RESUMO
RNA-binding motif 10 (Rbm10) is an RNA-binding protein that regulates alternative splicing, but its role in inflammation is not well defined. Here, we show that Rbm10 controls appropriate splicing of DNA (cytosine-5)-methyltransferase 3b (Dnmt3b), a DNA methyltransferase, to regulate the activity of NF-κB-responsive promoters and consequently inflammation development. Rbm10 deficiency suppressed NF-κB-mediated responses in vivo and in vitro. Mechanistic analysis showed that Rbm10 deficiency decreased promoter recruitment of NF-κB, with increased DNA methylation of the promoter regions in NF-κB-responsive genes. Consistently, Rbm10 deficiency increased the expression level of Dnmt3b2, which has enzyme activity, while it decreased the splicing isoform Dnmt3b3, which does not. These two isoforms associated with NF-κB efficiently, and overexpression of enzymatically active Dnmt3b2 suppressed the expression of NF-κB targets, indicating that Rbm10-mediated Dnmt3b2 regulation is important for the induction of NF-κB-mediated transcription. Therefore, Rbm10-dependent Dnmt3b regulation is a possible therapeutic target for various inflammatory diseases.
Assuntos
Artrite/imunologia , DNA (Citosina-5-)-Metiltransferases/genética , Inflamação/imunologia , Isoformas de Proteínas/genética , Proteínas de Ligação a RNA/metabolismo , Processamento Alternativo/genética , Animais , Artrite/genética , Células Cultivadas , Modelos Animais de Doenças , Humanos , Inflamação/genética , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Ativação Transcricional , DNA Metiltransferase 3BRESUMO
Lymphatic metastasis is common in advanced-stage carcinoma and is associated with a poor prognosis. However, few effective treatments to inhibit it are available. Z-100 is an immunomodulatory extract of Mycobacterium tuberculosis strain Aoyama B that contains polysaccharides such as arabinomannan and mannan. Here, we investigated the inhibitory effect of Z-100 on spontaneous lymphatic metastasis. C57BL/6N mice injected subcutaneously with B16-BL6 melanoma cells in the right hind footpad were administered Z-100 subcutaneously in the right inguinal region on a daily basis. On day twenty-one after the injection, the right inguinal lymph nodes were excised, and the extent of metastasis, the number of immune cells, and the amount of granzyme B protein in the lymph nodes were examined. We also investigated the combined effect of Z-100 and irradiation in this model. Results showed that Z-100 reduced number of animals with metastasis, with respective metastasis rates of 85.7%, 42.9%, 7.1% and 0.0% in saline, 0.1 mg/kg Z-100, 1 mg/kg Z-100 and 10 mg/kg Z-100 group. Further, mice that had been given Z-100 were found to have more immune cells and granzyme B protein in the lymph nodes than control mice. The combination of low dose Z-100 and irradiation also inhibited spontaneous lymph node metastases. These findings suggest that Z-100 may be beneficial in preventing lymphatic metastasis by enhancing the immune response.
Assuntos
Lipídeos/uso terapêutico , Metástase Linfática/prevenção & controle , Mananas/uso terapêutico , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Mycobacterium tuberculosis , Adjuvantes Imunológicos/farmacologia , Adjuvantes Imunológicos/uso terapêutico , Animais , Contagem de Células , Sobrevivência Celular/efeitos dos fármacos , Terapia Combinada , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Granzimas/metabolismo , Linfonodos/efeitos dos fármacos , Linfonodos/metabolismo , Linfonodos/patologia , Irradiação Linfática , Metástase Linfática/imunologia , Metástase Linfática/patologia , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Camundongos , Subpopulações de Linfócitos T/efeitos dos fármacosRESUMO
Macrophages are a major component of the innate immune system, and the cytokines they secrete are involved in antitumor responses. Z-100 is obtained from hot-water extract of human-type Mycobacterium tuberculosis strain Aoyama B and activates the innate immune response. However, while Z-100 is known to modulate macrophage activity, the mechanism behind this modulation is not fully understood. We evaluated the effects of Z-100 on the murine macrophage cell line RAW264.7. Tumor necrosis factor-alpha (TNF-α) production from RAW264.7 cells was strongly induced by Z-100 and interferon-gamma (IFN-γ) stimulation but only weakly induced by Z-100 alone. Quantitative gene expression analysis showed that nucleotide-binding oligomerization domain containing 2 (Nod2) expression was up-regulated by IFN-γ treatment in RAW264.7 cells while Z-100-induced TNF-α production was attenuated by Nod2 gene silencing. Further, componential analysis demonstrated that muramic acid and amino acids distinctive of muramyl dipeptide (MDP) were contained within Z-100 and Z-100Fr I, the low-molecular-weight fraction containing components <3 kDa in size. In addition, Z-100Fr I enhanced TNF-α production in RAW264.7 cells and promoted NOD2-dependent nuclear factor-kappa B (NF-κB) activation in murine NOD2-expressing SEAP reporter HEK293 (HEK-Blue-mNOD2) cells. Taken together, these results suggest that Z-100 contains MDP-like molecules and augments NF-κB signaling via the direct activation of Nod2 in macrophages, which might be one mechanism driving the innate immune responses induced by Z-100 in cancer immunotherapy.
Assuntos
Lipídeos/isolamento & purificação , Lipídeos/farmacologia , Macrófagos/metabolismo , Mananas/isolamento & purificação , Mananas/farmacologia , Mycobacterium tuberculosis/química , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Animais , Linhagem Celular , Lipídeos/química , Macrófagos/efeitos dos fármacos , Mananas/química , Camundongos , Peso Molecular , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
The survival of naïve T cells is believed to require signals from TCR-pMHC interactions and cytokines such as IL-7. In contrast, signals that negatively impact naïve T cell survival are less understood. We conducted a forward genetic screening of mice and found a mutant mouse line with reduced number of naïve T cells (T-Red mice). T-Red mice have a point mutation in the Kdelr1 gene, and their naïve T cells show enhanced integrated stress response (ISR), which eventually induces their apoptosis. Therefore, naïve T cells require a KDEL receptor-mediated mechanism that efficiently relieves cellular stress for their survival in vivo. Interestingly, naïve T cells expressing TCR with higher affinity/avidity to self-antigens survive in T-Red mice, suggesting the possible link between TCR-mediated survival and ISR-induced apoptosis. In this article, we discuss the regulation of naïve T cell homeostasis, keeping special attention on the ISR and TCR signal.
RESUMO
KDEL receptors are responsible for retrotransporting endoplasmic reticulum (ER) chaperones from the Golgi complex to the ER. Here we describe a role for KDEL receptor 1 (KDELR1) that involves the regulation of integrated stress responses (ISR) in T cells. Designing and using an N-ethyl-N-nitrosourea (ENU)-mutant mouse line, T-Red (naïve T-cell reduced), we show that a point mutation in KDELR1 is responsible for the reduction in the number of naïve T cells in this model owing to an increase in ISR. Mechanistic analysis shows that KDELR1 directly regulates protein phosphatase 1 (PP1), a key phosphatase for ISR in naïve T cells. T-Red KDELR1 does not associate with PP1, resulting in reduced phosphatase activity against eIF2α and subsequent expression of stress responsive genes including the proapoptotic factor Bim. These results demonstrate that KDELR1 regulates naïve T-cell homeostasis by controlling ISR.
Assuntos
Proteína Fosfatase 1/metabolismo , Receptores de Peptídeos/metabolismo , Linfócitos T/fisiologia , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Fator de Iniciação 2 em Eucariotos/metabolismo , Feminino , Homeostase , Memória Imunológica , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Fenótipo , Mutação Puntual , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Peptídeos/genética , Estresse FisiológicoRESUMO
The precise alignment of chromosomes on the metaphase plate prior to the onset of anaphase is essential for ensuring equal segregation of sister chromatids into two daughter cells, and defects in this process potentially cause chromosome instability and tumor progression [1-3]. NDR1 is an evolutionarily conserved serine/threonine kinase whose activity is regulated by MST kinases, Furry (Fry), and MOB [4]. Although the NDR1 signaling pathway is implicated in cell division and morphogenesis in yeast and invertebrates [4-16], the mechanisms of NDR1 activation and the functional significance of the NDR1 pathway in mammalian cells are largely unknown. Here, we show that NDR1 is required for accurate chromosome alignment at metaphase in HeLa cells; depletion of NDR1, Fry, or MST2 caused mitotic chromosome misalignment. Chromosome misalignment in MST2-depleted cells was corrected by expression of active NDR1. The kinase activity of NDR1 increased in early mitotic phase and was dependent on Fry and MST2. We also provide evidence that Fry binds to microtubules, localizes on the spindle, acts as a scaffold that binds to both NDR1 and MOB2, and synergistically activates NDR1 with MOB2. Our findings suggest that MST2-, Fry-, and MOB2-mediated activation of NDR1 is crucial for the fidelity of mitotic chromosome alignment in mammalian cells.