RESUMEN
Development of the metanephric kidney in mammals requires complex reciprocal tissue interactions between the ureteric epithelium and the mesenchyme. It is believed that Gdnf, produced in the metanephric mesenchyme, activates Ret signaling in the Wolffian duct to initiate the formation of the metanephros. However, the molecular mechanism for induction of Gdnf in the metanephric mesenchyme is not completely defined. Previous studies demonstrated that during the early stages of kidney development, loss of Osr1, Eya1, Pax2 or Wt1 gene function in the metanephric mesenchyme compromises the formation of the kidney. Moreover, it has been shown that the Hox11-Eya1-Pax2 complex activates the expression of Six2 and Gdnf in the metanephric mesenchyme to drive nephrogenesis. Here, we demonstrate that the orphan nuclear receptor chicken ovalbumin upstream promoter transcription factor II (COUP-TFII, also known as Nr2f2) is required for the specification of the metanephric mesenchyme. Deletion of COUP-TFII at E7.5 results in improper differentiation of the metanephric mesenchyme and absence of essential developmental regulators, such as Eya1, Six2, Pax2 and Gdnf. Importantly, we show that COUP-TFII directly regulates the expression of both Eya1 and Wt1 in the metanephric mesenchyme. Our findings reveal, for the first time, that COUP-TFII plays a central role in the specification of metanephric fate and in the maintenance of metanephric mesenchyme proliferation and survival by acting as a crucial regulator of Eya1 and Wt1 expression.
Asunto(s)
Factor de Transcripción COUP II/fisiología , Riñón/crecimiento & desarrollo , Células Madre Mesenquimatosas/fisiología , Mesodermo/crecimiento & desarrollo , Animales , Factor de Transcripción COUP II/genética , Diferenciación Celular/fisiología , Supervivencia Celular/fisiología , Desarrollo Embrionario , Femenino , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Factor Neurotrófico Derivado de la Línea Celular Glial/análisis , Proteínas de Homeodominio/biosíntesis , Péptidos y Proteínas de Señalización Intracelular , Riñón/metabolismo , Masculino , Mesodermo/metabolismo , Ratones , Proteínas Nucleares/biosíntesis , Organogénesis/fisiología , Factor de Transcripción PAX2/análisis , Embarazo , Proteínas Tirosina Fosfatasas/biosíntesis , Factores de Transcripción/biosíntesisRESUMEN
This study aimed to investigate the possible involvement of the orphan nuclear receptor chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) in the regulation of renin gene expression. COUP-TFII colocalized with renin in the juxtaglomerular cells of the kidney, which are the main source of renin in vivo. Protein-DNA binding studies demonstrated that COUP-TFII binds to an imperfect direct repeat COUP-TFII recognition sequence (termed hereafter proxDR) in the proximal renin promoter. Because cAMP signaling plays a central role in the control of the renin gene expression, we suggested that COUP-TFII may modulate this cAMP effect. Accordingly, knockdown of COUP-TFII in the clonal renin-producing cell lines As4.1 and Calu-6 diminished the stimulation of the renin mRNA expression by cAMP agonists. In addition, the mutation of the proxDR element in renin promoter reporter gene constructs abrogated the inducibility by cAMP. The proxDR sequence was found to be necessary for the function of a proximal renin promoter cAMP-response element (CRE). Knockdown of COUP-TFII or cAMP-binding protein (CREB), which is the archetypal transcription factor binding to CRE, decreased the basal renin gene expression. However, the deficiency of COUP-TFII did not further diminish the renin expression when CREB was knocked down. In agreement with the cell culture studies, mutant mice deficient in COUP-TFII have lower renin expression than their control strain. Altogether our data show that COUP-TFII is involved in the control of renin gene expression.
Asunto(s)
Factor de Transcripción COUP II/metabolismo , Renina/metabolismo , Animales , Western Blotting , Factor de Transcripción COUP II/genética , Pollos , Inmunoprecipitación de Cromatina , AMP Cíclico/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Inmunohistoquímica , Ratones , Ratones Noqueados , Unión Proteica/genética , Unión Proteica/fisiología , Interferencia de ARN , Renina/genéticaRESUMEN
N-methyl-D-aspartate (NMDA) receptors are essential for normal nervous system function, but their excessive activation can lead to neuronal degeneration. NMDA receptors are also expressed in peripheral tissues, where their properties and significance are not well understood. Here we show that functional NMDA receptors are expressed in podocytes, polarized cells that form an essential component of the glomerular filtration apparatus. Application of NMDA to podocyte cell lines or primary cultures of mouse podocytes evoked macroscopic currents mediated by cation channels, with significant permeability to Ca²(+). Podocyte NMDA receptors do not desensitize with prolonged exposure to NMDA. They are blocked by supraphysiological concentrations of external or internal Mg²(+) and, also, by the prototype antagonists MK-801 and D-2-aminophosphonovaleric acid. NMDA responses in podocytes were strongly potentiated by D-serine, but not by glycine, even at high concentrations. D-Aspartate and L-homocysteate are effective agonists of podocyte NMDA receptors. Surprisingly, L-glutamate and L-aspartate did not evoke robust ionic currents in podocytes, regardless of the concentration or membrane potential at which these amino acids were tested. NMDA application for 2 h caused activation of secondary signals through Erk and Akt pathways and, at higher concentrations, caused activation of RhoA. Exposure to NMDA for 6 h did not cause loss of cultured podocytes but did lead to a reduction in nephrin expression. NMDA receptors that respond to circulating ligands may play a role in regulation of glomerular function or dysfunction, but they are unlikely to be components of a local glutamate signaling system in glomeruli.
Asunto(s)
Regulación de la Expresión Génica/fisiología , N-Metilaspartato/farmacología , Podocitos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Calcio/metabolismo , Línea Celular , Maleato de Dizocilpina/farmacología , Relación Dosis-Respuesta a Droga , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Potenciales de la Membrana , Ratones , Proteínas Proto-Oncogénicas c-akt , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/genética , Transducción de Señal/fisiología , Factores de TiempoRESUMEN
Mechanosensitive large-conductance Ca(2+)-activated K(+) channels encoded by the Slo1 gene (BK(Ca) channels) are expressed in podocytes. Here we show that BK(Ca) channels reciprocally coimmunoprecipitate with synaptopodin (Synpo) in mouse glomeruli, in mouse podocytes, and in a heterologous expression system (HEK293T cells) in which these proteins are transiently expressed. Synpo and Slo1 colocalize along the surface of the glomerular basement membrane in mouse glomeruli. Synpo interacts with BK(Ca) channels at COOH-terminal domains that overlap with an actin-binding domain on the channel molecule that is necessary for trafficking of BK(Ca) channels to the cell surface. Moreover, addition of exogenous beta-actin to mouse podocyte lysates reduces BK(Ca)-Synpo interactions. Coexpression of Synpo increases steady-state surface expression of BK(Ca) channels in HEK293T cells. However, Synpo does not affect the stability of cell surface BK(Ca) channels, suggesting a primary effect on the rate of forward trafficking, and Synpo coexpression does not affect BK(Ca) gating. Conversely, stable knockdown of Synpo expression in mouse podocyte cell lines reduces steady-state surface expression of BK(Ca) channels but does not affect total expression of BK(Ca) channels or their gating. The effects of Synpo on surface expression of BK(Ca) are blocked by inhibition of Rho signaling in HEK293T cells and in podocytes. Functional cell surface BK(Ca) channels in podocytes are also reduced by sustained (2 h) but not acute (15 min) depolymerization of actin with cytochalasin D. Synpo may regulate BK(Ca) channels through its effects on actin dynamics and by modulating interactions between BK(Ca) channels and regulatory proteins of the podocyte slit diaphragm.
Asunto(s)
Actinas/fisiología , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/fisiología , Proteínas de Microfilamentos/fisiología , Podocitos/fisiología , Proteínas de Unión al GTP rho/fisiología , Citoesqueleto de Actina/fisiología , Animales , Línea Celular , Membrana Celular/fisiología , Humanos , Activación del Canal Iónico/fisiología , Ratones , Modelos Animales , Podocitos/citologíaRESUMEN
The cerebellum is essential for fine control of movement and posture, and it has been a useful model for studying many aspects of neural development because of its relatively simple anatomy and developmental program. However, the roles of nuclear receptors (NRs) underlying formation of the cerebellum and maintenance of cerebellar functions are still poorly characterized. As a contribution to the Nuclear Receptor Signaling Atlas (NURSA), we employed immunohistochemistry to investigate the expression pattern of 18 NRs in the cerebellum. Ten receptors were demonstrated to be expressed in the postnatal day 21 (P21) cerebellum. Among them, five receptors (COUP-TFI, COUP-TFII, RORalpha, ERbeta, and ERRgamma) were expressed at all stages (embryonic stage, P0, P7, and P21) examined. Interestingly, COUP-TFI and COUP-TFII show differential anterior-posterior expression patterns during cerebellar development. Taken together, our results suggest that members of the nuclear receptor superfamily might play importantly physiological roles in the cerebellum.
Asunto(s)
Cerebelo/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Factor de Transcripción COUP I/genética , Factor de Transcripción COUP I/metabolismo , Factor de Transcripción COUP II/genética , Factor de Transcripción COUP II/metabolismo , Cerebelo/embriología , Receptor beta de Estrógeno/genética , Receptor beta de Estrógeno/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Interferencia de ARN , Receptores Citoplasmáticos y Nucleares/genética , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismoRESUMEN
Nuclear receptors are transcriptional regulators that play important roles in embryonic development and organogenesis. To study the potential roles of nuclear receptors in kidney development, we examined the expression patterns of a subset of nuclear receptors in which specific antibodies are available for profiling using immunohistochemistry. As a prototype for our analysis, we investigated the expression patterns of chicken ovalbumin upstream promoter transcription factor (COUP-TF) -I and -II in more details during embryonic development and in the adult by immunohistochemistry. We showed that COUP-TFI is expressed in the stroma and mesenchymal cells at embryonic d 11.5 (E11.5) and expression persists throughout embryonic development. In the adult kidney, only mesangial cells show meaningful COUP-TFI expression. In contrast, COUP-TFII expression is detected as early as E9.5 and high expression is seen in the mesenchymal-derived epithelial cells but not in the ureteric buds through E12.5. At E13.5, COUP-TFII expression becomes regionalized with higher expression in the region that gives rise to the distal tubule. The proximal part of the S-shaped body that will become the glomerulus after endothelial cell migration shows COUP-TFII expression in podocyte precursor cells and epithelial cells of the Bowman's capsule. In the adult mouse kidney, COUP-TFII is detected in distal tubules, podocytes, and the epithelial cells of the Bowman's capsule. In addition to COUP-TFs, we also examined the expression profiles of eight other nuclear receptors (farnesoid X receptor, vitamin D receptor, hepatocyte nuclear factor 4alpha, retinoid X receptor alpha, mineralocorticoid receptor, steroidogenic factor 1, liver receptor homolog-1, and germ cell nuclear factor). Our results suggest that these nuclear receptors are likely to play important physiological roles in the kidney development.
Asunto(s)
Riñón/embriología , Factores de Transcripción/metabolismo , Animales , Anticuerpos/inmunología , Factor de Transcripción COUP I/análisis , Factor de Transcripción COUP I/metabolismo , Factor de Transcripción COUP II/análisis , Factor de Transcripción COUP II/metabolismo , Riñón/química , Riñón/metabolismo , Ratones , Factor Esteroidogénico 1 , Factores de Transcripción/análisisRESUMEN
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.
Asunto(s)
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
Anaplastic thyroid carcinomas are a highly aggressive and extremely lethal form of human cancer, but the biological characteristics related to their aggressive nature are not understood. Moreover, Gadd45 family proteins have been implicated in a variety of growth-regulatory mechanisms, including DNA replication and repair, G(2)/M checkpoint control, and apoptosis. In this study we found that Gadd45gamma RNA was present at significantly lower levels in anaplastic cancer cells, compared with normal primary cultured thyrocytes. In addition, the adenovirus-mediated reexpression of Gadd45gamma significantly inhibited the proliferation of anaplastic thyroid carcinoma cells, ARO, FRO, and NPA cells, which was attributed to apoptosis. Furthermore, the adenovirus-mediated delivery of Gadd45gamma gene in anaplastic thyroid cancer resulted in the inhibition of tumor growth in vivo. This in vitro and in vivo activity of the adenovirus-mediated transduction of CR6/Gadd45gamma, on anaplastic thyroid cancer cell growth suppression, was reminiscent of the effects of p53. This study demonstrates that the Gadd45gamma gene has potential use as a candidate gene for gene therapy in anaplastic thyroid cancer.
Asunto(s)
Apoptosis/genética , Carcinoma/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Biosíntesis de Proteínas , Neoplasias de la Tiroides/metabolismo , Adenoviridae/genética , Animales , Northern Blotting , Western Blotting , Carcinoma/genética , Genes p53/genética , Terapia Genética , Humanos , Inmunohistoquímica , Indicadores y Reactivos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Proteínas/genética , ARN Neoplásico/biosíntesis , Sales de Tetrazolio , Tiazoles , Neoplasias de la Tiroides/genética , Transducción Genética , Trasplante Heterólogo , Células Tumorales Cultivadas , Proteinas GADD45RESUMEN
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.
Asunto(s)
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.
Asunto(s)
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.
Asunto(s)
Carcinoma Papilar/metabolismo , Transformación Celular Neoplásica/genética , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Proteínas de Fusión Oncogénica/metabolismo , Neoplasias de la Tiroides/metabolismo , Transactivadores/metabolismo , Carcinoma Papilar/genética , Proteínas de Unión al ADN/genética , Humanos , Proteínas de Fusión Oncogénica/genética , Fosforilación , Proteínas Tirosina Quinasas , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Factor de Transcripción STAT3 , Transducción de Señal/genética , Neoplasias de la Tiroides/genética , Transactivadores/genética , Activación Transcripcional , Células Tumorales CultivadasRESUMEN
Anaplastic thyroid carcinomas (ATCs) are highly aggressive, extremely lethal human cancers with poor therapeutic response. Chemokines are a superfamily of small cytokine-like proteins that induce, through their interaction with G protein-coupled receptors, cytoskeletal rearrangement, firm adhesion to endothelial cells, and directional migration. In this study, we characterized the expression of CXC chemokine receptor 4 (CXCR4) and analyzed its functions in ARO cells, a human ATC cell. The normal primary cultured thyroid cells and ATC cell lines expressed CXCR4 and stromal cell-derived factor (SDF)-1 alpha transcripts, detected by RT-PCR. Fluorescence activated cell sorting analysis of CXCR4 expression in normal and ATC cells showed that ARO cells expressed significant levels of CXCR4. FRO, NPA, and normal thyroid cells did not express membrane CXCR4, as determined by fluorescence activated cell sorting analysis. To identify the functional role of CXCR4 in ARO cells, we treated ARO cells with SDF-1 alpha and analyzed the signaling pathways, cellular migration, and proliferation. SDF-1alpha enhanced the migration but did not affect the proliferation of ARO cells or activate the Janus kinase/signal transducer and activator of transcription signaling pathways. However, SDF-1 alpha/CXCR4 activation resulted in phosphorylation of the p70S6 kinase and its target protein, ribosomal S6 protein, and also activation of the ERK1/ERK2 signaling pathways. Furthermore, SDF-1 alpha/CXCR4- mediated activation of the p70S6 kinase and phosphorylation of the S6 protein were inhibited by treatment with an mTOR/FRAP inhibitor. The specificity of the CXCR4-mediated migration of ARO cells was demonstrated by the dose-dependent inhibition of migration by neutralizing anti-CXCR4. The ATC cells, FRO and NPA, which do not express CXCR4, did not demonstrate significant SDF-1 alpha-mediated migration in vitro. In addition, the CXCR4-mediated migration of ARO cells was inhibited by treatment with pertussis toxin (a Gi-protein inhibitor) and PD 98059 (a mitogen-activated ERK kinase inhibitor) but not by LY294002 and wortmanin, phosphatidylinositol 3-kinase inhibitors. These findings suggest that a subset of ATC cells expresses functional CXCR4, which may be important in tumor cell migration and local tumor invasion.
Asunto(s)
Carcinoma/metabolismo , Receptores CXCR4/metabolismo , Neoplasias de la Tiroides/metabolismo , Carcinoma/patología , Carcinoma/fisiopatología , División Celular/fisiología , Supervivencia Celular/fisiología , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , Quimiocinas CXC/fisiología , Proteínas de Unión al ADN/metabolismo , Activación Enzimática , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Receptores CXCR4/fisiología , Valores de Referencia , Proteínas Quinasas S6 Ribosómicas/metabolismo , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Neoplasias de la Tiroides/patología , Neoplasias de la Tiroides/fisiopatología , Transactivadores/metabolismo , Células Tumorales CultivadasRESUMEN
Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, known as statins, are widely used for primary and secondary prevention of coronary artery atherosclerosis. Pathogenesis of atherosclerosis is multistep processes where transendothelial migration of various leukocytes including monocytes is a crucial step. Interferon-gamma (IFN-gamma) contributes in this process by activating macrophages and T-lymphocytes, and by inducing adhesion molecules in vascular endothelial and smooth muscle cells. In this study we investigated the expression of intercellular cell adhesion molecule-1 (ICAM-1) in transformed endothelial cell line ECV304 cells as influenced by lovastatin, tumor necrosis factor-alpha (TNF-alpha) and IFN-gamma. Results show that lovastatin suppresses expression of ICAM-1 by inhibiting the IFN-gamma-induced extracellular signal-regulated kinase (ERK) p44/p42-STAT1 signaling pathway. In cells treated with lovastatin and IFN-gamma, ICAM-1 was expressed at a lower level than in cells treated with IFN-gamma alone. However, lovastatin does not reduce TNF-alpha induced expression of ICAM-1. A similar result was observed in cells treated with the MEKK inhibitor PD98059 and IFN-gamma. Cis-acting DNA sequence elements were identified in the 5'-flanking region of the ICAM-1 promoter that mediate inhibition by lovastatin; these sequences map to the IFN-gamma activated site which also binds the STAT1 homodimer. However, lovastatin did not inhibit IFN-gamma-mediated induction of the Y701 phosphorylated form of STAT1. But lovastatin does inhibit the IFN-gamma-mediated phosphorylation of ERK1/ERK2 (T202/Y204) and S727 phosphorylation of STAT1. TNF-alpha does not induce phosphorylation of ERK1/ERK2 and S727 in ECV304 and smooth muscle cells. The results provide the evidences that statins may have beneficial effects by inhibiting IFN-gamma action in atherosclerotic process