RESUMEN
The phenotypes associated with MED12 pathogenic variants are diverse. Male patients usually have missense variants, but the effects of base substitutions on mRNA splicing have not been investigated. Here, we report a Japanese brother with intellectual disability, characteristic facial appearance with blepharophimosis, cleft palate, Fallot tetralogy, vesicoureteral reflux, and deafness. A known missense pathogenic variant was detected in MED12, NM_005120.3:c.887G>A p.(Arg296Gln), and X-linked Ohdo syndrome was diagnosed in combination with their phenotype. mRNA splicing of MED12 was evaluated qualitatively and quantitatively using long-range PCR-based targeted RNA sequencing (reverse transcribed long amplicon sequencing), and it was shown that this missense variant simultaneously causes aberrant splicing of the 42-bp in-frame deletion in exon 7, r.847_888del, which accounts for approximately 30% of the mRNAs in both siblings. The X chromosome inactivation study showed that the X chromosome carrying the mutant allele was 100% inactivated in the carrier mothers. mRNA level analysis is essential for the accurate interpretation of the effects of variants. In this case, the MED12 protein function may be reduced by more than just an amino acid substitution, resulting in the patients with the most severe phenotype of MED12-related syndrome in males.
Asunto(s)
Blefarofimosis , Complejo Mediador , Empalme del ARN , Niño , Femenino , Humanos , Masculino , Anomalías Múltiples , Blefarofimosis/genética , Blefarofimosis/patología , Blefaroptosis , Fisura del Paladar/genética , Fisura del Paladar/patología , Sordera/genética , Sordera/patología , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Enfermedades Genéticas Ligadas al Cromosoma X/patología , Cardiopatías Congénitas , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Complejo Mediador/genética , Mutación Missense , Linaje , Fenotipo , Empalme del ARN/genética , Reflujo Vesicoureteral/genética , Reflujo Vesicoureteral/patología , Inactivación del Cromosoma X/genéticaRESUMEN
BACKGROUND: mRNA sequencing is a powerful technique, which is used to investigate the transcriptome status of a gene of interest, such as its transcription level and splicing variants. Presently, several RNA sequencing (RNA-Seq) methods have been developed; however, the relative advantage of each method has remained unknown. Here we used three commercially available RNA-Seq library preparation kits; the traditional method (TruSeq), in addition to full-length double-stranded cDNA methods (SMARTer and TeloPrime) to investigate the advantages and disadvantages of these three approaches in transcriptome analysis. RESULTS: We observed that the number of expressed genes detected from the TeloPrime sequencing method was fewer than that obtained using the TruSeq and SMARTer. We also observed that the expression patterns between TruSeq and SMARTer correlated strongly. Alternatively, SMARTer and TeloPrime methods underestimated the expression of relatively long transcripts. Moreover, genes having low expression levels were undetected stochastically regardless of any three methods used. Furthermore, although TeloPrime detected a significantly higher proportion at the transcription start site (TSS), its coverage of the gene body was not uniform. SMARTer is proposed to be yielded for nonspecific genomic DNA amplification. In contrast, the detected splicing event number was highest in the TruSeq. The percent spliced in index (PSI) of the three methods was highly correlated. CONCLUSIONS: TruSeq detected transcripts and splicing events better than the other methods and measured expression levels of genes, in addition to splicing events accurately. However, although detected transcripts and splicing events in TeloPrime were fewer, the coverage at TSS was highest. Additionally, SMARTer was better than TeloPrime with regards to the detected number of transcripts and splicing events among the understudied full-length double-stranded cDNA methods. In conclusion, for short-read sequencing, TruSeq has relative advantages for use in transcriptome analysis.
Asunto(s)
Perfilación de la Expresión Génica , Transcriptoma , Empalme Alternativo , ADN Complementario/genética , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN Mensajero/genética , ARN Mensajero/metabolismo , RNA-Seq , Análisis de Secuencia de ARN/métodosRESUMEN
Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by multiple dysplastic organ lesions and neuropsychiatric symptoms, caused by loss of function mutations in either TSC1 or TSC2. Genotype and phenotype analyses are conducted worldwide, but there have been few large-scale studies on Japanese patients, and there are still many unclear points. This study analyzed 283 Japanese patients with TSC (225 definite, 53 possible, and 5 genetic diagnoses). A total of 200 mutations (64 TSC1, 136 TSC2) were identified, of which 17 were mosaic mutations, 11 were large intragenic deletions, and four were splicing abnormalities due to deep intronic mutations. Several lesions and symptoms differed in prevalence and severity between TSC1 and TSC2 patients and were generally more severe in TSC2 patients. Moreover, TSC2 missense and in-frame mutations may attenuate skin and renal symptoms compared to other TSC2 mutations. Genetic testing revealed that approximately 20% of parents of a proband had mild TSC, which could have been missed. The patient demographics presented in this study revealed a high frequency of TSC1 patients and a low prevalence of epilepsy compared to global statistics. More patients with mild neuropsychiatric phenotypes were diagnosed in Japan, seemingly due to a higher utilization of brain imaging, and suggesting the possibility that a significant amount of mild TSC patients may not be correctly diagnosed worldwide.
Asunto(s)
Esclerosis Tuberosa , Humanos , Análisis Mutacional de ADN/métodos , Genotipo , Japón/epidemiología , Mutación , Fenotipo , Esclerosis Tuberosa/epidemiología , Esclerosis Tuberosa/genética , Proteína 1 del Complejo de la Esclerosis Tuberosa/genética , Proteína 2 del Complejo de la Esclerosis Tuberosa/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
Malignant pleural effusion (MPE) provides a liquid tumor microenvironment model that includes cancer cells and immune cells. However, the characteristics of tumor antigen-specific CD8+ T cells have not been investigated in detail. Here, we analyzed MPE samples taken from a patient with pancreatic cancer who received a dendritic cell vaccine targeting Wilms' Tumor 1 (WT1) antigen over the disease course (two points at MPE1st and 2nd, two months after MPE1st). Epithelial cell adhesion molecule (EpCAM)+ cancer cells (PD-L1- or T cell immunoglobulin mucin-3, TIM-3-), both PD-1 or TIM-3 positive CD8+ T cells, and CD14+CD68+CD163+TIM-3+ macrophages increased from the MPE1st to MPE2nd. The ratio of WT1-specific cytotoxic lymphocytes (WT1-CTLs) to MPE CD8+ T cells and IFN-γ secretion of WT1-CTLs were reduced with disease progression. Coincidentally, the fraction of central memory T (TCM) of WT1-CTLs was decreased. On the other hand, CD8+ T cells in response to SMAD4P130L, which is homogeneously expressed in EpCAM+ cancer cells, were detected using in vitro expansion with the HLA-A*11:01 restrictive SVCVNLYH neoantigen. Furthermore, the CD8+ T cell response to SMAD4P130L was diminished following remarkably decreased numbers of CD8+ TCM in MPE samples. In conclusion, CD8+ T cells responding to WT1 or SMAD4P130L neoantigen expressed in EpCAM+ pancreatic cancer cells were detected in MPE. A tumor antigen-specific immune response would provide novel insight into the MPE microenvironment.
Asunto(s)
Neoplasias Pancreáticas , Derrame Pleural Maligno , Vacunas , Humanos , Molécula de Adhesión Celular Epitelial/metabolismo , Linfocitos T CD8-positivos , Antígeno B7-H1/metabolismo , Receptor 2 Celular del Virus de la Hepatitis A/metabolismo , Proteínas WT1 , Receptor de Muerte Celular Programada 1/metabolismo , Mucina 3/metabolismo , Neoplasias Pancreáticas/patología , Inmunoglobulinas/metabolismo , Vacunas/metabolismo , Antígenos HLA-A , Microambiente Tumoral , Proteína Smad4/metabolismoRESUMEN
Elaborate analyses of the status of gene mutations in neurofibromatosis type 1 (NF1) are still difficult nowadays due to the large gene sizes, broad mutation spectrum, and the various effects of mutations on mRNA splicing. These problems cannot be solved simply by sequencing the entire coding region using next-generation sequencing (NGS). We recently developed a new strategy, named combined long amplicon sequencing (CoLAS), which is a method for simultaneously analysing the whole genomic DNA region and, also, the full-length cDNA of the disease-causative gene with long-range PCR-based NGS. In this study, CoLAS was specifically arranged for NF1 genetic analysis, then applied to 20 patients (five previously reported and 15 newly recruited patients, including suspicious cases) for optimising the method and to verify its efficacy and benefits. Among new cases, CoLAS detected not only 10 mutations, including three unreported mutations and one mosaic mutation, but also various splicing abnormalities and allelic expression ratios quantitatively. In addition, heterozygous mapping by polymorphisms, including introns, showed copy number monitoring of the entire NF1 gene region was possible in the majority of patients tested. Moreover, it was shown that, when a chromosomal level microdeletion was suspected from heterozygous mapping, it could be detected directly by breakpoint-specific long PCR. In conclusion, CoLAS not simply detect the causative mutation but accurately elucidated the entire structure of the NF1 gene, its mRNA expression, and also the splicing status, which reinforces its high usefulness in the gene analysis of NF1.
Asunto(s)
Biomarcadores de Tumor/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutación , Neurofibromatosis 1/genética , Alelos , Biomarcadores de Tumor/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Humanos , Neurofibromatosis 1/metabolismo , Neurofibromatosis 1/patología , Proyectos Piloto , Reacción en Cadena de la Polimerasa/métodos , Empalme del ARNRESUMEN
Alternative splicing is a regulated process by which eukaryotic genes may produce diverse biological products. Defects in the process typically affect cellular function and can lead to disease. Next-generation sequencing (NGS) technologies have been developed to detect alternative splicing events; however, the alternative splicing events detected by standard RNA-Seq may or may not be derived from full-length RNA. The SMARTer method provides full-length double-strand cDNA synthesis, and the resulting gene expression patterns correlate strongly with standard RNA-Seq. However, it also yields non-specific genomic DNA amplification. We improved the SMARTer method by employing a target-capture full-length double-strand cDNA sequencing method. High-fidelity, full-length cDNA is generated by the SMARTer method, followed by target-specific capture with exon probes. The expression pattern observed with this SMARTer Capture method was highly correlated with the results of the original SMARTer method. The number and accuracy of the detected splicing events were increased by eliminating non-specific genomic DNA amplification by the SMARTer Capture. Compared to the original SMARTer method, the SMARTer Capture provided 4-fold greater detection of alternative splicing events at the same read number, and it took less than 1/100 of read number to detect the same number of splicing events. The percent splicing in index (PSI) of the SMARTer Capture is highly correlated with the PSI of the SMARTer. These results indicate that the SMARTer Capture represents an improvement of the SMARTer method to accurately characterize alternative splicing repertories in targeted genes without biases.
Asunto(s)
Empalme Alternativo , ADN Complementario/genética , ADN/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN Mensajero/genética , Análisis de Secuencia de ARN/métodos , Esclerosis Tuberosa/genética , Humanos , Programas Informáticos , Esclerosis Tuberosa/sangreRESUMEN
Human hereditary malformation syndromes are caused by mutations in the genes of the signal transduction molecules involved in fetal development. Among them, the Sonic hedgehog (SHH) signaling pathway is the most important, and many syndromes result from its disruption. In this review, we summarize the molecular mechanisms and role in embryonic morphogenesis of the SHH pathway, then classify the phenotype of each malformation syndrome associated with mutations of major molecules in the pathway. The output of the SHH pathway is shown as GLI activity, which is generated by SHH in a concentration-dependent manner, i.e., the sum of activating form of GLI (GLIA) and repressive form of GLI (GLIR). Which gene is mutated and whether the mutation is loss-of-function or gain-of-function determine in which concentration range of SHH the imbalance occurs. In human malformation syndromes, too much or too little GLI activity produces symmetric phenotypes affecting brain size, craniofacial (midface) dysmorphism, and orientation of polydactyly with respect to the axis of the limb. The symptoms of each syndrome can be explained by the GLIA/R balance model.
Asunto(s)
Anomalías Craneofaciales/etiología , Proteínas Hedgehog/metabolismo , Deformidades Congénitas de las Extremidades/etiología , Cilios/fisiología , Anomalías Craneofaciales/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Deformidades Congénitas de las Extremidades/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Receptor Patched-1/genética , Receptor Patched-1/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Síndrome , Proteína Gli2 con Dedos de Zinc/genética , Proteína Gli2 con Dedos de Zinc/metabolismoRESUMEN
Cancer gene panel testing requires accurate detection of somatic mosaic mutations, as the test sample consists of a mixture of cancer cells and normal cells; each minor clone in the tumor also has different somatic mutations. Several studies have shown that the different types of software used for variant calling for next generation sequencing (NGS) can detect low-frequency somatic mutations. However, the accuracy of these somatic variant callers is unknown. We performed cancer gene panel testing in duplicate experiments using three different high-fidelity DNA polymerases in pre-capture amplification steps and analyzed by three different variant callers, Strelka2, Mutect2, and LoFreq. We selected six somatic variants that were detected in both experiments with more than two polymerases and by at least one variant caller. Among them, five single nucleotide variants were verified by CEL nuclease-mediated heteroduplex incision with polyacrylamide gel electrophoresis and silver staining (CHIPS) and Sanger sequencing. In silico analysis indicated that the FBXW7 and MAP3K1 missense mutations cause damage at the protein level. Comparing three somatic variant callers, we found that Strelka2 detected more variants than Mutect2 and LoFreq. We conclude that dual sequencing with Strelka2 analysis is useful for detection of accurate somatic mutations in cancer gene panel testing.
Asunto(s)
Genes Relacionados con las Neoplasias , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutación/genética , Neoplasias/genética , Secuencia de Bases , ADN Polimerasa Dirigida por ADN/metabolismo , Femenino , Frecuencia de los Genes/genética , Humanos , Persona de Mediana Edad , Reproducibilidad de los ResultadosRESUMEN
Signal-transducing adaptor family member-2 (STAP-2) is an adaptor protein that regulates various intracellular signaling pathways and promotes tumorigenesis in melanoma and breast cancer cells. However, the contribution of STAP-2 to the behavior of other types of cancer cells is unclear. Here, we show that STAP-2 promotes tumorigenesis of prostate cancer cells through up-regulation of EGF receptor (EGFR) signaling. Tumor growth of a prostate cancer cell line, DU145, was strongly decreased by STAP-2 knockdown. EGF-induced gene expression and phosphorylation of AKT, ERK, and STAT3 were significantly decreased in STAP-2-knockdown DU145 cells. Mechanistically, we found that STAP-2 interacted with EGFR and enhanced its stability by inhibiting c-CBL-mediated EGFR ubiquitination. Our results indicate that STAP-2 promotes prostate cancer progression via facilitating EGFR activation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proliferación Celular , Receptores ErbB/química , Fosfoproteínas/metabolismo , Neoplasias de la Próstata/patología , Animales , Receptores ErbB/metabolismo , Humanos , Masculino , Ratones Endogámicos BALB C , Fosforilación , Neoplasias de la Próstata/metabolismo , Estabilidad Proteica , Transducción de Señal , Células Tumorales Cultivadas , Ubiquitinación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Signal transducer and activator of transcription 3 (STAT3) is involved in cell proliferation, differentiation, and cell survival during immune responses, hematopoiesis, neurogenesis, and other biological processes. STAT3 activity is regulated by a variety of mechanisms, including phosphorylation and nuclear translocation. To clarify the molecular mechanisms underlying the regulation of STAT3 activity, we performed yeast two-hybrid screening. We identified ARL3 (ADP-ribosylation factor-like 3) as a novel STAT3-binding partner. ARL3 recognizes the DNA-binding domain as well as the C-terminal region of STAT3 in vivo, and their binding was the strongest when both proteins were activated. Importantly, small interfering RNA-mediated reduction of endogenous ARL3 expression decreased IL-6-induced tyrosine phosphorylation, nuclear accumulation, and transcriptional activity of STAT3. These results indicate that ARL3 interacts with STAT3 and regulates the transcriptional activation of STAT3 by influencing its nuclear accumulation of STAT3.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Núcleo Celular/metabolismo , Factor de Transcripción STAT3/metabolismo , Factores de Ribosilacion-ADP/genética , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/genética , Núcleo Celular/genética , Regulación de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Interleucina-6/farmacología , Fosforilación/efectos de los fármacos , Fosforilación/genética , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Factor de Transcripción STAT3/genéticaRESUMEN
Melanoma is the most serious type of skin cancer, with a highly metastatic phenotype. In this report, we show that signal transducing adaptor protein 2 (STAP-2) is involved in cell migration, proliferation, and melanogenesis as well as chemokine receptor expression and tumorigenesis in B16F10 melanoma cells. This was evident in mice injected with STAP-2 shRNA (shSTAP-2)-expressing B16F10 cells, which infiltrated organs in a completely different pattern from the original cells, showing massive colonization in the liver, kidney, and neck but not in the lung. The most important finding was that STAP-2 expression determined tyrosinase protein content. STAP-2 colocalized with tyrosinase in lysosomes and protected tyrosinase from protein degradation. It is noteworthy that B16F10 cells with knocked down tyrosinase showed similar cell characteristics as shSTAP-2 cells. These results indicated that tyrosinase contributed to some cellular events beyond melanogenesis. Taken together, one possibility is that STAP-2 positively regulates the protein levels of tyrosinase, which determines tumor invasion via controlling chemokine receptor expression.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Melanoma Experimental/metabolismo , Monofenol Monooxigenasa/metabolismo , Neoplasias Cutáneas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Línea Celular Tumoral , Movimiento Celular , Forma de la Célula , Supervivencia Celular , Técnicas de Silenciamiento del Gen , Lisosomas/metabolismo , Melaninas/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/secundario , Ratones , Ratones Endogámicos C57BL , Monofenol Monooxigenasa/antagonistas & inhibidores , Monofenol Monooxigenasa/genética , Invasividad Neoplásica , Especificidad de Órganos , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , ARN Interferente Pequeño/genética , Receptores de Quimiocina/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Neoplasias Cutáneas/genéticaRESUMEN
Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that regulates immune and inflammatory responses through interactions with a variety of signaling and transcriptional molecules. In the current study, we clarified the physiological role of STAP-2 in mast cell function, a key mediator of IgE-associated allergic responses. STAP-2 is constitutively expressed in mast cells. STAP-2 deficiency in mast cells greatly enhances FcεRI-mediated signals, resulting in the increased tyrosine phosphorylation of the phospholipase C-γ isoform, calcium mobilization, and degranulation. Of importance, STAP-2-deficient mice challenged with DNP-BSA after passive sensitization with anti-DNP IgE show more severe rectal temperature decrease than do wild-type mice. STAP-2-deficient mice also show increased vascular permeability and more severe cutaneous anaphylaxis after DNP-BSA injection. These regulatory functions performed by STAP-2 indicate that there is an interaction between STAP-2 and FcεRI. In addition, our previous data indicate that STAP-2 binds to the phospholipase C-γ isoform and IκB kinase-ß. Therefore, our data described in this article strongly suggest that manipulation of STAP-2 expression in mast cells may control the pathogenesis of allergic diseases and have the potential for treating patients with allergy.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Anafilaxia/inmunología , Anafilaxia/metabolismo , Inmunoglobulina E/inmunología , Mastocitos/inmunología , Mastocitos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Anafilaxia/genética , Animales , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Proliferación Celular , Citocinas/biosíntesis , Modelos Animales de Enfermedad , Ratones , Ratones Noqueados , Unión Proteica , Receptores de IgE/metabolismo , Transducción de SeñalRESUMEN
Integrins affect the motility of multiple cell types to control cell survival, growth, or differentiation, which are mediated by cell-cell and cell-extracellular matrix interactions. We reported previously that the α9 integrin splicing variant, SFα9, promotes WT α9 integrin-dependent adhesion. In this study, we introduced a new murine α4 integrin splicing variant, α4B, which has a novel short cytoplasmic tail. In inflamed tissues, the expression of α4B, as well as WT α4 integrin, was up-regulated. Cells expressing α4B specifically bound to VCAM-1 but not other α4 integrin ligands, such as fibronectin CS1 or osteopontin. The binding of cells expressing WT α4 integrin to α4 integrin ligands is inhibited by coexpression of α4B. Knockdown of α4B in metastatic melanoma cell lines results in a significant increase in lung metastasis. Expression levels of WT α4 integrin are unaltered by α4B, with α4B acting as a regulatory subunit for WT α4 integrin by a dominant-negative effect or inhibiting α4 integrin activation.
Asunto(s)
Adhesión Celular , Integrina alfa4/metabolismo , Empalme de Proteína , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Citoplasma/metabolismo , Cartilla de ADN , Fibronectinas/metabolismo , Integrina alfa4/química , Integrina alfa4/genética , Ratones , Células 3T3 NIH , Osteopontina/metabolismo , ARN Mensajero/genética , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA), which interacts with cellular proteins, plays a central role in modification of viral and/or cellular gene expression. Here, we show that LANA associates with glucocorticoid receptor (GR), and that LANA enhances the transcriptional activity of GR. Co-immunoprecipitation revealed a physical interaction between LANA and GR in transiently transfected 293T and HeLa cells. In human B-lymphoma cells, LANA overexpression enhanced GR activity and cell growth suppression following glucocorticoid stimulation. Furthermore, confocal microscopy showed that activated GR was bound to LANA and accumulated in the nucleus, leading to an increase in binding of activated GR to the glucocorticoid response element of target genes. Taken together, KSHV-derived LANA acts as a transcriptional co-activator of GR. Our results might suggest a careful use of glucocorticoids in the treatment of patients with KSHV-related malignancies such as Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman disease.
Asunto(s)
Antígenos Virales/metabolismo , Herpesvirus Humano 8/fisiología , Interacciones Huésped-Patógeno , Proteínas Nucleares/metabolismo , Receptores de Glucocorticoides/metabolismo , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/virología , Linfocitos B/metabolismo , Linfocitos B/virología , Línea Celular , Línea Celular Tumoral , Proliferación Celular , ADN/metabolismo , Células HeLa , Humanos , Receptores de Glucocorticoides/genética , Sarcoma de Kaposi/genética , Activación TranscripcionalRESUMEN
The promyelocytic leukemia protein PML acts as a tumor suppressor by forming transcription-regulatory complexes with a variety of repressor proteins. In the present study, we found that endogenous PML suppresses interleukin (IL)-6-induced gene expression as well as phosphorylation and transcriptional activation of STAT3 in hepatoma cells. We also found that PML-mediated suppression of IL-6-induced STAT3 activation by disrupting interactions between STAT3 and HDAC3. These results indicate that PML modulates IL-6-induced STAT3 activation and hepatoma cell growth by interacting with HDAC3.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Histona Desacetilasas/metabolismo , Interleucina-6/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas Nucleares/metabolismo , Factor de Transcripción STAT3/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/genética , Proteína de la Leucemia Promielocítica , Mapas de Interacción de Proteínas , Factor de Transcripción STAT3/genética , Activación TranscripcionalRESUMEN
Tyrosine kinase 2 (Tyk2), a member of the Jak kinase family, mediates signals triggered by various cytokines, which are related to the pathogenesis of psoriasis. In this study, we investigated the role of Tyk2 in IL-23-induced psoriasis-like skin inflammation. Tyk2(-/-) mice when injected with IL-23 showed significantly reduced ear skin swelling with epidermal hyperplasia and inflammatory cell infiltration compared with wild-type mice. In addition, Tyk2 deficiency reduced production of pro-inflammatory cytokines and psoriasis-relevant anti-microbial peptides. More noteworthy is that Tyk2 directly regulated IL-22-dependent inflammation and epidermal hyperplasia. Taken together with the inhibition of IL-23-induced inflammation by treatment with neutralizing antibodies against IL-17 or IL-22, Tyk2 participates in both IL-23 and IL-22 signal transduction to mediate psoriasis-like skin inflammation. On the basis of these findings, we demonstrated for the first time that a small-molecule Tyk2 inhibitor significantly inhibited IL-23-induced inflammation and cytokine production in the skin. These observations demonstrate the important role of Tyk2 in experimental skin inflammation and indicate the therapeutic potential of Tyk2 inhibition in human psoriasis.
Asunto(s)
Inflamación/inmunología , Psoriasis/inmunología , Piel/inmunología , TYK2 Quinasa/inmunología , Animales , Western Blotting , Calgranulina A/genética , Calgranulina A/inmunología , Línea Celular , Citocinas/inmunología , Citocinas/metabolismo , Defensinas/genética , Defensinas/inmunología , Inhibidores Enzimáticos/farmacología , Expresión Génica/inmunología , Humanos , Hiperplasia/inmunología , Inflamación/inducido químicamente , Inflamación/prevención & control , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Interleucina-17/inmunología , Interleucina-17/metabolismo , Interleucina-23 , Interleucinas/inmunología , Interleucinas/metabolismo , Interleucinas/farmacología , Queratinocitos/efectos de los fármacos , Queratinocitos/inmunología , Queratinocitos/metabolismo , Ratones Endogámicos BALB C , Ratones Noqueados , Psoriasis/inducido químicamente , Psoriasis/prevención & control , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Piel/metabolismo , Piel/patología , TYK2 Quinasa/antagonistas & inhibidores , TYK2 Quinasa/genética , Tirfostinos/farmacología , Interleucina-22RESUMEN
Although Y14 is known to be a component of the exon junction complex, we previously reported that Y14 regulates IL-6-induced STAT3 activation. In this study, we showed that endogenous Y14 positively regulated TNF-α-induced IL-6 expression in HeLa cells. Small interfering RNA-mediated Y14-knockdown reduced TNF-α-induced and NF-κB-mediated transcriptional activity, phosphorylation/degradation of IκBα, and nuclear localization of NF-κB/p65. As in the case of IL-6 stimuli, Y14 enhanced TNF-α-induced STAT3 phosphorylation, which is important for its nuclear retention. However, our manipulation of Y14 expression indicated that it is involved in TNF-α-induced IL-6 expression via both STAT3-dependent and -independent mechanisms. We screened signaling molecules in the TNF-α-NF-κB pathway and found that Y14 endogenously associated with receptor-interacting protein 1 (RIP1) and TNFR-associated death domain (TRADD). Overexpression of RIP1, but not TRADD, restored TNF-α-induced NF-κB activation in Y14-knockdown cells, and Y14 overexpression restored TNF-α-induced NF-κB activation in TRADD-knockdown cells, but not in RIP1-knockdown cells, indicating that Y14 lies downstream of TRADD and upstream of RIP1. Of importance, Y14 significantly enhanced the binding between RIP1 and TRADD, and this is a possible new mechanism for Y14-mediated modification of TNF-α signals. Although Y14 associates with MAGOH in the exon junction complex, Y14's actions in the TNF-α-NF-κB pathway are unlikely to require MAGOH. Therefore, Y14 positively regulates signals for TNF-α-induced IL-6 production at multiple steps beyond an exon junction complex protein.
Asunto(s)
FN-kappa B/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteína de Dominio de Muerte Asociada a Receptor de TNF/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Línea Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Proteínas I-kappa B/metabolismo , Interleucina-6/biosíntesis , Interleucina-6/genética , Inhibidor NF-kappaB alfa , FN-kappa B/genética , Proteínas de Complejo Poro Nuclear/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Interferente Pequeño , Proteínas de Unión al ARN/genética , Factor de Transcripción STAT1 , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Proteína de Dominio de Muerte Asociada a Receptor de TNF/genética , Factor 2 Asociado a Receptor de TNF/genética , Factor de Transcripción ReIA/metabolismo , Transcripción GenéticaRESUMEN
Here, we report a novel PROS1 splicing mutation in a patient with type I protein S deficiency. Qualitative and quantitative analysis of pathogenic splicing variants at the mRNA level was performed by long-range PCR-based targeted DNA and RNA sequencing. A base substitution in the exon 4 splicing donor site activates a potential splicing donor site in intron 4, resulting in an in-frame insertion of 48 bases (16 amino acids).
RESUMEN
X-linkded Ohdo syndrome is characterized mainly by intellectual disability, delays in reaching development, feeding difficulties, thyroid dysfunction, and dysmorphic appearance with blepharophimosis, immobile mask-like face and bulbous nose. The X-linked Ohdo syndrome is caused by loss of function mutation in MED12 gene on X chromosome. The peripheral blood mononuclear cells from a patient carrying missense mutation of the MED12 gene were reprogrammed using the CytoTune-iPS2.0 Sendai Reprogramming Kit. The missense mutation in MED12 gene causes the abnormal protein variant. The established human induced pluripotent cell line will enable proper in vitro disease modelling of X-linked Ohdo syndrome.
Asunto(s)
Células Madre Pluripotentes Inducidas , Complejo Mediador , Mutación Missense , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Complejo Mediador/genética , Complejo Mediador/metabolismo , Línea Celular , Masculino , Reprogramación Celular , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Enfermedades Genéticas Ligadas al Cromosoma X/patologíaRESUMEN
Signal-transducing adaptor protein-2 (STAP-2) is an adaptor molecule involved in several cellular signaling cascades. Here, we attempted to identify novel STAP-2 interacting molecules, and identified c-Cbl associated protein (CAP) as a binding protein through the C-terminal proline-rich region of STAP-2. Expression of STAP-2 increased the interaction between CAP and c-Cbl, suggesting that STAP-2 bridges these proteins and enhances complex formation. CAP/c-Cbl complex is known to regulate GLUT4 translocation in insulin signaling. STAP-2 overexpressed human hepatocyte Hep3B cells showed enhanced GLUT4 translocation after insulin treatment. Elevated levels of Stap2 mRNA have been observed in 3T3-L1 cells and mouse embryonic fibroblasts (MEFs) during adipocyte differentiation. The differentiation of 3T3-L1 cells into adipocytes was highly promoted by retroviral overexpression of STAP-2. In contrast, STAP-2 knockout (KO) MEFs exhibited suppressed adipogenesis. The increase in body weight with high-fat diet feeding was significantly decreased in STAP-2 KO mice compared to WT animals. These data suggest that the expression of STAP-2 correlates with adipogenesis. Thus, STAP-2 is a novel regulatory molecule that controls insulin signal transduction by forming a c-Cbl/STAP-2/CAP ternary complex.