antibodies (+) against the SREBP-1a, SREBP-1c, and SREBP-2 as described in MATERIALS AND METHODS. DNA was analyzed by PCR with the appropriate primers (Supplemental Table 1). The proximal promoter of the mouse Gapdh gene served as a control. Input DNA (precleared soluble chromatin) was used as a PCR control.

 

Figure 10
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  		Fig. 10. A and B: distinct effects of SREBP isoforms on the Abca3 promoter activity. Active forms of SREBP-1a (A) or SREBP-2 (B) were cotransfected with Abca3-Luc (0.5 µg) and increasing amounts pCMV-nSREBP-1c into MLE-15 cells. Results are expressed as the means ± SE of 3 experiments performed in triplicate. *P < 0.05 vs. (–) condition; **P < 0.01 vs. (–) condition.

 
SCAP regulates expression of SREBPs and ABCA3 in vivo. To determine whether Abca3 gene expression is regulated by the SREBP pathway in vivo, triple transgenic mice Scapflox/flox, SP-C-rtTAtg/–, and TetO-Cretg/tg were produced in which Scap was selectively deleted in the respiratory epithelium following administration of doxycycline to the dam. In the presence of doxycycline, rtTA binds to the TetO promoter and activates the expression of Cre recombinase, deleting the promoter and the first exon of the Scap gene (27) to produce Scap{Delta}/{Delta} mice. Genotype analysis demonstrated the transmission of the genes as predicted by Mendelian inheritance (data not shown). Scap gene deletion was assessed by quantitative RT-PCR on E18.5 lung mRNAs demonstrating a marked decrease in Scap mRNA in the lungs of Scap{Delta}/{Delta} mice (Fig. 11). Srebp-1c and Srebp-1a mRNAs were decreased by 70% and 50%, respectively. No statistical differences were observed for Srebp-2 mRNA. Abca3 mRNA levels in the lung were significantly reduced in theScap{Delta}/{Delta} mice (Fig. 11). To confirm that the SCAP pathway was altered, we analyzed the expression of known downstream targets of the SREBP transcription factors. Previous studies demonstrated that SREBPs regulate the Scd1 and Fas genes (2, 8, 9, 38, 42). In the present study, a marked decrease in Scd1 and Fas mRNAs was observed in the lungs of Scap{Delta}/{Delta} mice (Fig. 11).


Figure 11
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  		Fig. 11. SREBP cleavage-activating protein (SCAP) regulates both Srebps and Abca3 gene expression in vivo. Quantitative RT-PCR was performed to estimate Scap, Srebp-1c, Srebp-1a, Srebp-2, Abca3, stearoyl-CoA desaturase 1 (Scd1), and fatty acid synthase (Fas) mRNAs in lungs from triple transgenic Scap{Delta}/{Delta} (white bar) and control littermates (black bar) at embryonic day 18.5 (E18.5) and normalized by β-actin mRNA. Dams were treated with doxycycline from E6.5 to E12.5 to delete the Scap gene from respiratory epithelial cells. Results are expressed as the means ± SE of 5 animals per group. *P < 0.01 vs. control littermates. **P < 0.001 vs. control littermates.

 

   DISCUSSION
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 ABSTRACT
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The present work identifies two discrete cis-regulatory regions that modulate transcription of the mouse Abca3 gene. A distal region of the Abca3 promoter (bp –2591 to –1102) contains elements shared by other genes critical for normal surfactant homeostasis and epithelial cell differentiation in the lung, including FOXA2, C/EBP{alpha}, GATA-6, TTF-1, and NFATc3. A distinct proximal region (bp –1102 to +11) binds SREBPs and mediates SREBP-1c-dependant activation of the promoter. These distinct cis-acting regions are well-conserved in the mouse and human genes.

Developmental regulation of ABCA3. ABCA3, a member of the ABCA subfamily of transport proteins, is predominantly expressed in alveolar epithelial type II cells in the lung where it is localized to the limiting membrane of lamellar bodies (28, 52). Mutations in ABCA3 gene cause lethal respiratory distress in newborn infants due to impaired lamellar body formation and surfactant function. Study of ABCA3 mRNAs demonstrated a dramatic increase of ABCA3 mRNA before birth in a similar pattern to that of surfactant proteins B and C, proteins that are also required for surfactant function (41, 53). Previous studies established that TTF-1 (4, 14), FOXA2 (3, 44), GATA-6 (23, 24), C/EBP{alpha} (25), and NFATc3 (12, 13) are critical for perinatal lung function, regulating surfactant protein, and lipid expression. In the present study, we demonstrate that the Abca3 gene is also regulated by this group of transcription factors, indicating that a common transcriptional network influences various aspects of surfactant homeostasis in the lung. Comparison of the human and mouse ABCA3 genes demonstrated the presence of several potential binding sites for FOXA2, C/EBP{alpha}, GATA-6, TTF-1, and NFATc3 indicating the conservation of this regulatory network in the ABCA3 genes. In transgenic mouse models, changes in FOXA2, C/EBP{alpha}, GATA-6, TTF-1, and NFATc3 expression impaired lung maturation, resulting in respiratory distress at birth (3, 4, 13, 14, 23–25, 44). As seen in the Abca3–/– mice (1, 17), formation of lamellar bodies and tubular myelin was impaired in these models. Abca3 mRNA was decreased in mouse models where FOXA2, C/EBP{alpha}, and NFATc3 were deleted (13, 25, 45). DNA-binding sequences of FOXA2, C/EBP{alpha}, GATA-6, TTF-1, and NFATc3 form a cluster in the distal region of the Abca3 gene promoter, suggesting a lung epithelial-enriched regulatory element within the Abca3 gene. The Abca3 gene is expressed in many other tissues where it may be variably coexpressed with these transcription factors. Although each of these transcription factors can induce Abca3 promoter activity in vitro, it is likely that direct or indirect cooperation among them influences the expression of Abca3 in the lung. The present findings are consistent with recent observations demonstrating that the Abca3 promoter was synergistically activated by NFATc3 and TTF-1 in HeLa cells (13, 41).

SREBP family members regulate lipogenic pathways. Transcription of some genes encoding enzymes or proteins involved in fatty acid and cholesterol metabolism are regulated by the SREBPs (20). Lipid transporters, including ABCA1, ABCA7, ABCD2, ABCG5, and ABCG8, are regulated by SREBPs (20, 21, 46, 50, 54). Promoter analysis of ABCA1 demonstrated a direct interaction with SREBP-2 (35). In the present work, transfection experiments indicated that SREBP-1c, but not SREBP-1a and SREBP-2, enhanced Abca3 promoter activity. SREBP-1c expression in the rat lung increases between fetal days 17 and 21, whereas SREBP-1a and SREBP-2 do not change (55). Increased expression of SREBP-1c coincides with the prenatal increase in ABCA3 mRNA and protein staining that are dramatically induced before birth, the period during which the temporal and spatial pattern of ABCA3, SREBP-1c, surfactant proteins, and lipids are similarly increased before birth. Recent studies support the concept that SREBP-1c regulates fatty acid synthesis in alveolar type II cells in vitro (26). The coincident regulation of SREBP-1c, ABCA3, surfactant proteins, and lipids likely plays an important role in the regulation of surfactant homeostasis that is required for respiration after birth.

SREBP-1 preferentially activates fatty acid metabolism genes, whereas SREBP-2 is associated with the activation of gene-regulating cholesterol biosynthesis. In the present study, ChIP assay identified binding of both SREBP-1a and SREBP-1c on the Abca3 promoter. Because SREBP-1c contains a truncated activation domain compared with SREBP-1a, SREBP-1c is generally considered to be less active. In the present work, both SREBP-1c and SREBP-1a bound the Abca3 promoter, indicating that the distinct differences in Abca3 expression are not mediated by differences in binding affinities of the SREBPs. Although nSREBP-2 inhibited the activity of the Abca3 promoter in transfection assays, siRNA specific for SREBP-2 also inhibited Abca3 promoter activity. The mechanisms underlying this finding are unclear at present but may represent concentration-related differences between endogenously and exogenously produced SREBP-2. In addition, we were unable to detect any SREBP-2-DNA complexes by ChIP. SREBPs bind to their target genes in both hetero- and homodimerized forms through their basic helix-loop-helix/leucine zipper (bHLHLZ) domains (11). Thus it is possible that SREBP-2 competes with SREBP-1c for interactions with other cofactors to influence SREBP-1c activation. In support of this possibility, transfection of increasing amount of SREBP-1c enhanced Abca3 promoter activity in the presence of SREBP-2. Similar results were observed with SREBP-1 interfering with PGC-1-HNF-4a complexes in the regulation of hepatic gluconeogenic genes (51). Although SREBP activity is influenced by the presence of one or more coregulators, including SP1, YY1, cAMP response element-binding protein (CREB), and NF-Y in other organs (15, 34, 37), such coregulators have not yet been identified as active in the respiratory epithelium. In the present work, Abca3 promoter sequence analysis indicated the presence of multiple SP1 sites near SRE sites, suggesting that SP1 may serve as a potential coregulator with the SREBPs.

SCAP regulates SREBPs and ABCA3 in vivo. SREBP transcription factors are transported from the ER to the Golgi apparatus where they are sequentially cleaved by two proteases that release the cytosolic NH2-terminal transcription factor domains (5, 6). SCAP is a membrane carrier protein that forms a complex with SREBPs in the ER and stabilizes the SREBPs during transport to the Golgi apparatus. Because of potential embryonic lethality of the Scap homozygous knockout in the mouse, Scap was conditionally deleted in the liver (27). In the present study, the Scap gene was selectively deleted in the respiratory epithelium. The extent of Scap gene deletion in respiratory epithelial cells in Scap{Delta}/{Delta} mice was assessed at E18.5, indicating an ~60% decrease in Scap mRNA. SREBP-1c, SREBP-1a, and, to a lesser extent, SREBP-2 expression were markedly decreased in lungs of Scap{Delta}/{Delta} mice. In contrast to conditional Scap gene deletion in the liver (27), Scap mRNAs were still detectable in whole lung RNA using the SP-C-rtTA-TetO-Cre system that selectively deletes genes in alveolar type II epithelial cells. Both Abca3 and Srebps mRNAs were significantly decreased after deletion of Scap in the respiratory epithelium, supporting the regulatory role of SREBP on Abca3 expression in alveolar type II cells in vivo. Residual Abca3 mRNA seen after deletion of Scap may indicate its regulation by other lung-selective factors or the expression of Abca3 in nontargeted cells. Since no lung pathology was detected in SREBP-1 gene-targeted mice (39), it is likely that Abca3 expression is not entirely dependent on SREBP-1c since the absence of ABCA3 activity causes a lethal pulmonary disorder in infants and mice (1, 17, 40). Surfactant phospholipids are actively synthesized by alveolar type II cells. Fas and Scd1 are key enzymes in this process. Fas and Scd1 are regulated by SREBPs (2, 8, 9, 38, 42), and the SRE sites in the Fas and Scd1 gene promoters are directly bound by SREBPs. In the present work, Fas and Scd1 mRNA were markedly decreased in Scap{Delta}/{Delta} mice lungs demonstrating that the Scap gene deletion in the lung altered other genes regulating lipid metabolism.

The present work demonstrates that the expression of the Abca3 gene is influenced by a 5'-regulatory region containing elements for binding to a group of transcription factors FOXA2, C/EBP{alpha}, GATA-6, TTF-1, and NFATc3 known to regulate many other genes required for surfactant homeostasis in the respiratory epithelium, including surfactant proteins (A, B, C, and D). The Abca3 gene is also directly activated by SREBP-1c that binds to a distinct regulatory region of the Abca3 gene, providing a mechanism by which lipid homeostasis may be linked to lamellar body formation and lipid transport via ABCA3. Identification of the factors regulating the Abca3 gene provides additional insights into the transcriptional mechanism by which surfactant homeostasis is modulated during perinatal adaptation to air breathing.


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This work was supported by National Heart, Lung, and Blood Institute Grants HL-61646 and HL-85610 (J. A. Whitsett).


   ACKNOWLEDGMENTS
 
We acknowledge technical contribution from Sharon Dingle and Jean Clark.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. A. Whitsett, Cincinnati Children's Hospital Medical Center, Section of Neonatology, Perinatal and Pulmonary Biology, MLC 7029, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (e-mail: jeff.whitsett{at}cchmc.org)

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


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TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Ban N, Matsumura Y, Sakai H, Takanezawa Y, Sasaki M, Arai H, Inagaki N. ABCA3 as a lipid transporter in pulmonary surfactant biogenesis. J Biol Chem 282: 9628–9634, 2007.[Abstract/Free Full Text]
  2. Bennett MK, Lopez JM, Sanchez HB, Osborne TF. Sterol regulation of fatty acid synthase promoter. Coordinate feedback regulation of two major lipid pathways. J Biol Chem 270: 25578–25583, 1995.[Abstract/Free Full Text]
  3. Besnard V, Wert SE, Kaestner KH, Whitsett JA. Stage-specific regulation of respiratory epithelial cell differentiation by Foxa1. Am J Physiol Lung Cell Mol Physiol 289: L750–L759, 2005.[Abstract/Free Full Text]
  4. Bohinski RJ, Di Lauro R, Whitsett JA. The lung-specific surfactant protein B gene promoter is a target for thyroid transcription factor 1 and hepatocyte nuclear factor 3, indicating common factors for organ-specific gene expression along the foregut axis. Mol Cell Biol 14: 5671–5681, 1994.[Abstract/Free Full Text]
  5. Brown MS, Goldstein JL. A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood. Proc Natl Acad Sci USA 96: 11041–11048, 1999.[Abstract/Free Full Text]
  6. Brown MS, Goldstein JL. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 89: 331–340, 1997.[CrossRef][ISI][Medline]
  7. Burris TP, Eacho PI, Cao G. Genetic disorders associated with ATP binding cassette cholesterol transporters. Mol Genet Metab 77: 13–20, 2002.[CrossRef][ISI][Medline]
  8. Chang Y, Edeen K, Lu X, De Leon M, Mason RJ. Keratinocyte growth factor induces lipogenesis in alveolar type II cells through a sterol regulatory element binding protein-1c-dependent pathway. Am J Respir Cell Mol Biol 35: 268–274, 2006.[Abstract/Free Full Text]
  9. Chang Y, Wang J, Lu X, Thewke DP, Mason RJ. KGF induces lipogenic genes through a PI3K and JNK/SREBP-1 pathway in H292 cells. J Lipid Res 46: 2624–2635, 2005.[Abstract/Free Full Text]
  10. Cheong N, Madesh M, Gonzales LW, Zhao M, Yu K, Ballard PL, Shuman H. Functional and trafficking defects in ATP binding cassette A3 mutants associated with respiratory distress syndrome. J Biol Chem 281: 9791–9800, 2006.[Abstract/Free Full Text]
  11. Datta S, Osborne TF. Activation domains from both monomers contribute to transcriptional stimulation by sterol regulatory element-binding protein dimers. J Biol Chem 280: 3338–3345, 2005.[Abstract/Free Full Text]
  12. Davé V, Childs T, Whitsett JA. Nuclear factor of activated T cells regulates transcription of the surfactant protein D gene (Sftpd) via direct interaction with thyroid transcription factor-1 in lung epithelial cells. J Biol Chem 279: 34578–34588, 2004.[Abstract/Free Full Text]
  13. Davé V, Childs T, Xu Y, Ikegami M, Besnard V, Maeda Y, Wert SE, Neilson JR, Crabtree GR, Whitsett JA. Calcineurin/Nfat signaling is required for perinatal lung maturation and function. J Clin Invest 116: 2597–2609, 2006.[CrossRef][ISI][Medline]
  14. DeFelice M, Silberschmidt D, DiLauro R, Xu Y, Wert SE, Weaver TE, Bachurski CJ, Clark JC, Whitsett JA. TTF-1 phosphorylation is required for peripheral lung morphogenesis, perinatal survival, and tissue-specific gene expression. J Biol Chem 278: 35574–35583, 2003.[Abstract/Free Full Text]
  15. Dooley KA, Bennett MK, Osborne TF. A critical role for cAMP response element-binding protein (CREB) as a co-activator in sterol-regulated transcription of 3-hydroxy-3-methylglutaryl coenzyme A synthase promoter. J Biol Chem 274: 5285–5291, 1999.[Abstract/Free Full Text]
  16. Eberlé D, Hegarty B, Bossard P, Ferré P, Foufelle F. SREBP transcription factors: master regulators of lipid homeostasis. Biochimie 86: 839–848, 2004.[Medline]
  17. Fitzgerald ML, Xavier R, Haley KJ, Welti R, Goss JL, Brown CE, Zhuang DZ, Bell SA, Lu N, McKee M, Seed B, Freeman MW. ABCA3 inactivation in mice causes respiratory failure, loss of pulmonary surfactant, and depletion of lung phosphatidylglycerol. J Lipid Res 48: 621–632, 2007.[Abstract/Free Full Text]
  18. Flodby P, Barlow C, Kylefjord H, Ahrlund-Richter L, Xanthopoulos KG. Increased hepatic cell proliferation and lung abnormalities in mice deficient in CCAAT/enhancer binding protein alpha. J Biol Chem 271: 24753–24760, 1996.[Abstract/Free Full Text]
  19. Garmany TH, Moxley MA, White FV, Dean M, Hull WM, Whitsett JA, Nogee LM, Hamvas A. Surfactant composition and function in patients with ABCA3 mutations. Pediatr Res 59: 801–805, 2006.[CrossRef][ISI][Medline]
  20. Horton JD, Shah NA, Warrington JA, Anderson NN, Park SW, Brown MS, Goldstein JL. Combined analysis of oligonucleotide microarray data from transgenic and knockout mice identifies direct SREBP target genes. Proc Natl Acad Sci USA 100: 12027–12032, 2003.[Abstract/Free Full Text]
  21. Iwamoto N, Abe-Dohmae S, Sato R, Yokoyama S. ABCA7 expression is regulated by cellular cholesterol through the SREBP2 pathway and associated with phagocytosis. J Lipid Res 47: 1915–1927, 2006.[Abstract/Free Full Text]
  22. Kaminski WE, Piehler A, Wenzel JJ. ABC A-subfamily transporters: structure, function and disease. Biochim Biophys Acta 1762: 510–524, 2006.[Medline]
  23. Liu C, Ikegami M, Stahlman MT, Dey CR, Whitsett JA. Inhibition of alveolarization and altered pulmonary mechanics in mice expressing GATA-6. Am J Physiol Lung Cell Mol Physiol 285: L1246–L1254, 2003.[Abstract/Free Full Text]
  24. Liu C, Morrisey EE, Whitsett JA. GATA-6 is required for maturation of the lung in late gestation. Am J Physiol Lung Cell Mol Physiol 283: L468–L475, 2002.[Abstract/Free Full Text]
  25. Martis PC, Whitsett JA, Xu Y, Perl AK, Wan H, Ikegami M. C/EBPalpha is required for lung maturation at birth. Development 133: 1155–1164, 2006.[Abstract/Free Full Text]
  26. Mason RJ, Pan T, Edeen KE, Nielsen LD, Zhang F, Longphre M, Eckart MR, Neben S. Keratinocyte growth factor and the transcription factors C/EBP alpha, C/EBP delta, and SREBP-1c regulate fatty acid synthesis in alveolar type II cells. J Clin Invest 112: 244–255, 2003.[CrossRef][ISI][Medline]
  27. Matsuda M, Korn BS, Hammer RE, Moon YA, Komuro R, Horton JD, Goldstein JL, Brown MS, Shimomura I. SREBP cleavage-activating protein (SCAP) is required for increased lipid synthesis in liver induced by cholesterol deprivation and insulin elevation. Genes Dev 15: 1206–1216, 2001.[Abstract/Free Full Text]
  28. Mulugeta S, Gray JM, Notarfrancesco KL, Gonzales LW, Koval M, Feinstein SI, Ballard PL, Fisher AB, Shuman H. Identification of LBM180, a lamellar body limiting membrane protein of alveolar type II cells, as the ABC transporter protein ABCA3. J Biol Chem 277: 22147–22155, 2002.[Abstract/Free Full Text]
  29. Perl AK, Tichelaar JW, Whitsett JA. Conditional gene expression in the respiratory epithelium of the mouse. Transgenic Res 11: 21–29, 2002.[CrossRef][ISI][Medline]
  30. Perl AK, Wert SE, Loudy DE, Shan Z, Blair PA, Whitsett JA. Conditional recombination reveals distinct subsets of epithelial cells in trachea, bronchi, and alveoli. Am J Respir Cell Mol Biol 33: 455–462, 2005.[Abstract/Free Full Text]
  31. Rosen ED, Spiegelman BM. Molecular regulation of adipogenesis. Annu Rev Cell Dev Biol 16: 145–171, 2000.[CrossRef][ISI][Medline]
  32. Rosen ED, Walkey CJ, Puigserver P, Spiegelman BM. Transcriptional regulation of adipogenesis. Genes Dev 14: 1293–1307, 2000.[Free Full Text]
  33. Rugh R. The Mouse: Its Reproduction and Development. New York: Oxford Univ. Press, 1993.
  34. Sanchez HB, Yieh L, Osborne TF. Cooperation by sterol regulatory element-binding protein and Sp1 in sterol regulation of low density lipoprotein receptor gene. J Biol Chem 270: 1161–1169, 1995.[Abstract/Free Full Text]
  35. Santamarina-Fojo S, Peterson K, Knapper C, Qiu Y, Freeman L, Cheng JF, Osorio J, Remaley A, Yang XP, Haudenschild C, Prades C, Chimini G, Blackmon E, Francois T, Duverger N, Rubin EM, Rosier M, Denefle P, Fredrickson DS, Brewer HB Jr. Complete genomic sequence of the human ABCA1 gene: analysis of the human and mouse ATP-binding cassette A promoter. Proc Natl Acad Sci USA 97: 7987–7992, 2000.[Abstract/Free Full Text]
  36. Schmitz G, Kaminski WE, Orso E. ABC transporters in cellular lipid trafficking. Curr Opin Lipidol 11: 493–501, 2000.[CrossRef][ISI][Medline]
  37. Shea-Eaton WK, Trinidad MJ, Lopez D, Nackley A, McLean MP. Sterol regulatory element binding protein-1a regulation of the steroidogenic acute regulatory protein gene. Endocrinology 142: 1525–1533, 2001.[Abstract/Free Full Text]
  38. Shimano H, Horton JD, Hammer RE, Shimomura I, Brown MS, Goldstein JL. Overproduction of cholesterol and fatty acids causes massive liver enlargement in transgenic mice expressing truncated SREBP-1a. J Clin Invest 98: 1575–1584, 1996.[ISI][Medline]
  39. Shimano H, Shimomura I, Hammer RE, Herz J, Goldstein JL, Brown MS, Horton JD. Elevated levels of SREBP-2 and cholesterol synthesis in livers of mice homozygous for a targeted disruption of the SREBP-1 gene. J Clin Invest 100: 2115–2124, 1997.[ISI][Medline]
  40. Shulenin S, Nogee LM, Annilo T, Wert SE, Whitsett JA, Dean M. ABCA3 gene mutations in newborns with fatal surfactant deficiency. N Engl J Med 350: 1296–1303, 2004.[Abstract/Free Full Text]
  41. Stahlman MT, Besnard V, Wert SE, Weaver TE, Dingle S, Xu Y, von Zychlin K, Olson SJ, Whitsett JA. Expression of ABCA3 in developing lung and other tissues. J Histochem Cytochem 55: 71–83, 2007.[Abstract/Free Full Text]
  42. Tabor DE, Kim JB, Spiegelman BM, Edwards PA. Identification of conserved cis-elements and transcription factors required for sterol-regulated transcription of stearoyl-CoA desaturase 1 and 2. J Biol Chem 274: 20603–20610, 1999.[Abstract/Free Full Text]
  43. Takahashi K, Kimura Y, Nagata K, Yamamoto A, Matsuo M, Ueda K. ABC proteins: key molecules for lipid homeostasis. Med Mol Morphol 38: 2–12, 2005.[CrossRef][Medline]
  44. Wan H, Dingle S, Xu Y, Besnard V, Kaestner KH, Ang SL, Wert S, Stahlman MT, Whitsett JA. Compensatory roles of Foxa1 and Foxa2 during lung morphogenesis. J Biol Chem 280: 13809–13816, 2005.[Abstract/Free Full Text]
  45. Wan H, Xu Y, Ikegami M, Stahlman MT, Kaestner KH, Ang SL, Whitsett JA. Foxa2 is required for transition to air breathing at birth. Proc Natl Acad Sci USA 101: 14449–14454, 2004.[Abstract/Free Full Text]
  46. Weinhofer I, Kunze M, Rampler H, Bookout AL, Forss-Petter S, Berger J. Liver X receptor alpha interferes with SREBP1c-mediated Abcd2 expression. Novel cross-talk in gene regulation. J Biol Chem 280: 41243–41251, 2005.[Abstract/Free Full Text]
  47. Whitsett JA, Wert SE, Trapnell BC. Genetic disorders influencing lung formation and function at birth. Hum Mol Genet 13, Spec 2: R207–R215, 2004.
  48. Wikenheiser KA, Vorbroker DK, Rice WR, Clark JC, Bachurski CJ, Oie HK, Whitsett JA. Production of immortalized distal respiratory epithelial cell lines from surfactant protein C/simian virus 40 large tumor antigen transgenic mice. Proc Natl Acad Sci USA 90: 11029–11033, 1993.[Abstract/Free Full Text]
  49. Wilentz RE, Witters LA, Pizer ES. Lipogenic enzymes fatty acid synthase and acetyl-coenzyme A carboxylase are coexpressed with sterol regulatory element binding protein and Ki-67 in fetal tissues. Pediatr Dev Pathol 3: 525–531, 2000.[CrossRef][ISI][Medline]
  50. Wong J, Quinn CM, Brown AJ. SREBP-2 positively regulates transcription of the cholesterol efflux gene, ABCA1, by generating oxysterol ligands for LXR. Biochem J 400: 485–491, 2006.[CrossRef][ISI][Medline]
  51. Yamamoto T, Shimano H, Nakagawa Y, Ide T, Yahagi N, Matsuzaka T, Nakakuki M, Takahashi A, Suzuki H, Sone H, Toyoshima H, Sato R, Yamada N. SREBP-1 interacts with hepatocyte nuclear factor-4 alpha and interferes with PGC-1 recruitment to suppress hepatic gluconeogenic genes. J Biol Chem 279: 12027–12035, 2004.[Abstract/Free Full Text]
  52. Yamano G, Funahashi H, Kawanami O, Zhao LX, Ban N, Uchida Y, Morohoshi T, Ogawa J, Shioda S, Inagaki N. ABCA3 is a lamellar body membrane protein in human lung alveolar type II cells. FEBS Lett 508: 221–225, 2001.[CrossRef][ISI][Medline]
  53. Yoshida I, Ban N, Inagaki N. Expression of ABCA3, a causative gene for fatal surfactant deficiency, is up-regulated by glucocorticoids in lung alveolar type II cells. Biochem Biophys Res Commun 323: 547–555, 2004.[CrossRef][ISI][Medline]
  54. Zeng L, Liao H, Liu Y, Lee TS, Zhu M, Wang X, Stemerman MB, Zhu Y, Shyy JY. Sterol-responsive element-binding protein (SREBP) 2 down-regulates ATP-binding cassette transporter A1 in vascular endothelial cells: a novel role of SREBP in regulating cholesterol metabolism. J Biol Chem 279: 48801–48807, 2004.[Abstract/Free Full Text]
  55. Zhang F, Pan T, Nielsen LD, Mason RJ. Lipogenesis in fetal rat lung: importance of C/EBPalpha, SREBP-1c, and stearoyl-CoA desaturase. Am J Respir Cell Mol Biol 30: 174–183, 2004.[Abstract/Free Full Text]




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