Human SP-A 3'-UTR variants mediate differential gene
expression in basal levels and in response to dexamethasone
Guirong
Wang1,
Xiaoxuan
Guo1, and
Joanna
Floros1,2
Departments of 1 Cellular and Molecular Physiology
and 2 Pediatrics, The Pennsylvania State University
College of Medicine, Hershey, Pennsylvania 17033
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ABSTRACT |
Human surfactant
protein A (SP-A) is encoded by two genes (SP-A1, SP-A2), and
each is identified with several alleles. SP-A is involved in normal
lung function, innate immunity, inflammatory processes, and is
regulated by glucocorticoids. We investigated the role of
3'-untranslated region (UTR) of 10 SP-A variants on gene expression
using transient transfection of 3'-UTR constructs in the human lung
adenocarcinoma cell line NCI-H441. We found: 1) both basal
mRNA and protein levels of the reporter gene of SP-A 3'-UTR constructs
are significantly (P < 0.01) reduced compared with
controls (vector pGL3 and surfactant protein B pGL3) and that
differences exist among alleles; and 2) after dexamethasone (Dex) treatment (100 nM for 16 h), mRNA was reduced
(31-51%). Seven alleles showed a significant decrease
(P < 0.05) in mRNA, and three did not. Reporter
activity was also decreased, from 17% (1A1) to 38% (1A),
with six alleles showing a significant decrease. The data indicate that
the 3'-UTR of SP-As play a differential role in SP-A basal expression
and in response to Dex. Therefore, a careful consideration of
individual use of steroid treatment may be considered.
allele; gene regulation; surfactant protein A; 3'-untranslated
region
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INTRODUCTION |
PULMONARY
SURFACTANT, a lipoprotein complex, is essential for normal lung
function because deficiency of surfactant can lead to various diseases,
including respiratory distress syndrome (RDS), in the prematurely born
infant. Surfactant protein A (SP-A) is involved in surfactant
physiology (19) and plays a major role in innate host
defense and the regulation of inflammatory processes in the lung
(38, 51).
The human SP-A (hSP-A) locus consists of two functional genes,
SP-A1 and SP-A2, in opposite transcriptional
orientation, and a nonfunctional gene (25). A number of
alleles have been characterized for each SP-A gene (17).
On the basis of coding sequence differences to date, four alleles of
SP-A1 (6A, 6A2, 6A3,
6A4) and six alleles of SP-A2 (1A,
1A0, 1A1, 1A2, 1A3,
1A5) are found frequently (>1%) in the general population
(17). In addition, sequence variability within the
3'-untranslated region (3'-UTR) has been observed between the human
SP-A genes/alleles (26, 34, 53). Therefore, sequence
differences among human SP-A genes/alleles within both the coding and
noncoding regions hold the potential for functional and regulatory
differences, respectively. Our preliminary published information
indicates that both the coding region and the noncoding region may
result in functional (58, 59) and regulatory
(26) differences between the two SP-A genes and among the alleles.
Human SP-A is regulated by a variety of molecules, including several
hormones, some cytokines, growth factors, and oxygen (43).
For example, insulin, TNF-
, and glucocorticoids at high concentrations decrease human SP-A expression (8, 15, 18, 28, 31,
57). In clinical practice, steroid therapy is widely used for
mothers who are in danger of delivering prematurely and is also used
commonly in the management of preterm infants with chronic lung disease
[bronchopulmonary dysplasia (BPD)] to improve pulmonary
compliance and to wean infants from ventilators (6, 12, 33,
44). However, concerns have been raised about side effects of
steroid therapy, such as increased blood pressure, increased blood
sugar, transient adrenal suppression in infants, and impaired bone
growth in children (30, 48, 63). Recent findings from an
animal study indicate that dexamethasone (Dex; a synthetic
glucocorticoid) administration impairs normal postnatal lung growth in
rats (49). Therefore, a careful consideration of balancing
short- and long-term effects of steroid therapy may be warranted. In
this regard, weighing genetic and environmental factors together with
such untoward effects of steroid therapy may allow for a better
assessment of the optimal dose and duration of Dex treatment for a
given individual's therapy.
Glucocorticoids (GCs) play a role in the modulation of gene
transcription, posttranscription, and translation events by several different mechanisms. The glucocorticoid effect on human SP-A gene
expression is complex because it may involve transcriptional and
posttranscriptional events (8, 27, 28, 43, 52). In human
fetal lung explants, GCs reduce SP-A gene expression in a time- and
dose-dependent manner. At low concentrations (<10 nM), GCs increase
SP-A mRNA levels (5, 14, 28, 39, 43), and at high
concentrations (100 nM), GCs decrease SP-A expression (26-28, 31, 43). In contrast, Dex at 1-100 nM
has only an inhibitory effect on SP-A gene expression in the lung
adenocarcinoma cell line NCI-H441 (50, 52). The decay rate
of SP-A mRNA in the presence of Dex was shown to be biphasic in fetal
lung explants, with an initial rapid decrease followed later by a
slower decay rate (28). It was suggested that this
biphasic decay rate was a result of differential effects of Dex on
SP-A1 and SP-A2 mRNA stabilities. In fact, the
two SP-A genes have been shown to be differentially regulated by
certain agents, including GCs (27, 31, 35, 42, 55), and
certain SP-A alleles may be differentially regulated by Dex
(26).
Accumulating evidence indicates that the 3'-UTR of mRNA may play an
important role in regulating gene expression and may be essential for
the appropriate expression of many genes (10). Recent
findings show that the 3'-UTR of mRNA may affect several processes of
gene expression, such as mRNA stability (7, 37, 45, 46),
increased efficiency of mRNA formation (21), transport and
localization, posttranscriptional modification and degradation, and
translation (11, 16, 21, 22). GCs have been shown to
influence gene expression through the modulation of mRNA-protein interactions in the 3'-UTR of mRNA (20) and of the 3'-UTR
poly(A) of mRNA (23, 24). Moreover, studies of SP-A
indicate that certain regulatory regions, including the 3'-UTR of the
human SP-A gene, play a role in the GC-induced inhibition of gene
expression and may mediate differential allelic expression in response
to Dex (26). Allelic differences within regulatory regions
may account for differences in drug response among individuals, and these differences may help us to better understand some of the side
effects that may occur in the course of steroid therapy of prematurely
born infants with RDS, BPD, and/or other pulmonary diseases. Therefore,
a more in-depth understanding of the factors affecting the expression
of SP-A may lead to better strategies in the treatment and/or
prevention of pulmonary diseases of the prematurely born infant.
In the present study, we investigated the role of 3'-UTR on gene
expression of 10 hSP-A alleles 1) at the basal level and 2) in response to Dex treatment. We observed that the SP-A
3'-UTR leads to a decrease in basal gene expression compared with
controls and reduces expression levels after Dex treatment. Differences in basal level and in response to Dex exist among the 10 hSP-A alleles
studied. Nucleotide differences within the 3'-UTR region among SP-A
alleles and potential factors that may account for these observations
are discussed.
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MATERIALS AND METHODS |
Cell line and cell culture conditions.
The lung adenocarcinoma cell line NCI-H441 from American Type Culture
Collection (Manassas, VA) was used. The cells were grown in RPMI 1640 medium (Invitrogen, Carlsbad, CA) containing 10% heat-inactivated
fetal bovine serum (FBS), 1× antimycotic-antibiotic solution (Sigma,
St. Louis, MO), and 1% L-glutamine (Sigma) at 37°C
in 5% CO2 atmosphere. The cells were fed and passaged weekly.
SP-A 3'-UTR plasmid constructs.
Luciferase reporter constructs were generated by ligation of the pGL3
luciferase reporter vector (Promega, Madison, WI) with PCR products
spanning the entire 3'-UTR of SP-A, including the poly(A) addition
recognition signal (Fig. 1). First,
SP-A1- and SP-A2-specific segments were
amplified using oligonucleotide-specific primers for SP-A1
(primer pair 326/1,074) and SP-A2 (primer pair 327/1,075;
all primer information shown in Table 1)
together with genomic DNA from individuals of certain homozygous
genotypes (e.g.,
6A26A2/1A01A0, etc.).
PCR of gene-specific genomic segments was performed in duplicate and
treated as independent samples throughout to better control for PCR
error. PCR products were cloned and sequenced. Lack of PCR errors would
most likely result in clones with identical sequence from each
independent PCR.

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Fig. 1.
Firefly luciferase (Luci) vector pGL3/surfactant protein A (SP-A)
3'-untranslated region (UTR) construct. The firefly luciferase gene was
driven by the simian virus 40 (SV40) promoter. SP-A 3'-UTR of ~1.3-kb
fragments of alleles (6A, 6A2, 6A3,
6A4, 1A, 1A0, 1A1, 1A2,
1A3, 1A5) was inserted into XbaI
site of vector pGL3. A total of 10 constructs, each containing a
different 3'-UTR, were generated. In addition, a 1.3-kb fragment from
the human surfactant protein B (SP-B) gene cDNA was cloned into this
site to generate a control SP-B construct.
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The SP-A1 and SP-A2 gene-specific PCR products
were then used as templates for a second round of amplification with
the primer pairs 1073/1074 and 1073/1075, thus amplifying the entire
3'-UTR. Primer location and sequences are shown in Table 1. The
nucleotide locations are according to White et al. (60)
for SP-A1 and Katyal et al. (32) for
SP-A2. All primers at the 3'-UTR contain the specific enzyme
recognition site of XbaI. These XbaI PCR products were then cloned into the XbaI site of the pGL3 vector.
A total of 10 constructs, four SP-A1 alleles (6A,
6A2, 6A3, 6A4) and six
SP-A2 alleles (1A, 1A0, 1A1,
1A2, 1A3, 1A5), were generated. In
addition, a positive reporter control plasmid was prepared by cloning a
1.3-kb fragment of surfactant protein B (SP-B) cDNA consisting of the
entire coding region and ~0.3 kb of the 3'-UTR (29).
Recombinant DNA was performed according to standard methods
(54). Plasmid DNA for transfection was prepared using the
Qiagen plasmid Maxi kit (Qiagen, Hilden, Germany) according to the
manufacturer's instructions.
Transient transfection and Dex treatment.
Various constructs and reporter control plasmids were transiently
cotransfected into NCI-H441 cells, and expression of the reporter gene
was analyzed (Fig. 2). The reporter
control plasmid used as standardization in this study was
Renilla luciferase reporter plasmid pRL-SV40 (Promega) or
pCMV-SPORT-
-gal (Invitrogen). Our preliminary findings unexpectedly
indicated that Renilla luciferase reporter plasmid pRL-SV40
responds to Dex treatment, making it an unsuitable control for the Dex
experiments. Thus Renilla was used for assessment of basal
levels, and
-galactosidase (
-gal) was used where assessing levels
in response to Dex.

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Fig. 2.
Outline of the cotransfection protocol. The experimental construct
and transfection control vector were cotransfected in the H441 cells to
control for the efficiency of transfection. The results are normalized
to the transfection control. -gal, -galactosidase; Dex,
dexamethasone.
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NCI-H441 cells were grown to 80-90% confluency in 10-cm dishes
and subcultured into six-well culture plates with 1 × 106 cells/well 24 h before transfection. Four hours
before transfection, the RPMI 1640 medium plus 10% FBS was replaced
with DMEM (Invitrogen) containing neither FBS nor antibiotics.
The transfection procedure was performed using the Lipofectamine Plus
reagent kit (Invitrogen). In brief, 1 µg of DNA of experimental construct plus 0.05 µg of DNA of control pRL-SV40 plasmid or 0.3 µg
of DNA of pCMV-SPORT-
-gal plasmid were diluted into 100 µl of DMEM
without serum. Six microliters of Plus reagent were added, and the
mixture was incubated for 15 min at room temperature. In another tube,
4 µl of Lipofectamine reagent were diluted into 100 µl of DMEM
without serum. The components of the two tubes were then combined,
mixed, and incubated for 15 min at room temperature. The above
DNA-Lipofectamine complex was added to each well containing H441 cells
with 2.5 ml of fresh medium. Four hours after transfection, the DMEM
with 10% FBS was added to normal culture volume (5 ml/well). Transfection was carried out for 36 h or for the indicated time point at 37°C in 5% CO2 atmosphere for basal gene
expression assay. For Dex-treated experiments, the medium was changed
to RPMI 1640 plus 1% L-glutamine 24 h after
transfection, and then cells were treated with Dex (100 nM) 36 h
after transfection.
Enzyme activity assay.
Both dual luciferase assay (firefly/Renilla luciferase) and
firefly luciferase/
-gal activity were used in this study. The pRL-SV40 (Renilla luciferase) in dual luciferase assay or
pCMV-SPORT-
-gal was cotransfected with pGL3 reporter vector (firefly
luciferase) to H441 cells. Thus all values are expressed as the ratio
firefly-Renilla luciferase activity or firefly luciferase
activity-
-gal activity. For all experiments, transfection and
luciferase assay were performed in triplicate.
Dual luciferase assay.
Dual luciferase assay was performed with the Dual luciferase reporter
assay system (kit) (Promega). Transfected cells were harvested at
36 h after transfection or at other time points according to
statement in the experiments. The cells were washed with 1× PBS and
were dissolved in 500 µl of 1× passive lysis buffer. The culture
plates were rocked at room temperature for 15 min, and the lysate was
transferred to a tube and centrifuged for 1 min at 4°C to clear the
lysate. Twenty microliters of cell extract were transferred into a
luminometer tube containing 100 µl of luciferase Assay Reagent II
(LAR II). The tube was placed in an FB12 luminometer (Zylux, Maryville,
TN) to initiate firefly luciferase activity reading. Stop & Glo Reagent
(100 µl) was placed into the tube to initiate Renilla
luciferase activity reading. The ratio of firefly luciferase activity
to Renilla luciferase activity was calculated.
Luciferase assay and
-gal enzyme assay.
Luciferase assay and
-gal enzyme assay were performed with the
luciferase reporter assay system and the
-gal enzyme assay system
(Promega). Briefly, transfected cells were harvested at various time
points after transfection with or without Dex treatment. The cells were
washed with 1× PBS and were dissolved in 500 µl of 1× reporter
lysis buffer. The culture plates were rocked at room temperature for 15 min, and the lysate was transferred into a tube. The lysate was frozen
and thawed for several cycles and centrifuged for 1 min at 4°C to
remove cell fragments. Firefly luciferase was analyzed according to the
above statement. For the
-gal activity assay, 150 µl of diluted
(3:1) cell extract and 150 µl of assay buffer (2×) were put together
and mixed, and the mixture was incubated at 37°C for 30 min. The
reaction was stopped by the addition of 500 µl of 1 M sodium
carbonate. The absorbance at 420 nm was read with a spectrophotometer
(Ultrospec 4050, Cambridge, UK). The ratio of firefly luciferase
activity to
-gal activity was calculated.
Total mRNA preparation and measurement by real-time PCR method.
Total mRNA was prepared from cells according to the method of RNA-Bee
kit (Tel-Test, Friendswood, TX). Briefly, the culture medium in the
wells was removed, and the cells were washed with PBS buffer. One
milliliter of RNA-Bee solution was added, and the cells were
homogenized after addition of 0.2 ml of chloroform. The aqueous phase
was transferred to a clean tube, and total RNA was precipitated by
adding 0.5 ml of isopropanol for 5-10 min at room temperature and
centrifuging at 12,000 g for 5 min at 4°C. The pellet was
washed with 75% ethanol. The total RNA was dissolved in RNase-free
ddH2O. Any contaminating DNA was removed from RNA
preparations by using a DNA-free kit (Ambion, Austin, TX). Real-time
PCR was performed using the ABI Prism 7700 sequence detection system
and a kit of TaqMan one-step RT-PCR Master Mix Reagents (Applied
Biosystems, Foster City, CA). In brief, 100 ng of RNA were added into
50 µl of real-time PCR mix buffer. The buffer contained
forward/reverse primer pairs (each 50 nM), such as primers 1152/1153
targeting the reporter gene (luciferase gene) of pGL3, and a probe
primer, such as probe-pGL (see Table 1), and other enzyme and mixing
reagents provided by the manufacturer (Applied Biosystems). The
real-time PCR reaction was carried out through two steps: 1)
one cycle of 48°C for 30 min and 95°C for 10 min, and 2)
43 cycles of 95°C for 15 s and 60°C for 1 min. The mRNA of
luciferase from RNA preparation was standardized with control vector
and expressed as copies per nanogram of total RNA.
Statistical analysis.
In the present study, at least three independent experiments and
triplicate culture dishes for each experiment were performed. The data
were analyzed using the ANOVA test. Statistical significant differences
were considered when P < 0.05, and the results are expressed as means ± SD.
Alignment of SP-A 3'-UTR DNA sequences.
The alignment was performed using a megAlign program of DNASTAR
(version 5.0) by clustal V method and included entire 3'-UTR sequences
of all 10 alleles after the translation termination codon.
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RESULTS |
Comparison of 3'-UTR DNA sequences of 10 hSP-A alleles.
The entire 3'-UTR of 10 hSP-A alleles from human genomic DNA was cloned
using PCR amplification. To eliminate clones with PCR errors, two
independent PCR amplifications were performed for each SP-A allele, and
then three independent clones from each PCR reaction were sequenced.
The sequence identity and divergence of 10 SP-A 3'-UTR DNA sequences
are summarized in Table 2. Specific sequence differences between the SP-A genes and/or alleles include the
following: 1) an 11-bp insertion/deletion (indel) that is present at nucleotide positions 405-416 (26) in all
six SP-A2 alleles and the SP-A1, 6A2
allele; this 11-bp sequence is absent from the other three
commonly found alleles of SP-A1; 2) all
SP-A2 alleles lack 3 bp starting at the nucleotide position
713 after the stop codon compared with SP-A1; 3)
all SP-A1 alleles lack 5 bp starting at the nucleotide position 934 after the stop codon compared with SP-A2;
4) other single nucleotide transitions and transversions
among the alleles were identified. The nucleotide length of the 3'-UTR
for all six SP-A2 alleles is 1,308, for 6A2 is
1,304, and for the other three SP-A1 alleles (6A,
6A3, 6A4) is 1,293. Alignment results of the 10 3'-UTR DNA sequences indicate that the identities between
SP-A1 and SP-A2 ranged from 87.8 to 89.2%;
identities among SP-A1 alleles ranged from 98.4 to 99.8%, and among SP-A2 alleles are from 99.2 to 99.8% (Table 2).
Time course of mRNA and protein mediated by SP-A 3'-UTR.
The mRNA and protein (luciferase activity) contents were assessed from
6 to 52 h after transfection. The SP-A2 (1A allele) 3'-UTR construct was used for this experiment. As shown in Fig. 3, the results indicate that the mRNA
level increases starting at 10 h after transfection and reaches
the highest level at 18 h. From 18 to 24 h, the mRNA level
slowly deceases and then reaches a plateau that is maintained at least
up to 52 h from the start of the experiment. The time course of
luciferase activity follows the mRNA time course as it may be expected.
Therefore, a significant increase of luciferase activity starts at
24 h after transfection and peaks at 38 h (Fig. 3). On the
basis of these results, the 36-h time point was used for subsequent
experimentation.

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Fig. 3.
Time course of SP-A (1A) 3'-UTR-mediated expression. Time
courses of mRNA and protein (luciferase activity) were analyzed from 6 to 52 h after transfection using the SP-A (1A) 3'-UTR construct.
The mRNA level increases starting 10 h after transfection and
reaches the highest level at the 18-h time point. From 18 to 24 h,
the mRNA level slowly deceases and then stays at a plateau for up to
52 h of experimentation. The time course of luciferase activity is
similar to that of mRNA, but a significant increase of luciferase
activity starts at 24 h after transfection, reaching the highest
level at the 38-h time point. The experiments were repeated 3 times.
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Variation of 3'-UTR-mediated mRNA and protein among SP-A alleles.
Basal mRNA levels of the 10 experimental SP-A 3'-UTR constructs, SP-B
pGL3 (control), and vector pGL3 (control) were analyzed 36 h after
transfection (Fig. 4). The basal mRNA
levels of all 10 alleles (6A, 6A2, 6A3,
6A4, 1A, 1A0, 1A1, 1A2,
1A3, 1A5) are significantly reduced compared
with the SP-B pGL3 and pGL3 vector control (P < 0.01).
Variation in the basal mRNA level of various SP-A alleles is also
observed. The 6A3 and 6A2 alleles have higher
levels than the other (6A and 6A4) SP-A1
alleles; the 1A and 1A2 alleles of SP-A2 have
higher levels than the other four SP-A2 alleles.

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Fig. 4.
Comparison of SP-A 3'-UTR-mediated expression at the mRNA
level. This figure depicts basal mRNA levels of the experimental SP-A
3'-UTR constructs, SP-B pGL3 (control), and vector (pGL3) (control).
The basal levels of all 10 alleles (6A, 6A2,
6A3, 6A4, 1A, 1A0, 1A1,
1A2, 1A3, 1A5) are significantly
(P < 0.01) and differentially reduced compared with
the SP-B pGL3 and pGL3 vector. Of the 4 SP-A1 alleles, both
6A3 and 6A2 show higher (P < 0.05) mRNA content than the 6A4 allele; of the 6 SP-A2 alleles, 1A and 1A2 show higher
(P < 0.05) mRNA content than the other
SP-A2 alleles (n = 3). The symbols a, b, c,
and ab depict the statistical results (P < 0.05) from
ANOVA test. Significant difference exists between any 2 alleles when
they do not have the same symbols, for example, between 6A3
(ab) and 6A4 (c ) or between 6A (b) and 6A4
(c). In turn, if 2 alleles have any of the symbol(s) in common, no
significant difference exists between them, for example, between
6A2 (ab) and 6A (b) or between 6A2 (ab) and
6A3 (ab). Bar shows ± SD.
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The relative activities of firefly/Renilla luciferase
(indicating protein level) were assessed 36 h after transfection
(Fig. 5). The relative activities of
firefly/Renilla luciferase of all 10 alleles are
significantly reduced compared with SP-B pGL3 (control) and vector
alone (P < 0.01). As with mRNA levels, variation in reporter gene activity among the alleles is observed (Fig. 5). Of the
SP-A1 alleles, the 6A2 and 6A3 show
higher levels (P < 0.05) than the 6A and
6A4 alleles. The 1A allele shows a higher level than all
other SP-A2 alleles (P < 0.05), and the
1A1 and 1A5 alleles show lower levels than all
other SP-A2 alleles (P < 0.05; Fig. 5). In
addition, the SP-B pGL3 control was not significantly different from
the pGL3 vector. These results indicate that the SP-A 3'-UTR may play a
role in determining differentially, among alleles, both basal mRNA
levels and basal protein levels.

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Fig. 5.
Comparison of SP-A 3'-UTR-mediated expression at the
protein (luciferase activity) level. This figure depicts basal protein
(luciferase activity) levels of the experimental SP-A 3'-UTR
constructs, SP-B pGL3 (control), and vector (pGL3) (control). These
experiments were repeated 4 times. The luciferase activities of all 10 alleles (6A, 6A2, 6A3, 6A4, 1A,
1A0, 1A1, 1A2, 1A3,
1A5) are significantly (P < 0.01) reduced
compared with the SP-B pGL3 and pGL3 vector. Variation in basal levels
among alleles is also observed. From the 4 SP-A1 alleles,
6A2 and 6A3 exhibit higher (P < 0.05) activity than 6A and 6A4 alleles; from the 6 SP-A2 alleles, 1A allele shows higher (P < 0.05) activity than the others; 1A0, 1A2, and
1A3 have higher activity (P < 0.05) than
1A1 and 1A5. The symbols a, b, and c depict the
statistical results (P < 0.05) from ANOVA test (see
legend for Fig. 4). Bar shows ± SD.
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Comparison of luciferase activities with different transfection
controls.
Because the dual luciferase assay system with Renilla
luciferase as control is a simple, accurate, and easy system, all basal level experiments were performed using this system. In our preliminary experiments, we found that the Renilla control vector pRL
responded to Dex, i.e., Renilla luciferase activity of the
Renilla control vector pRL was strongly inhibited by Dex.
Therefore, we used the
-gal expression vector pCMV-SPORT-
-gal as
the control vector because this vector is not sensitive to Dex
treatment. To assess equity between the two cotransfection control
vectors (pRL and pCMV-SPORT-
-gal), we studied five representative
alleles with regards to basal level. The relative basal level of the
five alleles studied is similar with both control vectors pRL or
pCMV-SPORT-
-gal (Fig. 6). Therefore,
pCMV-SPORT-
-gal is an appropriate control vector in studies where
the response to Dex is investigated.

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Fig. 6.
Comparison of relative luciferase activity with different
transfection control vectors (pRL or pCMV-SPORT- -gal). Five
representative allele constructs [6A, 6A3, 1A,
1A0, and 1A1 were cotransfected with control
vector pRL (Renilla luciferase) or pCMV-SPORT- -gal ( -gal)].
These experiments were repeated 3 times. We observed that the relative
basal level of the 5 alleles studied is similar with both control
vectors, pRL or pCMV-SPORT- -gal. The symbols a, b, and c depict the
statistical results (P < 0.05) from ANOVA test (see
legend for Fig. 4). Bar shows ± SD.
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Time course of SP-A 3'-UTR-mediated response to Dex treatment.
On the basis of our previous studies (26, 27), we used 100 nM Dex to treat transfected cells and to investigate the time course
after Dex treatment with an SP-A2 allele (1A). Cells were treated with 100 nM Dex at 36 h after transfection, and luciferase activity was determined at several time points (0, 0.5, 1, 2, 4, 8, and
16 h) (Fig. 7). The results showed
no significant differences in luciferase activity in either the
presence or absence of Dex up to ~3 h from the start of the
experiment. From 3 to 8 h, the luciferase activity decreased and
reached a plateau after Dex treatment, but no decrease was observed in
the absence of Dex. On the basis of the time course experiment, we
decided to use the 16-h time point after Dex treatment (which
corresponds to 52 h after transfection) for further experiments
where we compared 3'-UTR-mediated differences among alleles in response
to Dex.

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Fig. 7.
Time courses of protein (luciferase activity) with and
without Dex treatment. The time course of protein (luciferase activity)
was analyzed from 0 to 16 h after Dex treatment. The cells at
36 h after transfection were treated with 100 nM Dex, and
luciferase activities at several time points, i.e., 0, 0.5, 1, 2, 4, 8, and 16 h, were analyzed. The results shown are from a
representative experiment (n = 3). No significant
differences in luciferase activity in the presence or absence of Dex up
to ~3 h were observed. From ~3 to 8 h, luciferase activity
decreased and reached a plateau after Dex treatment, but no decrease
was observed in the absence of Dex.
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Differential response to Dex among SP-A 3'-UTR alleles.
We determined SP-A 3'-UTR-mediated mRNA expression among alleles in the
absence or presence of 100 nM Dex at 16 h after Dex treatment
(Fig. 8). Luciferase mRNA in Dex-treated
cells was reduced 31-51% relative to untreated cells. Alleles
6A**, 6A4*, 1A**,
1A0**, 1A2**, 1A3**,
1A5* showed a significant decrease in luciferase mRNA
(*P < 0.05 or **P < 0.01) in the
presence of Dex compared with that observed in the absence of Dex.
However, three alleles (6A2, 6A3,
1A1) failed to show significant differences in response to
Dex (P > 0.05). The vector pGL3 and the control SP-B
pGL3 also failed to respond to Dex.

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Fig. 8.
SP-A 3'-UTR mediates mRNA expression in the presence or
absence of Dex. Total RNAs were extracted from the cells transfected
with SP-A 3'-UTR constructs in the absence or presence of 100 nM Dex
for 16 h after Dex treatment (see MATERIALS AND
METHODS). These experiments were performed 3 times. Dex treatment
decreases luciferase mRNA 31-51%. Alleles 6A**,
6A4*, 1A**, 1A0**,
1A2**, 1A3**, and 1A5* show a
significant decrease in luciferase mRNA (*P < 0.05 or
** P < 0.01) in the presence of Dex compared with that
observed in the absence of Dex. Three other alleles (6A2,
6A3, 1A1) show no significant differences in
response to Dex (P > 0.05). The mRNA level of vector
pGL3 shows no response to Dex treatment.
|
|
The relative luciferase activity in the presence or absence of 100 nM
Dex was also analyzed. Firefly luciferase activity was normalized using
-gal activity of pGL3 vector. We observed that Dex treatment
decreased luciferase activity ranging from 17% (1A1) to
38% (1A) in the 10 constructs, each containing a different 3'-UTR.
Alleles 6A2, 1A, 1A0, 1A2,
1A3, and 1A5 show a significant decrease in
luciferase activity (P < 0.05) in the presence of 100 nM Dex (Fig. 9). Vector pGL3 and control SP-B pGL3 activities showed no response to Dex treatment. These results
indicate that the SP-A 3'-UTR may differentially mediate the Dex
response among SP-A alleles (Fig. 9).

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 9.
SP-A 3'-UTR mediates protein expression (luciferase
activity) in the presence or absence of Dex. Luciferase activities were
measured using the cell lysate from transfected cells with SP-A 3'-UTR
constructs in the absence or presence of 100 nM Dex for 16 h after
Dex treatment (see MATERIALS AND METHODS). These
experiments were performed 4 times. Dex treatment decreased luciferase
activity from 17% (1A1) to 38% (1A) in the 10 SP-A
constructs. Alleles 6A2, 1A, 1A0,
1A2, 1A3, and 1A5 show a
significant decrease in luciferase activity (P < 0.05)
in the presence of Dex compared with that observed in the absence of
Dex. Vector pGL3 and a control SP-B pGL3 activity show no response to
Dex treatment.
|
|
 |
DISCUSSION |
Steroid therapy is widely used in the clinic to enhance lung
maturity and surfactant production, to improve pulmonary compliance, and to help wean infants from the ventilator. However, concerns about
the side effects of steroid therapy (30, 48, 63) and of
possible long-term consequences in lung development (41, 49) have been raised. SP-A, a Dex-responsive molecule, plays important roles in surfactant-related activities, innate host defense,
and in the regulation of inflammatory processes of the lung. Pilot
studies indicate that Dex may have a differential effect on expression
of SP-A variants and that this effect may be mediated via the 3'-UTR
(26). In the present study, we tested the effects of
3'-UTR on basal levels of gene expression as well as on expression
levels in response to Dex of the 10 most frequently found
SP-A1 and SP-A2 alleles. The results of these in
vitro transient transfection experiments indicate that the 3'-UTR of
SP-A differentially mediates both basal expression and expression in
response to Dex. Both basal mRNA and protein (activity) levels are
significantly reduced compared with either vector alone or control
construct, with some differences observed among alleles. Dex treatment
also decreased differentially mRNA and protein levels, with no such decrease observed with the control construct or vector alone. With some
of the SP-A1 3'-UTR constructs, an apparent discrepancy in
their response to Dex was observed, i.e., some showed a response to Dex
at the mRNA level but not at the protein and vice versa. No such
discrepancy was observed with the SP-A2 alleles.
In the present study, we observed that the basal mRNA level of
6A4 is significantly lower than those of 6A2
and 6A3, and the basal level of protein of both 6A and
6A4 are significantly lower than those of 6A2
and 6A3. DNA sequence analysis indicated that the 6A and
6A4 alleles differ from the 6A2 and
6A3 in 3'-UTR at positions 213/214 (CA to TC) and 249 (A to
C) (see Table 4). The nucleotide sequence changes at 213/214 generate, in 6A and 6A4, potential recognition binding sites for
several DNA binding factors, such as GATA-1, -2, -3, Sox-5, and Ik-2,
whereas the sequences at these positions for 6A2 and
6A3, although are lacking in recognition binding sites for
the aforementioned factors, are the sites for other factors such as
p300 and c-Myb. GATA family factors are of the zinc finger
protein family, and recently, it was found that the GATA-6 is required
for maturation of the lung in late gestation (40). Whether
or how these potential recognition binding sites for different DNA
binding factors between these two groups of SP-A1 alleles
may contribute to the changes observed in the basal level of SP-A
3'-UTR-mediated expression remains to be determined. Recently, Gehring
et al. (21) found that only one nucleotide mutation (G to
A) at the 3'-UTR of the prothrombin gene causes increased cleavage site
recognition, increased 3'-end processing, and increased mRNA
accumulation and protein synthesis. Similarly, a nucleotide insertion
(1484insG) at the 3'-UTR of the PTP1B gene increases specific gene
expression and may be responsible for insulin resistance
(16). Together, these findings indicate that the few
nucleotide differences between the two groups of SP-A alleles may, as
supported by the examples mentioned, cause a significant change in gene
expression levels. This change in turn may influence the efficiency of
mRNA 3'-UTR formation (21), transport and localization,
posttranscriptional modification and degradation, and translation
(10, 11, 16, 22).
GCs play a role in several processes of gene expression, including
transcription, posttranscription, and translation
(1-3). The action of GCs may occur via direct and
indirect mechanisms, such as GC-mediated destabilization of mRNA
(7, 37, 45, 46) and mRNA-protein interactions in 3'-UTR of
mRNA (20). GCs also influence both protein synthetic
(translational) and protein degradative pathways and are shown to
attenuate mRNA translation at two levels: translational efficiency
(i.e., translation initiation) and translational capacity (i.e.,
ribosome biogenesis) (20, 56). The present results
summarized in Table 3 show that
differences in response to Dex exist among the 10 SP-A 3'-UTR
constructs and that there are some apparent discrepancies between mRNA
and protein level among some of the SP-A1 3'-UTR constructs,
but not for the SP-A2. Whether these discrepancies are due
to a particular nucleotide difference either alone or in the context of
other surrounding nucleotide differences and/or as to which these
nucleotide differences may be are currently unknown and warrant further
investigation. Among the SP-A2 alleles, the 1A1
construct did not respond to Dex as assessed by measurements at either
the mRNA or protein level. The 1A1 allele has several
unique nucleotide differences in 3'-UTR from the other SP-A2
alleles at positions 29 (A to G), 275 (C to T), and 557 (C to G) (Table
4). These differences may cause a change in recognition binding sites for many factors. For example, potential binding sites for factors Ttk 69 and CF1 exist at the region that includes nucleotide position 29; binding sites for factors GRC1, C-Rel,
and Ik-2 exist at the region that includes nucleotide position 275. However, the precise factors and mechanisms responsible for the
difference between 1A1 and other SP-A2 alleles
remain to be determined.
Because an element of "pyrimidine-rich domain of 37 nucleotides"
and AUUUA (Au-rich) elements have been implicated in
GC-mediated mechanisms, we investigated the presence and/or similarity
of these elements between SP-A1 and SP-A2 alleles
in the context of the present study. At nucleotide positions
787-826, an element of pyrimidine-rich domain of 37 nucleotides
was observed in both SP-A1 and SP-A2 alleles, but
7 of the nucleotides in this region differ between SP-A1 and
SP-A2. Because this element is involved in RNA-protein
interaction of the GC-mediated destabilization of cyclin D3 mRNA in
murine T lymphoma cells (20), these seven nucleotide
differences may play a differential regulatory role between
SP-A1 and SP-A2 alleles, although our present
experimental conditions do not support this. However, a combination of
factors that may include the pyrimidine tract may provide allele
specificity. Moreover, the published literature indicates that AUUUA
sequences (AUUUA element) in the 3'-UTR of several mRNAs influence mRNA stability (9, 36, 62, 64), and Dex may affect mRNA
stability through the AUUUA element (4). The 3'-UTR of
SP-A2 contains two such AUUUA elements at positions
6-10 and 927-931, but the SP-A1 has only one at
position 6-10. However, because no discrete differences were
observed between SP-A1 and SP-A2 alleles, it is
unlikely that the AUUUA element plays, by itself, a role in the
processes studied here. But, it is possible that this element, in the
presence of other unknown elements/factors, contributes to the observed
SP-A allele differences. For example, the 1A1 3'-UTR has a
G at position 29, whereas all other SP-A2 alleles have an A. This nucleotide difference is located near the AUUUA element at the
nucleotide positions 6-10 after the translation stop codon (Table
5). Whether any interactions exist
between this AUUUA element and the region that contains nucleotide 29 that may explain the lack of Dex response of 1A1 compared
with other SP-A2 alleles remains to be determined. Further study is necessary to investigate the mechanisms involved in the 3'-UTR-mediated differential allele regulation, both in the basal level
and in the response to Dex.
Furthermore, in our present study, we observed for the SP-A1
alleles (but not for the SP-A2 alleles) some apparent
discrepancy in response to Dex. Some variants were only responsive at
the mRNA levels (6A, 6A4) or only at the protein level
(6A2). Recent studies indicate that the 3'-UTR and
associated proteins such as the poly(A)-binding protein (PABP) and the
PABP-interacting protein-1 may be involved in the regulation of
translation through interactions with translational factors, such as
elF4G, and thus influence mRNA stability or translational efficiency,
or both (11, 13, 22, 47). It would be of interest to
investigate whether such processes are involved in SP-A1
allele expression.
In summary, we have characterized the 3'-UTR sequences of 10 SP-A
alleles and compared the identities and divergence of 3'-UTR of 6 alleles of SP-A2 and 4 alleles of SP-A1. With the
use of transient transfection assays at the optimal time point and
duration of Dex treatment, we observed that: 1) the basal
mRNA and protein levels of all 10 alleles are significantly
(P < 0.01) and differentially reduced, compared with
those of vector alone or a control SP-B construct; and 2)
Dex treatment significantly and differentially decreases luciferase
mRNA and luciferase activity in some alleles but not in others compared
with controls. Given the allele-dependent differences, it is necessary
to carry out a detailed study of the mechanisms involved that may help
explain the differential gene expression of SP-A alleles for basal and
Dex-responsive regulation. We speculate that such studies may provide
the basis for a careful consideration of individual use of steroid treatment.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Myung-Ho Oh for contributions with constructs at the
initial stages of this study, Susan DiAngelo for expert technical
assistance, and Jeffrey Sandstrom for help and comments with the manuscript.
 |
FOOTNOTES |
This work is supported by National Heart, Lung, and Blood Institute
Grant R37 HL-34788.
Address for reprint requests and other correspondence: J. Floros, Dept. of Cellular and Molecular Physiology, H166, The
Pennsylvania State Univ. College of Medicine, 500 University Dr.,
Hershey, PA 17033 (E-mail: jfloros{at}psu.edu).
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.
First published January 10, 2003;10.1152/ajplung.00375.2002
Received 1 November 2002; accepted in final form 2 January 2003.
 |
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