Mature alveolar type II cells that produce pulmonary surfactant are essential for adaptation to extrauterine life and prevention of infant respiratory distress syndrome. We have developed a new in vitro model to further investigate regulation of type II cell differentiation. Epithelial cells isolated from human fetal lung were cultured in serum-free medium on plastic. Cells treated with dexamethasone + cAMP analog and isobutylmethylxanthine for 4 days exhibited increased phosphatidylcholine synthesis and content of disaturated phosphatidylcholine species, manyfold increases in all surfactant proteins with processing to mature forms, and abundant lamellar bodies. DNA microarray analysis identified ∼3,100 expressed genes, including subsets of genes induced 2- to >100-fold (∼2.5%) or repressed 2- to 18-fold (∼1.2%) by hormone treatment. Of the highly regulated genes, most were coregulated in an additive or synergistic manner by dexamethasone and cAMP agents. Approximately 90% of the regulated genes identified by this initial microarray analysis have not been previously recognized as hormone responsive. One newly identified hormone-induced gene is Nkx2.1 (thyroid transcription factor-1), which has a critical role in surfactant protein gene expression. Our findings indicate that glucocorticoid + cAMP is sufficient and necessary for precocious induction of functional type II cells in this in vitro system and that these hormones act primarily in combination to regulate expression of a subset of specific genes.

Silencing of ABCA3 expression also reduced vesicular uptake of surfactant lipids phosphatidylcholine, sphingomyelin, and cholesterol but not phosphatidylethanolamine. We conclude that ABCA3 is required for lysosomal loading of phosphatidylcholine and conversion of lysosomes to lamellar body-like structures. ATP binding cassette (ABC)2 transporters are a superfamily of highly conserved membrane proteins that transport a wide variety of substrates across cell membranes (1). Among the several subfamilies, the ABCA subclass has received considerable attention, because mutations of the ABCA1 gene cause Tangier disease and mutations of the ABCA4 gene cause Stargardt macular dystrophy in humans (2-5). ABCA1 and ABCA4 are proposed to be transmembrane transporters for intracellular cholesterol/phospholipids and N-retinylidene phosphatidylethanolamine, respectively (3-5). ABCA3, a member of the ABCA subfamily with unknown function (6 -10), is predominantly expressed in the lung and localized to the limiting membrane of lamellar bodies in alveolar epithelial type II cells (ATII) in both humans and rats (7,8).In the lung, development of structures for effective pulmonary gas exchange and production of pulmonary surfactant are necessary for successful adaptation to extrauterine life in the newborn infant. These key processes in lung maturation require differentiation of epithelium into ATII cells, the cellular source for surfactant. Pulmonary surfactant is a complex mixture of lipids, primarily phosphatidylcholine (60 -70% of which is dipalmitoylphosphatidylcholine) and specific proteins that line the alveolar surface of the lung, reducing surface tension at the air-liquid interface and preventing collapse of the lung on expiration (11). Surfactant is assembled and stored in lamellar bodies, the secretory organelles of ATII cells (11-13). Two other members of the ABCA subfamily, ABCA1 and ABCA4, have been implicated in lipid transport leading to the hypothesis that ABCA3 transports lipid into the lamellar bodies of ATII cells (7-9). Recently, it has been reported that mutations in ABCA3 are associated with defective assembly of lamellar bodies and fatal respiratory distress syndrome (RDS) in the newborn infant and interstitial lung disease (6, 10).To study the potential role of ABCA3 in RDS, we examined the subcellular trafficking and substrate specificity of ABCA3 in hATII cells and mammalian cell lines using green fluorescent protein (GFP)-tagged protein and fluorescent lipid analogs. Morphological and functional changes secondary to both loss-and gain-of-function experiments demonstrate that ABCA3 selectively transports phosphatidylcholine, sphingomyelin, and cholesterol to lamellar bodies in hATII cells. Our findings indicate that lipid trafficking by ABCA3 across lamellar body membranes is necessary for lamellar body biogenesis as a key step in assembly of lung surfactant in hATII cells.

Lamellar bodies are the specialized secretory organelles of alveolar type II (ATII) epithelial cells through which the cell packages pulmonary surfactant and regulates its secretion. Surfactant within lamellar bodies is densely packed as circular arrays of lipid membranes and appears to be the product of several trafficking and biosynthetic processes. To elucidate these processes, we reported previously on the generation of a monoclonal antibody (3C9) that recognizes a unique protein of the lamellar body membrane of 180 kDa, which we named LBM180. We report that mass spectrometry of the protein precipitated by this antibody generated a partial sequence that is identical to the ATP-binding cassette protein, ABCA3. Homology analysis of partial sequences suggests that this protein is highly conserved among species. The ABCA3 gene transcript was found in cell lines of human lung origin, in ATII cells of human, rat, and mouse, as well as different tissues of rat, but the highest expression of ABCA3 was observed in ATII cells. Expression of this transcript was at its maximum prior to birth, and hormonal induction of ABCA3 transcript was observed in human fetal lung at the same time as other surfactant protein transcripts were induced, suggesting that ABCA3 is developmentally regulated. Molecular and biochemical studies show that ABCA3 is targeted to vesicle membranes and is found in the limiting membrane of lamellar bodies. Because ABCA3 is a member of a subfamily of ABC transporters that are predominantly known to be involved in the regulation of lipid transport and membrane trafficking, we speculate that this protein may play a key role in lipid organization during the formation of lamellar bodies.

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)

Mutations in the surfactant protein (SP)-C gene are responsible for familial and sporadic interstitial lung disease (ILD). The consequences of such mutations on pulmonary surfactant composition and function are poorly understood. To determine the effects of a mutation in the SP-C gene on surfactant, we obtained lung tissue at the time of transplantation from a 14-mo-old infant with progressive ILD. An in-frame 9-bp deletion spanning codons 91-93 in Exon 3 of the SP-C gene was present on one allele; neither parent carried this deletion. SP-C mRNA was present in normal size and amount. By immunofluorescence, proSP-C was aggregated within alveolar Type II cells in a compartment separate from SP-B. In airway surfactant, there was little or no mature SP-B or SP-C; SP-A content was increased. Minimum surface tension was increased (20 mN/m, normal < 5 mN/m). Type II cells contained normal and disorganized appearing lamellar bodies by electron microscopy. This spontaneous deletion on one allele of the SP-C gene was associated with sporadic ILD and abnormalities in surfactant composition and function. We propose that a dominant negative effect on surfactant protein metabolism and function results from aggregation of misfolded proSP-C and subsequent cell injury and inflammation.

Tight junction proteins in the claudin family regulate epithelial barrier function. We examined claudin expression by human fetal lung (HFL) alveolar epithelial cells cultured in medium containing dexamethasone, 8-bromo-cAMP, and isobutylmethylxanthanine (DCI), which promotes alveolar epithelial cell differentiation to a type II phenotype. At the protein level, HFL cells expressed claudin-1, claudin-3, claudin-4, claudin-5, claudin-7, and claudin-18, where levels of expression varied with culture conditions. DCI-treated differentiated HFL cells cultured on permeable supports formed tight transepithelial barriers, with transepithelial resistance (TER) >1,700 ohm/cm(2). In contrast, HFL cells cultured in control medium without DCI did not form tight barriers (TER <250 ohm/cm(2)). Consistent with this difference in barrier function, claudins expressed by HFL cells cultured in DCI medium were tightly localized to the plasma membrane; however, claudins expressed by HFL cells cultured in control medium accumulated in an intracellular compartment and showed discontinuities in claudin plasma membrane localization. In contrast to claudins, localization of other tight junction proteins, zonula occludens (ZO)-1, ZO-2, and occludin, was not sensitive to HFL cell phenotype. Intracellular claudins expressed by undifferentiated HFL cells were localized to a compartment containing early endosome antigen-1, and treatment of HFL cells with the endocytosis inhibitor monodansylcadaverine increased barrier function. This suggests that during differentiation to a type II cell phenotype, fetal alveolar epithelial cells use differential claudin expression and localization to the plasma membrane to help regulate tight junction permeability.

Rationale: The pulmonary phenotype of Hermansky-Pudlak syndrome (HPS) in adults includes foamy alveolar type 2 cells, inflammation, and lung remodeling, but there is no information about ontogeny or early disease mediators. Objectives: To establish the ontogeny of HPS lung disease in an animal model, examine disease mediators, and relate them to patients with HPS1. Methods: Mice with mutations in both HPS1/pale ear and HPS2/ AP3B1/pearl (EPPE mice) were studied longitudinally. Total lung homogenate, lung tissue sections, and bronchoalveolar lavage (BAL) were examined for phospholipid, collagen, histology, cell counts, chemokines, surfactant protein D (SP-D), and S-nitrosylated SP-D. Isolated alveolar epithelial cells were examined for expression of inflammatory mediators, and chemotaxis assays were used to assess their importance. Pulmonary function test results and BAL from patients with HPS1 and normal volunteers were examined for clinical correlation. Measurements and Main Results: EPPE mice develop increased total lung phospholipid, followed by a macrophage-predominant pulmonary inflammation, and lung remodeling including fibrosis.

Claudins are a family of transmembrane proteins that are required for tight junction formation. Claudin (CLDN)-18.1, the only known lung-specific tight junction protein, is the most abundant claudin in alveolar epithelial type (AT) 1 cells, and is regulated by lung maturational agonists and inflammatory mediators. To determine the function of CLDN18 in the alveolar epithelium, CLDN18 knockout (KO) mice were generated and studied by histological, biochemical, and physiological approaches, in addition to whole-genome microarray. Alveolar epithelial barrier function was assessed after knockdown of CLDN18 in isolated lung cells. CLDN18 levels were measured by quantitative PCR in lung samples from fetal and postnatal human infants. We found that CLDN18 deficiency impaired alveolar epithelial barrier function in vivo and in vitro, with evidence of increased paracellular permeability and architectural distortion at AT1-AT1 cell junctions. Although CLDN18 KO mice were born without evidence of a lung abnormality, histological and gene expression analysis at Postnatal Day 3 and Week 4 identified impaired alveolarization. CLDN18 KO mice also had evidence of postnatal lung injury, including acquired AT1 cell damage. Human fetal lungs at 23-24 weeks gestational age, the highest-risk period for developing bronchopulmonary dysplasia, a disease of impaired alveolarization, had significantly lower CLDN18 expression relative to postnatal lungs. Thus, CLDN18 deficiency results in epithelial barrier dysfunction, injury, and impaired alveolarization in mice. Low expression of CLDN18 in human fetal lungs supports further investigation into a role for this tight junction protein in bronchopulmonary dysplasia.