Some ABCA3 mutations elevate ER stress and initiate apoptosis of lung epithelial cells

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.