RESUMEN
Measles virus (MeV), the causative agent of measles, is an enveloped RNA virus of the family Paramyxoviridae, which remains an important cause of childhood morbidity and mortality. MeV has two envelope glycoproteins, the hemagglutinin (H) and fusion (F) proteins. During viral entry or virus-mediated fusion between infected cells and neighboring susceptible cells, the head domain of the H protein initially binds to its receptors, signaling lymphocytic activation molecule family member 1 (SLAM) and nectin-4, and then the stalk region of the H protein transmits the fusion-triggering signal to the F protein. MeV may persist in the human brain and cause a fatal neurodegenerative disease, subacute sclerosing panencephalitis (SSPE). Recently, we showed, using in vitro cell culture, that cell adhesion molecule (CADM) 1 and CADM2 are host factors that trigger hyperfusogenic mutant F proteins, causing cell-to-cell fusion and the transfer of the MeV genome between neurons. Unlike conventional receptors, CADM1 and CADM2 interact in cis (on the same membrane) with the H protein and then trigger membrane fusion. Here, we show that alanine substitutions in part of the stalk region (positions 171-175) abolish the ability of the H protein to mediate membrane fusion triggered by CADM1 and CADM2, but not by SLAM. The recombinant hyperfusogenic MeV carrying this mutant H protein loses its ability to spread in primary mouse neurons as well as its neurovirulence in experimentally infected suckling hamsters. These results indicate that CADM1 and CADM2 are key molecules for MeV propagation in the brain and its neurovirulence in vivo. IMPORTANCE Measles is an acute febrile illness with skin rash. Despite the availability of highly effective vaccines, measles is still an important cause of childhood morbidity and mortality in many countries. The World Health Organization estimates that more than 120,000 people died from measles worldwide in 2021. Measles virus (MeV), the causative agent of measles, can also cause a fatal progressive neurological disorder, subacute sclerosing panencephalitis (SSPE), several years after acute infection. There is currently no effective treatment for this disease. In this study, using recombinant MeVs with altered receptor usage patterns, we show that cell adhesion molecule (CADM) 1 and CADM2 are host factors critical for MeV spread in neurons and its neurovirulence. These findings further our understanding of the molecular mechanism of MeV neuropathogenicity.
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Sarampión , Enfermedades Neurodegenerativas , Panencefalitis Esclerosante Subaguda , Cricetinae , Humanos , Ratones , Animales , Virus del Sarampión/fisiología , Panencefalitis Esclerosante Subaguda/genética , Hemaglutininas/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismo , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Proteínas Recombinantes/metabolismo , Neuronas , Molécula 1 de Adhesión Celular/metabolismoRESUMEN
Measles virus (MeV), an enveloped RNA virus in the family Paramyxoviridae, usually causes acute febrile illness with skin rash but in rare cases persists in the brain, causing a progressive neurological disorder, subacute sclerosing panencephalitis (SSPE). MeV bears two envelope glycoproteins, the hemagglutinin (H) and fusion (F) proteins. The H protein possesses a head domain that initially mediates receptor binding and a stalk domain that subsequently transmits the fusion-triggering signal to the F protein. We recently showed that cell adhesion molecule 1 (CADM1; also known as IGSF4A, Necl-2, and SynCAM1) and CADM2 (also known as IGSF4D, Necl-3, and SynCAM2) are host factors enabling cell-cell membrane fusion mediated by hyperfusogenic F proteins of neuropathogenic MeVs as well as MeV spread between neurons lacking the known receptors. CADM1 and CADM2 interact in cis with the H protein on the same cell membrane, triggering hyperfusogenic F protein-mediated membrane fusion. Multiple isoforms of CADM1 and CADM2 containing various lengths of their stalk regions are generated by alternative splicing. Here, we show that only short-stalk isoforms of CADM1 and CADM2 predominantly expressed in the brain induce hyperfusogenic F protein-mediated membrane fusion. While the known receptors interact in trans with the H protein through its head domain, these isoforms can interact in cis even with the H protein lacking the head domain and trigger membrane fusion, presumably through its stalk domain. Thus, our results unveil a new mechanism of viral fusion triggering by host factors. IMPORTANCE Measles, an acute febrile illness with skin rash, is still an important cause of childhood morbidity and mortality worldwide. Measles virus (MeV), the causative agent of measles, may also cause a progressive neurological disorder, subacute sclerosing panencephalitis (SSPE), several years after acute infection. The disease is fatal, and no effective therapy is available. Recently, we reported that cell adhesion molecule 1 (CADM1) and CADM2 are host factors enabling MeV cell-to-cell spread in neurons. These molecules interact in cis with the MeV attachment protein on the same cell membrane, triggering the fusion protein and causing membrane fusion. CADM1 and CADM2 are known to exist in multiple splice isoforms. In this study, we report that their short-stalk isoforms can induce membrane fusion by interacting in cis with the viral attachment protein independently of its receptor-binding head domain. This finding may have important implications for cis-acting fusion triggering by host factors.
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Molécula 1 de Adhesión Celular/metabolismo , Células Gigantes/virología , Hemaglutininas Virales/metabolismo , Interacciones Huésped-Patógeno , Virus del Sarampión/fisiología , Sarampión/metabolismo , Sarampión/virología , Animales , Encéfalo/metabolismo , Encéfalo/virología , Molécula 1 de Adhesión Celular/genética , Células Cultivadas , Cricetinae , Modelos Biológicos , Unión Proteica , Isoformas de Proteínas , Proteínas Virales de Fusión/metabolismoRESUMEN
The interruption of normal cell cycle execution acts as an important part to the development of leukemia. It was reported that microRNAs (miRNAs) were closely related to tumorigenesis and progression, and their aberrant expression had been demonstrated to play a crucial role in numerous types of cancer. Our previous study showed that miR-1246 was preferentially overexpressed in chemo-resistant leukemia cell lines, and participated in process of cell cycle progression and multidrug resistant regulation. However, the underlying mechanism remains unclear. In present study, bioinformatics prediction and dual luciferase reporter assay indicated that CADM1 was a direct target of miR-1246. Evidently decreased expression of CADM1 was observed in relapsed primary leukemia patients and chemo-resistant cell lines. Our results furtherly proved that inhibition of miR-1246 could significantly enhance drug sensitivity to Adriamycin (ADM), induce cell cycle arrest at G0/G1 phase, promote cell apoptosis, and relieve its suppression on CADM1 in K562/ADM and HL-60/RS cells. Interference with CADM1 could reduce the increased drug sensitivity induced by miR-1246 inhibition, and notably restore drug resistance by promoting cell cycle progression and cell survival via regulating CDKs/Cyclins complexes in chemo-resistant leukemia cells. Above all, our results demonstrated that CADM1 attenuated the role of miR-1246 in promoting cell cycle progression and cell survival, thus influencing multidrug resistance within chemo-resistant leukemia cells via CDKs/Cyclins. Higher expression of miR-1246 and lower expression of CADM1 might be risk factors for leukemia.
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Leucemia , MicroARNs , Humanos , MicroARNs/metabolismo , Células HL-60 , Doxorrubicina/farmacología , Ciclo Celular/genética , Leucemia/tratamiento farmacológico , Leucemia/genética , Ciclinas , Proliferación Celular , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Molécula 1 de Adhesión Celular/genéticaRESUMEN
T cell activation is a multifaceted process that depends on the activation of the T cell receptor (TCR). However, other coreceptors are also strictly necessary to provide co-signals and modulate the immune response. However, to date, most of these coreceptors are unknown in fish or their information is very limited. Therefore, in this work, we have identified the cytotoxic and regulatory T cell molecule, CRTAM, and its ligand, the cell adhesion molecule 1, CADM1, in European seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata); and evaluated their transcriptional levels. Both putative proteins showed the canonical architecture observed in mammals, where CRTAM exhibited two immunoglobulin domains and CADM1, both the a and b forms, exhibited three of these domains. In addition, phylogeny and synteny analyses showed their conservation throughout vertebrate evolution. We found constitutive expression of all three genes, with crtam and cadm1a being predominant in immune tissues such as spleen, thymus and head-kidney (HK), while cadm1b expression was more limited to the brain. In vitro, only the T cell mitogen phytohemagglutinin (PHA) up-regulated the transcription of crtam and cadm1a in HK leucocytes. Nodavirus (NNV) infection elicited an up-regulation of crtam and cadm1a in brain and HK, appearing earlier in seabream than in seabass, which could explain the resistance of seabream to the development of nodavirus disease. In addition, they are up-regulated during the innate cell-mediated cytotoxic response in seabream but not in seabass. Altogether, our data seem to indicate that CRTAM is more related to the innate cytotoxicity in seabream and more in the specific and T cell-mediated cytotoxicity in seabass. Our results highlight the importance of CRTAM and CADM1 as important molecules in the activation of T lymphocytes in seabass and seabream, but further studies are needed.
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Antineoplásicos , Lubina , Dorada , Animales , Molécula 1 de Adhesión Celular , Linfocitos T Reguladores , Ligandos , MamíferosRESUMEN
The initial step of organ infiltration of malignant cells is the interaction with host vascular endothelial cells, which is often mediated by specific combinations of cell adhesion molecules. Cell adhesion molecule 1 (CADM1) is overexpressed in adult T-cell leukemia/lymphoma (ATL) and provides a cell-surface diagnostic marker. CADM1 promotes the adhesion of ATL cells to vascular endothelial cells and multiple organ infiltration in mice. However, its binding partner on host cells has not yet been identified. In this study, we show that CADM1 promotes transendothelial migration of ATL cells in addition to the adhesion to vascular endothelial cells. Moreover, CADM1 enhances liver infiltration of mouse T-cell lymphoma cells, EL4, after tail vein injection, whereas a CADM1 mutant lacking adhesive activity did not. Among the known CADM1-binding proteins expressed in primary endothelial cells, only CADM1 and CADM4 could induce morphological extension of ATL cells when plated onto glass coated with these proteins. Furthermore, CADM1-mediated liver infiltration of EL4 cells was canceled in conventional and vascular endothelium-specific Cadm1 knockout mice, whereas it was not canceled in Cadm4 knockout mice. These results suggest that CADM1 on host vascular endothelial cells is required for organ infiltration of ATL and other T-cell lymphomas expressing CADM1.
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Molécula 1 de Adhesión Celular/metabolismo , Endotelio Vascular , Linfoma de Células T , Animales , Adhesión Celular , Molécula 1 de Adhesión Celular/genética , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Humanos , Inmunoglobulinas/química , Inmunoglobulinas/genética , Linfoma de Células T/genética , RatonesRESUMEN
INTRODUCTION AND OBJECTIVES: Long non-coding RNAs (lncRNAs) have great potential as therapeutic targets in hepatocellular carcinoma (HCC). In this study, we aimed to uncover the function and molecular mechanism of long intergenic non-protein coding RNA 1006 (LINC01006) in HCC. MATERIALS AND METHODS: Mice were injected with HCC cells in order to establish the HCC model. Quantitative reverse transcription polymerase chain reaction was used to determine the expression levels of LINC01006, cell adhesion molecule 1 (CADM1), and microRNA (miR)-194-5p in HCC tissues and cells. The cell proliferation, invasion, and migration abilities were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide, transwell, and wound healing assays. The interrelation between LINC01006, miR-194-5p, and CADM1 was confirmed by a dual-luciferase reporter assay. Western blotting was employed to assess the relative protein expression level of CADM1. RESULTS: LINC01006 and CADM1 displayed upregulation, but miR-194-5p exhibited downregulation in HCC cells and tissues. Short hairpin (sh)-LINC01006 and miR-194-5p mimics repressed the proliferative, migratory, and invasive capacities of HCC cells, and injection of sh-LINC01006 restrained the growth of HCC tumours in mice. LINC01006 served as a competing endogenous RNA of miR-194-5p and was inversely correlated with miR-194-5p. CADM1 was targeted by miR-194-5p, inversely correlated with miR-194-5p, and positively associated with LINC01006. Furthermore, transfection of pcDNA-CADM1 or the miR-194-5p inhibitor reversed the suppressive effects of sh-LINC01006 on the proliferation, invasion, and migration abilities of HCC cells. CONCLUSIONS: Downregulation of LINC01006 repressed the development of HCC by sponging miR-194-5p to modulate the expression of CADM1, implying its potential as a therapeutic target for HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroARNs , ARN Largo no Codificante , Animales , Carcinoma Hepatocelular/patología , Molécula 1 de Adhesión Celular/genética , Molécula 1 de Adhesión Celular/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/patología , Ratones , MicroARNs/genética , MicroARNs/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismoRESUMEN
Small-cell lung cancer (SCLC) is the most aggressive form of lung cancer and the leading cause of global cancer-related mortality. Despite the earlier identification of membrane-proximal cleavage of cell adhesion molecule 1 (CADM1) in cancers, the role of the membrane-bound fragment of CAMD1 (MF-CADM1) is yet to be clearly identified. In this study, we first isolated MF-CADM1-specific fully human single-chain variable fragments (scFvs) from the human synthetic scFv antibody library using the phage display technology. Following the selected scFv conversion to human immunoglobulin G1 (IgG1) scFv-Fc antibodies (K103.1-4), multiple characterization studies, including antibody cross-species reactivity, purity, production yield, and binding affinity, were verified. Finally, via intensive in vitro efficacy and toxicity evaluation studies, we identified K103.3 as a lead antibody that potently promotes the death of human SCLC cell lines, including NCI-H69, NCI-H146, and NCI-H187, by activated Jurkat T cells without severe endothelial toxicity. Taken together, these findings suggest that antibody-based targeting of MF-CADM1 may be an effective strategy to potentiate T cell-mediated SCLC death, and MF-CADM1 may be a novel potential therapeutic target in SCLC for antibody therapy.
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Neoplasias Pulmonares , Anticuerpos de Cadena Única , Carcinoma Pulmonar de Células Pequeñas , Molécula 1 de Adhesión Celular/genética , Técnicas de Visualización de Superficie Celular , Humanos , Anticuerpos de Cadena Única/farmacologíaRESUMEN
Cell adhesion molecule 1 (CADM1), which mediates intercellular adhesion between epithelial cells, is shown to be highly expressed in small-cell lung cancer (SCLC) and to enhance tumorigenicity of SCLC cells in nude mice. Here, we investigated the molecular mechanism underlying the oncogenic role of CADM1 in SCLC. CADM1 promoted colony formation of SCLC cells in soft agar. Analysis of deletion and point mutants of the conserved protein-binding motifs in CADM1 revealed that the 4.1 protein-binding motif in the cytoplasmic domain is responsible for the promotion of colony formation. Among the actin-binding 4.1 proteins, 4.1R was the only protein whose localization to the plasma membrane is dependent on CADM1 expression in SCLC cells. Knockdown of 4.1R suppressed the colony formation enhanced by CADM1, suggesting that 4.1R is required for the oncogenic role of CADM1 in SCLC. In primary SCLC, CADM1 expression was correlated with membranous localization of 4.1R, as was observed in a SCLC cell line. Moreover, membranous co-localization of CADM1 and 4.1R was associated with more advanced tumor stage. These results suggest that the formation of CADM1-4.1R complex would promote malignant features of SCLC.
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Molécula 1 de Adhesión Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de la Membrana/metabolismo , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Animales , Molécula 1 de Adhesión Celular/genética , Línea Celular Tumoral , Membrana Celular/metabolismo , Femenino , Neoplasias Pulmonares/patología , Ratones Endogámicos BALB C , Ratones Desnudos , Mutación , Carcinoma Pulmonar de Células Pequeñas/patologíaRESUMEN
Serum microRNA has been demonstrated as a noninvasive predictor for the progression of non-small-cell lung cancer (NSCLC). The role of microRNA-486-5p (miR-486-5p) in NSCLC seems to be paradoxical. On the one hand, elevated expression of miR-486-5p in serum is associated with unfavorable survival; on the other hand, miR-486-5p was notably reduced in NSCLC tissues and acted as a tumor-suppressor to inhibit NSCLC metastasis. The expression of miR-486-5p was analyzed in serum and tissue samples and their relationship was explored. The miR-486-5p-expressing cells were isolated by fluorescent-activated cell sorting. The downstream target of miR-486-5p was identified by bioinformatics prediction and experimental confirmation. Functional studies of miR-486-5p on NSCLC metastasis were determined by endothelial permeability assay and trans-endothelial invasion assay. We found that the expression of miR-486-5p was remarkably increased in serum, while dramatically downregulated in tumor tissues of NSCLC. However, the level of miR-486-5p in serum was positively correlated with that in tumor tissues. Next, we identified CD31+ vascular endothelial cells in the lung stroma as miR-486-5p-expressing cells. According to bioinformatics prediction, quantitative real-time reverse transcription PCR, luciferase reporter assay, and western blot, miR-486-5p directly targeted the cell adhesion molecule 1/tight junctions axis in vascular endothelial cells. In addition, endothelial permeability assay and trans-endothelial invasion assay confirmed that miR-486-5p promoted NSCLC metastasis. Highly elevated expression of miR-486-5p in CD31+ vascular endothelial cells increased vascular permeability and promoted NSCLC metastasis. In conclusion, stromal-derived miR-486-5p is responsible for the paradoxical effect of miR-486-5p in serum and tumor tissue.
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Carcinoma de Pulmón de Células no Pequeñas , Molécula 1 de Adhesión Celular/metabolismo , Células Endoteliales , Neoplasias Pulmonares , MicroARNs/fisiología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Células Endoteliales/metabolismo , Células Endoteliales/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologíaRESUMEN
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.
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Proteína Tirosina Quinasa CSK/metabolismo , Carcinogénesis/metabolismo , Molécula 1 de Adhesión Celular/metabolismo , Neoplasias del Colon/metabolismo , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Animales , Activación Enzimática , Femenino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
Cell adhesion molecule 1 (CADM1) is frequently silenced in lung, prostate, liver, stomach, pancreatic and breast carcinomas and other forms of human carcinomas. However, it is unclear regarding the role of CADM1 in irritable bowel syndrome with diarrhoea (IBS-D) that is the most common gastrointestinal diagnosis and may contribute to impaired intestinal barrier function. The aim of the present study is to explore the potential mechanism of CADM1 in regulating intestinal barrier function in IBS-D. A rat model with IBS-D induced by the combination method of mother-infant separation, acetic acid and restraint stress was initially established. The defecation frequency, faecal water content (FWC), total intestinal permeability, sIgA, endotoxin, D-lactic acid and diamine oxidase (DAO) were then measured. Next, positive expression of CADM1 protein was detected in distal colonic tissue of rats by immunohistochemistry. The expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in distal colonic mucosa, CADM1, Janus kinase 1 (JAK1), STAT3, p-JAK1, p-STAT3, Claudin-1and Claudin-2 were evaluated using ELISA, RT-qPCR and western blot analysis. IBS-D rats exhibited low CADM1 expression and activated STAT3 signaling pathway. Overexpression of CADM1 in rats was shown to increase Claudin-1 expression, while decreasing expression of STAT3, Claudin-2, TNF-α and IL-6. In addition, silencing of CADM1 or inhibition of the STAT3 signaling pathway was demonstrated to improve the intestinal barrier function. Our study provides evidence that CADM1 can potentially improve intestinal barrier function in rats with IBS-D by inhibiting the STAT3 signaling pathway.
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Moléculas de Adhesión Celular/metabolismo , Inmunoglobulinas/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Estudios de Casos y Controles , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/genética , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Células HeLa , Humanos , Inmunoglobulinas/biosíntesis , Inmunoglobulinas/genética , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Masculino , Ratas , Ratas Sprague-Dawley , Factor de Transcripción STAT3/antagonistas & inhibidores , Transducción de SeñalRESUMEN
Cell adhesion ability is one of the components to establish cell organization and shows a great contribution to human body construction consisting of various types of cells mixture to orchestrate tissue specific function. The cell adhesion molecule 1 (CADM1) is a molecule of cell adhesion with multiple functions and has been identified as a tumor suppressor gene. CADM1 has multifunctions on the pathogenesis of malignancies, and other normal cells such as immune cells. However, little is known about the function of CADM1 on cutaneous cells and cutaneous malignancies. CADM1 plays an important role in connecting cells with each other, contacting cells to deliver their signal, and acting as a scaffolding molecule for other immune cells to develop their immune responses. A limited number of studies reveal the contribution of CADM1 on the development of cutaneous malignancies. Solid cutaneous malignancies, such as cutaneous squamous cell carcinoma and malignant melanoma, reduce their CADM1 expression to promote the invasion and metastasis of the tumor. On the contrary to these cutaneous solid tumors except for Merkel cell carcinoma, cutaneous lymphomas, such as adult-T cell leukemia/lymphoma, mycosis fungoides, and Sézary syndrome, increase their CADM1 expression for the development of tumor environment. Based on the role of CADM1 in the etiology of tumor development, the theory of CADM1 contribution will desirably be applied to skin tumors according to other organ malignancies, however, the characteristics of skin as a multicomponent peripheral organ should be kept in mind to conclude their prognoses.
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Biomarcadores de Tumor/genética , Molécula 1 de Adhesión Celular/genética , Melanoma/genética , Neoplasias Cutáneas/genética , Adhesión Celular/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Melanoma/patología , Neoplasias Cutáneas/patología , Melanoma Cutáneo MalignoRESUMEN
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.
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Molécula 1 de Adhesión Celular/metabolismo , Moléculas de Adhesión Celular/metabolismo , Células Epiteliales/citología , Inmunoglobulinas/metabolismo , Animales , Apoptosis/genética , Células CACO-2 , Molécula 1 de Adhesión Celular/genética , Moléculas de Adhesión Celular/genética , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Línea Celular , Supervivencia Celular , Células Epiteliales/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Inmunoglobulinas/genética , Alveolos Pulmonares/citología , RatasRESUMEN
Cell adhesion molecule-1 (CADM1) is a member of the immunoglobulin superfamily that functions as a tumor suppressor of lung tumors. We herein demonstrated that CADM1 interacts with Hippo pathway core kinases and enhances the phosphorylation of YAP1, and also that the membranous co-expression of CADM1 and LATS2 predicts a favorable prognosis in lung adenocarcinoma. CADM1 significantly repressed the saturation density elevated by YAP1 overexpression in NIH3T3 cells. CADM1 significantly promoted YAP1 phosphorylation on Ser 127 and downregulated YAP1 target gene expression at confluency in lung adenocarcinoma cell lines. Moreover, CADM1 was co-precipitated with multiple Hippo pathway components, including the core kinases MST1/2 and LATS1/2, suggesting the involvement of CADM1 in the regulation of the Hippo pathway through cell-cell contact. An immunohistochemical analysis of primary lung adenocarcinomas (n = 145) revealed that the histologically low-grade subtype frequently showed the membranous co-expression of CADM1 (20/22, 91% of low-grade; 61/91, 67% of intermediate grade; and 13/32, 41% of high-grade subtypes; P < 0.0001) and LATS2 (22/22, 100% of low-grade; 44/91, 48% of intermediate-grade; and 1/32, 3% of high-grade subtypes; P < 0.0001). A subset analysis of disease-free survival revealed that the membranous co-expression of CADM1 and LATS2 was a favorable prognosis factor (5-year disease-free survival rate: 83.8%), even with nuclear YAP1-positive expression (5-year disease-free survival rate: 83.7%), whereas nuclear YAP1-positive cases with the negative expression of CADM1 and LATS2 had a poorer prognosis (5-year disease-free survival rate: 33.3%). These results indicate that the relationship between CADM1 and Hippo pathway core kinases at the cell membrane is important for suppressing the oncogenic role of YAP1.
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Molécula 1 de Adhesión Celular/metabolismo , Membrana Celular/metabolismo , Neoplasias Pulmonares/patología , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Proteínas de Ciclo Celular , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Vía de Señalización Hippo , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Células 3T3 NIH , Clasificación del Tumor , Fosforilación , Pronóstico , Análisis de Matrices TisularesRESUMEN
We recently took advantage of the universal expression of cell adhesion molecule 1 (CADM1) by CD4+ cells infected with HTLV-1 and the downregulation of CD7 expression that corresponds with the oncogenic stage of HTLV-1-infected cells to develop a flow cytometric system using CADM1 versus CD7 plotting of CD4+ cells. We risk-stratified HTLV-1 asymptomatic carriers (AC) and indolent adult T-cell leukemia/lymphoma (ATL) cases based on the CADM1+ percentage, in which HTLV-1-infected clones are efficiently enriched. AC and indolent ATL cases were initially classified according to their CADM1+ cell percentage. Follow-up clinical and flow cytometric data were obtained for 71 cases. In G1 (CADM1+ ≤ 10%) and G2 (10% < CADM1+ ≤ 25%) cases, no apparent clinical disease progression was observed. In G3 (25% < CADM1+ ≤ 50%) cases, five out of nine (55.5%) cases progressed from AC to smoldering-type ATL. In G4 (50% < CADM1+ ) cases, the cumulative incidence of receiving systemic chemotherapy at 3 years was 28.4%. Our results indicate that the percentage of the CD4+ CADM1+ population predicts clinical disease progression: G1 and G2 cases, including AC cases, are stable and considered to be at low risk; G3 cases, including advanced AC cases and smoldering-type ATL cases based on the Shimoyama criteria, are considered to have intermediate risk; and G4 cases, which are mainly indolent ATL cases, are unstable and at high risk of acute transformation.
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Linfocitos T CD4-Positivos/inmunología , Portador Sano/inmunología , Molécula 1 de Adhesión Celular/análisis , Infecciones por HTLV-I/inmunología , Leucemia-Linfoma de Células T del Adulto/inmunología , Adulto , Anciano , Progresión de la Enfermedad , Femenino , Infecciones por HTLV-I/tratamiento farmacológico , Humanos , Leucemia-Linfoma de Células T del Adulto/tratamiento farmacológico , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: Squamous intraepithelial lesions/cervical intraepithelial neoplasias (SIL/CIN) are high-risk human papilloma virus (hrHPV)-related lesions which are considered as high grade (HSIL/CIN2-3) or low grade (LSIL/CIN1) lesions according to their risk of progression to cervical cancer (CC). Most HSIL/CIN2-3 are considered as transforming hrHPV infections, so truly CC precursors, although some clear spontaneously. hrHPV testing has a high sensitivity for the detection of HSIL/CIN2-3 but a relatively low specificity for identifying transforming lesions. We aimed to determine whether the combination of CADM1, MAL and miR124 promoter methylation status assessed in histological samples can be used as a biomarker in the identification of transforming HSIL/CIN lesions. DESIGN: 131 cervical biopsies, including 8 cases with no lesion and a negative hrHPV test result (control group), 19 low-grade (L)SIL/CIN1, 30 HSIL/CIN2, 60 HSIL/CIN3, and 14 CC were prospectively collected. hrHPV was detected and genotyped using the polymerase chain reaction (PCR)-based technique SPF10 HPV LIPA. A multiplex quantitative methylation-specific PCR (qMSP) was used to identify the methylation status of the CADM1, MAL, and miR124 promoter genes. RESULTS: Significantly higher methylation levels of CADM1, MAL and miR-124 were found in HSIL/CIN2-3 and CC compared with normal and LSIL lesions. DNA methylation of at least one gene was detected in 12.5% (1/8) of normal samples, 31.5% (6/19) of LSIL/CIN1, 83.3% (25/30) of HSIL/CIN2, 81.6% (49/60) of HSIL/CIN3 and 100% (14/14) of CC (p < 0.001). The sensitivity and specificity for HSIL/CIN2-3 and CC of having at least one methylated gene were 84.6% and 74.0%, respectively. The sensitivity and specificity of the combination of at least one methylated gene and a positive hrHPV test were 80.7% and 85.1% for HSIL/CIN2-3 and CC, respectively. CONCLUSIONS: The methylation rate of CADM1, MAL and miR124 increases with the severity of the lesion. Further research is warranted to evaluate the usefulness of these biomarkers for the identification of transforming HSIL/CIN.
Asunto(s)
Biomarcadores de Tumor/genética , Molécula 1 de Adhesión Celular/genética , Metilación de ADN , MicroARNs/genética , Proteínas Proteolipídicas Asociadas a Mielina y Linfocito/genética , Lesiones Intraepiteliales Escamosas de Cuello Uterino/genética , Adolescente , Adulto , Anciano , Femenino , Humanos , Persona de Mediana Edad , Regiones Promotoras Genéticas , Lesiones Intraepiteliales Escamosas de Cuello Uterino/patologíaRESUMEN
Vein endothelial cells (VECs) constitute an important barrier for macromolecules and circulating cells from the blood to the tissues, stabilizing the colloid osmotic pressure of the blood, regulating the vascular tone, and rapidly changing the intercellular connection, and maintaining normal physiological function. Tight junction has been discovered as an important structural basis of intercellular connection and may play a key role in intercellular connection injuries or vascular diseases and selenium (Se) deficiency symptoms. Hence, we replicated the Se-deficient broilers model and detected the specific microRNA in response to Se-deficient vein by using quantitative real time-PCR (qRT-PCR) analysis. Also, we selected miR-128-1-5p based on differential expression in vein tissue and confirmed its target gene cell adhesion molecule 1 (CADM1) by the dual luciferase reporter assay and qRT-PCR in VECs. We made the ectopic miR-128-1-5p expression for the purpose of validating its function on tight junction. The result showed that miR-128-1-5p and CADM1 were involved in the ZO-1-mediated tight junction, increased paracellular permeability, and arrested cell cycle. We presumed that miR-128-1-5p and Se deficiency might trigger tight junction. Interestingly, miR-128-1-5p inhibitor and fasudil in part hinder the destruction of the intercellular structure caused by Se deficiency. The miR-128-1-5p/CADM1/tight junction axis provides a new avenue toward understanding the mechanism of Se deficiency, revealing a novel regulation model of tight junction injury in vascular diseases.
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Molécula 1 de Adhesión Celular/genética , MicroARNs/genética , Selenio/metabolismo , Uniones Estrechas/genética , Animales , Apoptosis/genética , Pollos , Células Endoteliales/metabolismo , Células Endoteliales/patología , MicroARNs/metabolismo , Selenio/deficiencia , Uniones Estrechas/patología , Venas/citología , Venas/metabolismoRESUMEN
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.
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Apoptosis , Molécula 1 de Adhesión Celular/metabolismo , Nefropatías Diabéticas/metabolismo , Células Epiteliales/metabolismo , Túbulos Renales/metabolismo , Nefroesclerosis/metabolismo , Fragmentos de Péptidos/metabolismo , Insuficiencia Renal Crónica/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Biomarcadores/sangre , Nitrógeno de la Urea Sanguínea , Molécula 1 de Adhesión Celular/genética , Línea Celular , Creatinina/sangre , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/patología , Progresión de la Enfermedad , Regulación hacia Abajo , Células Epiteliales/patología , Femenino , Humanos , Túbulos Renales/patología , Masculino , Persona de Mediana Edad , Nefroesclerosis/genética , Nefroesclerosis/patología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Conejos , Insuficiencia Renal Crónica/genética , Insuficiencia Renal Crónica/patología , Transducción de SeñalRESUMEN
This paper reports isolation of two monoclonal antibodies (mAbs) that bind to both a membrane protein and a cytoplasmic protein. Most Abs established as markers for autoimmune disease bind to cytoplasmic or nuclear substances. However, it remains unknown how these Abs are produced. On the other hand, there were examples where clones originally isolated as Abs that bind to membrane proteins also showed binding activity to cytoplasmic or nuclear substances. Based on these results, the following hypothesis has been proposed. The Abs that had been originally produced against a membrane protein showed cross-reactivity against cytoplasmic or nuclear substances. In the present study we reported isolation of Abs that bound to both a membrane protein, CADM1, and a cytoplasmic protein, α-actinin-4. The method adopted in the present study could be generally applicable to isolation of Abs showing such dual specificity. Firstly, we constructed a huge human Ab library using various organs including naïve B-cell-rich organs such as bone marrow and umbilical cords. Then, we developed a comprehensive screening method for isolation of Abs that bound to cell surface antigens. Through extensive screenings with many kinds of cell we newly obtained a library composed of around 4000 independent clones that bind to membrane proteins. We screened this library with α-actinin-4 and succeeded in isolating two Abs. They bound to α-actinin-4 and a membrane protein CADM1. Furthermore, they are encoded by naïve heavy and light chain variable genes (VH & VL). These results suggested that cross-reactive Abs to both a membrane protein and a cytoplasmic protein could be present in germline repertoire of Ab in humans. This methodology adopted in the present study could be applied to isolation of cross-reactive Abs possibly involved in autoimmune diseases.
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Actinina/inmunología , Anticuerpos Monoclonales/inmunología , Molécula 1 de Adhesión Celular/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Línea Celular , Reacciones Cruzadas , Células Hep G2 , Humanos , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , InmunoprecipitaciónRESUMEN
The central nervous system is widely known to exert control over our systemic physiology via several mechanisms including the regulation of skeletal metabolism. Neuronal circuits within the hypothalamus have been shown to impact bone mass via leptin-dependent and independent mechanisms; however, the full extent to which the brain controls bone homeostasis is not known. We previously identified cell adhesion molecule1 (Cadm1) as a regulator of body weight and energy homeostasis via its expression in multiple regions of the brain. Here, we show that loss of Cadm1 expression in excitatory neurons results in increased leptin sensitivity in addition to a concomitant reduction in bone mass. Femoral length, bone mineral content, diaphyseal cross-sectional area, and bone strength were all lower in Cadm1-deficient animals. Conversely, inducing expression of Cadm1 in excitatory neurons decreased leptin sensitivity and increased femoral length, bone mineral content, and diaphyseal cross-sectional area. Together, these results illustrate an essential role for this synaptic protein in the neuronal regulation of skeletal bone metabolism.