Human Fetal Lung Explants Research Articles

Effect of tyrosine kinase inhibition on surfactant protein A gene expression during human lung development

Epidermal growth factor (EGF) stimulates surfactant protein (SP) A synthesis in human fetal lung explants. Ligand binding to the EGF receptor stimulates an intrinsic receptor tyrosine kinase with subsequent activation of second messengers. We hypothesized that inhibition of EGF-receptor tyrosine kinase activity would block SP-A expression in spontaneously differentiating cultured human fetal lung tissue. Midtrimester fetal lung explants were exposed for 4 days to genistein (a broad-range inhibitor of tyrosine kinases) and tyrphostin AG-1478 (a specific inhibitor of EGF-receptor tyrosine kinase). Genistein significantly decreased SP-A and SP-A mRNA levels without affecting either tissue viability or the morphological differentiation of alveolar type II cells. Tyrphostin AG-1478 also decreased SP-A content and SP-A mRNA levels in cultured fetal lung explants. Treatment with EGF could not overcome the inhibitory effects of either genistein or tyrphostin on SP-A; however, only tyrphostin inhibited EGF-receptor tyrosine phosphorylation. We conclude that specific inhibition of EGF-receptor tyrosine kinase with tyrphostin AG-1478 blocks the expression of SP-A during spontaneous differentiation of cultured human fetal lung tissue. Furthermore, exposure to genistein also decreases SP-A expression and blocks the effects of EGF in human fetal lung tissue without inhibiting EGF-receptor tyrosine phosphorylation. These findings support the importance of tyrosine kinase-dependent signal transduction pathways in the regulation of SP-A during human fetal lung development.

Angiotensin II Type 1 (AT1) Receptor Expression and Regulation in Human Fetal Lung In Vitro 305

Specific angiotensin II (ANG II) receptors are present early in gestation and may play a role in organ growth and development. To investigate regulation of the AT1 receptor in human lung, we first characterized changes in AT1 mRNA levels in midtrimester human fetal lung explants, which spontaneously differentiate in culture. Northern blot analysis revealed that during four days in explant culture in serum-free medium, AT1 mRNA levels decreased to 19±3, 10±3, 8±2 and 5±2% (days 1, 2, 3, 4, respectively, all p<0.05) of control levels (day 0). The decrease in AT1 mRNA after 48 h in culture also occurred in the presence of 5% fetal calf serum. Immunostaining demonstrated a mesenchymal distribution of the AT1 receptor with limited expression in vascular smooth muscle. AT1 mRNA levels were unaltered after 48 h in culture with ANG II (10-5 to 10-9 M), the AT1 receptor antagonist losartan (10-5 to 10-9 M), or cortisol (10-6 and 10-9 M). Finally, abundance of AT1 mRNA was similarly decreased by incubation in 2% or 20% oxygen. These findings indicate that AT1 mRNA and protein are present in midtrimester fetal lung and that AT1 mRNA levels decrease in association with spontaneous differentiation of human fetal lung in vitro. AT1 mRNA in human fetal lung explants is not regulated by ligand, receptor antagonist, cortisol, or oxygen.

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.

Targeting type II and Clara cells for adenovirus-mediated gene transfer using the surfactant protein B promoter.

We examined the ability of the human surfactant protein B (SP-B) promoter to confer cell specificity of transgene expression in an adenoviral vector. Using similar replication-deficient adenoviruses (rAd), we compared lacZ reporter gene expression driven by the human SP-B promoter (rAd.SPBlacZ) with the ubiquitously expressed Rous sarcoma virus promoter (rAd.RSVlacZ). rAd.SPBlacZ expressed lacZ in H-441 and A549 lung epithelial cell lines and not in HeLa cells whereas rAd.RSVlacZ expressed in all three cell lines. In primary human fetal lung fibroblasts, beta-galactosidase activity from rAd.RSVlacZ transduction increased in a dose-dependent manner whereas activity from rAd.SPBlacZ remained low. In mixed cell cultures prepared from human fetal lung explants that contained fibroblasts and type II cells, X-Gal staining localized rAd.SPBlacZ expression to only type II cells whereas rAd.RSVlacZ expressed in both cell types. In 24-wk gestation human fetal tissue explants infected ex vivo, the RSV promoter directed lacZ expression in lung, trachea, heart, liver, and esophagus, whereas with the SP-B promoter lacZ was expressed only in lung, specifically in air space-lining cells. This specificity was maintained in vivo. lacZ expression was undetectable in lung and other tissues after intravenous administration of rAd.SPBlacZ whereas rAd.RSV-lacZ expressed primarily in liver. After intratracheal instillation of rAd.SPBlacZ into mice, X-Gal staining localized expression to type II and Clara cells. In contrast, rAd.RSVlacZ expressed in all pulmonary epithelial cell types. Our results indicate that the SP-B promoter may be useful in targeting type II and Clara cells for gene therapy of conditions such as inherited deficiency of SP-B.

Transcriptional regulation of surfactant proteins SP-A and SP-B by phorbol ester

Phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) activate protein kinase C and have been previously shown to down-regulate surfactant proteins SP-A and SP-B in H441 adenocarcinoma cells. We used H441 cells and human fetal lung to further study the mechanism of TPA action and to examine physiologic relevance. In H441 cells, TPA (10 nM) treatment for 24 h decreased SP-A mRNA content to ∼5% of control cells, with half-maximal effect at ∼0.5 nM, and reduced SP-A gene transcription rate to 28% of control after 8 h exposure. In cells cultured in the presence of dexamethasone, which increases the low basal level of SP-B expression, TPA decreased both SP-B mRNA content (∼8% of control) and rate of transcription (7% of control). In cultured human fetal lung explants, TPA decreased SP-A and SP-B protein and mRNA in a time- and dose-dependent fashion, with half-maximal effect on mRNAs at ∼3 nM and ∼50% inhibition after 24 h of exposure, and similarly reduced SP-A and SP-B gene transcription (∼55% of control at 8–24 h). We conclude that TPA acts primarily at the level of gene transcription to down-regulate both SP-A and SP-B in H441 and fetal lung cells, and we speculate that inflammatory and other agents that act through PKC may modulate expression of the surfactant proteins and alter surfactant function in vivo.

Regulation of surfactant protein gene expression by retinoic acid metabolites.

Surfactant-associated proteins (SP) play an important role in the function of pulmonary surfactant. We have previously shown that SP-B mRNA is increased whereas SP-A and SP-C mRNA are decreased by all-trans-retinoic acid (RA) in a dose-dependent manner in human fetal lung explants. All-trans-RA binds primarily to the retinoic acid receptors (RARs) and 9-cis-RA binds primarily to the retinoid X receptors (RXRs). Because the fetal lung contains RXRs, we hypothesized that 9-cis-RA regulates surfactant protein gene expression in lung epithelial cells. H441 human lung adenocarcinoma cells, which synthesize SP-A and SP-B mRNA and protein, were treated with either all-trans-RA or 9-cis-RA (10(-10) to 10(-6) M) for 24 h. Neither all-trans-RA nor 9-cis-RA had an effect on SP-A mRNA levels in the H441 cells. All-trans-RA (10(-6) M) significantly increased SP-B mRNA levels in the H441 cells and 9-cis-RA had a smaller, not statistically significant effect. Human fetal lung explants were treated with 9-cis-RA for 6 d. 9-cis-RA did not significantly increase SP-B mRNA levels, significantly inhibited SP-A mRNA levels at all concentrations tested, and significantly inhibited SP-C mRNA levels at 10(-6) M in the human fetal lung explants. Both all-trans-RA (10(-6) M) and 9-cis-RA (10(-6) M) significantly increased SP-B protein levels in the human fetal lung explants. Together, these results are suggestive that all-trans-RA directly regulates SP-B gene expression in human pulmonary epithelial cells. In addition, the inhibitory effect of all-trans-RA and 9-cis-RA on SP-A mRNA levels in pulmonary epithelial cells is probably an indirect effect mediated by other cell types present in fetal lung tissue.

Retinoic acid and glucocorticoids inhibit the expression of both surfactant protein A (SPA) genes in human fetal lung explants. † 273

Retinoic acid and glucocorticoids inhibit the expression of both surfactant protein A (SPA) genes in human fetal lung explants. † 273

SURFACTANT PROTEIN-B (SP-B) PROMOTER IN REPLICATION DEFICIENT ADENOVIRUS(rAd) DIRECTS PULMONARY EPITHELIAL CELL-SPECIFIC GENE EXPRESSION. • 1986

Gene therapy using rAd shows promise in treating genetic pulmonary diseases. Inherited deficiency of SP-B, a proteolipid critical to pulmonary surfactant, results in severe respiratory failure in term infants. SP-B is produced by alveolar type II cells and bronchiolar Clara cells and may be harmful within other cells due to its fusogenic properties. A SP-B promoter(SPB; -640/+319 relative to transcription start site) shows lung epithelial cell line specificity in transient transfection of H441 pulmonary adenocarcinoma cells with promoter strength similar to the promiscuous viral RSV promoter. To explore cell-type specific gene therapy for SP-B deficiency, we compared expression of the lacZ reporter gene driven by the RSV or SP-B promoters in rAd. Dose-response and time course experiments in H441 cells demonstrated efficient rAd.SPB.lacZ and rAd.RSV.lacZ infection with 24h of virus exposure at a multiplicity of infection (MOI) of≈1-10 with high-level β-galactosidase activity after 72h. X-gal staining showed specificity of rAd.SPB.lacZ for pulmonary derived cell lines (A549 and H441 versus HeLa) whereas all cell lines stained extensively with rAd.RSV.lacZ. This finding was confirmed by quantitative β-gal assay: the ratios of rAd.SPB.lacZ to rAd.RSV.lacZ β-gal activities(average ± SEM) were A549 = 1.96 ± 0.69; H441 = 0.66 ± 0.13; HeLa = 0.05 ± 0.01; p<0.05; n=3). Human fetal lung explants(24wk gestation) infected with rAd.SPB.lacZ had preferential X-gal staining of airway lining cells versus interstitial cells. Epithelia of explanted trachea showed minimal staining. All lung and trachea explant cell types stained extensively with rAd.RSV.lacZ. rAd.SPB.lacZ infection of mixed cells isolated from lung explants showed staining of most epithelial cells and rare fibroblasts. Primary fibroblasts infected with rAd.SPB.lacZ (MOI 1-300) had very low β-gal activities (7-94 A420/min/mg protein) whereas β-gal activities of fibroblasts infected with rAd.RSV.lacZ increased in a dose-dependent manner (480-55200 A420/min/mg protein). These results indicate that the SP-B promoter in rAd maintains cell line specificity and may be useful for limiting transgene expression to type II and Clara cells. (supported by 5 P30 HD28815)

RETINOIC ACID AND GLUCOCORTICOIDS REGULATE SURFACTANT PROTEIN B GENE EXPRESSION IN HUMAN LUNG EPITHELIAL CELLS. • 337

Surfactant protein B (SP-B) plays a vital role in the pulmonary function of neonates; infants with inherited SP-B deficiency follow a course of progressive and fatal pulmonary failure unless they undergo lung transplantation. In previous studies, we have shown that all-trans retinoic acid (RA), a ligand of the steroid hormone receptor family, increases SP-B mRNA levels in H441 human lung adenocarcinoma cells and in cultured human fetal lung explants. In order to determine the temporal nature of the RA effect on SP-B mRNA, H441 cells were incubated with 10-6M all-trans RA for 1, 2, 4, 8, 12 or 24 hours. The stimulatory effect of all-trans RA on SP-B mRNA levels was observed as early as after 4 hours of exposure to the hormone. We also found that other retinoids, including 9-cis retinoic acid, 13-cis retinoic acid and retinol, do not affect SP-B mRNA levels in the H441 cell line. Glucocorticoids have been shown to increase SP-B mRNA levels in H441 cells and in cultured human fetal lung tissue. We hypothesized that the combination of glucocorticoids and all-trans RA regulates SP-B gene expression in a manner different from the effect of either agent alone. In initial experiments, H441 cells were treated in serum-free medium for 24 hours with cortisol (10-10M to 10-6M) added alone. SP-B mRNA levels were increased compared to control levels in a dose-dependent manner from 1.8 times over control levels in 10-9M cortisol to 4.7 times over control levels in 10-6M cortisol; there was no effect of 10-10M cortisol. H441 cells were then exposed to varying concentrations of cortisol (10-10 M to 10-6M) in the presence of all-trans RA (10-6M). SP-B mRNA levels were further stimulated to 2.8 times control levels in 10-9M cortisol and to 7 times control levels in 10-6M cortisol. At each concentration of cortisol, 10-9M to 10-6M, there was an average 40% increase in the stimulatory effect of cortisol on the level of SP-B mRNA with the addition of 10-6M all-trans RA. These data are suggestive that there is an additive effect of all-trans retinoic acid on cortisol-stimulated surfactant protein B gene expression in human lung epithelial cells.

The combined effects of insulin and cortisol on surfactant protein mRNA levels.

Infants of diabetic mothers are frequently hyperinsulinemic and have an increased incidence of neonatal respiratory distress syndrome, a disease caused by a deficiency in the production of pulmonary surfactant by alveolar type II cells. It has been hypothesized that insulin inhibits fetal lung type II cell differentiation. We have shown previously that insulin inhibits the accumulation of surfactant protein (SP)-A and SP-B mRNA and has no effect on SP-C mRNA levels in human fetal lung tissue maintained in vitro. We hypothesized that treatment with glucocorticoids, which are used clinically to accelerate human fetal lung maturation, would overcome the inhibitory effects of insulin on human fetal lung development. In the present study, human fetal lung explants were maintained in the presence or absence of cortisol added alone, or in insulin plus cortisol added together. Cortisol significantly decreased SP-A mRNA levels by approximately 50% at the 100 nM concentration and significantly increased levels by approximately 20% at the 1 nM concentration. Cortisol increased SP-B and SP-C mRNA levels in a dose-dependent fashion (5- and 45-fold at 100 nM cortisol, respectively). The combination of 1 nM cortisol and insulin resulted in inhibition of mRNA levels for SP-A, SP-B, and SP-C at the high insulin concentrations (approximately 50% inhibition for SP-A and SP-B and approximately 25% inhibition of SP-C mRNA levels, in the presence of 40 pmol/L x 10(-3) insulin). Surprisingly, 100 nM cortisol plus inhibitory concentrations of insulin increased SP-A mRNA levels (2-fold at 40 pmol/L x 10(-3).(ABSTRACT TRUNCATED AT 250 WORDS)

Oxygen modulates surfactant protein mRNA expression and phospholipid production in human fetal lung in vitro

We studied the effect of 20-95% O2 on mRNA levels for the surfactant-associated proteins (SP)-A, SP-B, and SP-C and [3H]choline incorporation into total phosphatidylcholine and type II cell-specific disaturated phosphatidylcholine (DPPC) in human fetal lung in culture. SP-A mRNA levels were increased by 25 and 39% in lung explants incubated in 70 and 95% O2, respectively, compared with levels in tissues incubated in 20% O2. SP-B mRNA levels were unaffected by O2, whereas SP-C mRNA levels were increased by 85, 102, and 115% in atmospheres of 35, 50, and 70% O2, respectively. [3H]choline incorporation into total phosphatidylcholine and DPPC were both increased in human fetal lung explants incubated in increased O2 concentrations compared with tissues incubated in 20% O2. Tissue levels of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) activity were not affected by O2 concentration, implying that the changes observed in SP mRNA levels and [3H]choline incorporation may not be mediated through alterations in PKA enzyme activity. These findings demonstrate that O2 regulates SP mRNA expression and phospholipid production in human fetal lung in vitro. We speculate that surfactant composition and possibly function may be regulated by O2 in human lung.

Localization of epidermal growth factor receptor in alveolar epithelium during human fetal lung development in vitro.

Epidermal growth factor (EGF) enhances alveolar type II cell differentiation. In human fetal lung explants, EGF stimulates surfactant protein A (SP-A) synthesis. This effect may occur through a direct interaction of the ligand on EGF receptors located within distal pulmonary epithelium during alveolar type II cell differentiation. To determine if EGF receptor is present in alveolar epithelium, immunostaining for EGF receptor and in situ hybridization for EGF receptor mRNA were performed in human fetal lung explants undergoing alveolar type II cell differentiation in vitro. After 4 days in culture, EGF receptor immunostaining was present in alveolar epithelium from human fetal lung explants compared to minimal immunostaining in undifferentiated human fetal lung epithelium prior to culture. In situ hybridization revealed increased EGF receptor mRNA in differentiated type II cells from cultured explants, with minimal EGF receptor mRNA detected in undifferentiated epithelium from tissue prior to culture. Immunogold staining revealed EGF receptors on the cytoplasmic membranes of epithelial cells lining the prealveolar ducts in human fetal lung explants after 2 days in culture. Alveolar type II cell differentiation in vitro was confirmed ultrastructurally by the presence of lamellar bodies and biochemically by an increase in SP-A content. Thus, EGF receptor is found in alveolar epithelium during differentiation, which suggests an important role for EGF during human fetal lung development.

Glucocorticoid and cAMP increase fatty acid synthetase mRNA in human fetal lung explants

During late fetal development, synthesis of surfactant phospholipid requires a large supply of fatty acid precursor. Fatty acid synthetase is a regulatory enzyme for de novo fatty acid synthesis in lung as well as other lipogenic tissues. In this study, we report hormonal induction of FAS mRNA in human fetal lung explants (16–23 week gestation) cultured up to 7 days in Waymouth's medium (no serum) supplemented with dexamethasone (Dex, 10 nM) or agents that increase cAMP (8-Br-cAMP, 0.1 mM; isobutylmethylxanthine, 0.1 mM; forskolin, 0.01 mM; PGE 1, 0.01 mM). Exposure of explants to Dex or cAMP agents increased FAS mRNA content by 6 h and maximal stimulation occurred at 72 h for Dex (approx. 3-fold increase) and 24 h for cAMP (approx. 2-fold increase). In the presence of both Dex and cAMP there was a synergistic increase in FAS mRNA content at all times (approx. 11-fold increase at 72 h). Induction of FAS mRNA was specific for steroids with glucocorticoid activity, reversible on removal of hormone, and was half-maximal at 2–3 nM Dex consistent with receptor mediation. Actinomycin D blocked induction by Dex but not by cAMP suggesting a transcriptional effect by glucocorticoid but not by cAMP. T 3, which increases phosphatidylcholine synthesis, did not induce FAS mRNA. The findings indicate that both glucocorticoid and cAMP increase FAS gene expression consistent with an important role for FAS in regulating the supply of fatty acid for surfactant phospholipid synthesis.

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.

Retinoic acid differentially regulates expression of surfactant- associated proteins in human fetal lung

Retinoic acid is known to play an essential role in maintaining the differentiation of a wide variety of epithelial cell types. However, its effects on the differentiation of lung alveolar epithelium have not been described. In the present study, we examined the effects of retinoic acid on the differentiation of human fetal lung tissue maintained in vitro. Human fetal lung explants were cultured in serum-free medium for 6 days in the absence or presence of all-trans retinoic acid at concentrations from 0.3 nM to 3 microM. Explant content of the surfactant-associated protein SP-A was measured using a specific enzyme-linked immunosorbent assay. Retinoic acid reduced SP-A protein levels in a concentration-dependent manner [analysis of variance (ANOVA), P < 0.01]. To evaluate possible cytotoxic effects of retinoic acid, culture media were assayed for lactate dehydrogenase (LDH), a cytoplasmic enzyme. LDH levels in media from retinoic acid-treated explants were not significantly different than LDH levels in media from control explants, indicating that retinoic acid is not cytotoxic in human fetal lung explants. Changes in messenger RNA (mRNA) levels for surfactant-associated proteins SP-A, SP-B, and SP-C were measured by Northern blot analysis. Retinoic acid reduced SP-A mRNA levels in a concentration-dependent manner (ANOVA, P < 0.02) and reduced SP-C mRNA levels at 3 microM. In contrast, retinoic acid increased SP-B mRNA levels in a concentration-dependent manner (ANOVA, P < 0.03). Morphometric analysis showed that retinoic acid decreased epithelial volume density in the explants by approximately 17% and increased connective tissue volume density by approximately 20% when compared to dimethyl sulfoxide vehicle controls. These data indicate that retinoic acid regulates type II cell surfactant protein gene expression in human fetal lung tissue.

Retinoic acid differentially regulates expression of surfactant-associated proteins in human fetal lung.

Retinoic acid is known to play an essential role in maintaining the differentiation of a wide variety of epithelial cell types. However, its effects on the differentiation of lung alveolar epithelium have not been described. In the present study, we examined the effects of retinoic acid on the differentiation of human fetal lung tissue maintained in vitro. Human fetal lung explants were cultured in serum-free medium for 6 days in the absence or presence of all-trans retinoic acid at concentrations from 0.3 nM to 3 microM. Explant content of the surfactant-associated protein SP-A was measured using a specific enzyme-linked immunosorbent assay. Retinoic acid reduced SP-A protein levels in a concentration-dependent manner [analysis of variance (ANOVA), P < 0.01]. To evaluate possible cytotoxic effects of retinoic acid, culture media were assayed for lactate dehydrogenase (LDH), a cytoplasmic enzyme. LDH levels in media from retinoic acid-treated explants were not significantly different than LDH levels in media from control explants, indicating that retinoic acid is not cytotoxic in human fetal lung explants. Changes in messenger RNA (mRNA) levels for surfactant-associated proteins SP-A, SP-B, and SP-C were measured by Northern blot analysis. Retinoic acid reduced SP-A mRNA levels in a concentration-dependent manner (ANOVA, P < 0.02) and reduced SP-C mRNA levels at 3 microM. In contrast, retinoic acid increased SP-B mRNA levels in a concentration-dependent manner (ANOVA, P < 0.03). Morphometric analysis showed that retinoic acid decreased epithelial volume density in the explants by approximately 17% and increased connective tissue volume density by approximately 20% when compared to dimethyl sulfoxide vehicle controls. These data indicate that retinoic acid regulates type II cell surfactant protein gene expression in human fetal lung tissue.

Hormonal regulation of cholinephosphate cytidylyltransferase in human fetal lung

Cytidylyltransferase (CTP: cholinephosphate cytidylyltransferase, EC 2.7.7.15, CYT) is a regulatory enzyme for synthesis of pulmonary surfactant phosphatidylcholine (PC). The effects of glucocorticoid, T 3, and cAMP on CYT activity were studied in explants of human fetal lung (18–22 weeks gestation) cultured for 1–6 days in serum-free medium. Dexamethasone (Dex, 10 nM) treatment for 5 days increased homogenate CYT activity (+ 115%, P < 0.02) when assayed in the presence of added lipid co-factor ( l-α-phosphatidylglycerol, PG, 1.1 mM) and tended to increase activity in its absence (+ 77%, P = 0.12). Cytosolic activity was also significantly elevated in the presence of added co-factor (+124%, P < 0.01), but there was no effect of Dex on microsomal specific activity. Dex increased the recovery of CYT activity in the cytosolic fraction (75% vs. 43% (control) of the homogenate activity), but not in the microsomal, nuclear or mitochondrial fractions. Assayed in the presence of added co-factor, stimulation of CYT by Dex was apparent after 48 h exposure and maximal by 5–6 days exposure to ≤ 30 nM concentration. T 3 or agents that increase endogenous cAMP stimulated cytosolic activity by 40% and 36–74%, respectively, after 4–6 days exposure, but none produced an additive increase in the presence of Dex. We conclude that stimulation of CYT activity contributes to hormonal induction of surfactant lipids by each of these hormones. Glucocorticoids may increase the amount of CYT enzyme as well as activate the enzyme via increased synthesis of lipid co-factor.

Secretion of surfactant protein A and phosphatidylcholine from type II cells of human fetal lung.

Surfactant protein A (SP-A) appears to have a role in lung immune defense as well as generation and metabolism of the alveolar surface-active film. Previous studies indicated that lamellar bodies isolated from lung tissue had a relatively low content of SP-A and that exogenous SP-A was needed for rapid formation of a surface-active film in vitro. We therefore tested the hypothesis that SP-A was secreted from type II cells primarily by a pathway separate from lamellar bodies. Cells were isolated from explants of human fetal lung that had been cultured with hormones to promote differentiation of type II cells, and secretion of surfactant lipid and SP-A were compared. Cultured cells secreted labeled phosphatidylcholine in a nearly linear fashion for 48 h. Basal secretion of SP-A, assayed by enzyme-linked immunosorbent assay, was linear for only 12 h after plating of cells; during this time, there was no accumulation of intracellular SP-A. Addition of secretagogues (phorbol ester, calcium ionophore, and beta-adrenergic agonist) stimulated phosphatidylcholine secretion approximately 4-fold. In contrast, the secretion rate of SP-A was not significantly affected by secretagogues. These findings indicate that a relatively small amount of secreted SP-A (approximately 10%) is released with lamellar bodies. Most SP-A is released by constitutive secretion and may be important for both surfactant- and nonsurfactant-related functions.

Biphasic glucocorticoid regulation of pulmonary SP-A: characterization of inhibitory process.

Pulmonary surfactant, which is necessary for normal lung function, is under both developmental and hormonal regulation. Glucocorticoids induce all components of surfactant and have a unique biphasic effect on surfactant protein A (SP-A), either stimulating or inhibiting accumulation in cultured fetal lung depending on dose and time of exposure. In this study we further characterized glucocorticoid inhibition of SP-A in cultured explants of human fetal lung. Decreased content of SP-A mRNA was the dominant response to dexamethasone added either early or later during culture. Inhibition occurred at < or = 1 nM dexamethasone on prolonged exposure, was blocked by RU 486, and was observed with other glucocorticoids but not sex steroids. When cortisol was removed from the culture medium, inhibition was rapidly reversed. The immediate inhibitory effect of 100 nM dexamethasone on SP-A mRNA content was completely blocked in the presence of cycloheximide. SP-A gene transcription, measured by nuclear elongation assay, was decreased by 60% after 4- to 8-h exposure to 100 nM dexamethasone. Stability of SP-A mRNA, determined both by addition of actinomycin D and by label-chase experiments, was transiently decreased immediately after adding dexamethasone (t1/2 approximately 3 h). In tissue treated with dexamethasone for > or = 8 h the stability of SP-A mRNA in control and treated explants was not different (t1/2 approximately 8 h). Our findings indicate that inhibition of SP-A is the dominant response to glucocorticoid. This effect is receptor mediated and apparently involves induction of a labile protein(s) that decreases gene transcription and transiently reduces mRNA stability.

Characterization of the beta-adrenergic receptor in isolated human fetal lung type II cells.

Functioning of the beta-adrenergic response system is important for successful transition of the neonate from fetal life to breathing air. We characterized the beta-adrenergic receptors on human fetal lung type II cells, the cell type responsible for many pulmonary responses sensitive to beta-adrenergic stimulation. Type II cells were isolated from human fetal lung explants, and membrane particulates prepared from these cells were used for radioligand binding studies. 125I-iodocyanopindolol, a specific beta-adrenergic antagonist, bound to a single class of saturable, high-affinity binding sites on type II cell membranes with a receptor concentration of 78 +/- 9 fmol receptor/mg membrane protein, a kd of 79 +/- 18 nM, and 958 +/- 120 receptors per cell. Binding was stereoselective with l-propranolol binding with higher affinity than the inactive d-isomer. The binding site had the characteristics of a beta 2-adrenergic receptor. The order of potency of beta-adrenergic agonists was isoproterenol greater than epinephrine much greater than norepinephrine. The beta 2-selective antagonist ICI 118,551 competed for a single class of high-affinity sites. Agonist binding affinity was reduced in the presence of guanyl nucleotides, consistent with receptors coupled to guanine nucleotide binding proteins. beta-Adrenergic agonists also stimulated adenylyl cyclase in these membrane preparations. 125I-iodocyanopindolol binding to membranes prepared from human fetal lung fibroblasts indicated fewer receptors (404 +/- 68) than were present on type II cells. Work by others has suggested a difference in lung function and lung beta-adrenergic receptor concentration between males and females.(ABSTRACT TRUNCATED AT 250 WORDS)