RESUMO
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.
RESUMO
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.
Assuntos
Lagos , Nitrogênio , Nitratos/análise , Nitritos , SoloRESUMO
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.
RESUMO
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.
RESUMO
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.
Assuntos
Ciclopentanos/química , Água/química , Hidrocarbonetos , Mecânica , MétodosRESUMO
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.
Assuntos
Coloides/química , Reatores Biológicos , Catálise , Vidro , Microscopia/métodos , Microscopia Eletrônica de Varredura/métodos , Microscopia Eletrônica de Transmissão/métodos , Espectroscopia Fotoeletrônica/métodos , Poliestirenos/química , Porosidade , Propriedades de Superfície , Titânio/químicaRESUMO
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.
Assuntos
Polímeros/química , Coloides , Porosidade , Propriedades de SuperfícieRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Indóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidores , Pirróis/farmacologia , Administração Oral , Animais , Proliferação de Células/efeitos dos fármacos , Humanos , Camundongos , Células NIH 3T3 , Oxindóis , Fosforilação , Propionatos , Proteínas Proto-Oncogênicas c-ret/metabolismo , Fator de Transcrição STAT3/metabolismo , SunitinibeRESUMO
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.
Assuntos
Carcinoma Papilar/genética , Mutação Puntual , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias da Glândula Tireoide/genética , Fator A de Crescimento do Endotélio Vascular/biossíntese , Adulto , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patologia , Ciclina D1/metabolismo , Ciclo-Oxigenase 2/metabolismo , DNA de Neoplasias/química , DNA de Neoplasias/genética , Feminino , Galectina 3/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Estudos Retrospectivos , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Proteína Supressora de Tumor p53/metabolismo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
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.
Assuntos
Carcinoma Papilar/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-ret/metabolismo , Neoplasias da Glândula Tireoide/enzimologia , Animais , Carcinoma Papilar/genética , Células Cultivadas , Citoplasma/química , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/antagonistas & inibidores , Humanos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/análise , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-ret/análise , Glândula Tireoide/enzimologia , Neoplasias da Glândula Tireoide/genética , Tirosina/genética , Tirosina/metabolismoRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional/genética , Animais , Ciclo Celular , Proteínas de Ciclo Celular/química , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/genética , Subunidade alfa de Hormônios Glicoproteicos/farmacologia , Humanos , Camundongos , Mutação/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Ligação Proteica , Estrutura Terciária de Proteína , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/genética , Elementos de Resposta/genética , Fatores de Transcrição/genéticaRESUMO
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.
Assuntos
Carcinoma Papilar/enzimologia , Proteínas de Ligação a DNA/metabolismo , Proteínas Oncogênicas/fisiologia , Neoplasias da Glândula Tireoide/enzimologia , Transativadores/metabolismo , Ativação Transcricional , Carcinoma Papilar/imunologia , Proteínas de Ligação a DNA/genética , Genes MHC da Classe II/genética , Antígenos HLA-DR/análise , Antígenos HLA-DR/genética , Humanos , Fator Regulador 1 de Interferon , Neoplasia Endócrina Múltipla Tipo 2a/metabolismo , Neoplasia Endócrina Múltipla Tipo 2b/metabolismo , Proteínas Nucleares/genética , Proteínas de Fusão Oncogênica , Fosfoproteínas/genética , Fosforilação , Regiões Promotoras Genéticas/genética , Proteínas Inibidoras de STAT Ativados , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas c-ret , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Receptores Proteína Tirosina Quinases/fisiologia , Fator de Transcrição STAT1 , Transdução de Sinais , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/fisiologia , Neoplasias da Glândula Tireoide/imunologia , Transativadores/genética , Tirosina/metabolismoRESUMO
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.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas de Ligação a DNA/fisiologia , Regulação para Baixo/imunologia , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Nucleares , Proteínas/fisiologia , Proteínas Repressoras , Glândula Tireoide/metabolismo , Tireotropina/fisiologia , Transativadores/antagonistas & inibidores , Transativadores/biossíntese , Transativadores/fisiologia , Animais , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Bovinos , Linhagem Celular , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/genética , Antígenos de Histocompatibilidade/biossíntese , Humanos , Fator Regulador 1 de Interferon , Fator Gênico 3 Estimulado por Interferon , Interferon gama/genética , Interferon gama/farmacologia , Camundongos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Regiões Promotoras Genéticas/imunologia , Ligação Proteica/genética , Ligação Proteica/imunologia , Biossíntese de Proteínas , Proteínas/genética , Proteínas/metabolismo , Ratos , Elementos de Resposta/imunologia , Fator de Transcrição STAT3 , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Proteína 1 Supressora da Sinalização de Citocina , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina , Glândula Tireoide/citologia , Glândula Tireoide/imunologia , Transativadores/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
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.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Sequência de Aminoácidos , Animais , Células CHO , Carcinoma Papilar/enzimologia , Carcinoma Papilar/metabolismo , Cricetinae , Inibidores Enzimáticos/farmacologia , Fibroblastos/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Coativadores de Receptor Nuclear , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-ret , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais , Neoplasias da Glândula Tireoide/enzimologia , Neoplasias da Glândula Tireoide/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Tirosina/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
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.
Assuntos
Regulação Enzimológica da Expressão Gênica , Fosfatidilinositol 3-Quinases/biossíntese , Proteínas Quinases/biossíntese , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Quinases S6 Ribossômicas 90-kDa/biossíntese , Transdução de Sinais , Tireotropina/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Androstadienos/farmacologia , Animais , Western Blotting , Divisão Celular , Células Cultivadas , Cromonas/farmacologia , Relação Dose-Resposta a Droga , Citometria de Fluxo , Humanos , Imunoglobulina G/metabolismo , Imuno-Histoquímica , Microscopia Confocal , Microscopia de Fluorescência , Modelos Biológicos , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Testes de Precipitina , Ligação Proteica , Isoformas de Proteínas , Proteínas Quinases/genética , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ratos , Ratos Sprague-Dawley , Proteínas Quinases S6 Ribossômicas/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Sirolimo/farmacologia , Espectrometria de Fluorescência , Serina-Treonina Quinases TOR , Timidina/metabolismo , Glândula Tireoide/citologia , Glândula Tireoide/metabolismo , Fatores de Tempo , Transfecção , WortmaninaRESUMO
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.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Ciclo Celular , Proteínas de Ciclo Celular/química , Divisão Celular , DNA Complementar/metabolismo , Regulação para Baixo , Fase G1 , Glutationa Transferase/metabolismo , Humanos , Camundongos , Microscopia de Fluorescência , Dados de Sequência Molecular , Proteínas Nucleares , Fosforilação , Testes de Precipitina , Ligação Proteica , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/metabolismo , Proteína do Retinoblastoma/metabolismo , Fase S , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Distribuição Tecidual , Transfecção , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-Híbrido , Proteínas GADD45RESUMO
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.