RESUMO
When monocyte-derived immature dendritic cells (imDCs) were stimulated with LPS in the presence of anti-CD33/Siglec-3 mAb, the production of IL-12 and phosphorylation of NF-κB decreased significantly. The cell surface proteins of imDCs were chemically cross-linked, and CD33-linked proteins were analyzed by SDS-PAGE and immunoblotting. It was CD14 that was found to be cross-linked with CD33. A proximity ligation assay also indicated that CD33 was colocalized with CD14 on the cell surface of imDCs. Sialic acid-dependent binding of CD33 to CD14 was confirmed by a plate assay using recombinant CD33 and CD14. Three types of cells (HEK293T cells expressing the LPS receptor complex (Toll-like receptor (TLR) cells), and the LPS receptor complex plus either wild-type CD33 (TLR/CD33WT cells) or mutated CD33 without sialic acid-binding activity (TLR/CD33RA cells)) were prepared, and then the binding and uptake of LPS were investigated. Although the level of LPS bound on the cell surface was similar among these cells, the uptake of LPS was reduced in TLR/CD33WT cells. A higher level of CD14-bound LPS and a lower level of TLR4-bound LPS were detected in TLR/CD33WT cells compared with the other two cell types, probably due to reduced presentation of LPS from CD14 to TLR4. Phosphorylation of NF-κB after stimulation with LPS was also compared. Wild-type CD33 but not mutated CD33 significantly reduced the phosphorylation of NF-κB. These results suggest that CD14 is an endogenous ligand for CD33 and that ligation of CD33 with CD14 modulates with the presentation of LPS from CD14 to TLR4, leading to down-regulation of TLR4-mediated signaling.
Assuntos
Receptores de Lipopolissacarídeos/metabolismo , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Western Blotting , Membrana Celular/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Células HEK293 , Humanos , Interleucina-2/metabolismo , Ligantes , Receptores de Lipopolissacarídeos/genética , Lipopolissacarídeos/metabolismo , Microscopia Confocal , Modelos Biológicos , Mutação , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Receptor 4 Toll-Like/genéticaRESUMO
Mucin 1 (MUC1) is overexpressed in various human malignant tumors and its expression is correlated with a poor prognosis. MUC1 engages in signal transduction by interacting with receptors for growth and differentiation factors, which contributes to the growth and survival of cancer cells. However, the mechanism by which MUC1 promotes cancer cell invasion remains unclear. Microarray analysis revealed that expression of urokinase-type plasminogen activator (uPA) was elevated in MUC1-overexpressing cells. Furthermore, up- and down-modulation of MUC1 expression was clearly correlated with the change of uPA expression. An immunochemical study showed that the distribution of uPA coincided with that of MUC1 in various human cancer tissues. The MUC1 C-terminal domain (MUC1-CD) was associated with nuclear factor-κB (NF-κB) p65 in MUC1-expressing cells. Chromatin immunoprecipitation (ChIP) assays demonstrated that MUC1-CD existed with NF-κB p65 on the uPA promoter. Luciferase assays indicated that the uPA transcriptional activity was correlated with the level of MUC1 expression and that this MUC1-enhancing effect on the uPA transcription was abolished by introduction of mutations into the NF-κB binding sites on the uPA promoter. These results indicate that formation of the MUC1-CD and NF-κB p65 complex enhanced nuclear translocation of NF-κB p65 and subsequent occupancy of NF-κB binding region on the uPA promoter, leading to elevated transcription of uPA. We also demonstrated that uPA induced by MUC1 enhanced the matrix metalloproteinase (MMP)-2 and -9 activities, and consequently promoted cancer cell invasion. Thus, a MUC1 co-operating NF-κB signaling pathway plays a critical role in cancer cell invasion in MUC1-expressing cells.
Assuntos
Movimento Celular/genética , Mucina-1/genética , Regiões Promotoras Genéticas/genética , Fator de Transcrição RelA/genética , Ativador de Plasminogênio Tipo Uroquinase/genética , Sítios de Ligação/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Dipeptídeos/farmacologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Immunoblotting , Imuno-Histoquímica , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Mucina-1/metabolismo , Invasividade Neoplásica , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Fenilenodiaminas/farmacologia , Ligação Proteica/efeitos dos fármacos , Interferência de RNA , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
BACKGROUND: Galectin-3 is expressed in a variety of tumors and its expression level is related with tumor progression. Aberrant expression of MUC1 in various tumors is also associated with a poor prognosis. It has been reported that MUC1 is a natural ligand of galectin-3. METHODS: A stable MUC1 transfectant was produced by introducing MUC1 cDNA into mouse 3T3 fibroblasts (MUC1/3T3 cells). MUC1 was prepared from MUC1/3T3 cells; MUC1-N-terminal domain (MUC1-ND) and -C-terminal domain (MUC1-CD) were separated by CsCl ultracentrifugation, and then the galectin-3-binding domain was determined by co-immuniprecipitation assay. After ligation of galectin-3 to 3T3/MUC1 cells, MUC1-CD was immunoprecipitated from the cell lysate. The immunoprecipitate was subjected to SDS-PAGE and Western blotting, followed by detection of co-immunoprecipitated ß-catenin. RESULTS: Galectin-3 binds to the N-terminal domain of MUC1 but not to the C-terminal one. Galectin-3 present on the cell surface increased with the expression of MUC1 and is colocalized with MUC1. It should be noted that ß-catenin was detected in the immunoprecipitate with anti-MUC1-CD Ab from a lysate of galectin-3-treated 3T3/MUC1 cells. CONCLUSIONS: Galectin-3 binds to MUC1-ND and triggers MUC1-mediated signaling in 3T3/MUC1 cells, leading to recruitment of ß-catenin to MUC1-CD. GENERAL SIGNIFICANCE: This signaling may be another MUC1-mediated pathway and function in parallel with a growth factor-dependent MUC1-mediated pathway.
Assuntos
Galectina 3/fisiologia , Mucina-1/fisiologia , beta Catenina/metabolismo , Animais , Camundongos , Mucina-1/química , Células NIH 3T3 , Estrutura Terciária de Proteína , Transdução de Sinais/fisiologiaRESUMO
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.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Mucina-1/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Transdução de Sinais , beta Catenina/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Diferenciação de Linfócitos B/imunologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Células HEK293 , Humanos , Camundongos , Mucina-1/genética , Mucina-1/imunologia , Células NIH 3T3 , Ligação Proteica , Estrutura Terciária de Proteína , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/genética , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/imunologia , Nicho de Células-Tronco/genética , Nicho de Células-Tronco/imunologia , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , beta Catenina/genética , beta Catenina/imunologiaRESUMO
Previously we demonstrated that prohibitin-1 and -2 (prohibitins) were expressed on the surface of T cell leukemia cell lines and activated T lymphocytes. In the present study, we found that prohibitins play a role as counter receptors for Siglec-9 expressed on macrophages and dendritic cells. Siglec-9 bound to prohibitins in a sialic acid-independent manner. Mutated Siglec-9 with Arg(120) changed to Ala lost the binding activity, suggesting a specific ionic peptide-peptide interaction. Phosphorylation of ERK1/2 in Jurkat cells on treatment with anti-CD3 antibody immobilized beads was markedly diminished on treatment with anti-CD3 antibody and Siglec-9 co-immobilized beads, indicating that engagement of prohibitins with Siglec-9 inhibits ERK1/2 phosphorylation. Phosphorylation of c-Raf was also reduced, maybe due to inhibition of the c-Raf-prohibitin interaction by Siglec-9 ligation. In parallel with inhibition of the ERK cascade, IL-2 production was markedly decreased in Jurkat cells. Thus, this interaction may be a useful immunotherapeutic target.
Assuntos
Sistema de Sinalização das MAP Quinases , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Repressoras/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Animais , Complexo CD3/metabolismo , Células CHO , Cricetinae , Humanos , Células Jurkat , Ligantes , Ativação Linfocitária , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fosforilação , Proibitinas , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/genética , TransfecçãoRESUMO
Prohibitins (prohibitin-1 and -2) comprise a family of highly conserved proteins that are mainly localized to mitochondria. Recent studies showed that prohibitins are up-regulated upon T cell activation and play an essential role in maintaining mitochondrial homeostasis. In the present study, we found that a considerable proportion of prohibitin-1 and -2 induced in response to T cell activation was expressed on the surface of activated T cells. When mouse and human T cells were stimulated with PMA and ionomycin, prohibitins expressed on the cell surface were increased significantly, peaking at 48 h after stimulation. Stimulation of mouse T cells with anti-CD3 and anti-CD28 antibodies also remarkably induced the cell surface expression of prohibitins. Their expression on the cell surface was also detected in T cell leukemia cells such as Jurkat cells. In Jurkat cells, prohibitin-1 and -2 were co-localized with CD3 on the cell surface, and anti-CD3 antibody-induced signaling, the MAP kinase cascade, was inhibited on treatment with protein A magnetic beads co-conjugated with anti-CD3 antibody and anti-prohibitin-1 or anti-prohibitin-2 antibody. These results suggest that prohibitins expressed on the surface of activated T cells are involved in the T cell receptor-mediated signaling cascade.
Assuntos
Membrana Celular/imunologia , Ativação Linfocitária , Proteínas Repressoras/biossíntese , Linfócitos T/imunologia , Animais , Anticorpos , Feminino , Humanos , Células Jurkat , Camundongos , Camundongos Endogâmicos BALB C , Proibitinas , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/imunologiaRESUMO
MUC16/CA125 is over-expressed in human epithelial tumors including ovarian, breast and some other carcinomas. The purpose of this study is to investigate how cell surface MUC16 is functionally involved in tumor progression, with a special focus on the role of its cytoplasmic tail. Forced expression of C-terminal MUC16 fragment (MUC16C) in epithelial cancer cells increased cell migration. We found that MUC16C directly interacted with Src family kinases (SFKs). Notably, localizations of E-cadherin and ß-catenin at the cell-cell contacts were more diffuse in MUC16C transfectants compared with mock transfectants. Furthermore, MUC16C transfectants showed reduced Ca(2+)-dependent cell-cell adhesion, but the treatment of cells with PP2, a SFKs inhibitor, restored this. Because cell surface MUC16 is also associated with the E-cadherin/ß-catenin complex, the over-expression of MUC16 and its interaction with SFKs may enhance SFKs-induced deregulation of E-cadherin. Thus, our results suggest a role for cell surface MUC16 in cell-cell adhesion of epithelial cancer cells.