RESUMEN

Objective: A form of pathological social withdrawal which is also called hikikomori has been proved its existence in China. But the prevalence and characteristics of hikikomori in China remain unknown. Past studies had investigated the hikikomori phenomenon in three cities of China. The purpose of this study is to discover the prevalence of hikikomori in a convenient online sample in China as well as the difference in demographic characteristics and other possible traits between hikikomori sufferers and the general population. Methods: A total of 1,066 youths (mean age = 22.85 years) in China completed the online questionnaire, which consisted of questions about demographics, the 25-item Hikikomori Questionnaire (HQ-25), the Internet Addiction Test (IAT), the Loneliness Scale (UCLA), and the General Health Questionnaire (GHQ). SPSS is used to evaluate the data. Results: Of the 1,066 youths, 980 (91.9%) were identified as belonging to group A (be not social isolation nor withdrawn), 46 (4.3%) to group B (marked social isolation in one's home or withdrawn with a duration of at least 3 months), and 40 (3.8%) to group C (marked both social isolation in one's home and withdrawn with a duration of at least 3 months). The hikikomori group (combined group B and group C) accounted for 8.1%. The present data suggest that residence and loneliness are related to the occurrence of hikikomori. HQ-25 score of the hikikomori group was significantly higher than the comparison group. The UCLA score showed that those in the hikikomori group felt lonelier than those in the comparison. The regression model predicted hikikomori risk (χ2 = 38.658, P = 0.000), the Hosmer-Lemeshow test value is 7.114 and P = 0.524 > 0.05. Conclusion: The grouping criterion in our present study is reasonable and such a grouping criterion can screen out potential populations of hikikomori. When people develop into hikikomori sufferers in the present, their social withdrawal behaviors and feeling of loneliness are both much more severe than in the past. The possible relationships between hikikomori and loneliness reflect the need to give the youths more social support, to help them connect with society.

RESUMEN

To identify genes and signaling pathways that initiate Neurofibromatosis type 1 (NF1) neurofibromas, we used unbiased insertional mutagenesis screening, mouse models, and molecular analyses. We mapped an Nf1-Stat3-Arid1b/ß-catenin pathway that becomes active in the context of Nf1 loss. Genetic deletion of Stat3 in Schwann cell progenitors (SCPs) and Schwann cells (SCs) prevents neurofibroma formation, decreasing SCP self-renewal and ß-catenin activity. ß-catenin expression rescues effects of Stat3 loss in SCPs. Importantly, P-STAT3 and ß-catenin expression correlate in human neurofibromas. Mechanistically, P-Stat3 represses Gsk3ß and the SWI/SNF gene Arid1b to increase ß-catenin. Knockdown of Arid1b or Gsk3ß in Stat3(fl/fl);Nf1(fl/fl);DhhCre SCPs rescues neurofibroma formation after in vivo transplantation. Stat3 represses Arid1b through histone modification in a Brg1-dependent manner, indicating that epigenetic modification plays a role in early tumorigenesis. Our data map a neural tumorigenesis pathway and support testing JAK/STAT and Wnt/ß-catenin pathway inhibitors in neurofibroma therapeutic trials.

Asunto(s)

Carcinogénesis/genética , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Acetiltransferasa A N-Terminal/genética , Neurofibromatosis 1/genética , Neoplasias del Sistema Nervioso Periférico/genética , Factor de Transcripción STAT3/genética , beta Catenina/genética , Animales , Carcinogénesis/metabolismo , Carcinogénesis/patología , ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Femenino , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Ratones , Ratones Desnudos , Mutagénesis Insercional , Acetiltransferasa A N-Terminal/antagonistas & inhibidores , Acetiltransferasa A N-Terminal/metabolismo , Trasplante de Neoplasias , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Neurofibromatosis 1/metabolismo , Neurofibromatosis 1/patología , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias del Sistema Nervioso Periférico/metabolismo , Neoplasias del Sistema Nervioso Periférico/patología , Fosforilación , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismo , Células de Schwann/metabolismo , Células de Schwann/patología , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , beta Catenina/metabolismo

RESUMEN

Epstein-Barr Virus (EBV) causes infectious mononucleosis and establishes lifelong infection associated with cancer and autoimmune disease. To better understand immunity to EBV, we performed a prospective study of natural infection in healthy humans. Transcriptome analysis defined a striking and reproducible expression profile during acute infection but no lasting gene changes were apparent during latent infection. Comparing the EBV response profile to multiple other acute viral infections, including influenza A (influenza), respiratory syncytial virus (RSV), human rhinovirus (HRV), attenuated yellow fever virus (YFV), and Dengue fever virus (DENV), revealed similarity only to DENV. The signature shared by EBV and DENV was also present in patients with hemophagocytic syndromes, suggesting these two viruses cause uncontrolled inflammatory responses. Interestingly, while EBV induced a strong type I interferon response, a subset of interferon induced genes, including MX1, HERC5, and OAS1, were not upregulated, suggesting a mechanism by which viral antagonism of immunity results in a profound inflammatory response. These data provide an important first description of the response to a natural herpesvirus infection in humans.

Asunto(s)

Infecciones por Virus de Epstein-Barr/genética , Perfilación de la Expresión Génica , Herpesvirus Humano 4/fisiología , Linfohistiocitosis Hemofagocítica/genética , Linfohistiocitosis Hemofagocítica/virología , Linfocitos T CD8-positivos/inmunología , Dengue/patología , Dengue/virología , Infecciones por Virus de Epstein-Barr/sangre , Humanos , Mononucleosis Infecciosa/genética , Mononucleosis Infecciosa/virología , Inflamación/patología , Interferones/metabolismo , Cinética , Lupus Eritematoso Sistémico/genética , Linfohistiocitosis Hemofagocítica/sangre , Monocitos/inmunología , Regulación hacia Arriba/genética , Latencia del Virus , Adulto Joven

RESUMEN

UNLABELLED: Hepatocellular carcinoma (HCC) is one of the deadliest solid cancers and is the third leading cause of cancer-related death. There is a universal estimated male/female ratio of 2.5, but the reason for this is not well understood. The Sleeping Beauty (SB) transposon system was used to elucidate candidate oncogenic drivers of HCC in a forward genetics screening approach. Sex bias occurrence was conserved in our model, with male experimental mice developing liver tumors at reduced latency and higher tumor penetrance. In parallel, we explored sex differences regarding genomic aberrations in 235 HCC patients. Liver cancer candidate genes were identified from both sexes and genotypes. Interestingly, transposon insertions in the epidermal growth factor receptor (Egfr) gene were common in SB-induced liver tumors from male mice (10/10, 100%) but infrequent in female mice (2/9, 22%). Human single-nucleotide polymorphism data confirmed that polysomy of chromosome 7, locus of EGFR, was more frequent in males (26/62, 41%) than females (2/27, 7%) (P = 0.001). Gene expression-based Poly7 subclass patients were predominantly male (9/9) compared with 67% males (55/82) in other HCC subclasses (P = 0.02), and this subclass was accompanied by EGFR overexpression (P < 0.001). CONCLUSION: Sex bias occurrence of HCC associated with EGFR was confirmed in experimental animals using the SB transposon system in a reverse genetic approach. This study provides evidence for the role of EGFR in sex bias occurrences of liver cancer and as the driver mutational gene in the Poly7 molecular subclass of human HCC.

Asunto(s)

Carcinoma Hepatocelular/genética , Cromosomas Humanos Par 7 , Receptores ErbB/genética , Neoplasias Hepáticas/genética , Factores Sexuales , Animales , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Transformación Celular Neoplásica , Elementos Transponibles de ADN , Femenino , Hepatocitos/patología , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Transgénicos , Mutagénesis Insercional , beta Catenina/metabolismo

RESUMEN

INTRODUCTION: Progesterone receptors (PR) are emerging as important breast cancer drivers. Phosphorylation events common to breast cancer cells impact PR transcriptional activity, in part by direct phosphorylation. PR-B but not PR-A isoforms are phosphorylated on Ser294 by mitogen activated protein kinase (MAPK) and cyclin dependent kinase 2 (CDK2). Phospho-Ser294 PRs are resistant to ligand-dependent Lys388 SUMOylation (that is, a repressive modification). Antagonism of PR small ubiquitin-like modifier (SUMO)ylation by mitogenic protein kinases suggests a mechanism for derepression (that is, transcriptional activation) of target genes. As a broad range of PR protein expression is observed clinically, a PR gene signature would provide a valuable marker of PR contribution to early breast cancer progression. METHODS: Global gene expression patterns were measured in T47D and MCF-7 breast cancer cells expressing either wild-type (SUMOylation-capable) or K388R (SUMOylation-deficient) PRs and subjected to pathway analysis. Gene sets were validated by RT-qPCR. Recruitment of coregulators and histone methylation levels were determined by chromatin immunoprecipitation. Changes in cell proliferation and survival were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and western blotting. Finally, human breast tumor cohort datasets were probed to identify PR-associated gene signatures; metagene analysis was employed to define survival rates in patients whose tumors express a PR gene signature. RESULTS: 'SUMO-sensitive' PR target genes primarily include genes required for proliferative and pro-survival signaling. DeSUMOylated K388R receptors are preferentially recruited to enhancer regions of derepressed genes (that is, MSX2, RGS2, MAP1A, and PDK4) with the steroid receptor coactivator, CREB-(cAMP-response element-binding protein)-binding protein (CBP), and mixed lineage leukemia 2 (MLL2), a histone methyltransferase mediator of nucleosome remodeling. PR SUMOylation blocks these events, suggesting that SUMO modification of PR prevents interactions with mediators of early chromatin remodeling at 'closed' enhancer regions. SUMO-deficient (phospho-Ser294) PR gene signatures are significantly associated with human epidermal growth factor 2 (ERBB2)-positive luminal breast tumors and predictive of early metastasis and shortened survival. Treatment with antiprogestin or MEK inhibitor abrogated expression of SUMO-sensitive PR target-genes and inhibited proliferation in BT-474 (estrogen receptor (ER)+/PR+/ERBB2+) breast cancer cells. CONCLUSIONS: We conclude that reversible PR SUMOylation/deSUMOylation profoundly alters target gene selection in breast cancer cells. Phosphorylation-induced PR deSUMOylation favors a permissive chromatin environment via recruitment of CBP and MLL2. Patients whose ER+/PR+ tumors are driven by hyperactive (that is, derepressed) phospho-PRs may benefit from endocrine (antiestrogen) therapies that contain an antiprogestin.

Asunto(s)

Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Proteínas de Unión al ADN/genética , Progresión de la Enfermedad , Femenino , Perfilación de la Expresión Génica , Humanos , Células MCF-7 , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Neoplasias/genética , Fosforilación , Regiones Promotoras Genéticas , Receptor ErbB-2/metabolismo , Transducción de Señal , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/antagonistas & inhibidores , Sumoilación , Activación Transcripcional , Transcriptoma

RESUMEN

The Sleeping Beauty (SB) transposon mutagenesis system is a powerful tool that facilitates the discovery of mutations that accelerate tumorigenesis. In this study, we sought to identify mutations that cooperate with MYC, one of the most commonly dysregulated genes in human malignancy. We performed a forward genetic screen with a mouse model of MYC-induced liver cancer using SB-mediated mutagenesis. We sequenced insertions in 63 liver tumor nodules and identified at least 16 genes/loci that contribute to accelerated tumor development. RNAi-mediated knockdown in a liver progenitor cell line further validate three of these genes, Ncoa2/Src-2, Zfx, and Dtnb, as tumor suppressors in liver cancer. Moreover, deletion of Ncoa2/Src-2 in mice predisposes to diethylnitrosamine-induced liver tumorigenesis. These findings reveal genes and pathways that functionally restrain MYC-mediated liver tumorigenesis and therefore may provide targets for cancer therapy.

Asunto(s)

Carcinoma Hepatocelular/genética , Análisis Mutacional de ADN/métodos , Genes Supresores de Tumor , Neoplasias Hepáticas/genética , Coactivador 2 del Receptor Nuclear/genética , Transposasas/genética , Alquilantes/toxicidad , Animales , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/patología , Dietilnitrosamina/toxicidad , Modelos Animales de Enfermedad , Femenino , Genes myc/genética , Células HEK293 , Humanos , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Desnudos , Ratones Transgénicos , Trasplante de Neoplasias , Trasplante Heterólogo

RESUMEN

Patients with a t(9;11) translocation (MLL-AF9) develop acute myeloid leukemia (AML), and while in mice the expression of this fusion oncogene also results in the development of myeloid leukemia, it is with long latency. To identify mutations that cooperate with Mll-AF9, we infected neonatal wild-type (WT) or Mll-AF9 mice with a murine leukemia virus (MuLV). MuLV-infected Mll-AF9 mice succumbed to disease significantly faster than controls presenting predominantly with myeloid leukemia while infected WT animals developed predominantly lymphoid leukemia. We identified 88 candidate cancer genes near common sites of proviral insertion. Analysis of transcript levels revealed significantly elevated expression of Mn1, and a trend toward increased expression of Bcl11a and Fosb in Mll-AF9 murine leukemia samples with proviral insertions proximal to these genes. Accordingly, FOSB and BCL11A were also overexpressed in human AML harboring MLL gene translocations. FOSB was revealed to be essential for growth in mouse and human myeloid leukemia cells using shRNA lentiviral vectors in vitro. Importantly, MN1 cooperated with Mll-AF9 in leukemogenesis in an in vivo BM viral transduction and transplantation assay. Together, our data identified genes that define transcription factor networks and important genetic pathways acting during progression of leukemia induced by MLL fusion oncogenes.

Asunto(s)

Transformación Celular Neoplásica/genética , Redes Reguladoras de Genes/genética , Leucemia/genética , Mutagénesis Insercional , Proteína de la Leucemia Mieloide-Linfoide/fisiología , Proteínas de Fusión Oncogénica/fisiología , Animales , Animales Recién Nacidos , Células Cultivadas , Análisis Mutacional de ADN/métodos , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Leucemia/patología , Ratones , Ratones Endogámicos C57BL , Mutagénesis Insercional/fisiología , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Fusión Oncogénica/genética , Células U937

RESUMEN

Pathogens can substantially alter gene expression within an infected host depending on metabolic or virulence requirements in different tissues, however, the effect of these alterations on host immunity are unclear. Here we visualized multiple CD4 T cell responses to temporally expressed proteins in Salmonella-infected mice. Flagellin-specific CD4 T cells expanded and contracted early, differentiated into Th1 and Th17 lineages, and were enriched in mucosal tissues after oral infection. In contrast, CD4 T cells responding to Salmonella Type-III Secretion System (TTSS) effectors steadily accumulated until bacterial clearance was achieved, primarily differentiated into Th1 cells, and were predominantly detected in systemic tissues. Thus, pathogen regulation of antigen expression plays a major role in orchestrating the expansion, differentiation, and location of antigen-specific CD4 T cells in vivo.

Asunto(s)

Proteínas Bacterianas/inmunología , Linfocitos T CD4-Positivos/inmunología , Salmonelosis Animal/inmunología , Salmonella typhimurium/inmunología , Células Th17/inmunología , Animales , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos , Linfocitos T CD4-Positivos/microbiología , Diferenciación Celular/inmunología , Epítopos/análisis , Epítopos/inmunología , Flagelina/inmunología , Flagelina/metabolismo , Regulación Bacteriana de la Expresión Génica/inmunología , Interacciones Huésped-Patógeno , Ratones , Ratones Endogámicos C57BL , Salmonelosis Animal/microbiología , Salmonella typhimurium/patogenicidad , Células TH1/inmunología , Células TH1/microbiología , Células Th17/microbiología

RESUMEN

Androgen depletion for advanced prostate cancer (PCa) targets activity of the androgen receptor (AR), a steroid receptor transcription factor required for PCa growth. The emergence of lethal castration-resistant PCa (CRPCa) is marked by aberrant reactivation of the AR despite ongoing androgen depletion. Recently, alternative splicing has been described as a mechanism giving rise to COOH-terminally truncated, constitutively active AR isoforms that can support the CRPCa phenotype. However, the pathologic origin of these truncated AR isoforms is unknown. The goal of this study was to investigate alterations in AR expression arising in a cell-based model of PCa progression driven by truncated AR isoform activity. We show that stable, high-level expression of truncated AR isoforms in 22Rv1 CRPCa cells is associated with intragenic rearrangement of an approximately 35-kb AR genomic segment harboring a cluster of previously described alternative AR exons. Analysis of genomic data from clinical specimens indicated that related AR intragenic copy number alterations occurred in CRPCa in the context of AR amplification. Cloning of the break fusion junction in 22Rv1 cells revealed long interspersed nuclear elements (LINE-1) flanking the rearranged segment and a DNA repair signature consistent with microhomology-mediated, break-induced replication. This rearrangement served as a marker for the emergence of a rare subpopulation of CRPCa cells expressing high levels of truncated AR isoforms during PCa progression in vitro. Together, these data provide the first report of AR intragenic rearrangements in CRPCa and an association with pathologic expression of truncated AR isoforms in a cell-based model of PCa progression.

Asunto(s)

Reordenamiento Génico , Neoplasias de la Próstata/genética , Empalme del ARN , Receptores Androgénicos/genética , Algoritmos , Secuencia de Bases , Western Blotting , Línea Celular , Línea Celular Tumoral , Mapeo Cromosómico , Cromosomas Humanos X/genética , Progresión de la Enfermedad , Dosificación de Gen , Humanos , Elementos de Nucleótido Esparcido Largo/genética , Masculino , Modelos Genéticos , Datos de Secuencia Molecular , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Receptores Androgénicos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido Nucleico

RESUMEN

The usurping of translational control by sustained activation of translation initiation factors is oncogenic. Here, we show that the primary negative regulators of these oncogenic initiation factors--the 4E-BP protein family--operate as guardians of a translational control checkpoint in lung tumor defense. When challenged with the tobacco carcinogen 4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone (NNK), 4ebp1(-/-)/4ebp2(-/-) mice showed increased sensitivity to tumorigenesis compared with their wild-type counterparts. The 4E-BP-deficient state per se creates pro-oncogenic, genome-wide skewing of the molecular landscape, with translational activation of genes governing angiogenesis, growth, and proliferation, and translational activation of the precise cytochrome p450 enzyme isoform (CYP2A5) that bioactivates NNK into mutagenic metabolites. Our study provides in vivo proof for a translational control checkpoint in lung tumor defense.

Asunto(s)

Proteínas Portadoras/fisiología , Factores Eucarióticos de Iniciación/fisiología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Fosfoproteínas/fisiología , Biosíntesis de Proteínas , Proteínas Adaptadoras Transductoras de Señales , Animales , Carcinógenos/toxicidad , Proteínas de Ciclo Celular , Proliferación Celular , Sistema Enzimático del Citocromo P-450/metabolismo , Aductos de ADN/genética , Pulmón/metabolismo , Pulmón/patología , Neoplasias Pulmonares/inducido químicamente , Ratones , Ratones Endogámicos A , Ratones Endogámicos BALB C , Ratones Noqueados , Análisis por Micromatrices , Microsomas/metabolismo , Neovascularización Patológica , Nitrosaminas/toxicidad , Ribosomas/fisiología

RESUMEN

Regulatory elements in mRNA play an often pivotal role in post-transcriptional regulation of gene expression. However, a systematic approach to efficiently identify putative regulatory elements from sets of post-transcriptionally coregulated genes is lacking, hampering studies of coregulation mechanisms. Although there are several analytical methods that can be used to detect conserved mRNA regulatory elements in a set of transcripts, there has been no systematic study of how well any of these methods perform individually or as a group. We therefore compared how well three algorithms, each based on a different principle (enumeration, optimization, or structure/sequence profiles), can identify elements in unaligned untranslated sequence regions. Two algorithms were originally designed to detect transcription factor binding sites, Weeder and BioProspector; and one was designed to detect RNA elements conserved in structure, RNAProfile. Three types of elements were examined: (1) elements conserved in both primary sequence and secondary structure; (2) elements conserved only in primary sequence; and (3) microRNA targets. Our results indicate that all methods can uniquely identify certain known RNA elements, and therefore, integrating the output from all algorithms leads to the most complete identification of elements. We therefore developed an approach to integrate results and guide selection of candidate elements from several algorithms presented as a web service (https://dbw.msi.umn.edu:8443/recit). These findings together with the approach for integration can be used to identify candidate elements from genome-wide post-transcriptional profiling data sets.

Asunto(s)

Biología Computacional/métodos , Elementos Reguladores de la Transcripción , Algoritmos , Sitios de Unión/genética , Secuencia Conservada , Internet , MicroARNs/genética , MicroARNs/metabolismo , Procesamiento Postranscripcional del ARN , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN no Traducido/química , ARN no Traducido/genética , ARN no Traducido/metabolismo , Ribosomas/metabolismo , Factores de Transcripción/metabolismo

RESUMEN

BACKGROUND: As a group, fibroproliferative disorders of the lung, liver, kidney, heart, vasculature and integument are common, progressive and refractory to therapy. They can emerge following toxic insults, but are frequently idiopathic. Their enigmatic propensity to resist therapy and progress to organ failure has focused attention on the myofibroblast-the primary effector of the fibroproliferative response. We have recently shown that aberrant beta 1 integrin signaling in fibrotic fibroblasts results in defective PTEN function, unrestrained Akt signaling and subsequent activation of the translation initiation machinery. How this pathological integrin signaling alters the gene expression pathway has not been elucidated. RESULTS: Using a systems approach to study this question in a prototype fibrotic disease, Idiopathic Pulmonary Fibrosis (IPF); here we show organized changes in the gene expression pathway of primary lung myofibroblasts that persist for up to 9 sub-cultivations in vitro. When comparing IPF and control myofibroblasts in a 3-dimensional type I collagen matrix, more genes differed at the level of ribosome recruitment than at the level of transcript abundance, indicating pathological translational control as a major characteristic of IPF myofibroblasts. To determine the effect of matrix state on translational control, myofibroblasts were permitted to contract the matrix. Ribosome recruitment in control myofibroblasts was relatively stable. In contrast, IPF cells manifested large alterations in the ribosome recruitment pattern. Pathological studies suggest an epithelial origin for IPF myofibroblasts through the epithelial to mesenchymal transition (EMT). In accord with this, we found systems-level indications for TGF-beta -driven EMT as one source of IPF myofibroblasts. CONCLUSIONS: These findings establish the power of systems level genome-wide analysis to provide mechanistic insights into fibrotic disorders such as IPF. Our data point to derangements of translational control downstream of aberrant beta 1 integrin signaling as a fundamental component of IPF pathobiology and indicates that TGF-beta -driven EMT is one source for IPF myofibroblasts.

Asunto(s)

Genoma Humano , Biosíntesis de Proteínas , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/metabolismo , Anciano , Anciano de 80 o más Años , Femenino , Fibroblastos/metabolismo , Humanos , Integrina beta1/metabolismo , Masculino , Persona de Mediana Edad , Modelos Biológicos , Ribosomas/química , Ribosomas/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo

RESUMEN

We used computational algorithms to find conserved sequences in the 3' untranslated region (UTR) of transcripts that exhibited rapid decay in primary human T cells and found that the consensus sequence UGUUUGUUUGU, which we have termed a GU-rich element (GRE), was enriched in short-lived transcripts. Using a tet-off reporter system, we showed that insertion of GRE-containing sequences from c-jun, jun B, or TNF receptor 1B, but not mutated GRE sequences, into the 3'UTR of a beta-globin transcript conferred instability on the otherwise stable beta-globin transcript. CUG-binding protein 1 (CUGBP1) was identified as the major GRE-binding activity in cytoplasmic extracts from primary human T cells based on supershift and immunoprecipitation assays. siRNA-mediated knockdown of CUGBP1 in HeLa cells caused stabilization of GRE-containing transcripts, suggesting that CUGBP1 is a mediator of GRE-dependent mRNA decay. Overall, our results suggest that the GRE mediates coordinated mRNA decay by binding to CUGBP1.

Asunto(s)

Regiones no Traducidas 3'/metabolismo , Estabilidad del ARN/fisiología , Proteínas de Unión al ARN/metabolismo , Linfocitos T/metabolismo , Regiones no Traducidas 3'/genética , Proteínas CELF1 , Citoplasma/genética , Citoplasma/metabolismo , Globinas/genética , Globinas/metabolismo , Células HeLa , Humanos , Unión Proteica/fisiología , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/genética , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Linfocitos T/citología

RESUMEN

Aberrant activation of the translation initiation machinery is a common property of malignant cells, and is essential for breast carcinoma cells to manifest a malignant phenotype. How does sustained activation of the rate limiting step in protein synthesis so fundamentally alter a cell? In this report, we test the post transcriptional operon theory as a possible mechanism, employing a model system in which apoptosis resistance is conferred on NIH 3T3 cells by ectopic expression of eIF4E. We show (i) there is a set of 255 transcripts that manifest an increase in translational efficiency during eIF4E-mediated escape from apoptosis; (ii) there is a novel prototype 55 nt RNA consensus hairpin structure that is overrepresented in the 5'-untranslated region of translationally activated transcripts; (iii) the identified consensus hairpin structure is sufficient to target a reporter mRNA for translational activation under pro-apoptotic stress, but only when eIF4E is deregulated; and (iv) that osteopontin, one of the translationally activated transcripts harboring the identified consensus hairpin structure functions as one mediator of the apoptosis resistance seen in our model. Our findings offer genome-wide insights into the mechanism of eIF4E-mediated apoptosis resistance and provide a paradigm for the systematic study of posttranscriptional control in normal biology and disease.

Asunto(s)

Regiones no Traducidas 5'/química , Factor 4E Eucariótico de Iniciación/metabolismo , Regulación de la Expresión Génica , Biosíntesis de Proteínas , Secuencias Reguladoras de Ácido Ribonucleico , Animales , Apoptosis , Perfilación de la Expresión Génica , Genómica , Ratones , Células 3T3 NIH , Conformación de Ácido Nucleico , Osteopontina , Sialoglicoproteínas/biosíntesis , Sialoglicoproteínas/genética

RESUMEN

Natriuretic peptide receptor A (NPR-A/GC-A) and B (NPR-B/GC-B) are members of the transmembrane guanylyl cyclase family that mediate the effects of natriuretic peptides via the second messenger, cGMP. Despite numerous reports of these receptors being down-regulated in response to various pathological conditions, no studies have actually measured desensitization and receptor internalization in the same cell line. Furthermore, the ligand-dependent trafficking properties of NPR-A remain controversial, whereas nothing is known about the trafficking of NPR-B. In this report, we tested whether down-regulation explains the ligand-dependent desensitization of NPR-A and NPR-B and characterized their trafficking properties using a combination of hormone-binding and antibody-based assays. Quantitative partition analysis indicated that (125)I-atrial natriuretic peptide (ANP) was rapidly released into the medium after 293T cells stably expressing NPR-A were warmed from 4 degrees to 37 degrees C. High-performance liquid chromatography fractionation of medium supplemented with the protease inhibitor phosphoramidon indicated that the (125)I-ANP was mostly intact. In contrast, (125)I-ANP purified from medium bathing cells expressing NPR-C, a receptor known to internalize natriuretic peptides, was degraded. Cleavable biotinylation and noncleavable biotinylation assays indicated that neither NPR-A nor NPR-B was internalized or degraded in response to natriuretic peptide binding. In contrast, agonist-dependent internalization of a G protein-coupled receptor was clearly apparent in the same cell line. Finally, we show that NPR-A and NPR-B are desensitized in cells in which they are not internalized. We suggest that mechanisms other than receptor down-regulation account for the desensitization of NPR-A and NPR-B that occurs in response to various physiological and pathological stimuli.

Asunto(s)

Regulación hacia Abajo/fisiología , Guanilato Ciclasa/metabolismo , Guanilato Ciclasa/fisiología , Receptores del Factor Natriurético Atrial/fisiología , Factor Natriurético Atrial/farmacocinética , Transporte Biológico , Línea Celular , ADN Complementario , Glicopéptidos/farmacología , Humanos , Radioisótopos de Yodo , Transporte de Proteínas , Transfección