The intracellular domain of cell adhesion molecule 1 is present in emphysematous lungs and induces lung epithelial cell apoptosis

Abstract

Background: Pulmonary 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.

Results: The 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).

Conclusions: As 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.

Fig. 1

Mitochondrial localization of the cell adhesion molecule 1 intracellular domain (C-ICD) peptide in lung epithelial cell lines. a Full-length amino acid sequences of the α-C-terminal fragment (αCTF) and the αC-terminal fragment mutant (αCTFmut) of cell adhesion molecule 1. The C-ICD and the cell adhesion molecule 1 intracellular domain mutant (C-ICDmut) are shown in the boxed portions. The arrow indicates the γ-secretase cleavage site. Dots indicate the amino acid residues as above. The 7-amino acid substitution and the 2-amino acid deletion in C-ICDmut are shown in italics and –, respectively. The structural motifs are underlined. TM, transmembrane domain; P4.1-IS, protein 4.1 interaction sequence; PDZ-BM, PDZ type II domain-binding motif. Amino acid residues are numbered according to the NCBI database sequence (accession number, CCD32610). b The unlabeled and FITC-labeled C-ICD and C-ICDmut peptides were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and by silver staining (left) or Western blotting of the gels using antibodies against cell adhesion molecule 1 (CADM1) (middle) or FITC (right). c NCI-H441 (upper) and RLE-6TN (lower) cells were introduced with the FITC-labeled C-ICD or C-ICDmut peptide and stained with Mitotracker. Green (FITC) and red (Mitotracker) fluorescent images were merged, as shown in the right panels. Bar = 10 μm

Fig. 2

Induction of lung epithelial cell apoptosis by the cell adhesion molecule 1 intracellular domain (C-ICD) peptide. NCI-H441 and RLE-6TN cells were treated with Xfect solutions containing the unlabeled C-ICD or the cell adhesion molecule 1 intracellular domain mutant (C-ICDmut) peptide or were left untreated (−). The cells were analyzed the next day by Western blotting using anti-cell adhesion molecule 1 (CADM1) antibody (a). Arrowheads indicate immunoreactive bands corresponding to full-length CADM1, the C-ICD. The blots were reprobed with anti-β-actin antibody to indicate protein loading. Another set of cells was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay (b). The mean proportions of TUNEL-positive cells (depicted by arrows) and standard errors are indicated under the panels. The P-values were determined by Student s t-test. Bar = 50 μm. c Similar TUNEL assays were done using the FITC-labeled C-ICD peptide (C-ICD-FITC). The mean proportions of TUNEL-positive cells and standard errors were calculated in each of FITC-positive and -negative cells. Cells double positive for FITC and TUNEL are depicted by arrowheads. The P-values were determined by Student’s t-test. Bar = 20 μm

Fig. 3

Detection of the cell adhesion molecule 1 intracellular domain (C-ICD) in emphysematous lungs. a Western blot analysis of cell adhesion molecule 1 (CADM1) expression in normal and emphysematous lungs. Cases are numbered as in Table 1. Arrowheads indicate the immunoreactive bands corresponding to full-length CADM1, the β-C-terminal fragment (βCTF), the αCTF, and the C-ICD. Bands corresponding to the non-glycosylated full-length form and βCTF are depicted by one and two asterisks, respectively. The blot was reprobed with anti-β-actin antibody to indicate protein loading. b The lung lysates were subjected to immunoprecipitation using anti-CADM1 antibody and to Western blotting using the same antibody. Immunoreactive bands were detected after short and longer exposure times

Fig. 4

CADM1 isoforms expressed in human lungs and lung epithelial cells. Total RNAs were extracted from rat primary cultured type 1 and type 2 AECs, and were analyzed by RT-PCR using primer sets for calveolon-1 and surfactant protein B (a). Total RNAs from NCI-H441 and RLE-6TN human lung epithelial cell lines, rat type 1 and type 2 AECs and human lung tissues of cases indicated were analyzed by RT-PCR using a primer set encompassing the CADM1 extracellular juxtamembrane region, susceptible to alternative splicing (b). The PCR products were electrophoresed on 3 % agarose gels, together with CADM1 isoform size markers (rightmost lane in B). L, 100 base pair (bp) ladder. RNAs were also PCR-amplified using a primer set for G3PDH to indicate RNA loading per lane