RESUMEN
BACKGROUND: Canine models have proved to be predictive of clinical findings in human bone marrow (BM) transplantation; consequently, the utilization of dogs is an excellent tool for supporting therapeutic purposes. Considering the role of growth factors in homing and mobilization of hematopoietic progenitors, the aim of this work was to evaluate whether canine stem cell factor (cSCF) contributes to matrix metalloproteinase (MMP)-9 secretion by CD34 cells. METHODS: The study was carried out in a cell population selected by immunomagnetic techniques using the anti-canine CD34 monoclonal antibody (MAb) 3B4 produced by us. Secretion of MMP-9 was evaluated by zymography. RESULTS: Analyzes of canine CD34(+) cells guaranteed that the MAb 3B4 was optimum for selecting a subset population with defined characteristics of primitive hematopoietic cells. The isolated cells were able to proliferate onto irradiated pre-established stroma, giving rise to mature neutrophils. There was also a 20-fold enrichment in the long-term culture-initiating cell content when the isolated population was added to irradiated cultures, with respect to the starting mononuclear cell population. DISCUSSION: We have provided the first evidence that canine BM CD34(+) cells constitutively express MMP-9 and the role of cSCF in up-regulating the secretion of this enzyme. The fact that cSCF augments expression of MMP-9 together with the ability of the isolated CD34(+)cells to proliferate onto irradiated pre-established stroma enables further investigations to determine whether the secretion of MMP-9 mediated by cSCF is one of the factors that enhance migration, homing and repopulation of primitive hemopoietic cells.
Asunto(s)
Antígenos CD34/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Factor de Células Madre/metabolismo , Animales , Anticuerpos Monoclonales , Western Blotting , Línea Celular , Proliferación Celular , Ensayo de Unidades Formadoras de Colonias , Perros , Femenino , Citometría de Flujo , Células Madre/citología , Células Madre/metabolismoRESUMEN
This in vitro study has been conducted to determine the optimal experimental conditions under which to produce canine neutrophils in long-term bone marrow cultures (LTBMC), establish functional parameters of neutrophils obtained from LTBMC and peripheral blood and to ascertain whether these cells display physiological similarities. Our aim is to provide an experimental model, enabling a correlation between hemopoietic injury and neutrophil functionality. The authors demonstrate for the first time that canine neutrophils grown in cultures are able to produce oxyradicals capable of killing bacterial products. Moreover, culture-grown neutrophils contain gelatinase granules, a marker of terminal neutrophil differentiation, and express a specific surface antigen. The results described in this article illustrate the development of a dynamic system that mimics physiological hemopoiesis.
Asunto(s)
Células de la Médula Ósea/citología , Neutrófilos/citología , Neutrófilos/fisiología , Adenosina Trifosfato/análisis , Animales , Células Sanguíneas/citología , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Perros , Femenino , Gelatinasas/metabolismo , Hematopoyesis , Modelos Biológicos , Neutrófilos/inmunología , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/metabolismo , Factores de TiempoRESUMEN
The analysis of the expression of oxidative phosphorylation genes in the liver during development reveals the existence of two biological programs involved in the biogenesis of mitochondria. Differentiation is a short-term program of biogenesis that is controlled at post-transcriptional levels of gene expression and is responsible for the rapid changes in the bioenergetic phenotype of mitochondria. In contrast, proliferation is a long-term program controlled both at the transcriptional and post-transcriptional levels of gene expression and is responsible for the increase in mitochondrial mass in the hepatocyte. Recently, a specific subcellular structure involved in the localization and control of the translation of the mRNA encoding the beta-catalytic subunit of the H(+)-ATP synthase (beta-mRNA) has been identified. It is suggested that this structure plays a prominent role in the control of mitochondrial biogenesis at post-transcriptional levels. The fetal liver has many phenotypic manifestations in common with highly glycolytic tumor cells. In addition, both have a low mitochondrial content despite a paradoxical increase in the cellular representation of oxidative phosphorylation transcripts. Based on the paradigm provided by the fetal liver we hypothesize that the aberrant mitochondrial phenotype of fast-growing hepatomas represents a reversion to a fetal program of expression of oxidative phosphorylation genes by the activation, or increased expression, of an inhibitor of beta-mRNA translation.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Transformación Celular Neoplásica , Hígado/metabolismo , Mitocondrias/metabolismo , Animales , Carcinoma Hepatocelular/genética , Metabolismo Energético , Regulación de la Expresión Génica , Hígado/crecimiento & desarrollo , Fosforilación Oxidativa , ATPasas de Translocación de Protón/genéticaRESUMEN
Regulation of the expression of the nuclear-encoded beta-subunit of H(+)-ATP synthase (beta-F1-ATPase) gene of oxidative phosphorylation during differentiation of liver mitochondria is mainly exerted at two post-transcriptional levels affecting both the half-life [Izquierdo, Ricart, Ostronoff, Egea and Cuezva (1995) J. Biol. Chem. 270, 10342-10350] and translational efficiency [Luis, Izquierdo, Ostronoff, Salinas, Santarén and Cuezva (1993) J. Biol. Chem. 268, 1868-1875] of the transcript. Herein, we have studied the expression of the mitochondrial (mt) genome during differentiation of rat liver mitochondria in an effort to elucidate the mechanisms of nucleo-mitochondrial cross-talk during biogenesis of the organelle. Estimation of the relative cellular representation of met-DNA in liver reveals a negligible increase in mt-DNA copy number during organelle differentiation. Concurrently, the lack of changes in transcription rates of the mt-DNA "in organello', as well as in steady-state levels of the mt-transcripts, suggests that organelle differentiation is not controlled by an increase in transcription of the mt-genome. However, translation rates in isolated mitochondria revealed a transient 2-fold increase immediately after birth. Interestingly, the transient activation of mitochondrial translation at this stage of liver development is dependent on the synthesis of proteins in cytoplasmic polyribosomes. These findings support the hypothesis that the expression of nuclear and mitochondrial genes during biogenesis of mammalian mitochondria is developmentally regulated by a post-transcriptional mechanism that involves concerted translational control of both genomes.
Asunto(s)
Envejecimiento/metabolismo , ADN Mitocondrial/metabolismo , Regulación Enzimológica de la Expresión Génica , Mitocondrias Hepáticas/metabolismo , Biosíntesis de Proteínas , Animales , Animales Recién Nacidos , Femenino , Feto , Regulación del Desarrollo de la Expresión Génica , Hígado/embriología , Hígado/crecimiento & desarrollo , Mamíferos , Embarazo , ATPasas de Translocación de Protón/biosíntesis , ARN/biosíntesis , ARN Mitocondrial , Ratas , Ratas Wistar , Transcripción GenéticaRESUMEN
We have recently reported that regulation of the expression of the nuclear-encoded beta-F1-ATPase gene during development of rat liver is exerted also by the control of beta-F1-ATPase mRNA decay (Izquierdo, J.M., Ricart, J., Ostronoff, L.K., Egea, G. and Cuezva, J.M. (1995) J. Biol. Chem. 270, 10342-10350). In this paper, we report that high steady-state levels of the mitochondrial encoded mRNAs for subunits of the ATP synthase (ATP 6-8) in developing liver result from profound changes in the stability of the mitochondrial transcripts. The results strongly suggest that developmental regulation of nuclear and mitochondrial genes during biogenesis of mammalian mitochondria is concertedly controlled by a posttranscriptional mechanism that involves the regulation of mRNA degradation of both genomes.
Asunto(s)
Hígado/crecimiento & desarrollo , Mitocondrias Hepáticas/metabolismo , ARN/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Núcleo Celular/fisiología , Dactinomicina/farmacología , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , ATPasas de Translocación de Protón/genética , ARN Mensajero/genética , ARN Mitocondrial , Ratas , Ratas WistarRESUMEN
To elucidate the mechanisms that regulate the expression of nuclear genes during biogenesis of mammalian mitochondria, the expression pattern of the beta-subunit of the ATP synthase gene has been characterized in rat liver between day 20 in utero and 12 weeks postnatal. The parallelism existing between transcriptional activity of the gene and the amount of beta-F1-ATPase protein in liver indicates that proliferation of mitochondria is controlled at the transcriptional level. On the other hand, an increased stability (4-5-fold) of beta-F1-ATPase mRNA during early neonatal life as well as a rapid postnatal activation of translation rates affecting mitochondrial proteins appear to control mitochondrial differentiation. Immunoelectron microscopy of the F1-ATPase complex during liver development revealed that the rapid postnatal increase in the in vivo rate of F1-ATPase synthesis was mostly used for functional differentiation of pre-existing organelles (Valcarce, C., Navarrete, R. M., Encabo, P., Loeches, E., Satrústegui, J., and Cuezva, J. M. (1988) J. Biol Chem. 263, 7767-7775). The findings support that beta-F1-ATPase mRNA decay is developmentally regulated in liver, indicating that gene expression is also controlled at this level during physiological transitions that affect biogenesis of mitochondria.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Mitocondrias Hepáticas/enzimología , ATPasas de Translocación de Protón/genética , Procesamiento Postranscripcional del ARN , Transcripción Genética , Animales , Femenino , Regulación del Desarrollo de la Expresión Génica , Semivida , Microscopía Inmunoelectrónica , Mitocondrias Hepáticas/ultraestructura , Orgánulos , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas WistarRESUMEN
Postnatal (1-h) mitochondrial differentiation in normal neonatal rat liver is regulated at the translational level (Izquierdo, J. M., Luis, A. M., and Cuezva, J. M. (1990) J. Biol. Chem. 265, 9090-9097). The rapid postnatal increase in liver global rate of protein synthesis preferentially affects mitochondrial proteins (Valcarce, C., Navarrete, R. M., Encabo, P., Loeches, E., Satrústegui, J., and Cuezva, J. M. (1988) J. Biol. Chem. 263, 7767-7775). Analysis of polysome profiles and determination of both eukaryotic initiation factor 2 (eIF-2) activity and amount of eIF-2 beta protein in the liver of fetal and 1-h-old neonatal rats, indicate a rapid activation of translation initiation without changes in the amount of the translational machinery available between both stages of liver development. Appearance of a more acidic eIF-2 beta-subunit form in two-dimensional Western blots from 1-h-old rat livers suggests that covalently regulated modifications of the initiation factor phosphoproteins might be responsible for increased translation in the neonatal liver. On the other hand, preferential cytosolic translation of the mitochondrial nuclear-encoded beta-F1-ATPase mRNA at this stage of liver development is accomplished by (i) the antenatal accumulation of this mRNA in the fetal liver in 5-6-fold excess than that found in adults, although fetal liver beta-F1-ATPase mRNA shows negligible translational efficiency when compared to the adult counterpart; (ii) a 2-fold increase of the stored beta-F1-ATPase mRNA being rapidly mobilized into cytosolic polyribosomes, and (iii) a 3-fold increase in the in vitro determined translational efficiency of beta-F1-ATPase mRNA. Increased translational efficiency of beta-F1-ATPase mRNA at 1-h postnatal is specific for the nuclear-encoded template since beta-tubulin mRNA did not show any postnatal alteration in its translational efficiency. The results presented suggest that developmental changes in the poly(A)+ RNA fraction or in the reporter template itself are responsible for the increased and preferential translation of the nuclear-encoded mitochondrial mRNAs needed for mitochondrial differentiation and, thus, for mammalian adaptation to the extrauterine environment.