j. biol. chem.-1989-boggaram-11421-7

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 19, Issue of July 5, pp. 11421-11427, 1989 Printed in U. S. A.

Regulation of Expression of the Gene Encoding the Major Surfactant Protein (SP-A) in Human Fetal Lung in Vitro DISPARATE EFFECTS OF GLUCOCORTICOIDS ON TRANSCRIPTION AND ON mRNA STABILITY*

(Received for publication, October 12, 1988)

Vijayakumar Boggaram, Margaret E. Smith, and Carole R. Mendelson From the Departments of Biochemistry and Obstetrics-Gynecology, T h e Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, Texas 75235

We previously observed that dexamethasone had a biphasic effect on the levels of mRNA encoding the major surfactant protein (SP-A) in human fetal lung in vitro; at concentrations of 10"O and lo-' M, dexamethasone caused an increase in the levels of SP-A mRNA, whereas at concentrations of >lo-' M, the steroid had a pronounced inhibitory effect on SP-A mRNA levels. It was also found that dexamethasone antagonized the stimulatory effect of dibutyryl cyclic AMP (Bt2cAMP) on SP-A mRNA levels in human fetal lung in vitro. It was our objective, in the present study, to characterize further the effects of dexamethasone and Bt2cAMP on SP-A mRNA levels in human fetal lung tissue and to determine whether such effects are associated with comparable changes in the transcriptional activity of the SP-A gene. We found that the action of dexamethasone (lo" M) to reduce the levels of SP-A mRNA in control and Bt2cAMP-treated fetal lung explants was evident within 2 h of its addition to the culture medium; SP-A mRNA was reduced to barely detectable levels in control and in BtZcAMP-treated tissues after 24 h of dexamethasone treatment. The action of dexamethasone to reduce SP-A mRNA levels was not prevented by co-incubation with either actinomycin D or cycloheximide. In contrast to its dose-related biphasic effects on the levels of SP-A mRNA, we found that dexamethasone caused a dose-dependent stimulation of SP-A gene transcription. BtzcAMP also increased the transcriptional activity of the SP-A gene in the human fetal lung in vitro. In fetal lung explants incubated in the presence of dexamethasone plus Bt2cAMP, a synergistic induction of SP-A gene transcription was observed at concentrations of dexamethasone of lo-'- 10" M. Our findings are indicative that the stimulatory effects of dexamethasone (10"0-10-9 M) on SP-A mRNA levels are reflective of a stimulatory effect of the steroid on SP-A gene transcription, whereas the inhibitory effects of dexamethasone (lo" M) on SP-A mRNA levels are the result of a dominant effect of the steroid in elevated concentrations to reduce SP-A mRNA stability.

The synthesis of pulmonary surfactant, a lipoprotein com- prised of 80% glycerophospholipid and 5-10% protein, is developmentally regulated in fetal lung tissue. Surfactant

* This work was supported in part by National Institutes of Health Grant HD13912 and by Clinical Research Grant 6-365 from the March of Dimes Birth Defects Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

synthesis by the fetal lung, which is initiated during the latter 10-15% of gestation in all mammalian species, is believed to be hormonally regulated. A number of hormones, including glucocorticoids, prolactin, thyroid hormones, and estrogens, have been reported to enhance the synthesis of surfactant glycerophospholipids in fetal lung tissue (see Ref. 1 for review).

The surfactant-associated proteins also are subject to mul- tifactorial regulation. The major surfactant-associated protein, SP-A, a 35,000-dalton glycoprotein (2-4), binds strongly to surfactant glycerophospholipids promoting their aggrega- tion (5) and may serve a role together with calcium in the structural organization of tubular myelin (6, 7), a lattice-like structure that appears to facilitate the rapid formation of the phospholipid surface film at the alveolar air-liquid interface. SP-A may also bind to specific receptors on the lumenal surface of the type I1 cells and mediate the reutilization of secreted surfactant glycerophospholipids and proteins (8). Cyclic AMP analogues markedly stimulate the synthesis of SP-A and increase the levels of its mRNA in both rabbit (9, 10) and human (11,12) fetal lung tissue maintained in organ culture. In fetal rabbit lung in culture, the stimulatory effects of BtzcAMP1 on SP-A mRNA levels are associated with comparable increases in the transcriptional activity of the SP-A gene (13). The stimulatory effects of BtzcAMP on SP-A synthesis in human fetal lung in uitro are mimicked by the &adrenergic agonist, terbutaline (11). We also have observed that insulin causes a dose-dependent inhibition of the accumulation of immunoreactive SP-A in human fetal lung i n uitro (14).

Glucocorticoids also have been reported to regulate the synthesis of SP-A in fetal lung tissue in uitro and i n uiuo; however, the reported effects of glucocorticoids on SP-A synthesis and on the levels of its mRNA are conflicting. We observed that cortisol treatment of lung explants from 21-day gestational age fetal rabbits caused an increase in the levels of SP-A and its mRNA (9). Phelps et al. (15) recently reported that dexamethasone administration to fetal and neonatal rats increased SP-A synthesis and the levels of its mRNA. In studies with human fetal lung in uitro, Ballard and colleagues (16) reported that dexamethasone at lo-' M caused a marked stimulation of the levels of SP-A and its mRNA. In contrast, Whitsett et al. (12) reported that dexamethasone caused a dose-dependent inhibition of accumulation of SP-A and SP-A mRNA. On the other hand, we have found that dexamethasone has a biphasic effect on the levels of SP-A and its mRNA in human fetal lung in culture (17); at concentrations of 10"' and lo-' M, dexamethasone has a stimulatory effect, whereas at concentrations greater than lo-' M, the glucocor-

The abbreviation used is: Bt2cAMP, dibutyryl cyclic AMP.

11421

11422 Glucocorticoid Regulation of the SP-A Gene in Human Fetal Lung

ticoid is markedly inhibitory as compared with control tissues. These biphasic effects of dexamethasone are also observed in the presence of Bt2cAMP (17). Liley et al. (18) also recently reported that dexamethasone has dose-dependent biphasic effects on the levels of SP-A and its mRNA in human fetal lung in vitro; however, in those studies the levels of SP-A protein and mRNA in lung explants treated with dexamethasone at loT7 M were comparable with those of control tissues. These investigators also reported that the stimulatory effects of dexamethasone (lo-' M) on SP-A accumulation were manifest after relatively brief (24-72 h) periods of incubation; after longer incubation times, these stimulatory effects were no longer apparent (18).

In the present investigation, we have assessed the effects of dexamethasone on the transcriptional activity of the SP-A gene and on the associated levels of SP-A mRNA in human fetal lung in vitro to define the molecular mechanisms whereby glucocorticoids elicit their effects on the levels of SP-A mRNA in this tissue. We have observed that dexamethasone has a paradoxical effect on SP-A gene expression in human fetal lung. The glucocorticoid causes a rapid dose- dependent stimulation of SP-A gene transcription; however, at concentrations of >lo-' M, dexamethasone also causes a rapid decline in the levels of SP-A mRNA. The inhibitory effect of dexamethasone on SP-A mRNA levels is not dependent upon de novo mRNA and protein synthesis and, thus, appears to be mediated by a novel mechanism.

MATERIALS AND METHODS

Organ Culture-Lung tissues were obtained from human abortuses of 15-18 weeks of gestational age, in accordance with the Donors Anatomical Gift Act of the State of Texas. Consent procedures and protocols were approved by the Human Research Review Committee at the University of Texas Southwestern Medical Center at Dallas, TX. Lung tissues from 2 to 4 fetuses are commonly utilized for each experiment. The individual tissues were trimmed, rinsed, pooled, and then minced. Five to six explants were placed in each culture dish and maintained in organ culture in serum-free Waymouth's MB752/ 1 medium (GIBCO), as described in detail previously (19). Lung explants were incubated either in control medium, in medium con- tainingdexamethasone (10"0-10-7 M) , BtzcAMP (1 mM), or BtzcAMP plus dexamethasone for up to 6 days. The dexamethasone was solu- bilized in ethanol; however, the concentration of ethanol in the culture medium never exceeded 0.01%, and this concentration of ethanol had no effect on the levels of SP-A mRNA in the fetal lung explants (data not shown). In some experiments, fetal lung tissues were incubated in medium that also contained either actinomycin D (5 p M ) or cycloheximide (2 or 10 pg/ml) for time periods of up to 24 h. The media were changed daily.

Isolation of RNA and Northern Blot Analysis of SP-A mRNA- Total RNA was isolated from human fetal lung explants essentially by the method of Chirgwin et al. (20) and subjected to Northern analysis using a 32P-labeled cDNA insert encoding rabbit SP-A (lo), as described in detail previously (17). The blots were subjected to autoradiography using intensifying screens, and the relative amounts of SP-A mRNA were assessed by scanning densitometry of the autoradiograms.

Isolation of Nuclei and Transcription Elongation Assay-Isolation of nuclei and transcription elongation assays were performed as described previously using the rabbit SP-A cDNA as a hybridization probe (13). As a control for background, some filters either contained pUC18 DNA or no bound DNA. In both cases, the background radioactivity was found to be 110% of the values in control tissues. After the final wash step, the filters were subjected to autoradiography using intensifying screens, and the relative rates of transcription were assessed by one-dimensional scanning densitometry of the autoradiograms. Although this method can produce artifacts in the scanning of dot blots, in all cases the scanning beam was directed through the center of each dot. Furthermore, in some experiments the bound RNA was released by treating the filters with 0.25 ml of NaOH (0.4 M) for 30 min at room temperature and neutralized with 0.1 ml of acetic acid (1 M), and the radioactivity was determined by scintillation spectrometry. In those cases, the findings from densitometric scan-

ning of autoradiograms and from elution of radiolabeled RNA from filters were found to be similar (data not shown).

RESULTS

Effects of Dexamethasone in Various Concentrations and of BtzcAMP on the Levels of SP-A mRNA and on the Transcrip- tional Activity of the SP-A Gene-To define the mechanisms underlying the biphasic effect of dexamethasone on SP-A gene expression in human fetal lung in vitro (17), transcriptional activity of the SP-A gene was analyzed in nuclei isolated from human fetal lung explants maintained for 5 days in control medium or in medium containing BtzcAMP, in the absence or presence of dexamethasone at concentrations of 10-'0-10-7 M. Total RNA from the same tissues was analyzed for SP-A mRNA by Northern analysis. As shown in the autoradiogram of the Northern blot (Fig. lA, upper panel) and the plot of the scanned autoradiogram (Fig. lB, lower panel), a biphasic effect of dexamethasone on the levels of SP-A mRNA was observed that was consistent with our previous observations (17). We were surprised to find, however, that dexamethasone had a dose-dependent stimulatory effect on SP-A gene transcription (Fig. lA, center panel; Fig. lB, upper panel). A maximal stimulatory effect of dexamethasone was apparent at a concentration of M, with half- maximal stimulation occurring at concentrations between lo-' and lo-' M.

BtzcAMP increased both the levels of SP-A mRNA and the transcriptional activity of the SP-A gene. In tissues incubated with Bt2cAMP and dexamethasone (10-'0-10-7) in combination, dexamethasone at a concentration of 10"' M increased the levels of SP-A mRNA over those of explants incubated with BtzcAMP alone, whereas at M, dexamethasone caused a marked reduction in the levels of SP-A mRNA (Fig. lA, lowerpanel; Fig. lB, lowerpanel). In contrast, in the same tissues, dexamethasone caused a dose-dependent increase in SP-A gene transcription; a stimulatory effect of dexamethasone was apparent at concentrations as low as 10"' M. At concentrations of 10-9-10-7 M, dexamethasone in the presence of BtzcAMP caused an apparent synergistic increase in SP-A gene transcription (Fig. lA, centerpanel; Fig. lB, upperpanel). The effect of dexamethasone to alter SP-A mRNA levels appeared to be specific; when the same Northern blots were probed using a radiolabeled actin cDNA insert, it was found that the levels of actin mRNA were unaffected by dexamethasone at all concentrations tested (data not shown). Also, the amounts of total RNA isolated from the explants were unaffected by dexamethasone treatment (data not shown).

Effects of Dexamethasone on the Leuels of SP-A mRNA and on SP-A Gene Transcription as a Function of Incubation Time-To analyze the effects of dexamethasone on the levels of SP-A mRNA and on the transcriptional activity of the SP-A gene as a function of incubation time, human fetal lung explants were maintained in the absence or presence of BtzcAMP for 5 days; dexamethasone ( M) was then added to the media, and the incubation was continued for 2, 6, 24, or 48 h. The levels of SP-A mRNA and the transcriptional activity of the SP-A gene in these tissues were analyzed by Northern blotting of total RNA and transcription elongation assays, respectively. As shown in the autoradiogram in Fig. 2A and the corresponding.plot of the densitometric scan in Fig. 2B, the effect of BtzcAMP on SP-A gene transcription appears to be relatively modest at the start (day 5 of incubation) of the time course experiment (1.3-fold as compared with control). After an additional 48 h of incubation, however, SP-A gene transcription in BtzcAMP-treated tissues was increased to levels that were &fold greater than the corresponding control sample. Dexamethasone alone had no effect on

Glucocorticoid Regulation of the SP-A Gene in Human Fetal Lung 11423

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Dexamethasone [MI

FIG. 1. Effects of dexamethasone (Dex) in the absence or presence of Bt2cAMP (Bt2) on the transcriptional activity of the SP-A gene and on the levels of SP-A mRNA in human fetal lung in vitro. A, human fetal lung explants were maintained in culture for 5 days in the absence ( C ) or presence of dexamethasone (lO””-lO-i M) and in the absence or presence of BGcAMP (1 mM). Nuclei isolated from the tissues were subjected to transcription run- on analysis using the rabbit SP-A cDNA as a probe and an auto-

altering the transcriptional activity of the SP-A gene during the first 24 h of incubation; however, after 48 h of dexamethasone treatment, a 1.4-fold increase in SP-A transcription was detected. In contrast, in lung explants maintained in medium containing Bt2cAMP, a stimulatory effect of dexamethasone on SP-A gene transcription was evident within 2 h of its addition to the medium, and this stimulatory effect was increased as a function of incubation time. After 48 h of incubation, a synergistic effect of dexamethasone and BtcAMP was clearly apparent. At this time point, a 15-fold increase in SP-A gene transcription was observed in lung explants treated with dexamethasone and Bt2cAMP in combination, as compared with a 6- and 1.4-fold increase in SP-A gene transcription in lung explants incubated in the presence of either Bt2cAMP or dexamethasone alone, respectively.

On the other hand, dexamethasone was found to have a rapid effect in reducing the levels of SP-A mRNA in lung tissues maintained in either the absence or presence of Bt2cAMP (Fig. 2, A and B) . An inhibitory effect of dexamethasone on SP-A mRNA levels was apparent as early as 2 h after its addition to the culture medium; after 24 and 48 h of incubation in the presence of dexamethasone, the levels of SP-A mRNA were markedly reduced as compared with those of explants maintained in control or BtcAMP-containing medium.

Although there is some variability between experiments in the magnitude of the effects of BbcAMP and dexamethasone on SP-A gene transcription, we consistently have observed a stimulatory effect of BbcAMP and dexamethasone and a synergistic effect of Bt2cAMP plus dexamethasone on SP-A gene transcription in human fetal lung explants after 5-6 days of incubation. The data from several independent experiments are summarized in Table I. These effects on transcription are always associated with a stimulatory effect of Bt2cAMP on SP-A mRNA levels and a marked effect of dexamethasone to reduce (by >go%) the levels of SP-A mRNA in control and BtcAMP-treated fetal lung explants (Table I).

Effects of Actinomycin D and Cycloheximide on the Dexa- methasone-induced Inhibition of SP-A mRNA Levels-The finding that dexamethasone (lo” M) caused a rapid decline in SP-A mRNA levels despite its action to increase SP-A gene transcription suggests that the steroid acts to increase the rate of SP-A mRNA degradation. To determine whether this effect of dexamethasone to cause a rapid decline in the levels of SP-A mRNA is dependent upon de novo RNA and protein synthesis, the effects of cycloheximide (2 or 10 pg/ ml) and actinomycin D (5 p ~ ) on the action of dexamethasone ( M) to alter SP-A mRNA levels were studied in fetal lung tissues as a function of incubation time. We observed that antinomycin D at this concentration reduced RNA synthesis by 90% after 2 h and 97% after 24 h of incubation. Cyclohex- imide at 2 pg/ml inhibited protein synthesis by 66% after 2 h and 74% after 24 h of incubation; a t 10 pg/ml, protein synthesis was inhibited by 87% at each of these time points.

In the experiment to test the effect of cycloheximide (2 pg/ ml) on the dexamethasone-induced reduction of SP-A mRNA

radiogram was obtained (centerpanels). Total RNA isolated from the same tissues was analyzed by Northern blotting using 32P-labeled rabbit SP-A cDNA as a probe and an autoradiogram was obtained (upper and lower panels). B, the autoradiograms in panel A were subjected to scanning densitometry, and the relative rates of SP-A gene transcription and mRNA levels of treated tissues are plotted relative to control values. When nonrecombinant pUC18 DNA was used in transcription elongation assays as a hybridization control, the background radioactivity was found to be 510% of control values (data not shown).


A. Incubation time (h)

With Dex WhJ Dex Bt2 Start 2 6 24 48 48

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Incubation Time (h) FIG. 2. Effects of dexamethasone (Dex) and Bt2cAMP added

alone or in combination on SP-A gene transcription and mRNA levels as a function of incubation time. A, human fetal lung explants were maintained in culture in the absence or presence of BQAMP for 5 days; dexamethasone (lo-' M ) was then added to some of the dishes and the incubation was continued for 2-48 h. Nuclei were isolated from the tissues before (Start) and after the time of dexamethasone addition, and SP-A gene transcription was analyzed by transcription run-on analysis (upper panel); SP-A mRNA levels were assessed by Northern blotting (lower panel). B, the autoradiograms in panel A were subjected to scanning densitometry and SP-A gene transcription and mRNA levels of treated tissues are plotted relative to the control values at time 0. When nonrecombinant pUC18 DNA was used in transcription elongation assays as a hybridization control, the background radioactivity was found to be 110% of control values (data not shown).

levels, the fetal lung explants were preincubated in medium containing BbcAMP for 5 days before the addition of dexamethasone and cycloheximide to induce SP-A mRNA to readily detectable levels. As shown in Fig. 3, dexamethasone

M) had a rapid effect in reducing the levels of SP-A mRNA in the Bt2cAMP-treated explants; the levels of SP-A mRNA were detectably reduced within 2 h of the addition of

TABLE I Effects of BtSAMP and dexamethasone, added alone and in

combination, on the transcriptional activity of the SP-A gene and on the levels of SP-A mRNA in human fetal lung explants

Human fetal lung explants were maintained in organ culture for 5-6 days in control medium or in medium containing BbcAMP (1 mM), dexamethasone (lo-' M), or the two agents in combination. Total RNA was isolated from the tissues and analyzed for SP-A mRNA by Northern blotting and densitometric scanning of the autoradiograms. Nuclei were subjected to transcription elongation assays; the radiolabeled RNA transcripts were hybridized to filters with bound pUC18 containing the SP-A cDNA insert. Filters con- tainingpUC18 DNA were included in each incubation vial to measure background radioactivity. The autoradiograms were subjected to densitometric scanning, and the values were corrected by subtracting the value of the pUC 18 control in each case. Data are the mean f S.E. of the mean of three to four independent experiments and are ex- pressed relative to control values.

SP-A gene transcription

relative to control

SP-A mRNA levels relative to

control Treatment

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Control 1.00 ( n = 4) 1.00 ( n = 4) BbcAMP 2.58 f 0.44 ( n = 4) 3.61 f 0.62 ( n = 4) Dexamethasone 2.02 e 0.43 ( n = 3) 0.05 -+ 0.03 ( n = 4) BtSAMP plus 8.17 -+ 1.23 ( n = 3) 0.32 & 0.13 ( n = 4)

dexamethasone

Dex 110'7Ml CHX Dex+ CHX

Incubation Time (h) FIG. 3. Effects of cycloheximide (CHX) and dexamethasone

(Dex) added alone and in combination on the levels of SP-A mRNA in human fetal lung tissue as a function of incubation time. Human fetal lung explants were maintained in culture for 6 days in medium containing BtSAMP (Bt2, 1 mM). Either dexamethasone (lo-' M), cycloheximide (2 pg/ml), or the two agents in combination were added to some of the dishes from 2 to 24 h prior to harvest of the tissue on day 6. Total RNA was isolated, subjected to Northern blotting using the rabbit SP-A cDNA as a probe, and an autoradiogram was obtained.

dexamethasone to the culture medium, and SP-A mRNA was reduced to barely detectable levels within 6-24 h of its addition to the medium. In tissues incubated with cycloheximide (2 pg/ml) alone the levels of SP-A mRNA remained relatively constant for 6 h and then declined. When fetal lung explants were incubated with cycloheximide and dexamethasone in combination, the levels of SP-A mRNA were unaffected after 2 h, but were markedly reduced after 4-24 h of incubation in a manner similar to that of tissues maintained in the presence of dexamethasone alone.

In consideration of the possibility that our failure to observe an effect of cycloheximide in blocking the action of dexamethasone to reduce SP-A mRNA levels was due to an incomplete inhibition of protein synthesis, studies were performed using cycloheximide at a concentration of 10 pg/ml. In the same experiment, the effects of actinomycin D (5 p ~ )


Control Dex Act D CHX

Act D Dex CHX Dzx +

rl

0 6 24 6 24 6 24 6 24 6 24 6 24

hours FIG. 4. Effects of cycloheximide (CZfX) in elevated concen-

trations and actinomycin D (Act D) added alone or in combination with dexamethasone (Dex) on the levels of SP-A mRNA in human fetal lung explants. Human fetal lung explants were maintained in culture for 5 days in control medium. Dexamethasone (IO-' M) in the ahsence or presence of cycloheximide (10 pg/ml) or actinomycin D ( 5 ~ L M ) was then added to the medium, and the incuhation was continued for 6-24 h. Total RNA was isolated from the tissue and analyzed for SP-A mRNA by Northern blotting using the rahhit SP-A cDNA as a probe, and an autoradiogram was obtained.

also were investigated. In the study shown in Fig. 4, human fetal lung explants were maintained for 5 days in control medium; dexamethasone M) in the absence or presence of inhibitors was then added to the culture medium, and the levels of SP-A mRNA were analyzed after 6 and 24 h of further incubation. In this study, an inhibitory effect of dexamethasone was evident a t 6 h of incubation, and SP-A mRNA was reduced to barely detectable levels after 24 h. In lung explants incubated with actinomycin D alone, the levels of SP-A mRNA were unaffected after 6 h of incubation, but were markedly reduced after 24 h. Actinomycin D failed to block the inhibitory effect of dexamethasone on SP-A mRNA levels after 6 h of incubation; in fact, the levels of SP-A mRNA were lower than those observed after incubation with dexamethasone alone. In fetal lung explants incubated for 6 h with cycloheximide (10 pg/ml) alone, the levels of SP-A mRNA were reduced as compared with untreated tissues at the same time point and were barely detectable after 24 h of treatment. In fact, the levels of SP-A mRNA in cycloheximide-treated fetal lung explants were quite similar to those observed after treatment with dexamethasone alone. In tissues incubated with cycloheximide plus dexamethasone, the levels of SP-A mRNA were similar to those observed after treatment with either factor alone. This marked reduction in SP-A mRNA levels after treatment with cycloheximide may be caused by its effect to inhibit both SP-A gene transcription and mRNA stability. In previous studies using fetal rabbit lung tissue in vitro (13), we observed that cycloheximide (2 pg/ml) had a rapid effect on inhibiting SP-A gene transcription in control explants, as well as the stimulatory effect of BtcAMP, suggesting that a labile protein factor is required for regulation of the transcriptional activity of the SP-A gene.

DISCUSSION

In the present study, we have observed that dexamethasone has a paradoxical effect on the expression of the gene encoding the major surfactant-associated protein, SP-A, in human fetal

lung in vitro. The glucocorticoid caused a dose-dependent stimulation of SP-A gene transcription, with a maximum stimulatory effect evident a t concentrations of 10-R-lO" M. At these concentrations, dexamethasone acted synergistically with Bt2cAMP to increase the rate of SP-A gene transcription. On the other hand, dexamethasone caused a marked reduction in the levels of SP-A mRNA and reduced the magnitude of the stimulatory effect of BtcAMP on SP-A mRNA levels. The effect of dexamethasone (10" M) to reduce SP-A mRNA levels was not blocked by inhibitors of mRNA and protein synthesis. In preliminary studies, we observed that in fetal lung explants incubated with actinomycin D and dexamethasone (lo-' M) in combination, the rate of decline of SP-A mRNA levels was >3 times greater than that observed in explants incubated in the presence of actinomycin D alone.* From all of these findings, it is apparent that the effect of dexamethasone M) to reduce SP-A mRNA levels is not mediated by an inhibitory effect of the steroid on SP-A gene transcription. Rather, it appears that dexamethasone a t such concentrations is eliciting its effects by increasing the rate of degradation of SP-A mRNA. The present findings are suggestive that the stimulatory effect of dexamethasone on SP-A mRNA levels a t concentrations of 10-'n-lO-' M reflect effects of the steroid to increase SP-A gene transcription in the absence of an effect to decrease the stability of SP-A mRNA. At concentrations >lo-* M, however, the action of the glucocorticoid to increase the rate of SP-A mRNA degradation overrides its stimulatory effect on SP-A gene transcription, resulting in decreased levels of SP-A mRNA.

Although the majority of studies concerning the control of eucaryotic gene expression have been directed toward the regulation of gene transcription, it has become increasingly apparent that post-transcriptional mechanisms serve an equally important role in the regulation of mRNA levels (21). Such post-transcriptional events as mRNA processing and transport out of the nucleus, as well as mRNA degradation, and stabilization, have been reported to be regulated by hormones and cellular and environmental factors (22). Modula- tion of mRNA stability may provide an important homeo- static mechanism for the regulation of synthesis of a number of cellular proteins. Iron has been reported to negatively regulate the levels of mRNA for the human transferrin receptor; these effects on transferrin receptor mRNA levels are primarily due to a specific decrease in transferrin receptor mRNA stability (23). The iron-dependent regulation of transferrin receptor mRNA levels requires the presence of a stem- loop structure within the 3"untranslated region of the mRNA (23, 24) and is inhibited by actinomycin D or cycloheximide, suggesting that an iron-regulated RNase or other protein with a relatively short half-life binds to this structure and mediates transferrin receptor mRNA degradation (23).

A number of hormones and second messengers have been reported to exert effects on gene expression by altering the stability of specific mRNAs. Glucocorticoids selectively inhibit the transcription of the interleukin-18 gene and cause a marked decrease in the stability of its mRNA (25). This effect is blocked either by actinomycin D or cycloheximide (25), suggesting that a glucocorticoid-inducible protein may specif- ically act to reduce interleukin-18 mRNA stability. The mRNAs for interleukin-18 and a number of other protoon- cogenes and inflammatory mediators contain within their 3'- untranslated regions one or more copies of the sequence AUUUA, which is believed to mediate their selective degradation and resultant short half-life (26, 27). Although one

V. Boggaram. M. E. Smith, and C. R. Mendelson, unpublished observations.


might suggest that glucocorticoids increase interleukin-lp degradation by inducing the synthesis of an RNase or other protein that binds selectively to this A + U-rich sequence, in the same cells it was found that dexamethasone had no effect on the stability of C-fos mRNA, which contains several copies of such an A + U-rich sequence (25). Glucocorticoids also have been reported to reduce the levels of mRNAs for inter- feron (28) and granulocyte-macrophage colony-stimulating factor (29) in a variety of target cells, while having little effect on transcription of the respective genes.

Although the mechanisms whereby hormones and factors regulate the stability of specific mRNAs must differ signifi- cantly, it appears that in all reported examples the induction of mRNA destabilization is dependent upon ongoing protein synthesis, suggestive of the induction of protein factors that mediate mRNA degradation. In the present study, however, it was found that the effect of dexamethasone to cause a rapid decrease in the levels of SP-A mRNA was independent of ongoing mRNA and protein synthesis. In unpublished studies,' we found that the effect of glucocorticoids to reduce SP-A mRNA levels is rapidly reversible. Based on these observations, we suggest that glucocorticoids may effect a decrease in SP-A mRNA stability by inhibiting the synthesis of a labile protein(s) which is required for SP-A mRNA stability. This hypothesis is supported by the finding that in fetal lung explants incubated with cycloheximide (10 pg/ml) for 6 or 24 h, the levels of SP-A mRNA were reduced to levels comparable with those of explants incubated for these periods with dexamethasone M) alone (Fig. 4). It has recently been suggested that glucocorticoids decrease the stability of 3-hydroxymethylglutaryl-CoA reductase mRNA in rat liver tissue by counteracting a thyroid hormone-induced protein which stabilizes this mRNA species (30). Alternatively, dexamethasone may induce the post-translational modification of a stable protein factor resulting in an increase in its capacity to facilitate SP-A mRNA degradation.

The mechanisms underlying the biphasic effect of glucocorticoids on the levels of SP-A mRNA have not been defined. Possible explanations for the apparent biphasic effect could be differences in the sensitivities to dexamethasone or the time courses of its effects on SP-A gene transcription and mRNA destabilization. Thus, the inductive effect of dexamethasone on gene transcription may be manifest at a lower concentration of the steroid than its effect to reduce SP-A mRNA stability. In studies of Fu5-5 cells infected with mouse mammary tumor virus, it was found that the sensitivity of the endogenous tyrosine aminotransferase gene to dexamethasone was almost an order of magnitude greater than that of mouse mammary tumor virus (31). It also has been reported that glucocorticoid induction of tyrosine aminotransferase occurs at much lower concentrations than does glutamine synthetase in these cells (32). The inductive effect of dexamethasone on SP-A gene transcription and the inhibitory effect on SP-A mRNA stability also could be mediated through its actions on different cell types in the fetal lung explants with possibly differing sensitivities to glucocorticoids. It has been suggested that the stimulatory effects of glucocorticoids on surfactant glycerophospholipid biosyn- thesis are mediated by an action to stimulate the production of fibroblast pneumonocyte factor by lung mesenchyme (33). Therefore, the stimulatory effects of dexamethasone could be mediated through its action to increase the synthesis of a stimulatory factor by lung fibroblasts, whereas its inhibitory action on SP-A mRNA stability could be mediated by a direct action on the type I1 cell. We consider this to be rather unlikely, because both the stimulatory effect of dexametha-

sone on SP-A gene transcription and its effect to decrease SP-A mRNA stability are manifest within 2 h of its addition to the culture medium when lung explants are pretreated with BtzcAMP (Fig. 2).

It is important to note that this action of glucocorticoids to reduce SP-A mRNA stability appears to be unique to human fetal lung tissue. In studies with fetal rabbit lung tissue in vitro, we have observed that glucocorticoids cause a transient decrease in the levels of SP-A mRNA followed by a subse- quent induction of SP-A mRNA levels with increased time of incubation; however, these effects of glucocorticoids on SP-A expression in fetal rabbit lung tissue appear to be regulated primarily at the transcriptional level (13). In fetal rat lung tissue, glucocorticoids have been reported to increase the levels of SP-A mRNA when administered in vivo (15).

Another major finding of the present study is the synergistic action of dexamethasone and BtzcAMP to increase SP-A gene transcription. As shown in Fig. 2, when fetal lung explants were incubated for 48 h in the presence of either dexamethasone M) or Bt,cAMP, SP-A gene transcription was increased 1.4- and 6-fold, respectively. On the other hand, a >15-fold induction of SP-A gene transcription was detected in explants incubated for the same period in medium containing Bt,cAMP and dexamethasone in combination. In fetal lung explants incubated for 5 days in the presence of BtzcAMP and dexamethasone in combination, a synergistic effect of these agents on SP-A gene transcription was observed at concentrations of dexamethasone of 10-9-10-7 M (Fig. 1). Glucocorticoids and cyclic AMP analogues have been found to have either additive or synergistic effects on the induction of tyrosine aminotransferase mRNA in liver tissue (34) and in hepatoma cell lines (35). Although it is clear that cyclic AMP is acting to increase tyrosine aminotransferase mRNA levels, at least in part, at the transcriptional level (34), the molecular mechanisms for the combined effects of cyclic AMP and glucocorticoids on tyrosine aminotransferase gene expression have not been determined.

The actions of glucocorticoids to enhance eucaryotic gene transcription appear to be mediated by the interaction of the glucocorticoid receptor with specific sequences within the 5'- flanking regions of responsive genes (see Ref. 36 for review). Cyclic AMP is believed to mediate an increase in gene expression through the binding of a specific nuclear phosphoprotein to conserved cyclic AMP regulatory elements within the 5'- flanking regions of responsive genes (37). It will be of great interest to define the mechanisms whereby cyclic AMP and glucocorticoids act to increase SP-A gene transcription, as well as the molecular interactions that result in the synergistic effects of these factors to activate transcription of this gene.

Acknowledgments-We gratefully acknowledge the skilled techni- cal assistance of Gloria Vanderburg and Jo Smith.

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