Identification of LBM180, a Lamellar Body Limiting Membrane Protein of Alveolar Type II Cells, as the ABC Transporter Protein ABCA3
2002; Elsevier BV; Volume: 277; Issue: 25 Linguagem: Inglês
10.1074/jbc.m201812200
ISSN1083-351X
AutoresSurafel Mulugeta, Joseph M. Gray, Kathleen L. Notarfrancesco, Linda W. Gonzales, Michael Koval, Sheldon I. Feinstein, Philip L. Ballard, Aron B. Fisher, Henry Shuman,
Tópico(s)Neonatal Respiratory Health Research
ResumoLamellar 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. 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. alveolar type II ATP binding cassette multidrug-resistant surfactant protein reverse transcription dexamethasone isobutylmethylxanthine enhanced green fluorescent protein phosphatidylcholine monoclonal antibody Pulmonary surfactant is a complex mixture of phospholipids and proteins that functions to prevent atelectasis by reducing alveolar surface tension at low lung volumes (1Wright J.R. Dobbs L.G. Annu. Rev. Physiol. 1991; 53: 395-414Crossref PubMed Scopus (188) Google Scholar, 2Dobbs L.G. Am. J. Respir. Crit. Care Med. 1994; 150: S31-S32Crossref PubMed Google Scholar). Surfactant consists predominantly of phospholipids, synthesized by alveolar type II (ATII)1 cells, and several unique proteins. Surfactant protein (SP)-A, SP-B, SP-C, and SP-D are synthesized by ATII cells, but with the exception of SP-C, they are also produced by other cells (3Rooney S.A. Young S.L. Mendelson C.R. FASEB J. 1994; 8: 957-967Crossref PubMed Scopus (302) Google Scholar, 4Batenburg J.J. Haagsman H.P. Prog. Lipid Res. 1998; 37: 235-276Crossref PubMed Scopus (93) Google Scholar). The alveolar surfactant pool size appears to be controlled primarily by ATII cells that regulate both secretion to and re-uptake from the alveolar space. Prior to secretion, surfactant lipids along with surfactant proteins are stored in lamellar bodies as densely packed lamellae and are secreted into the alveolar lumen by regulated exocytosis (5Ryan U.S. Ryan J.W. Smith D.S. Tissue & Cell. 1975; 7: 587-599Crossref PubMed Scopus (69) Google Scholar). The average ATII cell normally contains 150 ± 30 lamellar bodies (6Crystal R.G. West J.B. Weibel E.R. Barnes P.J. Crystal R.G. The Lung. Lippincott-Raven, Philadelphia1999: 564Google Scholar) with an in vivobasal secretion rate of ∼15 lamellar bodies per h in rat lung (7Young S.L. Kremers S.A. Apple J.S. Crapo J.D. Brumley G.W. J. Appl. Physiol. 1981; 51: 248-253Crossref PubMed Scopus (61) Google Scholar). ATII cells also endocytose surfactant from the alveolar space, some of which is recycled to lamellar bodies (8Hallman M. Epstein B.L. Gluck L. J. Clin. Invest. 1981; 68: 742-751Crossref PubMed Scopus (96) Google Scholar, 9Chander A. Reicherter J. Fisher A.B. J. Clin. Invest. 1987; 79: 1133-1138Crossref PubMed Scopus (31) Google Scholar, 10Fisher A.B. Chander A. Annu. Rev. Physiol. 1985; 47: 789-802Crossref PubMed Google Scholar, 11Young S.L. Wright J.R. Clements J.A. J. Appl. Physiol. 1989; 66: 1336-1342Crossref PubMed Scopus (45) Google Scholar, 12Young S.L. Fram E.K. Larson E. Wright J.R. Am. J. Physiol. 1993; 265: L19-L26Crossref PubMed Google Scholar) and the remainder is degraded (9Chander A. Reicherter J. Fisher A.B. J. Clin. Invest. 1987; 79: 1133-1138Crossref PubMed Scopus (31) Google Scholar). Because of its central role in lung surfactant turnover, we have focused on the assembly of this organelle and the proteins that distinguish it from other secretory organelles. Lamellar bodies appear to be at the intersection of several membrane trafficking and vesicle sorting pathways. Surfactant lipids and proteins are targeted to lamellar bodies from both the secretory and endocytic pathways. SP-B and SP-C are delivered to lamellar bodies from the endoplasmic reticulum via the secretory pathway (13Voorhout W.F. Veenendaal T. Haagsman H.P. Weaver T.E. Whitsett J.A. van Golde L.M. Geuze H.J. Am. J. Physiol. 1992; 263: L479-L486PubMed Google Scholar, 14Beers M.F. Lomax C.A. Russo S.J. J. Biol. Chem. 1998; 273: 15287-15293Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), whereas SP-A is secreted to the airspace and subsequently internalized and trafficked to lamellar bodies via clathrin-coated pits through early and late endosomes (12Young S.L. Fram E.K. Larson E. Wright J.R. Am. J. Physiol. 1993; 265: L19-L26Crossref PubMed Google Scholar, 15Ryan R.M. Morris R.E. Rice W.R. Ciraolo G. Whitsett J.A. J. Histochem. Cytochem. 1989; 37: 429-440Crossref PubMed Scopus (82) Google Scholar). Newly synthesized lipids, such as phosphatidylcholine, are also delivered to the lamellar body from the endoplasmic reticulum via the secretory pathway (16Chevalier G. Collet A.J. Anat. Rec. 1972; 174: 289-310Crossref PubMed Scopus (231) Google Scholar, 17Jobe A. Ikegami M. Sarton-Miller I. Jones S. Yu G. Biochim. Biophys. Acta. 1981; 666: 47-57Crossref PubMed Scopus (28) Google Scholar, 18Magoon M.W. Wright J.R. Baritussio A. Williams M.C. Goerke J. Benson B.J. Hamilton R.L. Clements J.A. Biochim. Biophys. Acta. 1983; 750: 18-31Crossref PubMed Scopus (194) Google Scholar). Surfactant lipids are internalized and delivered to lamellar bodies by two pathways, one clathrin-dependent and another clathrin-independent but actin-dependent (19Muller W.J. Zen K. Fisher A.B. Shuman H. Am. J. Physiol. 1995; 269: L11-L19Crossref PubMed Google Scholar). Proteins essential for the formation of lamellar bodies have not been completely defined. SP-B appears to be one of the key proteins necessary for the proper organization of lamellar bodies. Newborns with hereditary SP-B deficiency have poorly formed lamellar bodies and abnormal surfactant (20deMello D.E. Heyman S. Phelps D.S. Hamvas A. Nogee L. Cole S. Colten H.R. Am. J. Respir. Cell Mol. Biol. 1994; 11: 230-239Crossref PubMed Scopus (98) Google Scholar, 21deMello D.E. Nogee L.M. Heyman S. Krous H.F. Hussain M. Merritt T.A. Hsueh W. Haas J.E. Heidelberger K. Schumacher R. J. Pediatr. 1994; 125: 43-50Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Similar abnormalities have been demonstrated in SP-B-deficient mice (22Clark J.C. Wert S.E. Bachurski C.J. Stahlman M.T. Stripp B.R. Weaver T.E. Whitsett J.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7794-7798Crossref PubMed Scopus (560) Google Scholar). However, other contributing factors must also exist because loss of lamellar bodies or poorly formed lamellar bodies have been observed in abnormalities associated with refractory neonatal respiratory failure and congenital alveolar proteinosis where SP-B appears normal (23Cutz E. Wert S.E. Nogee L.M. Moore A.M. Am. J. Respir. Crit. Care Med. 2000; 161: 608-614Crossref PubMed Scopus (42) Google Scholar, 24Tryka A.F. Wert S.E. Mazursky J.E. Arrington R.W. Nogee L.M. Pediatr. Dev. Pathol. 2000; 3: 335-345Crossref PubMed Scopus (50) Google Scholar). In an effort to understand the formation and organization of lamellar bodies, we began the process of identifying proteins that are uniquely expressed in this organelle. In our previous report (25Zen K. Notarfrancesco K. Oorschot V. Slot J.W. Fisher A.B. Shuman H. Am. J. Physiol. 1998; 275: L172-L183PubMed Google Scholar), we described the generation of a panel of 30 antibodies against the limiting membrane of lamellar bodies isolated from rat ATII cells. One of these antibodies, monoclonal antibody (mAb) 3C9, labels lamellar bodies of ATII cells with high specificity. Further characterization revealed that the antigen is an integral membrane protein with a molecular mass of 180 kDa, which we named LBM180. The present study identifies LBM180 as ABCA3, a member of ATP-binding cassette (ABC) transporter family, and characterizes temporal, spatial, and regulated expression. ABC transporters represent the largest family of transmembrane proteins, and its members have been found in every organism examined so far (26Dean M. Hamon Y. Chimini G. J. Lipid Res. 2001; 42: 1007-1017Abstract Full Text Full Text PDF PubMed Google Scholar). These proteins bind ATP and use the energy of its hydrolysis to drive the transport of various substrates across cell membranes (27Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3364) Google Scholar, 28Childs, S., and Ling, V. (1994) Important Adv. Oncol.21–36Google Scholar, 29Dean M. Allikmets R. Curr. Opin. Genet. & Dev. 1995; 5: 779-785Crossref PubMed Scopus (208) Google Scholar). The human ABC proteins are classified into subfamilies that include the ABCA subfamily of lipid transporters, multidrug resistance/transmembrane-associated proteins, adrenoleukodystrophy proteins, and the cystic fibrosis transmembrane conductance regulator (30Klein I. Sarkadi B. Varadi A. Biochim. Biophys. Acta. 1999; 1461: 237-262Crossref PubMed Scopus (511) Google Scholar). They transport a wide variety of substrates including lipids, ions, amino acids, peptides, sugars, vitamins, steroid hormones, and toxic compounds (30Klein I. Sarkadi B. Varadi A. Biochim. Biophys. Acta. 1999; 1461: 237-262Crossref PubMed Scopus (511) Google Scholar). Their distinguishing features are two nucleotide binding domains with conserved Walker A and B motifs, both involved in ATP binding (present in many other ATP-utilizing proteins), one conserved sequence diagnostic to the ABC unit called the “ABC signature” located between the Walker A and B sequences, and 12 membrane-spanning helices (31Schriml L.M. Dean M. Genomics. 2000; 64: 24-31Crossref PubMed Scopus (26) Google Scholar). Substrate specificity appears to be determined by the transmembrane domains, whereas coupling of the two nucleotide binding domains provides the energy required for transport. ABCA3 was originally cloned from a human medullary thyroid carcinoma cell line, and in humans, its message is expressed most highly in lung (32Klugbauer N. Hofmann F. FEBS Lett. 1996; 391: 61-65Crossref PubMed Scopus (59) Google Scholar, 33Connors T.D. Van Raay T.J. Petry L.R. Klinger K.W. Landes G.M. Burn T.C. Genomics. 1997; 39: 231-234Crossref PubMed Scopus (49) Google Scholar). Recently, Yamano et al. (34Yamano G. Funahashi H. Kawanami O. Zhao L.X. Ban N. Uchida Y. Morohoshi T. Ogawa J. Shioda S. Inagaki N. FEBS Lett. 2001; 508: 221-225Crossref PubMed Scopus (234) Google Scholar) cloned ABCA3 from human lung and showed, by immunohistochemical analyses, that ABCA3 is an ATII cell lamellar body membrane protein. They also demonstrated that ABCA3 protein had a molecular mass of 150 kDa in human lung but when expressed in HEK 293 cells, the protein had a molecular mass of 180 kDa. ABCA3 is a member of the ABCA subfamily of transporters and shares high homology with ABCA1. ABCA1, along with a number of other transporters belonging to the same subfamily, is believed to participate in the regulation of cellular lipid transport (35Borst P. Zelcer N. van Helvoort A. Biochim. Biophys. Acta. 2000; 1486: 128-144Crossref PubMed Scopus (261) Google Scholar, 36Kaminski W.E. Orso E. Diederich W. Klucken J. Drobnik W. Schmitz G. Biochem. Biophys. Res. Commun. 2000; 273: 532-538Crossref PubMed Scopus (122) Google Scholar). Consequently Yamano et al. (34Yamano G. Funahashi H. Kawanami O. Zhao L.X. Ban N. Uchida Y. Morohoshi T. Ogawa J. Shioda S. Inagaki N. FEBS Lett. 2001; 508: 221-225Crossref PubMed Scopus (234) Google Scholar) suggested that ABCA3 may function as a transporter of the phospholipid components of pulmonary surfactant. We report that ABCA3 was identified by immunoprecipitation from purified lamellar bodies of rat lung with mAb 3C9 confirming the previous results in another species. The ABCA3 transcript was found in cell lines of human lung origin, in human, rat, and mouse ATII cells with the highest expression of ABCA3 mRNA observed in ATII cells. The transcript was also observed in significant quantities in several non-lung tissues in rats. The protein appears to be developmentally and hormonally regulated with a pattern similar to other surfactant-related proteins. Antibodies generated against peptides derived from the human ABCA3 sequence recognized a 180-kDa lamellar body protein in human fetal ATII cells. These together with previous results establish the presence of a preferentially expressed and developmentally regulated ABC transporter in lamellar bodies of ATII cell. Preliminary reports of this work have been published as abstracts (37Mulugeta S. Annan R.S. Notarfancesco K. Fisher A.B. Shuman H. Am. J. Respir. Crit. Care Med. 2000; 161: 43Google Scholar, 38Mulugeta S. Gonzales L.W. Ballard P.L. Fisher A.B. Shuman H. FASEB J. 2001; 15: A496Google Scholar). The human full-length ABCA3 cDNA was prepared by amplifying three overlapping ∼2-kb segments of cDNA by reverse transcription (RT)-PCR method according to the manufacturer's recommendations (Advantage, CLONTECH, Palo Alto, CA). Total RNA of cells and tissues was transcribed by enzymatic reverse transcription followed by PCR amplification. The three segments chosen had unique restriction sites in their overlapping regions, namely BamHI,DraIII, and BsmI, and each were ligated into a TOPO-TA vector (pCR 2.1-TOPO, Invitrogen, Carlsbad, CA). Following amplification of the plasmids in competent bacterial cells (Invitrogen), the three segments were cut with the aforementioned restriction enzymes and were ligated to each other. The primers (primer nucleotide numbering is based on the data base, from the National Center for Biotechnology (NCBI), data accession number NM001089, of the human ABCA3) generated for these segments are as follows: forward primers, 5′-GACCACCTACTTCTCTAGCAGCACTGGGCG-3′, corresponding to nucleotides −47 to −18 from the START (ATG) codon; 5′-GGGCCATCTGGGATCTTCTTC-3′, corresponding to nucleotides 2661–2681; and 5′-GAGGGGGGCGGCTTTAATGAGCGGTGCCTTGTG-3′, corresponding to nucleotides 3602–3634; reverse primers, 5′-GGTAGGTCGAGGGACGGGACGTCATGGTCGTGC-3′, corresponding to nucleotides 3111–3143; 5′-CTGCACGCACGGAAGTGC-3′, corresponding to nucleotides 4061–4078; and 5′-CGGGAGTACGGGTCCTACGTCGTAGCGTAGAGG-3′, corresponding to nucleotides 5841–5873. The 310-bp ABCA3 cDNAs of cells and tissues were prepared by RT-PCR using the following primers: forward primer, 5′-GCGAGTGCGCGGCTTTTCCCTCCGAGAAGGACTT-3′, corresponding to nucleotides 874–906; and reverse primer, 5′-CCGGATGTACCCAGGTTCTCCGCCATCAGGGGA-3′ (complement to bases corresponding to the peptide SPDGGEPGYIR), corresponding to nucleotides 1151–1183. EGFP/ABCA3 cDNA fusion constructs were generated by ligating the full-length hABCA3 cDNA into a pEGFP-N1 amino-terminal protein fusion vector (CLONTECH) after removing the STOP (TGA) codon of ABCA3 by cutting at an internal BglI restriction site located near the STOP site of ABCA3. All resultant plasmids were transformed into competent bacterial cells (Invitrogen) for amplification. Two peptides from putative antigenic regions of the deduced amino acid sequence of human ABCA3 were synthesized and used to immunize rabbits. These regions of the protein have no homologies to any other proteins in the NCBI data base and no homologies to any known rabbit sequence. The peptides prepared were CQEKERRLKEYM (ABCA3 luminal loop) and CGKPRAVAGKE (ABCA3 cytosolic domain) producing antibody 1 and antibody 2, respectively. Protein samples were separated with SDS-PAGE under reducing conditions. Samples were solubilized in sample buffer (125 mm Tris/HCl, 0.32 m sucrose, 2% (w/v) SDS, 65 mm dithiothreitol, and 0.001% bromphenol blue, pH 6.8) at room temperature. The separated proteins were transferred electrophoretically onto nitrocellulose membranes (BA83, 0.32-μm pore size; Schleicher & Schuell) overnight at 20 mA in transfer buffer (12.5 mm Tris, pH 8.3, 96 mmglycine, 0.1% SDS, 15% (v/v) methanol, pH 8.0). Protein-binding sites were blocked by TBS containing 2–5% non-fat dry milk for 60 min at room temperature. The membrane was then incubated with primary antibody in TBS/milk solution for 2 h at room temperature. After three 15-min washes with TBS, the nitrocellulose was incubated for another 60 min at room temperature in TBS/milk solution containing horseradish peroxidase-conjugated anti-mouse, -rat, or, -rabbit IgG (1:2000 normal dilution). Blots were visualized by enhanced chemiluminescence (ECL System, Amersham Biosciences). Total RNA was prepared from cells and tissues using RNeasy Mini Kit (Qiagen, Valencia, CA) and separated by formaldehyde-agarose gel electrophoresis and transferred to nitrocellulose membrane (BA83, 0.32-μm pore size; Schleicher & Schuell). Blots were prehybridized in 50 mm sodium phosphate, pH 6.5, 5× SSC, 5× Denhardt's, 50% formamide, 0.1% SDS, and 100 μg/ml salmon sperm DNA for 4 h at 42 °C. Hybridization was carried out overnight in the same buffer containing 106 cpm/ml of a 32P-labeled 310-bp ABCA3 cDNA fragment. The filters were washed twice for 15 min in 1× SSC, 0.1% SDS at room temperature and then twice for 20 min in 0.2× SSC, 0.1% SDS at 55 °C, and the filters were exposed to a PhosphorImaging screen for 24–72 h. The intensities of signals on the autoradiogram were quantified on a PhosphorImager using the Quantity One computer software (Bio-Rad). To correct for RNA loading, the obtained signals were normalized with the densitometer quantified ethidium bromide-stained 28 S or 18 S bands. Animal protocols were reviewed and authorized by the Institutional Animal Care and Use Committees of both the University of Pennsylvania and The Children's Hospital of Philadelphia (Philadelphia, PA). “Timed-pregnant” Wistar rats (mating day = Gestational Day (GD) 1, term = GD 22) and newborn Wistar rats were used. Pregnant rats were delivered by Caesarian section at GD 17, 19, and 21 or were allowed to deliver naturally. Neonatal rats were designated to be 1-day-old postnatal day (PD) 1. PD 1, 4, 7, 14, and 16-week-old (as adult) rats were subjected to study. All animals were sacrificed by cutting the abdominal aorta after a surgical level (toe pinch) of anesthesia was induced with intraperitoneal injection of pentobarbital (<50 mg/kg). Cells in culture were fixed in 2% paraformaldehyde for 30 min, permeabilized with 0.3% Triton X-100, and washed. Lung tissues were cryosectioned (5–8 μm), and sections adhered to slides. The slides were incubated in NaBH4 to reduce tissue autofluorescence, permeabilized with Triton X-100, and washed in phosphate-buffered saline (PBS) (137 mm NaCl, 10 mm Na2HPO4, 2.7 mm KCl, 1.8 mm KH2PO4, pH 7.4). After washing, cells or sections were blocked with 5% normal goat serum, washed with PBS, incubated with primary antibody for 2 h at room temperature, washed, and incubated with fluorescent Texas Red-conjugated secondary IgG for 1 h. Specimens were mounted in Mowiol and examined by fluorescence microscopy. Antigen retrieval by anti-ABCA3 was carried out as described previously (39Robinson J.M. Vandre D.D. Histochem. Cell Biol. 2001; 116: 119-130Crossref PubMed Scopus (58) Google Scholar). Briefly, cultured cells on glass cover slips were rinsed in serum-free medium, fixed with 4% paraformaldehyde, and washed with PBS. Cells were permeabilized with 0.3% Triton X-100, washed with PBS, and treated with 1% SDS for 5 min before continuing with immunofluorescence described above. Samples were observed with inverted Nikon fluorescence microscopes equipped with either a cooled CCD camera and MetaMorph image analysis software (Universal Imaging, West Chester, PA) or Bio-Rad Microradiance 2000 confocal attachment (Bio-Rad). Human fetal lungs were obtained from 14- to 22-week gestation therapeutic abortions under protocols approved by the Committee on Human Research at The Children's Hospital of Philadelphia. Fetal lung parenchyma was minced into 1-mm3 pieces and placed in organ culture, as described previously (40Wagle S. Bui A. Ballard P.L. Shuman H. Gonzales J. Gonzales L.W. Am. J. Physiol. 1999; 277: L381-L390PubMed Google Scholar). Briefly, tissue pieces were distributed in two parallel strips on 60-mm culture dishes placed on a platform that rocks (3 oscillations/min) to expose the explants alternately to serum-free Waymouth medium (2 ml/dish) or an atmosphere of 95% air, 5% CO2. The explants were maintained for 1–5 days in medium without (control) or with dexamethasone (Dex, 10 nm) 8-bromo-cAMP (0.1 mm) plus isobutylmethylxanthine (IBMX, 0.1 mm), or both Dex and the cAMP agents. IBMX was added to all explants treated with 8-bromo-cAMP to maintain tissue cAMP levels. Fresh medium was added every 24 h. Tissue explants were harvested either before culture (preculture) or at various times during culture. Hormones were added to the medium of treated explants after 24 h of culture. The hormone concentrations used maximally stimulated phosphatidylcholine synthesis in the explants as described previously (41Gonzales L.W. Ballard P.L. Gonzales J. Biochim. Biophys. Acta. 1994; 1215: 49-58Crossref PubMed Scopus (23) Google Scholar). ATII cells were isolated using elastase essentially according to the method of Dobbs and Williams (42Dobbs L.G. Gonzalez R. Williams M.C. Am. Rev. Respir. Dis. 1986; 134: 141PubMed Google Scholar). The lungs of anesthetized Sprague-Dawley rats were perfused via the pulmonary artery and excised. The lungs were lavaged with elastase solution, chopped, placed in a flask, and shaken. The tissue was filtered through graded filters; the cells were centrifuged and then resuspended in minimal essential media without serum. The cells were “panned” on IgG plates to remove the macrophages and lymphocytes and replated in minimal essential media with 10% fetal calf serum. Every preparation of isolated ATII cells was identified by morphology and by staining of lamellar bodies with phosphine 3R (Pfaltz & Bauer, Inc.) or Nile Red (Sigma). Viability was monitored by exclusion of trypan blue. Rare cultures with more than l0% macrophages were discarded. Fetal ATII precursor cells were isolated from human (14- to 22-week gestational age) lung using collagenase-trypsin digestion and differential adhesion to remove fibroblasts and plated on coverslips coated with extracellular matrix produced by Madin-Darby canine kidney cells (43Alcorn J.L. Smith M.E. Smith J.F. Margraf L.R. Mendelson C.R. Am. J. Respir. Cell Mol. Biol. 1997; 17: 672-682Crossref PubMed Scopus (86) Google Scholar, 44Gonzales L.W. Angampalli S. Guttentag S.H. Beers M.F. Feinstein S.I. Matlapudi A. Ballard P.L. Pediatr. Pathol. Mol. Med. 2001; 20: 387-412Crossref PubMed Scopus (46) Google Scholar). Final cultures contained fewer than 10% fibroblasts. Cells were cultured in Waymouth's medium in 35-mm dishes. The following day, Dex (10 nm), cAMP (0.1 mm), and IBMX (0.1 mm) (DCI) were added to the media for the remainder of the culture period. Media were changed daily, and cells were studied at days 1 and 4 of culture. Fetal human lung treatment results in the differentiation of the precursor cells into ATII-like cells that contain lamellar bodies, express SP-A, -B, and –C mRNAs, process SP-B and -C proprotein to mature forms, and have regulated exocytosis of phospholipids (45Gonzales L.W. Ballard P.L. Pediatr. Res. 2001; 49: 262AGoogle Scholar). Lamellar bodies were isolated from rat lungs cleared of blood or from human lung explants using upward flotation on a sucrose density gradient, as described previously (46Chander A. Dodia C.R. Gil J. Fisher A.B. Biochim. Biophys. Acta. 1983; 753: 119-129Crossref PubMed Scopus (49) Google Scholar). The lamellar body fraction was collected between 0.35 and 0.50 m sucrose and pelleted in 0.20 m sucrose by centrifugation at 20,000 × g for 20 min. To isolate lamellar body membranes, the freshly isolated lamellar body fraction was suspended in hypotonic solution (10 mm Tris-HCl, 50 mm sucrose, pH 7.2) along with protease inhibitors (2 mmphenylmethylsulfonyl fluoride, 2 mg/mlN-acetyl-leucyl-leucyl-norleucinal, 5 mg/ml leupeptin, 10 mg/ml aprotinin, 10 mg/ml pepstatin A) and set on ice for 3 h or overnight at 4 °C. The suspension was loaded on a cushion of 0.50m sucrose and centrifuged for 1 h at 100,000 ×g using a swinging bucket rotor. The lamellar body membrane fraction was recovered from the pellet, whereas the lamellar body content fraction was recovered at the interface of buffer and 0.50m sucrose. Total RNA from day 4 control and hormone (DCI)-treated cells was used to prepare cDNAs and biotin-labeled cRNAs according to the protocol provided by the manufacturer (Affymetrix, Santa Clara, CA). We performed hybridization with microarray chip 9000183 (Affymetrix), which contains probes for ∼5,600 human genes, and analyzed data using Affymetrix software. Two separate experiments were performed with cells from lungs of 14- and 17-week gestation. Immunoprecipitation was carried out, as described previously (47Xie J. Drumm M.L., Ma, J. Davis P.B. J. Biol. Chem. 1995; 270: 28084-28091Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), with slight modifications. Lamellar body membrane fraction was resuspended in ice-cold PBS. To reduce nonspecific binding, the lamellar body membrane suspension was incubated with-end-over-end rotation for 90 min at 4 °C with 10 μl of preimmune serum (normal goat serum, Jackson ImmunoResearch) followed by 40 μl of protein A-Sepharose (Amersham Biosciences). After centrifugation, the supernatant was transferred into a clean tube and incubated with mAb 3C9 on end-to-end rotation for 90 min at 4 °C. Antibody complex was then precipitated with 50 μl of protein A-Sepharose by incubation for 90 min at 4 °C with end-over-end rotation. The immune complex protein A-Sepharose was collected by centrifugation and washed 4 times with ice-cold lysis buffer (150 mm NaCl, 50 mm Tris-Cl, pH 8.0, 1% Triton X-100, 1% deoxycholate, 0.1% SDS), and the bound proteins were solubilized with 25 μl of SDS-PAGE sample buffer (48Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207002) Google Scholar) (10% glycerol, 5% β-mercaptoethanol, 2.3% SDS, and 0.062 m Tris-HCl, pH 6.8). The Sepharose beads were removed by centrifugation, and the proteins were resolved by 10% SDS-PAGE. Transfection was performed by the FuGENE 6 method (Roche Molecular Biochemicals) according to the manufacturer's recommendation. 4 μg of pEGFP-N1-tagged ABCA3-containing plasmid was preincubated with 6 μl of FuGENE 6 in a total volume of 100 ml of serum-free medium at room temperature for 15–30 min. The complex mixture was added dropwise to 60–70% confluent cells in a 35-mm dish containing 3 ml of fresh medium with 10% fetal calf serum. Cells were incubated at 37 °C for various times, and medium was replaced 3 days after start of transfection. Experiments were analyzed by one-way analysis of variance. All values were expressed as mean ± S.E. All computations were performed using SigmaStat 2.0 statistical analysis software (Jandel Corp., Chicago, IL). The lamellar body membrane-specific protein LBM180 was identified previously using monoclonal antibody, mAb 3C9 (25Zen K. Notarfrancesco K. Oorschot V. Slot J.W. Fisher A.B. Shuman H. Am. J. Physiol. 1998; 275: L172-L183PubMed Google Scholar). To confirm this fin
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