Surfactant Protein mRNA Research Articles

Nitric oxide decreases surfactant protein A gene expression in H441 cells.

To study the effects of nitric oxide (NO) on surfactant protein A (SP-A) gene expression. In vitro study. A human lung tumor cell line (H441) representative of distal respiratory epithelium. Cells were treated with the NO donor S-nitroso-N-acetyl penicillamine (SNAP) at concentrations ranging from 0.3 to 3.0 mM for 24 hrs. Northern blot analyses using a radiolabeled cDNA probe for human SP-A demonstrated that SNAP modestly (approximately 30%) decreased SP-A mRNA expression in a dose-dependent manner. Western blot analyses using a polyclonal anti-human SP-A antibody demonstrated that SNAP also decreased SP-A peptide expression. mRNA stability assays demonstrated that SNAP did not affect the half-life of SP-A mRNA. Cell viability assays demonstrated that SNAP slightly decreased cell viability compared with control cells. There were no significant differences in cell viability among cells treated with the different concentrations of SNAP. NO decreases in vitro SP-A gene expression by approximately 30% in a human lung tumor cell line representative of distal respiratory epithelium. This effect does not occur at the posttranscriptional level and cannot be entirely accounted for by changes in cell viability. The inhibitory effect of NO demonstrated in this study is of relatively small magnitude and it is therefore difficult to make strong conclusions regarding biological relevance. However, these data, coupled with previous data demonstrating that NO negatively affects surfactant function, suggest that NO has the potential to negatively impact surfactant homeostasis.

Regulation of expression of human SP-A1 and SP-A2 genes in fetal lung explant culture

Human pulmonary surfactant protein A (SP-A) is genetically complex and its regulation may also be complex, reflecting genotypic variability. Fetal lung explants were used to study the regulation of the SP-A genes, SP-A1 and SP-A2, by dexamethasone, interferon γ (IFN γ), cyclic 3′,-5′ adenosine monophosphate (cAMP), and tumor necrosis factor α (TNF α). For comparison, the mRNA levels of surfactant protein B (SP-B) and its response to test substances were also examined. Results showed: (a) In control culture total SP-A mRNA varied widely among explants (C.V.=0.70) compared with SP-B (C.V.=0.26) (b) IFN γ significantly increased total SP-A mRNA but there were marked differences among fetal lungs in response to all treatments. (c) SP-A1 mRNA concentration is higher than SP-A2 in both control and treated explants. (d) SP-A1 alleles are inhibited to a greater degree by dexamethasone than SP-A2 alleles. The relative effect of cAMP and IFN γ on SP-A1 and SP-A2 mRNA varied widely among explants. We conclude that SP-A genotype may account in part for the marked differences in SP-A mRNA concentration among fetal lungs and that the SP-A genes and/or alleles may be differentially regulated.

Developmental and Glucocorticoid Regulation of Surfactant Protein mRNAs in Fetal Sheep † 310

We previously found that glucocorticoid treatment of pregnant sheep increases the content of surfactant proteins SP-A and SP-B in lung tissue and lavage of preterm lambs. To investigate the mechanism of this induction process, we quantitated SP mRNA levels after administration of betamethasone(0.5 mg/kg) to pregnant sheep prior to premature delivery of the fetus at 125 days. In the first protocol, 55 sheep were injected weekly with 1-4 doses of either saline (control) or betamethasone beginning at 104 days gestation with the last dose given 24 h prior to delivery. In a second protocol, 39 sheep were injected with 1-2 doses of saline or betamethasone at 24 and 48 h prior to delivery. Total RNA was extracted from fetal lung and hybridized with cDNAs for sheep SP-A, SP-B and SP-C and human β-actin. mRNA levels for control preterm lambs were 21%, 28% and 39% of the level in term lambs for SP-A, -B and -C, respectively. No increases in mRNA levels were demonstrated in sheep given 1-3 weekly doses of betamethasone vs. control. However, 4 doses of betamethasone, as well as both 48h treatment regimens, produced a 2- to 3-fold increase in each SP mRNA (p<0.01 by ANOVA), achieving levels equivalent to 60-75% of term control lambs. The magnitude of the betamethasone-induced increase in SP mRNA is similar to that for tissue SP-A and SP-B after 2-4 betamethasone doses. The observation that SP-A and SP-B proteins are increased 2 wk after betamethasone treatment, whereas mRNAs are not, implies that induction occurs with 2 doses, is reversible, and that the proteins have a longer half-life than the mRNAs. We conclude that in vivo betamethasone treatment rapidly induces a maximal and coordinated increase in SP-A, SP-B, and SP-C mRNAs which is fully reversible within 7 days. Similar increases in SP mRNA and protein content confirm that glucocorticoid regulation of SP genes in vivo is largely transcriptional.

Mechanism of all trans-retinoic acid and glucocorticoid regulation of surfactant protein mRNA.

The surfactant proteins (SPs) are required for the normal function of pulmonary surfactant, a lipoprotein substance that prevents alveolar collapse at end expiration. We characterized the effects of cortisol and all trans-retinoic acid (RA) on SP-A and SP-B gene expression in H441 cells, a human pulmonary adenocarcinoma cell line. Cortisol, at 10(-6) M, caused a significant inhibition of SP-A mRNA to levels that were 60-70% of controls and a five- to sixfold increase in the levels of SP-B mRNA. RA alone (10(-6) M) had no effect on SP-A mRNA levels and modestly reduced the inhibitory effect of cortisol. RA alone and the combination of cortisol and RA both significantly increased SP-B mRNA levels. RA had no effect on the rate of SP-A gene transcription or on SP-A mRNA stability. Cortisol alone and the combination of cortisol and RA significantly inhibited the rate of SP-A gene transcription but had no effect on SP-A mRNA half-life. RA at 10(-6) M had no effect on the rate of SP-B gene transcription but prolonged SP-B mRNA half-life. Cortisol alone and the combination of cortisol and RA caused a significant increase in the rate of SP-B gene transcription and also caused a significant increase in SP-B mRNA stability. We conclude that RA has no effect on SP-A gene expression and increases SP-B mRNA levels by an effect on SP-B mRNA stability and not on the rate of SP-B gene transcription. In addition, the effects of the combination of RA and cortisol were generally similar to those of cortisol alone.

Site specificity of surfactant protein expression in airways of baboons during gestation.

There are disparate reports concerning the presence of surfactant proteins in the airways of lung. The recent finding of SP-A in tracheobronchial epithelium and submucosal glands in lungs from second trimester humans has renewed interest in potential new functions of surfactant in lung biology. In situ hybridization studies were done to determine the distribution of SP-A, SP-B, and SP-C in baboon lung specimens from 60, 90, 120, 140, 160, and 180 (term) days of gestation and adults. Lungs from gestation controls were obtained at the time of hysterotomy and adult lungs at necropsy. Riboprobes used for in situ hybridization contained the entire coding regions for human SP-A, SP-B, and SP-C. At 60 days, SP-C mRNA expression was evident in focal portions of primitive tubular epithelium but not bronchi. This distal pattern of SP-C mRNA expression persisted and was present in some epithelial cells of respiratory bronchioles at term. At 90 days, SP-A mRNA expression was present in the epithelium of trachea and large bronchi. SP-B mRNA expression was found in small bronchi, bronchioles, and distal tubular epithelium at 120 days of gestation. SP-A mRNA bronchiolar localization became evident at 140 days of gestation and alveolar type 2 cellular expression at 160 days of gestation. Abrupt transitions of surfactant protein expression were identified (e.g., SP-A mRNA-positive cells in the epithelium of large bronchi with adjoining SP-B mRNA expression in small bronchi and bronchioles). Findings in the baboon indicate that there are well-delineated sites of surfactant protein mRNA expression in bronchial and bronchiolar epithelia. mRNA expressions of SP-A and SP-B are present in both bronchial and bronchiolar epithelium but at different sites, whereas SP-C expression is seen in loci of epithelial cells in respiratory bronchioles.

Differential regulation of SP-A1 and SP-A2 genes by cAMP, glucocorticoids, and insulin.

In the human fetal lung, surfactant protein A (SP-A) is encoded by two highly similar genes, SP-A1 and SP-A2, which are developmentally and hormonally regulated. Using primer extension analysis, we evaluated the levels of SP-A1 and SP-A2 mRNA transcripts in human fetal lung explants and in a human adult lung adenocarcinoma cell line (H441 cells) cultured in the absence or presence of either dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP, 1 mM), dexamethasone (10(-7) M), or insulin (2.5 micrograms/ml). In the human fetal lung explants, the content of SP-A1 mRNA was approximately four times that of SP-A2 mRNA. DBcAMP increased SP-A1 mRNA levels by 100% and SP-A2 mRNA levels by 500%, thus reducing the ratio of SP-A1 mRNA to SP-A2 mRNA to approximately 1:1. Dexamethasone inhibited all of the SP-A1 and SP-A2 mRNA transcripts to the same extent, by approximately 70%, whereas insulin inhibited all SP-A mRNA transcripts by approximately 60%. The ratio of SP-A1 to SP-A2 mRNA in dexamethasone- or insulin-treated explants was the same as the ratio observed in controls. In the H441 cells, SP-A1 mRNA levels were approximately 1.5 times that of SP-A2 mRNA levels. DBcAMP increased both SP-A1 and SP-A2 mRNA levels by 100%. Dexamethasone inhibited SP-A1 mRNA levels in the cell line by 60%, whereas SP-A2 mRNA levels were not significantly affected. Insulin inhibited SP-A1 mRNA levels in the cell line by 40% without affecting SP-A2 mRNA levels. These findings suggest that the two human SP-A genes are regulated differently in the two model systems.

KGF increases SP-A and SP-D mRNA levels and secretion in cultured rat alveolar type II cells.

Studies of secretion of surfactant proteins by alveolar type II cells have been limited because the expression of the genes for these proteins decreases rapidly in primary culture. We developed a culture system to investigate the regulation of lipid and protein secretion by alveolar type II cells and the genes involved in these processes. Rat type II cells were plated on membrane inserts coated with rat-tail collagen in medium containing 10% fetal bovine serum (FBS) for 1 d before being changed to medium containing 5 ng/ml keratinocyte growth factor (KGF) and 2% serum for 3 d and to medium with 5% Engelbreth-Holm-Swarm tumor matrix (EHS) but without serum for 2 d. From this time forward, the cells were placed on a rocking platform and cultured with 0.4 ml medium on the apical surface at the air-liquid interface (A/L) in four different, serum-free media: basal Dulbecco's modified Eagle's medium (DMEM)/F12 medium (DF12), basal medium plus EHS (DF12/EHS), basal medium plus KGF (DF12/KGF), and basal medium plus EHS and KGF (DF12/EHS/KGF). Cells cultured in DF12 and DF12/EHS assumed an attenuated, flattened morphology, whereas those in DF12/KGF and DF12/EHS/KGF were more cuboidal, contained numerous lamellar bodies, and had apical microvilli. Cells cultured in DF12 and DF12/EHS produced a relatively weak signal for the surfactant protein mRNAs (surfactant proteins [SP]-A, SP-B, SP-C, and SP-D, respectively), and secretion of SP-A and SP-D remained low. In contrast, cells maintained for 3 d at A/L and cultured in the presence of KGF showed strong signals for SP-A, SP-B, and SP-D mRNAs, and secreted SP-A, SP-D, and lysozyme into the apical medium. The combination of 12-O-tetradecanoyl-phorbol-11-acetate (TPA) and terbutaline stimulated secretion of [3H]phosphatidylcholine ([3H]PC), SP-A, and lysozyme, but not SP-D. This primary culture system should prove useful for mechanistic studies of the secretion of SP-A, SP-D, and surfactant lipids.

Influence of the cytoskeleton on surfactant protein gene expression in cultured rat alveolar type II cells.

We have investigated the role of the cytoskeleton in surfactant protein gene expression. Cytochalasin D (CD), colchicine (Col), or nocodazole (Noco) were tested on primary cultures of adult rat alveolar type II cells. Treatment with any of the drugs did not result in dramatic cell shape changes, but ultrastructural examination revealed that the cytoplasm of cells treated with CD was markedly disorganized; cells treated with Col did not exhibit such changes. Treatment with any of the three drugs resulted in a reduction in surfactant protein (SP) mRNAs. These decreases were not the result of cell toxicity, since overall protein synthesis was unimpaired by drug treatment. Washing the cells followed by an additional 2 days of culture resulted in a reaccumulation of SP mRNAs in CD-treated cells but not in Col-treated cells. Washing of Noco-treated cultures resulted in partial recovery. SP mRNA stability was estimated in the presence or absence of cytoskeleton-disrupting drugs. Disruption of either microfilaments or microtubules significantly affected the half-lives of mRNAs for SP-A, SP-B, and SP-C. These data support a role for the cytoskeleton in the maintenance of type II cell differentiation and suggest that the role of the cytoskeleton is at least in part to stabilize SP mRNAs.

Differential Effects in Vivo of Thyroid Hormone on the Expression of Surfactant Phospholipid, Surfactant Protein mRNA and Antioxidant Enzyme mRNA in Fetal Rat Lung

Antenatal administration of triiodo-L-thyronine (T3) to late gestation rats resulted in decreased lung antioxidant enzyme (AOE) activity but increased surfactant phospholipids. In fetal rat lung explant cultures, T3 decreased the expression of surfactant proteins (SP) A and B. There have been no reported studies of the simultaneous in vivo developmental influence of T3 on both pulmonary AOE and SP gene expression. We hypothesized that antenatal T3 treatment would cause differential regulation of surfactant phospholipid, SP, and AOE genes in the late gestation fetal rat. Timed pregnant rats received intramuscular injections of either T3 (7 mg/kg) or placebo on days 19 and 20 of gestation and fetuses were delivered on day 21. Fetal lung SP-A, SP-B, SP-C, and AOE mRNA levels were studied by Northern analysis. AOE mRNA levels were further quantitated by solution hybridization. Total lung phospholipids (TPL) and disaturated phosphatidylcholine (DSPC) content were quantitated by a phosphorus assay. T3 significantly increased TPL and DSPC content, and significantly decreased the expression of SP-A, SP-C, CuZnSOD, and catalase genes. Because of a crucial interplay of these factors for normal lung function at the time of birth, the molecular mechanisms by which these apparently opposing changes are accomplished warrant further investigation

Expression of hydrophilic surfactant proteins by mesentery cells in rat and man.

Human peritoneal dialysis effluent (PDE) contains a phosphatidylcholine-rich compound similar to the surfactant that lines lung alveoli. This material is secreted by mesothelial cells. Lung surfactant is also characterized by four proteins essential to its function. After having long been considered as lung-specific, some of them have been found in gastric and intestinal epithelial cells. To explore further the similarity between lung and peritoneal surfactants, we investigated whether mesothelial cells also produce surfactant proteins. We used rat transparent mesentery, human visceral peritoneum biopsies and PDE. Surfactant proteins were searched for after one- and two-dimensional SDS/PAGE and Western blotting. On a one-dimensional Western blot, bands at 38 and 66 kDa in rat mesentery, and at 38 and 66 kDa in human peritoneal mesothelial cells (in vivo and in vitro) and PDE, corresponded to monomeric and dimeric forms of lung surfactant protein A (SP-A). On two-dimensional Western blots, the 32 and 38 kDa spots in mesentery and PDE localized at the acidic pH appropriate to the SP-A monomer's isoelectric point. SP-D was also identified at the same 43 kDa molecular mass as in lung. SP-B was not detected in mesenteric samples. Expression of SP mRNA species was also assessed by reverse transcriptase-PCR, which was performed with specific primers of surfactant protein cDNA sequences. With primers of SP-A and SP-D, DNA fragments of the same size were amplified in lung and mesentery, indicating the presence of SP-A and SP-D mRNA species. These fragments were labelled by appropriate probes in a Southern blot. No amplification was obtained for SP-B. These results show that mesentery cells produce SP-A and SP-D, although they are of embryonic origin (mesodermal) and are different from those of the lung and digestive tract (endodermal) that secrete these surfactants.

Maintenance of the differentiated type II cell phenotype by culture with an apical air surface.

The study of differentiated functions of alveolar type II cells has been hampered because of the lack of good in vitro systems. We report that culture of type II cells on collagen gels with an apical surface exposed to air promotes expression of differentiated type II cell characteristics. Cells cultured in this manner are cuboidal, contain lamellar bodies, and produce tubular myelin; in addition, they secrete phosphatidylcholine in response to exogenous ATP. Cultures contain mRNA for surfactant proteins A, B, and C and surfactant proteins A, B, and D. In contrast, when type II cells are cultured with an apical surface exposed to liquid rather than to air, the cells are squamous, do not express surfactant proteins or their respective mRNA, and do not contain lamellar bodies or produce tubular myelin. Type II cells cultured on plastic for 7 days, which no longer express mRNA for surfactant proteins, can be induced to express these mRNA by changing culture conditions to that of an air surface. The culture system described in this paper should be useful for studies of surfactant metabolism, regulation of alveolar epithelial phenotypic expression, and the processing of transiently expressed transgenes.

Clearance of intra-amniotic lung surfactant: uptake and utilization by the fetal rabbit lung.

To investigate the metabolism of intra-amniotic surfactant, surfactant containing double-labeled dipalmitoylphosphatidylcholine (DPPC) was injected in amniotic fluid on days 23-27 of gestation. Within 44 h, DPPC was distributed to the gastrointestinal tract (45.9%), fetal membranes and placenta (8.2%), fetal lung (6.6%), and liver (1.9%). DPPC uptake was higher in the upper than in the lower lung lobes. The mixture of phosphatidylglycerol and DPPC increased the uptake of DPPC that was not saturable (range 15-60 mg phospholipid). There was no detectable metabolism of DPPC taken up by the fetal lung. Surfactant protein A, originating from intra-amniotic heterplogous surfactant, was detected immunohistochemically in alveolar epithelium. Intra-amniotic surfactants did not affect the expression of surfactant protein mRNAs. Intra-amniotic surfactant (1,500-2,000 mg/kg on day 25.3) improved lung compliance of ventilated 27.0-day premature rabbits less than intratracheal surfactant at birth (75-100 mg/kg). Reutilization by the alveolar epithelium of surfactant secreted to future airspaces, airways, and amniotic fluid may be a mechanism that increases intracellular surfactant pool before birth.

Intraamniotic interleukin-1 accelerates surfactant protein synthesis in fetal rabbits and improves lung stability after premature birth.

Intraamniotic infection is associated with increased IL-1 activity in amniotic fluid, increased incidence of preterm labor, and with decreased incidence of respiratory distress syndrome in infants born prematurely. We hypothesized that an elevated IL-1 in amniotic fluid promotes fetal lung maturation. On day 23 or 25 of gestation (term 31 d), either IL-1alpha (150 or 1,500 ng per fetus) or its antagonist IL-1 receptor antagonist (IL-1ra, 20 microg) was injected to the amniotic fluid sacs in one uterine horn, whereas the contralateral amniotic sacs were injected with vehicle. Within 40 h, IL-1alpha caused a dose-dependent increase in surfactant protein-A (SP-A) and SP-B mRNAs (maximally, fivefold), without affecting lung growth or increasing inflammatory cells in the lung. Both genders, and upper and lower lung lobes were similarly affected. IL-1ra did not modify SP-A, -B, or -C mRNA. IL-1 increased the intensity of staining of alveolar type II cells for SP-B, and the concentrations of SP-B, -A, and disaturated phosphatidylcholine in bronchoalveolar lavage. The dynamic lung compliance and the postventilatory expansion of lungs were increased two- to fourfold after IL-1alpha treatment. In fetal lung explants, IL-1alpha increased the expression of SP-A mRNA. IL-1 in amniotic fluid in preterm labor may promote lung maturation and thus be part of a host-defense mechanism that prepares the fetus for extrauterine life.

Hyperoxia suppresses maturational increases in surfactant protein mRNA accumulation in male and female. newborn rats. 1572

The lung surfactant system matures faster in females than in males in late gestation. We sought to determine whether hyperoxia in newborn rats influenced effects of maturation and sex on surfactant protein (SP) mRNA accumulation. Methods: newborn rat pups (8 litters) were exposed to air or 95% oxygen(O2) for 1, 4, and 8 days (d), anesthetized, sexed, had lungs removed and frozen. Total lung RNA was extracted and analyzed by Northem blots probed with 32P labeled SP-A, B, and C cDNA. Results: SP-A mRNA: signal increased in air and O2 treated pups with age, but air exposed females showed greater abundance than males at 8 d, while O2 exposed males and females showed similar, but lower levels. SP-B mRNA: signal increased from 1 d to 4 d in air exposed females, and by 8 d declined to 1 d levels. With O2 the rise was absent at 4 d. In males, age had no effect on signal, but O2 led to a decline by 8 d. SP-C mRNA: in contrast to SP-A and B, signal in air treated males decreased at 4 d but by 8 d exceeded levels at 1 d. O2 eliminated the 8 d increase. Air treated female signal at 1 d exceeded male, but declined at 4 and 8 d. O2 had no effect. Effects of maturation, sex, and O2 exposure were noncoordinate among the surfactant protein mRNAs. In general the female pups showed greater surfactant protein mRNA accumulation than males at 1 d and with age, but this was reduced by treatment with O2. Effects of O2 on SP mRNA in male pups were less marked. The suppressive effects of O2 on the maturational increases in surfactant protein mRNAs may have unexpected adverse effects on lung function in newborns, particularly females.

Study on the Mechanlsm of Interleukin-1 Induced Increase in Surfactant Protein A. † 237

Injection of interleukin-1α (IL- 1α) into the amniotic fluid accelerates the maturation of the surfactant system, improves the lung stability after premature birth, and upregulates the expression of surfactant protein A (SP-A) and SP-B. According to preliminary study, IL-1α increases SP-A mRNA in explants of the immature lung (Pediatr Res 37:61A, 1995). The aim of the present study was to investigate the mechanism of the IL-1 induced increase in SP-A mRNA.

IL-10 counteracts IL-1-induced surfactant protein A (SP-A) expression in rabbit lung explants. † 1472

The production of the proinflammatory cytokine IL-1 is increased in the fetal compartment in cases of preterm labor associated with intrauterine infection. The production of EGF, on the other hand, increases with advancing gestation. Both IL-1 and EGF upregulate the expression of SP-A. However, IL-1 may also have deleterious effects on the developing lung, since an elevated IL-1 concentration in lung effluent is associated with the development of chronic lung disease in the newborn. IL-10 is an anti-inflammatory cytokine that inhibits the production of IL-1 and of other proinflammatory cytokines.Aim. We investigated 1) whether IL-10 affects SP-A expression in lung explants and 2) whether IL-10 modulates SP-A expression in explants treated with IL-1 or EGF. Methods. Fetal rabbit lung explants(gestational age 22 days) were treated with IL-1 (570 ng/ml), EGF (50 ng/ml) or IL-10 (5 ng/ml) for 24 or 72 hours. Expression of SP-A mRNA was quantitated by Northern blotting. Results. SP-A expression at 24 and 72 h as percent of the respective controls (±SE) was:Table Thus, IL-1 and EGF enhanced SP-A expression, but did not potentiate each other's effects. IL-10 alone had no effect on SP-A expression. IL-10 suppressed the stimulatory effect of IL-1 in explants treated for 72 h, but not in those treated for 24 h. IL-10 did not suppress SP-A expression in EGF-treated explants. Conclusion. IL-10 selectively counteracts the effect of IL-1 but not that of EGF on SP-A expression. IL-10 may be of value in inhibiting lung inflammation caused by proinflammatory cytokines. The potential of IL-10 in preventing inflammation in the lungs without affecting maturational events unrelated to inflammation, should be further investigated.

Inhibition of tyrosine kinase activity decreases expression of surfactant protein A in a human lung adenocarcinoma cell line independent of epidermal growth factor receptor

Epidermal growth factor (EGF) enhances fetal lung development in vivo and in vitro. Ligand binding to the EGF receptor stimulates an intrinsic receptor tyrosine kinase initiating a signal transduction cascade. We hypothesized that blocking EGF receptor function with tyrosine kinase inhibitors would decrease the expression of surfactant protein A in human pulmonary epithelial cells. Human pulmonary adenocarcinoma cells (NCI-H441) were exposed to genistein (a broad range inhibitor of tyrosine kinases) and tyrphostin AG1478 (a specific inhibitor of EGF receptor tyrosine kinase). Genistein significantly decreased surfactant protein A (SP-A) and SP-A mRNA levels in H441 cells without affecting cell viability. The inhibitory effect of genistein on SP-A content was reversible. In contrast, tyrphostin AG1478 had no effect on SP-A levels despite a greater inhibitory effect than genistein on EGF receptor tyrosine autophosphorylation. Furthermore, treatment of H441 cells with exogenous EGF did not increase SP-A content or mRNA levels beyond baseline. We conclude that inhibition of tyrosine kinase activity other than the EGF receptor decreases the expression of surfactant protein A at a pretranslational level in human pulmonary adenocarcinoma cells. These results suggest the importance of tyrosine kinases in modulating human SP-A synthesis.

Effect of genotype on the levels of surfactant protein A mRNA and on the SP-A2 splice variants in adult humans.

Human pulmonary surfactant protein A (SP-A) is encoded by two genes, SP-A1 and SP-A2, that exhibit coding sequence (allelic) and 5' splicing variability. In this report we determine the effect of the genetic variability within the SP-A1 and SP-A2 genes on the level of SP-A mRNAs and on the SP-A2 splicing variants in different individuals. We analysed mRNA specimens from 23 unrelated adults using genotype analysis, Northern analysis and primer extension, and made the following observations. (1) The level of SP-A mRNA varies among individuals (coefficient of variation = 0.49). One SP-A genotype (6A(2)6A(2)1A(0)1A0) appears to be associated with a low to moderate level of SP-A mRNA. (2) The SP-A1/SP-A2 mRNA ratio varies among individuals, from 0.94 (lowest) to 6.80 (highest) within the study population. One genotype appears to be associated with a moderate to high SP-A1/SP-A2 mRNA ratio and another with a low to moderate ratio. (3) There is no correlation between the level of SP-A mRNA and the SP-A1/SP-A2 mRNA ratio. (4) Variability in the ratio of the major SP-A2 splice variants among individuals results from nucleotide differences in the splice-recognition sequence of specific SP-A2 alleles. The SP-A mRNA levels, the SP-A1/SP-A2 mRNA ratio, and the ratio of the major SP-A2 splice variants have a genetic basis in that they vary depending upon the specific SP-A alleles present.

Retinoic acid increases surfactant protein mRNA in fetal rat lung in culture.

Retinoic acid has both early or immediate (within hours) and late (after days) effects on gene expression. We studied the early effects of retinoic acid on the surfactant protein (SP) genes. Exposure of fetal rat lung explants to all trans-retinoic acid for 4 h resulted in a significant dose-dependent increase in SP-A, -B, and -C mRNA with markedly different dose-response characteristics. The maximal (2.5x) increase in SP-A mRNA was observed with 10(-10) M retinoic acid, whereas treatment with 10(-5) M resulted in a tendency to decreased levels. In contrast, maximal stimulation of SP-C (6x) was noted at 10(-5) M retinoic acid and that of SP-B (2x) at 10(-7) to 10(-5) M retinoic acid. Similar differences in the dose-response characteristics of SP-A and SP-C were observed with 9-cis-retinoic acid. A retinoic acid response element consensus sequence was identified in the rat SP-A gene; we hypothesize that retinoic acid-receptor complexes act directly on the SP-A gene via this response element.

Developmental and hormonal regulation of SP-A gene expression in baboon fetal lung.

In the present study, we found that surfactant protein A (SP-A) mRNA levels, which are barely detectable in baboon fetal lung at midgestation (92 days), are increased approximately four fold between 125 and 140 days gestation, approximately 7-fold between 140 and 160 days, and approximately 1.5-fold between 160 and 174 days gestation. We also investigated the effects of dibutyryl-adenosine 3',5'-cyclic monophosphate (DB-cAMP) and dexamethasone (Dex) on SP-A gene expression in lung explants from fetal baboons at 92, 125, 140, 160, and 174 days of gestation (term = 184 days). SP-A mRNA levels, which were barely detectable in lung tissues from 92- and 125-day fetal baboons before culture, were induced after incubation for 5 days in serum-free medium and were markedly stimulated by DBcAMP. Dex caused a dose-dependent inhibition of SP-A mRNA levels and antagonized the stimulatory effect of DBcAMP. SP-A mRNA was detectable in lung tissues from 140-day fetal baboons before culture; the levels were further induced after culture and were increased greatly by DBcAMP. Again, Dex antagonized the induction of SP-A mRNA by DBcAMP. The stimulatory effects of DBcAMP and inhibitory effects of Dex on SP-A mRNA levels in lung tissues of 92- to 140-day gestational age fetal baboons were highly similar to those observed in studies using lung explants of midgestation human abortuses. By contrast, SP-A mRNA was present in relatively high levels in lung tissues of 160- and 174-day fetal baboons before culture and was relatively unaffected after incubation for 5 days in control medium. In lung explants from 160- and 174-day fetal baboons, the stimulatory effect of DBcAMP and inhibitory effect of Dex on SP-A mRNA levels were relatively modest compared with the effects of these agents on SP-A mRNA in fetal lung tissues from 92-, 125-, and 140-day gestational age fetuses. These findings suggest that, with increased lung maturation and the developmental induction of SP-A gene expression, there is a decrease in responsiveness of the fetal lung to the stimulatory effects of cAMP and inhibitory effects of glucocorticoids on SP-A gene expression.