RESUMEN
Pig thyrocytes, either in the intact gland or cultured under conditions leading to thyroid follicle reconstitution, coexpress two gap junction proteins, connexin-32 (Cx32) and connexin-43 (Cx43). As thyrocytes cultured in the form of a monolayer only express Cx43, we hypothesized that Cx32 could play a role in thyroid folliculogenesis. In the present work, we analyzed the ability of polarized FRT cells (that are gap junction deficient) to form follicle-like structures after stable transfection with either Cx32 or Cx43 genes. Wild-type and transfected FRT cells, while growing, showed the capacity to form three-dimensional structures corresponding to domes that result from the accumulation of fluid underneath limited areas of the cell layer. The number of domes formed by FRT cells expressing Cx32 (FRT-Cx32) was 2- to 3-fold higher than that obtained with either wild-type or Cx43-transfected FRT cells (FRT-Cx43). Domes generated by FRT-Cx32 cells were stable (beyond 3 weeks of culture), whereas those formed from wild-type or FRT-Cx43 cells were transient, disappearing when cells reached confluence. Inspection of the cell organization within domes formed from FRT-Cx32 cells by phase contrast and confocal microscopy revealed a progressive transition from domes toward closed structures with a lumen. The tightness of the lumen was demonstrated by the retention of a fluorescent probe, lucifer yellow, introduced by microinjection. Electron microscope examinations showed that the neoformed follicle-like structures had an inside-out polarity. Analyses of cell motion and division with time, by fluorescence video microscopy, indicated that the transformation of domes into inside-out follicles brings into play the migration of cells and, to a lesser extent, cell multiplication underneath the domes. In conclusion, FRT cells forced to express Cx32 give rise to domes that transform into closed inside-out follicles. This gain of function appears Cx specific, as FRT-Cx43 cells did not form similar structures. Our data suggest that the formation and/or functioning of Cx32 gap junctions might represent a key event in thyroid epithelium morphogenesis, i.e. formation of a lumen from a tight epithelial cell layer.
Asunto(s)
Comunicación Celular/fisiología , Polaridad Celular/fisiología , Conexinas/fisiología , Glándula Tiroides/fisiología , Animales , Línea Celular/fisiología , Conexina 43/genética , Conexina 43/fisiología , Conexinas/genética , ADN Complementario/genética , Expresión Génica , Ratas , Ratas Endogámicas F344 , Glándula Tiroides/citología , Transfección , Proteína beta1 de Unión ComunicanteRESUMEN
Normal thyroid epithelial cells coexpress connexin-32 and connexin-43, which form distinct gap junctions. In primary culture, connexin-43 is expressed by thyrocytes in monolayers or reorganized into follicles, whereas the expression of connexin-32 is dependent upon the reconstitution of follicles. To study the functional impact of connexin-32 gap junctions in thyroid cells, we transfected connexin-32 cDNA in two thyroid-derived communication-deficient cell lines, FRT and FRTL-5. The selected clones, which stably expressed connexin-32 at high levels and exhibited high gap junction-mediated dye-coupling, presented a reduced proliferation rate as compared with that of the corresponding wild-type FRT and FRTL-5 cells; the mean population doubling time was increased by approximately 35%. The proliferation of connexin-32-transfected FRTL-5 cells remained thyrotropin-dependent; the range of thyrotropin concentrations that stimulated growth was the same in transfected and control cells. The expression of connexin-32 led to an increase of thyroglobulin gene expression in FRTL-5 cells. The expression of two other tissue-specific proteins, thyroid transcription factor-1 and Pax-8, was unchanged. These findings provide evidence that connexin-32 gap junction-mediated cell-to-cell communication participates in the control of growth and differentiation of thyroid cells.
Asunto(s)
Comunicación Celular , Conexinas/genética , Regulación de la Expresión Génica , Glándula Tiroides/metabolismo , Animales , División Celular/genética , Línea Celular , Ratas , Tiroglobulina/genética , Glándula Tiroides/citología , Regulación hacia Arriba , Proteína beta1 de Unión ComunicanteRESUMEN
We have previously shown that the signal peptideless cytokine interleukin 1 alpha (IL-1 alpha) may play a role as an intracellular regulator of human endothelial cell senescence (J. A. M. Maier, P. Voulalas, D. Roeder, and T. Maciag, Science 249:1570-1574, 1990). To investigate the potential intracellular function of IL-1 alpha, transformed endothelial cells were transfected with the human cDNAs that code for the two forms of IL-1 alpha, the precursor molecule IL-1(1-271) and the mature protein IL-1(113-271). The subcellular localization of the two different polypeptides was investigated directly or by using chimeric genes constructed by fusion of different fragments of the IL-1 alpha gene and the beta-galactosidase open reading frames. The IL-1(113-271) protein was cytoplasmic, while IL-1(1-271) was nuclear. The basic cluster at the NH2 terminus of IL-1, KVLKKRR, has been shown to mediate IL-1 alpha nuclear targeting. Moreover, nuclear localization of IL-1 alpha correlates with impaired cell growth and expression of some IL-1 alpha-inducible genes. These results suggest that transport of endogenous IL-1(1-271) into the nucleus is required for it to modulate endothelial cell function.
Asunto(s)
Endotelio Vascular/fisiología , Interleucina-1/metabolismo , Secuencia de Bases , Transporte Biológico , Compartimento Celular , División Celular , Núcleo Celular/metabolismo , Células Cultivadas , Colagenasas/genética , Cartilla de ADN/química , Técnica del Anticuerpo Fluorescente , Regulación Enzimológica de la Expresión Génica , Humanos , Técnicas In Vitro , Interleucina-1/fisiología , Datos de Secuencia Molecular , Inhibidor 1 de Activador Plasminogénico/genética , ARN Mensajero/genética , Proteínas Recombinantes , TransfecciónRESUMEN
Progressive pathophysiologic modifications of endothelial cells are associated with aging. In vitro, endothelial cell senescence is accompanied by the failure to proliferate as well as by perturbations in gene expression. Here we show that (i) senescence enhances monoblastoid U937 cell adhesion to the endothelial monolayer; (ii) the enhanced interaction between senescent endothelial cells and U937 cells is mediated, at least in part, by the overexpression of ICAM-1; and (iii) LPS and interleukin 1 alpha, but not tumor necrosis factor alpha, are unable to stimulate the adhesion of U937 to senescent endothelial cells. Since monocyte adhesion to the endothelium is an early event in atherosclerosis, the altered adhesive properties observed in senescent cells could give insights into the formation of atherosclerotic lesions.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Adhesión Celular , Senescencia Celular , Endotelio Vascular/citología , Monocitos/citología , Arteriosclerosis/patología , Secuencia de Bases , Línea Celular , Humanos , Técnicas In Vitro , Interleucina-1/farmacología , Lipopolisacáridos/farmacología , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Biopsies of human normal adrenal medulla, adrenal pheochromocytoma, and chemodectoma were studied for the presence of basic fibroblast growth factor (bFGF). An immunoreactive M(r) 18,000 bFGF-like molecule was detected both in normal and neoplastic tissues. This molecule was identified as bFGF on the basis of its molecular weight, its affinity for heparin, and its capacity to induce plasminogen activator production in cultured endothelial GM 7373 cells. The levels of immunoreactive and biologically active bFGF appeared to be significantly higher in the extracts of adrenal pheochromocytoma and chemodectoma than in the extracts of normal adrenal medulla. bFGF immunostaining was detectable in the nuclei of chief (Type-I) cells and of endothelial cells both in normal adrenal medulla and in pheochromocytoma. Cytoplasmic bFGF positivity of endothelial cells was also observed in pheochromocytoma but not in normal tissue. The data are in keeping with the hypothesis that bFGF may exert autocrine and paracrine functions in the growth and neovascularization of human pheochromocytoma.
Asunto(s)
Neoplasias de las Glándulas Suprarrenales/química , Médula Suprarrenal/química , Factor 2 de Crecimiento de Fibroblastos/análisis , Paraganglioma Extraadrenal/química , Feocromocitoma/química , Adulto , Anciano , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana EdadRESUMEN
Basic residues Arg-118, Lys-119, Lys-128, and Arg-129 within a putative heparin-binding and receptor-binding region of the 155-amino acid form of basic fibroblast growth factor (bFGF) have been changed to neutral glutamine residues by site-directed mutagenesis of the human bFGF cDNA. The bFGF mutant (M6B-bFGF) was expressed in E. coli and purified to homogeneity. When compared to wild type bFGF, M6B-bFGF showed in cultured endothelial cells a similar receptor-binding capacity and mitogenic activity, but a reduced affinity for heparin-like low affinity binding sites, a reduced chemotactic activity, and a reduced capacity to induce the production of urokinase-type plasminogen activator. In vivo, M6B-bFGF lacked a significant angiogenic activity. Modifications of both the primary and the tertiary structure of bFGF appear to be responsible for the modified biological properties of M6B-bFGF, thus confirming the possibility to dissociate at the structural level some of the biological activities exerted by bFGF on endothelial cells.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Heparina/metabolismo , Mutagénesis Sitio-Dirigida , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Bovinos , División Celular/efectos de los fármacos , Línea Celular Transformada , Membrana Celular/metabolismo , Quimiotaxis/efectos de los fármacos , Clonación Molecular , Endotelio Vascular , Inducción Enzimática , Factor 2 de Crecimiento de Fibroblastos/genética , Humanos , Cinética , Datos de Secuencia Molecular , Neovascularización Patológica , Oligodesoxirribonucleótidos , Plásmidos , Activadores Plasminogénicos/biosíntesis , Receptores de Factores de Crecimiento de FibroblastosAsunto(s)
Adhesión Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Endopeptidasas/metabolismo , Endotelio Vascular/fisiología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Secuencia de Aminoácidos , Animales , Bovinos , Línea Celular Transformada , Relación Dosis-Respuesta a Droga , Endopeptidasas/biosíntesis , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Feto , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Cinética , Datos de Secuencia Molecular , Unión Proteica , Secuencias Repetitivas de Ácidos NucleicosAsunto(s)
Factor 2 de Crecimiento de Fibroblastos/genética , Secuencia de Aminoácidos , Unión Competitiva , División Celular/efectos de los fármacos , Línea Celular , Deleción Cromosómica , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Humanos , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Receptores de Superficie Celular/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
A recombinant deletion mutant of the 155-amino acid form of human basic fibroblast growth factor (bFGF), lacking amino acid residues 27-32 (Lys-Asp-Pro-Lys-Arg-Leu), was expressed in Escherichia coli and purified to homogeneity by heparin-Sepharose affinity chromatography. When maintained in the presence of an equimolar concentration of soluble heparin, the bFGF mutant (M1-bFGF) is as potent as bFGF in stimulating cell proliferation in normal and transformed fetal bovine aortic endothelial cells, in adult bovine aortic endothelial cells, and in NIH 3T3 fibroblasts. However, under the same experimental conditions, M1-bFGF is at least 100 times less efficient than bFGF in stimulating plasminogen activator (PA) production in endothelial cells, as assayed by chromogenic PA assay, SDS/PAGE zymography, and Northern blot analysis of urokinase-type PA mRNA. In the presence of heparin, M1-bFGF binds to bFGF plasma membrane receptors present on endothelial cells in a manner undistinguishable from bFGF. It also induces the same tyrosine phosphorylation pattern when added to NIH 3T3 cells. The data suggest that the PA-inducing activity of bFGF may depend upon a functional domain that differs from those involved in the mitogenic activity of the growth factor and that the binding of bFGF to its plasma membrane receptor may not be sufficient to induce urokinase-type PA production in endothelial cells.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/genética , Mitógenos , Activadores Plasminogénicos , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , División Celular/efectos de los fármacos , Línea Celular , Deleción Cromosómica , Replicación del ADN/efectos de los fármacos , Regulación hacia Abajo , Factor 2 de Crecimiento de Fibroblastos/aislamiento & purificación , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Genes Sintéticos , Humanos , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Sondas de Oligonucleótidos , Plásmidos , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Superficie Celular/metabolismo , Receptores de Factores de Crecimiento de FibroblastosAsunto(s)
Factor 2 de Crecimiento de Fibroblastos/fisiología , Secuencia de Aminoácidos , División Celular/efectos de los fármacos , Endotelio/fisiología , Factor 2 de Crecimiento de Fibroblastos/química , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Oligopéptidos , Péptidos/química , Péptidos/farmacología , Receptores de Superficie Celular/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos , Transducción de Señal , Relación Estructura-ActividadAsunto(s)
Endotelio/metabolismo , Factor 2 de Crecimiento de Fibroblastos/química , Activadores Plasminogénicos/biosíntesis , Células 3T3 , Secuencia de Aminoácidos , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Quimiotaxis , Factor 2 de Crecimiento de Fibroblastos/farmacología , Expresión Génica , Genes fos/genética , Técnicas In Vitro , Ratones , Datos de Secuencia Molecular , Receptores de Superficie Celular/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos , Proteínas Recombinantes/farmacología , Transducción de Señal , Relación Estructura-Actividad , Transcripción GenéticaRESUMEN
A heparin-binding Mr 25,000 immunoreactive bFGF-like protein (ir-bFGF) is recognized in adult rat liver extract by affinity-purified polyclonal anti-human placental bFGF antibodies. Hepatic levels of this protein increase 4-fold in regenerating rat liver during the first 48 h after partial hepatectomy. Also, they appear to be higher in embryonic than in newborn or in adult rat liver. Mr 25,000 ir-bFGF from regenerating rat liver, partially purified by heparin-affinity chromatography, induces plasminogen activator activity and cell proliferation in transformed fetal bovine aortic endothelial GM 7373 cells and competes with Mr 18,000 [125I]bFGF for the binding to high affinity bFGF receptors. The data indicate the presence in rat liver of a high molecular weight form of bFGF whose expression is modulated during embryonic development and liver regeneration.