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3.
Horm Metab Res ; 34(6): 303-10, 2002 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-12173070

RESUMEN

APE/Ref-1 is a multifunctional protein possessing both redox and DNA repair functions. Through its redox activity, APE/Ref-1 controls the DNA-binding function of several transcriptional regulators (AP1, NF-kappaB, p53, Pax proteins). We have previously shown that APE/Ref-1 upregulates the transcriptional activity of the thyroid-specific transcription factor Pax8. In thyroid cells, APE/Ref-1 can be detected both in the nuclear and cytoplasmatic compartments. In this study regulatory mechanisms acting on APE/Ref-1 were revealed using the FRTL-5 cell line. TSH induces both cytoplasm-to-nucleus translocation and neosynthesis of APE/Ref-1 protein. Interestingly, only neosynthesis is dependent on cAMP signalling. In contrast, the cytoplasm-to-nucleus translocation is dependent on redox-mediated mechanisms. Based upon the data shown in this study and in others, a bimodal control of APE/Ref-1 by TSH can be delineated.


Asunto(s)
Liasas de Carbono-Oxígeno/análisis , Liasas de Carbono-Oxígeno/metabolismo , AMP Cíclico/farmacología , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Glándula Tiroides/metabolismo , Tirotropina/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Calcio/metabolismo , Liasas de Carbono-Oxígeno/biosíntesis , Línea Celular , Núcleo Celular/química , Núcleo Celular/metabolismo , AMP Cíclico/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Oxidación-Reducción , Ratas , Transducción de Señal , Glándula Tiroides/química , Glándula Tiroides/ultraestructura
4.
J Biol Chem ; 276(51): 47807-13, 2001 Dec 21.
Artículo en Inglés | MEDLINE | ID: mdl-11584016

RESUMEN

Thyroid transcription factor-1 (TTF-1, also known as NKX2.1 and T/EBP), a transcription factor belonging to the NKX-2 family of homeodomain-containing genes, plays an essential role in the organogenesis of the thyroid gland, lung, and ventral forebrain. Nestin is an intermediate filament protein strongly expressed in multipotential neuroepithelial stem cells and rapidly down-regulated during postnatal life. Here we show that stable fibroblastic clones expressing TTF-1 acquire a phenotype reminiscent of neuroepithelial cells in culture and up-regulate the endogenous nestin gene. TTF-1 transactivates in HeLa and NIH3T3 cells a reporter gene driven by a central nervous system-specific enhancer element from the second intron of the rat nestin gene, where it recognizes a DNA-binding site (NestBS) whose sequence resembles a nuclear hormone/cAMP-responsive element very different from canonical TTF-1 binding sites. Nuclear extracts from the head of mouse embryos form a retarded complex with NestBS of the same mobility of the extracts obtained from TTF1-expressing clones, which is either abolished or supershifted in the presence of two different antibodies recognizing the TTF-1 protein. Thus, the neuroepithelial marker nestin is a direct central nervous system-specific target gene of TTF-1, leading to the hypothesis that it might be the effector through which TTF-1 plays its role in the organogenesis of the forebrain.


Asunto(s)
Proteínas de Homeodominio/metabolismo , Proteínas de Filamentos Intermediarios/genética , Proteínas del Tejido Nervioso , Proteínas Nucleares/metabolismo , Prosencéfalo/embriología , Factores de Transcripción/metabolismo , Células 3T3 , Animales , Secuencia de Bases , Células COS , Cartilla de ADN , Elementos de Facilitación Genéticos , Epitelio/metabolismo , Ratones , Morfogénesis , Nestina , Proteínas Nucleares/genética , Unión Proteica , Ratas , Factor Nuclear Tiroideo 1 , Factores de Transcripción/genética , Regulación hacia Arriba
5.
J Biol Chem ; 276(50): 47320-8, 2001 Dec 14.
Artículo en Inglés | MEDLINE | ID: mdl-11584024

RESUMEN

Recognition of virally infected cells by CD8+ T cells requires differentiation between self and nonself peptide-class I major histocompatibility complexes (pMHC). Recognition of foreign pMHC by host T cells is a major factor in the rejection of transplanted organs from the same species (allotransplant) or different species (xenotransplant). AHIII12.2 is a murine T cell clone that recognizes the xenogeneic (human) class I MHC HLA-A2.1 molecule (A2) and the syngeneic murine class I MHC H-2 D(b) molecule (D(b)). Recognition of both A2 and D(b) are peptide-dependent, and the sequences of the peptides recognized have been determined. Alterations in the antigenic peptides bound to A2 cause large changes in AHIII12.2 T cell responsiveness. Crystal structures of three representative peptides (agonist, null, and antagonist) bound to A2 partially explain the changes in AHIII12.2 responsiveness. Using class I pMHC octamers, a strong correlation is seen between T cell activity and the affinity of pMHC complexes for the T cell receptor. However, contrary to previous studies, we see similar half-lives for the pMHC multimers bound to the AHIII12.2 cell surface.


Asunto(s)
Membrana Celular/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Linfocitos T/metabolismo , Secuencia de Aminoácidos , Animales , Biotinilación , Cristalografía por Rayos X , Dimerización , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Antígeno HLA-A2/metabolismo , Humanos , Cinética , Ligandos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Fenotipo , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Linfocitos T Citotóxicos/metabolismo , Factores de Tiempo
6.
Biochimie ; 82(12): 1107-14, 2000 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-11120352

RESUMEN

Vascular endothelial cells play an important role in coagulation regulation of vascular tone and in a variety of synthetic and metabolic functions. Endothelial cells also have a pivotal role in immunological diseases atherogenesis and tumor angiogenesis. Endothelial cells are often used as system to study the pathophysiology of late complications in diabetes mellitus atherosclerotic damages and leukocyte adhesion in inflammatory diseases. Most of the studies have been performed on primary arterial and venous endothelial cell cultures with problems such as availability of autoptic material and reproducibility of cell cultures. We have isolated and characterized a novel system of proliferating long-term cultures of human aortic endothelial cells that maintain their differentiated characteristics for many generations in vitro. They produce antithrombotic and thrombotic factors such as t-PA and PAI-1 and respond to TNFalpha, an important factor correlated with the inflammatory process by modifying growth characteristics by producing cytokines such as GM-CSF by expressing ICAM-1 on the surface and by producing large amounts of nitric oxide and endothelin. This new system may be very useful to understand and study the molecular mechanisms involved in many vascular alteration pathologies and in the aging process.


Asunto(s)
Aorta/citología , Endotelio Vascular/citología , División Celular/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Óxido Nítrico/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Receptores de LDL/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Factor de von Willebrand/metabolismo
7.
Endocrinology ; 141(3): 901-9, 2000 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-10698164

RESUMEN

Alteration of the redox potential has been proposed as a mechanism influencing gene expression. Reduced glutathione (GSH) is one of the cellular scavengers involved in the regulation of the redox potential. To test the role that GSH may play in thyroid cells, we cultured a differentiated rat thyroid cell strain (FRTL-5) in the presence of L-buthionine-(S,R)-sulfoximine (BSO). BSO affects GSH synthesis by irreversibly inhibiting gamma-glutamylcysteine synthetase (EC 6.3.2.2), a specific enzyme involved in GSH synthesis. BSO-treated FRTL-5 cells show a great decrease in the GSH level, whereas malondialdehyde increases in the cell culture medium as a sign of lipid peroxidation. In these conditions the activity of two thyroid-specific promoters, thyroglobulin (Tg) and thyroperoxidase (TPO), is strongly reduced in transient transfection experiments. As both Tg and TPO promoters depend upon the thyroid-specific transcription factors, thyroid-specific transcription factor-1 (TTF-1) and Pax-8 for full transcriptional activity, we tested whether reduction of GSH concentration impairs the activity of these transcription factors. After BSO treatment of FRTL-5 cells, both transcription factors fail to trans-activate the respective chimerical targets, C5 and B-cell specific activating protein promoters, containing, respectively, multimerized TTF-1- or Pax-8-binding sites only as well as the Tg and TPO natural promoters. Northern analysis revealed that endogenous Tg messenger RNA (mRNA) expression is also reduced by BSO treatment, whereas endogenous TPO expression is not modified. Furthermore, the Pax-8 mRNA steady state concentration does not change in BSO-treated cells, whereas TTF-1 mRNA slightly decreases. Immunoblotting analysis of FRTL-5 nuclear extracts does not show significant modification of the Pax-8 concentration in BSO-treated cells, whereas a decrease of 25% in TTF-1 protein is revealed. Furthermore, BSO treatment decreases the DNA-binding activity to the respective consensus sequence of both transcription factors. Finally, different mechanisms seem to act on TTF-1 and Pax-8 functional impairment in BSO-treated cells. Indeed, with a lowered GSH concentration, the overexpressed Pax-8 still activates transcription efficiently, whereas, on the contrary, the overexpressed TTF-1 does not recover its transactivation capability when the respective chimerical target sequences are used (C5 and BSAP). When the natural Tg and TPO promoter sequences are used, overexpression of Pax-8 parallels the effect on both promoters observed using the chimeric target sequences, whereas overexpression of TTF-1 increases TPO promoter transcriptional activity only.


Asunto(s)
Expresión Génica/genética , Glutatión/metabolismo , Glándula Tiroides/metabolismo , Animales , Antimetabolitos/farmacología , Northern Blotting , Butionina Sulfoximina/farmacología , Diferenciación Celular/fisiología , Línea Celular , Sondas de ADN/genética , Proteínas de Unión al ADN/genética , Densitometría , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Semivida , Peroxidación de Lípido/genética , Proteínas Nucleares/genética , Factor de Transcripción PAX8 , Factores de Transcripción Paired Box , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Ratas , Factor Nuclear Tiroideo 1 , Transactivadores/genética , Factores de Transcripción/genética , Activación Transcripcional/genética , Transfección/genética
8.
Biochem Biophys Res Commun ; 219(2): 412-7, 1996 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-8605001

RESUMEN

It has been reported that glucose may autooxidize generating free radicals which have been hypothesized to induce important cellular abnormalities. To investigate the cell damage induced by glucose-dependent oxidative stress, the FRTL5 cell strain was incubated in 10 or 20 mM glucose, either alone or in the presence of buthionine-sulfoximine, a transition state inhibitor that blocks glutathione synthesis. We found indeed that buthionine-sulfoximine greatly inhibited glutathione production and increased malondialdehyde (a marker of oxidative cell damage) levels, especially in 20mM glucose. We also found that, when glutathione production was inhibited, 10mM glucose induced apoptosis and 20 mM glucose induced necrosis. These data show that the glucose-dependent cell damage is a function of glutathione production. They also show that such glucose-dependent free radical production may be critical for determining cell damage, even for small variations as the ones we tested (from 10 to 20 mM glucose).


Asunto(s)
Muerte Celular/efectos de los fármacos , Glucosa/farmacología , Glutatión/metabolismo , Malondialdehído/metabolismo , Animales , Butionina Sulfoximina , Muerte Celular/fisiología , Línea Celular , ADN/efectos de los fármacos , Daño del ADN , Citometría de Flujo , Radicales Libres/metabolismo , Metionina Sulfoximina/análogos & derivados , Metionina Sulfoximina/farmacología , Protectores contra Radiación/farmacología , Ratas , Glándula Tiroides
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