Posttranscriptional regulation of surfactant protein-A messenger RNA in human fetal lung in vitro by glucocorticoids.

Posttranscriptional Regulation of Surfactant Protein-A Messenger RNA in Human Fetal Lung in Vitro by Glucocorticoids

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

methasone also was found to have dose-dependent effects on the degradation of SP-A mRNA. After 12 h of incubation in the presence of actinomycin-D and dexamethasone at 10~9 and 10~7 M, the levels of SPA mRNA were reduced by 50% and 80%, respectively, compared to those in tissue incubated with actinomycin-D alone. The inhibitory effects of glucocorticoids on SP-A mRNA levels were completely reversible and were blocked by the glucocorticoid antagonist RU486. Based on these findings, we suggest that the biphasic effects of glucocorticoids on SP-A mRNA levels in human fetal lung in vitro are caused by the differential actions of glucocorticoids on SP-A mRNA transcription and stability. (Molecular Endocrinology 5: 414-423, 1991)

Surfactant protein-A (SP-A), the major pulmonary surfactant-associated protein, is a developmentally and hormonally regulated sialoglycoprotein of about 35,000 mol wt. In previous studies we observed that dexamethasone has dose-dependent biphasic effects on the levels of SP-A and its mRNA in human fetal lung in vitro. At concentrations of 10~10-10"9 M, dexamethasone increased the levels of SP-A and its mRNA over those of control tissues, whereas at concentrations >10~8 M, the steroid was markedly inhibitory. Our findings suggest that the inhibitory action of dexamethasone (>10~8 M) on SP-A mRNA levels was mediated by an effect to reduce SP-A mRNA stability, since the steroid caused a dosedependent increase in the rate of transcription; however, an effect to increase transcription with premature termination leading to instability of mRNA transcripts could not be ruled out. In the present investigation we have studied in detail the mechanisms underlying the biphasic effects of glucocorticoids on SP-A mRNA levels in human fetal lung tissues in vitro. Our findings indicate that dexamethasone (10~7 M) has no adverse effect on the elongation of nascent mRNA transcripts throughout the SP-A gene; elongation of SP-A mRNA transcripts in dexamethasone-treated tissue explants was similar to that observed in tissues incubated in control medium or medium containing (Bu)2cAMP. Therefore, premature termination of SP-A mRNA transcription leading to the instability of SP-A mRNA can be ruled out. On the other hand, we found that dexamethasone (10~7 M) had a pronounced effect to reduce the apparent half-life of SP-A mRNA; in control explants maintained in the presence of actinomycin-D to block gene transcription, the SP-A mRNA half-life was estimated to be 11.4 h, whereas in tissues also treated with dexamethasone, the SP-A mRNA halflife was reduced by more than 60% to 5.0 h. Dexa-

INTRODUCTION

Pulmonary surfactant is a unique lipoprotein (1) that is synthesized and secreted by the type II epithelial cells of the pulmonary alveolus, where it acts to reduce surface tension at the alveolar air-liquid interface. Inadequate synthesis and secretion of surfactant by the lungs of prematurely bom infants can result in respiratory distress syndrome (2), also known as hyaline membrane disease, the leading cause of neonatal morbidity and mortality in developed countries. Surfactant synthesis is developmentally and hormonally regulated in the fetal lung; its synthesis is initiated during the latter part of gestation in mammalian species and appears to be under multifactorial regulation (3, 4). Surfactant protein A (SP-A), the major protein of pulmonary surfactant, is a sialoglycoprotein of about 35,000 mol wt (Mr) (5-8). SP-A has been shown to possess unique biochemical and biophysical properties; it causes aggregation of surfactant phospholipids and in the presence of calcium acts to promote the structural transformation of the lamellar body into tubular myelin (9-12), a lattice-like structure that is formed upon

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Glucocorticoid Regulation of SP-A mRNA Stability

lamellar body secretion into the alveolar lumen. SP-A also is known to act in a cooperative and calciumdependent manner with surfactant proteins SP-B and SP-C to promote rapid formation of phospholipid surface films and, thus, to reduce alveolar surface tension (13). SP-A may mediate the recycling of surfactant lipids and proteins through binding to specific high affinity receptors that are localized on the lumenal surface of type II cells (14). Expression of the SP-A gene in human lung tissue appears to occur exclusively in the type II pneumonocyte (15); however, in rabbits (16) and rats (17) SP-A mRNA has been localized in both type II cells and nonciliated bronchiolar epithelial cells. SP-A gene expression has been found to be developmentally and hormonally regulated in fetal lung tissues of a number of species (3,4). Among the variety of factors that have been reported to regulate the synthesis of SP-A and its mRNA in fetal lung tissues, cAMP and glucocorticoids have been found to exert major effects (4). In human fetal lung in vitro, cAMP analogs act to increase the accumulation of SP-A and its mRNA (18, 19). The stimulatory effect of cAMP on SP-A mRNA levels appears to be mediated primarily by its action to increase SP-A gene transcription (20). Glucocorticoids have been reported to exert doseand time-dependent stimulatory and inhibitory effects on the accumulation of SP-A and its mRNA in human fetal lung in vitro (19, 21-23). We previously observed that dexamethasone has dose-dependent biphasic effects on the levels of SP-A and its mRNA in human fetal lung in vitro; at concentrations of 10~10-10~9 M, dexamethasone increases SP-A mRNA levels, whereas at concentrations greater than 10~8 M, the steroid acts to markedly reduce the levels of SP-A mRNA compared to those in control tissues (22). These stimulatory and inhibitory effects of dexamethasone also are observed in the presence of (Bu)2cAMP (Bt2cAMP) (22). We also found that dexamethasone acts in a dose-dependent manner to increase SP-A gene transcription and that dexamethasone and Bt2cAMP act synergistically to increase the rate of SP-A gene transcription (20). These findings, therefore, suggest that the dose-dependent biphasic effects of dexamethasone on SP-A mRNA levels in human fetal lung in vitro are the result of disparate effects on SP-A gene transcription and mRNA stability (20). The action of dexamethasone at concentrations >10~8 M to reduce SP-A mRNA levels in human fetal lung in vitro was not found to be affected by coincubation with either actinomycin-D or cycloheximide, suggesting that de novo synthesis of either RNA or protein is not required for the action of dexamethasone to reduce SP-A mRNA levels (20). The stimulatory effects of dexamethasone (10~10-10~9 M) on SP-A mRNA levels are, therefore, probably the result of its action to increase SP-A gene transcription, whereas the inhibitory effects of dexamethasone (>10~8 M) on SP-A mRNA levels are possibly caused by the dominant action of the steroid at elevated concentrations to reduce SP-A mRNA stability.

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In the present investigation we have characterized further the mechanisms by which dexamethasone acts to reduce SP-A mRNA levels in human fetal lung in vitro. We have observed that in the presence of actinomycin-D the effect of dexamethasone to reduce SPA mRNA is manifest at concentrations as low as 10~9 M. Furthermore, we have found that the inhibitory actions of dexamethasone on SP-A gene expression are indeed caused by an effect to decrease SP-A mRNA half-life and are not the result of an effect of the steroid to cause premature termination of SP-A gene transcription. We also have found that the actions of glucocorticoids to inhibit SP-A mRNA levels are rapidly reversible and are blocked by the glucocorticoid receptor antagonist RU486.

RESULTS Effects of Dexamethasone and Bt2cAMP on the Transcriptional Activity of SP-A Gene Processes affecting the elongation of nascent RNA transcripts into completed primary transcripts have been shown to play an important role in the regulation of gene expression (24, 25). To determine whether the effect of dexamethasone (10~7 M) to increase SP-A gene transcription but reduce SP-A mRNA levels could be due to an increase in the initiation of prematurely terminated transcripts, the effect of dexamethasone (1CT7 M) in the absence or presence of Bt2cAMP on the rate of elongation of nascent RNA transcripts throughout the SP-A gene was examined. Transcriptional activity of the SP-A gene was analyzed in nuclei isolated from human fetal lung explants maintained for 5 days in control medium, in medium containing dexamethasone (10~7 M), or in medium containing Bt2cAMP in the absence or presence of dexamethasone (10~7 M), using fragments of SP-A gene that spanned its entire length (-81 to 5590; Fig. 1). Using the 1.9-kilobase (kb) SP-A cDNA as a probe, we observed that both Bt2cAMP and dexamethasone increased the rate of SP-A gene transcription; the rate of SP-A gene transcription was increased synergistically in explants incubated with Bt2cAMP and dexamethasone in combination (data not shown). This is in accordance with the findings of our previous studies with human fetal lung in vitro (20). Similar results were obtained using probes 1-4, which spanned the entire SP-A structural gene (Fig. 1, A and B). The blot shown in Fig. 1B was scanned densitometrically, and the arbitrary units obtained for each treatment were calculated relative to the appropriate control value using each probe. These values, calculated as a function of probe length, are presented in Table 1. As indicated in Table 1, the sizes of probes 1-4 are 0.57, 1.7, 1.0, and 2.3 (contains~ 1.3 kb of mRNA coding sequence) kb, respectively. Although the intensity of the signal using probe 2 was considerably greater than that obtained using probes 1, 3, and 4 (Fig. 1B), probe 2 was 2-3


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TATAAA

I

Sstl

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ATG

TGA

Sstl

Sstl

AATAAA EcoR I

EcoRI

-81

5590 Probe 1-

Probe 2

-Probe 3

Probe 4

10 kb

Probe

Bt 2

Dex |Bt 2 +Dex)

B 1234-

Fig. 1. A, Schematic Diagram of the Human SP-A Gene and Genomic Fragments Used as Probes to Study the Effects of Dexamethasone and Bt2cAMP on SP-A Gene Transcription Elongation Intron-exon organization of the human SP-A gene as well as the locations of the TATAA sequence, the translation initiation codon, ATG, the translation termination codon, TGA, and the polyadenylation signal, AATAAA, are shown. Also shown in the diagram are the locations of the restriction fragments of the human SP-A gene that served as probes in analysis of SP-A mRNA transcription rates in nuclei isolated from human fetal lung tissue. The derived structure of the human SP-A gene is based on previously published findings of White et al. (46) and on our own findings (Boggaram, V., and C. R. Mendelson, unpublished observations). B, Effects of Bt2cAMP (Bt2) and dexamethasone (Dex) treatment of human fetal lung explants on the relative rates of SP-A gene transcription using genomic probes 1-4. Nuclei were isolated from human fetal lung explants that were incubated for 5 days in the absence [control (C)] or presence of Bt2cAMP (1 mM) or dexamethasone (10~7 M), added alone or in combination, and SP-A gene transcription was analyzed. The 32P-labeled RNA (7.0 x 106 cpm) was hybridized to nitrocellulose filters that contained equimolar amounts (6.0 pmol) of various fragments of the human SP-A gene in pUC (probes 1-4) or nonspecific DNA (pUC), and an autoradiogram was obtained and scanned. The background signals obtained in transcription run-on assays using nitrocellulose filters containing pUC DNA alone were subtracted from those of the corresponding control and treated samples that were assayed using probes 1 - 4 , before calculation of the fold induction of transcription. C, Effects of Bt2cAMP and dexamethasone, added alone and in combination, on the levels of SP-A mRNA in human fetal lung explants. Total RNA (30.0 ^g) isolated from the lung explants was analyzed by Northern blotting using 32P-labeled rabbit SP-A cDNA, and an autoradiogram was obtained.

times larger than the others. When the relative densities

Bt2cAMP, dexamethasone, and Bt2cAMP plus dexa-

of the spots were calculated as a function of probe

methasone, respectively. Comparable fold inductions

length, the fold stimulations of SP-A gene transcription

by Bt2cAMP and dexamethasone alone and in combi-

by Bt2cAMP, dexamethasone, and Bt2cAMP plus dex-

nation were observed using probes 1, 3, and 4. These

amethasone treatment were similar using each of the

findings are indicative that the rate of elongation of

probes. By use of probe 2, the induction of SP-A gene

nascent SP-A mRNA transcripts was unaffected by

transcription compared to the control value was 7.0-,

dexamethasone or Bt2cAMP added alone or in combi-

7.6-, and 49-fold in nuclei from tissues treated with

nation. In accordance with our previous findings (20,


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Table 1. Effects of Bt2cAMP, Dexamethasone (Dex), and Bt2cAMP plus Dex on the Relative Rates of Transcription of the SP-A Gene using Genomic Probes 1-4 Probe 1 Probe 2 Probe 3 Probe 4 - 8 1 to 639 639-2289 2289-3284 3284-4590

Control Bt2cAMP

Dex Bt2cAMP + Dex

1.0 3.4 2.8 33.5

1.0 7.1 7.6 49.5

1.0 5.3 3.9 43.4

1.0 9.5 4.2 38.3

22), we observed that Bt2cAMP increased the levels of SP-A mRNA by more than 5-fold compared to control values, whereas dexamethasone caused a pronounced inhibition of SP-A mRNA levels in tissues incubated in the absence or presence of Bt2cAMP; the levels of SPA mRNA were reduced by more than 90% in tissues incubated with dexamethasone plus Bt2cAMP as compared to those in tissues incubated with Bt2cAMP alone

Effect of Dexamethasone on SP-A mRNA Turnover In an effort to analyze the effects of dexamethasone on the half-life of SP-A mRNA, we initally used [3H]uridine to radiolabel the RNA pool and performed pulse-chase analysis and filter hybridization to assess the rate of degradation of SP-A mRNA. We found, however, that the amount of tritium-labeled SP-A mRNA recovered was too low to accurately determine the mRNA halflife. In previous studies we observed that dexamethasone (10~7 M) had a rapid effect to reduce the levels of SP-A mRNA in human fetal lung in culture, and that this action was apparently unaffected by actinomycin-D (20). As an alternative approach, we, therefore, decided to use Northern analysis of SP-A mRNA in fetal lung explants at various times after treatment with actinomycin-D in the absence or presence of dexamethasone as a means of analyzing the relative effects of dexamethasone on SP-A mRNA stability. This approach was possible, since the effect of dexamethasone (10~7 M) to reduce SP-A mRNA levels was not prevented by actinomycin-D treatment. In consideration of the likelihood that actinomycin-D itself may alter SP-A mRNA stability, this analysis cannot provide an accurate assessment of the absolute value of the SP-A mRNA half-life. Rather, this study was designed to analyze the effects of dexamethasone on the apparent rate of SP-A mRNA decay in actinomycin-D-treated tissues compared to that in tissues treated with actinomycin-D alone. Human fetal lung explants were maintained in Bt2cAMP-containing medium for 5 days in order to induce SP-A mRNA. Afterward, the lung explants were rinsed and placed in medium containing actinomycin-D (5 MM) in the absence or presence of dexamethasone (10~7 M). We observed previously that actinomycin-D at a concentration of 5 ^M inhibited RNA synthesis by more than 90% after 24 h of incubation (20). Lung explants were processed for isolation of RNA after various times of treatment ranging from 2-24 h, and

the levels of SP-A mRNA were analyzed by Northern blotting. Autoradiograms of Northern blots from five independent experiments were analyzed by scanning densitometry to determine the relative effect of dexamethasone on SP-A mRNA half-life (Fig. 2). From the data presented in Fig. 2, it is apparent that the relative half-life of SP-A mRNA was decreased by more than 60% in lung explants incubated in the presence of dexamethasone plus actinomycin-D (5.0 h) compared to that in explants incubated with actinomycin-D alone (11.4 h). In other experiments we observed that dexamethasone (10~7 M) in the presence of actinomycin-D did not alter actin mRNA levels (data not shown). Effects of Dexamethasone in Various Concentrations on SP-A mRNA Levels In previous studies we observed that dexamethasone at 10~10 and 10~9 M increased the level of SP-A mRNA in human fetal lung in vitro, whereas at dexamethasone concentrations >10~8 M, SP-A mRNA levels were markedly reduced (20). Since dexamethasone also caused a dose-dependent increase in SP-A gene transcription (20), we suggested that the inhibitory effect of dexa-

t S

0

5

10

15

20

25

TIME AFTER ADDITION OF ACTINOMYCIN D (h)

Fig. 2. Effect of Dexamethasone (Dex) on the Rate of SP-A mRNA Degradation Human fetal lung explants were maintained in Bt2cAMPcontaining medium for 5 days in order to induce the levels of SP-A mRNA. Afterward, the lung explants were rinsed and placed in medium containing actinomycin-D (5 HM) in the absence or presence of dexamethasone (10~7 M). Total RNA (30.0 Mg) isolated from lung explants harvested at times ranging from 2-24 h after the addition of actinomycin-D was analyzed for SP-A mRNA levels using Northern blotting. Autoradiograms were quantitated, and data from five independent experiments were plotted using regression analysis. The apparent half-life of SP-A mRNA in tissues treated with actinomycin-D in the absence or presence of dexamethasone was determined. The half-life of SP-A mRNA was calculated from the relationship U = In2/k, where tV2 is the half-life, and k is the first order degradation rate constant, which is determined from the relationship k = -(2.303) (slope). Open symbols and dashed lines represent data from explants incubated wtih actinomycin-D alone; filled symbols and solid line are data from explants incubated with actinomycin-D plus dexamethasone (10~7 M).


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methasone at concentrations >10~8 M was caused by its dominant effect to reduce SP-A mRNA stability. To determine whether the effect of dexamethasone on SP-A mRNA stability is biphasic and manifest only at pharmacological concentrations, or dose dependent and apparent in the physiological range, the effects of dexamethasone in various concentrations on SP-A mRNA levels were analyzed under conditions where SP-A transcription was blocked. Lung explants were first incubated for 5 days in the presence of Bt2cAMP to induce SP-A mRNA. Afterward, the lung explants were rinsed and maintained in medium containing actinomycin-D (5 JUM) in the absence or presence of dexamethasone (1(r 10 -10- 7 M) for 12-24 h. Total RNA isolated from the explants was analyzed for SP-A mRNA levels by Northern blotting. From the results presented in Fig. 3, it can be seen that dexamethasone in the presence of actinomycin-D acted in a dose-dependent manner to decrease the levels of SP-A mRNA in human fetal lung explants. Densitometric scanning analysis of the autoradiograms revealed that after 12 h of incubation in the presence of dexamethasone at 10~10,10~9, and 10~7 M, the levels of SP-A mRNA were reduced by 25%, 50%, and 80%, respectively, compared to those in tissues incubated for those periods with actinomycin-D alone. Reversibility of Glucocorticoid Inhibition of SP-A mRNA Levels and Effect of the Glucocorticoid Receptor Antagonist RU486 We observed previously that the effect of dexamethasone (10~7 M) to decrease the levels of SP-A mRNA in human fetal lung explants is rapid; a marked inhibition of SP-A mRNA levels is frequently observed as early as 2 h after the addition of dexamethasone to the culture medium (20). To determine whether the effect

of glucocorticoids to decrease SP-A mRNA levels is reversible, human fetal lung explants were maintained in the presence of Bt2cAMP for 5 days to induce the levels of SP-A mRNA. Afterward the lung explants were rinsed and incubated for an additional 24 h in the presence of cortisol (10~6 M). Cortisol was used instead of dexamethasone because the former binds with lower affinity to the glucocorticoid receptor and can presumably be removed from the cells more readily. After incubation for 24 h in the presence of cortisol, the cortisol-containing medium was removed, and lung explants were rinsed several times with cortisol-free medium and then incubated in the presence of Bt2cAMP alone for up to 24 h. The levels of SP-A mRNA in lung explants at various time points were analyzed by Northern blotting. Densitometric scanning of the autoradiogram shown in Fig. 4 revealed that incubation of lung explants in the presence of 10~6 M cortisol for 24 h resulted in an 80% decrease in the levels of SP-A mRNA. Subsequent removal of cortisol and continued incubation in the presence of Bt2cAMP resulted in an increase in SP-A mRNA levels; within 24 h, SP-A mRNA levels were comparable to those present in the tissue before cortisol treatment. The results of the preceding experiments indicate that glucocorticoids cause a dose-dependent decrease in the stability of SP-A mRNA in human fetal lung in vitro. A - 5 0 % inhibition of SP-A mRNA levels was observed in tissues coincubated with actinomycin-D and dexamethasone at a concentration of 10~9 M, similar to the Kd for binding of dexamethasone to the glucocorticoid receptor. To provide further evidence that the effect of dexamethasone to decrease SP-A mRNA levels is mediated by its interaction with the

+F «-F>Bt> 24h 2h 24h I *

I

i

I

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o 1(3°169167 o id°i6 9 i6 8 id 7 2.1 kb

2.V kb

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24 h

Fig. 3. Effect of Dexamethasone (Dex; 10~10-10~7 M) in the Presence of Actinomycin-D on SP-A mRNA Levels in Human Fetal Lung Explants Human fetal lung explants were incubated in the presence of Bt2cAMP for 5 days (1 ITIM). Afterward, the medium was removed, and the explants were incubated in the absence [control (C)] or presence of various concentrations of dexamethasone (10~10-10"7 M) and actinomycin-D (5 MM) for 12 and 24 h. Total RNA (30.0 ng) isolated from the lung explants was analyzed using Northern blotting, and an autoradiogram was obtained.

Fig. 4. Reversibility of the Glucocorticoid Inhibition of SP-A mRNA Levels in Human Fetal Lung Explants Human fetal lung explants were incubated in the presence of 1 HIM Bt2cAMP (Bt2) for 5 days. Afterward, the lung explants were rinsed and incubated in the presence of 10"6 M cortisol (F) for 24 h. After incubation for 24 h, the cortisol-containing medium was removed, and the lung explants were rinsed several times with cortisol-free medium and incubated in the presence of Bt2cAMP for 24 h. Total RNA (30.0 ng) isolated from lung explants was subjected to Northern blot analysis, and an autoradiogram was obtained.



glucocorticoid receptor, the effects of the glucocorticoid receptor antagonist RU486 (26) were investigated. Human fetal lung explants were maintained in the presence of Bt2cAMP (1 ITIM) for 5 days in order to increase the level of SP-A mRNA. Afterward, the tissues were incubated for 48 h in medium containing RU486 at concentrations of 10~10-10~6 M in the absence or presence of dexamethasone (10~7 M). The levels of SP-A mRNA in the lung explants were then analyzed by Northern blotting. Densitometric scanning of the autoradiogram shown in Fig. 5 revealed that when fetal lung explants were incubated with RU486 alone, the levels of SP-A mRNA were increased by - 5 0 % over those of control tissues at all concentrations of antagonist tested. In tissues incubated with dexamethasone (10~7 M) alone, SP-A mRNA levels were reduced by 80% compared to those in control tissues. RU486 at 10~10-10~8 M had little effect to block the inhibitory action of dexamethasone (10~7 M) on SP-A mRNA; however, in tissues treated with dexamethasone and RU486 at concentrations of 10~7 and 10~6 M, SP-A mRNA was increased to levels that were 90% and 274%, respectively, of those in control tissues.

DISCUSSION In previous studies of the regulation of SP-A gene expression in human fetal lung in vitro, we found that glucocorticoids have dose-dependent biphasic effects on the levels of SP-A mRNA (18, 20). We consistently have observed that dexamethasone, at concentrations of 10"10 and 10" 9 M, increases the levels of SP-A mRNA over those in control tissues, whereas at concentrations of 10~8 and 10~7 M, the steroid has marked inhibitory

RU 486 [Ml

C

i6 1 o i6 9 io 8 id 7 id 6

RU 486IMI+ Dex[107M)

o 161O169168167166

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Fig. 5. Effect of the Glucocorticoid Antagonist RU486 on the Dexamethasone-Mediated Inhibition of SP-A mRNA Levels in Human Fetal Lung Explants Human fetal lung explants were incubated in the presence of Bt2cAMP (1 HIM) for 5 days. Medium containing Bt2cAMP was then removed, and the explants were incubated further in medium containing various concentrations of the glucocorticoid receptor antagonist RU486 (10"10-10"6 M) in the absence [control (C)] or presence of dexamethasone (10~7 M) for 48 h. Total RNA (30.0 jig) isolated from explants was subjected to Northern blot analysis, and an autoradiogram was obtained.

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effects on SP-A mRNA accumulation (18, 20). Quite contrary to these effects on SP-A mRNA levels, by use of transcription run-on analysis, we found that dexamethasone has a dose-dependent stimulatory effect on SP-A gene transcription (20). Based on these findings, we suggested that at concentrations of 10~10 and 10~9 M, dexamethasone acts to increase SP-A gene transcription without markedly affecting SP-A mRNA stability, whereas at concentrations >10~8 M, its action to reduce SP-A mRNA stability predominates. In those studies we also found that the effects of dexamethasone to reduce SP-A mRNA levels were not prevented by simultaneous incubation with inhibitors of de novo mRNA and protein synthesis (20), suggesting that the action of dexamethasone is not mediated by the induction of protein factors that cause SP-A mRNA degradation. The finding in those studies that the translational inhibitor cycloheximide had time-dependent effects to reduce SP-A mRNA levels similar to those observed with dexamethasone alone suggests that glucocorticoids exert their inhibitory effects on SP-A gene expression by reducing the synthesis of a protein(s) that acts to stabilize SP-A mRNA. It is suggested that the effects of dexamethasone on SP-A mRNA stability are specific, since it has been reported that this steroid increases the levels of mRNA for the surfactant proteolipid SP-B by increasing both SP-B gene transcription and mRNA stability (27). In the present investigation we have examined further the mechanisms by which dexamethasone acts to reduce SP-A mRNA levels in human fetal lung in vitro. We considered that the effects we observed could possibly be explained by an action of dexamethasone to increase the rate of transcription, but at the same time to cause premature termination of transcripts, rendering them unstable. Premature termination of nascent RNA transcripts beyond the promoter proximal region of mammalian genes has been suggested to provide a mechanism for the regulation of gene expression (24). A number of eukaryotic genes transcribed by RNA polymerase II have been shown to undergo transcription termination soon after initiation, and in the case of certain genes, the process of premature termination of transcription has been found to be subject to regulation (24). For example, the promoter in the long terminal repeat of the human immunodeficiency virus genome is subject to premature termination, leading to the generation of transcripts of - 6 0 nucleotides, when the viral tat gene is defective, suggestive that tat protein is a factor that functions to alleviate transcription termination (28). Erythropoietic differentiation of the Friend erythroleukemia cell line induced by treatment with dimethylsulfoxide has been shown to be associated with high levels of expression of the mouse /3-major globin gene (29). The high level of /3-major globin gene expression is caused by abolition of the premature termination of transcription that otherwise occurs in untreated cells (29). Transcription of human and mouse c-myc genes has been shown to prematurely terminate at the end of the first exons in cell lines expressing low


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levels of c-myc after induced differentiation (24, 30). More recently, similar mechanisms were found to operate in the tissue-dependent regulation of expression of mouse adenosine deaminase gene; premature termination of transcription preferentially occurred in tissues that expressed physiologically low levels of adenosine deaminase (25). In our previous studies on dexamethasone regulation of SP-A gene expression in human fetal lung in vitro (20), we assessed SP-A gene transcription using an SP-A cDNA insert that was nearly full-length. Use of a full-length cDNA or gene probe in transcription run-on assays would not provide information concerning elongation of transcripts to discrete regions throughout the gene. In the present study we, therefore, have analyzed the effects of dexamethasone on SP-A gene transcription using DNA probes that correspond to four regions encompassing the entire human SP-A gene. By use of these genomic probes, we observed that the relative rate of elongation of nascent SP-A mRNA transcripts across the SP-A gene was similar in nuclei isolated from dexamethasone- or dexamethasone- plus Bt2cAMPtreated tissues as compared to nuclei isolated from control or Bt2cAMP-treated fetal lung explants. These results clearly indicate that dexamethasone does not cause premature termination of SP-A gene transcription; therefore, this can be ruled out as an explanation for the disparate effects of glucocorticoids on SP-A gene transcription and mRNA levels. A number of hormones and factors have been reported to regulate gene expression by altering the stability of specific mRNAs (31, 32). Glucocorticoids, in particular, have been reported to exert both positive and negative effects on mRNA stability. Glucocorticoids enhance the stability of GH (33, 34), fibronectin (35), and phosphoenolpyruvate carboxykinase (PEPCK) mRNAs (36) and decrease the stability of mRNA for interleukin-1j8 (37), 3-hydroxy-3-methylglutaryl-coenzyme-A reductase (38), type I procollagen (39), and granulocyte-macrophage colony-stimulating factor (40). In the present investigation we chose to investigate the posttranscriptional effects of dexamethasone on SP-A mRNA in fetal lung tissues treated with the transcriptional inhibitor actinomycin-D. This was necessitated by the very low levels of incorporation of [3H]uridine into SP-A mRNA in pulse-chase studies designed to analyze the effects of dexamethasone on SP-A mRNA half-life. Since actinomycin-D could have differential effects on SP-A mRNA stability, this analysis may not provide information regarding the absolute half-life of SP-A mRNA in fetal lung tissue. For example, if actinomycinD blocks the transcription of genes encoding factors that stabilize SP-A mRNA, the calculated value for the SP-A mRNA half-life may be an underestimate. It was our objective in this study, however, to compare the rates of disappearance of SP-A mRNA in actinomycinD-treated tissues incubated in the absence or presence of dexamethasone (10~7 M). In lung explants incubated in medium containing actinomycin-D, the apparent halflife of SP-A mRNA was calculated to be 11.4 h, whereas

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in lung explants incubated in medium containing actinomycin-D plus dexamethasone (10~7 M) this was reduced by more than 60% to 5.0 h. The question arises of whether a 60% decline in the apparent half-life of SP-A mRNA is sufficient to explain the effect of the steroid to cause a 90% reduction of SP-A mRNA levels in control and Bt2cAMP-treated tissues. We can think of several explanations for this discrepancy. First, in the experiment shown in Fig. 1C, the human fetal lung tissues were incubated for 5 days in the absence or presence of dexamethasone and Bt2cAMP, added alone or in combination. The lung tissue at the start of culture has no differentiated type II cells, and SP-A gene expression is undetectable; within several days of organ culture the tissue spontaneously differentiates, and SPA gene expression is initiated (22). If the effect of dexamethasone to decrease SP-A mRNA stability has a more rapid onset than the effects of dexamethasone and Bt2cAMP to increase SP-A gene transcription, the apparent inhibitory effects of dexamethasone on SP-A mRNA half-life may appear to be an underestimate compared to its action to reduce SP-A mRNA levels. Alternatively, as discussed above, the apparent half-life of SP-A mRNA in fetal lung tissues treated with actinomycin-D may be underestimated because of an action of actinomycin-D to inhibit the synthesis of specific factors that stabilize SP-A mRNA. Since we postulate that dexamethasone may act specifically to inhibit the synthesis of such factors, the effect of dexamethasone to reduce SP-A mRNA half-life also may be underestimated. Finally, actinomycin-D could act to inhibit the synthesis of mRNA-destabilizing factors and may, therefore, antagonize the effects of dexamethasone to reduce SP-A mRNA stability. In the present study we also observed that the effects of dexamethasone on SP-A mRNA stability are dose dependent. In human fetal lung explants incubated in the presence of actinomycin-D, dexamethasone at concentrations as low as 10~9 M caused ~a 50% decrease in the levels of SP-A mRNA compared to those in tissues incubated with actinomycin-D alone. SP-A mRNA levels were markedly reduced at concentrations >10~8 M. The inhibitory effects of glucocorticoids on SP-A mRNA levels also were found to be rapidly reversible. In previous studies we found that the effect of dexamethasone (10~7 M) to reduce SP-A mRNA levels was manifest as early as 2 h after addition to the culture medium (20). Furthermore, in the present study the inhibitory effect of dexamethasone on SP-A mRNA accumulation was completely blocked by the glucocorticoid receptor antagonist RU486, suggesting that the dexamethasone-induced destabilization of SP-A mRNA is mediated by the action of the glucocorticoid receptor. The complete reversal of the glucocorticoid-mediated inhibition of SP-A mRNA accumulation after removal of hormone from the medium suggests that the inhibition of SP-A mRNA levels is not due to a deleterious effect of steroid on the viability of fetal lung tissue. It is of interest to note that in a human pulmonary adenocarcinoma cell line, dexamethasone (10~7 M) caused a



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marked inhibition of SP-A mRNA accumulation, whereas a significant increase in the accumulation of mRNA for another surfactant-associated protein, namely SP-B, was observed (41). It is evident from the findings of the present investigation and those of our previous studies (20) that dexamethasone has differential dose-dependent effects on SP-A gene transcription and SP-A mRNA stability that result in the observed biphasic effects on SP-A mRNA accumulation. At a concentration of 10~7 M, dexamethasone causes a more than 60% reduction in the apparent half-life of SP-A mRNA. Our findings further suggest that dexamethasone acts through the glucocorticoid receptor to reduce the SP-A mRNA halflife by inhibiting the synthesis of a labile protein factor(s) that acts to stabilize SP-A mRNA. Alternatively, dexamethasone could posttranslationally modify a protein factor and, thereby, alter its action to regulate SP-A mRNA stability.

were placed in each culture dish on lens paper covered stainless steel grids and maintained in organ culture in serum-free Waymouth's MB752/1 medium (Gibco, Grand Island, NY), as described in detail previously (42). Lung explants were maintained in culture for up to 6 days in either control medium or medium containing Bt2cAMP (1 HIM), dexamethasone, and/or RU486 (Roussel-UCLAF, Romainville, France; kindly provided by Dr. Etienne-Emile Baulieu) at the concentrations indicated in the figure legends. Media were replaced every 24 h. Dexamethasone and RU486 were dissolved in ethanol before addition to the the culture medium. The concentration of ethanol in culture medium never exceeded 0.01 %, and at this concentration, ethanol had no effect on SP-A mRNA levels in the fetal lung explants (data not shown). Each of the studies presented was performed using pooled lung tissues from two to four human abortuses. Each experiment was repeated two to four times, and the data presented are representative of these experiments.

At present, the mechanisms by which glucocorticoids and other steroid hormones regulate the stability of specific mRNAs are not well understood. The findings of Nielsen and Shapiro (32) indicate that the nuclear estrogen-receptor complex of Xenopus liver cells is essential for the estrogen-induced increase in vitellogenin mRNA stability. It was suggested that this action of estrogen may be mediated by the increased transcription of a gene encoding a stable protein factor(s). Alternatively, it was suggested that the estrogen-receptor complex may act as a protein kinase and mediate the posttranslational modification of a protein(s) that facilitates ribosomal loading of vitellogenin mRNA (32).

Total RNA was isolated from human fetal lung explants essentially by the method of Chirgwin et a/. (43) and quantitated by determining its absorbance at 260 nm. Equal amounts of total RNA from fetal lung tissues were size-fractionated in formaldehyde agarose gels and blotted onto Zeta-Probe membranes (Promega, Madison, Wl) by capillary transfer. The RNA blot was hybridized to 32P-labeled rabbit SP-A cDNA (44). The conditions for gel electrophoresis, transfer, hybridization, and washings were essentially as described in detail previously (44). After hybridization, the blots were subjected to autoradiography using intensifying screens. The autoradiographic signals were quantitated using a Molecular Dynamics (Sunnyvale, CA) Computing Densitometer.

Sequences contained within mRNAs, particularly in the 3'-untranslated regions, have been implicated to serve important roles in determining the degradation rates of mRNAs by acting as recognition sites for specific RNases or other destabilizing factors (31, 32). Recently, in studies of glucocorticoid regulation of PEPCK gene expression in the rat hepatoma line H4IIE, it was determined that the glucocorticoid-induced stabilization of PEPCK mRNA occurs by interaction of an inducible factor with the 3'-untranslated region (36). In this regard it is interesting to note that the human SPA mRNA contains more than 1 kb of 3'-untranslated sequence. Studies directed toward delineation of the molecular mechanisms underlying glucocorticoid-mediated destabilization of SP-A mRNA in human fetal lung tissue are in progress in our laboratory.

MATERIALS AND METHODS Organ Culture of Human Fetal Lung Lung tissues were obtained from human abortuses of 15-18 weeks gestational age in accordance with the Donors Anatomical Gift Act of the State of Texas. Consent forms and protocols were approved by the Human Research Review Committee at the University of Texas Southwestern Medical Center (Dallas, TX). The individual lung tissues from several fetuses were trimmed, rinsed, pooled, and minced. Five or six tissue pieces

Isolation of RNA and Northern Blot Analysis of SP-A mRNA

Isolation of Nuclei and Transcription Elongation Assay Methods for isolation of nuclei and transcription run-on assays, hybridization, and washing conditions were described previously (45). Transcription rates were analyzed using restriction fragments of the human SP-A gene (46) (Boggaram, V., and C. R. Mendelson, unpublished observations) immobilized on nitrocellulose. Nitrocellulose containing pUC DNA served as a control. After hybridization to 32P-labeled RNA, the filters were washed and autoradiographed using intensifying screens. Autoradiographic signals were quantitated using a Molecular Dynamics Computing Densitometer. Acknowledgments The authors acknowledge the excellent technical assistance of Ms. Jo Smith, and the editorial assistance of Ms. Darlene Tutton.

Received September 25, 1990. Revision received December 26,1990. Accepted December 31,1990. Address requests for reprints to: Dr. Carole R. Mendelson, Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9038. This work was supported in part by NIH Grant HD-13912, Basic Research Grant 1 -1183 from the March of Dimes Birth Defects Foundation, and American Heart Association, Texas Affiliate, Grant 89G-103. * Present address: Department of Molecular Biology, University of Texas Health Science Center, P.O. Box 2003, Tyler, Texas 75710.


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REFERENCES 21. 1. Clements JA, King RJ 1976 Composition of surface-active material. In: Crystal RG (ed) The Biochemical Basis of Pulmonary Function. Marcel Dekker, New York, pp 363387 2. Avery ME, Mead J 1959 Surface properties in relation to atelectasis and hyaline membrane disease. Am J Dis Child 97:517-523 3. Ballard PL 1989 Hormonal regulation of pulmonary surfactant. Endocr Rev 10:165-181 4. Mendelson CR, Boggaram V 1989 Regulation of pulmonary surfactant protein synthesis in fetal lung: a major role of glucocorticoids and cyclic AMP. Trends Endocrinol Metab 1:20-26 5. King RJ, Klass DJ, Gikas EG, Clements JA 1973 Isolation of apoproteins from canine surface active material. Am J Physiol 224:788-795 6. Possmayer F 1988 Pulmonary perspective: a proposed nomenclature for pulmonary surfactant-associated proteins. Am Rev Respir Dis 138:990-998 7. Ng VL, Herndon VL, Mendelson CR, Snyder JM 1983 Characterization of rabbit surfactant-associated proteins. Biochim Biophys Acta 754:218-226 8. Phelps DS, Taeusch HW, Benson B, Hawgood S 1984 An electrophoretic and immunochemical characterization of human surfactant-associated proteins. Biochim Biophys Acta 791.226-238 9. King RJ, MacBeth MC 1981 Interaction of the lipid and protein components of pulmonary surfactant: role of phosphatidylglycerol and calcium. Biochim Biophys Acta 647:159-168 10. King RJ, Carmichael MC, Horowitz PM 1983 Reassembly of lipid-protein complexes of pulmonary surfactant. Proposed mechanism of interaction. J Biol Chem 258:1067210680 11. Hawgood S, Benson BJ, Hamilton RL 1985 Effects of a surfactant-associated protein and calcium ions on the structure and surface activity of lung surfactant lipids. Biochemistry 24:184-190 12. Suzuki Y, Fujita Y, Kogishi Y 1989 Reconstitution of tubular myelin from synthetic lipids and proteins associated with pig pulmonary surfactant. Am Rev Respir Dis 140:75-81 13. Hawgood S, Benson BJ, Schilling J, Damm D, Clements JA, White RT 1987 Nucleotide and amino acid sequences of pulmonary surfactant protein SP18 and evidence for cooperation between SP18 and SP28-36 in surfactant lipid absorption. Proc Natl Acad Sci USA 84:66-70 14. Kuroki Y, Mason RJ, Voelker DR 1988 Alveolar type II cells express a high-affinity receptor for pulmonary surfactant protein A. Proc Natl Acad Sci USA 85:5566-5570 15. Phelps DS, Floras J 1988 Localization of surfactant protein synthesis in human lung by in situ hybridization. Am Rev Respir Dis 137:939-942 16. Auten RL, Watkins RH, Shapiro DL, Horowitz S 1991 Surfactant apoprotein A (SP-A) is synthesized in airway cells. Am J Respir Cell Mol Biol 3:491-496 17. Phelps DS, Floras J 1990 Differential localization of surfactant protein mRNAs in rat and human lung. Am Rev Respir Dis 141:A694 18. Odom MJ, Snyder JM, Mendelson CR 1987 Adenosine 3',5'-monophosphate analogs and /3-adrenergic agonists induce the synthesis of the major surfactant apoprotein in human fetal lung in vitro. Endocrinology 121:11551163 19. Whitsett JA, Pilot T, Clark JC, Weaver TE 1987 Induction of surfactant protein in fetal lung: effects of cAMP and dexamethasone on SAP-35 RNA and synthesis. J Biol Chem 262:5256-5261 20. Boggaram V, Smith ME, Mendelson CR 1989 Regulation of expression of the gene encoding the major surfactant

22.

23.

24.

25.

26.

27.

28.

29.

30.

31. 32.

33.

34.

35.

36.

37.

38.

39.

40.

protein (SP-A) in human fetal lung in vitro. J Biol Chem 264:11421-11427 Ballard PL, Hawgood S, Liley H, Wellenstein G, Gonzales LW, Benson B, Cordell B, White RT 1986 Regulation of pulmonary surfactant apoprotein SP28-36 gene in fetal human lung. Proc Natl Acad Sci USA 83:9527-9531 Odom MJ, Snyder JM, Boggaram V, Mendelson CR 1988 Glucocorticoid regulation of the major surfactant-associated protein (SP-A) and its mRNA and of morphologic development of human fetal lung in vitro. Endocrinology 123:1712-1720 Liley HG, White RT, Benson BJ, Ballard PL 1988 Glucocorticoids both stimulate and inhibit production of pulmonary surfactant protein A in fetal human lung. Proc Natl Acad Sci USA 85:9096-9100 Proudfoot NJ 1989 How RNA polymerase II terminates transcription in higher eukaryotes. Trends Biochem Sci 14:105-110 Chinsky JM, Maa M-C, Ramamurthy V, Kellems RE 1989 Adenosine deaminase gene expression. Tissue-dependent regulation of transcriptional elongation. J Biol Chem 264:14561-14565 Baulieu E-E 1989 RU-486 as an antiprogesterone steroid: from receptor to contragestion and beyond. JAMA 262:1808-1814 O'Reilly MA, Clark JC, Whitsett JA 1990 Transcriptional and post-transcriptional regulation of human pulmonary surfactant protein B expression by glucocorticoids. Am Rev Respir Dis 141:A692 Kao S-Y, Caiman AF, Luciw PA, Peterlin BM 1987 Antitermination of transcription within the long terminal repeat of HIV-1 by tat gene product. Nature 330:489-493 Proudfoot NJ, Whitelaw E 1988 Termination and 3' end processing of eukaroytic RNA. In: Hes BD, Glover DM (eds) Frontiers in Molecular Biology; Transcription and Splicing. IRL Press, Oxford and New York, pp 97-130 Bentley DL, Groudine M 1988 Sequence requirements for premature termination of transcription in the human cmyc gene. Cell 53:245-256 Ross J 1988 Messenger RNA turnover in eukaryotic cells. Mol Biol Med 5:1-14 Nielsen DA, Shapiro DJ 1990 Insights into hormonal control of messenger RNA stability. Mol Endocrinol 4:953957 Pack I, Axel R 1987 Glucocorticoids enhance stability of human growth hormone mRNA. Mol Cell Biol 7:14961507 Diamond DJ, Goodman HM 1985 Regulation of growth hormone messenger RNA synthesis by dexamethasone and triiodothyronine. Transcriptional rate and mRNA stability changes in pituitary tumor cells. J Mol Biol 81:4162 Dean C, Newly RF, Bourgeois S 1988 Regulation of fibronectin biosynthesis by dexamethasone, transforming growth factor /3 and cAMP in human cell lines. J Cell Biol 106:2159-2170 Peterson DD, Koch SR, Granner DK 1989 3' Noncoding region of phosphoenolpyruvate carboxykinase mRNA contains a glucocorticoid-responsive mRNA-stabilizing element. Proc Natl Acad Sci USA 86:7800-7804 Lee SW, Tsov A-P, Chan H, Thomas J, Petrie K, Eugni EM, Allison AC 1988 Glucocorticoids selectively inhibit the transcription of the interleukin 1/? gene and decrease the stability of interleukin 1(8 mRNA. Proc Natl Acad Sci USA 85:1204-1208 Simonet WS, Ness GC 1989 Post-transcriptional regulation of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA in rat liver. Glucocorticoids block the stabilization caused by thyroid hormones. J Biol Chem 264:569-573 Raghow R, Gossage DL, Kang AN 1986 Pretranslational regulation of type I collagen, fibronectin, and a 50-kilodalton noncollagenous extracellular protein by dexamethasone in rat fibroblasts. J Biol Chem 261:4677-4684 Thorens B, Mermod J-J, Vassalli P 1987 Phagocytosis



and inflammatory stimuli induce GM-CSF mRNA in macrophages through post-transcriptional regulation. Cell 48:671-679 41. O'Reilly MA, Gazdar AF, Morins RE, Whitsett JA 1988 Differential effects of glucocorticoids on expression of surfactant proteins in a human lung adenocarcinoma cell line. Biochim Biophys Acta 970:194-204 42. Mendelson CR, Chen C, Boggaram V, Zacharias C, Snyder J 1986 Regulation of the synthesis of the major surfactant apoprotein in fetal rabbit lung tissue. J Biol Chem 261:9938-9943 43. Chirgwin JM, Przbyla AE, MacDonald RJ, Rutter WJ 1979 Isolation of biologically active ribonucleic acid from

423

sources enriched in ribonuclease. Biochemistry 18:52945299 44. Boggaram V, Qing K, Mendelson CR 1988 The major apoprotein of rabbit pulmonary surfactant. Elucidation of primary sequence and cyclic AMP and developmental regulation. J Biol Chem 263:2939-2947 45. Boggaram V, Mendelson CR 1988 Transcriptional regulation of the gene encoding the major surfactant protein (SP-A) in rabbit fetal lung. J Biol Chem 263:19060-19065 46. White RT, Damm D, Miller J, Spratt K, Schilling J, Hawgood S, Benson B, Cordell B 1985 Isolation and characterization of the human pulmonary surfactant apoprotein gene. Nature 316:361-363


Insulin regulation of messenger ribonucleic acid for the surfactant-associated proteins in human fetal lung in vitro.

Role of messenger RNA subcellular localization in the posttranscriptional regulation of human histone gene expression.

Regulation of CYP3A genes by glucocorticoids in human lung cells.

Prostaglandins regulate surfactant protein A (SP-A) gene expression in human fetal lung in vitro.

Coordinate regulation of fibrinogen subunit messenger RNA levels by glucocorticoids in primary cultures of Xenopus liver parenchymal cells.

Glucocorticoids and fetal lung development.

Fetal lung development and the influence of glucocorticoids on pulmonary surfactant.

Messenger RNA regulation in humam diploid fibroblasts.

Posttranscriptional Regulation and RNA Binding Proteins in Cancer Biology.

Regulation of lung surfactant secretion.

Hormonal and developmental regulation of pulmonary surfactant synthesis in fetal lung.

Glucocorticoids stimulate resorption in fetal rat parietal bones in vitro.

Interferon-gamma and synthesis of surfactant components by cultured human fetal lung.

Conjugated glucocorticoids in amniotic fluid and fetal lung maturation.

Posttranscriptional regulation of albumin and alpha-fetoprotein messenger RNA by transforming growth factor-beta 1 requires de novo RNA and protein synthesis.

Hormonal control of the surfactant system in fetal lung.

Regulation of vasopressin messenger RNA levels in the small cell lung carcinoma cell line GLC-8: interactions between glucocorticoids and second messengers.

Messenger activity of RNA transcribed in vitro by DNA-RNA polymerase associated to vaccinia virus cores.

The globalization of messenger RNA regulation.

Isolation and in vitro translation of human placental lactogen messenger RNA from human term placenta.

Androgens delay human fetal lung maturation in vitro.

Negative regulation of angiotensinogen gene expression by glucocorticoids in fetal sheep liver.

Competing endogenous RNA: the key to posttranscriptional regulation.

Glucocorticoids inhibit fibronectin synthesis and messenger ribonucleic acid levels in cultured fetal rat parietal bones.