Introduction
Idiopathic interstitial pneumonias (IIPs) are a diverse group of diffuse parenchymal lung diseases of unknown etiology characterized by various degrees of acute or chronic inflammation and progressive fibrosis of the lung parenchyma. IIPs comprise several entities, including acute interstitial pneumonia (AIP), nonspecific interstitial pneumonia (NSIP), cryptogenic organizing pneumonia (COP), and usual interstitial pneumonia (UIP) or idiopathic pulmonary fibrosis (IPF) [
1]. These entities share many features but are sufficiently different from one another in terms of their typical histological patterns. Accordingly, NSIP is further subclassified into cellular (c-NSIP) and fibrosing (f-NSIP) types [
2]. This subtyping is of clinical importance, because the 5-year survival rate of c-NSIP is nearly 100 %, while that of f-NSIP is intermediate between those of c-NSIP and UIP, showing the worst rate among all of the entities [
2]. Although the etiology of IIP remains enigmatic, there is a growing body of evidence supporting the theory that alveolar epithelial cell (AEC) injury and apoptosis contribute to the development and progression of IIP [
3,
4]. In UIP, apoptotic AECs are found in areas of morphologically unaffected alveoli, suggesting that AEC apoptosis is a primary event prior to the onset of fibrosis and subsequently disrupts the basement membrane integrity, leading to various degrees of interstitial responses characterized by inflammatory cell infiltration and fibroblast recruitment [
5]. Additionally, apoptosis in AIP is reportedly responsible for the resolution of type II AECs (AECIIs) during the early phase [
6]. Molecular pathways that induce AEC apoptosis have been analyzed mainly in the setting of UIP or IPF. Intrinsic apoptotic markers (p53, p21, Bax, and caspase-3) and antiapoptotic markers (bcl-2) are increased and decreased, respectively, within AECs [
7]. The extrinsic apoptotic factors Fas and Fas ligand are upregulated in AECs and infiltrating leukocytes, respectively [
8]. Other mechanisms include increased oxidative stress products (hydrogen peroxide, myeloperoxidase, and nitric oxide) [
9,
10], increased endoplasmic reticulum stress [
11], and activation of hypoxia-inducible factor-1α [
12]. Although AEC apoptosis is assumed to be a key event regardless of the IIP subtype, it remains unclear whether there is one AEC apoptosis mechanism common to all entities versus several mechanisms specific to each entity.
Cell adhesion molecule 1 (CADM1), also known as tumor suppressor in lung cancer 1 (TSLC1), is an intercellular adhesion molecule that belongs to the immunoglobulin (Ig) superfamily [
13]. This membrane-spanning glycoprotein comprises three extracellular Ig-like domains, a single transmembrane region, and a short carboxy-terminal intracytoplasmic tail with a protein 4.1 interaction sequence and a PDZ type II domain-binding motif [
13]. Various types of cells express CADM1, including pulmonary cells, biliary cells, renal tubular epithelial cells, neurons, mast cells, and pancreatic endocrine cells [
14‐
17]. In epithelia, CADM1 is located on the lateral cell membrane and mediates neighboring cell–cell adhesion via
trans-homophilic binding [
18,
19]. Recent studies have shown that CADM1 expression is regulated by post-transcriptional mechanisms, including glycosylation and proteolytic cleavage, referred to as shedding [
20,
21]. The ectodomain of CADM1 is cleaved at one of two sites, yielding two membrane-associated C-terminal fragments, αCTF and βCTF [
21]. We recently identified CADM1 shedding as a key event in the development of pulmonary emphysema, a representative chronic obstructive pulmonary disease characterized by alveolar wall destruction and enlarged air spaces [
22]. CADM1 ectodomain shedding rates increase in emphysematous lungs, and the mitochondrial localization of αCTF contributes to lung epithelial cell apoptosis [
22].
In the present study, we evaluated the data of 39 patients diagnosed with IIP from the autopsy record archive of Kinki University Hospital and histologically classified them into four subtypes: AIP, f-NSIP, COP, and UIP. According to a previously described method [
23], protein extracts were prepared from the autopsied lung paraffin sections and examined for CADM1 expression by Western blotting. We found that CADM1 ectodomain shedding increased and the full-length CADM1 level decreased in IIP lungs, with statistical significance being different among the subtypes. We examined whether these alterations in expression might be involved in AEC apoptosis in IIP, both
in vivo and
in vitro. This study identifies CADM1 shedding as a possible pathogenic event common to the four subtypes of IIP.
Discussion
In the present study, we examined CADM1 expression in four histologic subtypes of IIP and found that CADM1 α-shedding was increased in all four subtypes and that the full-length CADM1 level was decreased in f-NSIP. We also found that the α- or (α + β)-shedding rate and the full-length CADM1 level were correlated with one another and with the proportion of ssDNA-positive AECIIs, suggesting a significant contribution of CADM1 shedding to AECII apoptosis in IIP lungs. Two mechanisms for these phenomena are proposed. One involves αCTF, an α-shedding product of CADM1. As we previously showed in emphysematous lungs, once αCTF is produced in AECIIs, it accumulates preferentially in the mitochondria and consequently depolarizes the mitochondrial membrane potential, resulting in activation of the mitochondrial apoptotic pathway in AECIIs [
22]. As shown in Fig.
3, CADM1 was often detected in the cytoplasm, but not on the cell membrane, of detaching AECIIs, suggesting accumulation of αCTF in the mitochondria. The notion that the mitochondrial apoptotic pathway is activated in IIP lung epithelial cells was described as early as 2002 by Kuwano et al. [
32]. The present study reinforces this notion and proposes αCTF as a key molecule in the activation of this pathway. Another mechanism involves the decrease in full-length CADM1 levels as a result of increased CADM1 shedding. As evidenced by the siRNA transfection experiment, decreased full-length CADM1 expression in lung epithelial cells appeared to be associated with increased apoptosis. We previously performed similar experiments and reported that downregulation of CADM1 by siRNA abrogates the formation of epithelial cell structure, suggesting an essential role for full-length CADM1 in the maintenance of epithelial cell polarity [
33]. Because loss of cell polarity can trigger apoptosis to eliminate damaged epithelial cells [
34,
35], full-length CADM1 downregulation supposedly causes AECII apoptosis through loss of epithelial polarity. Considering the present Western blot data, these two mechanisms may contribute differently to the pathogenesis of individual IIP subtypes; the αCTF mechanism profoundly impacts AIP, f-NSIP, and COP and relatively mildly impacts UIP, while the full-length CADM1 mechanism acts particularly on f-NSIP.
Among the four IIP subtypes examined, f-NSIP was notable, because it exhibited the highest shedding rates and expressed the lowest level of full-length CADM1 per epithelial cell, suggesting strong involvement of CADM1 shedding in the pathogenesis of f-NSIP. NSIP has some degree of overlap with a variety of other IIP subtypes [
2]. Yang et al. [
36] examined the gene expression profiles of NSIP and UIP and concluded that the two entities are similar transcriptionally. However, they identified eight transcripts that best differentiate the two, including the serine protease cathepsin G gene, which was upregulated in NSIP [
36]. Another study by Takahashi et al. [
37] reported that matrix metalloproteinase-2 (MMP-2) mRNA levels are higher in NSIP than in UIP. The fact that these two proteases are released from neutrophils and macrophages [
38,
39] is consistent with our clinical experiences showing that NSIP behaves as a more inflammatory process, in distinct contrast to UIP [
1,
2]. Both cathepsin G and MMP-2, once released, are executers of ectodomain shedding of Ig superfamily adhesion molecules, such as ICAM-1 or 5 and VCAM-1 expressed on bone marrow cells or neurons [
40‐
42]. The particularly increased CADM1 shedding in f-NSIP may be attributable to the high expression levels of these proteases.
The f-NSIP group also differed substantially from the UIP group with respect to the incidence of AECII apoptosis. Some previous studies have demonstrated that in UIP, AECII apoptosis occurs actively in morphologically unaffected lung parenchyma around the remodeled fibrotic lesion, but only occasionally within the fibrotic lesion, where the percentage of apoptotic AECIIs is approximately 1–5 % (in contrast, it is practically 0 % in the normal lung) [
5,
32,
43]. These values are consistent with our histological findings in the UIP fibrotic lesions and control lungs. Only a few referable studies on NSIP are available; these studies reported that 1-2 % of AECIIs were apoptotic in NSIP (whether the cellular or fibrosing subtype was not specified) [
32,
43]. Compared with this estimation, our measured values (10.4 ± 3.8 %) are quite high. This may represent a limitation of the present study, as we analyzed only autopsied cases. This sampling method might have introduced significant bias in the patient pool compositions. In fact, six of ten patients in the f-NSIP group and three of nine patients in the COP group died of IIP itself, although these IIP entities reportedly have fairly good 5-year survival rates [
2]. Our f-NSIP and COP groups may have comprised more patients with severe pathophysiology. Another interpretation of the present results is that active AECII apoptosis may contribute to treatment resistance and poor prognoses in patients with f-NSIP and COP.
Among the four subtypes, the incidence of AECII apoptosis was the highest in AIP, which is consistent with several past studies [
6,
44]. Serial activation of macrophages and neutrophils has long been believed to be an important pathogenic process for AECII injury in AIP, but how these cells act on AECIIs is not entirely clear. When activated, these inflammatory cells potently release a variety of products, including proteases such as cathepsin G and MMP-2, as described above. Inflammatory cell infiltration may cause local increases in proteolytic activities and thereby promote AECII apoptosis by increasing CADM1 shedding in AIP lungs.
In conclusion, the present study demonstrated a close link between increased CADM1 ectodomain shedding and increased AECII apoptosis in IIP and suggests a pathophysiologic commonality between IIP and pulmonary emphysema at the molecular level. Following our previous characterization of CADM1 as a human pancreatic islet cell adhesion molecule involved in hormone secretion [
18], we recently found that CADM1 shedding was increased in type two diabetes mellitus pancreata and involved in β cell apoptosis [
23]. Taken together, our findings indicate that CADM1 shedding may trigger apoptosis in cells that require CADM1 to fulfill their proper function. Although the precise mechanisms of increased CADM1 shedding in individual diseases remain to be clarified, it is noteworthy that ectodomain shedding is a proteolytic process mediated by proteases. As shown in pulmonary emphysema [
22], protease-over-antiprotease activity imbalance may be a precedent key event in lesions in which CADM1-expressing cell apoptosis occurs. Although this study identified the molecular distinctions among IIP subtypes, it also suggests that IIP, regardless of the subtype, may share a pathogenic molecular process with more common diseases, such as type two diabetes mellitus and pulmonary emphysema.
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Competing Interests
The authors declare that they have no competing interests.
Author’s Contributions
AY carried out the Western blotting and immunohistochemical examinations, and performed the statistical analysis. MH and TK participated in the Western blot analysis. TI participated in the immunohistochemistry. TM and MO carried out the cell biological experiments and performed the statistical analysis. EE provided the human samples and participated in the pathological examination. AI conceived and designed the study, carried out the pathological examination, and drafted the manuscript. All authors read and approved the final manuscript.