Departments of 1 Pediatrics and 2 Anatomy and Cell Biology, University of Iowa, Iowa City, Iowa 52242
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ABSTRACT |
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The surfactant
proteins (SPs) are required for the normal function of pulmonary
surfactant, a lipoprotein substance that prevents alveolar collapse at
end expiration. We characterized the effects of cortisol and all
trans-retinoic acid (RA) on SP-A and
SP-B gene expression in H441 cells, a human pulmonary adenocarcinoma cell line. Cortisol, at 106
M, caused a significant inhibition of SP-A mRNA to levels that were
60-70% of controls and a five- to sixfold increase in the levels
of SP-B mRNA. RA alone (10
6
M) had no effect on SP-A mRNA levels and modestly reduced the inhibitory effect of cortisol. RA alone and the combination of cortisol
and RA both significantly increased SP-B mRNA levels. RA had no effect
on the rate of SP-A gene transcription or on SP-A mRNA stability.
Cortisol alone and the combination of cortisol and RA significantly
inhibited the rate of SP-A gene transcription but had no effect on SP-A
mRNA half-life. RA at 10
6 M
had no effect on the rate of SP-B gene transcription but prolonged SP-B
mRNA half-life. Cortisol alone and the combination of cortisol and RA
caused a significant increase in the rate of SP-B gene transcription
and also caused a significant increase in SP-B mRNA stability. We
conclude that RA has no effect on SP-A gene expression and increases
SP-B mRNA levels by an effect on SP-B mRNA stability and not on the
rate of SP-B gene transcription. In addition, the effects of the
combination of RA and cortisol were generally similar to those of
cortisol alone.
H441 cell line; pulmonary surfactant; lung; surfactant protein A mRNA; surfactant protein B mRNA
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INTRODUCTION |
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SURFACTANT-ASSOCIATED PROTEINS (SPs) play a vital role in the function of pulmonary surfactant, a lipoprotein that prevents alveolar collapse at end expiration (14, 31). Four SPs, SP-A, SP-B, SP-C, and SP-D, have been identified and characterized (31). SP gene expression is regulated developmentally as well as by hormones, growth factors, and other regulatory substances, e.g., glucocorticoids, insulin, epidermal growth factor, and cAMP (24). Glucocorticoids are used clinically to accelerate human fetal lung maturation, and their use has resulted in decreased morbidity and mortality of preterm infants who have been exposed to maternally administered glucocorticoids (12). Glucocorticoids have been shown to inhibit SP-A gene expression and to stimulate SP-B and SP-C gene expression in human fetal lung explants (23, 26). Glucocorticoids regulate surfactant gene expression via effects on both mRNA transcription and stability (5, 6, 20, 30). All trans-retinoic acid (RA), a metabolite of retinol (vitamin A), has recently been identified as a regulator of SP gene expression (7, 8, 17, 25) and surfactant phospholipid synthesis (15). The molecular mechanisms by which RA regulates SP gene expression in the lung have not been described.
H441 cells are a human pulmonary adenocarcinoma cell line that expresses SP-A and SP-B (16). The H441 cell line has been shown to respond to regulatory factors that influence the SPs in a manner similar to that observed in human fetal lung explants maintained in vitro (17, 27, 28, 32). Because both glucocorticoids and RA have been shown to be regulators of SP gene expression, we hypothesized that the effect of the combination of cortisol and RA on SP levels would be different from the effect of either hormone added alone. The results of these experiments are of interest because both glucocorticoids and retinoids are used clinically to accelerate lung development, and it is likely that they might be used in combination (12, 22). To understand the mechanism of gene regulation by these factors, we also evaluated the effects of cortisol, RA, and the combination of RA+cortisol on SP-A and SP-B gene transcription and on SP-A and SP-B mRNA stability.
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MATERIALS AND METHODS |
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Cell culture.
NCI-H441 cells, a human pulmonary adenocarcinoma cell line, were
cultured in RPMI 1640 medium (GIBCO
BRL) that contained 10% fetal bovine serum (vol/vol; Hyclone, Logan,
UT), 100 U/ml penicillin G, 100 µg/ml streptomycin, and 0.25 µg/ml
amphotericin B. The cells were grown to confluence in 100-mm-diameter
plastic tissue culture dishes that contained 10 ml of media and were
maintained at 37°C in a humidified atmosphere of 95% air-5%
CO2. The serum-containing medium
was then replaced by serum-free medium for 24 h (to reduce the possible
influence of mediators present in serum), and then the cells were
incubated for an additional 24 h in serum-free medium containing either
no additions (control), RA
(106 M), varying
concentrations of cortisol
(10
10 to
10
6 M), or varying
concentrations of cortisol in the presence of RA
(10
6 M). Culture of the
H441 cells in serum-free medium for longer than ~48 h results in
detachment of the cells from the culture dish. Maximal effects of RA on
SP-B are observed after 24 h of treatment (17). All
trans-RA (Sigma, St. Louis, MO) stock
solutions were aliquoted as
10
3 M stocks in DMSO in
light-proof containers and kept frozen at
70°C until use.
Stock solutions of cortisol (0.5 mM) were dissolved in ethanol and
stored at
20°C. Media that contained RA and/or cortisol were made immediately before each experiment. After 24 h of
exposure to the regulatory substances, the H441 cells were harvested by
scraping with a rubber policeman, quickly frozen in a microfuge tube
with 1 ml of RNA extraction mixture, and then stored at
70°C.
RNA isolation. Total RNA was isolated from the cells as described previously (9, 17). After quantitation of total RNA by determination of the ultraviolet (UV) absorbency at 260 nm, equal amounts of total RNA from each sample were separated by gel electrophoresis, capillary transferred to Nytran membranes (Schleicher & Schuell, Keene, NH), and then cross-linked to the Nytran membrane by UV irradiation for 2 min in a Bio-Rad GS Gene Linker (Bio-Rad, San Jose, CA). The rRNA bands in the agarose gel were stained with ethidium bromide and photographed on a UV light box.
Northern blot analysis.
The human cDNA probes for SP-A and SP-B (a kind gift of Dr. J. Whitsett, Univ. of Cincinnati) were radiolabeled with
[32P]cytosine
triphosphate (~3,000 Ci/mmol; Amersham, Bedford, MA) using a
random-primer kit (Boehringer Mannheim, Indianapolis, IN). Northern
blot analysis was performed as described previously (13). After
hybridization, the membranes were wrapped in plastic wrap and exposed
to X-ray film (Eastman Kodak, Rochester, NY) for up to 48 h at
70°C. A scanner and PDI software (Protein and DNA Imageware
Systems, Huntington Station, NY) were used to quantify the reactive
bands on the X-ray film. Densitometry was also performed on the 18S
rRNA bands on the photograph of the agarose gel taken before transfer
to the membrane. This allowed correction for possible loading artifacts
by correcting the density of the reactive mRNA band on the Northern
blot with the density of the 18S rRNA band from the same lane.
Isolation of nuclei and nuclear transcriptional
run-on assay.
Cells used for transcription assays were cultured in 150-mm dishes that
contained 20 ml of media. After treatment, H441 cells were released
from the culture dish by trypsinization (27). The cell suspension was
centrifuged at 800 g for 3 min and
then washed twice with Hanks' balanced salt solution. The cells were lysed by incubation on ice for 5 min in 12 ml of lysis buffer (10 mM
Tris · HCl, pH 7.4, 10 mM NaCl, 3 mM
MgCl2, and 0.5% Polydet-40). The
lysate was then centrifuged at 600 g
for 5 min to pellet the nuclei. The isolated nuclei were washed once
with fresh lysis buffer and then resuspended in 4 ml of ice-cold
glycerol storage buffer (40% glycerol, 50 mM
Tris · HCl, pH 8.3, 5 mM
MgCl2,, and 0.1 mM EDTA). The
nuclear suspension was centrifuged at 600 g for 5 min, and then the nuclei were
counted and stored at 70°C until further analysis.
Assay for mRNA stability.
NCI-H441 cells were grown to confluence in RPMI 1640 media that
contained serum as described in Cell
culture. The cells were then exposed to
serum-free RPMI 1640 media for 4-6 h. After this period, cells
were incubated in serum-free media with either no additions (control),
RA (106 M), cortisol
(10
6 M), or cortisol
(10
6 M) plus RA
(10
6 M) for an additional
18 h. The cells were then further incubated in the presence or absence
of the regulators plus actinomycin D (10 µg/ml) for up to 24 h. Cells
were harvested at 0, 4, 8, 12, or 24 h after addition of actinomycin D. RNA was isolated and immobilized on Nytran membranes using a slot-blot
apparatus, and Northern blot analysis was performed as described in
Northern blot analysis. Slot blots
were analyzed using scanning densitometric analysis with NIH Image
software as described in Isolation of nuclei and
nuclear transcriptional run-on assay. The amount of SP
mRNA present at each time point was calculated by assigning a value of
one to the value at the zero time point and then normalizing the data
from the other time points to the zero time point for that condition.
The experiment was repeated five times. To calculate the mRNA half-life
in each condition, the log of the mRNA concentration was plotted vs.
time in hours for each experiment. Linear regression was used to
calculate the slope of the resulting line, and the following equation
was used to calculate the mRNA half-life:
t1/2 (h) = ln
2/(
2.303 × slope) (5). Mean ± SE values
of the half-life values were calculated for each condition
(n = 4-6) and compared using
Dunnett's test and paired t-tests
(34). In addition, mRNA data for each time point within each condition
were pooled, and a regression line was calculated from the pooled data.
The slope of the pooled data line was also used to calculate an mRNA
half-life.
Statistical analysis. Statistical analysis of data was performed using a computer software package (SigmaStat; Jandel, San Rafael, CA). Data from the treated conditions were normalized relative to the control value in that experiment, which was assigned a value of one. Data are expressed as means ± SE. Densitometric data from three to six independent experiments were pooled and analyzed by one-way ANOVA, with significant values noted by P values < 0.05. A two-tailed Dunnett's test, a two-factor ANOVA, and a paired Student's t-test were also used to make statistical comparisons between the various conditions (34).
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RESULTS |
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There was a significant, dose-dependent inhibition of SP-A mRNA levels
by 24 h of cortisol treatment (P < 0.01, ANOVA; Fig. 1,
A and
B). The maximal inhibition was
~60% at 106 M cortisol.
Densitometric data from six experiments showed that cortisol at
10
8,
10
7, and
10
6 M inhibited SP-A mRNA
levels significantly compared with controls (Fig.
1B). In contrast, cortisol caused a
significant, dose-dependent stimulation of SP-B mRNA levels
(P < 0.01, ANOVA; Fig.
2, A and B). Maximal stimulation was observed
at 10
6 and
10
7 M cortisol, at which
point SP-B mRNA levels were about seven times the levels in controls.
The stimulatory effect of cortisol on SP-B mRNA levels at
10
8,
10
7, and
10
6 M was statistically
significant compared with controls (Fig. 2B).
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We have previously shown that
106 M RA causes a
significant stimulation of SP-B mRNA levels in the H441 cell line (17).
We have noted toxic effects of RA at concentrations
>10
6 M (25). Consistent
with our previous results (17), RA
(10
6 M) had no effect on
SP-A mRNA levels in the H441 cells (Fig. 1). Cortisol, in the presence
of 10
6 M RA, caused a
significant, dose-dependent inhibitory effect on SP-A mRNA levels
(P < 0.05, ANOVA; Fig. 1). However,
the significant inhibition of SP-A mRNA that was caused by
10
8,
10
7, and
10
6 M cortisol when added
alone only occurred at 10
7
and 10
6 M cortisol in the
presence of RA (Fig. 1B). In
addition, the inhibitory effect of the combination of cortisol
(10
6 M) plus RA
(10
6 M) on SP-A mRNA levels
was less than that of cortisol
(10
6 M) added alone
(P < 0.05, paired
t-test; Fig.
1B). Thus RA
(10
6 M) blunted the
inhibitory effect of cortisol on SP-A mRNA levels, although the effect
was of relatively small magnitude, ~15%.
RA (106 M) stimulated SP-B
mRNA levels about two times (Fig. 2, A
and B). Densitometric data from six
experiments revealed a significant, dose-dependent stimulatory effect
of cortisol on SP-B mRNA levels in the presence of RA
(10
6 M; Fig.
2B). This stimulatory effect was
significant at 10
8,
10
7, and
10
6 M cortisol plus
10
6 M RA (Fig.
2B). The stimulatory effect of the
combination of cortisol
(10
6 M) and RA
(10
6 M) was greater (by
~20%) than the effect of cortisol added alone (P < 0.05, paired
t-test; Fig.
2B).
RA (106 M) slightly
inhibited the rate of SP-A gene transcription (by 23%); however, the
effect did not reach statistical significance (P = 0.16, paired
t-test; Fig.
3A).
Cortisol (10
6 M) caused a
significant (~60%) inhibition of the rate of SP-A gene transcription
compared with the control condition (Fig.
3A). The combination of cortisol and
RA also significantly inhibited the rate of SP-A gene transcription (by
~50%) compared with controls (Fig.
3A). The effect of cortisol added
alone was not different from the effect of cortisol+RA
(P = 0.54, paired
t-test). Although there was no effect
of RA (10
6 M) on the rate
of SP-B gene transcription, both cortisol alone and the combination of
cortisol+RA increased the rate of SP-B gene transcription 310 and
330%, respectively, relative to controls (P < 0.05, Dunnett's test; Fig.
3B). The effect of cortisol added alone was not different from the effect of cortisol+RA
(P = 0.83, paired
t-test). There was no significant
effect of either RA or cortisol or the combination of the two
regulators on actin gene transcription in the H441 cells (data not
shown).
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The half-life of SP-A mRNA was 17 ± 2.8 h and was increased ~80% in the presence of RA, although the effect was not significant (P = 0.12, paired t-test; Fig. 4A; Table 1). Cortisol and the combination of cortisol+RA also did not affect SP-A mRNA half-life (Fig. 4A; Table 1). The half-life of SP-B mRNA was 14.3 ± 2.1 h in control cells and was significantly increased in all treated conditions (Fig. 4B; Table 1). The increase was ~65% in the presence of RA, ~110% in the presence of cortisol, and ~120% in the presence of the combination of cortisol and RA (P < 0.05, Dunnett's test).
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DISCUSSION |
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Antenatal dexamethasone and postnatal surfactant replacement therapy have reduced, but not eliminated, the morbidity and mortality of preterm infants with respiratory distress syndrome (21). Efforts to improve the pulmonary status of these premature infants could further decrease the impact of pulmonary immaturity on long-term outcome. To this end, many studies have been performed to evaluate the effects of hormones and combinations of hormones on lung development (24). For example, Ballard et al. (3) found that treatment with prenatal thyrotropin-releasing hormone reduced the incidence of chronic lung disease among infants who also received betamethasone. However, more recent evidence suggests that this specific combination may not clinically improve outcomes and, in fact, may worsen morbidity (1, 11).
Glucocorticoids have been shown to be potent regulators of SP gene
expression in cultured human fetal lung explants and in human pulmonary
epithelial cell lines (6, 20, 23, 26-28, 30). In the present
study, cortisol caused a significant, dose-dependent inhibition in the
levels of SP-A mRNA and a dose-dependent increase in SP-B mRNA levels
in H441 cells. The half-maximal inhibitory and stimulatory effects of
cortisol occurred at a concentration of
108 to
10
9 M.
RA has been shown to regulate the growth and differentiation of many
epithelial cell types and to play an active role in lung development
(10, 18, 33). Previous studies have demonstrated that in the developing
lung and in human pulmonary epithelial cells, RA regulates the
expression of the SPs, which are required for the function of pulmonary
surfactant (7, 8, 17, 25). We hypothesized that the interactive effects
of RA and glucocorticoids on SP gene expression would be different from
the effect of either agent added alone. Our studies showed that there
was no interactive effect of cortisol and RA in the regulation of SP-A
mRNA except at 106 M
cortisol, at which point the inhibitory effect of the combination of
regulatory agents was slightly less (~15%) than the inhibitory effect of cortisol added alone. RA
(10
6 M) had a further small
stimulatory effect (~20%) on SP-B mRNA levels in cells treated with
10
6 M cortisol compared
with cells treated with RA alone. These results suggest that all
trans-RA and cortisol, at high
concentrations, may have an additive effect on SP-B mRNA levels in H441
cells.
Glucocorticoids have been shown to regulate SP mRNA levels by their effects on the transcription rates of the SP genes and by changes in mRNA stability (2, 5, 6, 20, 27, 30). In the present study, RA added alone had no significant effect on SP-A gene transcription. This is consistent with data showing that all trans-RA had no effect on SP-A mRNA levels in the H441 cell line (17). Both cortisol added alone and the combination of all trans-RA and cortisol significantly inhibited the rate of SP-A transcription. These results are consistent with the inhibitory effect of glucocorticoids on SP-A mRNA levels observed in the H441 cells.
Boggaram et al. (6) reported that dexamethasone
(107 M) had no effect on
the transcription rate of SP-A mRNA in human fetal lung explants after
a 24-h exposure to the hormone and increased the rate of SP-A gene
transcription 1.4 times after 48-h exposure to the hormone. In
contrast, Ianuzzi et al. (20) found that a 4- to 8-h incubation of
cultured human fetal lung explants with dexamethasone
(10
7 M) inhibited SP-A gene
transcription by ~50%. In our experiments, the exposure of H441
cells to cortisol (10
6 M)
for 24 h caused a significant inhibition of SP-A gene transcription that was correlated with a decrease in SP-A mRNA content. Thus our
results are consistent with those of Ianuzzi et al. in human fetal lung
explants; however, because the effects of glucocorticoids in fetal lung
explant systems may represent indirect effects on pulmonary epithelial
cells, it may be incorrect to compare our results using the H441 cell
line with explant studies.
RA had no effect on the rate of SP-B gene transcription in the H441
cells. In contrast, cortisol and the combination of cortisol+RA caused
a threefold increase in the rate of SP-B mRNA transcription. O'Reilly
et al. (27) noted a two- to fourfold increase in the rate of SP-B mRNA
transcription in H441 cells treated for 48 h with dexamethasone (0.5 × 107 M). Both
dexamethasone (10
7 M) for 8 h and cortisol (10
6 M) for
24 h have been shown to stimulate the transcription of SP-B mRNA about
threefold in human fetal lung explants (2, 30). Thus the results of our
experiments with respect to regulation of SP-B gene transcription by
glucocorticoids are in good agreement with the results of other
investigators. In addition, we have shown that RA apparently does not
regulate SP-B mRNA levels by effects on gene transcription.
Two experimental approaches have been used in previous studies to
determine SP mRNA stability, namely actinomycin D and the pulse-chase
method (20, 30). The half-life data obtained using the two methods are
similar. Because the actinomycin D method is more straightforward, we
used it to perform our studies. Neither RA, cortisol, nor the
combination of cortisol and RA had a significant effect on SP-A mRNA
stability. These data suggest that glucocorticoids do not regulate SP-A
gene expression at the level of mRNA stability and that the inhibitory
effects of glucocorticoids on SP-A mRNA levels are achieved primarily
via effects on transcription. In human fetal lung explants, Ianuzzi et
al. (20) found that dexamethasone (107 M) had a short-term
effect to decrease SP-A mRNA stability; however, a 4-day exposure to
dexamethasone had no effect on SP-A mRNA stability. Interestingly, the
SP-A mRNA half-lives observed in controls in the human fetal lung
explant model ranged from 7 to 11 h, whereas, in the H441 cell line, we
found that SP-A mRNA had a much longer half-life of ~17 h (5, 20).
RA significantly increased SP-B mRNA stability; this, together with the
transcription run-on data, suggests that RA causes an increase in SP-B
mRNA levels primarily by its effects on increasing SP-B mRNA stability.
Cortisol caused a greater than twofold increase in SP-B mRNA half-life.
O'Reilly et al. (27) have previously reported that treatment of H441
cells for 48 h with 0.5 × 107 M dexamethasone had no
effect on SP-B mRNA stability in H441 cells. We used
10
6 M cortisol for 24 h in
the present study and found a significant effect of cortisol to
increase SP-B mRNA half-life. The discrepancy may be the result of the
glucocorticoid concentration used, the exposure time, or the use of
cortisol vs. dexamethasone. In agreement with our observations,
Venkatesh et al. (30) found that treatment of human fetal lung explants
with dexamethasone (10
8 M)
significantly increased SP-B mRNA stability from ~7 h in controls to
~19 h. The half-life of SP-B mRNA observed in our studies using the
H441 cell line (~14 h) is longer than the half-life observed in human
fetal lung explant studies, which ranged from 6 to 7.5 h (30).
Treatment of the H441 cells with the combination of cortisol and RA
also caused a prolongation of SP-B mRNA half-life, although the effect
was not different from the effect of cortisol added alone. We suggest
that the small stimulatory effect of the combination of cortisol and RA
on SP-B mRNA levels over that caused by treatment with cortisol or RA
alone probably occurs via a combination of effects on the rate of SP-B
gene transcription and SP-B mRNA stability, although we were unable to
demonstrate a significant effect on either parameter in the combination
condition.
Glucocorticoids are used to treat and/or prevent bronchopulmonary dysplasia (BPD) in human newborns (19). It has been proposed that vitamin A, the metabolic precursor to all trans-RA, may prevent the development of BPD in human newborns (22). Premature human newborns are frequently vitamin A deficient (29). In a metanalysis of four small clinical trials in infants with very low birth weight, a reduced risk of BPD and death was associated with vitamin A supplementation (22). Because current clinical practice frequently includes the use of pre- and postnatal glucocorticoid therapy, it is likely that some infants will be treated with both vitamin A and glucocorticoids. Our data suggest that with respect to SP-B gene expression in particular, the combination of glucocorticoids and vitamin A should be beneficial because SP-B levels would probably be increased. Further experiments in an in vivo model would address this issue.
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ACKNOWLEDGEMENTS |
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We thank Rose Marsh for preparation of the manuscript and Dr. Jonathan Klein for helpful discussions.
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FOOTNOTES |
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This research was supported, in part, by a Clinical Research Grant (6-FY-95-0978) from the March of Dimes Birth Defects Foundation, by the Children's Miracle Network, and by Diabetes Endocrinology Research Center, National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-95295. T. N. George was supported by an Iowa Cardiovascular Interdisciplinary Research Fellowship (HL-07121).
Address for reprint requests: J. M. Snyder, Dept. of Anatomy and Cell Biology, Univ. of Iowa College of Medicine, Iowa City, IA 52242.
Received 14 July 1997; accepted in final form 15 January 1998.
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