RESUMEN
Anaplastic large cell lymphomas (ALCLs) represent a relatively common group of T-cell non-Hodgkin lymphomas (T-NHLs) that are unified by similar pathologic features but demonstrate marked genetic heterogeneity. ALCLs are broadly classified as being anaplastic lymphoma kinase (ALK)+ or ALK-, based on the presence or absence of ALK rearrangements. Exome sequencing of 62 T-NHLs identified a previously unreported recurrent mutation in the musculin gene, MSC E116K, exclusively in ALK- ALCLs. Additional sequencing for a total of 238 T-NHLs confirmed the specificity of MSC E116K for ALK- ALCL and further demonstrated that 14 of 15 mutated cases (93%) had coexisting DUSP22 rearrangements. Musculin is a basic helix-loop-helix (bHLH) transcription factor that heterodimerizes with other bHLH proteins to regulate lymphocyte development. The E116K mutation localized to the DNA binding domain of musculin and permitted formation of musculin-bHLH heterodimers but prevented their binding to authentic target sequence. Functional analysis showed MSCE116K acted in a dominant-negative fashion, reversing wild-type musculin-induced repression of MYC and cell cycle inhibition. Chromatin immunoprecipitation-sequencing and transcriptome analysis identified the cell cycle regulatory gene E2F2 as a direct transcriptional target of musculin. MSCE116K reversed E2F2-induced cell cycle arrest and promoted expression of the CD30-IRF4-MYC axis, whereas its expression was reciprocally induced by binding of IRF4 to the MSC promoter. Finally, ALCL cells expressing MSC E116K were preferentially targeted by the BET inhibitor JQ1. These findings identify a novel recurrent MSC mutation as a key driver of the CD30-IRF4-MYC axis and cell cycle progression in a unique subset of ALCLs.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Linfoma Anaplásico de Células Grandes/genética , Quinasa de Linfoma Anaplásico/genética , Ciclo Celular/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , MutaciónRESUMEN
Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is a recently characterized T-cell malignancy that has raised significant patient safety concerns and led to worldwide impact on the implants used and clinical management of patients undergoing reconstructive or cosmetic breast surgery. Molecular signatures distinguishing BIA-ALCL from other ALCLs have not been fully elucidated and classification of BIA-ALCL as a WHO entity remains provisional. We performed RNA sequencing and gene set enrichment analysis comparing BIA-ALCLs to non-BIA-ALCLs and identified dramatic upregulation of hypoxia signaling genes including the hypoxia-associated biomarker CA9 (carbonic anyhydrase-9). Immunohistochemistry validated CA9 expression in all BIA-ALCLs, with only minimal expression in non-BIA-ALCLs. Growth induction in BIA-ALCL-derived cell lines cultured under hypoxic conditions was proportional to up-regulation of CA9 expression, and RNA sequencing demonstrated induction of the same gene signature observed in BIA-ALCL tissue samples compared to non-BIA-ALCLs. CA9 silencing blocked hypoxia-induced BIA-ALCL cell growth and cell cycle-associated gene expression, whereas CA9 overexpression in BIA-ALCL cells promoted growth in a xenograft mouse model. Furthermore, CA9 was secreted into BIA-ALCL cell line supernatants and was markedly elevated in human BIA-ALCL seroma samples. Finally, serum CA9 concentrations in mice bearing BIA-ALCL xenografts were significantly elevated compared to control serum. Together, these findings characterize BIA-ALCL as a hypoxia-associated neoplasm, likely attributable to the unique microenvironment in which it arises. These data support classification of BIA-ALCL as a distinct entity and uncover opportunities for investigating hypoxia-related proteins such as CA9 as novel biomarkers and therapeutic targets in this disease.
Asunto(s)
Implantes de Mama , Neoplasias de la Mama , Linfoma Anaplásico de Células Grandes , Animales , Implantes de Mama/efectos adversos , Femenino , Humanos , Hipoxia/genética , Inmunohistoquímica , Linfoma Anaplásico de Células Grandes/genética , Ratones , Microambiente TumoralRESUMEN
Anaplastic large cell lymphomas (ALCLs) are CD30-positive T-cell non-Hodgkin lymphomas broadly segregated into ALK-positive and ALK-negative types. Although ALK-positive ALCLs consistently bear rearrangements of the ALK tyrosine kinase gene, ALK-negative ALCLs are clinically and genetically heterogeneous. About 30% of ALK-negative ALCLs have rearrangements of DUSP22 and have excellent long-term outcomes with standard therapy. To better understand this group of tumors, we evaluated their molecular signature using gene expression profiling. DUSP22-rearranged ALCLs belonged to a distinct subset of ALCLs that lacked expression of genes associated with JAK-STAT3 signaling, a pathway contributing to growth in the majority of ALCLs. Reverse-phase protein array and immunohistochemical studies confirmed the lack of activated STAT3 in DUSP22-rearranged ALCLs. DUSP22-rearranged ALCLs also overexpressed immunogenic cancer-testis antigen (CTA) genes and showed marked DNA hypomethylation by reduced representation bisulfate sequencing and DNA methylation arrays. Pharmacologic DNA demethylation in ALCL cells recapitulated the overexpression of CTAs and other DUSP22 signature genes. In addition, DUSP22-rearranged ALCLs minimally expressed PD-L1 compared with other ALCLs, but showed high expression of the costimulatory gene CD58 and HLA class II. Taken together, these findings indicate that DUSP22 rearrangements define a molecularly distinct subgroup of ALCLs, and that immunogenic cues related to antigenicity, costimulatory molecule expression, and inactivity of the PD-1/PD-L1 immune checkpoint likely contribute to their favorable prognosis. More aggressive ALCLs might be pharmacologically reprogrammed to a DUSP22-like immunogenic molecular signature through the use of demethylating agents and/or immune checkpoint inhibitors.
Asunto(s)
Metilación de ADN , Fosfatasas de Especificidad Dual/genética , Regulación Neoplásica de la Expresión Génica , Reordenamiento Génico , Linfoma Anaplásico de Células Grandes/genética , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/genética , Antígenos de Neoplasias/genética , Fosfatasas de Especificidad Dual/inmunología , Femenino , Humanos , Linfoma Anaplásico de Células Grandes/diagnóstico , Linfoma Anaplásico de Células Grandes/inmunología , Linfoma Anaplásico de Células Grandes/patología , Masculino , Persona de Mediana Edad , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/inmunología , Fosforilación , Pronóstico , Factor de Transcripción STAT3/análisis , Transcriptoma , Escape del TumorRESUMEN
Peripheral T-cell lymphomas (PTCLs) represent a heterogeneous group of T-cell malignancies that generally demonstrate aggressive clinical behavior, often are refractory to standard therapy, and remain significantly understudied. The most common World Health Organization subtype is PTCL, not otherwise specified (NOS), essentially a "wastebasket" category because of inadequate understanding to assign cases to a more specific diagnostic entity. Identification of novel fusion genes has contributed significantly to improving the classification, biologic understanding, and therapeutic targeting of PTCLs. Here, we integrated mate-pair DNA and RNA next-generation sequencing to identify chromosomal rearrangements encoding expressed fusion transcripts in PTCL, NOS. Two of 11 cases had novel fusions involving VAV1, encoding a truncated form of the VAV1 guanine nucleotide exchange factor important in T-cell receptor signaling. Fluorescence in situ hybridization studies identified VAV1 rearrangements in 10 of 148 PTCLs (7%). These were observed exclusively in PTCL, NOS (11%) and anaplastic large cell lymphoma (11%). In vitro, ectopic expression of a VAV1 fusion promoted cell growth and migration in a RAC1-dependent manner. This growth was inhibited by azathioprine, a clinically available RAC1 inhibitor. We also identified novel kinase gene fusions, ITK-FER and IKZF2-ERBB4, as candidate therapeutic targets that show similarities to known recurrent oncogenic ITK-SYK fusions and ERBB4 transcript variants in PTCLs, respectively. Additional novel and potentially clinically relevant fusions also were discovered. Together, these findings identify VAV1 fusions as recurrent and targetable events in PTCLs and highlight the potential for clinical sequencing to guide individualized therapy approaches for this group of aggressive malignancies.
Asunto(s)
Linfoma de Células T Periférico/genética , Proteínas de Fusión Oncogénica/genética , Anciano , Animales , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Células Jurkat , Linfoma de Células T Periférico/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Células 3T3 NIH , Proteínas de Fusión Oncogénica/metabolismoAsunto(s)
Enfermedades Gastrointestinales/diagnóstico , Enfermedades Gastrointestinales/genética , Fusión Génica , Janus Quinasa 2/genética , Trastornos Linfoproliferativos/diagnóstico , Trastornos Linfoproliferativos/genética , Factor de Transcripción STAT3/genética , Linfocitos T/metabolismo , Enfermedades Gastrointestinales/metabolismo , Reordenamiento Génico , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Janus Quinasa 2/metabolismo , Trastornos Linfoproliferativos/metabolismo , Factor de Transcripción STAT3/metabolismo , Linfocitos T/patologíaRESUMEN
Cytokines are deregulated in cancers and can contribute to tumor growth. In patients with diffuse large-cell lymphoma (DLBCL), we observed higher levels of JAK/STAT pathway-related serum cytokines (ie, IL-6, IL-10, epidermal growth factor, and IL-2) compared with controls. Of these, only IL-10 activated the JAK2 pathway in lymphoma cells in vitro. Patients with high serum IL-10 had shorter event-free survival (EFS) than patients with low levels (P > .01) and high IL-10 was correlated with high lactase dehydrogenase (P = .0085) and higher International Prognostic Index scores (P = .01). To explore the mechanism by which IL-10 may contribute to an inferior EFS, we investigated the effect of IL-10 on the JAK2 pathway and found that the IL-10/IL-10 receptor complex up-regulated JAK2 signaling. Neutralizing Ab to IL-10 inhibited constitutive and IL-10-induced JAK2/STAT3 phosphorylation. JAK2 inhibition dephosphorylated JAK2 and STAT3 and caused an inhibitory effect on phospho-JAK2-positive DLBCL cells; there was a minimal effect on phospho-JAK2-negative cells. Apoptosis induced by JAK2 inhibition was dependent on inhibition of autocrine IL-10 and c-myc expression and independent of Bcl-2 family expression. These results provide the rationale for testing JAK2 inhibitors in DLBCL patients, and indicate that serum IL-10 may be a biomarker to identify patients more likely to respond to JAK2-targeted therapy.
Asunto(s)
Biomarcadores de Tumor/sangre , Interleucina-10/sangre , Janus Quinasa 2/metabolismo , Linfoma de Células B Grandes Difuso/sangre , Western Blotting , Activación Enzimática/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Inmunohistoquímica , Inmunoprecipitación , Janus Quinasa 2/inmunología , Estimación de Kaplan-Meier , Linfoma de Células B Grandes Difuso/inmunología , Linfoma de Células B Grandes Difuso/mortalidad , ARN Interferente Pequeño , Receptores de Interleucina-10/inmunología , Receptores de Interleucina-10/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT3/inmunología , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/inmunologíaRESUMEN
Heterotrimeric G protein knock-out mutants have no phenotypic defect in chloroplast development, and the connection between the G protein signaling pathway and chloroplast development has only been inferred from pharmaceutical evidence. Thus, whether G protein signaling plays a role in chloroplast development remains an open question. Here, we present genetic evidence, using the leaf-variegated mutant thylakoid formation 1 (thf1), indicating that inactivation or activation of the endogenous G protein alpha-subunit (GPA1) affects chloroplast development, as does the ectopic expression of the constitutively active Galpha-subunit (cGPA1). Molecular biological and genetic analyses showed that FtsH complexes, which are composed of type-A (FtsH1/FtsH5) and type-B (FtsH2/FtsH8) subunits, are required for cGPA1-promoted chloroplast development in thf1. Furthermore, the ectopic expression of cGPA1 rescues the leaf variegation of ftsh2. Consistent with this finding, microarray analysis shows that ectopic expression of cGPA1 partially corrects mis-regulated gene expression in thf1. This overlooked function of G proteins provides new insight into our understanding of the integrative signaling network, which dynamically regulates chloroplast development and function in response to both intracellular and extracellular signals.
Asunto(s)
Proteasas ATP-Dependientes/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Cloroplastos/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Proteínas de la Membrana/metabolismo , Proteasas ATP-Dependientes/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Cloroplastos/genética , Cloroplastos/ultraestructura , Subunidades alfa de la Proteína de Unión al GTP/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Proteínas de la Membrana/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , ARN de Planta/genéticaRESUMEN
Stomata are essential for efficient gas and water-vapor exchange between the atmosphere and plants. Stomatal density and movement are controlled by a series of signal molecules including phytohormones and peptides as well as by environmental stimuli. It is known that heterotrimeric G-proteins play an important role in the ABA-inhibited stomatal opening. In this study, the G-protein signaling pathway was also found to regulate stomatal density on the lower epidermis of Arabidopsis cotyledons. The loss-of-function mutation of the G-protein alpha-subunit (GPA1) showed a reduction in stomatal density, while overexpression of the constitutively active form of GPA1(QL) increased stomatal density, indicating a positive role of the active form of GPA1 in stomatal development. In contrast, stomatal density increased in the null mutant of the G-protein beta-subunit (AGB1) but decreased in transgenic lines that overexpressed AGB1. Stomatal analysis of the gpa1 agb1 double mutants displayed an average value of stomatal density compared to the single mutants. Taken together, these results suggest that the stomatal density in Arabidopsis is modulated by GPA1 and AGB1 in an antagonistic manner.
Asunto(s)
Arabidopsis/fisiología , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Estomas de Plantas/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades beta de la Proteína de Unión al GTP/genética , Regulación de la Expresión Génica de las Plantas , Mutación , Transducción de SeñalRESUMEN
The heterotrimeric guanine nucleotide-binding protein (G-protein) has been demonstrated to mediate various signaling pathways in plants. However, its role in phytochrome A (phyA) signaling remains elusive. In this study, we discover a new phyA-mediated phenotype designated far-red irradiation (FR) preconditioned cell death, which occurs only in the hypocotyls of FR-grown seedlings following exposure to white light (WL). The cell death is mitigated in the Ga mutant gpa1 but aggravated in the Gbeta mutant agb1 in comparison with the wild type (WT), indicative of antagonistic roles of GPA1 and AGB1 in the phyA-mediated cell-death pathway. Further investigation indicates that FR-induced accumulation of nonphotoconvertible protochlorophyllide (Pchlide633), which generates reactive oxygen species (ROS) on exposure to WL, is required for FR-preconditioned cell death. Moreover, ROS is mainly detected in chloroplasts using the fluorescent probe. Interestingly, the application of H2O2 to dark-grown seedlings results in a phenotype similar to FR-preconditioned cell death. This reveals that ROS is a critical mediator for the cell death. In addition, we observe that agb1 is more sensitive to H2O2 than WT seedlings, indicating that the G-protein may also modify the sensitivity of the seedlings to ROS stress. Taking these results together, we infer that the G-protein may be involved in the phyA signaling pathway to regulate FR-preconditioned cell death of Arabidopsis hypocotyls. A possible mechanism underlying the involvement of the G-protein in phyA signaling is discussed in this study.
Asunto(s)
Arabidopsis/citología , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Hipocótilo/citología , Fitocromo A/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/enzimología , Proteínas de Arabidopsis/metabolismo , Muerte Celular/efectos de los fármacos , Cloroplastos/efectos de los fármacos , Cloroplastos/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Hipocótilo/efectos de los fármacos , Hipocótilo/enzimología , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente , Imitación Molecular/efectos de los fármacos , Mutación/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Protoclorofilida/metabolismoRESUMEN
Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma characterized by rapid disease progression. The needs for new therapeutic strategies for MCL patients call for further understanding on the molecular mechanisms of pathogenesis of MCL. Recently, long noncoding RNAs (lncRNAs) have been recognized as key regulators of gene expression and disease development, however, the role of lncRNAs in non-Hodgkin lymphoma and specifically in MCL is still unknown. Next generation RNA-sequencing was carried out on MCL patient samples along with normal controls and data was analyzed. As a result, several novel lncRNAs were found significantly overexpressed in the MCL samples with lncRNA ROR1-AS1 the most significant one. We cloned the ROR1-AS1 lncRNA in expression vector and ectopically transfected in MCL cell lines. Results showed that overexpression of ROR1-AS1 lncRNA promoted growth of MCL cells while decreased sensitivity to the treatment with drugs ibrutinib and dexamethasone. ROR-AS1 overexpression also decreased the mRNA expression of P16 (P = 0.21), and SOX11 (p = 0.017), without much effect on P53, ATM and P14 mRNA. RNA-immunoprecipitation assays demonstrated high affinity binding of lncRNA ROR1-AS1 with EZH2 and SUZ12 proteins of the polycomb repressive complex-2 (PRC2). Suppressing EZH2 activity with pharmacological inhibitor GSK343 abolished binding of ROR1-AS1 with EZH2. Taken together, this study identified a functional lncRNA ROR-AS1 involved with regulation of gene transcription via associating with PRC2 complex, and may serve as a novel biomarker in MCL patients.
RESUMEN
Organic pollutants are major contaminants of soil, water and air whereas phytoremediation is an efficient way to clean up the polluted environment. In recent years, a number of exogenous genes encoding the proteins with remediation activities have been introduced into plants, and transgenic plants with enhanced ability of bioremediation obtained. In this paper, recent achievements in the research on phytoremediation of environments polluted by organic pollutants, especially the persistent organic pollutants (POPs), is briefly described, and recent achievements and potential uses of transgenic plants in pollution remediation are discussed.
Asunto(s)
Contaminantes Ambientales/metabolismo , Compuestos Orgánicos/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Biodegradación Ambiental , Contaminación Ambiental/prevención & controlRESUMEN
PTPN6 (SHP1) is a tyrosine phosphatase that negatively controls the activity of multiple signaling pathways including STAT signaling, however role of mutated PTPN6 is not much known. Here we investigated whether PTPN6 might also be a potential target for diffuse large B cell lymphoma (DLBCL) and performed Sanger sequencing of the PTPN6 gene. We have identified missense mutations within PTPN6 (N225K and A550V) in 5% (2/38) of DLBCL tumors. Site directed mutagenesis was performed to mutate wild type (WT) PTPN6 and stable cell lines were generated by lentiviral transduction of PTPN6(WT), PTPN6(N225K) and PTPN6(A550V) constructs, and effects of WT or mutated PTPN6 on STAT3 signaling were analyzed. WT PTPN6 dephosphorylated STAT3, but had no effect on STAT1, STAT5 or STAT6 phosphorylation. Both PTPN6 mutants were unable to inhibit constitutive, as well as cytokines induced STAT3 activation. Both PTPN6 mutants also demonstrated reduced tyrosine phosphatase activity and exhibited enhanced STAT3 transactivation activity. Intriguingly, a lack of direct binding between STAT3 and WT or mutated PTPN6 was observed. However, compared to WT PTPN6, cells expressing PTPN6 mutants exhibited increased binding between JAK3 and PTPN6 suggesting a more dynamic interaction of PTPN6 with upstream regulators of STAT3. Consistent with this notion, both the mutants demonstrated increased resistance to JAK3 inhibitor, WHIP-154 relative to WT PTPN6. Overall, this is the first study, which demonstrates that N225K and A550V PTPN6 mutations cause loss-of-function leading to JAK3 mediated deregulation of STAT3 pathway and uncovers a mechanism that tumor cells can use to control PTPN6 substrate specificity.
Asunto(s)
Janus Quinasa 3/metabolismo , Linfoma de Células B Grandes Difuso/enzimología , Linfoma de Células B Grandes Difuso/genética , Mutación Missense , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , Factor de Transcripción STAT3/metabolismo , Antineoplásicos/farmacología , Resistencia a Antineoplásicos , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Janus Quinasa 3/antagonistas & inhibidores , Janus Quinasa 3/genética , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/patología , Mutagénesis Sitio-Dirigida , Fosforilación , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Factor de Transcripción STAT3/genética , Transducción de Señal , Especificidad por Sustrato , TransfecciónAsunto(s)
Neoplasias Gastrointestinales/complicaciones , Janus Quinasa 2/antagonistas & inhibidores , Linfoma de Células T/tratamiento farmacológico , Trastornos Linfoproliferativos/tratamiento farmacológico , Pirazoles/farmacología , Factor de Transcripción STAT3/antagonistas & inhibidores , Linfocitos T/efectos de los fármacos , Animales , Apoptosis , Proliferación Celular , Humanos , Linfoma de Células T/metabolismo , Linfoma de Células T/patología , Trastornos Linfoproliferativos/etiología , Trastornos Linfoproliferativos/metabolismo , Trastornos Linfoproliferativos/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Nitrilos , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas , Linfocitos T/metabolismo , Linfocitos T/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoAsunto(s)
Linfoma de Células del Manto/genética , ARN Largo no Codificante/genética , Estudios de Casos y Controles , Línea Celular Tumoral , Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4F Eucariótico de Iniciación/genética , Factor 4F Eucariótico de Iniciación/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Linfoma de Células del Manto/metabolismo , Biosíntesis de Proteínas , ARN Largo no Codificante/metabolismo , Análisis de Secuencia de ARNRESUMEN
Long noncoding RNAs (lncRNAs) are important regulators of transcription; however, their involvement in protein translation is not well known. Here we explored whether the lncRNA GAS5 is associated with translation initiation machinery and regulates translation. GAS5 was enriched with eukaryotic translation initiation factor-4E (eIF4E) in an RNA-immunoprecipitation assay using lymphoma cell lines. We identified two RNA binding motifs within eIF4E protein and the deletion of each motif inhibited the binding of GAS5 with eIF4E. To confirm the role of GAS5 in translation regulation, GAS5 siRNA and in vitro transcribed GAS5 RNA were used to knock down or overexpress GAS5, respectively. GAS5 siRNA had no effect on global protein translation but did specifically increase c-Myc protein level without an effect on c-Myc mRNA. The mechanism of this increase in c-Myc protein was enhanced association of c-Myc mRNA with the polysome without any effect on protein stability. In contrast, overexpression of in vitro transcribed GAS5 RNA suppressed c-Myc protein without affecting c-Myc mRNA. Interestingly, GAS5 was found to be bound with c-Myc mRNA, suggesting that GAS5 regulates c-Myc translation through lncRNA-mRNA interaction. Our findings have uncovered a role of GAS5 lncRNA in translation regulation through its interactions with eIF4E and c-Myc mRNA.
Asunto(s)
Factor 4E Eucariótico de Iniciación/metabolismo , Regulación de la Expresión Génica , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Largo no Codificante/genética , Secuencia de Aminoácidos , Western Blotting , Células Cultivadas , Factor 4E Eucariótico de Iniciación/genética , Humanos , Inmunoprecipitación , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Polirribosomas , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/genética , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/metabolismo , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Some chemicals used in consumer products or manufacturing (e.g. plastics, surfactants, pesticides, resins) have estrogenic activities; these xenoestrogens (XEs) chemically resemble physiological estrogens and are one of the major categories of synthesized compounds that disrupt endocrine actions. Potent rapid actions of XEs via nongenomic mechanisms contribute significantly to their disruptive effects on functional endpoints (e.g. cell proliferation/death, transport, peptide release). Membrane-initiated hormonal signaling in our pituitary cell model is predominantly driven by mERα with mERß and GPR30 participation. We visualized ERα on plasma membranes using many techniques in the past (impeded ligands, antibodies to ERα) and now add observations of epitope proximity with other membrane signaling proteins. We have demonstrated a range of rapid signals/protein activations by XEs including: calcium channels, cAMP/PKA, MAPKs, G proteins, caspases, and transcription factors. XEs can cause disruptions of the oscillating temporal patterns of nongenomic signaling elicited by endogenous estrogens. Concentration effects of XEs are nonmonotonic (a trait shared with natural hormones), making it difficult to design efficient (single concentration) toxicology tests to monitor their harmful effects. A plastics monomer, bisphenol A, modified by waste treatment (chlorination) and other processes causes dephosphorylation of extracellular-regulated kinases, in contrast to having no effects as it does in genomic signaling. Mixtures of XEs, commonly found in contaminated environments, disrupt the signaling actions of physiological estrogens even more severely than do single XEs. Understanding the features of XEs that drive these disruptive mechanisms will allow us to redesign useful chemicals that exclude estrogenic or anti-estrogenic activities.
Asunto(s)
Congéneres del Estradiol/farmacología , Congéneres del Estradiol/toxicidad , Transducción de Señal/efectos de los fármacos , Animales , Compuestos de Bencidrilo/farmacología , Caveolina 1/metabolismo , Mezclas Complejas/farmacología , Receptor alfa de Estrógeno/metabolismo , Estrona/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Fenoles/farmacología , Hipófisis/citología , RatasRESUMEN
Persistent STAT3 activation has been found in activated B-cell like diffuse large B cell tumors (DLBCL). To investigate whether genetic mutations play a role in aberrant STAT3 signaling in DLBCL, we bi-directionally sequenced all 24 exons of the STAT3 gene in DLBCL tumors (nâ=â40). We identified 2 novel point mutations in 2 separate (2/40; 5%) patients at exon 7 and 24. Point mutation 2552G>A was a silent mutation in the stop codon. Another heterozygous mutation 857T>A encoded a methionine substitution by lysine at codon 206 (M206K) in the coiled-coil domain of STAT3. We performed site directed mutagenesis to mutate wild type (WT) STAT3α and STAT3ß at codon 206 and constructed stable cell lines by lentiviral transfection of STAT3α(WT), STAT3α(M206K), STAT3ß(WT) and STAT3ß(M206K) plasmids. The mutation was found to increase STAT3 phosphorylation in STAT3α mutant cell lines with no effect on the STAT3ß mutant cell line. Transcriptional activation was also increased in the STAT3α mutant cells compared with STAT3α WT cells as detected by a luciferase reporter assay. Moreover, STAT3α(M206K) mutant cells were resistant to JAK2 pathway inhibition compared to STAT3α WT cells. These results indicate that missense mutations in STAT3 increase signaling through the JAK/STAT pathway. JAK2 inhibitors may be useful in the patient with this STAT3 mutation as well as those with pathway activation by other mechanisms.
Asunto(s)
Exones , Janus Quinasa 2/genética , Linfoma de Células B Grandes Difuso/genética , Mutación Missense , Isoformas de Proteínas/genética , Factor de Transcripción STAT3/genética , Transducción de Señal , Secuencia de Aminoácidos , Linfocitos B/metabolismo , Linfocitos B/patología , Línea Celular Tumoral , Genes Reporteros , Vectores Genéticos , Humanos , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/metabolismo , Lentivirus/genética , Luciferasas , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Datos de Secuencia Molecular , Isoformas de Proteínas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Factor de Transcripción STAT3/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Activación TranscripcionalRESUMEN
The secretory pathway is responsible for the transport of newly synthesized transmembrane proteins from the endoplasmic reticulum to their destinations via the Golgi/trans-Golgi network (TGN). Cargo proteins at each station are actively sorted by specific sorting signals on the cargo and the corresponding coat complexes. Here, we used the Arabidopsis regulator of G-protein signaling (AtRGS1), which contains an N-terminal potentially sensing glucose seven-transmembrane domain and a C-terminal RGS domain, as a model to uncover sorting motifs required for its cell surface expression. Expression of wild-type and truncated or mutated AtRGS1 fluorescent fusion proteins identified two cysteine residues in the extracellular N-terminus that are essential for endoplasmic reticulum exit and/or correct folding of AtRGS1. The linker between the seven-transmembrane and RGS domains contains an endoplasmic reticulum export signal, whereas the C-terminus is dispensable for the plasma membrane expression of AtRGS1. Interestingly, deletion of the RGS domain results in Golgi/TGN localization of the truncated AtRGS1. Further analysis using site-directed mutagenesis showed that a tyrosine-based motif embedded in the RGS domain is essential for Golgi/TGN export of AtRGS1. These results reveal a new role for the RGS domain in regulating AtRGS1 trafficking from the Golgi/TGN to the plasma membrane and explain the interaction between the seven-transmembrane and RGS domains.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/metabolismo , Proteínas RGS/fisiología , Vías Secretoras , Red trans-Golgi/fisiología , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Cisteína/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Señales de Exportación Nuclear , Proteínas RGS/química , Proteínas RGS/metabolismoRESUMEN
UNLABELLED: Multiple physiologic estrogens (estradiol, estriol, and estrone), as well as xenoestrogenic compounds (including alkylphenols and bisphenol A), can act via nongenomic signaling initiated by liganding of the plasma membrane estrogen receptor-α (mERα). We examined heterotrimeric G protein involvement leading to extracellular-regulated kinase (ERK) activation in GH3/B6/F10 rat anterior pituitary tumor cells that express abundant mERα, and smaller amounts of mERß and GPR30. A combination of microarrays, immunoblots, and quantitative immunoassays demonstrated the expression of members of all α, ß, and γ G protein classes in these cells. Use of selective inhibitors showed that the G(αi) subtype was the primary initiator of downstream ERK signaling. Using antibodies against the GTP-bound form of G(α) protein subtypes i and s, we showed that xenoestrogens (bisphenol A, nonylphenol) activated G(αi) at 15-30s; all alkylphenols examined subsequently suppressed activation by 5min. GTP-activation of G(αi) for all estrogens was enhanced by irreversible cumulative binding to GTPγS. In contrast, G(αs) was neither activated nor deactivated by these treatments with estrogens. ERα and G(αi) co-localized outside nuclei and could be immuno-captured together. Interactions of ERα with G(αi) and caveolin I were demonstrated by epitope proximity ligation assays. An ERα/ß antagonist (ICI182780) and a selective disruptor of caveolar structures (nystatin) blocked estrogen-induced ERK activation. CONCLUSIONS: Xenoestrogens, like physiologic estrogens, can evoke downstream kinase signaling involving selective interactions of ERα with G(αi) and caveolin I, but with some different characteristics, which could explain their disruptive actions.