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The regression relationship between water discharge rates and nutrient concentrations can provide a quick and straightforward way to estimate nutrient loads. However, recent studies indicated that the relationship might produce large biases in load estimates and, therefore, may not be applicable in certain types of cases. The goal of this study is to explore the theoretical reasons behind the selective applicability of the regression relationship between flow rates and nitrate + nitrite concentrations. For this study, we examined daily flow and nitrate + nitrite concentration observations made at the outlets of 22 watersheds monitored by the Heidelberg Tributary Loading Program (HTLP). The statistical relationship between the flow rates and concentrations was explored using regression equations offered by the LOAD ESTimator (LOADEST). Results demonstrated that the use of the regression equations provided nitrate + nitrite load estimates at acceptable accuracy levels (NSE≥0.35 and |PBIAS|≤30.0%) in 14 watersheds (64 % of 22 study watersheds). The regression relationships provided highly biased results at eight watersheds (36 %), implying their limited applicability. The heteroscedasticity of the residuals led to the high bias and resulting inaccurate regression, which was commonly found in watersheds where low flow had high nitrate + nitrite concentration variations. Conversely, the regression relationships provided acceptable accuracy for watersheds that had a relatively constant variance of the nitrate + nitrite concentrations. The results indicate that the homoscedasticity of residuals is the key assumption to be satisfied to estimate nitrate + nitrite loads from a statistical regression between flow discharge and nitrate + nitrite concentrations. The transport capacity (capacity-limited) concept implicitly assumed in the regression relationship between flow discharge and nitrate + nitrite concentrations is not always applicable, especially to agricultural areas in which nitrate + nitrite loads are highly variable depending on management practices (supply-limited). The findings suggest that the regression relationship should be carefully applied to areas in which intensive agricultural activities, including crop management and conservation practices, are implemented. Thus, the transport capacity concept is reasonably regarded to contribute to the homoscedasticity of residuals.
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Soluble nitrogen is highly mobile in soil and susceptible to leaching. It is important to identify nitrogen transport pathways so that the sources can be efficiently targeted in environment management. This study quantified the contribution of direct runoff and baseflow to nitrate + nitrite loading by separating flow and nitrate + nitrite concentration measurements into two periods depending on whether only baseflow was present or not using baseflow separation methods. When both direct runoff and baseflow were present in streamflow, their nitrate + nitrite concentrations were assumed based on the hydrological reasoning that baseflow does not change rapidly, and streamflow mostly consists of direct runoff within a rainfall event. For this study, we obtained and investigated daily flow and nitrate + nitrite concentration observations made at the outlets of 22 watersheds located in the Western Lake Erie area. Results showed that baseflow was responsible for 26 to 77% of the nitrate + nitrite loads. The relative nitrate + nitrite load contributions of direct runoff and baseflow substantially varied with the sizes of drainage areas and agricultural land uses. Increases in drainage areas tend to prolong the travel time of surface runoff and thus help its reinfiltration into soil, which then could increase the baseflow contribution. In addition, the artificial drainage networks common in the agricultural fields of the study areas would promote the drainage of nutrient-laden excess water from soils. Such findings suggest the need for environmental management customized considering nitrogen transport pathways.
Asunto(s)
Lagos , Nitrógeno , Nitratos/análisis , Nitritos , SueloRESUMEN
Three types of iron oxide Janus particles are obtained by varying the deposition rate of iron in a 3:1 Ar/O(2) atmosphere during physical vapor deposition. Each type of iron oxide Janus particle shows a distinct assembly behavior when an external magnetic field is applied, i.e., formation of staggered chains, double chains, or no assembly. A detailed deposition rate diagram is obtained to identify the relationship between deposition rate and assembly behavior. The extent of iron oxidation is identified as the key parameter in determining the assembly behavior. In addition, the effects of particle volume fraction, thickness of the iron oxide cap, and assembly time on the final assembly behavior are studied. Cap thickness is shown not to influence the assembly behavior, while particle volume fraction and assembly time affect the chain growth rate and the chain length, but not the overall assembly behavior. The samples are characterized by optical, scanning electron, and atomic force microscopies.
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A facile and efficient technique for synthesizing rapid and large-scale colloidal monolayer is introduced to obtain surface anisotropic particles. Silica particles grafted with long alkyl chains are rapidly organized into monolayer assemblies by implementing water-film climbing and convective particle assembly on glass slides. Assembled particles are modified into surface-anisotropic particles utilizing physical vapor deposition with a magnetically active material. The magnetic hemisphere enables separation of modified and unmodified surface-anisotropic particles. The proposed methodology can lead to a large-scale production opportunity for surface-anisotropic particles.
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Macroscopic interfacial interactions between cyclopentane (CP) hydrates and various surfactants droplets are examined in a CP/n-decane oil mixture. Initial contact force and subsequent z-axis dependent retraction force are measured utilizing a high-resolution microbalance integrated with a micrometer-precision stage. The resulting retraction force is utilized to determine the overall adhesion energy of the system. In addition, interfacial tensions and contact angles of the system are examined to further understand the effect of surface-active agents and substrates on the initial contact and retraction forces.
RESUMEN
A method for precise and reproducible initial contact force measurements is introduced utilizing an apparatus fabricated with a microbalance and z-axis stage to study the interaction behavior between cyclopentane (CP) hydrate and water in a temperature controlled hydrocarbon environment. CP hydrate probes are prepared using hydrate slurries composed of 5 wt % CP and Wilhelmy rods. The CP hydrate probe is slowly brought into contact with water to determine the initial contact force. The effect of substrate morphology on the initial contact force is reported through employing aluminum substrates prepared using physical vapor deposition (PVD) and milling. Accurate and facile measurements are performed by applying a high-resolution microbalance with 0.1 microN resolution to provide repeatable and consistent results of initial contact force between hydrate and water.
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Ciclopentanos/química , Agua/química , Hidrocarburos , Mecánica , MétodosRESUMEN
A polymeric catalytic membrane reactor (CMR) is fabricated using alternating assemblies of surface-anisotropic (sa-) and plain (p-) polystyrene (PS) colloids as a template. We report the preparation of TiO2 sa-PS colloids by physical vapor deposition of titanium onto a colloidal monolayer in an oxygen-rich environment and employ the modified colloids as a means to deliver the TiO2 catalyst to the CMR pores. sa-PS and p-PS colloids are assembled into alternating cylindrical sections inside a microcapillary followed by infiltration and curing of a liquid polymer precursor in the interstitial space of the assembly. Subsequent organic solvent treatment results in a cylindrical porous CMR with embedded TiO2 caps. TiO2 cap embedment, composition and surface morphology, surface pore structure, and cross-sectional integrity are analyzed using variable-pressure scanning electron microscopy, transmission electron microscopy, and X-ray photoelectron spectroscopy.
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Coloides/química , Reactores Biológicos , Catálisis , Vidrio , Microscopía/métodos , Microscopía Electrónica de Rastreo/métodos , Microscopía Electrónica de Transmisión/métodos , Espectroscopía de Fotoelectrones/métodos , Poliestirenos/química , Porosidad , Propiedades de Superficie , Titanio/químicaRESUMEN
A fabrication method for porous polymeric fibers (PPFs) is reported. We show that a multisectional colloidal crystal can be assembled within a microcapillary by alternating dipping into colloidal solutions of varying size. Subsequent infiltration with curable polymer and washing with suitable solvents results in porous fibers with a cylindrical cross section. Along the length of the fiber, alternating sections of controlled length, pore size, and pore size distribution exist. These fibers present interesting materials for neural scaffolding, catalysis, and possibly photonics if produced with a high degree of crystallinity. The surface pores and bulk porosity of the fibers are characterized by variable-pressure scanning electron microscopy (vp-SEM). Careful analysis shows that the surface pores vary with the colloidal template diameter and polymer infiltration time.
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Polímeros/química , Coloides , Porosidad , Propiedades de SuperficieRESUMEN
CONTEXT: The oncogenic RET/PTC tyrosine kinase causes papillary thyroid cancer (PTC). The use of inhibitors specific for RET/PTC may be useful for targeted therapy of PTC. OBJECTIVE: The objective of the study was to evaluate the efficacies of the recently developed kinase inhibitors SU11248, SU5416, and SU6668 in inhibition of RET/PTC. DESIGN: SU11248, SU5416, and SU6668 were synthesized, and their inhibitory potencies were evaluated using an in vitro RET/PTC kinase assay. The inhibitory effects of the compounds on RET/PTC were evaluated by quantifying the autophosphorylation of RET/PTC, signal transducer and activator of transcription (STAT)-3 activation, and the morphological reversal of RET/PTC-transformed cells. RESULTS: An in vitro kinase assay revealed that SU5416, SU6668, and SU11248 inhibited phosphorylation of the synthetic tyrosine kinase substrate peptide E4Y by RET/PTC3 in a dose-dependent manner with IC(50) of approximately 944 nm for SU5416, 562 nm for SU6668, and 224 nm for SU11248. Thus, SU11248 effectively inhibits the kinase activity of RET/PTC3. RET/PTC-mediated Y705 phosphorylation of STAT3 was inhibited by addition of SU11248, and the inhibitory effects of SU11248 on the tyrosine phosphorylation and transcriptional activation of STAT3 were very closely correlated with decreased autophosphorylation of RET/PTC. SU11248 caused a complete morphological reversion of transformed NIH-RET/PTC3 cells and inhibited the growth of TPC-1 cells that have an endogenous RET/PTC1. CONCLUSION: SU11248 is a highly effective tyrosine kinase inhibitor of the RET/PTC oncogenic kinase.
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Antineoplásicos/farmacología , Indoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-ret/antagonistas & inhibidores , Pirroles/farmacología , Administración Oral , Animales , Proliferación Celular/efectos de los fármacos , Humanos , Ratones , Células 3T3 NIH , Oxindoles , Fosforilación , Propionatos , Proteínas Proto-Oncogénicas c-ret/metabolismo , Factor de Transcripción STAT3/metabolismo , SunitinibRESUMEN
CONTEXT: The BRAF mutation may influence the expression patterns of molecular markers that are related to the development and progression of thyroid cancer. OBJECTIVE: The objective of the study was to investigate the effects of the BRAF V600E mutation on expression of galectin-3, cyclooxygenase-2, cyclin D1, p53, and vascular endothelial growth factor (VEGF) in papillary thyroid cancer (PTC). DESIGN, SETTING, AND SUBJECTS: One hundred sixty-three PTC and 28 nodular hyperplasia patients were selected retrospectively. The presence of the BRAF V600E mutation and the level of expression of the molecular markers were determined. RESULTS: Of 161 PTC patients, 102 patients (63.4%) were BRAF V600E(+), and these cases had significantly larger tumor sizes (P = 0.01), compared with V600E(-) cases (n = 59, 36.6%). Although PTC tissues had higher expression levels of the selected molecular markers than nodular hyperplasia tissues, expression levels of several molecular markers in BRAF V600E(+) PTC were not significantly different from those of BRAF V600E(-) PTC. But VEGF was significantly up-regulated in BRAF V600E(+) PTC, compared with BRAF V600E(-) PTC. VEGF expression levels were strongly positively correlated to tumor size (P < 0.001), extrathyroidal invasion (P = 0.02), and tumor stage (P = 0.04). Multivariate analysis clearly showed that VEGF expression was up-regulated in BRAF V600E(+) PTC (odds ratio 2.5, confidence interval 1.1-5.6; P = 0.03). CONCLUSIONS: BRAF V600E(+) PTC tended to have larger tumor volumes and higher expression of VEGF. The level of VEGF expression was closely correlated with tumor size, extrathyroidal invasion, and stage. The relatively high levels of VEGF expression may be related to poorer clinical outcomes and recurrences in BRAF V600E(+) PTC.
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Carcinoma Papilar/genética , Mutación Puntual , Proteínas Proto-Oncogénicas B-raf/genética , Neoplasias de la Tiroides/genética , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Adulto , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patología , Ciclina D1/metabolismo , Ciclooxigenasa 2/metabolismo , ADN de Neoplasias/química , ADN de Neoplasias/genética , Femenino , Galectina 3/metabolismo , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Estudios Retrospectivos , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Proteína p53 Supresora de Tumor/metabolismo , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Papillary thyroid carcinoma (PTC) is a heterogenous disorder characterized by unique gene rearrangements and gene mutations that activate signaling pathways responsible for cellular transformation, survival, and antiapoptosis. Activation of protein kinase B (PKB) and its downstream signaling pathways appears to be an important event in thyroid tumorigenesis. In this study, we found that the thyroid-specific oncogenic RET/PTC tyrosine kinase is able to phosphorylate PKB in vitro and in vivo. RET/PTC-transfected cells showed tyrosine phosphorylation of endogenous and exogenous PKB, which was independent of phosphorylation of T308 and S473 regulated by the upstream kinases phosphoinositide-dependent kinase-1 and -2, respectively. The PKB Y315 residue, which is known to be phosphorylated by Src tyrosine kinase, was also a major site of phosphorylation by RET/PTC. RET/PTC-mediated tyrosine phosphorylation results in the activation of PKB kinase activity. The activation of PKB by RET/PTC blocked the activity of the forkhead transcription factor, FKHRL1, but a Y315F mutant of PKB failed to inhibit FKHRL1 activity. In summary, these observations suggest that RET/PTC is able to phosphorylate the Y315 residue of PKB, an event that results in maximal activation of PKB for RET/PTC-induced thyroid tumorigenesis.
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Carcinoma Papilar/enzimología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-ret/metabolismo , Neoplasias de la Tiroides/enzimología , Animales , Carcinoma Papilar/genética , Células Cultivadas , Citoplasma/química , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/antagonistas & inhibidores , Humanos , Fosforilación , Proteínas Proto-Oncogénicas c-akt/análisis , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-ret/análisis , Glándula Tiroides/enzimología , Neoplasias de la Tiroides/genética , Tirosina/genética , Tirosina/metabolismoRESUMEN
CR6-interacting factor 1 (CRIF1) was recently identified as a nuclear protein that interacts with the Gadd45 (growth arrest and DNA damage inducible 45) family of proteins and participates in the regulation of the G1/S phase of the cell cycle. However, the nuclear action of CRIF1 is largely unknown. In this study, we demonstrate that CRIF1 acts as a novel coregulator of transactivation of the orphan nuclear receptor Nur77. Both in vitro and in vivo studies show that CRIF1 interacts with Nur77 via the Nur77 AB domain and that it dramatically inhibits the AB domain-mediated transactivation of Nur77. Transient transfection assays demonstrate that CRIF1 inhibits steroid receptor coactivator-2-mediated Nur77 transactivation, and silencing of endogenous CRIF1 by small interfering RNA relieves this repression. CRIF1 possesses intrinsic repressor activities that are not affected by the histone deacetylase inhibitor Trichostatin A. In addition, overexpression of CRIF1 inhibits TSH/protein kinase A-induced Nur-responsive element promoter activity. CRIF1 inhibited Nur77-dependent induction of E2F1 promoter activity, mRNA expression, and Nur77-mediated G1/S progression in cell cycle. These results suggest that CRIF1 acts as a repressor of the orphan nuclear receptor Nur77 by inhibiting AB domain-mediated transcriptional activity.
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Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional/genética , Animales , Ciclo Celular , Proteínas de Ciclo Celular/química , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Unión al ADN/genética , Hormonas Glicoproteicas de Subunidad alfa/farmacología , Humanos , Ratones , Mutación/genética , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Unión Proteica , Estructura Terciaria de Proteína , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores de Esteroides/genética , Elementos de Respuesta/genética , Factores de Transcripción/genéticaRESUMEN
Chimeric RET/PTC (rearranged in transformation/papillary thyroid carcinoma) oncoproteins are constitutively active tyrosine kinases found in thyroid papillary carcinoma and nonneoplastic Hashimoto's thyroiditis. Although several proteins have been identified to be substrates of RET/PTC kinases, the pathogenic roles played by RET/PTC in malignant and benign thyroid diseases and the molecular mechanisms that are involved are not fully understood. We found that RET/PTC expression phosphorylates the Y701 residue of STAT1, a type II interferon (IFN)-responsive protein. RET/PTC-mediated signal transducer and activator of transcription 1 (STAT1) phosphorylation requires RET/PTC kinase activity to be intact but other tyrosine kinases, such as Janus kinases or c-Src, are not involved. RET/PTC-induced STAT1 transcriptional activation was not inhibited by suppressor of cytokine signaling-1 or -3, or protein inhibitors of activated STAT3 [(protein inhibitor of activated STAT (PIAS3)], but PIAS1 strongly repressed the RET/PTC-induced transcriptional activity of STAT1. RET/PTC-induced STAT1 activation caused IFN regulatory factor-1 expression. We found that STAT1 and IFN regulatory factor-1 cooperated to significantly increase transcription from type IV IFN-gamma responsive promoters of class II transactivator genes. Significantly, cells stably expressing RET/PTC expressed class II transactivator and showed enhanced de novo membrane expression of major histocompatibility complex (MHC) class II proteins. Furthermore, RET/PTC1-bearing papillary thyroid carcinoma cells strongly expressed MHC class II (human leukocyte-associated antigen-DR alpha) genes, whereas the surrounding normal tissues did not. Thus, RET/PTC is able to phosphorylate and activate STAT1. This may lead to enhanced MHC class II expression, which may explain why the tissues surrounding RET/PTC-positive cancers are infiltrated with lymphocytes. Such immune response-promoting activity of RET/PTC may also relate to the development of Hashimoto's thyroiditis.
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Carcinoma Papilar/enzimología , Proteínas de Unión al ADN/metabolismo , Proteínas Oncogénicas/fisiología , Neoplasias de la Tiroides/enzimología , Transactivadores/metabolismo , Activación Transcripcional , Carcinoma Papilar/inmunología , Proteínas de Unión al ADN/genética , Genes MHC Clase II/genética , Antígenos HLA-DR/análisis , Antígenos HLA-DR/genética , Humanos , Factor 1 Regulador del Interferón , Neoplasia Endocrina Múltiple Tipo 2a/metabolismo , Neoplasia Endocrina Múltiple Tipo 2b/metabolismo , Proteínas Nucleares/genética , Proteínas de Fusión Oncogénica , Fosfoproteínas/genética , Fosforilación , Regiones Promotoras Genéticas/genética , Proteínas Inhibidoras de STAT Activados , Proteínas Tirosina Quinasas , Proteínas Proto-Oncogénicas c-ret , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Proteínas Tirosina Quinasas Receptoras/fisiología , Factor de Transcripción STAT1 , Transducción de Señal , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/fisiología , Neoplasias de la Tiroides/inmunología , Transactivadores/genética , Tirosina/metabolismoRESUMEN
It has been suggested that class I and class II MHC are contributing factors for numerous diseases including autoimmune thyroid diseases, type 1 diabetes, rheumatoid arthritis, Alzheimer's disease, and multiple sclerosis. The class II trans-activator (CIITA), which is a non-DNA-binding regulator of class II MHC transcription, regulates the constitutive and inducible expression of the class I and class II genes. FRTL-5 thyroid cells incubated in the presence of IFN-gamma have a significantly higher level of cell surface rat MHC class II RTI.B. However, the IFN-gamma-induced RT1.B expression was suppressed significantly in cells incubated in the presence of thyrotropin. Thyrotropin (TSH) represses IFN-gamma-induced CIITA expression by inhibiting type IV CIITA promoter activity through the suppression of STAT1 activation and IFN regulatory factor 1 induction. This study found that TSH induces transcriptional activation of the STAT3 gene through the phosphorylation of STAT3 and CREB activation. TSH induces SOCS-1 and SOCS-3, and TSH-mediated SOCS-3 induction was dependent on STAT3. The cell line stably expressing the wild-type STAT3 showed a higher CIITA induction in response to IFN-gamma and also exhibited TSH repression of the IFN-gamma-mediated induction of CIITA. However, TSH repression of the IFN-gamma-induced CIITA expression was not observed in FRTL-5 thyroid cells, which stably expresses the dominant negative forms of STAT3, STAT3-Y705F, and STAT3-S727A. This report suggests that TSH is also engaged in immunomodulation through signal cross-talk with the cytokines in thyroid cells.
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Proteínas Portadoras/fisiología , Proteínas de Unión al ADN/fisiología , Regulación hacia Abajo/inmunología , Péptidos y Proteínas de Señalización Intracelular , Proteínas Nucleares , Proteínas/fisiología , Proteínas Represoras , Glándula Tiroides/metabolismo , Tirotropina/fisiología , Transactivadores/antagonistas & inhibidores , Transactivadores/biosíntesis , Transactivadores/fisiología , Animales , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Bovinos , Línea Celular , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo/genética , Antígenos de Histocompatibilidad/biosíntesis , Humanos , Factor 1 Regulador del Interferón , Factor 3 de Genes Estimulados por el Interferón , Interferón gamma/genética , Interferón gamma/farmacología , Ratones , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Regiones Promotoras Genéticas/inmunología , Unión Proteica/genética , Unión Proteica/inmunología , Biosíntesis de Proteínas , Proteínas/genética , Proteínas/metabolismo , Ratas , Elementos de Respuesta/inmunología , Factor de Transcripción STAT3 , Transducción de Señal/genética , Transducción de Señal/inmunología , Proteína 1 Supresora de la Señalización de Citocinas , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas , Glándula Tiroides/citología , Glándula Tiroides/inmunología , Transactivadores/genética , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
Thyroid cancers are a leading cause of death due to endocrine malignancies. RET/PTC (rearranged in transformation/papillary thyroid carcinomas) gene rearrangements are the most frequent genetic alterations identified in papillary thyroid carcinoma. Although the oncogenic potential of RET/PTC is related to intrinsic tyrosine kinase activity, the substrates for this enzyme are yet to be identified. In this report, we show that phosphoinositide-dependent kinase 1 (PDK1), a pivotal serine/threonine kinase in growth factor-signaling pathways, is a target of RET/PTC. RET/PTC and PDK1 colocalize in the cytoplasm. RET/PTC phosphorylates a specific tyrosine (Y9) residue located in the N-terminal region of PDK1. Y9 phosphorylation of PDK1 by RET/PTC requires an intact catalytic kinase domain. The short (iso 9) and long forms (iso 51) of the RET/PTC kinases (RET/PTC1 and RET/PTC3) induce Y9 phosphorylation of PDK1. Moreover, Y9 phosphorylation of PDK1 by RET/PTC does not require phosphatidylinositol 3-kinase or Src activity. RET/PTC-induced phosphorylation of the Y9 residue results in increased PDK1 activity, decrease of cellular p53 levels, and repression of p53-dependent transactivation. In conclusion, RET/PTC-induced tyrosine phosphorylation of PDK1 may be one of the mechanisms by which it acts as an oncogenic tyrosine kinase in thyroid carcinogenesis.
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Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Secuencia de Aminoácidos , Animales , Células CHO , Carcinoma Papilar/enzimología , Carcinoma Papilar/metabolismo , Cricetinae , Inhibidores Enzimáticos/farmacología , Fibroblastos/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Coactivadores de Receptor Nuclear , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Proteínas Tirosina Quinasas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-ret , Proteínas Tirosina Quinasas Receptoras/genética , Transducción de Señal , Neoplasias de la Tiroides/enzimología , Neoplasias de la Tiroides/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Tirosina/metabolismo , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
Thyroid-stimulating hormone (TSH) regulates the growth and differentiation of thyrocytes by activating the TSH receptor (TSHR). This study investigated the roles of the phosphatidylinositol 3-kinase (PI3K), PDK1, FRAP/mammalian target of rapamycin, and ribosomal S6 kinase 1 (S6K1) signaling mechanism by which TSH and the stimulating type TSHR antibodies regulate thyrocyte proliferation and the follicle activities in vitro and in vivo. The TSHR immunoprecipitates exhibited PI3K activity, which was higher in the cells treated with either TSH or 8-bromo-cAMP. TSH and cAMP increased the tyrosine phosphorylation of TSHR and the association between TSHR and the p85alpha regulatory subunit of PI3K. TSH induced a redistribution of PDK1 from the cytoplasm to the plasma membrane in the cells in a PI3K- and protein kinase A-dependent manner. TSH induced the PDK1-dependent phosphorylation of S6K1 but did not induce Akt/protein kinase B phosphorylation. The TSH-induced S6K1 phosphorylation was inhibited by a dominant negative p85alpha regulatory subunit or by the PI3K inhibitors wortmannin and LY294002. Rapamycin inhibited the phosphorylation of S6K1 in the cells treated with either TSH or 8-bromo-cAMP. The stimulating type TSHR antibodies from patients with Graves disease also induced S6K1 activation, whereas the blocking type TSHR antibodies from patients with primary myxedema inhibited TSH- but not the insulin-induced phosphorylation of S6K1. In addition, rapamycin treatment in vivo inhibited the TSH-stimulated thyroid follicle hyperplasia and follicle activity. These findings suggest an interaction between TSHR and PI3K, which is stimulated by TSH and cAMP and might involve the downstream S6K1 but not Akt/protein kinase B. This pathway may play a role in the TSH/stimulating type TSH receptor antibody-mediated thyrocyte proliferation in vitro and in the response to TSH in vivo.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Fosfatidilinositol 3-Quinasas/biosíntesis , Proteínas Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Quinasas S6 Ribosómicas 90-kDa/biosíntesis , Transducción de Señal , Tirotropina/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Androstadienos/farmacología , Animales , Western Blotting , División Celular , Células Cultivadas , Cromonas/farmacología , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Humanos , Inmunoglobulina G/metabolismo , Inmunohistoquímica , Microscopía Confocal , Microscopía Fluorescente , Modelos Biológicos , Morfolinas/farmacología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Pruebas de Precipitina , Unión Proteica , Isoformas de Proteínas , Proteínas Quinasas/genética , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Ratas , Ratas Sprague-Dawley , Proteínas Quinasas S6 Ribosómicas/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Sirolimus/farmacología , Espectrometría de Fluorescencia , Serina-Treonina Quinasas TOR , Timidina/metabolismo , Glándula Tiroides/citología , Glándula Tiroides/metabolismo , Factores de Tiempo , Transfección , WortmaninaRESUMEN
The Gadd45 family of proteins includes Gadd45alpha, MyD118/Gadd45beta, and CR6/OIG37/Gadd45gamma. These proteins play important roles in maintaining genomic stability and in regulating the cell cycle. This study reports the cloning of a novel protein called CR6-interacting factor 1 (CRIF1) which interacts with Gadd45alpha, MyD118/Gadd45beta, and CR6/OIG37/Gadd45gamma. CRIF1 binds specifically to the Gadd45 family proteins, as determined by an in vitro glutathione S-transferase pull-down assay and an in vivo mammalian cell two-hybrid assay along with coimmunoprecipitation assays. CRIF1 mRNA is highly expressed in the thyroid gland, heart, lymph nodes, trachea, and adrenal tissues. CRIF1 localizes exclusively to the nucleus and colocalizes with Gadd45gamma. Recombinant CRIF1 inhibits the histone H1 kinase activity of immunoprecipitated Cdc2-cyclin B1 and Cdk2-cyclin E, and the inhibitory effects were additive with Gadd45 proteins. Overexpression of CRIF1 increases the percentage of cells in G1, decreases the percentage of cells in S phase, and suppresses growth in NIH3T3 cells. The down-regulation of endogenous CRIF1 by the transfection of the small interfering RNA duplexes resulted in the inactivation of Rb by phosphorylation and decreased the G1 phase cell populations. Expression of CRIF1 is barely detectable in adrenal adenoma and papillary thyroid cancer and much lower than in adjacent normal tissue. The results presented here suggest that CRIF1 is a novel nuclear protein that interacts with Gadd45 and may play a role in negative regulation of cell cycle progression and cell growth.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/biosíntesis , Péptidos y Proteínas de Señalización Intracelular , Proteínas/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Ciclo Celular , Proteínas de Ciclo Celular/química , División Celular , ADN Complementario/metabolismo , Regulación hacia Abajo , Fase G1 , Glutatión Transferasa/metabolismo , Humanos , Ratones , Microscopía Fluorescente , Datos de Secuencia Molecular , Proteínas Nucleares , Fosforilación , Pruebas de Precipitina , Unión Proteica , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/metabolismo , Proteína de Retinoblastoma/metabolismo , Fase S , Homología de Secuencia de Aminoácido , Factores de Tiempo , Distribución Tisular , Transfección , Células Tumorales Cultivadas , Técnicas del Sistema de Dos Híbridos , Proteinas GADD45RESUMEN
Thyroid papillary carcinomas are characterized by RET/PTC (rearranged in transformation/papillary thyroid carcinoma) rearrangements that result in fusion of the tyrosine kinase domain of the RET receptor to the N-terminal sequences encoded by heterologous genes. This thyroid-specific rearrangement causes aberrant expression of RET/PTC and results in constitutive ligand-independent activation of RET kinase. However, it is unclear how RET/PTC activates the specific signaling pathways for cellular transformation. In this study, we show that RET/PTC associates with signal transducer and activator of transcription 3 (STAT3) and activates it by the specific phosphorylation of the tyrosine 705 residue. Activation of STAT3 requires the intrinsic kinase activity of RET/PTC; Janus tyrosine kinase and c-Src kinase are not involved in the RET/PTC-mediated activation of STAT3. RET/PTC-induced activation of STAT3 induces the STAT3-responsive genes, vascular endothelial growth factor, cyclin D1, and intercellular adhesion molecule 1. In addition, RET/PTC-mediated cellular transformation and proliferation of transformed cells require tyrosine 705 phosphorylation of STAT3 in NIH3T3 cells. We conclude that STAT3 activation by the RET/PTC tyrosine kinase is one of the critical signaling pathways for the regulation of specific genes, such as cyclin D1, vascular endothelial growth factor, and intercellular adhesion molecule 1, and for cellular transformation.