RESUMEN
The US dairy industry has made substantial gains in reducing the greenhouse gas emission intensity of a gallon of milk. At the same time, consumer and investor interest for improved environmental benefits or reduced environmental impact of food production continues to grow. Following a trend of increasing greenhouse gas emission commitments for businesses across sectors of the economy, the US dairy industry has committed to a goal of net zero greenhouse gas emissions by 2050. The Paris Climate Accord's goal is to reduce warming of the atmosphere to less than 1.5 to 2°C based on preindustrial levels, which is different from emission goals of historic climate agreements that focus on emission reduction targets. Most of the emissions that account for the greenhouse gas footprint of a gallon of milk are from the short-lived climate pollutant CH4, which has a half-life of approximately 10 yr. The relatively new accounting system Global Warming Potential Star and the unit CO2 warming equivalents gives the industry the appropriate metrics to quantify their current and projected warming impact on future emissions. Incorporating this metric into potential future emissions pathways can allow the industry to understand the magnitude of emissions reductions needed to no longer contribute additional warming. Deterministic modeling was performed across the dairy industry's emission areas of enteric fermentation, manure management, feed production, and other upstream emissions necessary for dairy production. By reducing farm-level absolute emissions by 23% based on current levels, there is the opportunity for the US dairy industry to realize climate neutrality within the next few decades.
Asunto(s)
Gases de Efecto Invernadero , Animales , Dióxido de Carbono/análisis , Bovinos , Industria Lechera , Gases de Efecto Invernadero/metabolismo , Estiércol/análisis , Leche/químicaRESUMEN
Circadian and metabolic systems are interlocked and reciprocally regulated. To determine if the circadian system regulates glucose homeostasis and mammary development, the function of the circadian system was disrupted by exposing cattle to chronic light-dark cycle phase shifts from 5 wk before expected calving (BEC) to parturition. Multiparous Holstein cows were exposed to 16 h of light and 8 h of dark (CON, n = 8) or phase shifting (PS, n = 8) the light cycle 6 h every 3 d beginning 35 d BEC. After calving, both treatments were exposed to CON lighting. Mammary biopsies were taken at 21 d BEC and 21 d in milk (DIM), and histological analysis indicated PS treatment decreased the ratio of lumen to alveolar area and percentage of proliferating epithelial cells in the prepartum period. Intravenous glucose tolerance test was performed at 14 d BEC and 7 DIM by administering 50% dextrose. Blood glucose, ß-hydroxybutyrate, insulin, and nonesterified fatty acids were consequently measured over 3 h. At 14 d BEC no treatment differences were observed in baseline glucose or insulin. Treatment had no effect on blood glucose or glucose area under the curve at 14 d BEC and 7 DIM. Insulin area under the curve was higher in PS versus CON at 14 d BEC and 7 DIM. The PS cows produced less milk than CON cows through 60 DIM (40.3 vs. 42.6 kg/d). Exposure to chronic light-dark PS in late gestation decreased mammary development and increased insulin resistance in periparturient cows, which may have caused subsequent lower milk yield.
Asunto(s)
Bovinos , Relojes Circadianos , Resistencia a la Insulina , Glándulas Mamarias Animales/crecimiento & desarrollo , Leche , Periodo Posparto , Ácido 3-Hidroxibutírico/sangre , Animales , Glucemia/metabolismo , Oscuridad , Ácidos Grasos no Esterificados/sangre , Femenino , Prueba de Tolerancia a la Glucosa/veterinaria , Insulina/sangre , Lactancia/fisiología , Luz , Parto , EmbarazoRESUMEN
Pituitary tumor-transforming gene 1-binding factor (PTTG1IP; PBF) is a multifunctional glycoprotein, which is overexpressed in a wide range of tumours, and significantly associated with poorer oncological outcomes, such as early tumour recurrence, distant metastasis, extramural vascular invasion and decreased disease-specific survival. PBF transforms NIH 3T3 fibroblasts and induces tumours in nude mice, while mice harbouring transgenic thyroidal PBF expression show hyperplasia and macrofollicular lesions. Our assumption that PBF becomes an oncogene purely through increased expression has been challenged by the recent report of mutations in PBF within the Catalogue of Somatic Mutations in Cancer (COSMIC) database. We therefore sought to determine whether the first 10 PBF missense substitutions in human cancer might be oncogenic. Anisomycin half-life studies revealed that most mutations were associated with reduced protein stability compared to wild-type (WT) PBF. Proliferation assays narrowed our interest to two mutational events which significantly altered cell turnover: C51R and R140W. C51R was mainly confined to the endoplasmic reticulum while R140W was apparent in the Golgi apparatus. Both C51R and R140W lost the capacity to induce cellular migration and significantly reduced cell invasion. Colony formation and soft agar assays demonstrated that, in contrast to WT PBF, both mutants were unable to elicit significant colony formation or anchorage-independent growth. However, C51R and R140W retained the ability to repress radioiodide uptake, a functional hallmark of PBF. Our data reveal new insight into PBF function and confirm that, rather than being oncogenic, mutations in PBF are likely to be passenger effects, with overexpression of PBF the more important aetiological event in human cancer.
Asunto(s)
Proteínas de la Membrana/genética , Animales , Proliferación Celular , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/metabolismo , Ratones , Mutación , Proto-Oncogenes Mas , TransfecciónRESUMEN
The proto-oncogene PTTG and its binding partner PBF have been widely studied in multiple cancer types, particularly thyroid and colorectal, but their combined role in tumourigenesis is uncharacterised. Here, we show for the first time that together PTTG and PBF significantly modulate DNA damage response (DDR) genes, including p53 target genes, required to maintain genomic integrity in thyroid cells. Critically, DDR genes were extensively repressed in primary thyrocytes from a bitransgenic murine model (Bi-Tg) of thyroid-specific PBF and PTTG overexpression. Irradiation exposure to amplify p53 levels further induced significant repression of DDR genes in Bi-Tg thyrocytes (P=2.4 × 10-4) compared with either PBF- (P=1.5 × 10-3) or PTTG-expressing thyrocytes (P=NS). Consistent with this, genetic instability was greatest in Bi-Tg thyrocytes with a mean genetic instability (GI) index of 35.8±2.6%, as well as significant induction of gross chromosomal aberrations in thyroidal TPC-1 cells following overexpression of PBF and PTTG. We extended our findings to human thyroid cancer using TCGA data sets (n=322) and found striking correlations with PBF and PTTG expression in well-characterised DDR gene panel RNA-seq data. In addition, genetic associations and transient transfection identified PBF as a downstream target of the receptor tyrosine kinase-BRAF signalling pathway, emphasising a role for PBF as a novel component in a pathway well described to drive neoplastic growth. We also showed that overall survival (P=1.91 × 10-5) and disease-free survival (P=4.9 × 10-5) was poorer for TCGA patients with elevated tumoural PBF/PTTG expression and mutationally activated BRAF. Together our findings indicate that PBF and PTTG have a critical role in promoting thyroid cancer that is predictive of poorer patient outcome.
Asunto(s)
Daño del ADN , Proteínas de la Membrana/metabolismo , Securina/metabolismo , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos , Pronóstico , Proto-Oncogenes Mas , Securina/genética , Tasa de Supervivencia , Neoplasias de la Tiroides/patología , Transfección , Resultado del TratamientoRESUMEN
INTRODUCTION: Incomplete human extravillous trophoblast (EVT) invasion of the decidua and maternal spiral arteries is characteristic of pre-eclampsia, a condition linked to low maternal vitamin D status. It is hypothesized that dysregulated vitamin D action in uteroplacental tissues disrupts EVT invasion leading to malplacentation. METHODS: This study assessed the effects of the active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25-D3), and its precursor, 25-hydroxyvitamin D3 (25-D3), on primary human EVT isolated from first trimester pregnancies. Expression of EVT markers (cytokeratin-7, HLA-G), the vitamin D-activating enzyme (CYP27B1) and 1,25-D3 receptor (VDR) was assessed by immunocytochemistry. EVT responses following in vitro treatment with 1,25-D3 (0-10 nM) or 25-D3 (0-100 nM) for 48-60 h were assessed using quantitative RT-PCR (qRT-PCR) analysis of key target genes. Effects on EVT invasion through Matrigel(®) were quantified alongside zymographic analysis of secreted matrix metalloproteinases (MMPs). Effects on cell viability were assessed by measurement of MTT. RESULTS: EVT co-expressed mRNA and protein for CYP27B1 and VDR, and demonstrated induction of mRNA encoding vitamin D-responsive genes, 24-hydroxylase (CYP24A1) and cathelicidin following 1,25-D3 treatment. EVT could respond to 1,25-D3 and 25-D3, both of which significantly increased EVT invasion, with maximal effect at 1 nM 1,25-D3 (1.9-fold; p < 0.01) and 100 nM 25-D3 (2.2-fold; p < 0.05) respectively compared with untreated controls. This was accompanied by increased pro-MMP2 and pro-MMP9 secretion. The invasion was independent of cell viability, which remained unchanged. DISCUSSION: These data support a role for vitamin D in EVT invasion during human placentation and suggest that vitamin D-deficiency may contribute to impaired EVT invasion and pre-eclampsia.
Asunto(s)
Calcifediol/metabolismo , Calcitriol/metabolismo , Catelicidinas/agonistas , Placentación , Trofoblastos/metabolismo , Regulación hacia Arriba , Vitamina D3 24-Hidroxilasa/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/metabolismo , Biomarcadores/metabolismo , Catelicidinas/genética , Catelicidinas/metabolismo , Movimiento Celular , Supervivencia Celular , Células Cultivadas , Femenino , Humanos , Metaloproteinasas de la Matriz Secretadas/metabolismo , Microscopía Fluorescente , Embarazo , Primer Trimestre del Embarazo , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trofoblastos/citología , Vitamina D3 24-Hidroxilasa/química , Vitamina D3 24-Hidroxilasa/genéticaRESUMEN
STUDY QUESTION: Does triiodothyronine (T3) regulate the secretion of angiogenic growth factors and cytokines by human decidual cells isolated from early pregnancy? SUMMARY ANSWER: T3 modulates the secretion of specific angiogenic growth factors and cytokines, with different regulatory patterns observed amongst various isolated subpopulations of human decidual cells and with a distinct change between the first and second trimesters of pregnancy. WHAT IS KNOWN ALREADY: Maternal thyroid dysfunction during early pregnancy is associated with complications of malplacentation including miscarriage and pre-eclampsia. T3 regulates the proliferation and apoptosis of fetal-derived trophoblasts, as well as promotes the invasive capability of extravillous trophoblasts (EVT). We hypothesize that T3 may also have a direct impact on human maternal-derived decidual cells, which are known to exert paracrine regulation upon trophoblast behaviour and vascular development at the uteroplacental interface. STUDY DESIGN, SIZE, DURATION: This laboratory-based study used human decidua from first (8-11 weeks; n = 18) and second (12-16 weeks; n = 12) trimester surgical terminations of apparently uncomplicated pregnancies. PARTICIPANTS/MATERIALS, SETTING, METHODS: Primary cultures of total decidual cells, and immunomagnetic bead-isolated populations of stromal-enriched (CD10+) and stromal-depleted (CD10-) cells, uterine natural killer cells (uNK cells; CD56+) and macrophages (CD14+) were assessed for thyroid hormone receptors and transporters by immunocytochemistry. Each cell population was treated with T3 (0, 1, 10, 100 nM) and assessments were made of cell viability (MTT assay) and angiogenic growth factor and cytokine secretion (immunomediated assay). The effect of decidual cell-conditioned media on EVT invasion through Matrigel(®) was evaluated. MAIN RESULTS AND THE ROLE OF CHANCE: Immunocytochemistry showed the expression of thyroid hormone transporters (MCT8, MCT10) and receptors (TRα1, TRß1) required for thyroid hormone-responsiveness in uNK cells and macrophages from the first trimester. The viability of total decidual cells and the different cell isolates were unaffected by T3 so changes in cell numbers could not account for any observed effects. In the first trimester, T3 decreased VEGF-A secretion by total decidual cells (P < 0.05) and increased angiopoietin-2 secretion by stromal-depleted cells (P < 0.05) but in the second trimester total decidual cells showed only increased angiogenin secretion (P < 0.05). In the first trimester, T3 reduced IL-10 secretion by total decidual cells (P < 0.05), and reduced granulocyte macrophage colony stimulating factor (P < 0.01), IL-8 (P < 0.05), IL-10 (P < 0.01), IL-1ß (P < 0.05) and monocyte chemotactic protein -1 (P < 0.001) secretion by macrophages, but increased tumour necrosis factor-α secretion by stromal-depleted cells (P < 0.05) and increased IL-6 by uNK cells (P < 0.05). In contrast, in the second trimester T3 increased IL-10 secretion by total decidual cells (P < 0.01) but did not affect cytokine secretion by uNK cells and macrophages. Conditioned media from first trimester T3-treated total decidual cells and macrophages did not alter EVT invasion compared with untreated controls. Thus, treatment of decidual cells with T3 resulted in changes in both angiogenic growth factor and cytokine secretion in a cell type-specific and gestational age-dependent manner, with first trimester decidual macrophages being the most responsive to T3 treatment, but these changes in decidual cell secretome did not affect EVT invasion in vitro. LIMITATIONS, REASONS FOR CAUTION: Our results are based on in vitro findings and we cannot be certain if a similar response occurs in human pregnancy in vivo. WIDER IMPLICATIONS OF THE FINDINGS: Optimal maternal thyroid hormone concentrations could play a critical role in maintaining a balanced inflammatory response in early pregnancy to prevent fetal immune rejection and promote normal placental development through the regulation of the secretion of critical cytokines and angiogenic growth factors by human decidual cells. Our data suggest that there is an ontogenically determined regulatory 'switch' in T3 responsiveness between the first and second trimesters, and support the notion that the timely and early correction of maternal thyroid dysfunction is critical in influencing pregnancy outcomes. STUDY FUNDING/COMPETING INTEREST(S): This study is funded by Wellbeing of Women (RG/1082/09 to S.Y.C., M.D.K., J.A.F., L.S.L., G.E.L.) and Action Medical Research - Henry Smith Charity (SP4335 to M.D.K., S.Y.C., L.S.L., J.A.F.). The authors have no conflicts of interest to disclose.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Citocinas/metabolismo , Decidua/efectos de los fármacos , Placenta/efectos de los fármacos , Triyodotironina/farmacología , Factores de Edad , Angiopoyetina 2/metabolismo , Decidua/citología , Decidua/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Edad Gestacional , Humanos , Células Asesinas Naturales/citología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/metabolismo , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Placenta/citología , Placenta/metabolismo , Embarazo , Primer Trimestre del Embarazo , Segundo Trimestre del Embarazo , Receptores de Hormona Tiroidea/metabolismo , Triyodotironina/administración & dosificación , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
CONTEXT: The clinical effectiveness of ablative radioiodine treatment of thyroid tumors is limited by the availability of the sodium iodide symporter (NIS) at the plasma membrane (PM) for uptake of ¹³¹I. A significant proportion of well-differentiated thyroid tumors are unable to concentrate sufficient radioiodine for effective therapy, and in other tumor models such as breast tumors, where radioiodine uptake would be an attractive therapeutic option, uptake is insufficient. OBJECTIVE: Pituitary tumor-transforming gene-binding factor (PBF; PTTG1IP) is overexpressed in multiple cancers and significantly decreases NIS expression at the PM. The goal of this study was to identify a method by which PBF repression of NIS may be overcome in human tumors. RESULTS: Here, we identify PBF as a tyrosine phosphoprotein that specifically binds the proto-oncogene tyrosine protein kinase Src in mass spectrometry, glutathione S-transferase pulldown and coimmunoprecipitation assays. Src induction leads to phosphorylation at PBF residue Y174. Abrogation of this residue results in PM retention and a markedly reduced ability to bind NIS. The Src inhibitor PP1 inhibits PBF phosphorylation in multiple cell lines in vitro, including human primary thyroid cells. Of direct clinical importance to the treatment of thyroid cancer, PP1 stimulates iodide uptake by transfected NIS in TPC1 thyroid carcinoma cells and entirely overcomes PBF repression of iodide uptake in human primary thyroid cells. CONCLUSIONS: We propose that targeting PBF phosphorylation at residue Y174 via tyrosine kinase inhibitors may be a novel therapeutic strategy to enhance the efficacy of ablative radioiodine treatment in thyroid and other endocrine and endocrine-related tumors.
Asunto(s)
Membrana Celular/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Simportadores/metabolismo , Glándula Tiroides/metabolismo , Neoplasias de la Tiroides/metabolismo , Sustitución de Aminoácidos , Animales , Transporte Biológico/efectos de los fármacos , Células COS , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/patología , Células Cultivadas , Chlorocebus aethiops , Humanos , Péptidos y Proteínas de Señalización Intracelular , Radioisótopos de Yodo/metabolismo , Proteínas de la Membrana/genética , Proteínas Mutantes/metabolismo , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas pp60(c-src)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Radiofármacos/metabolismo , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Simportadores/agonistas , Simportadores/genética , Glándula Tiroides/citología , Glándula Tiroides/efectos de los fármacos , Glándula Tiroides/patología , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/patología , Neoplasias de la Tiroides/radioterapiaRESUMEN
Within the basolateral membrane of thyroid follicular epithelial cells, two transporter proteins are central to thyroid hormone (TH) biosynthesis and secretion. The sodium iodide symporter (NIS) delivers iodide from the bloodstream into the thyroid, and after TH biosynthesis, monocarboxylate transporter 8 (MCT8) mediates TH secretion from the thyroid gland. Pituitary tumor-transforming gene-binding factor (PBF; PTTG1IP) is a protooncogene that is up-regulated in thyroid cancer and that binds NIS and modulates its subcellular localization and function. We now show that PBF binds MCT8 in vitro, eliciting a marked shift in MCT8 subcellular localization and resulting in a significant reduction in the amount of MCT8 at the plasma membrane as determined by cell surface biotinylation assays. Colocalization and interaction between PBF and Mct8 was also observed in vivo in a mouse model of thyroid-specific PBF overexpression driven by a bovine thyroglobulin (Tg) promoter (PBF-Tg). Thyroidal Mct8 mRNA and protein expression levels were similar to wild-type mice. Critically, however, PBF-Tg mice demonstrated significantly enhanced thyroidal TH accumulation and reduced TH secretion upon TSH stimulation. Importantly, Mct8-knockout mice share this phenotype. These data show that PBF binds and alters the subcellular localization of MCT8 in vitro, with PBF overexpression leading to an accumulation of TH within the thyroid in vivo. Overall, these studies identify PBF as the first protein to interact with the critical TH transporter MCT8 and modulate its function in vivo. Furthermore, alongside NIS repression, PBF may thus represent a new regulator of TH biosynthesis and secretion.
Asunto(s)
Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica , Hormonas Tiroideas/metabolismo , Animales , Biotinilación , Células COS , Chlorocebus aethiops , ADN Complementario/metabolismo , Glutatión Transferasa/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas de Transporte de Membrana/metabolismo , Ratones , Modelos Biológicos , Transportadores de Ácidos Monocarboxílicos , Fenotipo , Procesamiento Proteico-Postraduccional , Simportadores , Tetraspanina 30/biosíntesis , Transcripción GenéticaRESUMEN
The pituitary tumor transforming gene (PTTG) is a multifunctional proto-oncogene that is over-expressed in various tumors including thyroid carcinomas, where it is a prognostic indicator of tumor recurrence. PTTG has potent transforming capabilities in vitro and in vivo, and many studies have investigated the potential mechanisms by which PTTG contributes to tumorigenesis. As the human securin, PTTG is involved in critical mechanisms of cell cycle regulation, whereby aberrant expression induces aneuploidy. PTTG may further contribute to tumorigenesis through its role in DNA damage response pathways and via complex interactions with hormones and growth factors. Furthermore, PTTG over-expression negatively impacts upon the efficacy of radioiodine therapy in thyroid cancer, through repression of expression and function of the sodium iodide symporter. Given its various roles at all disease stages, PTTG appears to be an important oncogene in thyroid cancer. This review discusses the current knowledge of PTTG with particular focus on its role in thyroid cancer.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/metabolismo , Animales , Carcinoma/genética , Carcinoma/metabolismo , Humanos , Recurrencia Local de Neoplasia , Pronóstico , Proto-Oncogenes Mas , Proto-Oncogenes/genética , SecurinaRESUMEN
Associations of neurological impairment with mutations in the thyroid hormone (TH) transporter, MCT8, and with maternal hypothyroxinaemia, suggest that THs are crucial for human fetal brain development. It has been postulated that TH transporters regulate the cellular supply of THs within the fetal brain during development. This study describes the expression of TH transporters in the human fetal cerebral cortex (720 weeks gestation) and during retinoic acid induced neurodifferentiation of the human N-Tera-2 (NT2) cell line, in triiodothyronine (T3) replete and T3-depleted media. Compared with adult cortex, mRNAs encoding OATP1A2, OATP1C1, OATP3A1 variant 2, OATP4A1, LAT2 and CD98 were reduced in fetal cortex at different gestational ages, whilst mRNAs encoding MCT8, MCT10, OATP3A1 variant 1 and LAT1 were similar. From the early first trimester, immunohistochemistry localised MCT8 and MCT10 to the microvasculature and to undifferentiated CNS cells. With neurodifferentiation, NT2 cells demonstrated declining T3 uptake, accompanied by reduced expressions of MCT8, LAT1, CD98 and OATP4A1. T3 depletion significantly reduced MCT10 and LAT2 mRNA expression at specific time points during neurodifferentiation but there were no effects upon T3 uptake, neurodifferentiation marker expression or neurite lengths and branching. MCT8 repression also did not affect NT2 neurodifferentiation. In conclusion, many TH transporters are expressed in the human fetal cerebral cortex from the first trimester, which could regulate cellular TH supply during early development. However, human NT2 neurodifferentiation is not dependent upon T3 or MCT8 and there were no compensatory changes to promote T3 uptake in a T3-depleted environment.
Asunto(s)
Feto Abortado/metabolismo , Proteínas Portadoras/metabolismo , Diferenciación Celular/fisiología , Corteza Cerebral/embriología , Neuronas/metabolismo , Células Madre Pluripotentes/metabolismo , Hormonas Tiroideas/metabolismo , Feto Abortado/embriología , Sistema de Transporte de Aminoácidos y+/genética , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Proteínas Portadoras/genética , Diferenciación Celular/efectos de los fármacos , Línea Celular , Corteza Cerebral/metabolismo , Cadenas Ligeras de la Proteína-1 Reguladora de Fusión/genética , Proteína-1 Reguladora de Fusión/genética , Proteína-1 Reguladora de Fusión/metabolismo , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Edad Gestacional , Humanos , Proteínas de Filamentos Intermediarios/genética , Transportador de Aminoácidos Neutros Grandes 1/genética , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas del Tejido Nervioso/genética , Nestina , Proteínas de Neurofilamentos/genética , Neuronas/citología , Factor 3 de Transcripción de Unión a Octámeros/genética , Transportadores de Anión Orgánico/genética , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , ARN Interferente Pequeño/genética , Simportadores , Tretinoina/farmacología , Triyodotironina/metabolismo , Triyodotironina/farmacologíaRESUMEN
CONTEXT: Abnormal placentation in human pregnancy is associated with intrauterine fetal growth restriction (IUGR). Our group has previously reported the association between severe IUGR, lower fetal circulating concentrations of thyroid hormones (THs), and altered expression of TH receptors and TH transporters within human placental villi. We postulate that altered TH bioavailability to trophoblasts may contribute to the pathogenesis of IUGR. DESIGN AND OBJECTIVE: Cytotrophoblasts were isolated from normal and IUGR human placentae to compare their responsiveness to T(3) and their capability for T(3) transport. RESULTS: Compared with normal cytotrophoblasts, the viability of IUGR cytotrophoblasts (assessed by methyltetrazoleum assay) was significantly reduced (P < 0.001), whereas apoptosis (assessed using caspase 3/7 activity and M30 immunoreactivity) was significantly increased after T(3) treatment for 48 h (P < 0.001 and P < 0.01, respectively). The secretion of human chorionic gonadotropin was significantly increased by IUGR cytotrophoblasts compared with normal cytotrophoblasts (P < 0.001), independently of T(3) treatment. Net transport of [(125)I]T(3) was 20% higher by IUGR cytotrophoblasts compared with normal cytotrophoblasts (P < 0.001), and this was accompanied by a 2-fold increase in the protein expression of the TH transporter, monocarboxylate transporter 8, as assessed by Western immunoblotting (P < 0.01). CONCLUSIONS: IUGR cytotrophoblasts demonstrate altered responsiveness to T(3) with significant effects on cell survival and apoptosis compared with normal cytotrophoblasts. Increased monocarboxylate transporter 8 expression and intracellular T(3) accumulation may contribute to the altered T(3) responsiveness of IUGR cytotrophoblasts.
Asunto(s)
Retardo del Crecimiento Fetal/patología , Triyodotironina/metabolismo , Triyodotironina/farmacología , Trofoblastos/efectos de los fármacos , Trofoblastos/metabolismo , Adulto , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Apoptosis/efectos de los fármacos , Transporte Biológico/fisiología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Gonadotropina Coriónica/metabolismo , Femenino , Retardo del Crecimiento Fetal/metabolismo , Humanos , Masculino , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Placenta/metabolismo , Placenta/patología , Embarazo , Simportadores , Triyodotironina/farmacocinética , Trofoblastos/patología , Adulto JovenRESUMEN
Thyroid hormones (TH) are important for the development of the human fetus and placenta from very early gestation. The transplacental passage of TH from mother to fetus and the supply of TH into trophoblasts require the expression of placental TH plasma membrane transporters. We describe the ontogeny of the TH transporters MCT8, MCT10, LAT1, LAT2, OATP1A2 and OATP4A1 in a large series (n = 110) of normal human placentae across gestation and describe their expression changes with intrauterine fetal growth restriction (IUGR n = 22). Quantitative RT-PCR revealed that all the mRNAs encoding TH transporters are expressed in human placenta from 6 weeks gestation and throughout pregnancy. MCT8, MCT10, OATP1A2 and LAT1 mRNA expression increased with gestation. OATP4A1 and CD98 (LATs obligatory associated protein) mRNA expression reached a nadir in mid-gestation before increasing towards term. LAT2 mRNA expression did not alter throughout gestation. Immunohistochemistry localised MCT10 and OATP1A2 to villous cytotrophoblasts and syncytiotrophoblasts, and extravillous trophoblasts while OATP4A1 was preferentially expressed in the villous syncytiotrophoblasts. Whilst MCT8 protein expression was increased, MCT10 mRNA expression was decreased in placentae from IUGR pregnancies delivered in the early 3rd trimester compared to age matched appropriately grown for gestational age controls. No significant change was found in the mRNA expression of the other transporters with IUGR. In conclusion, several TH transporters are present in the human placenta from early 1st trimester with varying patterns of expression throughout gestation. Their coordinated effects may regulate both transplacental TH passage and TH supply to trophoblasts, which are critical for the normal development of the fetus and placenta. Increased MCT8 and decreased MCT10 expression within placentae of pregnancies complicated by IUGR may contribute to aberrant development of the fetoplacental unit.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/biosíntesis , Retardo del Crecimiento Fetal/metabolismo , Transportadores de Ácidos Monocarboxílicos/biosíntesis , Hormonas Tiroideas/metabolismo , Femenino , Humanos , Transportador de Aminoácidos Neutros Grandes 1/biosíntesis , Transportadores de Anión Orgánico/biosíntesis , Placenta/metabolismo , Embarazo , ARN Mensajero/metabolismo , Simportadores , Trofoblastos/metabolismoRESUMEN
Thyroid hormones are essential for the normal growth and development of the fetus, and even small alterations in maternal thyroid hormone status during early pregnancy may be associated with neurodevelopmental abnormalities in childhood. Mutations in the novel and specific thyroid hormone transporter monocarboxylate transporter 8 (MCT8) have been associated with severe neurodevelopmental impairment. However, the mechanism by which MCT8 influences neural development remains poorly defined. We have therefore investigated the effect of wild-type (WT) MCT8, and the previously reported L471P mutant, on the growth and function of human neuronal precursor NT2 cells as well as MCT8-null JEG-3 cells. HA-tagged WT MCT8 correctly localized to the plasma membrane in NT2 cells and increased T(3) uptake in both cell types. In contrast, L471P MCT8 was largely retained in the endoplasmic reticulum and displayed no T(3) transport activity. Transient overexpression of WT and mutant MCT8 proteins failed to induce endoplasmic reticular stress or apoptosis. However, MCT8 overexpression significantly repressed cell proliferation in each cell type in both the presence and absence of the active thyroid hormone T(3) and in a dose-dependent manner. In contrast, L471P MCT8 showed no such influence. Finally, small interfering RNA depletion of endogenous MCT8 resulted in increased cell survival and decreased T(3) uptake. Given that T(3) stimulated proliferation in embryonic neuronal NT2 cells, whereas MCT8 repressed cell growth, these data suggest an entirely novel role for MCT8 in addition to T(3) transport, mediated through the modulation of cell proliferation in the developing brain.
Asunto(s)
Transportadores de Ácidos Monocarboxílicos/fisiología , Neuronas/citología , Neuronas/metabolismo , Transporte Biológico/genética , Transporte Biológico/fisiología , Western Blotting , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Línea Celular , Proliferación Celular , Supervivencia Celular/genética , Cristalinas/genética , Retículo Endoplásmico , Técnica del Anticuerpo Fluorescente , Vectores Genéticos , Humanos , Transportadores de Ácidos Monocarboxílicos/genética , Proteínas Musculares/genética , Proteínas Musculares/fisiología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/fisiología , Simportadores , Triyodotironina/metabolismo , Cristalinas muRESUMEN
It is now 10 years since PTTG was first cloned and isolated. Perhaps the major story of the intervening decade of work performed by numerous groups around the world is the sheer multifunctionality ascribed to this gene. PTTG has been implicated in mechanisms of gene transactivation, cell transformation, angiogenesis, metabolism, apoptosis, DNA repair, genetic instability and mitotic control, both in endocrine and non-endocrine settings. In the current review, we cast a critical eye over a decade of PTTG research within the field of endocrine neoplasia.
Asunto(s)
Transformación Celular Neoplásica/genética , Proteínas de Neoplasias/fisiología , Neoplasias Hipofisarias/genética , Animales , Humanos , Ratones , Proteínas de Neoplasias/genética , Ratas , SecurinaRESUMEN
The key mitotic regulator securin is expressed at low levels in fetal brain compared with adult, and modulates the proliferation of human embryonic neuronal N-Tera2 (NT2) cells. We now examine the function and expression of securin's interacting partner separase, along with Rad21, the functional component of cohesin, which is cleaved by separase following interaction with securin. In contrast to securin, the cleaved forms of separase and Rad21 were highly expressed in human fetal cerebral cortex compared with adult. In a murine model of absent securin expression - the PTTG knock-out mouse - separase and Rad21 were over-expressed in multiple brain regions. In addition, cDNA array analysis of other key mitotic regulators additionally identified cyclin C and sestrin 2 to be induced in the brains of securin-null mice compared with wild type. Further, Rad21 mRNA expression was highly correlated with that of securin, separase, cyclin C and sestrin 2 in fetal brains. In embryonic neuronal NT2 cells, siRNA repression of separase failed to significantly alter cell turnover, whereas repression of securin expression resulted in increased levels of the activated forms of Rad21 and separase, and promoted cell proliferation. Our data suggest that the co-ordinated expression of separase, securin and Rad21 is fundamental for the developing brain.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Endopeptidasas/metabolismo , Proteínas de Neoplasias/metabolismo , Neuronas/citología , Neuronas/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Animales , Secuencia de Bases , Encéfalo/citología , Encéfalo/embriología , Encéfalo/metabolismo , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular , Proliferación Celular , Ciclina C , Ciclinas/genética , Cartilla de ADN/genética , Proteínas de Unión al ADN , Endopeptidasas/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Ratones Noqueados , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Securina , SeparasaRESUMEN
The ability of the thyroid to accumulate iodide provides the basis for radioiodine ablation of differentiated thyroid cancers and their metastases. Most thyroid tumours exhibit reduced iodide uptake, although the mechanisms accounting for this remain poorly understood. Pituitary tumour transforming gene (PTTG) is a proto-oncogene implicated in the pathogenesis of thyroid tumours. We now show that PTTG and its binding factor PBF repress expression of sodium iodide symporter (NIS) messenger RNA (mRNA), and inhibit iodide uptake. This process is mediated at least in part through fibroblast growth factor-2. In detailed studies of the NIS promoter in rat FRTL-5 cells, PTTG and PBF demonstrated specific inhibition of promoter activity via the human upstream enhancer element (hNUE). Within this approximately 1 kb element, a complex PAX8-upstream stimulating factor 1 (USF1) response element proved critical both to basal promoter activity and to PTTG and PBF repression of NIS. In particular, repression by PTTG was contingent upon the USF1, but not the PAX8, site. Finally, in human primary thyroid cells, PTTG and PBF similarly repressed the NIS promoter via hNUE. Taken together, our data suggest that the reported overexpression of PTTG and PBF in differentiated thyroid cancer has profound implications for activity of the NIS gene, and hence significantly impacts upon the efficacy of radioiodine treatment.
Asunto(s)
Proteínas de la Membrana/fisiología , Proteínas de Neoplasias/fisiología , Proteínas Represoras/fisiología , Simportadores/antagonistas & inhibidores , Adulto , Anciano , Femenino , Factor 2 de Crecimiento de Fibroblastos/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Yoduros/metabolismo , Masculino , Persona de Mediana Edad , Regiones Promotoras Genéticas , Proto-Oncogenes Mas , ARN Mensajero/análisis , Securina , Simportadores/genética , Neoplasias de la Tiroides/genéticaRESUMEN
Genetic instability (GI) is a hallmark feature of tumor development. Securin, also known as pituitary tumor transforming gene (PTTG), is a mitotic checkpoint protein which is highly expressed in numerous cancers, is associated with tumor invasiveness, and induces GI in thyroid cells. We used fluorescence inter-simple sequence repeat PCR to assess GI caused primarily by DNA breakage events in 19 colorectal tumors. GI values ranged significantly, with Dukes' stage C&D colorectal tumors exhibiting greater GI and higher securin expression than Dukes' stage A&B tumors. Consistent with these findings, we observed a dose-dependent increase in GI in HCT116 cells in response to securin overexpression, as well as in non-transformed human fibroblasts. As securin has been implicated in a novel DNA repair pathway in fission yeast, we investigated its potential role in chemotoxic DNA damage response pathways in mammalian cells, using host cell reactivation assays. Securin overexpression in HCT116 cells inhibited etoposide-induced double-stranded DNA damage repair activity, and repressed Ku heterodimer function. Additionally, we observed that securin and Ku70 showed a reciprocal cytosol-nuclear translocation in response to etoposide-induced dsDNA damage. Our data suggest that, by repressing Ku70 activity and inhibiting the non-homologous end-joining dsDNA repair pathway, securin may be a critical gene in the development of GI in colorectal cancer.
Asunto(s)
Neoplasias Colorrectales/genética , Inestabilidad Genómica/fisiología , Proteínas de Neoplasias/fisiología , Animales , Antígenos Nucleares/fisiología , Línea Celular Tumoral , Reparación del ADN , Proteínas de Unión al ADN/fisiología , Fibroblastos/fisiología , Fase G1/fisiología , Humanos , Autoantígeno Ku , Mamíferos , SecurinaRESUMEN
CONTEXT: Vascular endothelial growth factor (VEGF) exerts its biological effects by binding to the tyrosine kinase receptors VEGF receptor type 1 (VEGFR1/Flt-1) and VEGFR2 (Flk-1/KDR). Kinase insert domain receptor (KDR) is the critical receptor controlling proliferation and migration of endothelial cells and has been shown to be expressed in some nonendothelial cells. We recently reported that the proangiogenic pituitary tumor transforming gene (PTTG) stimulates VEGF and up-regulates inhibitor of DNA binding-3 (ID3), an important gene in VEGF-dependent angiogenesis. OBJECTIVE: Our objective was to test whether VEGF, ID3, and KDR confer a PTTG-mediated effect on thyroid cell growth. DESIGN: Gene expression, MAPK stimulation, and cell proliferation were assessed in follicular thyroid cancer FTC133 cells. Gene expression and clinical associations were determined in 21 normal and 38 tumorous thyroid specimens (nine follicular and 29 papillary). RESULTS: ID3 correlated with VEGF mRNA expression in our series of thyroid cancers, which also showed up-regulated KDR mRNA. Stimulation of FTC133 cells with exogenous VEGF enhanced ID3 expression, which could be abrogated by the KDR-specific inhibitor ZM323881, suggesting that VEGF regulation of ID3 is KDR dependent. PTTG significantly correlated with KDR mRNA expression in our thyroid cancer cohort and up-regulated KDR and VEGF expression in FTC133 cells. Finally, cells transfected with PTTG demonstrated increased cell proliferation and phosphorylation of MAPK, which was abrogated by ZM323881. CONCLUSIONS: We report the presence of a VEGF/KDR/ID3-dependent autocrine pathway in FTC133 thyroid cells. By up-regulating both VEGF and KDR expression, we propose a novel PTTG-mediated proliferative pathway that may be critical to thyroid cancer growth and progression.
Asunto(s)
Comunicación Autocrina , Proliferación Celular , Proteínas Inhibidoras de la Diferenciación/fisiología , Proteínas de Neoplasias/fisiología , Glándula Tiroides/fisiología , Factor A de Crecimiento Endotelial Vascular/fisiología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/fisiología , Adenocarcinoma Folicular/metabolismo , Carcinoma Papilar/metabolismo , Regulación Neoplásica de la Expresión Génica , Sustancias de Crecimiento/metabolismo , Humanos , Proteínas Inhibidoras de la Diferenciación/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Neoplasias/metabolismo , Comunicación Paracrina , Fosforilación , Securina , Glándula Tiroides/metabolismo , Neoplasias de la Tiroides/metabolismo , Células Tumorales Cultivadas , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Thyroid hormones (THs) are essential for normal fetal development, with even mild perturbation in maternal thyroid status in early pregnancy being associated with neurodevelopmental delay in children. Transplacental transfer of maternal THs is critical, with increasing evidence suggesting a role for 3,3',5-tri-iodothyronine (T3) in development and function of the placenta itself, as well as in development of the central nervous and other organ systems. Intrauterine growth restriction (IUGR) is associated with fetal hypothyroxinaemia, a factor that may contribute to neurodevelopmental delay. The recent description of monocarboxylate transporter 8 (MCT8) as a powerful and specific TH membrane transporter, and the association of MCT8 mutations with profound neurodevelopmental delay, led us to explore MCT8 expression in placenta. We describe the expression of MCT8 in normal human placenta throughout gestation, and in normal third-trimester placenta compared with that associated with IUGR using quantitative reverse transcriptase PCR. MCT8 mRNA was detected in placenta from early first trimester, with a significant increase with advancing gestation (P=0.007). In the early third trimester, MCT8 mRNA was increased in IUGR placenta compared with normal samples matched for gestational age (P<0.05), but there was no difference between IUGR and normal placenta in the late third trimester. Western immunoblotting findings in IUGR and normal placentae were in accord with mRNA data. MCT8 immunostaining was demonstrated in villous cytotrophoblast and syncytiotrophoblast as well as extravillous trophoblast cells from the first trimester onwards with increasingly widespread immunoreactivity seen with advancing gestation. In conclusion, expression of MCT8 in placenta from early gestation is compatible with an important role in TH transport during fetal development and a specific role in placental development. Altered expression in placenta associated with IUGR may reflect a compensatory mechanism attempting to increase T3 uptake by trophoblast cells.
Asunto(s)
Retardo del Crecimiento Fetal/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Placenta/metabolismo , Adulto , Análisis de Varianza , Western Blotting/métodos , Estudios de Casos y Controles , Membranas Extraembrionarias/química , Membranas Extraembrionarias/metabolismo , Femenino , Edad Gestacional , Humanos , Inmunohistoquímica/métodos , Transportadores de Ácidos Monocarboxílicos/análisis , Transportadores de Ácidos Monocarboxílicos/genética , Placenta/química , Embarazo , Tercer Trimestre del Embarazo , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Simportadores , Hormonas Tiroideas/metabolismo , Trofoblastos/química , Trofoblastos/metabolismoRESUMEN
CONTEXT: Pituitary tumor-transforming gene (PTTG) is a multifunctional protein involved in several tumorigenic mechanisms, including angiogenesis. PTTG has been shown to promote angiogenesis, a key rate-limiting step in tumor progression, by up-regulation of fibroblast growth factor-2 and vascular endothelial growth factor. OBJECTIVE: To investigate whether PTTG regulates other angiogenic genes in thyroid cells, we performed angiogenesis-specific cDNA arrays after PTTG transfection. Two of the genes [inhibitor of DNA binding-3 (ID3) and thrombospondin-1 (TSP-1)] which showed differential expression in primary thyroid cells were validated in vitro and in vivo. RESULTS: TSP-1 showed a 2.5-fold reduction and ID3 showed a 3.5-fold induction in expression in response to PTTG overexpression in vitro. Conversely, suppression of PTTG with small interfering RNA was associated with a 2-fold induction of TSP-1 and a 2.2-fold reduction in ID3 expression. When we examined TSP-1 and ID3 expression in 34 differentiated thyroid cancers, ID3 was significantly increased in tumors compared with normal thyroid tissue. Furthermore, ID3 expression was significantly higher in follicular thyroid tumors than in papillary tumors. Although mean TSP-1 expression was not altered in cancers compared with normal thyroids, we observed a significant independent association between TSP-1 expression and early tumor recurrence, with recurrent tumors demonstrating 4.2-fold lower TSP-1 expression than normal thyroid tissues. CONCLUSION: We have identified ID3 and TSP-1 as two new downstream targets of PTTG in thyroid cancer. We propose that PTTG may promote angiogenesis by regulating the expression of multiple genes with both pro- and antiangiogenic properties and may thus be a key gene in triggering the angiogenic switch in thyroid tumorigenesis.