Am J Physiol Lung Cell Mol Physiol 288: L625–L632, 2005.
First published December 3, 2004; doi:10.1152/ajplung.00250.2004.
The murine SP-C promoter directs type II cell-specific expression
in transgenic mice
Stephan W. Glasser, Susan K. Eszterhas, Emily A. Detmer,
Melissa D. Maxfield, and Thomas R. Korfhagen
Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio
Submitted 6 July 2004; accepted in final form 24 November 2004
PULMONARY SURFACTANT is a phospholipid and protein mixture
that lines the gas-exchanging regions of the lung and prevents
alveolar collapse during respiration (10). Pulmonary surfactant
is synthesized and secreted in the alveolus by distinct cuboidal
type II cells. The composition, alveolar concentration, and
intracellular reservoir of pulmonary surfactant is precisely
maintained in the normal lung. Alterations in surfactant composition or homeostasis can inhibit normal respiratory function. Neonatal respiratory distress is due to inadequate surfactant levels. Surfactant dysfunction can also result from microbial, particulate, or gaseous injury causing vascular leak that
inhibits surfactant function. Surfactant inactivation contributes
to the adult respiratory distress syndrome (16). Mutations that
alter surfactant protein (SP) components result in interstitial
lung disease or surfactant dysfunction (19).
Although phospholipids comprise the bulk of surfactant,
essential functions are fulfilled by surfactant-associated proteins. Whereas SP-A and SP-D are components of innate
immunity (13, 15), the hydrophobic proteins SP-B and SP-C
augment the surface properties of surfactant phospholipids
(25). SP-C is a highly hydrophobic 34-amino acid peptide that
is proteolytically processed from a 197-amino acid precursor.
The processed form of SP-C is associated with surfactant
phospholipids through an ␣-helical structure spanning a lipid
bilayer. Hydrophobic properties of SP-C are further enhanced
by palmitoylation of two NH2-terminal cysteine residues at
positions 5 and 6. Several clinical studies and laboratory
investigations indicate that SP-C contributes to surfactant function. Surfactant replacement extracts enriched for SP-C are
highly effective in the treatment of neonatal respiratory distress
syndrome. A synthetic replacement surfactant consisting of
surfactant phospholipids supplemented with a recombinant
SP-C peptide as the only protein component restores pulmonary function in several animal models of surfactant-depleted
acute lung injury (11). Addition of purified SP-C to synthetic
phospholipid preparations lowers the surface activity to levels
approximating whole native surfactant preparations (24).
The identification of naturally occurring SP gene mutations
in humans and the development of gene knockout models in
mice have provided insight into “nonsurfactant” functions.
Correct temperospatial expression of the SP-C gene and subsequent processing of SP-C and its release from the type II cell
is essential to normal pulmonary homeostasis. Mutations that
alter normal intracellular pro-SP-C processing have been identified in cases of familial idiopathic interstitial pneumonitis
(19, 23). Affected individuals within a family can present with
highly variable onset and severity of clinical disease suggesting
a complex etiology due to environmental influences or unknown modifier genes. SP-C-deficient mice generated by targeted gene inactivation develop a progressive interstitial lung
disease consisting of cellular infiltrates and alveolar remodeling with fibrotic lesions in the most severely affected animals (8).
In the adult lung, SP-C mRNA expression is detected at high
levels only in type II alveolar epithelial cells, whereas other SP
genes are expressed in bronchiolar epithelial cells (SP-A and
SP-B) and subsets of tracheal gland cells (SP-A and SP-D) as
well as type II cells. Thus SP-C is the only gene expressed only
in type II cells and has served as a marker of type II cell
differentiation in the mammalian lung. In cell transfection
experiments, the SP-C promoters are activated by an overlapping set of transcription factors that include thyroid transcription factor (TTF)-1, hepatocyte nuclear factors, GATA-6, and
nuclear factor I (NFI). Analysis of SP gene promoters in
transgenic mice has been used to correlate the arrangement of
in vitro cis-active sites to actual in vivo regulation as well as to
identify new cis-active regions, such as developmental control
Address for reprint requests and other correspondence: S. W. Glasser,
Cincinnati Children’s Hospital Medical Center, Div. of Pulmonary Biology, 3333
Burnet Ave., Cincinnati, OH 45229-3039 (E-mail: [email protected]).
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.
surfactant protein C; type II cells
http://www.ajplung.org
1040-0605/05 $8.00 Copyright © 2005 the American Physiological Society
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Glasser, Stephan W., Susan K. Eszterhas, Emily A. Detmer,
Melissa D. Maxfield, and Thomas R. Korfhagen. The murine SP-C
promoter directs type II cell-specific expression in transgenic mice.
Am J Physiol Lung Cell Mol Physiol 288: L625–L632, 2005. First
published December 3, 2004; doi:10.1152/ajplung.00250.2004.—
Genomic DNA from the mouse pulmonary surfactant protein C
(SP-C) gene was analyzed in transgenic mice to identify DNA
essential for alveolar type II cell-specific expression. SP-C promoter
constructs extending either 13 or 4.8 kb upstream of the transcription
start site directed lung-specific expression of the bacterial chloramphenicol acetyl transferase (CAT) reporter gene. In situ hybridization
analysis demonstrated alveolar cell-specific expression in the lungs of
adult transgenic mice, and the pattern of 4.8 SP-C-CAT expression
during development paralleled that of the endogenous SP-C gene.
With the use of deletion constructs, lung-specific, low-level CAT
activity was detected in tissue assays of SP-C-CAT transgenic mice
retaining 318 bp of the promoter. In transient and stable cell transfection experiments, the 4.8-kb SP-C promoter was 90-fold more
active as a stably integrated gene. These findings indicate that 1) the
4.8-kb SP-C promoter is sufficient to direct cell-specific and developmental expression, 2) an enhancer essential for lung-specific expression maps to the proximal 318-bp promoter, and 3) the activity of
the 4.8-kb SP-C promoter construct is highly dependent on its chromatin environment.
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SP-C PROMOTER ACTIVITY IN TRANSGENIC MICE
MATERIALS AND METHODS
SP-C-CAT constructs and transgenic mice. A 4.8-kb fragment of
murine SP-C genomic DNA was isolated from a 129/J library and
subcloned into the promoterless pBLCAT 6 expression plasmid. The
⫺318- and ⫺230-bp deletion constructs were generated by restriction
digestion of the 4.8-kb fragment. The 13-kb SP-C-CAT construct was
generated by ligation of an 8.2-kb SalI-BamHI genomic fragment to a
unique 5⬘ BamHI site in the proximal 4.8-kb SP-C promoter fragment
(12). Restriction digestion at unique sites in the flanking multicloning
sites was used to liberate the transgene constructs. Transgene constructs were purified from plasmid vector DNA by electrophoresis
through low-melt agarose gels. DNA was recovered and purified for
microinjection using Magic PCR columns (Promega, Madison, WI).
DNA was resuspended in 5 mM Tris 䡠 HCl, pH 7.9, and provided to the
Cincinnati Children’s Hospital transgenic core facility. Founder (F0)
SP-C-CAT mice were identified by genomic Southern blot using a
550-bp fragment of the CAT gene as probe or by PCR of genomic
DNA isolated from tail clips. F0 mice were bred to FVB/N mice
(Taconic, Germantown, NY) to establish heterozygous F1 animals that
were bred to produce mice homozygous for the transgene. All mice
were maintained in microisolator housing within a barrier facility.
Sentinel mice were negative for both bacterial and viral pathogens.
Studies were reviewed and approved by the Cincinnati Children’s
Hospital Institutional Animal Use and Care Committee.
CAT enzyme assay of tissue extracts. Organs were removed and
homogenized in 500 ␮l of 1 M Tris 䡠 HCl, pH 7.4 (adult mice), 200 ␮l
embryonic day 16 (ed 16) or 100 ␮l (ed 13 animals). Organs from
adult or ed 16 mice were homogenized with a conventional tissue
homogenizer or with a microgrinder fitted to 1.5-ml Eppendorf vials
for the organs of ed 13 mice. Homogenates were heated at 65°C for
5 min and centrifuged to remove cell debris. CAT assays were
normalized to uniform protein concentration from organ extracts and
run as previously described in detail (9).
Cell culture and selection for stable transfection. MLE-15 cells
were maintained in HITES media supplemented with 4% FBS.
MLE-15 cells are mouse lung epithelial tumor cells with features of
type II cells. Cells were plated in six-well dishes at a density of 105
cells/well. Cotransfections included 0.01 pmol of the plasmid pGKneo, 0.125 pmol of pGL-2, or 0.18 pmol of SP-C-luciferase (luc)
constructs complexed with Fugene-6 transfection reagent (Boehringer
Mannheim). Cells were held at 37°C and 5% CO2 overnight, and the
media were removed and replaced with selection media (HITES, 4%
FBS, and 350 ␮g/ml G418, a neomycin analog). Media were changed
every 2 days. Cells were harvested at 6 days for luciferase assays or
trypsinized and replated in the G418 selection media to expand the
stable, transfected cell populations. Passage 2 of the mixed populations of these G418-resistant colonies were frozen as stock cultures.
AJP-Lung Cell Mol Physiol • VOL
In situ hybridization. Lungs of adult mice were inflation fixed at
20-cm pressure with freshly prepared 4% paraformaldehyde. Animals
taken for developmental time points were fixed by immersion after the
thoracic cavity was opened. Tissue was embedded in paraffin, and
4-␮m sections were cut. 35S-labeled riboprobes for bacterial CAT and
murine SP-C were synthesized from pGEM plasmids. Probe synthesis, hybridization, and wash conditions have been previously described (26). Specificity of CAT hybridization was established by
comparison of antisense CAT probe hybridization to nontransgenic
lung and hybridization signal from CAT and SP-C sense strand
probes. Equivalent total counts of CAT or SP-C riboprobes were used
in individual hybridizations.
Relative copy number estimation. Genomic DNA was isolated from
tail clips of control and candidate SP-C-CAT transgenic mice. Five
micrograms of DNA were digested with the restriction enzyme EcoRI
and separated on 0.8% agarose gel for Southern blot analysis. A
550-bp CAT fragment was labeled with ␣-dCTP by nick translation
and used as the hybridization probe. Blots were hybridized overnight
and washed at a final stringency of 0.5⫻ SSC at 65°C and subjected
to autoradiography. Films were scanned, and relative densitometer
values of the SP-C-CAT diagnostic bands were compared.
RESULTS
Transgenic mouse lines were generated with two large
fragments of genomic DNA from the mouse SP-C gene directing CAT reporter gene expression. One construct incorporated
4.8 kb of DNA upstream of the basal promoter. By sequence
comparison, the mouse 4.8-kb DNA fragment was equivalent
to a 3.7-kb human SP-C promoter fragment used in previous
transgenic studies. Analysis of the 4.8-kb SP-C promoter in
vivo could potentially distinguish expression differences due to
species-specific variation between mouse and human sequences. A second construct was made with 13 kb upstream of
the promoter to functionally identify any distal enhancer sequence that would contribute to type II cell specificity or that
Fig. 1. Organ-specific expression of the mouse 13- and 4.8-kb surfactant
protein C-chloramphenicol acetyl transferase (SP-C-CAT) transgenes. CAT
enzyme assays were performed as described in MATERIALS AND METHODS with
tissue extracts prepared from organs of the 4.8 SP-C-CAT transgene-positive
mice line 4.4 (A), age-matched nontransgenic FVB/N mice (B), and 13-kb
SP-C-CAT line (C). Lane 1, lung; lane 2, heart; lane 3, liver; lane 4, kidney;
lane 5, spleen; lane 6, muscle; lane 7, salivary gland; lane 8, no extract,
negative control (C only). Significant CAT enzyme activity is detected only in
lung extract of transgene-positive lung from 4.8 SP-C-CAT and 13 SP-C-CAT
mice (arrows). Nearly identical lung-specific expression was obtained with
tissue extracts of a second 4.8 SP-C-CAT transgenic line 6.1 (not shown).
Presented data are representative of four 4.8 SP-C-CAT mice and eight 13
SP-C-CAT mice.
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and chromatin-dependent enhancers that may go undetected in
transient cell experiments.
In transgenic mice generated with human SP-C genomic
DNA, 3.7 kb of the human SP-C promoter directed lungspecific expression of a diphtheria toxin A or chloramphenicol
acetyl transferase (CAT) reporter gene in transgenic mice (9,
14). Subsequent experiments demonstrated that this region of
DNA directed developmental transgene expression similar to
the endogenous gene and that expression was sustained in
bronchiolar and alveolar type II cells of the adult lung (26).
Further dissection of the elements critical for type II cellspecific transcription have proven complicated by the bronchoalveolar pattern of expression. To discern what elements
are essential for accurate temporal spatial expression in the
lung, we have analyzed segments of the mouse SP-C gene
promoter in transgenic mice.
SP-C PROMOTER ACTIVITY IN TRANSGENIC MICE
absent, whereas CAT expression was restricted to alveolar
cells and overlapped expression of SP-C. These findings from
two independent founder lines indicate that the 4.8-kb region is
sufficient to restrict expression to the adult type II cells.
Development expression of 4.8-kb SP-C-CAT transgenes.
Tissue CAT assays and in situ analysis were used to assess
whether developmental expression was encoded by the 4.8-kb
SP-C-CAT transgene. Extremely low levels of lung-specific
CAT activity was detected in extracts from ed 13 embryos.
Significantly higher levels of transgene CAT activity was
detected in lung extracted from ed 16 embryos (Fig. 3).
Low-level 4.8-kb SP-C-CAT expression was localized by in
situ hybridization over the epithelial cells at the distal boundary
of the developing tubules in ed 13 lungs (Fig. 4). CAT or SP-C
expression was not detected over large proximal airways or
mesenchymal tissue. At ed 16, the relative level of CAT signal
was increased relative to ed 13 and was confined to the
numerous small distal airway epithelia, consistent with apparent increased branching (Fig. 5). Whereas the developmental
pattern of 4.8-kb SP-C-CAT transgene expression was similar
to the pattern of SP-C expression, in situ signals of 4.8-kb
Fig. 2. 13- and 4.8-kb SP-C promoter regions direct
SP-C-CAT transgene expression in alveolar epithelial cells of adult transgenic mice. CAT transgene
mRNA and endogenous SP-C mRNA expression
was detected using in situ hybridization. A and B:
corresponding dark- and bright-field images of SP-C
antisense hybridization. C–F: images of CAT antisense hybridization. Arrowheads indicate bronchiolar structures that are negative for SP-C or CAT
hybridization. C and D: dark- and bright-field images of 4.8 SP-C-CAT antisense hybridization results. E and F: images of 13 SP-C-CAT results. Both
CAT and SP-C probes hybridize in a focal pattern to
alveolar sites consistent with the location of type II
cells. Presented micrographs are representative of
sections from 4 mice.
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would silence bronchiolar expression seen with the human
3.7-kb SP-C promoter.
Tissue-specific expression of 4.8-kb SP-C-CAT transgenes.
Three 4.8-kb SP-C-CAT transgene founder mice were identified by genomic Southern blot. Two of the 4.8-kb SP-C-CAT
founders were bred to establish lines 4.4 and 6.1. Tissue
extracts were prepared from organs of adult mice homozygous
for the transgene from both lines and assayed for CAT enzyme
activity. High levels of CAT activity were detected only in
lung extracts of both 4.4 SP-C-CAT and 6.1 SP-C-CAT mice
and not in other tissues. Lung and other organ extracts were
uniformly negative from nontransgenic control mice (Fig. 1).
The murine 4.8-kb promoter segment targeted lung-specific
gene expression.
Cell-specific expression of 4.8-kb SP-C-CAT transgenes.
The cellular sites of 4.8-kb SP-C-CAT expression from lines
4.4 and 6.1 in the lung were determined by in situ hybridization
with CAT riboprobes. Sections of adult lung and ed 13 and ed
16 lung from both 4.4 and 6.1 mice were compared with the
pattern of expression for the endogenous SP-C gene. Strong
focal alveolar CAT expression was detected in sections of adult
lung from both lines (Fig. 2). Bronchiolar expression was
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SP-C PROMOTER ACTIVITY IN TRANSGENIC MICE
SP-C-CAT expression were modest in comparison with the
intensity of endogenous SP-C signal.
Lung- and cell-specific expression of 13-kb SP-C-CAT transgenes. Four transgenic lines were established with the 13-kb
SP-C-CAT construct. CAT reporter gene activity was lung
specific (Fig. 1C). Transgene expression was detected in a
focal pattern throughout the parenchyma. No aberrant bronchiolar expression was detected (Fig. 2, E and F). The number
of alveolar sites of transgene expression varied between 13
SP-C-CAT lines similar to the findings obtained from the
4.8-kb SP-C-CAT lines. The extended 13 kb of SP-C DNA,
therefore, do not appear to harbor enhancers that confer uniform expression among all type II cells. Whereas the cis-active
elements in the SP-C sequences confer lung- and type II
cell-specific expression, other chromatin-dependent elements
associated with the SP-C locus may be required to elicit
uniform transcription in all type II cells.
Specificity of truncated 0.32 SP-C-CAT transgene expression. To map essential cis-active regulatory regions, eight
founder transgenic lines were established from constructs consisting of 318 or 230 bp of 5⬘ DNA. Tissue CAT activity was
not detected from the 230-bp SP-C-CAT lines. Very weak
CAT activity was detected in lung extracts from the 318-bp
SP-C-CAT line 1.3 (Fig. 6). This finding localizes an essential
cis-active determinant of SP-C transcription to the proximal
⫺318 bp of the SP-C promoter. The relative transgene copy
number for 4.8 SP-C-CAT and 318 SP-C-CAT lines was
compared. Both 4.8 SP-C-CAT lines 4.4 and 6.1, expressed at
high levels, were extremely low copy number (line 4.4, 1 copy)
or low copy number (line 6.4, 3 copies). The only 318 SP-CCAT line to express had 30 copies relative to 4.8 SP-C-CAT
line 4.4. Therefore, the high-level 4.8 SP-C-CAT expression
did not reflect an increased transgene copy number.
AJP-Lung Cell Mol Physiol • VOL
Increased 4.8-kb promoter activity is due to chromatin.
Whereas the 4.8-kb promoter was far more active in transgenic
mice than the 318-bp promoter, the 4.8-kb promoter construct
was only slightly more active than the 318-bp promoter in
transient cell transfection experiments (12). To determine
whether the increased activity of 4.8-kb SP-C promoter activity
in vivo could be explained by a chromatin-dependent mechanism, 4.8-kb SP-C-luc and 318-bp SP-C-luc gene expression
was compared when stably integrated in cells. Stable transfected MLE-15 cells were generated by cotransfection with a
pGKneo plasmid and selection of neomycin (G418)-resistant
colonies. Equal molar amounts of 318-luc or 4.8-luc DNA
were used in transfections. A similar number of G418-resistant
colonies were obtained in each of seven independent transfections, indicating that efficiency of integration was nearly identical with both 318-SP-C-luc and 4.8-SP-C-luc constructs. In
each experiment, between 200 and 500 G418-resistant colonies
were harvested per plate so that measured reporter activity
reflected numerous independent integrations. Luciferase assays
were normalized to total cell protein. Luciferase reporter activity was 90-fold higher in extracts from 4.8-kb SP-C-luctransfected colonies than in the 318-bp SP-C-luc cell extracts.
In contrast, the 4.8-kb SP-C-luc luciferase activity was only
seven- to eightfold higher than 318-bp SP-C-luc activity in
transient transfection experiments (Fig. 7). This finding indicates that incorporation of the 4.8-kb SP-C DNA into chromatin structure stimulates enhancer activity by more than an order
of magnitude. Virtually no luciferase activity was detected in
cells transfected with the promoterless luciferase plasmid,
indicating that potential stimulation of luciferase activity due to
integration of test DNA adjacent to active but unrelated cellular
promoters was minimal.
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Fig. 3. CAT activity in tissue extracts during mouse embryonic
development. CAT assays were performed using extracts prepared from 4.8 SP-C-CAT transgenic mice at embryonic day 13
(A) and embryonic day 16 (B) and nontransgenic mice (C).
Arrows indicate specific acetylated chloramphenicol reaction
products. CAT activity was barely detected in day 13 lung and
was abundant in day 16 lung extracts. Organ extracts in A and
C: lane 1, liver; lane 2, heart; lane 3, intestine; lane 4, lung.
Organ extracts in B: lane 1, liver; lane 2, heart; lane 3, intestine;
lane 4, kidney; lane 5, stomach; lane 6, lung. Presented data are
representative of 4 mice from 4.8-kb SP-C-CAT transgenic line.
SP-C PROMOTER ACTIVITY IN TRANSGENIC MICE
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DISCUSSION
SP-C is distinguished from other SP by being expressed only
in alveolar type II cells and serves as a specific type II cell
marker during lung maturation or injury response. SP-C gene
expression has also been analyzed in vitro in cell transcription
assays and in vivo transgenic models to ascertain mechanisms
regulating SP-C gene transcription. The mouse SP-C gene
promoter was analyzed in transgenic mice with the goal of
identifying cis-active regions that support alveolar type II cell
expression. Previous transgenic studies using the human SP-C
promoter demonstrated lung-specific expression but were complicated by the finding that human 3.7-kb SP-C transgenes did
not recapitulate the expression pattern of the endogenous SP-C
gene. The human SP-C transgene was expressed in alveolar
type II cells and robustly in bronchiolar cells (7, 26). Analysis
of the mouse SP-C promoter constructs in transgenic mice
identified promoter regions that direct cell-appropriate expression, a proximal region essential for in vivo promoter activity,
and a distal chromatin-dependent regulatory domain that functions as an enhancer.
Tissue CAT assays and CAT in situ hybridization experiments demonstrated that 4.8 kb of mouse SP-C promoter and
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5⬘-flanking DNA targeted lung and alveolar cell-specific expression in adult mice. In an independent study, the mouse
4.8-kb SP-C promoter was used to restore SP-B expression in
lungs of SP-B⫺/⫺ mice. SP-B mRNA and protein expression
was type II cell specific for two of the three lines and was
detected only in a subset of the alveolar type II cells (17). The
murine 4.8-kb promoter segment is the sequence equivalent to
the 3.7-kb human promoter wherein three regions of homology
are conserved between the two species (7). The bronchiolar
expression obtained from the human promoter likely results
from altered assembly of murine transcription complexes on
human binding sites or from displacement of complexes that
silence SP-C expression in bronchiolar cells. The number of
alveolar cells expressing 4.8 SP-C-CAT in the transgenic lines
was reduced relative to sites of endogenous SP-C expression,
recapitulating results obtained with the human SP-C promoter.
This consistent finding suggested either that additional enhancers with type II cell activity were missing or that uniform type
II cell expression is context dependent, requiring sequences
and higher-ordered chromatin unique to the SP-C locus. Such
modifiers have been documented and include matrix attachment regions that tether chromatin loops at highly transcribed
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Fig. 4. Expression of 4.8 SP-C-CAT transgene at
embryonic day 13. Dark-field images of sense (A)
and antisense (B) SP-C hybridization. Sense (C) and
antisense (D) images of CAT hybridization. Magnification, ⫻4. Weak CAT and strong SP-C signals are
detected by antisense probes in peripheral tubules. E
and F are high magnification. Arrowheads indicate
central tubule that is negative for CAT expression,
and small arrows indicate peripheral airways positive for CAT expression. Magnification, ⫻20.
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SP-C PROMOTER ACTIVITY IN TRANSGENIC MICE
gene loci, locus control regions that modify gene expression
over distance and/or insulator elements that limit active gene
transcription and selective alteration of histone acetylation (6).
Transgenic mice were generated with extended 13 kb of SP-C
promoter and upstream DNA to test whether extended sequences harbored such type II cell normalizing elements. Each
of four separate 13-kb SP-C-CAT founder lines supported
alveolar (type II) cell-specific expression. Surprisingly, the
number of CAT-positive alveolar cells varied between each
founder line, with one line having 79% of cells expressing the
CAT gene compared with the number of cells expressing SP-C.
Expression from the other three 13-kb SP-C-CAT lines had
diminished number of sites of expression similar to the 4.8
SP-C-CAT lines. The current analysis with the extended promoter does not rule out intron or 3⬘ enhancer(s) that would
confer uniform type II cell expression. The structural features
of the SP-C locus limit the possibility of 3⬘ regulatory elements
wherein the SP-C gene terminates adjacent to the promoter of
a new gene. The sequence immediately 3⬘ of SP-C exon 6 is
G-C rich (82%) and serves as the promoter of the bone
morphogenetic protein (BMP)-1 gene, with the BMP-1 transcription start site only 700 bp downstream of the SP-C gene
AJP-Lung Cell Mol Physiol • VOL
(22). The BMP-1 gene encodes an essential developmental
procollagenase C gene. The BMP-1 exon 1 is followed by
extended CA dinucleotide repeats that are highly polymorphic.
The structural features of the unusual sequence immediately 3⬘
of the SP-C gene suggest that 3⬘ cis-active regulation of the
SP-C gene is unlikely.
Extremely low level but lung-specific CAT activity was
detected when the mouse 4.8-kb SP-C promoter was deleted to
318 bp. This finding indicates that significant enhancer activity
is located in the deleted distal region. The nature of the
enhancer is unclear, but initial cell transfection experiments
suggest that it is responsive to higher-ordered DNA structure.
When previously tested in transient cell transfection, only
modestly elevated 4.8 SP-C-CAT expression levels were seen
compared with 318 SP-C-CAT activity (12). By selecting for
stable chromosome integration, the relative expression of the
4.8 SP-C to the ⫺318-bp SP-C promoter was 90-fold greater
than differences detected in transient assays. This finding is
consistent with strong 4.8 SP-C vs. 318 SP-C expression in
transgenic mice and indicates chromatin association is necessary for activity of an enhancer upstream of 318 SP-C sequence. The 318 SP-C-CAT transgenes were present at ⬎10288 • APRIL 2005 •
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Fig. 5. Expression of 4.8 SP-C-CAT transgene at
embryonic day 16. CAT antisense hybridization (A,
C, and E) and SP-C antisense hybridization (B, D,
and F) were performed as described in MATERIALS
AND METHODS. CAT expression was detected in developing distal epithelium similar to the pattern of
SP-C transcripts. At high magnification, CAT and
SP-C signals are concentrated over peripheral airways and extinguished in central airway epithelia
(arrowheads in C–F, ⫻20). E and F are the corresponding bright-field images for the dark-field images in C and D to illustrate proximal to distal CAT
transgene and endogenous SP-C expression. CAT
transgene expression is significantly increased over
levels seen on embryonic day 13. Photomicrographs
are representative of sections of line 4.4 mice.
SP-C PROMOTER ACTIVITY IN TRANSGENIC MICE
fold higher copy number than in 4.8 SP-C-CAT lines. The
high-level expression of 4.8 SP-C-CAT lines was, therefore,
not simply due to increased number of transgenes. It is unknown whether integration increases the efficiency of the weak
enhancer activity detected in transient expression or if integration activates a separate enhancer. Analysis of the human 3.7
SP-C promoter produced similar results wherein the 3.7-kb
SP-C promoter was extremely weak in transient assays but
directs vigorous expression as an integrated transgene. Two
regions of homology to the human 3.7-kb sequence are found
in the upstream region at ⬃⫺4,700 to ⫺4,100 bp and ⫺3,500
to ⫺2,800 bp (7). These conserved sequences between species
are potential candidates for the chromatin enhancer element(s).
In related studies, a transgenic approach has been used to
localize cis-active regions that direct lung-specific expression
of the SP-A and SP-B promoters. These studies indicate that
the arrangement and nature of enhancer activity are different
for each SP gene. A 4-kb segment of the rabbit SP-A promoter
directed lung-specific transgene expression. Deletion to ⫺991
bp resulted in transgene expression in several additional organs. This result suggests that unlike SP-C, a distal silencing
element actively contributes to correct SP-A gene expression.
A second region that mediates bronchoalveolar SP-A expression is mapped to the ⫺378- to ⫺47-bp region (2). The authors
were unable to separate alveolar and bronchiolar regulation of
SP-A expression. Three recent reports have assessed SP-B
promoter activity in transgenic mice. A ⫺1,039- to ⫹431-bp
segment of the human SP-B promoter generated bronchoalveolar expression and low-level thyroid, trachea, and intestine
expression (21). A separate study of the human SP-B promoter
demonstrated that 1.5 kb directs lung and intestine expression
in mice. An enhancer was mapped to the ⫺500- to ⫺331-bp
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region (27). The ⫺236- to ⫹39-bp region of the rabbit SP-B
promoter directed bronchoalveolar selective expression and
transcriptional repression mapped in the distal ⫺2,176- to
⫺730-bp region (1). In all of these studies, ontogenic expression was shown to parallel the endogenous gene. It will be
intriguing to learn whether in vivo mutational analysis can map
regulatory regions that determine ontogenic expression distinct
from cell-selective expression. Although the SP-A and SP-B
promoter studies provide initial insights into elements required
for bronchoalveolar expression, identification of cis-elements
may be affected by species sequence variation as seen with the
SP-C promoters.
The essential 318-bp SP-C sequence conferring lung-specific activity identified in this study spans a region where
multiple factors interact to influence SP-C transcription in
vitro. The homeobox transcription factor TTF-1 (also termed
Nkx2.1) trans-activates the 318-bp SP-C promoter (12). With
the use of mutagenesis assays, NFI binding sites were identified and shown to be required for SP-C promoter activity (3).
Cotransfection experiments demonstrated that NFI and TTF-1
synergistically activate 318 SP-C promoter activity (4). TTF-1
also directly interacts with the transcription factor TAZ to
enhance 318 SP-C promoter activity (20) and interacts cooperatively with GATA-6 to increase SP-C expression (18). In
addition, TTF-1/GATA-6 interactions enhance SP-A activity
(5). Deletion mapping of the human SP-C promoter in transgenic mice showed that the equivalent TTF-1-responsive region was required for lung-specific expression in vivo, similar
to the current findings (7). Thus the combined in vitro promoter
activation studies and functional in vivo mapping implicate a
critical role for the TTF-1-responsive region in controlling the
level and cell-specific expression of SP-C.
The current study demonstrates that 4.8 kb of promoter
sequence activates and sustains alveolar cell-specific expression in the lungs of transgenic mice. A proximal 318-bp region
containing known cis-active NFI, TTF-1, and GATA-6 binding
sites is critical for lung-specific expression. High-level expression from the 4.8-kb promoter is mediated by an element that
Fig. 7. Comparison of 4.8-kb and 318-bp promoters in permanent MLE cell
lines and transiently transfected MLE cells. Luciferase (luc) activity from 318
SP-C-luc transfections was set at 1. 4.8 SP-C-luc activity is reported as fold
increase over 318 SP-C-luc activity in transient expression experiments (n ⫽
5) or stable transfected cells (n ⫽ 7). P ⬍ 0.01 comparing 4.8-kb to 318-bp
values.
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Fig. 6. Lung-specific CAT expression in mice carrying the 318 SP-C-CAT
transgene. CAT assays were performed using tissue extracts from adult 318
SP-C-CAT transgenic mice (A) and nontransgenic extracts (B). Tissue extract:
lane 1, liver; lane 2, muscle; lane 3, kidney; lane 4, heart; lane 5, lung; lane
6, no extract. Arrows indicate acetylated chloramphenicol due to CAT enzyme
activity.
L631
L632
SP-C PROMOTER ACTIVITY IN TRANSGENIC MICE
is dependent on a chromatin environment. The pattern of CAT
expression directed by 4.8 SP-C promoter was alveolar type II
cell specific. The aberrant bronchiolar component of expression obtained with the human 3.7 SP-C promoter in transgenic
mice is due to species/sequence differences. The 3.7 SP-C
promoter has been used successfully to express a variety of
bioactive reporter molecules in lungs of transgenic mice. The
4.8-kb murine SP-C promoter may have further utility in
directing expression restricted to just type II cells in vivo and
thus provides a useful tool for developing transgenic models.
ACKNOWLEDGMENTS
GRANTS
This work was supported by National Heart, Lung, and Blood Institute
Grants HL-50046 and HL-58795.
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