Abstract
BackgroundPulmonary emphysema is characterized histologically by destruction of alveolar walls and enlargement of air spaces due to lung epithelial cell apoptosis. Cell adhesion molecule 1 (CADM1) is an immunoglobulin superfamily member expressed in lung epithelial cells. CADM1 generates a membrane-associated C-terminal fragment, αCTF, through A disintegrin- and metalloprotease-10-mediated ectodomain shedding, subsequently releasing the intracellular domain (ICD) through γ-secretase-mediated intramembrane shedding of αCTF. αCTF localizes to mitochondria and induces apoptosis in lung epithelial cells. αCTF contributes to the development and progression of emphysema as a consequence of increased CADM1 ectodomain shedding. The purpose of this study was to examine whether the ICD makes a similar contribution.ResultsThe ICD was synthesized as a 51-amino acid peptide, and its mutant was synthesized by substituting seven amino acids and deleting two amino acids. These peptides were labeled with fluorescein isothiocyanate and were introduced into various cell lines. ICD peptide-derived fluorescence was well visualized in lung epithelial cells at the site of Mitotracker mitochondrial labeling, but was detected in locations other than mitochondria in other cell types. Mutant peptide-derived fluorescence was detected in locations other than mitochondria, even in lung epithelial cells. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays revealed that transduction of the ICD peptide increased the proportion of apoptotic cells 2- to 5-fold in the lung epithelial cell lines, whereas the mutant peptide did not. Abundance of the ICD was below the Western blot detection limit in emphysematous (n = 4) and control (n = 4) human lungs. However, the ICD was detected only in emphysematous lungs when it was immunoprecipitated with anti-CADM1 antibody (4/4 vs. 0/4, P = 0.029).ConclusionsAs the abundance of ICD molecules was sparse but present, increased CADM1 shedding appeared to contribute to the development of emphysema by generating αCTF and the ICD in lung epithelial cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12929-015-0173-8) contains supplementary material, which is available to authorized users.
Highlights
Pulmonary emphysema is characterized histologically by destruction of alveolar walls and enlargement of air spaces due to lung epithelial cell apoptosis
Amino acid residues are numbered according to the NCBI database sequence. b The unlabeled and fluorescein isothiocyanate (FITC)-labeled C-intracellular domain (ICD) and C-ICDmut peptides were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and by silver staining or Western blotting of the gels using antibodies against cell adhesion molecule 1 (CADM1) or FITC. c NCI-H441 and RLE-6TN cells were introduced with the FITC-labeled C-ICD or C-ICDmut peptide and stained with Mitotracker
The C-ICD localized in mitochondria, induced apoptosis in lung epithelial cells, and was scarce in abundance but present in emphysematous lungs
Summary
Pulmonary emphysema is characterized histologically by destruction of alveolar walls and enlargement of air spaces due to lung epithelial cell apoptosis. Pulmonary emphysema is a representative chronic obstructive pulmonary disease characterized by destruction of alveolar walls and enlargement of air spaces [1] These histological characteristics evolve from alveolar and bronchiolar epithelial cell apoptosis and a local imbalance in protease over anti-protease activities [2, 3]. CADM1 is an intercellular adhesion molecule in the immunoglobulin superfamily This membrane-spanning glycoprotein is composed of three extracellular Ig-like domains, a single transmembrane region, and a short carboxy-terminal intracytoplasmic tail with a protein 4.1 interaction sequence (P4.1-IS) and a PDZ type II domain-binding motif (PDZ-BM) [5]. We found that CADM1 shedding increases in emphysematous lungs, and αCTF contributes to apoptosis of lung epithelial cells by localizing in mitochondria [4]. A mutant form of αCTF (αCTFmut) carrying amino acid substitutions and deletions in the intervening region between the P4.1-IS and the PDZ-BM did not localize to mitochondria, suggesting that the intervening region may carry the mitochondrial localization signal [4]
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