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Weichert, Nina (2011): Some ABCA3 mutations elevate ER stress and initiate apoptosis of lung epithelial cells. Dissertation, LMU München: Faculty of Medicine
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Abstract

Mutations in the gene coding for the ATP binding cassette protein A3 (ABCA3) are known as the most frequent genetic cause of fatal neonatal respiratory distress syndrome and chronic interstitial lung disease (ILD) of children. ABCA3 transporter is localized to the limiting membrane of lamellar bodies, organelles for assembly and storage of pulmonary surfactant in alveolar epithelial type II cells. It transports surfactant phospholipids into lamellar bodies and is essential for their biogenesis. 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). This study showed previously not examined cellular dysfunction in cultured lung epithelial A549 cells overexpressing the three ABCA3 mutations R43L, R280C and L101P. All three mutations were found in children with ABCA3-associated lung disease either with fatal neonatal respiratory distress syndrome (L101P and R43L) or chronic pediatric ILD (R280C). Cell biology of R43L and R280C mutations was studied here for the first time. L101P mutation was used as a known example of the trafficking/folding defect leading to the ER retention of ABCA3 protein. Human lung epithelial A549 cells were transfected with vectors containing 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- and apoptosis-proteins, Annexin V surface staining and determination of the intracellular glutathion level. Induction of epithelial-mesenchymal transition (EMT) was assessed by immunoblotting. It was demonstrated 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 apoptosis in A549 cells. A549 cells expressing L101P additionally gain a mesenchymal phenotype. R43L mutation, resulting in a functional defect of the properly localized ABCA3, had no effect on intracellular stress and apoptotic signaling. These data suggest that expression of partially or completely ER localized ABCA3 mutant proteins induce raised intracellular stress and apoptotic cell death of the affected cells, which are factors that might contribute to the pathogenesis of genetic ILD via a fatal ER-stress/apoptosis/fibrogenesis-axis.