1 Department of Biochemistry, 2 The Howard Hughes Medical Institute, 3 Department of Obstetrics-Gynecology, and 4 The Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, Texas 75235
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ABSTRACT |
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The gene encoding surfactant protein (SP) A, a developmentally regulated pulmonary surfactant-associated protein, is expressed in a lung-specific manner, primarily in pulmonary type II cells. SP-A gene transcription in the rabbit fetal lung is increased by cAMP. To delineate the genomic regions involved in regulation of SP-A gene expression, lines of transgenic mice carrying fusion genes composed of various amounts of 5'-flanking DNA from the rabbit SP-A gene linked to the human growth hormone structural gene as a reporter were established. We found that as little as 378 bp of 5'-flanking DNA was sufficient to direct appropriate lung cell-selective and developmental regulation of transgene expression. The same region was also sufficient to mediate cAMP induction of transgene expression. Mutagenesis or deletion of either of two DNA elements, proximal binding element and a cAMP response element-like sequence, previously found to be crucial for cAMP induction of SP-A promoter activity in transfected type II cells, did not affect lung-selective or temporal regulation of expression of the transgene; however, overall levels of fusion gene expression were reduced compared with those of wild-type transgenes.
type II cell; lung; developmental regulation; hormonal regulation
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INTRODUCTION |
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THE SYNTHESIS OF PULMONARY SURFACTANT, a developmentally regulated lipoprotein, is restricted to type II pneumonocytes of the lung alveoli where surfactant is stored as lamellated cytoplasmic inclusion bodies termed lamellar bodies. Surfactant synthesis in the fetal lung appears to be regulated by a number of hormones and factors, including glucocorticoids, prolactin, and hormones that increase cAMP (5, 29). To define the mechanisms for the type II cell-specific, developmental, and hormonal regulation of surfactant synthesis in the fetal lung, we studied the gene encoding surfactant protein (SP) A, a major surfactant-associated protein, which has been reported to have important actions in the conversion of secreted surfactant into tubular myelin (16), in surfactant function and reutilization by type II cells, and in the modulation of host immune defense mechanisms within the lung alveolus (20, 25). SP-A gene expression is lung specific (7), occurs primarily in type II cells and, to a lesser extent, in nonciliated bronchioalveolar epithelial (Clara) cells (4, 47), and is regulated by cAMP and glucocorticoids (8, 9, 29). Expression of the SP-A gene is developmentally regulated in fetal lung tissue and is initiated only after 70% of gestation is completed (7, 43).
The cDNAs and gene encoding rabbit SP-A have previously been cloned and characterized in our laboratory (7, 12). Rabbit SP-A is encoded by a single-copy gene (7). In the rabbit, SP-A gene transcriptional activity is first detectable on day 24 of the 31-day gestation period (6, 30). The levels of SP-A gene transcription reach a maximum by day 28 of gestation and are decreased slightly in neonatal and adult lung tissues (6). Lung explants from 19- and 21-day gestational age fetal rabbits differentiate spontaneously when maintained in organ culture; after several days of incubation in serum-free defined medium, the epithelial cells lining the prealveolar ducts develop the phenotypic properties of type II cells (44). The spontaneous appearance of type II cells is associated with an induction of SP-A gene transcription and increased steady-state levels of SP-A mRNA (6, 7).
SP-A gene transcription in fetal
rabbit lung in organ culture is induced by cAMP analogs and by a number
of hormones and factors that increase cAMP formation, including
catecholamines, acting through
2-adrenergic
receptors (29, 33), prostaglandin
E2 (1), and vasoactive intestinal
peptide (V. Boggaram, M. E. Smith, and C. R. Mendelson, unpublished
observations). In studies of fetal rabbit lung explants maintained in
organ culture, we have found that glucocorticoids have both stimulatory
and inhibitory effects on SP-A gene
transcription. Treatment of lung explants from 21-day gestational age
fetal rabbits with cortisol or dexamethasone (Dex;
10
7 M) caused an acute
(6-24 h) inhibition of SP-A gene
transcription and reduced the magnitude of the stimulatory effect of
cAMP. However, after 48-72 h of incubation, a stimulatory effect
of glucocorticoids on SP-A gene
transcription was observed, and there was found to be an additive
stimulatory effect with dibutyryl cAMP (DBcAMP) (6).
In type II cell transfection studies, we observed that 378 bp of rabbit
SP-A 5'-flanking DNA mediated
cAMP induction of SP-A promoter
activity (2). Elevated levels of basal expression were observed only in
type II cells or in type II-derived lung adenocarcinoma cell lines (2,
49). Furthermore, cAMP induction of
SP-A promoter activity was evident
only in primary cultures of type II cells and not in type II-derived
lung adenocarcinoma cell lines (2, 49). These findings suggest that the
cell lines either have lost the capacity for cAMP responsiveness or lack a transcription factor(s) that is an essential mediator of cAMP
responsiveness. Our laboratory has also found that a cAMP response element (CRE)-like sequence [CRE for the
SP-A promoter (CRESP-A); TGACCTC/TA]
present in the 5'-flanking regions of the rabbit (2, 31) and
human (49) SP-A genes at 261
and
242 bp, respectively, is required for basal and cAMP
induction of SP-A promoter activity.
Michael et al. (31) have observed that the protein(s) that binds to
this element is distinct from CRE binding protein (CREB)/activating
transcription factor family members and is likely a member of the
nuclear-receptor family.
A lung-specific DNase I-hypersensitive site at 100 bp from the
SP-A gene transcription start site
also has been identified (12). This hypersensitive site was detectable
in chromatin isolated from lung tissues of 22- and 28-day gestational
age fetal rabbits as well as from adults. Another DNase
I-hypersensitive site at
1,300 bp was detected in the lung as
well as in a number of other tissues of fetal and adult rabbits (12).
Interestingly, these two hypersensitive sites are in close proximity to
two elements that we previously found to be important in basal and
cAMP-induced transcription of the rabbit
SP-A gene; these are denoted as the proximal binding element (PBE) at
80 bp and the distal binding element (DBE) at
980 bp (13, 14). The DBE contains the core sequence CACGTG, which corresponds to an E-box motif to which members
of the basic helix-loop-helix-leucine zipper family of gene regulatory
proteins are known to bind. The PBE contains a related sequence,
CTCGTG. Our laboratory has recently found that the basic
helix-loop-helix-leucine zipper family members upstream stimulatory
factor-1 (14) and -2 (Gao and Mendelson, unpublished observations) bind
to both of these elements. Our laboratory has also observed that cAMP
induction of SP-A promoter activity is mediated by increased phosphorylation and binding of the homeodomain factor thyroid transcription factor-1 (26) and through the binding of
Sp1 and related factors to a GT box (50) in the 5'-flanking regions of the baboon and human SP-A2
genes, respectively.
Despite the fact that glucocorticoids stimulate rabbit SP-A transcription and have an additive stimulatory effect with cAMP (6), we previously observed that glucocorticoids antagonized the stimulatory effect of cAMP on the expression of SP-A:human growth hormone (hGH) fusion genes containing up to 1,754 bp of SP-A 5'-flanking DNA in transfected type II cells (2). An inhibitory effect of glucocorticoids was also observed when 650 bp of an SP-A downstream sequence (containing the first exon, intron, and second exon) were also included in the fusion gene constructs (2). To date, we have been unable to identify a DNA element that mediates a stimulatory effect of glucocorticoids on rabbit SP-A gene transcription.
To begin to define the regions within and surrounding the rabbit SP-A gene involved in developmental, lung cell-specific, and multifactorial regulation of expression, we have created transgenic mice carrying SP-A:hGH fusion genes composed of 47-4,000 bp of rabbit SP-A 5'-flanking DNA linked to the hGH structural gene as a reporter. We have observed that as little as 378 bp of SP-A 5'-flanking sequence is sufficient to mediate appropriate lung cell-selective, developmental, and hormonal regulation of SP-A promoter activity in the transgenic mice.
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EXPERIMENTAL PROCEDURES |
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Construction of plasmids containing fusion
genes. DNA purification, restriction endonuclease
digestion, ligation, agarose gel electrophoresis, transformation, and
maintenance of Escherichia coli were
carried out according to methods of Sambrook et al. (37). The plasmid
p0GH (40), which contains a promoterless hGH structural gene subcloned into
pUC12, was used to construct the rabbit
SP-A:hGH fusion genes. Schematic
diagrams of the fusion genes used to create transgenic mice are shown
in Fig. 1. To construct SP-A47:hGH,
a Hind
III-Sau 3AI fragment of rabbit genomic DNA containing
47 bp of DNA flanking the 5'-end of the
SP-A gene transcription initiation
site (including the TATA box) and the first 20 bp of exon I (12) was
fused to the first exon of the hGH
structural gene in p0GH by ligation to a
BamH I site.
SP-A
378:hGH was constructed by fusing an
EcoR
I-Sau 3AI fragment of rabbit genomic
DNA that included
378 bp of 5'-flanking DNA and 20 bp of
exon I to the hGH structural gene by
subcloning into the BamH I site of
p0GH.
SP-A
4000:hGH
was constructed by ligating an Xba
I-Sau 3AI fragment containing
4,000 bp of SP-A
5'-flanking DNA and 20 bp of exon I to the
BamH I site of p0GH.
SP-A
991:hGH was constructed by digestion of
SP-A
4000:hGH
with Xba I and
BamH I, releasing a DNA fragment
containing the sequences from
4,000 to
991 bp; the
remaining vector and fusion gene sequences were religated. Construction
of the
SP-A
378CRE
:hGH mutant, in which the CRE element was changed from TGACCTCA to TGACGACA
(CRE
), has been previously described (2). Construction of the
SP-A
378PBE
:hGH
mutant, in which the PBE was changed from CCCTCGTG to ACTCTAGA
(PBE
), was accomplished by replacing the
BstE
II-Hind III fragment of
SP-A
378:hGH containing the wild-type PBE with the corresponding
BstE
II-Hind III fragment of the
SP-A
991PBE
:hGH
fusion gene (13) containing the mutated PBE. Construction of the
SP-A
991CRE-S:hGH mutant in which the CRE element was changed from TGACCTCA to TTCTAGAA, thereby completely "scrambling" the CRE sequence (CRE-S), was accomplished by site-directed mutagenesis of the element in
SP-A
991:hGH with the Altered Sites II in vitro mutagenesis kit (Promega, Madison, WI).
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Production and identification of transgenic mice. After digestion of recombinant plasmids with the appropriate restriction endonucleases to release the fusion genes from vector sequences, the fusion genes were isolated on agarose gels and purified as described by Short et al. (41). Fusion gene DNA was microinjected into the male pronucleus of fertilized F2 hybrid mouse eggs (obtained by mating C57B1/6 × SJL hybrid adults), which then were cultured to the two-cell stage, reimplanted into pseudopregnant mice (10, 34), and allowed to develop to term. Transgenic progeny were identified, and the number of fusion gene copies per genome was determined by dot blot analysis of tail DNA, with hGH DNA as a probe. Copy number was determined by comparison of the hGH-specific signal present in the tail DNA with the signal of a known amount of hGH structural gene that was added to the tail DNA of a nontransgenic mouse.
Culture of fetal mouse lung tissue.
Lung tissue explants from 15-day gestational age fetal transgenic and
nontransgenic mice were maintained in organ culture in serum-free
Waymouth MB752/1 medium (GIBCO BRL, Life Technologies, Grand Island,
NY) in the absence and presence of DBcAMP (1 mM), Dex
(107 M), or both agents in
combination for up to 5 days (42, 44). The explants were maintained in
a humidified atmosphere of 95% air-5%
CO2.
Production of recombinant adenoviruses. Recombinant adenoviruses containing rabbit SP-A:hGH fusion genes were produced and characterized as described previously (2, 3).
hGH assays. Medium from cultured mouse lung explants was collected at 24-h intervals. The concentration of hGH present in the medium was quantitated by radioimmunoassay with an Allegro hGH kit (Nichols Institute Diagnostics, San Juan Capistrano, CA).
Northern analysis of SP-A and hGH mRNAs in transgenic fetal mouse tissues. Total RNA was extracted from the tissues by homogenization in guanidinium isothiocyanate (4.0 M) with a Teflon glass homogenizer. The extracts were centrifuged through a cesium chloride gradient (5.7 M), and the pelleted RNA was resuspended in water (28). Total RNA (15-30 µg) was electrophoresed, transferred to nitrocellulose, and probed with either 32P-labeled rabbit SP-A cDNA, hGH cDNA, or an 18S oligonucleotide as previously described in detail (7).
Immunocytochemical analysis of SP-A and hGH proteins in transgenic mouse lung tissue. Lungs of adult transgenic mice were fixed in 4% paraformaldehyde and sectioned with a Leitz cryostat (model 1720, Leitz, Oberkochen, Germany). Sections were adhered to charged slides and subjected to immunocytochemical analysis as previously described (3). Guinea pig anti-rabbit SP-A antibody was raised in our laboratory with standard protocols (19). Rabbit anti-hGH antibody was purchased (Dako, Carpenteria, CA). Immunocytochemical localization of SP-A and hGH was achieved with these antibodies and a Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Visualization of the complexes as red granules was accomplished by use of the aminoethyl carbazole substrate kit from Zymed Laboratories (San Francisco, CA).
In situ hybridization analysis of hGH mRNA in transgenic fetal mouse lung tissue. Lungs of adult transgenic mice were fixed in 4% paraformaldehyde and embedded in paraffin. The embedded tissues were sectioned and adhered to charged slides. In situ analysis of the sections was performed after deparaffinization with the method described by Hackett and Gitlin (18). 35S-labeled sense and antisense hGH cRNAs were used for hybridization to the fixed sections. After autoradiography in Kodak NTB2 emulsion (Eastman Kodak, Rochester, NY), the sections were counterstained with hematoxylin and eosin. The resulting slides were visualized and photographed under bright- and dark-field microscopy.
Experimental animals. Mice used in this research were treated in accordance with the guidelines set by the Animal Welfare Information Center and approved by the Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center at Dallas.
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RESULTS |
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The 378-bp sequence flanking the 5'-end of the
rabbit SP-A gene is sufficient to direct expression of the SP-A:hGH
fusion genes in lung tissues of transgenic
mice. Five
SP-A:hGH fusion genes containing
various amounts of SP-A
5'-flanking DNA
(SP-A47:hGH, SP-A
378:hGH,
SP-A
991:hGH,
SP-A
1765:hGH,
and SP-A
4000:hGH)
were constructed (Fig. 1) and introduced into the genomes of mice as
described in EXPERIMENTAL PROCEDURES. The founder mice were bred to eliminate the effects of mosaicism; thus
the analysis was conducted in established lines. Table
1 shows the copy numbers (as determined by
Southern blot analysis described in EXPERIMENTAL
PROCEDURES) and expression (as determined by Northern
analysis of mRNA described in EXPERIMENTAL
PROCEDURES) of the fusion genes in lung tissues of
the established transgenic mouse lines. The lack of hGH expression in
the lung tissues of 13 of the 13 lines of mice carrying the
SP-A
47:hGH
fusion gene and the presence of hGH expression in the lung tissues of 9 of 14 lines of mice carrying the
SP-A
378:hGH
fusion gene indicate that SP-A
5'-flanking sequences between
47 and
378 bp are
required for expression in the lung. Expression in the lung of the
SP-A:hGH transgenes containing
991 bp (expression in 15 of 15 lines) and
4,000 bp
(expression in 2 of 6 lines) of the
SP-A 5'-flanking sequence was
also evident (Table 1). The relatively low proportion of mice
expressing the 4,000-bp-containing fusion gene compared with that of
the other constructs may be caused by the possible presence of silencer
elements within the 4,000-bp 5'-flanking region that recruit
transcriptional repressors and promote a "closed" chromatin
structure. On the other hand, this merely could be due to the fact
that, in comparison with the other transgenic constructs studied, a
relatively small number of
SP-A
4000:hGH
lines were analyzed, and three-fourths of the nonexpressing founders
carried only two copies of the transgene (Table 1). It should be noted
that an absence of transgene expression can also be due to an in vivo
rearrangement of the construct, and this possibility cannot be
excluded.
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The 378-bp 5'-flanking sequence of the rabbit
SP-A gene is sufficient to direct lung-selective expression of SP-A:hGH
fusion genes in transgenic mice. To determine which
genomic regions are required for lung-specific expression of the rabbit
SP-A gene, expression of the
transgenes in lungs and other tissues was analyzed. Total RNA isolated
from various tissues of adult transgenic mice carrying the
SP-A378:hGH,
SP-A
991:hGH,
and
SP-A
4000:hGH transgenes that were found to be expressed in lung was analyzed for hGH
mRNA by Northern blotting. Equivalent amounts of RNA from the lungs and
other tissues to be analyzed from each mouse line were electrophoresed
on the same gel and probed for hGH transcripts on the same Northern
blot. The levels of hybridizable hGH mRNA in various tissues were
determined by scanning densitometry of the autoradiograms (computing
densitometer model 300A and ImageQuant software version 3.3, Molecular
Dynamics, Sunnyvale, CA) and corrected for loading and transfer of RNA
by comparison to levels of hybridizable 18S RNA present in each lane of
the blots. For each transgenic line, the corrected value of hGH mRNA
expressed in the lung was given an arbitrary number of 100, and the
levels of hGH mRNA in the other tissues (muscle, fat, intestine, brain,
thymus, spleen, mammary tissue, kidney, liver, and heart) are presented
as a percentage of expression in the lung. The results of these
analyses are presented in Table 2.
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In the two lines of mice that expressed the
SP-A4000:hGH
fusion gene in the lung, expression was undetectable in all other
tissues examined. Expression of the
SP-A
991:hGH
transgene was detected in the lung tissues of all 15 lines of
transgenic mice established. However, in two of the five randomly
chosen lines studied for lung-specific expression, substantial levels of expression were detected in certain other tissues. In two lines, expression of the transgene in heart tissue was ~40-50% of that detected in lung tissue. In one of these lines, transgene expression also was detected in the kidney, mammary gland, and spleen at levels
between 2 and 7% of those in lung and at <1% of those in the other
tissues studied. In the three other lines of mice carrying the
SP-A
991:hGH
transgene, expression in the brain, thymus, spleen, mammary gland,
kidney, and liver was either undetectable or
1% of that in lung. In
two of four of the transgenic lines studied carrying the
SP-A
378:hGH
transgene, substantial levels of hGH expression were detected in the
spleen and thymus; in one of these lines, a considerable level of
expression was detected in the heart as well. Relatively low levels of
transgene expression were also detected in fat, small intestine,
kidney, and liver tissues of each of these lines. These findings
suggest that although SP-A
5'-flanking sequences between
378 and
47 bp are
sufficient to mediate transgene expression in a lung-selective manner,
sequences upstream of
991 bp appear to be required for lung-specific expression.
In a previous study with transfected type II cells, Alcorn et al. (2)
observed that basal and DBcAMP-stimulated levels of expression of
SP-A:hGH fusion genes containing 378 bp of SP-A 5'-flanking DNA were
considerably lower than those of fusion genes containing 991 bp of the
5'-flanking sequence. In the present study, equivalent amounts of
lung RNA from a number of transgenic lines containing the different
fusion gene constructs were analyzed on the same Northern blot. We were
surprised to find that the corrected values for hGH mRNA in transgenic
lines carrying the SP-A378:hGH
and
SP-A
991:hGH
fusion genes were roughly equivalent (data not shown).
The 378-bp 5'-flanking sequence of the rabbit
SP-A gene is sufficient for appropriate lung cell-selective expression
of SP-A:hGH fusion genes in transgenic mice. To analyze
cellular localization of transgene expression, immunocytochemical and
in situ hybridization analyses were performed as described in
EXPERIMENTAL PROCEDURES. Adjacent
sections of lung tissue from an adult transgenic mouse carrying the
SP-A4000:hGH
fusion gene (line 3-3) were immunostained for SP-A and hGH. As can be
seen in Fig. 2, the same alveolar epithelial cells that contained hGH immunoreactivity
(B) also immunostained for SP-A
(A); however, other cells that were
immunoreactive for SP-A did not contain detectable hGH protein. The
decreased number of cells immunopositive for hGH compared with SP-A may be due to different secretory pathways of the two proteins. SP-A is
primarily associated with lamellar bodies in type II cells (16),
whereas hGH may be constitutively secreted (40). During sectioning of
the tissue, there is also a potential for removing a portion of the
cell in which one of the proteins is localized while the other protein
remains.
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By in situ hybridization analyses of lung tissues of transgenic mice
containing the
SP-A991:hGH
(line 1699) and
SP-A
378:hGH (line 62-9) fusion genes with an hGH antisense cRNA probe, it is
apparent that hGH mRNA transcripts are present both in alveolar epithelial cells and in the cells of the bronchiolar epithelium (Fig.
3). In situ hybridization analysis of lung
tissue of a nontransgenic adult mouse lung with an antisense hGH cRNA
probe is shown in Fig. 3, A and
B. As can be seen, no hybridization of
the hGH cRNA was detectable. By contrast, hGH mRNA transcripts were
readily detectable in the lung tissues of transgenic mice carrying the SP-A
378:hGH
(Fig. 3, C and
D) and
SP-A
991:hGH
(Fig. 3, E and
F) fusion genes. The finding that
silver grains were localized to cells in the "corners" of alveoli
(Fig. 3, C and E, arrowheads) is suggestive of
transgene expression in type II cells. Expression of the transgene in
bronchiolar epithelial cells (Fig. 3,
C and
E, arrows) of transgenic mice carrying
either the SP-A
378:hGH
(Fig. 3C) or
SP-A
991:hGH
(Fig. 3E) fusion genes was also
apparent. These patterns of expression are identical to in situ
analyses previously performed by others for both mouse (23) and rabbit
(4, 47) SP-A mRNA transcripts. No hybridization signal was detected in
lung sections from the transgenic mice when a sense hGH probe was
utilized (data not shown). These findings suggest that the fusion genes
are expressed in type II cells and in bronchiolar epithelial cells and
that 378 bp of the sequence flanking the 5'-end of the rabbit
SP-A gene is sufficient for appropriate lung cell-selective expression in transgenic mice.
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The 378-bp 5'-flanking sequence from the rabbit
SP-A gene is sufficient to direct appropriate developmental regulation
of SP-A:hGH fusion gene expression in transgenic mouse lung
tissue. To begin to analyze
SP-A 5'-flanking sequences that
mediate appropriate temporal regulation of
SP-A promoter activity, the
developmental regulation of
SP-A4000:hGH
(line 3-3) and
SP-A
378:hGH (line 62-9) fusion genes was analyzed in lung tissues of fetal, neonatal, and adult transgenic mice. Figure
4A shows
Northern blots of RNA isolated from lung tissues of 16- to 19-day
gestational age transgenic fetuses carrying the
SP-A
4000:hGH
fusion gene and their nontransgenic littermates. The blots were probed either with hGH cDNA to analyze transgene expression or with an SP-A
cDNA to analyze endogenous SP-A expression. As can be seen, the
expression of the
SP-A
4000:hGH
transgene was detectable as early as day
17 and increased through day
19. Transgene expression was developmentally regulated
in concert with the endogenous SP-A gene (Fig. 4A). In transgenic mice
carrying
SP-A
378:hGH fusion genes, transgene expression was detectable as early as day 17 (day
16 tissues were not analyzed in this group) and
appeared to reach maximal levels by day
19 (Fig. 4B). These
findings suggest that the genomic regions responsible for appropriate
developmental regulation of the rabbit
SP-A gene lie within the 378-bp region upstream of the SP-A transcription
initiation site.
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Transgene expression in fetal mouse lung tissue in
organ culture is regulated by cAMP and glucocorticoids.
In previous type II cell transfection studies, Alcorn et al. (2)
observed that cAMP induced expression of
SP-A:hGH fusion genes containing
378 to
1,765 bp of SP-A
5'-flanking DNA, whereas Dex, which had little effect on basal
expression, antagonized the stimulatory effects of cAMP. In a study
using fetal rabbit lung explants, Boggaram and Mendelson (6) have found
that glucocorticoids have stimulatory effects on
SP-A gene transcription after
24-72 h of incubation; however, acutely, glucocorticoids inhibit
SP-A gene transcription and antagonize
the stimulatory effects of DBcAMP. In the present study, the expression
of
SP-A
4000:hGH
(line 3-3) and
SP-A
378:hGH (line 62-9) fusion genes was analyzed in transgenic fetal mouse lung
explants incubated for up to 5 days in the absence and presence of
DBcAMP and Dex. Transgene expression was assessed by the accumulation of hGH in the culture medium over each 24-h period. As can be seen in
Fig. 5, expression of the
SP-A
4000:hGH
fusion gene in 16-day fetal mouse lungs in organ culture increased as a
function of time in culture. Treatment of fetal lung explants with
DBcAMP (1 mM) resulted in a three- to sixfold increase in fusion gene expression. The inductive effect of DBcAMP on transgene expression was
of a similar magnitude to the stimulatory effect of cAMP on the
expression of the SP-A gene in rabbit
fetal lung explants (6). On the other hand, Dex
(10
7 M) caused a slight
decrease in hGH expression compared with that in control tissues and
antagonized the stimulatory effect of DBcAMP on fusion gene expression,
reducing the levels of expression two- to fourfold.
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Similar findings of the effects of DBcAMP and Dex on
SP-A promoter activity were obtained
with fetal lung explants from a transgenic mouse line carrying the
SP-A378:hGH
fusion gene. Figure
6A
shows the effects of DBcAMP and of DBcAMP and Dex in combination on the
expression of
SP-A
4000:hGH
and
SP-A
378:hGH fusion genes in lung explants of 16-day gestational age fetal transgenic mice after 5 days of organ culture. Shown are the levels of
hGH that accumulated in the culture medium over a 24-h period between
days 4 and
5 of culture. As can be seen, DBcAMP
stimulated expression of both fusion genes approximately threefold
compared with fusion gene expression in lung explants maintained in
control medium, whereas Dex antagonized the stimulatory effect of
DBcAMP. The effect of Dex to antagonize cAMP induction of
SP-A promoter activity is similar to
that observed in type II cells transiently transfected with
SP-A:hGH fusion gene constructs (2,
3). These findings suggest that sequences responsible for cAMP
stimulation and glucocorticoid inhibition of rabbit
SP-A gene promoter activity lie within
the 378-bp SP-A 5'-flanking
region.
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To compare the effects of cAMP and Dex on transgene expression with
those on expression of the endogenous
SP-A gene in the same lung tissue
samples, lung explants isolated from 16-day gestational age transgenic
mice carrying the
SP-A4000:hGH
fusion gene were maintained in organ culture for 5 days in the absence
and presence of DBcAMP, Dex, or DBcAMP plus Dex at various
concentrations. Figure 6B shows a
Northern blot of RNA isolated from the transgenic fetal lung explants
that was probed for hGH and SP-A mRNA transcripts. As can be seen,
although DBcAMP increased transgene expression, there was no detectable
effect of cAMP on expression of the endogenous mouse
SP-A gene. Although Dex
(10
7 M) inhibited transgene
expression and had a dose-dependent effect to antagonize the
stimulatory effect of cAMP, Dex caused a dose-dependent increase in the
levels of endogenous mouse SP-A mRNA. These findings indicate that
species-specific differences in the effects of cAMP and glucocorticoids
on SP-A transcription are due to
species differences in cis-acting
elements rather than to differences in
trans-acting factors.
Mutation of the PBE and CRESP-A elements
within the 5'-flanking sequence of the rabbit
SP-A gene has no effect on tissue-specific
or developmental regulation of SP-A:hGH
transgene expression.
Previously, our laboratory (2, 13, 31) observed that an E box and a
putative nuclear-receptor binding site, termed PBE and
CRESP-A, respectively, are
required for basal and cAMP-induced expression of rabbit
SP-A:hGH fusion genes transfected into
rat, human, and rabbit type II cells. To investigate the regulatory influences of these elements on the levels of expression in the lung as
well as on lung-specific and developmental regulation of transgene
expression, transgenic mice were created carrying SP-A:hGH fusion genes in which one of
these elements, PBE or CRESP-A, was mutated (Fig. 1). The CRESP-A
sequence TGACCTCA was mutated either to TGACGACA (CRE) or to the
sequence TTCTAGAA (CRE-S). These mutations were previously found to
markedly reduce cAMP induction of rabbit
SP-A:hGH fusion gene expression in
transfected type II cells (2, 31). The PBE (CCTCGTGA) was mutated to ACTCTAGA, a sequence found to markedly reduce basal and prevent cAMP
induction of SP-A promoter activity in
transfected type II cells (13). The CRE
and PBE
mutations
were created in the SP-A
378:hGH
fusion gene
(SP-A
378CRE
:hGH
and SP-A
378PBE
:hGH,
respectively), whereas, the CRE-S mutation was created in the
SP-A
991:hGH
fusion gene
(SP-A
991CRE-S:hGH). These mutated fusion genes were then introduced into transgenic mice;
levels of expression of the
SP-A
378:hGH
transgenes with and without the mutations were determined in five to
nine independently derived lines of mice by Northern analysis. The blots were reprobed for 18S rRNA to correct for differences in loading
and transfer. As can be seen in Fig. 7,
top, when transgene expression levels were analyzed without
regard to transgene copy number in each of the lines, the PBE mutation
was found to have little effect on expression of the
SP-A
378:hGH
fusion gene compared with mice carrying the wild-type
SP-A
378:hGH
fusion gene. By contrast, the CRE
mutation was found to decrease
expression by ~50%. As can be seen in Fig. 7, bottom,
when transgene copy number was taken into account, the PBE
mutation was found to cause a 50% reduction in expression of the
SP-A
378:hGH fusion gene, whereas the inhibitory effect of the CRE
mutation was more pronounced, resulting in a 65% reduction compared with wild-type levels. These results indicate that these elements do act as
enhancers of rabbit SP-A gene
expression. Surprisingly, neither of the CRE
nor PBE
mutations had an apparent effect on lung-specific expression (Table 2).
In fact, in the case of the CRE-S and PBE
mutations in the
context of the
SP-A
991:hGH and
SP-A
378:hGH
fusion genes, respectively, there was generally greater lung
specificity of expression compared with the corresponding wild-type
fusion gene constructs. Furthermore, in transgenic mice carrying the
SP-A
991:hGH
fusion gene containing the CRE-S mutation, we observed that fusion gene
expression appeared to be developmentally regulated in a manner similar
to that of the SP-A
4000:hGH
and
SP-A
378:hGH
fusion genes and to the endogenous mouse
SP-A gene (data not shown).
|
![]() |
DISCUSSION |
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---|
In the present study, transgenic mice were used to define genomic
regions upstream of the rabbit SP-A
gene that mediate lung type II cell-specific, developmental, and
hormonal regulation of SP-A gene
expression. We found that SP-A:hGH
fusion genes containing 4,000 bp of the sequence flanking the
5'-end of the rabbit SP-A gene
were expressed in transgenic mice in a lung-specific manner and were
developmentally regulated in concert with the endogenous mouse
SP-A gene. To begin to define the
minimum genomic region required for lung cell-specific and appropriate
developmental regulation of SP-A
promoter activity, transgenic mice were also created carrying
SP-A991:hGH,
SP-A
378:hGH,
and
SP-A
47:hGH fusion genes. In three of the five lines of transgenic mice carrying SP-A
991:hGH
that were analyzed for tissue-specific expression, fusion gene
expression was essentially lung specific. However, in the two other
lines examined, considerable levels of transgene expression were also
detected in the heart. In two of five lines of transgenic mice carrying
SP-A
378:hGH
fusion genes, expression was essentially lung specific, whereas in
three other lines, there were also substantial levels of transgene
expression detected in the heart, thymus, and spleen. Expression in the
lung was always greater than in the other tissues. The variability in
ectopic expression from one line of mice to the other is likely due to positional effects of transgene integration (34).
Expression of the SP-A gene is
essentially lung specific (7); however, low levels of SP-A mRNA have
been reported in epithelium of the small intestine (36) and the thymus
and prostate (27). In the present study, there was no evidence for
transgene expression in the small intestine; however, when ectopic
expression of SP-A:hGH transgenes
containing 991 bp of SP-A
5'-flanking DNA was observed, the highest levels were found in
the heart, thymus, and spleen. The finding that in mice carrying
SP-A
991:hGH
transgenes with a scramble mutation in
CRESP-A
(SP-A
991CRE-S:hGH) there was little or no ectopic expression in the heart suggests that
transcription factors in the heart may bind to this element and
activate the SP-A promoter in the
absence of constraints imposed by upstream sequences. Similarly, the
finding that expression of
SP-A
378:hGH
transgenes with a mutation in the PBE
(SP-A
378PBE
:hGH) was essentially lung specific suggests that transcription factors in
the heart, spleen, and thymus may activate the
SP-A promoter by binding to this
element in the absence of constraints imposed by upstream sequences.
Evidence for the role of upstream sequences in conferring binding
specificity to CRESP-A was
obtained in studies with type II cells transfected with
SP-A378:hGH
and
SP-A
991:hGH fusion genes with and without mutations in
CRESP-A. When
CRESP-A (TGACCTCA) was mutated to
the canonical palindromic CRE
(CREpal) sequence (TGACGTCA),
known to bind the transcription factor CREB, in the context of the
SP-A
378:hGH
fusion gene, basal and cAMP-induced expression were increased compared
with that of fusion genes containing the wild-type sequence (2). In
contrast, in the context of the
SP-A
991:hGH
fusion gene, mutagenesis of
CRESP-A to
CREpal resulted in a marked
decrease in basal and cAMP-induced expression (31). These findings
suggest that in the absence of upstream sequences, mutagenesis of
CRESP-A to a sequence that binds
CREB can increase expression and cAMP responsiveness, whereas in the
context of the 991-bp 5'-flanking sequence, CREB homodimers may
be prevented from binding to
CREpal because of restraints
imposed by other trans-acting factors
bound to upstream response elements. We have obtained evidence that
CRESP-A does not bind CREB;
rather, a member of the nuclear-receptor family appears to bind to this
site (31).
By in situ hybridization, the
SP-A378:hGH
fusion gene was found to be expressed in alveolar epithelial cells that
have characteristics of type II cells in terms of their localization at
the corners of the alveoli. Expression also was evident
in bronchiolar epithelial cells. These patterns of expression
correspond to those of the endogenous
SP-A gene (4, 23, 47). The finding that the
SP-A
378:hGH
fusion gene was developmentally regulated in association with the
endogenous mouse SP-A gene suggests
that the genetic elements required for accurate developmental timing of
expression are also contained within the 378-bp region. Based on these
findings, we suggest that the regulatory elements required for lung
cell-selective and developmental regulation of
SP-A gene expression are localized
within the 378-bp 5'-flanking region and that sequences upstream
of this region are required for preventing activation of the
SP-A promoter in other tissues.
Transgenic technology has been used to begin to define sequences that
mediate lung cell-specific and developmental timing of expression of
SP-C and the Clara cell secretory
protein/Clara cell 10-kDa
protein
(CCSP/CC10)
genes. In studies of the rat
CCSP/CC10 gene, it was found that 2.25 kb of the 5'-flanking sequence
directed lung- and Clara cell-specific expression of an
hGH reporter gene (17); however,
transgene expression was detected as early as embryonic
day 12.5, at least 4 days before the
time that expression of the endogenous
CCSP/CC10
gene is initiated (18). In subsequent studies, it was found that
although 166 bp of the mouse CCSP/CC10 5'-flanking region were
sufficient to mediate Clara cell-specific expression, enhancer
sequences between 166 and
803 bp were required to achieve
maximal levels of transgene expression (35).
In transgenic mouse studies to define the genomic elements required for lung cell-specific and developmental timing of expression of the human SP-C gene, it was found that 3.7 kb of the SP-C 5'-flanking region linked to bacterial chloramphenicol acetyltransferase mediated high levels of chloramphenicol acetyltransferase expression in cells of the alveolar and bronchiolar epithelia (15, 46). Interestingly, although SP-C expression is restricted to type II cells in the mouse lung, proSP-C in the human lung is detectable both in alveolar and in distal bronchiolar cells (22). These findings suggest the presence of sequences within the 5'-flanking region of the human SP-C gene that mediate bronchiolar as well as type II-specific expression.
The effects of cAMP and Dex on SP-A
gene expression differ among species (29). Whereas expression of rabbit
(7), human (33), and baboon (39) SP-A
genes is markedly stimulated by cAMP, expression of rat (32) and mouse
(11) SP-A genes is relatively
unaffected by cAMP treatment. In the present study, we observed that
DBcAMP treatment of lung explants from 15- to 16-day gestational age
fetal transgenic mice increased expression of rabbit
SP-A4000:hGH
and
SP-A
378:hGH
integrated transgenes in a manner similar to its effects on
transcription of the endogenous rabbit
SP-A gene in fetal rabbit lung
explants (7) and on expression of rabbit
SP-A:hGH fusion genes in transfected type II cells (2, 3, 31). Because the endogenous mouse SP-A gene is not responsive to cAMP,
the present findings suggest that mouse type II cells contain the
cellular proteins and transcription factors required to mediate cAMP
induction of rabbit SP-A promoter activity. Therefore, it is likely that species-specific differences in
cAMP responsiveness are due to genetic differences in enhancer elements
that confer cAMP responsiveness to the promoter rather than to
species-specific differences in
trans-acting factors. In contrast to
the stimulatory effects of cAMP on transgene expression, Dex treatment
of the transgenic fetal mouse lung explants inhibited fusion gene
expression and antagonized the stimulatory effects of DBcAMP. The
inhibitory effects of glucocorticoids on cAMP induction of
SP-A:hGH fusion gene expression are
similar to those observed previously with type II cells transfected
with rabbit SP-A:hGH fusion genes (2).
However, these findings are in contrast to the stimulatory effects of
glucocorticoids when added alone and in combination with cAMP on the
transcriptional activity of the endogenous
SP-A gene in fetal rabbit lung
explants (7). In the present study, we observed that Dex caused a
dose-dependent increase in the levels of endogenous mouse SP-A mRNA in
the transgenic fetal lung explants; this was coordinate with a
dose-dependent decrease in hGH mRNA levels.
The mechanisms whereby glucocorticoids enhance transcriptional activity of the endogenous SP-A gene have not been defined. From the findings of the present study and from those with transfected type II cells (2), it is likely that the stimulatory effects of glucocorticoids on expression of the rabbit SP-A gene are mediated by response elements that lie >4,000 bp upstream of the transcription initiation site, within the structural gene, or in the 3'-flanking region. In cell transfection studies, Alcorn et al. (2) also observed that sequences within the first exon and intron of the rabbit SP-A gene failed to mediate glucocorticoid induction of SP-A promoter activity. The stimulatory effects of glucocorticoids may be mediated by glucocorticoid-receptor binding to a glucocorticoid response element(s) or by induction or activation of another transcription factor. If the latter is the case, such a transcription factor must also act through an element(s) that lies outside the confines of the SP-A promoter constructs used in these studies. Because the observed effects of glucocorticoids on expression of SP-A:hGH transgenes are inhibitory, it is possible that in the absence of a functional glucocorticoid response element, the glucocorticoid receptor represses cAMP induction of SP-A promoter activity by an inhibitory interaction with a cAMP-responsive transcription factor or by competition for binding to an essential coactivator. Glucocorticoids have been reported to inhibit transcription factor activator protein-1 activation by direct interaction of the glucocorticoid receptor with c-Jun (38, 48) and by competition for binding to limiting amounts of the coactivator CBP/p300 (21).
In previous studies using transfected type II cells, Alcorn et al. (2)
and Gao and colleagues (13, 14) observed that basal and cAMP induction
of rabbit SP-A promoter activity
requires the cooperative interaction of transcription factors bound to two E boxes, termed DBE (980 bp) and PBE (
80 bp), and to
a CRE-like element (CRESP-A;
261 bp).
SP-A
976:hGH
and
SP-A
378:hGH fusion genes, which lack the DBE, manifested a marked reduction in
basal and cAMP-stimulated expression compared with that of SP-A
991:hGH
fusion genes that contain this sequence; however, an inductive effect
of cAMP was still observed. Mutagenesis of CRESP-A within the
SP-A
991:hGH
fusion gene had similar effects to reduce overall levels of basal and
cAMP-induced expression; again, an ~10-fold inductive effect of cAMP
on SP-A promoter activity was still
evident (2, 31). On the other hand, mutagenesis of the PBE caused a
marked decrease in basal expression and loss of cAMP-inducible
expression (13). In contrast to our findings using transfected type II
cells, we were surprised to find that SP-A
378:hGH
fusion genes, which lack the DBE, were expressed in lung tissues of
transgenic mice at levels roughly equivalent to those of
SP-A:hGH fusion genes containing
991
bp of the SP-A 5'-flanking
sequence. Furthermore, an
SP-A
378PBE
:hGH fusion gene (which lacks both DBE and PBE) and
SP-A
378CRE
:hGH and
SP-A
991CRE-S:hGH
fusion genes (containing mutations in the
CRESP-A) were also expressed in
the lung and in a lung-specific manner, albeit at lower levels than the
wild-type constructs.
These findings suggest a redundancy of response elements within the SP-A 5'-flanking region that have the capacity to mediate high levels of SP-A promoter activity in the context of multiple inserted copies of a transgene in tandem arrays. Similar results were obtained in studies of the pancreatic elastase I gene in which it was found, with transfected pancreatic acinar cells, that each of three genomic domains, which bind different nuclear proteins, were required for enhanced levels of expression in an acinar cell-specific manner (24). By contrast, in transgenic mice, it was found that any two of the three domains were sufficient to direct pancreas-specific expression (45). A similar mechanism may apply to the rabbit SP-A gene; redundancy of dissimilar elements allows for appropriate expression of SP-A promoter activity in transgenic mice when single elements are eliminated.
In type II cell transfection studies, our laboratory has found that
basal and cAMP-induced expression of
SP-A promoter activity are mediated by
the cooperative interaction of ubiquitously expressed [e.g.,
upstream stimulatory factor-1 (13, 14), Sp1 (50)] and
tissue-selective [e.g., thyroid transcription factor-1
(26)] transcription factors as well as unidentified members of
the nuclear-receptor family (2, 31, 49). We suggest that increased
ectopic expression of SP-A:hGH fusion
genes containing 991 bp of SP-A
5'-flanking DNA compared with
SP-A
4000:hGH
transgenes may be due to the presence of binding sites for ubiquitously
expressed transcription factors within the 991-bp
SP-A 5'-flanking region and to
the absence of putative inhibitory elements that block expression in
other tissues. Random integration of the transgenes into chromosomal regions adjacent to other enhancer/promoter elements may also cause
their inappropriate expression; however, this would not explain why the
shorter SP-A:hGH transgenes manifest a
higher degree of ectopic expression than the
SP-A
4000:hGH transgene.
In summary, the results of this study indicate that a 378-bp genomic
sequence upstream of the transcription initiation site of the rabbit
SP-A gene directs expression of the
SP-A promoter in lung type II and
bronchiolar epithelial cells. This genomic region is also sufficient
for appropriate developmental regulation of
SP-A promoter activity in transgenic
mice. Sequences between 991 and
4,000 bp appear to
prevent ectopic expression of the transgene, which was found to occur
predominantly in the heart, thymus, and spleen. In subsequent studies,
we will attempt to more fully define the regulatory elements within
this 378-bp region that are responsible for the temporal and spatial
regulation of SP-A gene expression. In
this manner, a better understanding of the molecular mechanisms
involved in developmental and tissue-specific regulation of the genes
expressed in pulmonary epithelium can be achieved.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Jo F. Smith for expert help with tissue culture.
![]() |
FOOTNOTES |
---|
This research was supported in part by National Heart, Lung, and Blood Institute Grant R37-HL-50022 (to C. R. Mendelson) and Grant-in-Aid 92G-083 from the American Heart Association Texas Affiliate (to J. L. Alcorn).
J. L. Alcorn was supported in part by National Heart, Lung, and Blood Institute Research Training Grant 5-T32-HL-07362.
Present addresses: J. L. Alcorn, Dept. of Pediatrics, UT-Houston Medical School, 6431 Fannin, Suite 3.222, Houston, TX 77030; L. F. Michael, Joslin Diabetes Center, One Joslin Place, Boston, MA 10015; E. Gao, Dept. of Anesthesiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: C. R. Mendelson, Dept. of Biochemistry, Univ. of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9038 (E-mail: cmende{at}biochem.swmed.edu).
Received 16 September 1998; accepted in final form 29 March 1999.
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