Mechanical distention modulates alveolar epithelial cell phenotypic expression by transcriptional regulation.

The development of a normal pulmonary alveolar epithelium, essential for gas exchange, is critical for the successful adaptation to extrauterine life. From observations of natural and experimental developmental abnormalities, it has been hypothesized that mechanical factors may play a role in regulating differentiation of the pulmonary alveolar epithelium. To test this hypothesis directly, we have investigated the in vitro effects of mechanical distention on the expression of specific markers for the type I and type II cell phenotypes. Fetal rat lung (18-d) explants were mechanically distended in culture for 18 h. Mechanical distention caused an increase in RTI 40 messenger RNA (mRNA), a marker of the type I cell phenotype, of 10.6 times (n = 3, P < 0.05) that of undistended controls. In contrast, mechanical distention resulted in a decrease in mRNA content of two markers of the type II cell phenotype, surfactant protein (SP)-B and SP-C. SP-B was reduced to 10 +/- 9% (n = 3, P < 0.005) and of SP-C to 12 +/- 7% (n = 3, P < 0.0001) of undistended controls. Mechanical distention had no effect on content of mRNA for SP-A or 18S ribosomal RNA. Examined by nuclear run-on assays, mechanical distention caused changes in transcriptional rates of RTI 40, SP-B, and SP-C. These data show that mechanical distention stimulates expression of a type I cell marker and inhibits expression of markers for the type II phenotype; these effects occur at least in part at the transcriptional level. These studies support the hypothesis that mechanical distention of fetal lung tissue stimulates expression of the type I cell phenotype and inhibits expression of the type II phenotype.

Human surfactant proteins A1 and A2 are differentially regulated during development and by soluble factors.

An RT-PCR method for the relative quantitation of the mRNAs for human surfactant protein (SP) A1 and SP-A2 was developed, verified, and then utilized to determine the relative levels of these mRNAs in fetal and adult lung samples in vivo, as well as in cultured human fetal lung explants and H441 cells. For the cultured tissue and cells, we assessed the effects of a variety of soluble factors known to modulate total SP-A. Comprehensive analysis revealed many significant findings, including the following: both mRNAs were expressed as early as 15 wk of gestation; throughout midgestation, SP-A1 was present at higher levels than SP-A2, with an average ratio of 30:1. In the adult lung, SP-A1 mRNA was present at lower levels than SP-A2, with a ratio of 0.4:1, whereas in H441 cells, the ratio was 0.85:1. In fetal lung cultured for 4 days, both mRNAs increased, with a greater increase in SP-A2 (97-fold) than in SP-A1 (15-fold), resulting in a final ratio of 4:1. Differential regulation was demonstrated for 8-(4-chlorophenylthio)-cAMP, interferon (IFN)-gamma, tumor necrosis factor-alpha, and transforming growth factor (TGF)-beta in the human fetal lung explant system, with SP-A2 being more affected, and for IFN-gamma and TGF-beta in the H441 cells, where SP-A1 showed greater regulation. Of the soluble factors tested, IFN-gamma and TGF-beta had the most potent and consistent effects in both systems.

Apoptosis in the development of rat and human fetal lungs.

The establishment of an effective pulmonary alveolar-capillary interface occurs during mid to late gestation. This requires an expansion of endothelial, epithelial, and air space compartments with relative thinning of the interstitial compartment. Traditionally, these changes have been attributed to differences in the rate of cell growth in the respective compartments. We hypothesized that apoptosis also participates in this lung remodeling. Using light and electron microscopy, the nucleosomal ladder pattern of DNA digestion, and the detection of apoptotic cells in situ by the TUNEL method (Gavrieli, et al. J. Cell Biol. 1992;119:493-501), we demonstrated the occurrence of apoptosis in fetal lungs in vivo and in explant culture. In the rat fetal lung (RFL) in vivo we detected apoptosis from 16 through 22 d gestation. There was variation in the amount of DNA digestion between fetal lungs, but no correlation with gestational age. The findings in human fetal lungs (HFL) from 15 through 24 wk gestation were similar to those of the RFL; the apoptotic indices for both were about 2 apoptotic cells per thousand, suggesting that a significant percentage of cells are eliminated by this mechanism. In the HFL explant culture system, a rapid and massive wave of apoptosis occurred. In all samples of RFL and HFL examined, apoptosis was restricted to interstitial cells. This work has demonstrated for the first time that apoptosis is a feature of normal fetal lung development and that the process is accelerated in lung explant culture.

Coverslip mounted-immersion cycled in situ RT-PCR for the localization of mRNA in tissue sections.

An improved method was developed for in situ reverse transcription-polymerase chain reaction (RT-PCR) to detect and localize mRNA in tissue sections. The coverslip mounted-immersion cycled (COSMIC) in situ RT-PCR technique combines the advantages of solution-phase PCR with the tissue immobilization necessary for in situ analysis. The tissue specimen is mounted on an AES-silane-coated coverslip, excess glass is removed and the sample is immersed in reaction mixture in a PCR tube and subjected to thermal cycling. Processing the section on the coverslip is efficient, the thin glass withstands the high temperature cycling and the tissue adheres securely through the process. The specimen is fully exposed to the reagents, and is heated uniformly and accurately according to temperatures programmed into the thermal cycler. An application is described for the detection and localization of the mRNA for surfactant protein A (SP-A) in fetal rat lung tissue.

Transcriptional regulation of human pulmonary surfactant proteins SP-B and SP-C by glucocorticoids.

Expression of the pulmonary surfactant-associated proteins SP-B and SP-C is under both developmental and hormonal regulation. We used human fetal lung to investigate developmental changes and the mechanism of glucocorticoid stimulation of SP-B and SP-C gene expression. There were similar approximately 3-fold increases in SP-B cytoplasmic mRNA content and transcription rate comparing lung samples of 24 wk versus 16 wk gestation. During 5 days of lung explant culture without hormones, the transcription rate increased for SP-B and decreased for SP-C, paralleling changes in mRNA content. Treatment with 100 nM dexamethasone maximally increased transcription of the SP-B gene (approximately 3-fold) and SP-C gene (approximately 11-fold) after 2 and 8 h, respectively, similar to changes in mRNA content. In dose-response studies, the maximal increase in transcription rate occurred at approximately 10 nM dexamethasone for SP-B and at > or = 100 nM for SP-C. Induction of SP-B mRNA content and transcription rate were not affected by prior cycloheximide exposure, whereas induction of SP-C mRNA was decreased by as little as 1 h exposure to inhibitor. We conclude that glucocorticoids, acting directly in type II cells, regulate the SP-B and SP-C genes primarily at the level of transcription. Induction of SP-C, but not SP-B, requires ongoing protein synthesis which likely reflects involvement of a labile transcription factor. The difference in glucocorticoid sensitivity may indicate that the two surfactant protein genes contain glucocorticoid response elements with different affinities for receptor.

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.

Differential glucocorticoid regulation of the pulmonary hydrophobic surfactant proteins SP-B and SP-C.

Glucocorticoids increase expression of the genes for the pulmonary surfactant-associated proteins SP-B and SP-C in fetal lung both in vivo and in vitro. To examine the mechanism of these effects, we studied induction of SP-B and SP-C mRNAs in human fetal lung cultured as explants. Both mRNA levels rose rapidly in response to 100 nM dexamethasone (Dex), with a faster response for SP-B: maximal levels of induction were achieved in < or = 12 h for SP-B (3.5-fold versus control) versus approximately 24 h for SP-C mRNA (35-fold versus control). Cycloheximide (2.5 micrograms/ml) did not affect glucocorticoid induction of SP-B mRNA but markedly decreased induction of SP-C mRNA. In control cultures, cycloheximide did not significantly reduce levels of either transcript. In nuclear run-on assays, Dex increased the rate of gene transcription for both SP-B (2.8 +/- 0.3-fold versus control, n = 4) and SP-C (10- to 30-fold). Using actinomycin D to assess mRNA stability, the t1/2 of SP-B mRNA was increased from 7.5 +/- 0.4 h to 18.8 +/- 2.9 h by Dex treatment (P < 0.05), whereas the t1/2 of SP-C mRNA was not affected (9.3 +/- 1.7 h versus 8.1 +/- 1.2 h; NS). A similar increase in SP-B mRNA t1/2 with Dex (from 6 h to 19 h) was observed in label-chase studies with [3H]uridine. We conclude that glucocorticoids regulate the hydrophobic surfactant proteins of alveolar type II cells by different mechanisms: induction of SP-B is a primary response and includes an increase in both transcription rate and mRNA stability, whereas induction of SP-C is a secondary process, requiring ongoing protein synthesis, involving increased transcription rate without a change in mRNA stability.

Hormonally regulated proteins in cultured human fetal lung: analysis by two-dimensional gel electrophoresis.

We used high-resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) to identify hormonally regulated proteins in cultured human fetal lung. Proteins labeled with [35S]methionine were separated by 2-D PAGE, and fluorograms were analyzed by computer-assisted analysis of densitometric scans. Dexamethasone (10 nM) and gamma-interferon (10 ng/ml) induced (2- to 22-fold vs. control) distinct sets of proteins (comprising approximately 2% of approximately 1,000 resolved proteins). Treatment with forskolin (10 microM) plus 3-isobutyl-1-methylxanthine (IBMX, 100 microM), which increases intracellular adenosine 3',5'-cyclic monophosphate (cAMP), induced both unique proteins and several proteins induced by dexamethasone. One protein (Mr 40,000, pI 4.4) was induced only with combined dexamethasone and cAMP treatment. Dexamethasone repressed four proteins, but inhibition was not observed with other hormones. Some of the regulated proteins were enriched in either fibroblasts or type II cells isolated from lung explants. We found no proteins that were consistently regulated by triiodothyronine (T3) (2 nM) or transforming growth factor-beta (10 ng/ml). Additionally, none of the hormonal treatments substantially altered the rate of methionine incorporation into total protein. Thus we have identified separate subsets of proteins that are regulated by glucocorticoids, gamma-interferon, and cAMP; these proteins may be important mediators of hormonal effects in the developing fetal lung.

Glucocorticoid stimulation of fatty acid synthesis in explants of human fetal lung.

We examined the effects of glucocorticoids and thyroid hormone (T3) on fatty acid synthesis, fatty acid composition and fatty acid synthetase activity in explants of human fetal lung (16-23 wk gestation). Explants were cultured 1-7 days in the absence (control) or presence of dexamethasone (10 nM) and/or T3 (2 nM). In control explants fatty acid synthesis and fatty acid synthetase activity increased 200% and 455%, respectively, between 1 and 5 days. Dexamethasone (10 nM) stimulated fatty acid synthesis (tritiated water incorporation) 155% and fatty acid synthetase activity 117% after 5 days in culture. T3 (2 nM) was not stimulatory, either alone or in the presence of dexamethasone. Dexamethasone increased the proportion of newly synthesized fatty acid recovered in phosphatidylcholine from 72% (control) to 90% (P less than 0.02) of total fatty acid. Dexamethasone stimulation of fatty acid synthetase activity was consistent with a receptor-mediated process: (1) stimulation was saturable and dose-dependent (Kd = 1.5 +/- 0.3 nM); (2) the potency of glucocorticoid analogs and other steroids reflected their glucocorticoid activity; (3) stimulation was reversible when cortisol was removed from the medium. Stimulation by dexamethasone was apparent within 24 h of hormone exposure, and increased to a maximum between 4 and 6 days. Fatty acid synthetase activity was higher in Type II cells (3.54 +/- 0.58 nmol malate/min per mg protein) than in fibroblasts from treated explants. Although both cell types responded to hormone treatment the stimulation was greater for Type II cells (200% vs. 75% increase). The fatty acid composition of PC showed increases in 14:0 and 16:1 with culture alone which were further stimulated by dexamethasone but not T3. These results indicate glucocorticoid stimulation of fatty acid synthesis and are consistent with a key role for fatty acid synthetase in the hormonal induction of pulmonary surfactant phosphatidylcholine synthesis in cultured fetal lung.

Synthesis of surfactant components by cultured type II cells from human lung.

We examined the effect of monolayer culture on surfactant phospholipids and proteins of type II cells isolated from human adult and fetal lung. Type II cells were prepared from cultured explants of fetal lung (16-24 weeks gestation) and from adult surgical specimens. Cells were maintained for up to 6 days on plastic tissue culture dishes. Although incorporation of [methyl-3H]choline into phosphatidylcholine (PC) by fetal cells was similar on day 1 and day 5 of culture, saturation of PC fell from 35 to 26%. In addition, there was decreased distribution of labeled acetate into PC, whereas distribution into other phospholipids increased or did not change. The decrease in saturation of newly synthesized PC was not altered by triiodothyronine (T3) and dexamethasone treatment or by culture as mixed type II cell/fibroblast monolayers. The content of surfactant protein SP-A (28-36 kDa) in fetal cells, as measured by ELISA and immunofluorescence microscopy, rose during the first day and then fell to undetectable levels by the fifth. Synthesis of SP-A, as measured by [35S]methionine labeling and immunoprecipitation, was detectable on day 1 but not thereafter. Levels of mRNAs for SP-A and for the two lipophilic surfactant proteins SP-B (18 kDa) and SP-C (5 kDa) fell with half-times of maximally 24 h. In contrast, total protein synthesis measured by [35S]methionine incorporation increased and then plateaued. In adult cells, the content of SP-A and its mRNA decreased during culture, with time-courses similar to those for fetal cells. We conclude that in monolayer culture on plastic culture dishes, human type II cells lose their ability to synthesize both phospholipids and proteins of surfactant. The control of type II cell differentiation under these conditions appears to be at a pretranslational level.

Isolation and characterization of differentiated alveolar type II cells from fetal human lung.

A method has been developed for isolating differentiated type II cells from human lung of 18-24-week gestation. The procedure involves an initial 4-day culture of lung explants in the presence of dexamethasone (10 nM) and triiodothyronine (2 nM). Type II cells (and fibroblasts) are isolated by trypsin digestion of the explants, two differential adherence steps and incubation overnight in primary culture. This method provides a high yield of type II cells ((50 +/- 15) X 10(6) cells/g wet weight of explant) with a purity of 85 +/- 5% in 16 experiments. The type II cells contain numerous perinuclear granules which stain darkly with toluidine blue and Papanicolaou stain; electron microscopy showed these inclusions to be lamellar bodies with tightly stacked, well defined lamellae. Type II cells, but not fibroblasts, were positive by immunofluorescence histology for surfactant apoprotein and binding of Maclura pomifera lectin which binds to the surface of type II but not type I cells in vivo. The rate of both [3H]acetate and [3H]choline incorporation into phosphatidylcholine (PC) was several-fold greater in type II cells than fibroblasts; the saturation of PC was 36.2 and 25.9%, respectively. Release of saturated PC was stimulated by terbutaline, the ionophore A23187, and tetradecanoyl phorbol acetate in type II cells but not fibroblasts. We conclude that differentiated type II cells can be isolated in relatively high yield and purity from hormone-treated explants of fetal human lung.

Glucocorticoids and thyroid hormones stimulate biochemical and morphological differentiation of human fetal lung in organ culture.

We characterized the stimulatory effects of both glucocorticoids and thyroid hormones on the surfactant system in human fetal lung. Synthesis of phosphatidylcholine (PC) and morphology were examined in explant cultures (15-24 weeks gestation) maintained 1-7 days in serum-free Waymouth's medium in a 95%-air-5% CO2 atmosphere. Control explants (no hormones) had the same rate of choline incorporation into PC between 1 and 7 days, but a significant increase in tissue PC content [82 +/- 21%, (+/- SEM), day 6 vs. 1], consistent with slow turnover of PC. [3H]Choline incorporation was stimulated 36%, 137%, and 192% by T3 (2 nM), dexamethasone (Dex; 10 nM), and T3 plus Dex, respectively, after 6 days of exposure (optimal response) compared to 19%, 38%, and 84% after 2 days of exposure. Thus, a supra-additive response occurred in the presence of both hormones and was greater at a shorter exposure time. Dex increased the percent saturation of newly synthesized PC (28.9 +/- 0.9% vs. 17.8 +/- 0.8% for control), but T3 did not, whereas both hormones increased tissue PC content (74.4 +/- 7.3% and 18.7 +/- 7.8% increase vs. control, respectively). Pulse-chase experiments with [3H]choline suggest that remodeling of unsaturated PC to saturated PC occurred during culture and was stimulated by Dex. Incorporation of [3H]acetate and [3H]glycerol into PC was stimulated by Dex (830% and 77%, respectively), but not by T3; the distribution of incorporated radioactivity among phospholipids was changed by Dex (increased counts per min into PC and phosphatidylglycerol with acetate and glycerol, respectively), but not by T3. Half-maximal stimulation of choline incorporation occurred at concentrations of Dex (2.1 nM) and T3 (0.03 nM) that are similar to the Kd values for receptor binding (5 and 0.05 nM, respectively). The relative potencies of thyroid hormones were T3 greater than T4 greater than rT3, and for corticosteroids, Dex much greater than corticosterone greater than 11-dehydrocorticosterone = cortisol greater than cortisone. Stimulation by either T3 or cortisol was reversed within 24-48 h of hormone removal. Initial treatment of explants with Dex enhanced the subsequent response to T3, but not vice versa. Culture for 4-5 days in the absence of hormones produced some morphological maturation of the epithelial cells, whereas treatment with T3 plus Dex markedly increased the number and size of lamellar bodies in epithelial cells, caused extensive proliferation of apical microvilli, and reduced glycogen deposits. Our findings are consistent with receptor-mediated stimulation of surfactant synthesis in human lung by both glucocorticoids and thyroid hormones.(ABSTRACT TRUNCATED AT 400 WORDS)

Recessive 2-(methylamino)-isobutyrate (MeAIB)-resistant mutant of Chinese hamster ovary cells (CHO-K1) with increased transport through ASC system.

Mutants of Chinese hamster ovary cells (CHO-K1 Pro-), resistant to the proline transport antagonist 2-(methylamino)-isobutyrate (MeAIB) were isolated by a single-step procedure. Mutation rates to Pro+ and to Pro- MeAIB resistance (MeAIBr) are 1.7 X 10(-6) and 2.4 X 10(-5), respectively. Several Pro- MeAIBr mutants were tested by measuring the uptake of 0.05 mM proline through the various amino acid transport systems: some showed increases in one transport system only; others revealed pleiotropic changes affecting two or more systems; still others had no apparent change in proline transport. One Pro- MeAIBr mutant analyzed in detail (MeAIBr22) was isolated after EMS treatment as resistant to 5 mM MeAIB, is Pro-, stable, and shows a 1.6-fold increase in the initial velocity of transport of 0.05 mM proline. There appears to be no change in the velocity of proline transport through the amino acid transport systems A, P, and L, and the "glutamine inhibitable fraction." In contrast, there is a 5.5-fold increase in the velocity of transport of 0.05 mM proline through the ASC system. Kinetic studies reveal a sixfold increase in the Vm and a slight increase in the Km of the transport of serine through the ASC system. Hybrids between MeAIBr22 and CHO-K1 Pro-, OUAr, HPRT- showed the parental phenotype. These results indicate that the mutant ASC phenotype of MeAIBr22 is recessive and is probably the result of a regulatory gene mutation.

Alanine-resistant mutants of Chinese hamster ovary cells, CHO-K1, producing increases in velocity of proline transport through the A, ASC, and P systems.

We have developed a method for the isolation of transport mutants with increases in velocity of transport through the A and ASC systems and through a newly discovered P system utilizing the amino acid antagonism between A system amino acids and proline in CHO-K1 pro- cells. Mutants alar2 and alar3, isolated in a single-step procedure, resistant to 25 mM alanine in MEM-10 plus 0.05 mM proline are pro-, stable, cross resistant to alpha-(methylamino)isobutyric acid (MeAIB) and show an approximately twofold increase in the initial velocity of proline uptake. Ethyl methane sulfonate (EMS) increases the frequency of pro- alar clones in the population by at least 50 times the spontaneous frequency. The increased velocity of proline transport by alar2 and alar3 can be attributable to the 1.5 to 3 times increase in velocity of transport of proline through systems A, ASC, and P. The Vmax for proline transport through the A system has increased two times for alar2 while the Km and Vmax for alar3 has increased by 1.4 and 2.3 times that of CHO-K1. There is a corresponding increase in Vmax of proline transport by alar2 through the P system. The P system is defined operationally as that portion of the Na+-dependent velocity that remains when the A, ASC, and glutamine-inhibitable fraction are eliminated. The system is concentrative. Proline appears to be the preferred substrate. Li+ cannot be substituted for Na+. The system is moderately dependent upon pH. It obeys Michaelis-Menten kinetics and is not derepressible by starvation. There is no evidence for an N system in CHO-K1.