Potential Contribution of Cell Adhesion Molecule 1 to the Binding of SARS-CoV-2 Spike Protein to Mouse Nasal Mucosa.

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 hour. After 3 hours, 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.

Cell Adhesion Molecule 1 Contributes to Cell Survival in Crowded Epithelial Monolayers.

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.

Cell adhesion molecule-1 shedding induces apoptosis of renal epithelial cells and exacerbates human nephropathies.

Chronic kidney disease (CKD) is an important problem throughout the world, associated with the increase of blood urea nitrogen (BUN) and serum creatinine (sCre) and with renal tubular injuries. It is crucial to elucidate the molecular mechanisms of renal injuries to identify the new therapeutics and early diagnostic methods. We focused on cell adhesion molecule-1 (CADM1) protein. CADM1, its isoform SP4, is expressed in the epithelial cells of various tissues, including renal distal tubules, localized on the lateral cell membrane, mediates cell-cell adhesion via trans-homophilic binding, and interacts with various proteins. We previously reported that its expression was downregulated by post-proteolytic cleavage (α- and ß-shedding) in pulmonary diseases. To investigate whether CADM1 α-shedding occurs in human nephropathies, we performed Western blotting and immunohistochemical analysis of specimens with arterionephrosclerosis (AS) and diabetic nephropathy (DN) from autopsied kidneys. CADM1 α-shedding was induced in AS and DN kidneys and derived from the decrease in full-length CADM1 (FL-CADM1) and increase of the COOH-terminal fragment (α-CTF). In particular, the reduced FL-CADM1 level was correlated with tubular and tubulointerstitial injuries and the increases in BUN and sCre levels. Apoptosis of renal tubular epithelial cells (TECs) was promoted in both nephropathies, and it was significantly correlated with the decrease in the FL-CADM1. Furthermore, FL-CADM1 knockdown by small interfering RNA downregulated anti-apoptotic Bcl-2 protein and promoted apoptosis of cultured renal TECs. The present study suggests that the reduction of FL-CADM1 leads to renal TEC apoptosis and could exacerbate renal tubular and tubulointerstitial injuries, which contribute to the development of CKD.

Manifestation of osteoblastic phenotypes in the sarcomatous component of epithelial carcinoma and sarcomatoid carcinoma.

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.

Heart-bound adiponectin, not serum adiponectin, inversely correlates with cardiac hypertrophy in stroke-prone spontaneously hypertensive rats.

NEW FINDINGS: What is the central question of this study? An inverse correlation between circulating adiponectin and many diseases has been reported, but some studies have found no correlation. To evaluate this controversy, we investigated the relationship between heart-bound adiponectin and hypertension or cardiac hypertrophy, compared with serum adiponectin. What is the main finding and its importance? Using hypertensive and normotensive rats, we found that heart-bound adiponectin was inversely correlated with cardiac hypertrophy, suggesting that heart-bound adiponectin has a more important function in preventing cardiac hypertrophy than circulating adiponectin. Our study provides new insights regarding the role of adiponectin in diseases. The inverse correlation between circulating adiponectin concentration and hypertension or cardiac hypertrophy is still controversial. In addition to circulating adiponectin, adiponectin is also bound to tissues such as the heart and skeletal muscle. In this study, we investigated the relationship of serum adiponectin and heart-bound adiponectin with hypertension and cardiac hypertrophy. Four types of hypertensive rats presenting different blood pressure levels were used at different ages, as follows: normotensive Wistar-Kyoto rats (WKYs); two sub-strains (strains C and B2, having low and high blood pressure, respectively) of spontaneously hypertensive rats (SHRs); and stroke-prone SHRs (SHRSPs). Blood pressure, heart-to-body weight ratio, serum adiponectin and heart-bound adiponectin were determined. Histopathological analysis of the heart was carried out to evaluate the relationship with heart-bound adiponectin. Serum adiponectin concentration was not inversely correlated with blood pressure or heart-to-body weight ratio. In contrast, heart-bound adiponectin levels were significantly lower in SHRSPs than in other strains at respective ages. This resulted from a decrease in T-cadherin expression, which induced adiponectin binding to tissues. No significant difference in heart-bound adiponectin among WKYs and SHRs (C and B2) was detected, indicating that heart-bound adiponectin is not related to hypertension. In addition, differences in heart-bound adiponectin did not affect AMP-activated protein kinase in the traditional adiponectin activation cascade. Histopathological analysis revealed that heart-bound adiponectin was inversely correlated with cardiomyocyte hypertrophy and left ventricular wall thickness and, in part, with cardiac fibrosis. These results suggest that the decreased level of heart-bound adiponectin in SHRSPs is more related to their cardiac hypertrophy than circulating adiponectin.

Time-Course Statistical Evaluation of Intercellular Adhesion Maturation by Femtosecond Laser Impulse.

The maturation of intercellular adhesion is an essential process for establishing the signal transduction network in living cells. Although the maturation is naturally considered to enhance the signal transduction, the relationship between the signal transduction and the maturation process has not been revealed in detail using time-course data. Here, using a coculture of mast cells and neurites, differences in maturation between individual cells were estimated as a function of the adhesion strength by our original single-cell measurement method utilizing a laser-induced impulsive force. When an intense femtosecond laser is focused into a culture medium under a microscope, shock and stress waves are generated at the laser focal point that exert an impulsive force on individual cells. In our method, this impulse is used to break the adhesion between a mast cell and a neurite. The magnitude of the impulse is then quantified by a local force-measurement system utilizing an atomic force microscope, and the adhesion strength is estimated from the threshold of the impulse required to break the adhesion. The measurement is conducted within 1 min/cell, and thus, data on the individual differences of the adhesion strength can be obtained within only a few hours. Coculturing of neurites and mast cells for 4 h resulted in a specific adhesion that was stronger than the nonspecific adhesions between the substrate and mast cells. In the time-course investigation, we identified two distinct temporal patterns of adhesion: 1) the strength at 24 h was the same as the initial strength; and 2) the strength increased threefold from baseline and became saturated within 24 h. Based on these results, the distribution of CADM1 adhesion molecules in the neurites was suggested to be inhomogeneous, and the relationship between adhesion maturation and the signal-transduction process was considered.

Increased ectodomain shedding of cell adhesion molecule 1 as a cause of type II alveolar epithelial cell apoptosis in patients with idiopathic interstitial pneumonia.

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.

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

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.

Increased ectodomain shedding of lung epithelial cell adhesion molecule 1 as a cause of increased alveolar cell apoptosis in emphysema.

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 ßCTF, 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.

α-Parvin, a pseudopodial constituent, promotes cell motility and is associated with lymph node metastasis of lobular breast carcinoma.

Invasive lobular carcinoma (ILC) is more frequently lymph node positive than is invasive ductal carcinoma (IDC), and ILC cell infiltration shows distinctive histological characteristics, suggesting the action of ILC-specific invasion molecules. To identify such a molecule, we used a proteomic approach in the pseudopodia of MDA-MB-231 breast cancer cells. A pseudopodial constituent was identified using excimer laser ablation, two-dimensional difference gel electrophoresis, mass spectroscopy, and immunocytofluorescence. MDA-MB-231 cells were modified to express various levels of this constituent by transient transfection and were examined for pseudopodia formation and migratory abilities using wound healing and two-chamber assays. Immunohistochemical positivity of human breast cancer cells (56 ILCs and 21 IDCs) was compared with clinicopathological variables. An actin-binding adaptor protein, α-parvin, was found to localize to pseudopodia and to form focal adhesions in cells not induced to extend pseudopodia. Pseudopodial length and density and migratory abilities correlated with α-parvin expression. Twenty-one (37.5 %) ILCs stained positive for α-parvin, whereas the results were negative for all 21 IDCs (P < 0.001). α-Parvin positivity in ILC was significantly associated with lymphatic invasion (P = 0.038) and lymph node metastasis (P = 0.003) in univariate analyses and to lymph node metastasis (P = 0.020) in multivariate analyses. α-Parvin, a pseudopodial constituent, was found to promote migration of breast cancer cells and to be expressed exclusively by ILC, suggesting that α-parvin is an ILC-specific invasion molecule that may have clinical utility as a biomarker for aggressive subsets of ILC.

Noncontact estimation of intercellular breaking force using a femtosecond laser impulse quantified by atomic force microscopy.

When a femtosecond laser pulse (fsLP) is focused through an objective lens into a culture medium, an impulsive force (fsLP-IF) is generated that propagates from the laser focal point (O(f)) in a micron-sized space. This force can detach individual adherent cells without causing considerable cell damage. In this study, an fsLP-IF was reflected in the vibratory movement of an atomic force microscopy (AFM) cantilever. Based on the magnitude of the vibration and the geometrical relationship between O(f) and the cantilever, the fsLP-IF generated at O(f) was calculated as a unit of impulse [N-s]. This impulsive force broke adhesion molecule-mediated intercellular interactions in a manner that depended on the adhesion strength that was estimated by the cell aggregation assay. The force also broke the interactions between streptavidin-coated microspheres and a biotin-coated substrate with a measurement error of approximately 7%. These results suggest that fsLP-IF can be used to break intermolecular and intercellular interactions and estimate the adhesion strength. The fsLP-IF was used to break intercellular contacts in two biologically relevant cultures: a coculture of leukocytes seeded over on an endothelial cell monolayer, and a polarized monolayer culture of epithelial cells. The impulses needed to break leukocyte-endothelial and interepithelial interactions, which were calculated based on the geometrical relationship between O(f) and the adhesive interface, were on the order of 10(-13) and 10(-12) N-s, respectively. When the total impulse at O(f) is well-defined, fsLP-IF can be used to estimate the force required to break intercellular adhesions in a noncontact manner under biologically relevant conditions.

Efficient intracellular drug delivery by co-administration of two antibodies against cell adhesion molecule 1.

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.

Enhanced nerve-mast cell interaction by a neuronal short isoform of cell adhesion molecule-1.

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.

Somatic mutations of CADM1 in aldosterone-producing adenomas and gap junction-dependent regulation of aldosterone production.

Aldosterone-producing adenomas (APAs) are the commonest curable cause of hypertension. Most have gain-of-function somatic mutations of ion channels or transporters. Herein we report the discovery, replication and phenotype of mutations in the neuronal cell adhesion gene CADM1. Independent whole exome sequencing of 40 and 81 APAs found intramembranous p.Val380Asp or p.Gly379Asp variants in two patients whose hypertension and periodic primary aldosteronism were cured by adrenalectomy. Replication identified two more APAs with each variant (total, n = 6). The most upregulated gene (10- to 25-fold) in human adrenocortical H295R cells transduced with the mutations (compared to wildtype) was CYP11B2 (aldosterone synthase), and biological rhythms were the most differentially expressed process. CADM1 knockdown or mutation inhibited gap junction (GJ)-permeable dye transfer. GJ blockade by Gap27 increased CYP11B2 similarly to CADM1 mutation. Human adrenal zona glomerulosa (ZG) expression of GJA1 (the main GJ protein) was patchy, and annular GJs (sequelae of GJ communication) were less prominent in CYP11B2-positive micronodules than adjacent ZG. Somatic mutations of CADM1 cause reversible hypertension and reveal a role for GJ communication in suppressing physiological aldosterone production.

Novel application for pseudopodia proteomics using excimer laser ablation and two-dimensional difference gel electrophoresis.

We developed a novel application to conduct pseudopodia proteomics. Pseudopodia are ventral actin-rich protrusions and play functional roles in cell migrations. Identification of pseudopodia proteins leads to a further understanding of malignant phenotypes of tumor cells and novel therapeutic strategies. In our application, tumor cells were placed on a fibronectin-coated porous membrane to form pseudopodia. According to the motile potentials of the cells, the cells formed pseudopodial microprocesses in the pores. An excimer laser, which was used for ophthalmic refractive surgeries, horizontally ablated cells at the membrane surface to remove the cell body. The microscopic observations and the protein expression studies suggested that the laser treatment caused no apparent damages to pseudopodia. Proteins in whole cells and pseudopodia fractions were individually solubilized, labeled with a highly sensitive fluorescent dye, and separated using two-dimensional difference gel electrophoresis. Among 2508 protein spots observed, 211 had different intensity between whole cells and pseudopodia fractions (more than fourfold differences and P-value of <0.05). The protein enrichment depended on the pore size. Mass spectrometric protein identification revealed 46 pseudopodia-localizing proteins. The localization of novel pseudopodia-localizing proteins such as RAB1A, HSP90B, TDRD7, and vimentin was confirmed using immunohistochemical examinations. The previous studies demonstrated that these four proteins may function in the cell migration process. This method will provide insights into the molecular details of pseudopodia and a further understanding of malignant phenotypes of tumor cells and novel therapeutic strategies.

Tumor suppressor cell adhesion molecule 1 (CADM1) is cleaved by a disintegrin and metalloprotease 10 (ADAM10) and subsequently cleaved by γ-secretase complex.

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.

Transcriptional regulation of the CADM1 gene by retinoic acid during the neural differentiation of murine embryonal carcinoma P19 cells.

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.

Indigo plant leaf extract inhibits the binding of SARS-CoV-2 spike protein to angiotensin-converting enzyme 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses its S1 spike protein to bind to angiotensin-converting enzyme 2 (ACE2) on human cells in the first step of cell entry. Tryptanthrin, extracted from leaves of the indigo plant, Polygonum tinctorium, using d-limonene (17.3 µg/ml), is considered to inhibit ACE2-mediated cell entry of another type of coronavirus, HCoV-NL63. The current study examined whether this extract could inhibit the binding of the SARS-CoV-2 spike protein to ACE2. Binding was quantified as cell-bound fluorescence intensity in live cell cultures in which canine kidney MDCK cells overexpressing ACE2 were incubated with fluorescein-labeled S1 spike protein. When indigo extract, together with S1 protein, was added at 8,650x and 17,300x dilutions, fluorescence intensity decreased in a dose- and S1 extract-dependent manner, without affecting cell viability. When 4.0-nM tryptanthrin was added instead of the indigo extract, fluorescence intensity also decreased, but to a lesser degree than with indigo extract. Docking simulation analyses revealed that tryptanthrin readily bound to the receptor-binding domain of the S1 protein, and identified 2- and 7-amino acid sequences as the preferred binding sites. The indigo extract appeared to inhibit S1-ACE2 binding at high dilutions, and evidently contained other inhibitory elements as well as tryptanthrin. This extract may be useful for the prevention or treatment of SARS-CoV-2 infection.

Possible Therapeutic Utility of anti-Cell Adhesion Molecule 1 Antibodies for Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is a highly aggressive malignant tumor, and the effective therapeutic drugs are limited. Thus, the establishment of novel therapeutic method is desired. Considerable proportion of MPMs are shown to express cell adhesion molecule 1 (CADM1), and to use CADM1 to bind to and proliferate on the pleural mesothelial surface, suggesting that CADM1 is a possible therapeutic target. Here, anti-CADM1 ectodomain chicken monoclonal antibodies, 3E1 and 9D2, were examined for their possible therapeutic utility. The full-length form of CADM1 was expressed in eight out of twelve human MPM cell lines. MPM cell lines were cultured on a confluent monolayer of mesothelial MeT-5A cells in the presence of 9D2, the neutralizing antibody. 9D2 suppressed the cell growth of CADM1-positive MPM cells with the loss and aggregation of CADM1 molecules on the MPM cell membrane, but not of CADM1-negative MPM cells. Co-addition of 3E1, lacking the neutralizing action, enhanced the growth-suppressive effect of 9D2. The two antibodies were tested as drug delivery vectors. 3E1 was converted into a humanized antibody (h3E1) and conjugated with monomethyl auristatin E (MMAE), a tubulin polymerization inhibitor. When the resulting h3E1-MMAE antibody-drug conjugate (ADC) was added to the standard cultures of CADM1-positive MPM cells, it suppressed the cell growth in a dose-dependent manner. Co-addition of 9D2 enhanced the growth-suppressive effect of h3E1-MMAE ADC. Anti-CADM1 ectodomain antibodies were suggested to serve as both antibody drugs and drug vectors in the treatment of MPM.

Expression of cell adhesion molecule 1 in human and murine endometrial glandular cells and its increase during the proliferative phase by estrogen and cell density.

AIMS: 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. MAIN METHODS: 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. KEY FINDINGS: 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. SIGNIFICANCE: Two (proximal and distal) promoter regions are suggested to function additively to transactivate CADM1 in endometrial glandular cells that crowd in the proliferative phase.