Transcriptional Regulation of the Gene Encoding the Major Surfactant Protein (SP-A) in Rabbit Fetal Lung*

The expression of the major protein of rabbit pulmonary surfactant (SP-A), a glycoprotein of M, = 29,000-36,000, is regulated during development and by hormones. In the present study, utilizing a cDNA insert complementary to mRNA coding for SP-A and nuclear transcription elongation assays, we have investigated the developmental and hormonal regulation of transcription of the SP-A gene in rabbit fetal lung tissue. The relative rates of transcription of SP-A mRNA increased as a function of the gestational age of the fetus. The rate of transcription reached a maximum level in lung tissues of 28-day gestational age fetuses and declined slightly in those of neonatal rabbits. The relative rate of transcription of SP-A mRNA increased in rabbit fetal lung explants maintained in organ culture in control medium as a function of incubation time. Dibutyryl cyclic rate of transcription of SP-A several-fold; >4-fold the of transcription of was determined by liquid scintillation spectrometry. When the results obtained by scintillation spectrometry were compared to those obtained by scanning densitometry, it was found that relative changes in SP-A gene transcription in control and treated samples were similar.

The expression of the major protein of rabbit pulmonary surfactant (SP-A), a glycoprotein of M, = 29,000-36,000, is regulated during development and by hormones. In the present study, utilizing a cDNA insert complementary to mRNA coding for SP-A and nuclear transcription elongation assays, we have investigated the developmental and hormonal regulation of transcription of the SP-A gene in rabbit fetal lung tissue. The relative rates of transcription of SP-A mRNA increased as a function of the gestational age of the fetus. The rate of transcription reached a maximum level in lung tissues of 28-day gestational age fetuses and declined slightly in those of neonatal rabbits. The relative rate of transcription of SP-A mRNA increased in rabbit fetal lung explants maintained in organ culture in control medium as a function of incubation time. Dibutyryl cyclic AMP (Bt2cAMP) treatment of fetal lung explants increased the rate of transcription of SP-A mRNA over that of control tissues by several-fold; after 12 h of incubation in the presence of Bt2cAMP, there was >4-fold increase in the rate of transcription of SP-A mRNA as compared to control lung explants. In contrast, glucocorticoids had a rapid effect to decrease the rate of SP-A mRNA transcription. The rapid effect of glucocorticoids to inhibit the transcription of SP-A mRNA was transient; in fetal lung explants incubated in the presence of dexamethasone for >24 h, there was an increase in the rate of transcription of SP-A mRNA over that of control explants. Cycloheximide caused an inhibition of both basal as well as Bt2cAMP-stimulated rates of transcription of SP-A mRNA in the rabbit fetal lung tissue in vitro. This finding is suggestive of a role of labile protein factor@) in mediating transcription of the SP-A gene as well as its induction by Bt2cAMP. The magnitude of changes in the relative rates of transcription of SP-A mRNA during development of rabbit fetal lung in uitro as well as those effected by hormones in vitro were similar to changes in the steady-state levels of SP-A mRNA, suggestive that the regulation of the levels of SP-A mRNA in fetal rabbit lung tissue both in vivo and in vitro occurs primarily at the transcriptional level.
The major pulmonary surfactant-associated protein (SP-* This research 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. A),' a 29-36-kDa glycoprotein synthesized by type I1 pneumonocytes, has been reported to bind to surfactant glycerophospholipids and to enhance the rate of adsorption of dipalmitoylphosphatidylcholine to an air-liquid interface (1). Also, it has been suggested that SP-A and calcium may function in the structural organization of tubular myelin (2, 3), a latticelike structure that may serve as an intermediate between the secreted lamellar body and the mono-molecular phospholipid film at the alveolar air-liquid interface. Through these actions, it is believed that SP-A facilitates the reduction of surface tension at the alveolar air-liquid interface and thereby promotes normal respiration. Inadequate production of surfactant at birth results in respiratory distress syndrome of the newborn. SP-A may also facilitate the recycling of surfactant glycerophospholipids and, thus, may serve as a feedback regulator of surfactant glycerophospholipid synthesis and secretion (4, 5).
Using polyclonal antibodies, as well as a cDNA complementary to mRNA encoding rabbit SP-A, we have previously observed that the levels of SP-A and its mRNA are induced in rabbit fetal lung tissue during development and also that Bt,cAMP and glucocorticoids increase the accumulation of SP-A and its mRNA in rabbit fetal lung in uitro (6,7). Also, we have reported that the induction of SP-A mRNA by BtzcAMP is dependent upon on-going protein synthesis (7). Cyclic AMP analogues (8,9) and @-adrenergic agonists (8) also increase the accumulation of SP-A and its mRNA in human fetal lung in uitro. The presence of @-adrenergic receptors on alveolar epithelial cells (10) and the action of catecholamines to stimulate adenylate cyclase activity of fetal lung tissue (11) suggest a key role for cyclic AMP as a mediator of regulation of SP-A gene expression in uivo. Glucocorticoids have been reported to have both inhibitory (9) and stimulatory (12) effects on the accumulation of SP-A and its mRNA in human fetal lung in uitro. We recently have observed that glucocorticoids have a biphasic effect on the levels of SP-A and its mRNA in human fetal lung in culture; at concentrations of 10"O and lo-' M, dexamethasone increased the levels of SP-A and SP-A mRNA over those of control tissues, whereas at concentrations >lo-* M, the steroid was found to be markedly inhibitory (13).
In the present study, we have utilized a cDNA complementary to the mRNA encoding rabbit SP-A and nuclear transcription elongation assays to investigate the molecular mechanisms that mediate the regulation of SP-A mRNA levels in rabbit fetal lung tissue during development, as well as the effects of BtzcAMP and glucocorticoids on SP-A mRNA levels in rabbit fetal lung tissue in organ culture. To date, no information is available on the developmental and hormonal regulation of the SP-A gene at the transcriptional level.

EXPERIMENTAL PROCEDURES
Organ Culture of Rabbit Fetal Lung Tissue-Lung tissues of fetal rabbits of 21-days gestational age were maintained in organ culture in serum-free Waymouth's MB752/1 medium (GIBCO) as described previously (14). Lung explants were maintained in organ culture for up to 3 days in control medium or in medium containing BhcAMP (1 mM), cortisol (lo" M), or dexamethasone (lo" M). Prior to the addition of hormones or BtSAMP, lung explants were maintained in culture in control medium for 1 day. Methods for isolation of RNA and Northern analysis have been published previously (7). Isolation of Nuclei and Transcription Elongation Assay-Methods for isolation of nuclei, transcription elongation assay, and isolation of 32P-labeled RNA were essentially as described by Sasaki et al. (15). All procedures for isolation of nuclei were performed on ice a t 4 "C. Fetal lung explants, after various treatments, were homogenized in 10 volumes/gram tissue of homogenization buffer (0.25 M sucrose, 10 mM Hepes, pH 8.0, 10 mM MgC12, 2 mM dithiothreitol, and 0.1% (v/ v) Triton X-100) using a glass/glass Dounce homogenizer and 10 hand-driven strokes. The homogenate was filtered through six layers of gauze, and a crude nuclear pellet was isolated by centrifugation at 600 X g for 5 min. The crude nuclei were washed twice in homogenization buffer and finally centrifuged through 1.3 M sucrose in homogenization buffer at 10,000 x g for 10 min (1 volume of nuclei to 3 volumes of 1.3 M sucrose). The nuclei were suspended in 50 mM Hepes, pH 8.0, containing 40% (v/v) glycerol, 5 mM MgCIz, 0.1 mM EDTA, and 2 mM dithiothreitol. Typically, nuclei from 1 g of tissue were suspended in 1.0 ml of glycerol-containing buffer. The suspended nuclei were divided into 100-pl aliquots, immediately frozen in liquid nitrogen, and stored a t -70 "C for future use. Nuclei were quantitated using a hemacytometer.
Transcription elongation assays were performed in a total volume of 200 pl that contained 20 mM Hepes, pH 8.0, 5 mM MgC12,90 mM NH,CI, 0.5 mM MnC12, 16% (v/v) glycerol, 0.04 mM EDTA, 2 mM dithiothreitol, 0.4 mM each of ATP, CTP, GTP, and 300 pCi of [a-"'PIUTP (3000 Ci/mmol) and 20 x lo6 nuclei. The reaction was carried out for 20 min a t 26 "C. The reaction was terminated by digestion with 100 units of DNase I (Worthington) for 20 min at 37 "C followed by digestion with proteinase K (100 pg/ml) in the presence of 15 mM EDTA, 0.5% SDS, and 30 pg of yeast tRNA for 30 min a t 37 "C. Samples were extracted twice with an equal volume of phenol/chloroform ( L l , v/v), and the RNA was precipitated with ammonium acetate (2.5 M) and 2 volumes of ethanol. The RNA precipitate was subjected t o digestions with DNase I, and proteinase K again, phenol/chloroform extracted, and precipitated with ammonium acetate and ethanol. The final RNA precipitate was dissolved in 100 pl of 1 mM Tris-HCI, pH 7.5, 0.5 mM EDTA and 0.1% SDS, and radioactivity was determined. Equivalent amounts of radioactive RNA from each treatment group or gestational age sample were precipitated again as described before, dissolved directly in hybridization buffer (50% formamide containing 4 X SSC, 2 X Denhardt's solution, 50 mM Pipes pH 7.0, 2 mM EDTA, 0.1% SDS, 200 pg/ml yeast tRNA, 100 pg/ml poly(A), and 200 pg/ml denatured salmon sperm DNA) and added t o vials containing two nitrocellulose filters; one contained immobilized cDNA for rabbit SP-A (10.0 pg of pUC18 DNA containing SP-A cDNA), the other contained nonspecific DNA (5.7 pg of pUC18 DNA) or else no added DNA. Plasmid DNAs were previously immobilized to nitrocellulose according to the method described by Diamond and Goodman (16). Hybridization was performed a t 45 "C for 4 days in a shaking water bath. The nitrocellulose filters had been prehybridized for 12 h under identical conditions. Following hybridization, the filters were washed four times in 5 ml of wash buffer (0.3 M NaCI, 2 mM EDTA, 10 mM Tris-HC1, pH 7.5) containing 0.1% SDS at 45 "C for 30 min each time. The filters were then washed once in 5.0 ml of wash buffer at 45 "C for 30 min and incubated in 1.0 ml of wash buffer containing 10.0 pg of RNase "A" and 1.0 pg of RNase "TI" a t 37 "C for 30 min. They were then incubated in 1.0 ml of wash buffer containing 0.1% SDS and 100 pg of proteinase K for 20 min at 37 "C. The filters were washed twice with 5.0 ml of wash buffer containing 0.1% SDS at 45 "C for 30 min each time. After the washes, the filters were subjected to autoradiography using intensifying screens. Relative rates of transcription were assessed by scanning densitometry of the autoradiograms. In all cases, the background (signal obtained after hybridization of radiolabeled RNA to filters containing pUC18 DNA or to blank filters) was subtracted from the signal obtained using the corresponding filters containing the rabbit SP-A cDNA plasmid. In some cases, following autoradiography the bound RNA was released by treating filters with 0.25 ml of 0.4 M NaOH for 30 min a t room temperature, neutralized with 0.1 ml of 1 M acetic acid, and the radioactivity was determined by liquid scintillation spectrometry. When the results obtained by scintillation spectrometry were compared to those obtained by scanning densitometry, it was found that relative changes in SP-A gene transcription in control and treated samples were similar.

RESULTS
Transcription of SP-A mRNA during Development-We have previously observed that SP-A mRNA is undetectable in rabbit fetal lung tissue until day 26 of gestation, the levels of SP-A mRNA reach a maximum on day 31 of gestation and decline slightly in the neonate (7). To determine whether the changes in the levels of SP-A mRNA during development are regulated at the transcriptional level, the rate of transcription of SP-A mRNA was determined in nuclei isolated from lung tissues of fetal rabbits of 21-28-days gestational age and from neonates. It can be seen (Fig. 1) that the transcription rate of SP-A mRNA is detectably increased in nuclei from 24-day rabbit fetal lung tissue (2.0-fold increase as compared to that of nuclei from 21-day fetal lung tissue) and that the rate is increased further as a function of gestational age of the fetus, reaching a maximum level in lung nuclei from 28-day fetal rabbits (11.5-fold increase as compared to that of nuclei from 21-day fetal lung tissue) and declined slightly after birth (8.8fold increase as compared to that of nuclei from 21-day fetal lung tissue). It should be noted that the overall transcription rate was similar in nuclei obtained from lung tissues at different developmental stages. A similar pattern was observed when the levels of SP-A mRNA were analyzed by Northern blot analysis of total RNA isolated from the same tissues (data not shown).
Effect of Bt2cAMP and Dexamethasone on the Rate of Transcription of SP-A mRNA-In previous studies, we found that incubation of rabbit fetal lung explants in the presence of BhcAMP (1 mM) or cortisol (loT7 M ) resulted in increased accumulation of SP-A and the levels of translatable SP-A mRNA (6). In other studies, we found that BhcAMP (1 mM) treatment of rabbit fetal lung explants resulted in an increased accumulation of cytoplasmic and nuclear RNAs encoding SP-A as rapidly as 2 h after its addition to the culture medium (7). In the present study, we compared the effects of BtgAMP and cortisol on the levels of SP-A mRNA in rabbit fetal lung explants as a function of incubation time (Fig. 2). As we have found previously (7), levels of the two species of SP-A mRNA of 2.0 and 3.0 kilobases in size were increased in control explants as a function of incubation time. BtZcAMP caused a rapid induction of SP-A mRNA in fetal lung explants; a stimulatory effect was observed as early as 6 h after its addition to the culture medium. In explants incubated with cortisol for 48 and 72 h, SP-A mRNA levels were greater than those of control tissues. We were surprised to find, however, that after 6 and 24 h of incubation in the presence of cortisol, the levels of SP-A mRNA were reduced as compared to those of control explants. This rapid effect of glucocorticoids to inhibit accumulation of SP-A mRNA also was observed in two other experiments.
To determine whether the effects of cyclic AMP analogues and glucocorticoids to alter the levels of SP-A mRNA are the result of changes in the rate of transcription of the SP-A gene, nuclei were isolated from 21-day rabbit fetal lung explants after 12 h of incubation in the absence or presence of BtzcAMP (1 mM) or the synthetic glucocorticoid, dexamethasone M), added alone or in combination, and nuclear transcription elongation assays were performed. Also, cytoplasmic RNA from the same tissues was isolated and subjected to Northern blot analysis. Incubation of lung explants in the presence of BtzcAMP (1 mM) for 12 h caused a 4.7-fold increase in the rate of transcription of SP-A mRNA over that of tissues incubated in control medium (Fig. 3). In contrast, when lung explants were incubated with dexamethasone M) for 12 h, there was a 50% decrease in the rate of transcription of SP-A mRNA as compared to that of untreated tissues. When explants were incubated with dexamethasone

M)
and BtzcAMP (1 mM) in combination, the rate of transcription of SP-A mRNA was reduced to a level that was -50% of that observed in nuclei incubated with BtzcAMP alone (2.1fold greater than that of control explants). Northern blot analysis (Fig. 3) of cytoplasmic RNAs isolated from the same tissues was indicative of similar effects of BtzcAMP and dexamethasone on the levels of SP-A mRNA (hybridizable SP-A mRNA relative to control: BtzcAMP-treated, 5.0-fold; dexamethasone-treated, 0.4-fold; BtzcAMP plus dexamethasone, 3.5-fold).
Treatment of fetal lung explants with BtzcAMP or with dexamethasone had no effect either on the overall transcriptional activity of the nuclei or on the amount of total RNA isolated per gram wet weight of tissue.
The rate of transcription of SP-A mRNA also was determined in nuclei isolated from 21-day rabbit fetal lung explants incubated in the absence or presence of either BtzcAMP (1 mM) or dexamethasone M) for 24-72 h. It was observed  (prSP-A). This is an autoradiogram of the filters from this experiment. that the rate of SP-A mRNA transcription increased in control explants as a function of incubation time (Fig. 4). After 24 h of incubation, the inhibitory effect of dexamethasone M) on SP-A mRNA transcription was no longer apparent, whereas in fetal lung explants incubated for 24 h in the presence of BtzcAMP (1 mM), a 4.8-fold increase in the rate of transcription of SP-A mRNA as compared to control tissues was observed. After 48 h of incubation, dexamethasone M) clearly increased the rate of transcription of SP-A mRNA; a 1.5-fold increase in the rate of transcription was observed compared to that of untreated tissues. BtzcAMP (1 mM) caused a %fold increase in the rate of SP-A mRNA transcription over that of control explants at this time point. By 72 h of incubation, however, the stimulatory effects of BtzcAMP (1 mM) and dexamethasone M) on SP-A mRNA transcription as compared to control explants were diminished, presumably because of the increased rate of transcription of SP-A mRNA in control tissues. Effect of Cycloheximide on the Rate of Transcription of SP-A RNA-We previously observed that treatment of rabbit fetal lung explants with cycloheximide for 6 h inhibited the accumulation of SP-A mRNA in control as well as in Bt2cAMP-treated lung explants. We suggested that these findings were indicative of a requirement for on-going protein synthesis for accumulation of SP-A mRNA (7).
To determine whether the action of cycloheximide to decrease the levels of SP-A mRNA occurs at the transcriptional level, the rate of transcription of SP-A mRNA was evaluated in nuclei that were isolated from tissues incubated in the absence or presence of Bt2cAMP (1 mM) in the absence or presence of cycloheximide (2 pg/ml) for 6 h. Also, the rate of transcription of actin mRNA was determined in the same set of nuclei (Fig. 5). Treatment of fetal lung explants with cycloheximide for 6 h resulted in a 30% decrease in the rate of transcription of SP-A mRNA in explants maintained in control medium. BtzcAMP caused a %fold increase in the rate of SP-A transcription; cycloheximide when present in combination with BhcAMP inhibited the stimulatory effect of Bt2cAMP by >80%. Cycloheximide treatment, however, did not have any significant inhibitory effect on the overall rate of RNA transcription (data not shown) or on the rate of transcription of actin mRNA.

DISCUSSION
In the present study, we have investigated the transcriptional regulation of expression of the gene encoding the major surfactant-associated protein, SP-A, in rabbit fetal lung tissue during development, as well as the effects of BtzcAMP and glucocorticoids on SP-A gene transcription in 21-day gestational age fetal rabbit lung tissue in vitro. We observed that Bt2cAMP caused a detectable increase in the rate of transcription of SP-A mRNA in nuclei obtained from the rabbit fetal lung explants within 12 h of its addition to the culture medium. These relatively rapid changes in the rates of transcription of SP-A mRNA caused by BtzcAMP were found to be associated with similar changes in the steady-state levels of SP-A mRNA. In a number of experiments, the fold-induction of SP-A mRNA levels in lung explants incubated with BbcAMP for periods 224 h were found to be greater than the fold-increase in SP-A gene transcription.2 These findings are V. Boggaram and C. R. Mendelson, unpublished observations. suggestive that BbcAMP may also act in the long term to increase SP-A mRNA stability. It has recently been reported by Hod and Hanson (17) that cyclic AMP acts to increase phosphoenolpyruvate carboxykinase mRNA stability in addition to its action to increase the transcriptional activity of the phosphoenolpyruvate carboxykinase gene (15,17,18).
In contrast to the stimulatory effect of Bt2cAMP on SP-A gene transcription and mRNA levels, we observed that when lung explants were incubated in the presence of dexamethasone M), there was a rapid effect of the steroid to reduce both the rate of transcription of SP-A mRNA and SP-A mRNA levels as compared to explants maintained in control medium. After 24 h of incubation, however, the inhibitory effect of dexamethasone on the rate of transcription of SP-A mRNA was no longer observed, and by 48 h, a stimulatory effect of the steroid on the rate of transcription of SP-A mRNA was clearly apparent. After 72 h of incubation, the stimulatory effects of both BtzcAMP and dexamethasone were diminished, presumably because the transcriptional activity of SP-A gene in fetal lung explants maintained in control medium had increased to comparable levels. We have previously observed that 21-day rabbit fetal lung explants differentiate in vitro (14); the levels of SP-A and its mRNA increase as a function of incubation time, concomitant with the appearance of increased numbers of type I1 cells (6). The cellular mechanisms that underly this spontaneous differentiation of the fetal lung tissue and the induction of SP-A gene transcription are not understood.
In the present study, we found that treatment of lung explants for 6 h with cycloheximide caused a decrease in the rate of SP-A mRNA transcription as compared to that of untreated lung explants, as well as an inhibition of the BhcAMP-induced increase in the rate of transcription of SP-A mRNA. These changes in rates of transcription of SP-A mRNA were associated with comparable changes in steadystate levels of SP-A mRNA. Cycloheximide treatment did not have an inhibitory effect on the overall transcriptional activity of fetal lung nuclei nor on the rate of transcription of actin mRNA. These results taken together indicate that the effects of cycloheximide to inhibit SP-A mRNA transcription are relatively specific and are not due to a general toxic effect of cycloheximide on the fetal lung tissue. In previous studies, we have found that the inhibitory effects of cycloheximide on SP-A mRNA accumulation are reversible (7). Also, the present findings support the concept that a rapidly turning over protein factor(s) is required for transcription of the SP-A gene and its induction by BtcAMP.
Cyclic AMP has been found to increase the transcription of a number of eucaryotic genes including phosphoenolpyruvate carboxykinase (15, 17, 18), vasoactive intestinal peptide (19), somatostatin (20), prolactin (21, 22), plasminogen activator (23), tyrosine hydroxylase (24), and a number of steroidogenic forms of cytochrome P-450 (25). In the cases of phosphoenolpyruvate carboxykinase (15) and plasminogen activator (23), the induction of gene transcription has been shown to be independent of new protein synthesis. In the case of cyclic AMP regulation of cytochrome P-450, (25), cytochrome P-45OI7, (26), and aromatase cytochrome P-450 (27), the induction of mRNA levels were found to be blocked by cycloheximide, indicating that a labile protein factor(s) is required for the transcriptional activation of these genes. The mechanisms by which cyclic AMP regulates eucaryotic gene expression are not clear. Putative cyclic AMP regulatory elements have been identified in the 5"flanking regions of a number of genes including somatostatin (20), vasoactive intestinal peptide (19), tyrosine hydroxylase (24), and phos-Transcriptional Regulation of Rabbit Surfactant Protein (SP-A) G e m phoenolpyruvate carboxykinase (28). Recently, a nuclear protein of M, = 43,000 was isolated that binds selectively to the cyclic AMP response element of the somatostatin gene (29). This protein is phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase i n uitro, and its phosphorylation state is increased i n uiuo in response to forskolin treatment. In eucaryotes, cyclic AMP, after binding to cyclic AMP-dependent protein kinase, induces the phosphorylation of cellular proteins, some of which might directly interact with target genes to modulate their expression. Recently, it has been demonstrated that in rat liver, increases in intracellular cyclic AMP levels cause increased association of the catalytic and regulatory subunits of cyclic AMP-dependent protein kinase with transcriptionally active chromatin (30).
In mutant PC12 cells, which lack cyclic AMP-dependent protein kinase type I1 activity, cyclic AMP fails to regulate somatostatin gene expression (20). Glucocorticoids have been reported to act both as positive (31) and negative (32,33) regulators of gene expression. The rapid but transient action of glucocorticoids to inhibit the transcription of SP-A mRNA observed in the present study may be mediated by an interaction of the glucocorticoid receptor complex with the regulatory elements of the SP-A gene. The glucocorticoid-receptor complex has been reported to have a direct effect to inhibit the transcription of the proopiomelanocortin gene by interacting with a regulatory element that is located upstream of the start site of transcription (34). The mechanisms whereby glucocorticoids increase the transcription of SP-A mRNA after longer periods of incubation are not clear. The results of recent studies on the hormonal regulation of prolactin gene expression have revealed that positive and negative transcriptional effects of estrogens and glucocorticoids, respectively, are mediated by distinct functional domains of the steroid receptors (35). Of particular interest are the findings of studies of the regulation of expression of the human glycoprotein hormone &-subunit gene, which are suggestive that the glucocorticoid receptor complex may negatively regulate a-subunit expression by interference with the binding of specific transcription factors to cyclic AMP regulatory elements within the 5"flanking region of the gene (36). The mechanisms whereby glucocorticoids exert both positive and negative regulatory effects on SP-A gene expression remain to be determined. The identification of putative glucocorticoid regulatory DNA-sequence elements in both human (37) and the rabbit SP-A genes3 is suggestive that these actions may be mediated by the interaction of the glucocorticoid receptor with distinct regulatory regions of the SP-A gene. Alternatively, glucocorticoids may increase SP-A gene transcription through their action to elevate intracellular levels of cyclic AMP. Glucocorticoids have been reported to cause inhibition of phosphodiesterase in cultured HTC hepatoma cells (38) and in rat fetal lung tissue (39) and to increase the levels of Gs, the stimulatory guanine nucleotide binding protein of adenylate cyclase, in ROS17/2.8 cells (40).
In the present study, we found that the transcriptional activity of the SP-A gene is increased in rabbit fetal lung tissue during development. Appreciable rates of transcription were observed in nuclei from 24-day rabbit fetal lung. The transcriptional activity of the SP-A gene increased with gestational age and reached a maximum level in lung tissues of 28-day fetal rabbits. The transcription rate of the SP-A gene was slightly decreased in the lungs of neonates as compared t o lungs of 28-day fetal rabbits. Again, the developmental Q. Kuang, V. Boggaram, and C. R. Mendelson, unpublished observations. changes in the relative transcription rate of this gene were similar to the changes in steady-state levels of SP-A mRNA (6,7). The factors that regulate the expression of SP-A mRNA during rabbit fetal lung development in uiuo are not known.
The findings of this and our previous investigations (6, 7) are suggestive that cyclic AMP may serve an important role in the transcriptional activation of the SP-A gene in developing fetal lung and that glucocorticoids may have both stimulatory and inhibitory effects that are dependent upon the stage of type I1 cell differentiation. The results of our studies are further suggestive that changes in the levels of SP-A mRNA in rabbit fetal lung tissue during development i n uiuo or after treatment with cyclic AMP or glucocorticoids in uitro are regulated primarily at the transcriptional level.