Mechanism of all trans-retinoic acid and glucocorticoid regulation of surfactant protein mRNA

Thomas N. George1, Olga L. Miakotina2, Kelli L. Goss2, and Jeanne M. Snyder2

Departments of 1 Pediatrics and 2 Anatomy and Cell Biology, University of Iowa, Iowa City, Iowa 52242

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 10-6 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

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 (10-6 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.

Nuclear run-on transcription assays were performed using modifications of previously described methods (4, 20). The transcription reaction was performed using 20 × 106 nuclei resuspended in 200 µl of transcriptional buffer [20 mM Tris · HCl, pH 8.0, 5 mM MgCl2, 0.5 mM MnCl2, 90 mM NH4Cl, 0.04 mM EDTA, 2 mM dithiothreitol, 200 units of RNasin (Promega, Madison, WI)], 10 µl [alpha -32P]UTP (3,000 Ci/mol), and 0.4 mM of each nucleotide triphosphate for 20 min at 37°C. The reaction mixture was then incubated for 20 min at 37°C with 100 U of RNase-free DNase. Proteinase K solution (100 µg/ml of proteinase K, 30 µg of yeast tRNA, 0.5% SDS, and 15 mM EDTA) was then added, and the solution was incubated for 30 min at 37°C. Phenol-isoamyl alcohol-chloroform extraction followed by ethanol precipitation was used to isolate labeled total RNA. The resulting labeled RNA pellet was dissolved in 85 µl of 10 mM Tris buffer, pH 8.0, that contained 1 mM EDTA, 100 mM NaCl, and 120 units of RNasin and then purified using an RNA G-50 Quick Spin column (Boerhinger Mannheim, Indianapolis, IN).

Five micrograms of linearized human SP-A, SP-B, or actin cDNA in a Bluescript vector or Bluescript alone were immobilized on a Nytran membrane. The membrane was prehybridized in buffer containing 50% formamide, 5× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0), 100 µg/ml of denatured herring sperm DNA, 5× Denhardt's solution (Sigma), 0.5 units/µl of RNasin, and 0.1% SDS for 4 h at 45°C. The labeled RNA, resuspended in 1 ml of hybridization buffer, was added to the membranes (~6 × 106 counts · min-1 · membrane-1) and hybridized at 45°C for 60 h. Hybridized membranes were then washed twice in buffer that contained 0.2× SSC and 0.1% SDS at 55°C for 1 h, in 2× SSC buffer at room temperature for 15 min, in 2× SSC buffer with 10 µg/ml of heat-treated RNase A at 37°C for 30 min, and, finally, twice in 2× SSC buffer at room temperature for 15 min. The membranes were dried and exposed to X-ray film for 4-7 days at -70°C. The resulting autoradiographs were analyzed by densitometry with NIH Image software.

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 (10-6 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).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 10-6 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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Fig. 1.   Combined effect of cortisol and retinoic acid (RA) on surfactant protein (SP) A mRNA levels in H441 cells. Confluent monolayers of H441 cells were exposed to serum-free medium containing either no additions [control (cont)], RA (10-6 M), cortisol (10-10 to 10-6 M), or varying concentrations of cortisol (10-10 to 10-6 M) in presence of RA (10-6 M). A: representative autoradiogram of SP-A Northern blot. Equal amounts of total RNA (20 µg) were loaded into each lane for each condition. B: densitometric data from 6 experiments. Data are means ± SE. Densitometric data from each individual experiment were normalized to control condition, which was made equal to 1. There was no significant effect of all trans-RA on SP-A mRNA levels. There was a significant (P < 0.05, ANOVA), dose-dependent inhibitory effect of cortisol on SP-A mRNA levels. There was also a significant (P < 0.05, ANOVA), dose-dependent inhibitory effect of cortisol in presence of all trans-RA (10-6 M). * Significant difference from controls (P < 0.05, Dunnett's test). + Significant difference between cortisol (10-6 M) alone condition and cortisol (10-6 M) in presence of RA (10-6 M) (P < 0.05, Student's paired t-test).


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Fig. 2.   Combined effects of cortisol and RA on SP-B mRNA levels in H441 cells. Confluent monolayers of H441 cells were exposed to serum-free medium containing either no additions (cont), RA (10-6 M), cortisol at various concentrations (10-10 to 10-6 M), or varying concentrations of cortisol (10-10 to 10-6 M) in presence of RA (10-6 M). A: representative autoradiogram of SP-B Northern blot. Equal amounts of total RNA (40 µg) were loaded into each lane for each condition. B: densitometric data from 6 experiments. Data are means ± SE. Densitometric data from each individual experiment were normalized to control condition, which was made equal to 1. There was a significant (P < 0.05, ANOVA) stimulatory effect of RA on SP-B mRNA levels. There was a significant (P < 0.05, ANOVA), dose-dependent stimulatory effect of cortisol on SP-B mRNA levels. There was also a significant (P < 0.05, ANOVA), dose-dependent stimulatory effect of cortisol in presence of RA (10-6 M). * Significant difference from control condition (P < 0.05, Dunnett's test). + Significant difference between effect of cortisol (10-6 M) added alone and cortisol added together with RA (10-6 M) (P < 0.05, Student's paired t-test).

We have previously shown that 10-6 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 (10-6 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 (10-6 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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Fig. 3.   Effect of RA, cortisol, and combination of cortisol+RA on rates of SP-A and SP-B gene transcription. Confluent monolayers of H441 cells were exposed to serum-free medium containing either no additions (control), RA (10-6 M), cortisol (10-6 M), or a combination of cortisol+RA for 24 h. Densitometric data representing rate of gene transcription from each individual experiment were normalized to control condition, which was made equal to 1. Densitometric data were obtained from 4-5 experiments and are means ± SE. A: SP-A transcription data. There was no effect of RA on rate of SP-A gene transcription. There was a significant inhibitory effect of cortisol on rate of SP-A gene transcription compared with control values (P < 0.05, Dunnett's test). Combination of cortisol and RA also had a significant inhibitory effect on SP-A gene transcription compared with controls (P < 0.05, Dunnett's test). B: SP-B transcription data. RA had no effect on rate of SP-B gene transcription. There was a significant stimulatory effect of cortisol added alone and the combination of cortisol and RA on rate of SP-B gene transcription compared with control condition (P < 0.05, Dunnett's test). * Significant difference from control condition (A and B; P < 0.05, Dunnett's test).

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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Fig. 4.   Effect of RA, cortisol, and combination of cortisol+RA on SP-A and SP-B mRNA stability. Confluent monolayers of H441 cells were exposed for 18 h to serum-free media containing either no additions (control), RA (10-6 M), cortisol (10-6 M), or a combination of cortisol+RA. These cells were then further incubated with actinomycin D (10 µg/ml) and harvested after 0, 4, 8, 12, or 24 h. A: SP-A mRNA stability. Time points on graph represent means of SP-A mRNA levels relative to 0 time point, which was made equal to 1 (means of 4-5 experiments). None of regulatory substances alone or in combination had a significant effect on SP-A mRNA stability. SP-A mRNA half-life values calculated from these pooled data were: control, 15.2 h; RA, 26.2 h; cortisol, 15.5 h; and RA+cortisol, 16.0 h. B: SP-B mRNA stability. Points on graph represent densitometric data of SP-B mRNA levels relative to 0 time point, which was made equal to 1 (means of 5-6 experiments). RA alone, cortisol alone, and combination of cortisol+RA all significantly increased half-life of SP-B mRNA. SP-B mRNA half-life values calculated from these pooled data were: control, 13.2 h; RA, 22.7 h; cortisol, 25.1 h; and RA+cortisol, 29.6 h.

                              
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Table 1.   Effect of all trans-retinoic acid, cortisol, and combination of all trans-retinoic acid + cortisol on SP mRNA stability

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 10-8 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 10-6 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 (10-7 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 × 10-7 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 (10-7 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 × 10-7 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.

    ACKNOWLEDGEMENTS

We thank Rose Marsh for preparation of the manuscript and Dr. Jonathan Klein for helpful discussions.

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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