Regulation of mRNA levels for pulmonary surfactant-associated proteins in developing rabbit lung

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Biochimica et Biophysica Acta 1254 (1995) 302-310

Biochi~ic~a et BiophysicaA~ta

Regulation of mRNA levels for pulmonary surfactant-associated proteins in developing rabbit lung Jiejing Xu a,b,1,Li-Juan Yao b, Fred Possmayer a,b,* a MRC Group in Fetal and Neonatal Health and Development and Departments of Biochemistry, The University of Western Ontario, 339 Windermere Road, London, Ontario, N6A 5A5, Canada b Obstetrics and Gynaecology, The University of Western Ontario, 339 Windermere Road, London, Ontario, N6A 5A5, Canada Received 23 February 1994; revised 16 September 1994

Abstract

Gene transcriptional activities and steady-state mRNA levels have been examined for the surfactant-associated proteins SP-A, SP-B and SP-C in developing rabbit lung. It was observed SP-C mRNA levels increase early in gestation, while SP-A and SP-B mRNA levels increase rapidly between 26 and 30 days gestation. Transcriptional activities for all three surfactant apoproteins increase between 26 and 30 days. Studies conducted with fetal lung explants of 26 days gestation demonstrated exposure to low doses of dexamethasone increases SP-A and SP-C mRNA levels, while high doses stimulate transcription, although this was only significant for SP-C. Time course studies revealed different temporal patterns and glucocorticoid responses for SP-A and SP-C mRNAs. SP-A and SP-C mRNA production and steady-state levels were reduced after treatment with cycloheximide. In contrast, SP-B gene transcription was selectively stimulated, suggesting involvement of a labile negative regulatory factory. It is concluded that expression of the three surfactant apoproteins is independently regulated. Early in gestation, SP-C mRNA levels may be regulated in vivo through message stabilization. Glucocorticoids can affect SP-A and SP-C mRNA levels in culture at both transcriptional and post-transcriptional levels. The ability of glucocorticoids to influence these processes declines during fetal development. Keywords: Pulmonary suffactant; Surfactant-associated protein; Glucocorticoid; Transcription; mRNA level; Fetal development; Respiratory Distress Syndrome

I. Introduction

Pulmonary surfactant stabilizes the lung by reducing surface tension at the air/liquid interface of the alveolus and prevents alveolar collapse, particularly at low lung volumes [1]. A deficiency of surfactant is a major cause of respiratory distress syndrome (RDS) and mortality in premature infants [2]. Surfactant contains phosphatidylcholine and other phospholipids, and at least three distinct proteins, surfactant-protein A (SP-A), SP-B and SP-C, which are considered important for surfactant structure and function [3,4]. SP-A enhances phospholipid adsorption in the

Abbreviations: EDTA, ethylenediaminetetraacetic acid; ELISA, Enzyme-linked immunosorbant assay; SP-, surfactant-associated protein-. * Corresponding author. Fax: + 1 (519) 663 3388. i Present Address: Institute for Biological Sciences, National Research Council, 1500 Montreal Road and 100 Sussex Drive, Ottawa, ON K1A OR6, Canada. 0005-2760/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 5 - 2 7 6 0 ( 9 4 ) 0 0 1 9 1 - X

presence of SP-B, and is involved in the formation of a unique alveolar surfactant complex known as tubular myelin [5,6]. SP-A may also affect surfactant secretion and uptake and play a role in host immunodefence mechanisms [4,7]. SP-B and SP-C are low molecular weight hydrophobic proteins that promote the adsorption of phospholipids to form a surface active monolayer and the squeeze-out of unsaturated components from the phospholipid monolayer, resulting in an enrichment in dipalmitoylphosphatidylcholine [8,9]. Pulmonary surfactant is synthesized in alveolar type II cells, accumulated in lamellar bodies and secreted into the alveolar subphase. Ontological studies with rat [10,11], human [12,13] and rabbit [14-16] lung have revealed SP-A and SP-A mRNA become detectable about the time that differentiated (i.e., lamellar body containing) type II cells are first noted (see [17] for review). In contrast, studies with the rat [18], human [12] and rabbit [19-21] reveal SP-C mRNA can be detected early in gestation prior to the morphological appearance of differentiated type II cells

J. Xu et aL /Biochimica et Biophysica Acta 1254 (1995) 302-310

and the developmental increases in SP-A and surfactant phospholipid levels which occur near term [4,17,22,23]. These observations indicate expression of surfactant apoproteins is independently regulated. Glucocorticoids enhance pulmonary maturation in vitro and in vivo [4,17,22,24]. Previous studies have revealed that glucocorticoid treatment of rabbit fetal lung explants in early gestation (day 21 of gestation, term 31 days) influences SP-A gene transcription and mRNA levels [14,16,23,24]. Whether glucocorticoids affect SP-A expression throughout gestation or influence SP-C mRNA production and levels has not been reported for this species. The present studies were conducted to further investigate the control of surfactant apoprotein expression in the rabbit throughout the perinatal period. SP-A and SP-C gene transcriptional rates and mRNA steady-state levels were expressed relative to adult values and the results compared with those previously reported by our group for SP-B [25]. In order to determine whether glucocorticoids influence surfactant apoprotein expression in a similar manner throughout fetal development, the effect of this steroid on gene transcription and steady-state mRNA levels was examined with fetal lung explants of 26 and 30 days gestation. Day 26 is prior to the appearance of lamellar bodies and the surge in surfactant accumulation while by day 30 fetal rabbit lungs are functionally mature [23]. Since it had previously been shown that cycloheximide can block SP-A mRNA induction in rabbit fetal lung explants at 21 days [16], the effects of the protein synthesis inhibitor on surfactant apoprotein mRNA expression was examined with explants of 26 and 30 days gestation.

2. Materials and methods

For the most part, the materials and methods used were identical to those described in our previous publication [19,25]. Timed pregnant New Zealand White rabbits were purchased from Reiman's Fur Ranch (St. Agatha, ON, Canada). For the organ culture studies lung tissues of 26 and 30 days gestation were diced with a Mcllwain chopper to pieces of approx. 1 mm 3 and cultured as explants in Waymouth's medium at 37°C in 95% air-5% CO 2 on a rocker platform [25]. Additives were dissolved in ethanol and added to the culture after the tissues were established for 24 h and changed daily thereafter. Cytoplasmic RNA was isolated from the supernatants of the nuclear isolations using the guanidinium isothiocyanate procedure [25] except for one of the experiments in Fig. 7 where total cytoplasmic RNA was purified by the acid/guanidinium/phenol/chloroform procedure [26]. For each experiment RNA was examined by gel electrophoresis to assess intactness. Quantitative determinations of the relative amounts of mRNA were conducted by slot blot analysis [25] using as probes (1) a 1.4-kb rabbit SP-A cDNA [19]; (2) a 1.7-kb rabbit SP-B cDNA [27]; (3) a

303

rabbit SP-C cDNA [28]; (4) a rabbit 18S cDNA fragment [25] or a mouse /3-actin cDNA (pAL41) [25]. Under the conditions used, signals obtained for the surfactant apoprotein mRNAs with liver RNA were consistently lower than 5% of the positive samples. Nuclear run-on transcriptional assays were conducted as previously described [25]. Control plasmid pUC19, which carried the specific cDNA sequences, consistently produced signals on X-ray film considerably less than 5% of the positive signals. Transcripts made with adult rabbit liver nuclei produced essentially undetectable signals for the surfactant apoprotein mRNAs. Data are expressed as mean ___S.E. Statistical analyses were performed on raw or log-transformed data by one or two-way analysis of variance followed by Duncan's multiple comparisons. Two-way analysis was used for the dexamethasone time course studies.

3. Results 3.1. Developmental patterns o f SP-A, SP-C gene transcriptional activities and mRNA levels

The development changes in the steady-state levels of SP-A and SP-C mRNAs, determined with total RNA samples by slot blot hybridization and the transcriptional rates of the SP-A and SP-C genes are presented relative to adult values in Fig. 1. Alterations in SP-B gene transcription and its steady-state mRNA levels (published in our recent report, [25]) are also included to allow comparison of the changing patterns for all three surfactant proteins. SP-A mRNA was not detected in lungs of 22 days gestation, but became detectable at a very low level on day 24 of gestation. The levels then increased markedly between 26 days and 28 days gestation and reached a peak on day 30 (the last day of gestation examined), then fell after birth. This pattern is similar to that previously observed for SP-B mRNA except the latter mRNA was detected at a very low level several days earlier. SP-C mRNA differed from SP-A and SP-B mRNA in being readily detected on day 22 of gestation, the earliest day examined in this study, and increased steadily but rather gradually thereafter. SP-C mRNA levels at term were only slightly greater than in the adult. SP-A and SP-C gene transcription was readily detectable in nuclei isolated from 22 day fetal lung and increased gradually until 26 days of gestation, at which time it reached levels approximately double the levels at 22 days. With both genes, there was a 4-fold increase in transcription rate between 26 days and 28 days gestation. This increase was reflected in a corresponding elevation in SP-A message but SP-C mRNA was only slightly increased. SP-B gene transcription was relatively constant between days 22 to 26 of gestation but then increased markedly on day 28. Transcription levels reached a maxi-

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J. Xu et al. / Biochimica et Biophysica Acta 1254 (1995) 302-310

mum in nuclei from 30 day fetal lung (approx. 6.5-fold increase over day 22 for the SP-A gene and 5-fold for the SP-C gene) and remained the same after birth. Comparison of the transcriptional activities of the genes with their corresponding mRNA levels during development revealed a similar overall pattern for SP-A and SP-B, indicating that for these apoproteins that the message levels could be controlled primarily by gene transcription. However, SP-C mRNA levels were significantly increased at days 24 and 26 of gestation prior to a significant increase in SP-C gene transcription. 3.2. D e x a m e t h a s o n e concentration studies

In order to determine whether dexamethasone affects the induction a n d / o r accumulation of SP-A and SP-C mRNA in fetal rabbit lungs at early and late stages of lung maturation, fetal lung tissues of day 26 and day 30 of gestation were examined in cultures for 72 h with dexamethasone added for the last 48 h. In preliminary studies, a portion of the cultured tissue was fixed in 10% formalin

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Fig. 1. Comparisons of SP-A, SP-B and SP-C gene transcriptional activities and their mRNA levels in rabbit lung during development. Nuclei isolated from rabbit lungs of gestational age 22 to 30 days (22-30), neonatal 1 day (N1D) and adult (Ad) animals were allowed to transcribe in vitro in the presence of [c~-32p]GTP. cDNAs for rabbit SP-A, SP-B and SP-C were immobilizedon a filter to hybridizewith the corresponding 32P-labelled nuclear transcripts. A mouse /3-actin cDNA was also included on the filter as a referencemessage.Total cytoplasmic RNA (duplicate with 5 /Lg/slot) isolated from the same lung2 tissues as for nuclear run-on assays was blotted and hybridized with P-labelled cDNA probes indicated. The autoradiogramsobtained from both nuclear run-on (solid bars) and slot blot (hatched bars) analyseswere subjected to densitometry. The data obtained are normalizedand presented relative to respective adult values (= 1) as mean-I-S.E. (n= 3 to 4). * Indicates values are significantly different from those on 22 or 24 days gestation (e < 0.05).

followed by routine light microscopy. As seen in Fig. 2A, pre-culture lung tissue of day 26 gestational age rabbit was in the pseudoglandular or early canalicular stage of development. Within 48 h in organ culture, the lumen of the ducts enlarged (Fig. 2B). The epithelium and connective tissue appeared viable and basically intact. The addition of dexamethasone at 10 -8 M to the media had no discernible effect on tissue morphology when compared to control explants (Fig. 2C). The absence of glucocorticoid effects on fetal rabbit lung morphology with light microscopy has been reported by Snyder et al. [29] who examined 19 day fetal lung explants maintained in culture for 5 days. However, in other studies electron microscopic examination showed cortisol increased the proportion of type II cells from 42 to 66% in 21 day explants [30]. Preculture lung tissue of 30 days gestation rabbit, shown in Fig. 2D, was in the saccular stage of development which appears as large irregularly-formed lumina with thin walls composed of both cuboidal and flattened cells. After 48 h explant culture, with (Fig. 2F) or without (Fig. 2E) dexamethasone, the luminal walls became thicker. Some cells were observed within the alveolar spaces. Extensive cell death was not observed in any of the conditions studied. The mRNA levels for SP-A and SP-C and their gene transcriptional activities were measured by slot blot analysis, and nuclear run-on assays, respectively. Exposure of lung explants from fetuses of 26 days gestation to dexamethasone at concentrations from 10 -1° M to 10 - 6 M led to 2-4-fold increases in SP-A mRNA levels and a 5-6-fold increase ( P < 0.05) in SP-C mRNA levels relative to tissues incubated in the absence of steroid (Fig. 3). These increases are similar to the 3-8-fold elevations observed for SP-B mRNA [25]. The lowest concentration (10 - t ° M) of glucocorticoid used produced significant increases in SP-A and SP-C but not SP-B mRNAs. At the higher concentrations used in this study, dexamethasone produced an approximately doubling of SP-A and SP-C gene transcription. However, the increase in SP-A transcription did not reach statistical significance ( P < 0.059). This contrasts with SP-B gene transcription where dexamethasone had no apparent effect [25]. Experiments conducted using fetal rabbit lung explant of 30 days gestation revealed similar but lesser effects on surfactant-associated protein mRNA levels (Fig. 4) than observed on day 26. SP-A mRNA levels were responsive to dexamethasone at 10 -1°, 10 - 9 and 10 - 6 M but were not elevated by this steroid at 10 -8 o r 10 - 7 M. Interestingly, a considerably smaller, although still significant effect, was noted when these concentrations of steroids were applied to explants of 26 days gestation (Fig. 3). We have previously reported exposure of 30 day explants to dexamethasone led to an increase in SP-B mRNA levels which was significant at 10 - 6 and 10 - 7 M [25]. Only a small non-significant increase in SP-C mRNA (Fig. 4) was observed. In contrast to fetal explant cultures of 26 days gestation,

J. Xu et al. / Biochimica et Biophysica A cta 1254 (1995) 302-310

dexamethasone produced highly variable effects in S P - A and SP-C transcription with explants of 30 days gestation, It should be noted that the control transcription rates and

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the control m R N A levels observed with fetal lung explants of 30 days gestation were greater than those observed with 26 day explants (see Fig. 1).

Fig. 2. Light microscopic histology of 26 and 30 day fetal rabbit lung tissue after various times in explant culture. The sections were stained with hemotoxin and eosin (45 × ). (A) 26 day fetal rabbit lung (preculture). (B) Explants of 26 day fetal lung maintained for 3 days in control medium. (C) Explants of 26 day fetal lung maintained for 1 day in control medium and 2 days in medium containing 10 -8 M dexamethasone. (D) 30 day fetal rabbit lung (preculture). (E) Explants of 30 day fetal lung maintained for 3 days in control medium. (F) Explants of 30 day fetal lung maintained for 1 day in control medium and 2 days in medium containing 10-8 M dexamethasone.

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Fig. 4. Dexamethasone dose response for SP-A and SP-C gene transcription and mRNA levels in 30 day fetal lung explants. Experiments were conducted as in Fig. 3. Solid bars represent transcriptional activity and hatched bars the mRNA levels. Data are mean + S.E. with n = 3. * Indicates values are significantly different from control values ( P < 0.05).

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Days in Culture Fig. 5. Day 26 lung SP-A and SP-C mRNA and run-on transcript levels in response to dexamethasone treatment. Lung explants were cultured without hormone (dashed lines, open symbols) or with 10 -8 M dexamethasone (solid lines, closed symbols) added on day l. Nuclei and cytoplasmic RNA were isolated from the tissues before (point 0) and after the indicated times of culture and subjected to nuclear run-on assays and slot blot analyses. Signals on the autoradiograms were quantified by densitometry and plotted relative to the respective adult values. The data are presented as the mean__+S.E. for three separate experiments. * indicates values are significantly different from the corresponding control values ( P < 0.05). The relative transcriptional activities of the genes measured by nuclear run-on assays are shown by the triangles and the mRNA levels by the circles.

rapid increase during the first day in culture, followed by a more gradual elevation which accelerated mainly between days 4 and 7, resulting in levels approx. 8-fold the adult level. SP-C mRNA content was not altered until the 4th day in culture when an increase became apparent and the levels increased gradually thereafter yielding a final level approx. 3-fold the adult value. The presence of dexamethasone (10 -8 M) produced a rapid increase ( P < 0.05) in SP-C mRNA steady-state levels to approx. 4-fold the adult level within 48 h of treatment with little change in level thereafter. This latter pattern was similar to that previously observed with SP-B mRNA abundance [25]. In contrast, dexamethasone at this concentration produced only a small non-significant increase in SP-A mRNA levels. Except for starting at a higher initial level, the alterations in SP-A mRNA levels observed in fetal lung cultures of 30 days gestation in the absence of dexamethasone (Fig. 6) showed a similar pattern to that observed with tissues of 26 days gestation. This was true for both the magnitude and the duration of the changes. At this gestational age, the hormone again had no discernible effect on SP-A mRNA. In contrast, SP-C mRNA levels in fetal lung of 30 days showed a somewhat different profile from that observed with 26 days lung tissue. Incubation in the absence of dexamethasone led to a fall during the first day

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Fig. 6. Day 30 lung SP-A and SP-C mRNA and run-on transcript levels in response to dexamethasone treatment. The experiments were conducted as for Fig. 5. Triangles represent the gene transcription and circles represent the message levels. Closed symbols indicate data obtained with lung explants in the presence of dexamethasone and open symbols in the absence of the hormone. Data are mean___S.E. with n = 3. * Indicates values are significantly different from respective control values (P < O.05).

3.4. Effects o f cycloheximide

In order to obtain potential insights into the manner in which dexamethasone might be affecting the levels of the mRNAs for the surfactant proteins, experiments were conducted in which protein synthesis was inhibited by the addition of cycloheximide. In these studies, lung explants from 26 day and 30 day fetal rabbits were treated for 24 h with 10 -8 M dexamethasone (added on day 1 of culture) in the presence or absence of 10 /xM cycloheximide and the levels of the mRNAs for SP-A, SP-B and SP-C and their gene transcriptional activities were determined 24 hours later. As illustrated in Fig. 7, dexamethasone (10 -8 M) had no detectable effect on the synthesis or accumulation of SP-A m R N A in 26 or 30 day explants. In both 26 day and 30 day explant tissues incubated with cycloheximide (10 ~ M ) alone, the levels of SP-A gene transcription were reduced to less than half of that detected in untreated explants. This was accompanied by a similar decrease in SP-A m R N A level. Essentially identical patterns were observed when the explants were cultured in medium containing both dexamethasone and cycloheximide. 2.5

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in culture followed by a slow moderate increase in SP-C m R N A during the next 5 days, resulting in SP-C m R N A levels approx. 3-fold those of adult lung. Steroid at 10 -8 M resulted in a very small increase in the m R N A levels during the first few days of treatment. Thus, dexamethasone at 10 -8 M showed stimulatory effects on SP-C m R N A levels in 26 day lung explants, but these effects were not evident with SP-C m R N A levels in 30 day lung explants. At the concentration employed, th.e hormone did not have any significant effects on SP-A m R N A content with lung explants of either gestational age. These results contrast with SP-B m R N A which was significantly increased by dexamethasone at both gestational ages. Initial transcription rates for the SP-A and SP-C genes were low in fetal lung of 26 days gestation (Fig. 5) and remained at low levels during culture. Addition of dexamethasone at 10 -8 M did not produce a significant increase in the transcription rates of the genes for either surfactant protein. With explants from 30 days gestation (Fig. 6), the transcription rates of the SP-A and SP-C genes fell to low values during the first day in culture. Transcriptional activities for the SP-A and SP-C genes increased gradually during culture, but the final rates remained lower than those prior to culture. As in the case of 26 day lung, transcription rates of the SP-A and SP-C genes in 30 day gestation lung explants were slightly greater in the presence of 10 -8 M dexamethasone but, with the possible exception of SP-C on day 7 in culture, the increases were not significant. Similar results have been observed with SP-B gene transcription [25].

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J. Xu et al. / Biochimica et Biophysica Acta 1254 (1995) 302-310

In the case of SP-C mRNA levels (Fig. 7), dexamethasone at 10 -8 M resulted in small 1.2-fold and 1.7-fold increases in 26 day lung and 30 day lung explants, respectively. There was no significant effect of the hormone on the SP-C gene transcription in explants of either gestational ages. As in the case of SP-A mRNA, cycloheximide at 10/~M alone caused decreases in both the synthesis and accumulation of SP-C mRNA to less than half of that determined with untreated explants. The effect was more pronounced in 30 day explants than in 26 day explants. Combined cycloheximide and dexamethasone treatment resulted in similar alterations in SP-C mRNA levels and gene transcription as those observed with the inhibitor alone. Somewhat different effects were observed with SP-B. Exposure to 10 -8 M dexamethasone for 24 h resulted in significant increases in steady state SP-B mRNA levels with explants of both gestational ages. As indicated earlier, SP-B gene transcription was not affected. As with SP-A and SP-C, treatment with cycloheximide alone resulted in a depression of SP-B message levels, but this was only significant with explants of 30 days gestation. Surprisingly, with explants of 26 days gestation SP-B nuclear transcription run-on activity was doubled in the presence of cycloheximide. This effect was not observed with explants from 30 day fetuses. When the explants were exposed to both dexamethasone and cycloheximide SP-B mRNA levels and transcription rates were indistinguishable from the controls. Thus cycloheximide alone promoted an increase in SP-B gene transcription with 26 day explants which was not observed in the presence of dexamethasone plus cycloheximide. Cycloheximide treatment did not have a significant inhibitory effect on the overall transcriptional activity of fetal lung nuclei nor on the rate of transcription of 18s RNA or 18s RNA levels in lung explants (data not shown).

4. Discussion In this report, we compared the ontogenies of SP-A, SP-B and SP-C mRNA levels and their relative gene transcription rates in developing rabbit lung. SP-A and SP-B mRNA levels remained relatively low until 26 days gestation when they increase dramatically to a maximum on day 30 (term 31 days). Although SP-B gene transcription was relatively higher on days 22-26 of gestation, with both SP-A and SP-B similar profiles were evident for gene transcription and mRNA levels, suggesting that in vivo mRNA levels could be controlled primarily at the transcriptional level. In contrast, SP-C mRNA levels increased significantly early in gestation prior to the marked increase in SP-C gene transcription noted between days 26 and 28. This suggests that in early gestation SP-C mRNA levels are influenced through message stabilization. The developmental patterns for surfactant apoprotein

mRNA levels presented in this paper are in excellent overall agreement with previous studies with this species [16,19,20,31]. The transcriptional pattern for SP-A is in good agreement with previous studies by Boggaram and Mendelson [16]. A possible interpretation for the distinct developmental pattern for SP-C arises from in situ hybridization studies which revealed the presence of SP-C mRNA in all cells of the rounded distal portions of extending alveolar ducts early in gestation with a more distinct localization of SP-C message in type II pneumonocytes on day 27 and thereafter [20]. These observations suggest those epithelial cells destined to become type I or type II cells express SP-C early in gestation but once type II differentiate around day 26, SP-C expression is limited to these cells [20,32]. In contrast to SP-C, SP-B and SP-A mRNAs are first detected on days 24 and 26 respectively in pre-type II and type II cells [21]. From day 28 onwards, SP-A and SP-B mRNA can be observed in bronchiolar epithelial cells but this appears to account for only a small proportion of total. The basis for the differences in the spatial and temporal relationships between these apoproteins and the mechanisms involved in the apparent stabilization of SP-C in pre-alveolar regions are unknown. Boggaram and Mendelson [16] have observed exposure of 21 day fetal rabbit lung explants to 10 - 7 M dexamethasone produces a transient decrease (approx. 24 h) in SP-A gene transcription followed by increased transcription at 48 h. By 72 h the stimulatory effect over control was no longer evident. A stimulatory effect of dexamethasone on SP-A gene transcription was also observed in preliminary experiments conducted in our laboratory using 22 day fetal lung (data not shown). At this gestational age basal transcription levels were low and the overall increase small relative to adult levels. It was considered important to determine whether the effects of dexamethasone noted early in gestation remain evident at latter stages of development. Therefore the effects of dexamethasone were examined at 26 and 30 days gestation. We chose 26 days because gene transcription for all three apoproteins increases between 26 and 28 days of gestation. Phosphatidylcholine and disaturated phosphatidylcholine synthesis and accumulation increases at this time [23]. By day 30 apoprotein transcription and mRNA levels are high and stored surfactant is abundant [21]. Dose response studies revealed low levels of dexamethasone produced significant increases in SP-A and SP-C mRNA levels with lung explants from 26 day fetuses. Similar, albeit smaller, effects were observed with day 30 explants. With 26 day explants, dexamethasone treatment resulted in a dose-dependent progressive but not significant increase in SP-A and a significant increase in SP-C gene transcription with 26 day explants. The discordance observed between the dose-responses for mRNA synthesis and levels can best be explained through enhanced message stabilization at low steroid levels. Message stabilization was not observed with actin or 18S

J. Xu et aL / Biochimica et Biophysica Acta 1254 (1995) 302-310

mRNA. Further investigations are required to identify the potential stabilization factors involved and whether the same putative factors affect all three apoproteins. Culture studies with other species have been limited to early gestation. Investigations with second trimester human lung have observed dexamethasone produced a dose-dependent increase in SP-A gene transcription [33]. However, while mRNA stabilization was noted at low steroid concentrations, destabilization effects on SP-A mRNA were evident at high steroid and with prolonged incubation [33,34]. A biphasic effect on SP-A mRNA levels was observed with the type II cell-like NCI-H820 adenocarcinoma cells but only the destabilization occurred with Clara cell-like NCI-H441 cells [35,36]. Only positive effects of glucocorticoids on mRNA levels have been reported for fetal rat lung explants [37]. These studies emphasize the complexity of SP-A regulation. Investigations using human fetal lung explants and human lung tumour cells indicate glucocorticoids have a small effect on SP-B transcription but a prominent effect on SP-B mRNA stabilization [38,39]. In contrast, studies using second trimester human lung suggest glucocorticoids influence SP-C mRNA levels primarily through promoting gene transcription [39]. Glucocorticoids also enhance SP-C gene transcription with explants from fetal rat lung, but whether this is primarily responsible for the elevated SP-C mRNA levels is not clear [40]. Glucocorticoids affect surfactant apoproteins in vivo as well as in vitro. Treatment of pregnant rabbit does of 25 days gestation with betamethasone led to a 6- to 10-fold increase in SP-A mRNA in the fetal lungs, a 2-fold increase in SP-B mRNA but a 50% decrease in SP-C mRNA [19]. Treatment of pregnant rats with dexamethasone produced increases in surfactant apoprotein mRNA levels with relatively greater alterations in mRNA levels being observed early in gestation [10,11,41]. However, postnatal and adult rats remain responsive [41,42]. Whether glucocorticoids influence surfactant apoprotein mRNA levels in vivo at the pre- or post-transcriptional level has not been determined. Although cycloheximide depressed SP-A and SP-C gene transcription and mRNA accumulation with 26 day and 30 day fetal rabbit lung explants, a selective increase in SP-B gene transcription was observed with 26 day explants. This effect was not observed in the presence of dexamethasone. The basis of the stimulation of SP-B transcription is not understood, but could be explained by the involvement of a labile negative regulatory factor in the initiation of SP-B gene transcription. Negative regulatory factors have been implicated in the cycloheximide-dependent synthesis of interferon, c-myc, c-fos and metallothionine and in the calcium-induced gene transcription of the urokinase-type plasminogen activator [43,44]. Venkatesh et al. [39] observed cycloheximide inhibited the dexamethasone-induced increase in SP-C gene transcription in human lung explants without affecting the steroid-dependent increase in SP-B

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mRNA. O'Reilly et al. [38] found cycloheximide depressed SP-B mRNA levels in NCI-H441 cells. These observations demonstrate differences in the regulation of SP-B and SP-C. The present investigations attempted to relate the developmental regulation of surfactant apoprotein mRNA levels in vivo with the effects of glucocorticoids on mRNA levels in vitro. Enhanced transcriptional activities could explain, at least in part, the developmental increases in SP-A and SP-B mRNA which take place during the critical period of lung development in the rabbit. SP-C mRNA levels could be influenced by stabilization early in gestation, but enhanced transcription, apparently in type II cells, also occurs during the critical period. The observation that gene transcription increased for all three apoproteins suggested the possibility that at 26 days lung tissue becomes primed for enhanced apoprotein production. However, explant studies showed that, while dexamethasone elevates surfactant apoprotein levels at low concentrations, SP-C and possibly SP-A gene transcription was stimulated only at higher concentrations. Time course studies revealed considerably different patterns for the individual mRNAs, suggesting independent regulation and, as in the dose-response curves, a considerably dampened effect at 30 days compared to 26 days gestation. Alterations in gene transcription in vitro were small compared to the developmental increases observed in vivo (Fig. 1 vs. Figs. 3-6). This indicates an in vivo component, probably multifactorial, which is not evident in vitro. It has been shown that serum enhances the ability of glucocorticoids to accelerate rabbit lung maturation with respect to morphology and phosphatidylcholine biosynthesis but the effects on surfactant apoproteins have not been elucidated [29]. The present investigations served to confirm the complex nature of the regulatory controls for surfactant apoproteins and support the view that different mechanisms contribute to their expression. They also emphasize differences in regulation related to the developmental stage.

Acknowledgements We thank Dr. C. Ketola and Mr. K. Inchley for their productive discussion on the manuscript, and Mr. Gerald Barbe for his statistical assistance. Ms Barbara Lowery provided excellent editorial assistance. This work was supported by a Medical Research Council of Canada Group grant.

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