Search

When epithelial cells in vivo are stimulated to proliferate, they crowd and often grow in height. These processes are likely to implicate dynamic interactions among lateral membranous proteins, such as cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member. Pulmonary epithelial cell lines that express CADM1, named NCI-H441 and RLE-6TN, were grown to become overconfluent in the polarized 2D culture system, and were examined for the expression of CADM1. Western analyses showed that the CADM1 expression levels increased gradually up to 3 times in a cell density-dependent manner. Confocal microscopic observations revealed dense immunostaining for CADM1 on the lateral membrane. In the overconfluent monolayers, CADM1 knockdown was achieved by two methods using CADM1 -targeting siRNA and an anti-CADM1 neutralizing antibody. Antibody treatment experiments were also done on 6 other epithelial cell lines expressing CADM1. The CADM1 expression levels were reduced roughly by half, in association with cell height decrease by half in 3 lines. TUNEL assays revealed that the CADM1 knockdown increased the proportion of TUNEL-positive apoptotic cells approximately 10 folds. Increased expression of CADM1 appeared to contribute to cell survival in crowded epithelial monolayers.

Epithelial carcinomas occasionally have sarcomatous components that consist primarily of spindle and cuboidal cells, which often resemble osteoblasts. Sarcomatoid carcinomas consist of similar cells. Recent studies have characterized these phenomena as a manifestation of epithelial-mesenchymal transition in carcinoma cells, but the mesenchymal phenotypes that manifest in sarcomatous cells of epithelial carcinomas are not well understood. Here, we examined the expression profiles of four osteoblastic differentiation biomarkers in the sarcomatous components of multiple carcinoma types, including five renal clear cell, four breast invasive ductal, two esophageal, one maxillary squamous cell, three larynx, three lung, one liver, and one skin sarcomatoid carcinoma. Expression was analyzed by immunohistochemistry using antibodies against cell adhesion molecule 1, a member of the IgCAM superfamily, osterix transcription factor (Osterix), cluster of differentiation 151, a transmembrane 4 superfamily member, and alkaline phosphatase. Immunostaining intensity was rated in scale 0 (negative), 0.5 (weak), and 1 (strong) for each marker, and the four scale values were summed to calculate osteoblastic scores. In all, 10 cases had a osteoblastic score ≥3, and all of these 10 cases were cell adhesion molecule 1- and Osterix-positive. Eight and five of the nine samples with a osteoblastic score <3 were negative for cell adhesion molecule 1 ( p < 0.0001) and Osterix ( p = 0.006), respectively. The other markers showed no statistical significance. These results indicate that osteoblastic differentiation can occur in carcinoma cells and that cell adhesion molecule 1 could be a useful marker for identifying this phenomenon in carcinoma tissues.

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.

Background: Lung alveolar epithelial cell (AEC) apoptosis has attracted attention as an early pathogenic event in the development of idiopathic interstitial pneumonia (IIP); however, the causative mechanism remains unclear. Cell adhesion molecule 1 (CADM1) is an AEC adhesion molecule in the immunoglobulin superfamily. It generates a membrane-associated C-terminal fragment, αCTF, through protease-mediated ectodomain shedding, termed α-shedding. Increased CADM1 α-shedding contributes to AEC apoptosis in emphysematous lungs., Methods: Formalin-fixed, paraffin-embedded lung lobes (n = 39) from 36 autopsied patients with IIP were classified as acute IIP (n = 10), fibrosing-type nonspecific IIP (f-NSIP, n = 10), cryptogenic organizing IIP (n = 9), and usual IIP (n = 10). CADM1 expression in the lung sections was examined by western blotting and compared with control lungs (n = 10). The rate of CADM1 α-shedding was calculated as the relative amount of αCTF to full-length CADM1, and the full-length CADM1 level was estimated per epithelial cell by normalization to cytokeratin 7, a lung epithelial marker. Apoptotic AECs were detected by immunohistochemistry for single-stranded DNA (ssDNA). NCI-H441 and A549 human lung epithelial cells were transfected with small interfering RNA (siRNA) to silence CADM1 expression and analyzed by terminal nucleotide nick end labeling assays., Results: The rate of CADM1 α-shedding was higher in all IIP subtypes than in the control (P ≤ 0.019), and the full-length CADM1 level was lower in f-NSIP (P = 0.007). The α-shedding rate and full-length CADM1 level were correlated with each other (P = 0.015) and with the proportion of ssDNA-positive AECs (P ≤ 0.024). NCI-H441 cells transfected with siRNA exhibited a 61 % lower rate of expression of full-length CADM1 and a 17-fold increased proportion of apoptotic cells. Similar results were obtained with A549 cells., Conclusions: CADM1 α-shedding appeared to be increased in all four IIP subtypes and consequently contributed to AEC apoptosis by decreasing the full-length CADM1 level. This mechanism particularly impacted f-NSIP. The molecular mechanism causing AEC apoptosis may be similar between IIP and emphysema.

Pulmonary emphysema and type 2 diabetes mellitus (T2DM), both caused by lifestyle factors, frequently concur. Respectively, the diseases affect lung alveolar and pancreatic islet cells, which express cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member. Protease-mediated ectodomain shedding of full-length CADM1 produces C-terminal fragments (CTFs) with proapoptotic activity. In emphysematous lungs, the CADM1 shedding rate and thus the level of CTFs in alveolar cells increase. In this study, CADM1 expression in islet cells was examined by western blotting. Protein was extracted from formalin-fixed, paraffin-embedded sections of pancreata isolated from patients with T2DM (n = 12) or from patients without pancreatic disease (n = 8) at autopsy. After adjusting for the number of islet cells present in the adjacent section, we found that full-length CADM1 decreased in T2DM islets, while ectodomain shedding increased. Hemoglobin A1c levels, measured when patients were alive, correlated inversely with full-length CADM1 levels (P = 0.041) and positively with ectodomain shedding rates (P = 0.001). In immunofluorescence images of T2DM islet cells, CADM1 was detected in the cytoplasm, but not on the cell membrane. Consistently, when MIN6-m9 mouse beta cells were treated with phorbol ester and trypsin to induce shedding, CADM1 immunostaining was diffuse in the cytoplasm. When a form of CTFs was exogenously expressed in MIN6-m9 cells, it localized diffusely in the cytoplasm and increased the number of apoptotic cells. These results suggest that increased CADM1 ectodomain shedding contributes to blood glucose dysregulation in T2DM by decreasing full-length CADM1 and producing CTFs that accumulate in the cytoplasm and promote apoptosis of beta cells. Thus, this study has identified a molecular alteration shared by pulmonary emphysema and T2DM.

Rationale: Alveolar epithelial cell apoptosis and protease/antiprotease imbalance based proteolysis play central roles in the pathogenesis of pulmonary emphysema but molecular mechanisms underlying these two events are not yet clearly understood. Cell adhesion molecule 1 (CADM1) is a lung epithelial cell adhesion molecule in the immunoglobulin superfamily. It generates two membrane associated C terminal fragments (CTFs), αCTF and βCTF, through protease mediated ectodomain shedding., Objective: To explore the hypothesis that more CADM1-CTFs are generated in emphysematous lungs through enhanced ectodomain shedding, and cause increased apoptosis of alveolar epithelial cells., Methods and Results: Western blot analyses revealed that CADM1-CTFs increased in human emphysematous lungs in association with increased ectodomain shedding. Increased apoptosis of alveolar epithelial cells in emphysematous lungs was confirmed by terminal nucleotide nick end labelling (TUNEL) assays. NCI-H441 lung epithelial cells expressing mature CADM1 but not CTFs were induced to express αCTF both endogenously (by shedding inducers phorbol ester and trypsin) and exogenously (by transfection). Cell fractionation, immunofluorescence, mitochondrial membrane potentiometric JC-1 dye labelling and TUNEL assays revealed that CADM1-αCTF was localised to mitochondria where it decreased mitochondrial membrane potential and increased cell apoptosis. A mutation in the intracytoplasmic domain abrogated all three abilities of αCTF., Conclusions: CADM1 ectodomain shedding appeared to cause alveolar cell apoptosis in emphysematous lungs by producing αCTF that accumulated in mitochondria. These data link proteolysis to apoptosis, which are two landmark events in emphysema.

Aims: An immunohistochemical screen for mouse embryos showed that cell adhesion molecule 1 (CADM1), which is an immunoglobulin superfamily member, was expressed in developing bones. Here, we determined the cell types expressing CADM1 and examined its usefulness in the differential diagnosis of osteosarcoma., Main Methods: Serial sections of murine developing mandibles were stained with anti-CADM1 antibody, by a coloring substrate reactive to alkaline phosphatase (ALP), a broad osteoblastic marker for preosteoblasts to osteoblasts, and by in situ hybridization for osteopontin (OPN), a marker for mature osteoblasts. CADM1 immunohistochemistry was also performed on human remodeling bones, osteosarcomas and other soft tissue tumors., Key Findings: CADM1 immunohistochemistry for the mandible revealed that morphologically identifiable osteoblasts expressed CADM1 on their plasma membranes, but neither osteocytes nor bone lining cells did. At the mandibular margin, not only OPN-positive cells but also OPN-negative, ALP-positive cells were CADM1-positive, whereas inside the mandible, OPN-positive cells were often CADM1-negative. Clear membranous staining was detected in the majority of osteosarcomas (46/57), whereas only 13% (6/46) of the other soft tissue tumors were CADM1-positive (P<0.001)., Significance: These results indicated that CADM1 was a novel osteoblastic adhesion molecule that is expressed transiently during osteoblastic maturation, and a useful diagnostic marker for osteosarcoma cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cell adhesion molecule 1 (CADM1) on mast cells promotes attachment and communication with neurons by homophilic binding. However, we found that mast cell CADM1 was responsible for both the attachment of mast cells to dorsal root ganglia (DRG) neurites and their calcium responses to activated DRG neurites, despite the low expression of CADM1 in DRG. Instead, nectin-3 was expressed on DRG neurons and localized to regions of cell-cell contact. A neutralizing antibody to nectin-3 inhibited both mast cell attachment and subsequent calcium responses. This suggests that heterophilic binding between CADM1 and nectin-3 mediates functional DRG-mast cell interactions in vitro., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: The tumor suppressor genes CADM1/TSLC1 and DAL-1/4.1B are frequently inactivated by promoter methylation in non-small cell lung cancer. The proteins they encode, CADM1 and 4.1B, form a complex in human epithelial cells and are involved in cell-cell adhesion., Methods: Expression of CADM1 and 4.1B proteins was examined by immunohistochemistry in 67 primary breast cancer and adjacent noncancerous tissues. CADM1 and 4.1B messenger RNA (mRNA) was detected by reverse-transcription polymerase chain reaction (RT-PCR). The methylation status of the CADM1 and 4.1B promoters was determined quantitatively by bisulfite treatment followed by pyrosequencing., Results: CADM1 and 4.1B protein signals were detected along the cell membrane in normal mammary epithelia. By contrast, 47 (70%) and 49 (73%) of 67 primary breast cancers showed aberrant CADM1 and 4.1B staining, respectively. Aberrant CADM1 staining was more frequently observed in pT2 and pT3 tumors and for stages II and III (P = 0.045 and P = 0.020, respectively), while aberrant 4.1B staining was more often observed in tumors with lymph node metastasis, for pT2 and pT3 tumors, and for stages II and III (P = 0.0058, P = 0.0098, and P = 0.0007, respectively). Furthermore, aberrant CADM1 and 4.1B expression was preferentially observed in invasive relative to noninvasive lesions from the same specimen (P = 0.036 and P = 0.0009, respectively). Finally, hypermethylation of CADM1 and 4.1B genes was detected in 46% and 42% of primary breast cancers, respectively., Conclusions: Our findings suggest that aberrant CADM1 and 4.1B expression is involved in progression of breast cancer, especially in invasion into the stroma and metastasis.

Renal clear cell carcinoma (RCCC) is the most frequent subpopulation of renal cell carcinoma and is derived from the proximal uriniferous tubules. We have previously reported that an actin-binding protein, 4.1B/DAL-1, is expressed in renal proximal tubules, whereas it is inactivated in 45% of RCCC by promoter methylation. In the lung and several epithelial tissues, 4.1B is shown to associate with a tumor suppressor protein, CADM1, belonging to the immunoglobulin-superfamily cell adhesion molecules. Here, we demonstrate by immunohistochemistry that another member of the CADM-family protein, CADM4, as well as 4.1B is expressed specifically in human proximal tubules, while CADM1 and 4.1N, another member of the 4.1 proteins, are expressed in the distal tubules. Immunoprecipitation analysis coupled with Western blotting revealed that CADM4 associated with 4.1B, while CADM1 associated with 4.1N in the lysate from normal human kidney, implicating that a cascade of CADM4 and 4.1B plays an important role in normal cell adhesion of the proximal tubules. On the other hand, CADM4 expression was lost or markedly reduced in 7 of 10 (70%) RCC cell lines and 28 of 40 (70%) surgically resected RCCC, including 10 of 16 (63%) tumors with T1a. CADM4 expression was more preferentially lost in RCCC with vascular infiltration (p = 0.04), suggesting that loss of CADM4 is involved in tumor invasion. Finally, introduction of CADM4 into an RCC cell line, 786-O, dramatically suppressed tumor formation in nude mice. These findings suggest that CADM4 is a novel tumor suppressor candidate in RCCC acting with its binding partner 4.1B., (Copyright © 2011 UICC.)

Cell adhesion molecule 1 (CADM1) is a type I transmembrane glycoprotein expressed in various tissues. CADM1 is a cell adhesion molecule with many functions, including roles in tumor suppression, apoptosis, mast cell survival, synapse formation, and spermatogenesis. CADM1 undergoes membrane-proximal cleavage called shedding, but the sheddase and mechanisms of CADM1 proteolysis have not been reported. We determined the cleavage site involved in CADM1 shedding by LC/MS/MS and showed that CADM1 shedding occurred in the membrane fraction and was inhibited by tumor necrosis factor-α protease inhibitor-1 (TAPI-1). An siRNA experiment revealed that ADAM10 mediates endogenous CADM1 shedding. In addition, the membrane-bound fragment generated by shedding was further cleaved by γ-secretase and generated CADM1-intracellular domain (ICD) in a mechanism called regulated intramembrane proteolysis (RIP). These results clarify the detailed mechanism of membrane-proximal cleavage of CADM1, suggesting the possibility of RIP-mediated CADM1 signaling., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Cell adhesion molecule-1 (CADM1) is a recently identified adhesion molecule of pancreatic islet α-cells that mediates nerve-α-cell interactions via trans-homophilic binding and serves anatomical units for the autonomic control of glucagon secretion. CADM1 also mediates attachment between adjacent α-cells. Since gap junctional intercellular communication (GJIC) among islet cells is essential for islet hormone secretion, we examined whether CADM1 promotes GJIC among α-cells and subsequently participates in glucagon secretion regulation. Dye transfer assays using αTC6 mouse α-cells, which endogenously express CADM1, supported this possibility; efficient cell-to-cell spread of gap junction-permeable dye was detected in clusters of αTC6 cells transfected with nonspecific, but not with CADM1-targeting, siRNA. Immunocytochemical analysis of connexin 36, a major component of the gap junction among αTC6 cells, revealed that it was localized exclusively to the cell membrane in CADM1-non-targeted αTC6 cells, but diffusely to the cytoplasm in CADM1-targeted cells. Next, we incubated CADM1-targeted and non-targeted αTC6 cells in a medium containing 1 mM glucose and 200 mM arginine for 30 min to induce glucagon secretion, and found that the targeted cells secreted three times more glucagon than did the non-targeted. We conducted similar experiments using pancreatic islets that were freshly isolated from wild-type and CADM1-knockout mice, and expressed glucagon secretion as ratios relative to baseline values. The increase in ratio was larger in CADM1-knockout islets than in wild-type islets. These results suggest that CADM1 may serve as a volume limiter of glucagon secretion by sustaining α-cell attachment necessary for efficient GJIC.

CADM1 is a multifunctional cell adhesion molecule expressed predominantly in the nerve system, testis and lung. The expression of the Cadm1 gene is induced during the neural differentiation of murine embryonal carcinoma P19 cells by treatment with retinoic acid (RA). Here, we show that the suppression of CADM1 expression using RNAi interfered with P19 cell aggregation and reduced cell populations expressing MAP2 after RA treatment. Nonaggregated P19 cells were not differentiated into neurons, suggesting that CADM1 participates in the aggregate formation and neuronal differentiation of P19 in vitro. A luciferase assay of a series of deletion mutants of the CADM1 promoter localized an RA-responsive cis-acting element to an approximately 90-bp fragment upstream of the translational start site. This element contains a putative binding site for transcription factor Sp1, named Sp1-binding site-1 (Sp1BS-1). Sp1BS-1 and adjacent Sp1-binding sites (Sp1BS-2 and Sp1BS-3) showed enhanced transcriptional activity by RA. Moreover, a chromatin immunoprecipitation showed that RA receptor (RAR)α was associated with a DNA fragment containing Sp1BS-1, whereas suppression of RARα expression using siRNA reduced the responsiveness of the CADM1 promoter to RA. These results suggest that Sp1 plays a critical role in RA-induced CADM1 expression through possible interaction with RARα in the neural differentiation of P19., (© 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.)

Close apposition of nerve and mast cells is viewed as a functional unit of neuro-immune mechanisms, and it is sustained by trans-homophilic binding of cell adhesion molecule-1 (CADM1), an Ig superfamily member. Cerebral nerve-mast cell interaction might be developmentally modulated, because the alternative splicing pattern of four (a-d) types of CADM1 transcripts drastically changed during development of the mouse cerebrum: developing cerebrums expressed CADM1b and CADM1c exclusively, while mature cerebrums expressed CADM1d additionally and predominantly. To probe how individual isoforms are involved in nerve-mast cell interaction, Neuro2a neuroblastoma cells that express CADM1c endogenously were modified to express additionally either CADM1b (Neuro2a-CADM1b) or CADM1d (Neuro2a-CADM1d), and they were cocultured with mouse bone marrow-derived mast cells (BMMCs) and BMMC-derived cell line IC-2 cells, both of which expressed CADM1c. BMMCs were found to adhere to Neuro2a-CADM1d neurites more firmly than to Neuro2a-CADM1b neurites when the adhesive strengths were estimated from the femtosecond laser-induced impulsive forces minimally required for detaching BMMCs. GFP-tagging and crosslinking experiments revealed that the firmer adhesion site consisted of an assembly of CADM1d cis-homodimers. When Neuro2a cells were specifically activated by histamine, intracellular Ca(2+) concentration was increased in 63 and 38% of CADM1c-expressing IC-2 cells that attached to the CADM1d assembly site and elsewhere, respectively. These results indicate that CADM1d is a specific neuronal isoform that enhances nerve-mast cell interaction, and they suggest that nerve-mast cell interaction may be reinforced as the brain grows mature because CADM1d becomes predominant.

The malignant Hodgkin/Reed-Sternberg (HRS) cells of Hodgkin lymphoma (HL) are believed to derive from germinal center (GC) B cells, but lack expression of a functional B cell receptor. As apoptosis is the normal fate of B-cell receptor-negative GC B cells, mechanisms that abrogate apoptosis are thus critical in HL development, such as epigenetic disruption of certain pro-apoptotic cancer genes including tumor suppressor genes. Identifying methylated genes elucidates oncogenic mechanisms and provides valuable biomarkers; therefore, we performed a chemical epigenetic screening for methylated genes in HL through pharmacological demethylation and expression profiling. IGSF4/CADM1/TSLC1, a pro-apoptotic cell adhesion molecule of the immunoglobulin superfamily, was identified together with other methylated targets. In contrast to its expression in normal GC B cells, IGSF4 was down-regulated and methylated in HL cell lines, most primary HL, and microdissected HRS cells of 3/5 cases, but not in normal peripheral blood mononuclear cells and seldom in normal lymph nodes. We also detected IGSF4 methylation in sera of 14/18 (78%) HL patients but seldom in normal sera. Ectopic IGSF4 expression decreased HL cells survival and increased their sensitivity to apoptosis. IGSF4 induction that normally follows heat shock stress treatment was also abrogated in methylated lymphoma cells. Thus, our data demonstrate that IGSF4 silencing by CpG methylation provides an anti-apoptotic signal to HRS cells important in HL pathogenesis.

The cell adhesion protein immunoglobulin superfamily 4A (IGSF4A) is expressed on the surfaces of spermatogenic cells in the mouse testis. During spermatogenesis, IGSF4A is considered to bind to the surface of Sertoli cells in a heterophilic manner. To identify this unknown partner of IGSF4A, we generated rat monoclonal antibodies against the membrane proteins of mouse Sertoli cells grown in primary culture. Using these monoclonal antibodies, we isolated a clone that immunostained Sertoli cells and reacted with the product of immunoprecipitation of the homogenate of mouse testis with anti-IGSF4A antibody. Subsequently, to identify the Sertoli cell membrane protein that is recognized by this monoclonal antibody, we performed expression cloning of a cDNA library from the mouse testis. As a result, we identified poliovirus receptor (PVR), which is another IGSF-type cell adhesion molecule, as the binding partner of IGSF4A. The antibodies raised against PVR and IGSF4A immunoprecipitated both antigens in the homogenate of mouse testis. Immunoreactivity for PVR was present in Sertoli cells but not in spermatogenic cells at all stages of spermatogenesis. Overexpression of PVR in TM4, a mouse Sertoli cell line, increased more than three-fold its capacity to adhere to Tera-2, which is a human cell line that expresses IGSF4A. These findings suggest that the heterophilic binding of PVR to IGSF4A is responsible, at least in part, for the interaction between Sertoli and spermatogenic cells during mouse spermatogenesis.

Unlabelled: Spermatogenic immunoglobulin superfamily (SgIGSF) is an intercellular adhesion molecule of the nectin-like family. While screening its tissue distribution, we found that it was expressed in fetal liver but not adult liver. In the present study, we examined which cells in developing and regenerating liver express SgIGSF via immunohistochemistry and Western blot analysis. In developing mouse liver, SgIGSF expression was transiently upregulated at perinatal ages and was restricted to the lateral membrane of biliary epithelial cells (BECs). In regenerating rat livers from the 2-acetylaminofluorene/partial hepatectomy model, SgIGSF was detected exclusively in oval cells that aligned in ductal and trabecular patterns by the second week posthepatectomy. In human livers, fetal and newborn bile ducts and cirrhotic bile ductules were clearly positive for SgIGSF, whereas disease-free adult bile ducts were negative. To investigate the role of SgIGSF in bile duct/ductule formation, we used an in vitro model in which rat hepatocyte aggregates embedded in collagen gels containing insulin and epidermal growth factor extend epithelial sheets and processes in the first week and form ductules within a month. The process and ductular cells were continuously positive for SgIGSF and cytokeratin 19, a BEC marker. When the aggregate culture was started in the presence of a function-blocking anti-SgIGSF antibody, the number of epithelial processes per aggregate was reduced by 80%., Conclusion: We propose that SgIGSF is a novel and functional BEC adhesion molecule that is expressed for a limited time during active bile duct/ductule formation.

Accumulating evidence has so far indicated that cross-talk between the nervous and immune systems plays a pivotal role in the pathophysiology of various diseases. As a prototypic demonstration of neuro-immune systems, the interaction between nerves and mast cells has been examined intensively. Anatomically, mast cells are often located in close proximity to nerves. Functionally, both cells communicate with each other in a bi-directional manner. Substance P released from nerves and proteases and cytokines from mast cells have proved to be important mediators in such communication. On the other hand, the molecules involved in membrane-membrane contacts between nerves and mast cells were largely unknown. In 2003, both cells were found to express the identical adhesion molecule, named SynCAM (synaptic cell adhesion molecule) or SgIGSF (spermatogenic immunoglobulin superfamily). Since SgIGSF/SynCAM binds homophilically, its involvement in nerve-mast cell interaction was examined in vitro. Superior cervical ganglia expressed SgIGSF/SynCAM along their neurites. Adhesion to these neurites of mast cells lacking SgIGSF/SynCAM was poor, and this was normalized by ectopic expression of SgIGSF/SynCAM. Moreover, SgIGSF/SynCAM-expressing mast cells were more competent in communicating with the neurites. Further understanding of the adhesion molecule-dependent interaction will be expected to open a new avenue in the field of neuro-immune cross-talk.

Microphthalmia transcription factor (MITF) is a basic-helix-loop-helix-leucine zipper-type transcription factor. The mutant mi and Mi(wh) alleles encode MITFs with deletion and alteration of a single amino acid, respectively, whereas the tg is a null mutation. In coculture with NIH/3T3 fibroblasts, the numbers of cultured mast cells (CMCs) derived from C57BL/6 (B6)(mi/mi), B6(Miwh/Miwh), and B6(tg/tg) mice that adhered to NIH/3T3 fibroblasts were one third as large as the number of B6(+/+) CMCs that adhered to NIH/3T3 fibroblasts. From a cDNA library of B6(+/+) CMCs, we subtracted messenger RNAs expressed by B6(mi/mi) CMCs and found a clone encoding SgIGSF, a recently identified member of the immunoglobulin superfamily. Northern and Western blot analyses revealed that SgIGSF was expressed in B6(+/+) CMCs but not in CMCs derived from MITF mutants. Immunocytochemical analysis showed that SgIGSF localized to the cell-to-cell contact areas between B6(+/+) CMCs and NIH/3T3 fibroblasts. Transfection of B6(mi/mi) and B6(tg/tg) CMCs with SgIGSF cDNA normalized their adhesion to NIH/3T3 fibroblasts. NIH/3T3 fibroblasts did not express SgIGSF, indicating that SgIGSF acts as a heterophilic adhesion molecule. Transfection of B6(tg/tg) CMCs with normal MITF cDNA elevated their SgIGSF expression to normal levels. These results indicated that SgIGSF mediated the adhesion of CMCs to fibroblasts and that the transcription of SgIGSF was critically regulated by MITF.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) first infects the host nasal mucosa, where the viral spike protein binds to angiotensin-converting enzyme 2 (ACE2) on the mucosal cells. This study aimed at searching host cell surface molecules that could contribute to the infection in two views; abundance on host cells and affinity to the spike protein. Since the nasal mucosa is lined by respiratory and olfactory epithelia, and both express an immunoglobulin superfamily member cell adhesion molecule 1 (CADM1), whether CADM1 would participate in the spike protein binding was examined. Immunohistochemistry on the mouse nasal cavity detected CADM1 strongly in the olfactory epithelium at cell-cell contacts and on the apical surface but just faintly in the respiratory epithelium. In contrast, ACE2 was detected in the respiratory, not olfactory, epithelium. When mice were administered intranasally with SARS-CoV-2 S1 spike protein and an anti-CADM1 ectodomain antibody separately, both were detected exclusively on the olfactory, not respiratory, epithelium. Then, the antibody and S1 spike protein were administered intranasally to mice in this order with an interval of 1 h. After 3 h, S1 spike protein was detected as a protein aggregate floating in the nasal cavity. Next, S1 spike protein labeled with fluorescein was added to the monolayer cultures of epithelial cells exogenously expressing ACE2 or CADM1. Quantitative detection of fluorescein bound to the cells revealed that S1 spike protein bound to CADM1 with affinity half as high as to ACE2. Consistently, docking simulation analyses revealed that S1 spike protein could bind to CADM1 three-quarters as strongly as to ACE2 and that the interface of ACE2 was similar in both binding modes. Collectively, intranasal S1 spike protein appeared to prefer to accumulate on the olfactory epithelium, and CADM1 was suggested to contribute to this preference of S1 spike protein based on the molecular abundance and affinity. [ABSTRACT FROM AUTHOR]

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the human gastrointestinal tract. Small intestinal GISTs appear to be associated with poorer prognosis and higher metastasis rate than gastric GISTs of the same size and mitotic index. Recently, we reported that cell adhesion molecule 1 (CADM1) is expressed specifically in most small intestinal GISTs, but not in most gastric GISTs, suggesting that this difference in CADM1 expression between gastric GISTs and small intestinal GISTs might influence the difference in clinical behavior between them. The aim of the present study was to examine whether high CADM1 expression affected proliferation, migration, invasion, adhesion to endothelial cells and transendothelial migration of cultured GIST cells by comparing original GIST-T1 cells with very low CADM1 expression with GIST-T1 cells with high CADM1 expression induced by CADM1 cDNA transfection (GIST-T1-CAD cells). GIST-T1-CAD cells had reduced ability to proliferate, migrate and invade compared with the original GIST-T1 cells, but showed significantly higher ability to adhere to human umbilical vein endothelial cells and migrate through endothelial cell monolayers. Thus, CADM1 may contribute to higher metastasis rates in small intestinal GISTs facilitating tumor cell adhesion to vascular endothelial cell and transendothelial migration of tumor cells. CADM1 might serve as a potential target for inhibition of metastasis in small intestinal GISTs. [ABSTRACT FROM AUTHOR]

Cell adhesion molecule 1 (CADM1), a single-pass transmembrane protein, is involved in oncogenesis. We previously demonstrated the therapeutic efficacy of anti-CADM1 ectodomain monoclonal antibodies against mesothelioma; however, the underlying mechanism is unclear. In the present study, we explored the molecular behavior of anti-CADM1 antibodies in CADM1-expressing tumor cells. Sequencing analyses revealed that the anti-CADM1 chicken monoclonal antibodies 3E1 and 9D2 are IgY and IgM isotype antibodies, respectively. Co-administration of 3E1 and 9D2 altered the subcellular distribution of CADM1 from the detergent-soluble fraction to the detergent-resistant fraction in tumor cells. Using recombinant chicken–mouse chimeric antibodies that had been isotype-switched from IgG to IgM, we demonstrated that the combination of the variable region of 3E1 and the constant region of IgM was required for CADM1 relocation. Cytochemical studies showed that 3E1 colocalized with late endosomes/lysosomes after co-administration with 9D2, suggesting that the CADM1–antibody complex is internalized from the cell surface to intracellular compartments by lipid-raft mediated endocytosis. Finally, 3E1 was conjugated with the antimitotic agent monomethyl auristatin E (MMAE) via a cathepsin-cleavable linker. Co-administration of 3E1–monomethyl auristatin E and 9D2 suppressed the growth of multiple types of tumor cells, and this anti-tumor activity was confirmed in a syngeneic mouse model of melanoma. 3E1 and 9D2 are promising drug delivery vehicles for CADM1-expressing tumor cells. Proposed mechanism of the effects of 3E1 and 9D2 on CADM1 relocation. (1) 9D2-IgM interferes with CADM1 homophilic trans -binding in cell–cell interactions. (2) 3E1-IgG (along with 9D2-IgM) strongly binds to cell-surface CADM1, inducing CADM1 clustering by the combined effect of 3E1 epitope binding and the IgM structure, leading to CADM1 relocation to detergent-resistant microdomains (DRM). (3) As a result of clustering in DRMs, the CADM1–antibody complex is internalized by lipid raft-mediated endocytosis. [Display omitted] [ABSTRACT FROM AUTHOR]

Cell adhesion molecules act as tumor suppressors primarily by cell attachment activity, but additional mechanisms modifying signal transduction are suggested in some cases. Cell adhesion molecule 1 (CADM1), a membrane-spanning immunoglobulin superfamily, mediates intercellular adhesion by trans-homophilic interaction and acts as a tumor suppressor. Here, we investigated CADM1-associated proteins comprehensively using proteomic analysis of immune-precipitates of CADM1 by mass spectrometry and identified a transmembrane adaptor protein, Csk-binding protein (Cbp), known to suppress Src-mediated transformation, as a binding partner of CADM1. CADM1 localizes to detergent-resistant membrane fractions and co-immunoprecipitated with Cbp and c-Src. Suppression of CADM1 expression using siRNA reduces the amount of co-immunoprecipitated c-Src with Cbp and activates c-Src in colon cancer cells expressing both CADM1 and Cbp. On the other hand, co-replacement of CADM1 and Cbp in colon cancer cells lacking CADM1 and Cbp expression suppresses c-Src activation, wound healing and tumorigenicity in nude mice. Furthermore, expression of Cbp and CADM1 was lost in 55% and 83% of human colon cancer, respectively, preferentially in tumors with larger size and/or lymph node metastasis. CADM1 would act as a colon tumor suppressor by intervening oncogenic c-Src signaling through binding with Cbp besides its authentic cell adhesion activity. • CADM1 associates with Cbp and stabilizes c-Src-Cbp complex on the cell membrane. • Suppression of CADM1 by siRNA activates c-Src in colon cancer cells. • Co-replacement of CADM1 and Cbp suppresses c-Src and tumor formation in mice. • Loss of CADM1 and Cbp was observed preferentially in advanced colon cancer. • CADM1 suppresses colon cancer in both Src-dependent and independent manner. [ABSTRACT FROM AUTHOR]

Abstract Aims To determine cellular distribution of cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member, in the human oxyntic gastric mucosa, and to explore possible involvement in the development and peritoneal dissemination of signet ring cell (SRC) gastric carcinoma, which often develops in the oxyntic mucosa. Main methods Immunohistochemistry and double immunofluorescence were conducted on surgical specimens of normal and SRC-bearing stomachs and peritoneal metastatic foci of SRCs. KATO-III (lacking CADM1) and HSC-43 (expressing CADM1) SRC cell lines were cocultured on a Met-5A mesothelial or TIG-1 fibroblastic cell monolayer. Key findings In the oxyntic gland, some neck and nearly all base glandular cells were CADM1-positive, and mucin 5AC-positive cells were CADM1-negative, while some mucin 6-positive neck cells were CADM1-positive. Foveolar-epithelial, parietal, and endocrine cells were CADM1-negative. CADM1 was negative in all SRC carcinomas that were confined within the submucosa (n = 11) and all but one of those invading deeper (n = 15). In contrast, peritoneal metastatic foci of SRCs were CADM1-positive in five out of eleven cases (P < 0.01). In the cocultures, exogenous CADM1 made KATO-III cells adhere more and grow faster on a Met-5A monolayer, not on TIG-1 monolayers. HSC-43 cells adhered more and grew faster on Met-5A than on TIG-1 monolayers, which were partly counteracted by a function-neutralizing anti-CADM1 antibody. Significance Nearly all chief cells and a part of mucous neck cells express CADM1. SRC gastric carcinoma appears to emerge as a CADM1-negative tumor, but CADM1 may help SRCs develop peritoneal dissemination through promoting their adhesion and growth in the serosal tissue. [ABSTRACT FROM AUTHOR]

CADM1 encodes a multifunctional immunoglobulin-like cell adhesion molecule whose cytoplasmic domain contains a type II PSD95/Dlg/ZO-1 (PDZ)-binding motif (BM) for associating with other intracellular proteins. Although CADM1 lacks expression in T lymphocytes of healthy individuals, it is overexpressed in adult T-cell leukemia-lymphoma (ATL) cells. It has been suggested that the expression of CADM1 protein promotes infiltration of leukemic cells into various organs and tissues, which is one of the frequent clinical manifestations of ATL. Amino acid sequence alignment revealed that Tiam1 (T-lymphoma invasion and metastasis 1), a Rac-specific guanine nucleotide exchange factor, has a type II PDZ domain similar to those of membrane-associated guanylate kinase homologs (MAGUKs) that are known to bind to the PDZ-BM of CADM1. In this study, we demonstrated that the cytoplasmic domain of CADM1 directly interacted with the PDZ domain of Tiam1 and induced formation of lamellipodia through Rac activation in HTLV-I-transformed cell lines as well as ATL cell lines. Our results indicate that Tiam1 integrates signals from CADM1 to regulate the actin cytoskeleton through Rac activation, which may lead to tissue infiltration of leukemic cells in ATL patients. [ABSTRACT FROM AUTHOR]

Cell adhesion molecule 1 (CADM1) mediates interepithelial adhesion and is upregulated in crowded epithelial monolayers. This study aimed to examine CADM1 expression in the human endometrium of proliferative and secretory phases, and its transcriptional regulation in terms of estrogen stimuli and higher cellularity. CADM1 immunohistochemistry was conducted on endometrial tissues from women in their 40s and adult mice subcutaneously injected with estradiol following ovariectomy. Dual-luciferase reporter assays were conducted using human endometrial HEC-50B and HEC-1B cells and reporter plasmids harboring the human CADM1 3.4-kb promoter and its deleted and mutated forms. Cells were transfected with estrogen receptor α cDNA and reporter plasmids, and treated with estradiol before luciferase activity measurement. Immunohistochemistry revealed that CADM1 was clearly expressed on the lateral membranes of the simple columnar glandular cells in the proliferative phase, but not in the secretory phase, from both women and the mouse model. The glandular cell density increased two-fold in the proliferative phase. Reporter assays identified three Sp1-binding sites as estradiol-responsive elements in the proximal region (from −223 to −84) of the transcription start site (+1) in HEC-50B cells. When the cell culture was started at eight-fold higher cell density, the CADM1 3.4-kb promoter was transactivated at a two-fold higher level in HEC-50B cells. This cell density effect was not detected for the CADM1 2.3-kb or 1.6-kb promoter. Two (proximal and distal) promoter regions are suggested to function additively to transactivate CADM1 in endometrial glandular cells that crowd in the proliferative phase. [ABSTRACT FROM AUTHOR]