Identification of a novel DNA regulatory element in the rabbit surfactant protein B (SP-B) promoter that is a target for ATF/CREB and AP-1 transcription factors.

Surfactant protein B (SP-B) is required for the maintenance of biophysical properties and physiological function of pulmonary surfactant. SP-B is expressed in a cell/tissue-specific manner by the alveolar type II and bronchiolar (Clara) epithelial cells of the lung and is developmentally and hormonally regulated. We previously identified a minimal promoter region containing -236/+39 base pairs (bp) of rabbit SP-B gene that is necessary and sufficient for high level promoter activity in NCI-H441 cells, a cell line with characteristics of Clara cells. In this study, we have characterized the functional importance of a novel DNA regulatory element, termed SP-B CRE, with the sequence TGAGGTCA in the SP-B minimal promoter. The SP-B CRE sequence shared homology to cyclic AMP responsive element (CRE) binding sequence and contained an overlapping nuclear receptor element binding half-site. Mutation of SP-B CRE into a scrambled sequence reduced promoter activity by greater than 70%, whereas mutation into a palindromic consensus CRE increased the promoter activity by 100%. Electrophoretic mobility shift assay (EMSA) and Western immunoblot analysis of affinity purified proteins interacting with SP-B CRE showed that it is a target for binding of members of the activating transcription factor (ATF)/cyclic AMP response element binding protein (CREB) family of transcription factors, such as CREB, CREM, ATF-1, ATF-2 as well as c-Jun and TTF-1. Overexpression of CREB, ATF-2 and c-Jun inhibited SP-B promoter activity in NCI-H441 cells. These data have shown that members of the ATF/CREB family of transcription factors and c-Jun play important roles in mediating the transcriptional regulation of the SP-B gene.

Cell-specific and developmental regulation of rabbit surfactant protein B promoter in transgenic mice.

Surfactant protein B (SP-B) is expressed tissue specifically in the lung and is developmentally regulated. To identify genomic regions that control SP-B expression, we analyzed SP-B promoter activity in transgenic mice containing rabbit SP-B 5'-flanking DNA fragments linked to the chloramphenicol acetyltransferase (CAT) reporter gene. Results showed that whereas the -2,176/+39-bp fragment failed to express CAT, shorter fragments of -730/+39 and -236/+39 bp expressed CAT tissue specifically in the lung. Further deletion of 5'-flanking DNA to -136 bp resulted in no expression of CAT. Immunostaining demonstrated that both -730/+39- and -236/+39-bp regions expressed CAT specifically in alveolar type II and Clara cells. The -236/+39-bp region expressed CAT at a significantly lower level than the -730/+39-bp region. CAT expression in mice containing the -730/+39-bp region was detected in embryonic day 14 lung and attained maximum levels in day 18 lung, indicating that the developmental expression of CAT was similar to that of SP-B. These data show that the DNA elements necessary for cell type-specific expression are located within -236/+39 bp of the SP-B gene. Additionally, these data suggest that the -2,176/-730- and -730/-236-bp regions contain the DNA elements that repress and enhance SP-B gene transcription, respectively.

Characterization of rabbit SP-B promoter region responsive to downregulation by tumor necrosis factor-alpha.

Surfactant protein B (SP-B) is essential for the maintenance of biophysical properties and physiological function of pulmonary surfactant. Tumor necrosis factor-alpha (TNF-alpha), an important mediator of lung inflammation, inhibits surfactant phospholipid and surfactant protein synthesis in the lung. In the present study, we investigated the TNF-alpha inhibition of rabbit SP-B promoter activity in a human lung adenocarcinoma cell line (NCI-H441). Deletion experiments indicated that the TNF-alpha response elements are located within -236 bp of SP-B 5'-flanking DNA. The TNF-alpha response region contained binding sites for nuclear factor-kappa B (NF-kappa B), Sp1/Sp3, thyroid transcription factor (TTF)-1, and hepatocyte nuclear factor (HNF)-3 transcription factors. Inhibitors of NF-kappa B activation such as dexamethasone and N-tosyl-L-phenylalanine chloromethyl ketone and mutation of the NF-kappa B element did not reverse TNF-alpha inhibition of SP-B promoter, indicating that TNF-alpha inhibition of SP-B promoter activity occurs independently of NF-kappa B activation. TNF-alpha treatment decreased the binding activities of TTF-1 and HNF-3 elements without altering the nuclear levels of TTF-1 and HNF-3 alpha proteins. Pretreatment of cells with okadaic acid reversed TNF-alpha inhibition of SP-B promoter activity. Taken together these data indicated that in NCI-H441 cells 1) TNF-alpha inhibition of SP-B promoter activity may be caused by decreased binding activities of TTF-1 and HNF-3 elements, 2) the decreased binding activities of TTF-1 and HNF-3 alpha are not due to decreased nuclear levels of the proteins, and 3) okadaic acid-sensitive phosphatases may be involved in mediating TNF-alpha inhibition of SP-B promoter activity.

Identification of functional TTF-1 and Sp1/Sp3 sites in the upstream promoter region of rabbit SP-B gene.

Surfactant protein B (SP-B) is essential for the maintenance of biophysical properties and physiological function of pulmonary surfactant. SP-B mRNA is expressed in a cell type-restricted manner in alveolar type II and bronchiolar (Clara) epithelial cells of the lung and is developmentally induced. In NCI-H441 cells, a lung cell line with characteristics of Clara cells, a minimal promoter region comprising -236 to +39 nucleotides supports high-level expression of chloramphenicol acetyltransferase reporter activity. In the present investigation, we characterized the upstream promoter region, -236 to -140 nucleotides, that is essential for promoter activity. Deletion mapping identified two segments, -236 to -170 and -170 to -140 nucleotides, that are important for promoter activity. Mutational analysis and gel mobility shift experiments identified thyroid transcription factor-1, Sp1, and Sp3 as important trans-acting factors that bind to sequences in the upstream promoter region. Our data suggest that SP-B promoter activity is dependent on interactions between factors bound to upstream and downstream regions of the promoter.

Tissue-specific and developmental stage-dependent expression of a novel rat Dopa/tyrosine sulfotransferase.

Tissue-specific and developmental stage-dependent expression of a novel Dopa/tyrosine sulfotransferase in Sprague-Dawley rats was examined. Both immunoblot and Northern blot analyses showed that the enzyme was expressed predominantly in liver and to a lesser extent in kidney. Its expression could not be detected in nine other organs tested. Livers from different age groups of male or female rats were examined for the developmental regulation of the expression of the Dopa/tyrosine sulfotransferase. Results from immunoblot and Northern blot analyses revealed that the enzyme was present at a very low level in livers of 1-day-old to 2-week-old rats, and gradually increased to a maximum level in rats older than 2 months. Data from the enzymatic assays also showed a similar trend of expression in both male and female rats. The Dopa/tyrosine sulfotransferase activities detected in liver samples of the 8-week-old male and female rats were, respectively, 8.6 and 6.6 times that of the activities detected in liver samples of the 1-day-old male and female rats. These data provide a foundation for the future investigation of the cis- and trans-acting factors involved in the regulation of the tissue-specific and developmental stage-dependent expression of this enzyme.

Functional analysis of surfactant protein B (SP-B) promoter. Sp1, Sp3, TTF-1, and HNF-3alpha transcription factors are necessary for lung cell-specific activation of SP-B gene transcription.

Surfactant protein B (SP-B) is essential for maintenance of biophysical properties and physiological function of pulmonary surfactant. SP-B mRNA expression is restricted to alveolar type II epithelial cells and bronchiolar epithelial cells (Clara cells) of adult lung. We previously (Margana, R. K., and Boggaram, V. (1996) Am. J. Physiol. 270, L601-L612) found that a minimal promoter region (-236 to +39) of rabbit SP-B gene is sufficient for high level expression of chloramphenicol acetyltransferase reporter gene in NCI-H441 cells, a cell line with characteristics of Clara cells. In the present study we used mutational analysis, electrophoretic mobility shift assays, and DNase I footprinting to identify cis-DNA regulatory elements and trans-acting protein factors required for lung cell-specific expression of SP-B gene. We found that in addition to thyroid transcription factor 1 (TTF-1) and hepatocyte nuclear factor 3alpha (HNF-3alpha) binding sites, two spatially separate DNA sequences that bind Sp1 and Sp3 factors are necessary for the maintenance of SP-B promoter activity. Mutation of any one of the transcription factor binding sites caused a significant reduction in SP-B promoter activity suggesting that Sp1, Sp3, and TTF-1 and HNF-3alpha interact cooperatively with SP-B promoter to activate gene transcription.

Rabbit surfactant protein B gene: structure and functional characterization of the promoter.

Surfactant protein B (SP-B) is essential for physiological function of pulmonary surfactant. In the present investigation, we isolated rabbit SP-B gene and determined its nucleotide sequence, transcription start site, and exon/intron organization. The coding region of rabbit SP-B gene is comprised of 6.8 kb of sequence and is organized into 10 introns and 11 exons. By deletion analysis we determined that a region of SP-B gene extending from -236 to +39 nucleotides is sufficient for high-level expression of CAT reporter gene in a cell type-specific manner in the pulmonary adenocarcinoma cell line NCI-H441. Deletion of 5'-flanking sequence to -140 and -71 nucleotides significantly reduced SP-B promoter activity, suggesting that the -236 to +39 region contains cis-DNA elements required for cell type-specific expression. The proximal promoter region of SP-B gene contained DNA sequence motifs for binding thyroid transcription factor 1 (TTF-1) (-113 to -97) and hepatocyte nuclear factor 3(HNF-3) (-91 to -81). Although SP-B gene sequence -140 to +39 did not support high level expression of CAT gene in NCI-H441 cells, it was capable of activation by TTF-1 in HeLa cells, suggesting that the -236 to -140 sequence plays an important role in cell type-specific activation of SP-B promoter.

A rapid protocol for isolation of RNA from mycobacteria.

Isolation of total cellular RNA from members of mycobacteria has been a labour-intensive task involving large volumes of cells, multiple extractions of cell lysates with phenol-chloroform followed by caesium chloride centrifugation. A simple and rapid procedure is reported for isolation of RNA from mycobacteria using as few as 1 x 10(7) cells. The RNA thus isolated when analysed on ethidium bromide gels contained 16S and 23S RNA as major species. Further, the RNA was used for amplification of an internal segment of hsp65 gene by reverse transcription followed by PCR.

Transcription and mRNA stability regulate developmental and hormonal expression of rabbit surfactant protein B gene.

Surfactant protein B (SP-B), a hydrophobic protein of pulmonary surfactant, is essential for the surface tension-reducing properties of surfactant. In the present study, we isolated and characterized cDNAs encoding rabbit SP-B, and used transcription run-on assays and Northern blot analysis to investigate the role of transcriptional and posttranscriptional mechanisms in the developmental and cAMP and dexamethasone induction of SP-B mRNA. We found two forms of SP-B cDNAs that differed by an insertion of 69 nucleotides in the 3' untranslated regions. We found that transcription across the SP-B gene is nonequimolar and the 3' end of the gene has high levels of antisense transcription. SP-B gene transcription and SP-B mRNA levels increased during fetal lung development. However, increased SP-B mRNA levels could not be accounted for primarily on the basis of increased transcription. These results suggested that enhanced SP-B gene transcription and enhanced SP-B mRNA stability mediate developmental induction of SP-B gene. In rabbit fetal lung in vitro, both dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) and dexamethasone increased SP-B mRNA levels. DBcAMP-dependent increase in SP-B mRNA levels resulted from increased SP-B gene transcription, whereas dexamethasone-dependent increase resulted from combined effects on increased SP-B gene transcription and SP-B mRNA stability. In tissues treated with dexamethasone the half-life (t1/2) of SP-B mRNA increased > 2.5-fold (t1/2 control = 9 h; t1/2 dex-treated = 25 h). These data show that both transcription and mRNA stability regulate induction of SP-B gene expression during fetal lung development and by cAMP and dexamethasone in fetal lung in vitro.

Developmental and hormonal regulation of surfactant protein C (SP-C) gene expression in fetal lung. Role of transcription and mRNA stability.

Pulmonary surfactant protein C (SP-C) gene expression is developmentally and hormonally regulated in fetal lung. In the present study, we investigated the role of transcriptional and posttranscriptional mechanisms in the developmental, cAMP, and dexamethasone induction of SP-C mRNA. We found that developmental induction of SP-C mRNA was not coincident with induction of SP-C gene transcription. SP-C mRNA levels reached approximately 90% of levels in adult lung on day 24 of gestation, whereas SP-C gene transcription was only approximately 4% of level in adult lung and did not increase until day 28 of gestation (term in rabbit = 31 days). Treatment of fetal lung tissues in vitro with dibutyryl cyclic AMP (Bt2cAMP) and dexamethasone increased SP-C mRNA accumulation by different mechanisms. Increase in SP-C mRNA accumulation by Bt2cAMP was the result of increased SP-C gene transcription, whereas increased SP-C mRNA accumulation by dexamethasone was due to stabilization of RNA. In control tissues the SP-C mRNA half-life (t1/2) was 11.2 h, and after dexamethasone treatment it increased to 30 h. These data show that both transcriptional and mRNA stabilization mechanisms regulate induction of SP-C gene expression during fetal lung development and by cAMP and dexamethasone in fetal lung in vitro.

Characterization of mRNA transcripts and organization of human SP-A1 and SP-A2 genes.

In the present study, we have characterized the mRNA transcripts and intron-exon organization of the human surfactant protein (SP)A1 and SP-A2 genes. By primer extension analysis of mRNA isolated from human fetal lung explants using an oligonucleotide primer to exon II (as delineated in the SP-A1 gene), a minimum of nine primer extended transcripts was observed. Rapid amplification of cDNA ends was used to amplify the primer extended transcripts for sequence analysis. Sequence analysis of 47 full-length primer extended cDNAs and comparison with the sequences of the genes encoding SP-A1 and SP-A2 revealed four different classes of transcripts of the SP-A2 gene and five different classes of transcripts of the gene encoding SP-A1. A major difference between SP-A2 and SP-A1 mRNA transcripts is that SP-A2 transcripts are always comprised of sequences contained within six exons; the extra exon in SP-A2 (exon II of VI) encodes additional 5'-untranslated sequence and is located between exons I and II of SP-A1. By contrast, the majority of transcripts of the SP-A1 gene are comprised of sequences contained within five exons. In the cases of both SP-A1 and SP-A2 genes, a small proportion of the mRNA transcripts contain sequences present in alternate exons. In addition, the majority of the SP-A1 mRNA transcripts are initiated 5 bp downstream of the transcription initiation site of SP-A2. In our companion paper [McCormick and Mendelson. Am. J. Physiol. 266 (Lung Cell. Mol. Physiol. 10): L367-L374, 1994], we report that the SP-A1 and SP-A2 genes are differentially regulated during development and by adenosine 3',5'-cyclic monophosphate and glucocorticoids in human fetal lung in culture.

Insulin-like growth factor I modulates hypothalamic somatostatin through a growth hormone releasing factor increased somatostatin release and messenger ribonucleic acid levels.

Insulin-like growth factor I (IGF-I) has been shown to participate in feedback inhibition of growth hormone (GH) secretion at the level of both the pituitary and hypothalamus. Therefore, we tested the possible involvement of IGF-I on somatostatin (SRIF) and GH-releasing factor (GRF) release in median eminence (ME) fragments and periventricular nucleus (PeN) of male rats. The levels of SRIF messenger ribonucleic acid (mRNA) were also determined in PeN incubated in vitro with IGF-I. The ME's were incubated in Krebs-Ringer bicarbonate glucose buffer in the presence of various concentrations of IGF-I (10(-7) to 10(-11) M) for 30 min. SRIF and GRF released into the medium were quantitated by RIA. The release of SRIF and GRF from the ME's was stimulated significantly (P < 0.025 and P < 0.05, respectively) by 10(-9) M IGF-I. To determine whether the effect of IGF-I on SRIF release is mediated by GRF release in the ME, a specific GRF antibody (ab) (1:500) was used concomitantly with IGF-I (10(-9) M). The release of SRIF induced by IGF-I was blocked by the GRF ab (P < 0.001), but not by normal rabbit serum used at the same dilution. To determine the effect of IGF-I on the regulation of SRIF mRNA levels, SRIF mRNA was determined in PeN explants incubated in the presence of IGF-I (10(-8) to 10(-10) M) for 2 to 6 h. Levels of SRIF mRNA were determined by a S1 nuclease protection assay using a 32P-labelled rat SRIF riboprobe.(ABSTRACT TRUNCATED AT 250 WORDS)

Rabbit surfactant protein C: cDNA cloning and regulation of alternatively spliced surfactant protein C mRNAs.

Surfactant protein C (SP-C), a hydrophobic protein of pulmonary surfactant is essential for surfactant function. Toward elucidating molecular mechanisms that mediate regulation of SP-C gene expression in rabbit lung, we isolated and characterized cDNAs encoding rabbit SP-C and studied the regulation of SP-C gene expression during fetal lung development and by adenosine 3',5'-cyclic monophosphate (cAMP) and dexamethasone in fetal lung tissues in vitro. We found that rabbit SP-C is highly homologous to SP-C of other species and is encoded by two mRNAs that differ by an insertion of 31 nucleotides in the 3' untranslated regions. SP-C mRNAs were classified into two types based on the nucleotide sequence; type I represents RNA without the 31 nucleotide insert and comprises approximately 80-90% of total SP-C mRNA content, whereas type II represents RNA containing the insert and comprises approximately 10-20% of total SP-C mRNA content. SP-C mRNAs were induced in a coordinate manner during fetal lung development and by cAMP and dexamethasone in fetal lung tissues in vitro. Southern hybridization analysis of genomic DNA suggested that SP-C mRNAs are encoded by a single gene. Polymerase [corrected] chain reaction-amplification of genomic DNA with oligonucleotide primers flanking the insertional sequence and sequence analysis of amplified DNA showed that SP-C mRNAs are produced by alternative use of 3' splice sites of intron 5 of SP-C gene.

Rabbit lung surfactant protein A gene: identification of a lung-specific DNase I hypersensitive site.

Expression of the gene encoding pulmonary surfactant protein A, SP-A, is lung specific and developmentally and hormonally regulated. Previously, we observed that SP-A gene transcription is initiated in fetal rabbit lung after day 21 of gestation and reaches maximal levels by day 28. In the present study, a cDNA specific for rabbit SP-A was used to isolate the SP-A gene from a rabbit genomic library. A 7.6-kb fragment containing the entire structural gene and approximately 380 bp of 5'-flanking DNA was isolated and characterized. The transcription initiation site, mapped by primer extension analysis, was localized 23 bp downstream of a putative TATA element. The structural gene is composed of five exons and four introns. The first exon encodes the 5'-untranslated region of the mRNA; the translation initiation site is in exon II, and exon V contains the two polyadenylation sites that give rise to the 2.0- and 3.0-kb species of SP-A mRNA. A potential adenosine 3',5'-cyclic monophosphate (cAMP)-regulatory element (CRE) was identified at -261 bp, and sequences with homology to glucocorticoid-regulatory element (GRE) half-sites were found at -150 and -190 bp upstream of the transcription initiation site and within the first intron. A DNase I hypersensitive site was identified in genomic DNA isolated from 21- and 28-day fetal and adult rabbit lung tissues. This site was mapped within the 5'-flanking region of the SP-A gene, at approximately -80 to -180 bp upstream of the transcription initiation site. The absence of this hypersensitive site in genomic DNA of liver, kidney, and heart tissues suggests that altered chromatin structure may serve a role in lung-specific SP-A gene expression. The presence of this tissue-specific DNase I hypersensitive site in lung nuclei from 21-day gestational age fetal rabbits suggests that the SP-A gene may exist in an accessible conformation prior to the time of transcription initiation.

Expression and transport of rabbit surfactant protein A in COS-1 cells.

SV40-transformed green monkey kidney (COS-1) cells were transfected with expression plasmids that contained either the structural gene or cDNA for surfactant protein A (SP-A), a major protein of rabbit lung surfactant. The transfected COS-1 cells synthesized several isoforms of SP-A that were found to be less acidic than those produced in rabbit lung tissue. SP-A species with apparent molecular weight (M(r)) approximately equal to 29,000-33,000 were detected in the transfected cells, whereas glycosylated forms with apparent M(r) approximately equal to 33,000-38,000 were detectable only in the culture medium. Analysis of transfected cells by indirect immunofluorescence revealed that SP-A was localized in punctate bodies throughout the cytoplasm. Expressed SP-A was not detectable on the cell surface nor was there evidence that secreted SP-A was endocytosed by COS-1 cells. After subcellular fractionation of the transfected COS-1 cells, SP-A was found to be localized predominantly in the 5,000- and 18,000-g pellet fractions; little or no immunoreactive SP-A was detectable in cytosolic fractions. Treatment of transfected cells with the glycosylation inhibitor tunicamycin prevented secretion of SP-A into the medium, suggesting a role of glycosylation in secretion of SP-A. On the other hand, treatment of transfected cells with inhibitors of proline hydroxylation, which may cause destabilization of the collagen-like domain of SP-A, reduced but did not prevent secretion of SP-A into the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)

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

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 greater than or equal to 10(-8) M, the steroid was markedly inhibitory. Our findings suggest that the inhibitory action of dexamethasone (greater than 10(-8) M) on SP-A mRNA levels was mediated by an effect to reduce SP-A mRNA stability, since the steroid caused a dose-dependent 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 half-life was reduced by more than 60% to 5.0 h. Dexamethasone 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 SP-A 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Developmental and hormonal regulation of surfactant protein A (SP-A) gene expression in fetal lung.

Pulmonary surfactant is a developmentally-regulated lipoprotein synthesized and secreted by the type II cells of the pulmonary alveolus where surfactant glycerophospholipids and proteins act to reduce surface tension at the alveolar air-liquid interface. Surfactant protein A (SP-A), the major surfactant-associated protein, appears to serve an important role in surfactant function and reutilization by type II cells. SP-A synthesis and gene expression are initiated in fetal lung tissue in concert with the developmental induction of surfactant glycerophospholipid synthesis. In studies using midtrimester human fetal lung explants maintained in organ culture, we have observed that cyclic AMP and glucocorticoids have pronounced effects on morphologic development and on the levels of SP-A gene expression. Cyclic AMP analogues act primarily to induce SP-A gene transcription; whereas, glucocorticoids have complex effects at both the transcriptional and posttranscriptional levels. We also have found that human fetal lung in vitro secretes into the culture medium relatively large amounts of prostaglandins (PG) PGE2 and PGF2 alpha and the PGI2 and thromboxane A2 metabolites, 6-keto-PGF1 alpha and TxB2, respectively. The prostaglandin synthesis inhibitor, indomethacin, markedly inhibits SP-A gene expression and cyclic AMP formation by human fetal lung in culture; the inhibitory effect of indomethacin on SP-A gene expression can be prevented by simultaneous incubation with either Bt2cAMP or PGE2. These findings are suggestive that prostaglandins acting through cyclic AMP also may serve an important role in the regulation of SP-A gene expression in human fetal lung tissue.

Hormonal control of the surfactant system in fetal lung.

The synthesis of surfactant glycerophospholipids and proteins is under multifactorial control and is regulated by a number of hormones and factors, including glucocorticoids, prolactin, insulin, growth factors, estrogens, androgens, thyroid hormones and catecholamines acting through beta-adrenergic receptors, and cAMP. In studies with human fetal lung in organ culture, glucocorticoids, in combination with prolactin and/or insulin, were found to increase the rate of lamellar body PC synthesis and increase the molar ratio of surfactant PG to PI to a value similar to that of surfactant secreted by the human fetal lung at term. Recognition of the potential importance of the surfactant proteins SP-A, SP-B, and SP-C in the reduction of alveolar surface tension and in endocytosis and reutilization of secreted surfactant by type II cells has stimulated rapid advancement of knowledge concerning the structures of these proteins and their genes, as well as their developmental and hormonal regulation in fetal lung tissue. The genes encoding the surfactant proteins are expressed in a lung-specific manner and appear to be regulated independently during fetal development. SP-A gene expression is initiated in fetal lung tissue after 75-85% of gestation is completed in all mammalian species studied to date. In the human fetus, however, expression of the SP-B and SP-C genes is detectable prior to mid-gestation. In situ hybridization studies of human lung tissue indicate that the SP-A gene is expressed only in type II cells, whereas SP-B gene expression is detectable in bronchioalveolar epithelial cells as well. Cyclic AMP and glucocorticoids have pronounced effects on the regulation of SP-A gene expression in human and rabbit fetal lung in culture. In human fetal lung in vitro, the effects of cAMP are primarily at the level of gene transcription. By contrast, glucocorticoids have stimulatory effects on SP-A gene transcription and inhibitory effects on SP-A mRNA stability. Furthermore, the combined effects of cAMP and glucocorticoids on SP-A gene transcription in human fetal lung in vitro are synergistic. Glucocorticoids appear to be of primary importance in the regulation of the genes encoding SP-B and SP-C. Elucidation of the molecular mechanisms involved in the regulation of expression of the surfactant protein genes in developing fetal lung will be of fundamental importance to our understanding of the developmental and tissue-specific regulation of eukaryotic gene expression.

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

Pulmonary surfactant, a unique developmentally regulated, phospholipid-rich lipoprotein, is synthesized by the type II cells of the pulmonary alveolus, where it is stored in organelles termed lamellar bodies. The principal surface-active component of surfactant, dipalmitoylphosphatidylcholine, a disaturated form of phosphatidylcholine, acts in concert with the surfactant-associated proteins to reduce alveolar surface tension. Relatively large amounts of phosphatidylglycerol also are present in lung surfactants of a number of species, including man. The role of phosphatidylglycerol in surfactant function has not been elucidated; however, its presence in increased amounts in pulmonary surfactant is correlated with enhanced fetal lung maturity. Surfactant glycerophospholipid synthesis in fetal lung tissue is regulated by a number of hormones and factors, including glucocorticoids, prolactin, insulin, oestrogens, androgens, thyroid hormones, and catecholamines acting through cyclic AMP. In studies with human fetal lung in organ culture, we have observed that glucocorticoids, in combination with prolactin and/or insulin, increase the rate of lamellar body phosphatidylcholine synthesis and alter lamellar body glycerophospholipid composition to one reflective of surfactant secreted by the human fetal lung at term. Four surfactant-associated proteins, SP-A, SP-B, SP-C and SP-D, have recently been characterized. Recognition of their potential importance in the reduction of alveolar surface tension and in endocytosis and reutilization of secreted surfactant by type II cells has stimulated rapid advancement of knowledge concerning the structures of the surfactant proteins and their genes, as well as their developmental and hormonal regulation in fetal lung tissue. The genes encoding SP-A, SP-B and SP-C are expressed in a cell-specific manner and are independently regulated in fetal lung tissue during development. SP-A gene expression occurs exclusively in the type II cell and is initiated after 75% of gestation is complete. In the human fetus, expression of the SP-B and SP-C genes is detectable much earlier in development than SP-A, before the time of appearance of differentiated type II cells. It is apparent from studies using human and rabbit fetal lung in culture that cyclic AMP and glucocorticoids serve important roles in the regulation of SP-A gene expression. While the effects of cyclic AMP are exerted primarily at the level of gene transcription in human fetal lung tissue, glucocorticoids have stimulatory effects on SP-A gene transcription and inhibitory effects on SP-A mRNA stability. In addition, cyclic AMP and glucocorticoids act synergistically to increase SP-A gene transcription in human fetal lung in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of pulmonary surfactant protein synthesis in fetal lung: a major role of glucocorticoids and cyclic AMP.

Augmented synthesis of the lipoprotein, pulmonary surfactant, is initiated in fetal lung toward the end of-gestation. Inadequate surfactant synthesis by the lungs of premature infants can result in respiratory distress syndrome, the leading cause of neonatal morbidity and mortality in developed countries. The surfactant-associated proteins act with surfactant glycerophospholipids to reduce alveolar surface tension, and mediate the reutilization of secreted surfactant components by type II cells. Genes encoding the surfactant proteins SP-A, SP-B, and SP-C have been isolated and characterized. Recent findings suggest that surfactant protein gene expression in fetal lung is under multifactortal control and is regulated by glucocorticoids, cAMP, growth factors, and insulin.