RESUMEN
Because MUC1 carries a variety of sialoglycans that are possibly recognized by the siglec family, we examined MUC1-binding siglecs and found that Siglec-9 prominently bound to MUC1. An immunochemical study showed that Siglec-9-positive immune cells were associated with MUC1-positive cells in human colon, pancreas, and breast tumor tissues. We investigated whether or not this interaction has any functional implications for MUC1-expressing cells. When mouse 3T3 fibroblast cells and a human colon cancer cell line, HCT116, stably transfected with MUC1cDNA were ligated with recombinant soluble Siglec-9, ß-catenin was recruited to the MUC1 C-terminal domain, which was enhanced on stimulation with soluble Siglec-9 in dose- and time-dependent manners. A co-culture model of MUC1-expressing cells and Siglec-9-expressing cells mimicking the interaction between MUC1-expressing malignant cells, and Siglec-9-expressing immune cells in a tumor microenvironment was designed. Brief co-incubation of Siglec-9-expressing HEK293 cells, but not mock HEK293 cells, with MUC1-expressing cells similarly enhanced the recruitment of ß-catenin to the MUC1 C-terminal domain. In addition, treatment of MUC1-expressing cells with neuraminidase almost completely abolished the effect of Siglec-9 on MUC1-mediated signaling. The recruited ß-catenin was thereafter transported to the nucleus, leading to cell growth. These findings suggest that Siglec-9 expressed on immune cells may play a role as a potential counterreceptor for MUC1 and that this signaling may be another MUC1-mediated pathway and function in parallel with a growth factor-dependent pathway.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos B/metabolismo , Mucina-1/metabolismo , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Transducción de Señal , beta Catenina/metabolismo , Animales , Antígenos CD/genética , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos B/genética , Antígenos de Diferenciación de Linfocitos B/inmunología , Línea Celular Tumoral , Técnicas de Cocultivo , Células HEK293 , Humanos , Ratones , Mucina-1/genética , Mucina-1/inmunología , Células 3T3 NIH , Unión Proteica , Estructura Terciaria de Proteína , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/genética , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/inmunología , Nicho de Células Madre/genética , Nicho de Células Madre/inmunología , Microambiente Tumoral/genética , Microambiente Tumoral/inmunología , beta Catenina/genética , beta Catenina/inmunologíaRESUMEN
We investigated a physiological role for ERK, a member of the MAPK family, in the hypotonic stimulation of epithelial Na(+) channel (ENaC)-mediated Na(+) reabsorption in renal epithelial A6 cells. We show that hypotonic stress causes a major dephosphorylation of ERK following a rapid transient phosphorylation. PD98059 (a MEK inhibitor) increases dephosphorylated ERK and enhances the hypotonic-stress-stimulated Na(+) reabsorption. ERK dephosphorylation is mediated by MAPK phosphatase (MKP). Hypotonic stress activates p38, which in turn induces MKP-1 and to a lesser extent MKP-3 mRNA expression. Inhibition of p38 suppresses MKP-1 induction, preventing hypotonic stress from dephosphorylating ERK. Inhibition of MKP-1 and -3 by the inhibitor NSC95397 also suppresses the hypotonicity-induced dephosphorylation of ERK. NSC95397 reduces both ß- and γ-ENaC mRNA expression and ENaC-mediated Na(+) reabsorption stimulated by hypotonic stress. In contrast, pretreatment with PD98059 significantly enhances mRNA and protein expression of ß- and γ-ENaC even under isotonic conditions. However, PD98059 only stimulates Na(+) reabsorption in response to hypotonic stress, suggesting that ERK inactivation by itself (i.e., under isotonic conditions) is not sufficient to stimulate Na(+) reabsorption, even though ERK inactivation enhances ß- and γ-ENaC expression. Based on these results, we conclude that hypotonic stress stimulates Na(+) reabsorption through at least two signaling pathways: 1) induction of MKP-1 that suppresses ERK activity and induces ß- and γ-ENaC expression, and 2) promotion of translocation of the newly synthesized ENaC to the apical membrane.
Asunto(s)
Fosfatasa 1 de Especificidad Dual/metabolismo , Células Epiteliales/efectos de los fármacos , Canales Epiteliales de Sodio/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Soluciones Hipotónicas/farmacología , Riñón/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Animales , Células Cultivadas , Fosfatasa 6 de Especificidad Dual/metabolismo , Inhibidores Enzimáticos/farmacología , Células Epiteliales/metabolismo , Flavonoides/farmacología , Riñón/citología , MAP Quinasa Quinasa 4/metabolismo , Modelos Animales , Naftoquinonas/farmacología , Transducción de Señal/efectos de los fármacos , Xenopus laevis , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Renal epithelial Na+ transport plays an important role in homeostasis of our body fluid content and blood pressure. Further, the Na+ transport in alveolar epithelial cells essentially controls the amount of alveolar fluid that should be kept at an appropriate level for normal gas exchange. The epithelial Na+ transport is generally mediated through two steps: (1) the entry step of Na+ via epithelial Na+ channel (ENaC) at the apical membrane and (2) the extrusion step of Na+ via the Na+, K+-ATPase at the basolateral membrane. In general, the Na+ entry via ENaC is the rate-limiting step. Therefore, the regulation of ENaC plays an essential role in control of blood pressure and normal gas exchange. In this paper, we discuss two major factors in ENaC regulation: (1) activity of individual ENaC and (2) number of ENaC located at the apical membrane.
Asunto(s)
Células Epiteliales/metabolismo , Canales Epiteliales de Sodio/metabolismo , Sodio/metabolismo , Animales , Humanos , Transporte Iónico , RatonesRESUMEN
Dendritic cells (DCs) play an essential role in the induction and maintenance of an effective immune response and express multiple siglecs. In the present study, we investigated whether or not the ligation of tumor-produced mucins with Siglec-9 expressed on immature DCs is related to escape from immunosurveillance in the tumor-bearing state. Expression of Siglec-9 was up-regulated on the development of monocytes into immature DCs and was decreased in mature DCs. Binding of various mucins and artificial glycopolymers carrying poly (NeuAc α2,6 LacNAc) or poly (NeuAc α2,3 LacNAc) to Siglec-9 was demonstrated by means of a plate assay. These mucins also bound to the surface of immature DCs. When immature DCs were treated with LPS in the presence of these mucins or artificial glycopolymers, the production of IL-12 was significantly reduced, but that of IL-10 was not. Furthermore, IL-12 production was decreased to a similar level on treatment with anti-Siglec-9 mAb. Mucins prepared from serum of cancer patients actually could bind to Siglec-9. These results suggest that Siglec-9 expressed on DCs is involved in immunoregulation through ligation with mucins in an epithelial cancer patient.
Asunto(s)
Antígenos CD/metabolismo , Carcinoma/inmunología , Células Dendríticas/inmunología , Lectinas/metabolismo , Monocitos/inmunología , Mucinas/metabolismo , Neoplasias/inmunología , Escape del Tumor , Línea Celular Tumoral , Humanos , Inmunomodulación , Mucinas/sangre , Lectinas Similares a la Inmunoglobulina de Unión a Ácido SiálicoRESUMEN
The effect of hydrostatic pressure on the paracellular ion conductance (Gp) composed of the Na(+) conductance (G(Na)) and the Cl(-) conductance (G(Cl)) has been Investigated. Gp, G(Na) and G(Cl) were time-dependently increased after applying an osmotic gradient generated by NaCl with basolateral hypotonicity. Hydrostatic pressure (1-4cm H2O) applied from the basolateral side enhanced the osmotic gradient-induced increase in Gp, G(Na) and G(Cl) in a magnitude-dependent manner, while the hydrostatic pressure applied from the apical side diminished the osmotic gradient-induced increase in Gp, G(Na) and G(Cl). How the hydrostatic pressure influences Gp, G(Na) and G(Cl) under an isosmotic condition was also investigated. Gp, G(Na) and G(Cl) were stably constant under a condition with basolateral application of sucrose canceling the NaCl-generated osmotic gradient (an isotonic condition). Even under this stable condition, the basolaterally applied hydrostatic pressure drastically elevated Gp, G(Na) and G(Cl), while apically applied hydrostatic pressure had little effect on Gp, G(Na) or G(Cl). Taken together, these observations suggest that certain factors controlled by the basolateral osmolality and the basolaterally applied hydrostatic pressure mainly regulate the Gp, G(Na) and G(Cl).
Asunto(s)
Cloruros/metabolismo , Presión Hidrostática , Cloruro de Sodio/metabolismo , Sodio/metabolismo , Uniones Estrechas/fisiología , Animales , Línea Celular , Presión Osmótica/fisiología , Uniones Estrechas/metabolismo , Xenopus laevisRESUMEN
Many tumors arising from epithelial tissues produce mucins, which readily come into contact with infiltrating cells in cancer tissues. MUC2 mucins were purified from the conditioned medium of a colorectal cancer cell line, LS180 cells. It is known that in cancer patients, the number of dendritic cells (DCs) is reduced and their function is impaired. Mature DCs were generated from human peripheral blood monocytes through successive treatments with GM-CSF and IL-4, and then with proinflammatory mediators. When monocytes were cultured in the presence of MUC2 mucins in addition to GM-CSF and IL-4 at an early stage of development, mature DCs expressing CD83 decreased and apoptotic cells increased in a dose-dependent manner. During the development of DCs, sialic acid-binding Ig-like lectin (Siglec)-3 was constantly expressed. We prepared recombinant soluble Siglec-3 corresponding to the ectodomain of Siglec-3 and confirmed the binding of soluble Siglec-3 to the MUC2 mucins, probably through alpha2,6-sialic acid-containing O-glycans including a sialyl Tn antigen, which is known to bind to Siglec-3. Apoptosis was partially inhibited by anti-Siglec-3 mAb or recombinant soluble Siglec-3. These results suggest that apoptosis was partially induced through the ligation of the MUC2 mucins with Siglec-3.
Asunto(s)
Apoptosis/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Monocitos/efectos de los fármacos , Mucina 2/farmacología , Anticuerpos Monoclonales/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Células Dendríticas/citología , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Interleucina-4/farmacología , Lectinas/genética , Lectinas/inmunología , Lectinas/metabolismo , Monocitos/citología , Mucina 2/metabolismo , Unión Proteica , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico , Células U937RESUMEN
Microsporidian spores were isolated from two squirrel monkeys (Saimiri sciureus) that had been bred at an animal-breeding colony in Japan. The spores were identified as Encephalitozoon cuniculi on the basis of nucleotide sequence analysis of the small-subunit (SSU) rRNA gene. The internal transcribed spacer (ITS) gene sequence revealed that these isolates were classified into genotype III because it contained tetrarepeats of 5'-GTTT-3'. However, the sequences of the polar tube protein (PTP) gene of the monkey isolates were not identical to a reported sequence of genotype III but were quite similar to a reported sequence of genotype II. On the other hand, sequence analysis of the spore wall protein 1 (SWP-1) gene revealed that the monkey isolates did not belong to any of genotypes I, II and III. These results suggest that the present E. cuniculi isolates of squirrel monkey origin are a new subtype of E. cuniculi ITS genotype III that can cause a disseminated infection.