title: Some ABCA3 mutations elevate ER stress and initiate apoptosis of lung epithelial cells.
abstract: BACKGROUND
  ABCA3 transporter (ATP-binding cassette transporter of the A subfamily) is localized to the limiting membrane of lamellar bodies, organelles for assembly and storage of pulmonary surfactant in alveolar epithelial type II cells (AECII). It transports surfactant phospholipids into lamellar bodies and absence of ABCA3 function disrupts lamellar body biogenesis. Mutations of the ABCA3 gene lead to fatal neonatal surfactant deficiency and chronic interstitial lung disease (ILD) of children. ABCA3 mutations can result in either functional defects of the correctly localized ABCA3 or trafficking/folding defects where mutated ABCA3 remains in the endoplasmic reticulum (ER).
  
  
  METHODS
  Human alveolar epithelial A549 cells were transfected with vectors expressing wild-type ABCA3 or one of the three ABCA3 mutant forms, R43L, R280C and L101P, C-terminally tagged with YFP or hemagglutinin-tag. Localization/trafficking properties were analyzed by immunofluorescence and ABCA3 deglycosylation. Uptake of fluorescent NBD-labeled lipids into lamellar bodies was used as a functional assay. ER stress and apoptotic signaling were examined through RT-PCR based analyses of XBP1 splicing, immunoblotting or FACS analyses of stress/apoptosis proteins, Annexin V surface staining and determination of the intracellular glutathion level.
  
  
  RESULTS
  We demonstrate that two ABCA3 mutations, which affect ABCA3 protein trafficking/folding and lead to partial (R280C) or complete (L101P) retention of ABCA3 in the ER compartment, can elevate ER stress and susceptibility to it and induce apoptotic markers in the cultured lung epithelial A549 cells. R43L mutation, resulting in a functional defect of the properly localized ABCA3, had no effect on intracellular stress and apoptotic signaling.
  
  
  CONCLUSIONS
  Our data suggest that expression of partially or completely ER localized ABCA3 mutant proteins can increase the apoptotic cell death of the affected cells, which are factors that might contribute to the pathogenesis of genetic ILD.
authors.affiliation: Pediatric Pneumology, Dr. von Hauner Children's Hospital, Ludwig-Maximilians University, Munich, Germany.
authors.forename: Nina
authors.initials: N
authors.lastname: Weichert
authors.forename: Eva
authors.initials: E
authors.lastname: Kaltenborn
authors.forename: Andreas
authors.initials: A
authors.lastname: Hector
authors.forename: Markus
authors.initials: M
authors.lastname: Woischnik
authors.forename: Andrea
authors.initials: A
authors.lastname: Schams
authors.forename: Andreas
authors.initials: A
authors.lastname: Holzinger
authors.forename: Sunčana
authors.initials: S
authors.lastname: Kern
authors.forename: Matthias
authors.initials: M
authors.lastname: Griese
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism; D017398:Alternative Splicing; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000235:genetics; D053148:Caspase 3 / Q000378:metabolism; D053471:Caspases, Initiator / Q000378:metabolism; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002469:Cell Separation / Q000379:methods; D004268:DNA-Binding Proteins / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000473:pathology*; D000091342:Endoplasmic Reticulum Chaperone BiP; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005978:Glutathione / Q000378:metabolism; D006031:Glycosylation; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D008168:Lung / Q000378:metabolism / Q000473:pathology*; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D009154:Mutation; D009363:Neoplasm Proteins / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000071701:Regulatory Factor X Transcription Factors; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013312:Stress, Physiological / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection; D000071717:X-Box Binding Protein 1; D059666:Lysosomal-Associated Membrane Protein 3
chemical_list: D018528:ATP-Binding Cassette Transporters; D017304:Annexin A5; D053148:Caspase 3; D053471:Caspases, Initiator; D004268:DNA-Binding Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D005978:Glutathione; D006360:Heat-Shock Proteins; D051907:Lysosomal Membrane Proteins; D009363:Neoplasm Proteins; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D000071717:X-Box Binding Protein 1; C441524:ABCA3 protein, human; C114233:LAMP3 protein, human; D059666:Lysosomal-Associated Membrane Protein 3; C000605543:XBP1 protein, human; C505407:CASP3 protein, human; C093516:CASP4 protein, human