RESUMEN

INTRODUCTION: Preeclampsia (PE) is highly associated with the presence of autoantibodies that activate the major angiotensin receptor, AT1R. These autoantibodies (termed AT1-AA) cause most features of PE when introduced into pregnant mice and are believed to contribute to disease pathogenesis. Tissue transglutaminase (tTG) is a prominent member of a family of enzymes that crosslink proteins by catalyzing the formation of an isopeptide bond between the amide group of glutamine and the free amino group of lysine and is believed to play a role in AT1R activation. OBJECTIVES: To test the hypothesis that tTG plays a role in autoantibody-mediated AT1R activation and thereby contributes to the pathophyhsiology of PE. METHODS: Western blotting was used to quantify tTG in placentas of normotensive pregnant women and those with PE. Immunohistochemistry was used to determine cellular localization of tTG, AT1R and glutamyl-lysine isopeptides in placenta tissue. Co-immunoprecipitation experiments were used to explore the possible association of tTG and AT1R. Finally, in an effort to determine the role of tTG in AT1-AA-induced features of PE in vivo, we treated autoantibody-injected pregnant mice with cystamine, a potent tTG inhibitor. RESULTS: Western blot analysis showed elevated tTG in placentas of women with PE. Immunohistochemical analysis revealed that increased levels of tTG, glutamyl-lysineisopeptides, AT1R co-localized in the microvillous membrane of PE placentas. Immunoprecipitation of tTG from human trophoblast cell (HTR) lysates resulted in the co-immunoprecipitation of AT1R, thereby providing evidence for an interaction between tTG and AT1R in HTR cells. Finally, we found that cystamine treatment significantly attenuated key features associated with PE including hypertension and proteinuria in the PE-IgG-injected pregnant mice. CONCLUSION: Taken together, we identified a previously unrecognized role of elevated tTG in PE likely by activating or stabilizing AT1Rs. These studies provide novel mechanisms of autoantibody-induced AT1R activation by elevated placental tTG, highlight the role of tTG in the pathogenesis of PE and suggest novel therapeutic strategies for the disease.

RESUMEN

INTRODUCTION: Preeclampsia (PE) is associated with alterations in the renin-angiotensin system due to the presence of autoantibodies that activate the major angiotensin receptor, AT1R. The resulting chronic activation of the AT1 receptor is believed to contribute to a pro-inflammatory state that is also characteristic of PE. Microarray analysis revealed a pronounced increase in the abundance of LIGHT, a potent member of the TNF superfamily that has emerged as a key factor mediating strong T-cell inflammatory responses and a contributor to autoimmune disease pathogenesis. OBJECTIVES: The goal of research described here is to determine the contribution of LIGHT to the pathophysiology of PE. METHODS: An ELISA, specific for LIGHT was used to validate the results of the initial microarray analysis. Infusion experiments were used to determine the effects of elevated LIGHT in pregnant and non-pregnant mice. An adoptive transfer model of PE in pregnant mice was used to determine if AT1R agonistic autoantibodies (AT1AA) stimulate LIGHT production. The potential contribution of increased LIGHT to clinical features of PE in pregnant mice was evaluated by the use of neutralizing antibodies directed to the LIGHT receptors, HVEM and LTßR. RESULTS: The use of a LIGHT-specific ELISA showed that the cytokine was significantly increased in the circulation and placentas of women with PE. The infusion of recombinant LIGHT into pregnant mice induced major clinical features of PE, including hypertension and proteinuria. Such changes were not observed when LIGHT was infused into not non-pregnant mice. LIGHT was significantly elevated in a pregnant mouse model of PE based on the introduction of IgG from women with PE. In this autoantibody-injection model of PE, the use of neutralizing antibodies to the LIGHT receptors, HVEM and LTßR, blocked autoantibody-induced hypertension, proteinuria, elevated sFlt-1 production, impaired placental angiogenesis and endothelial dysfunction. CONCLUSION: Overall, our studies show that LIGHT is significantly elevated in women with PE and that elevated LIGHT contributes to pathophysiology of PE in an antibody-injection model of PE in pregnant mice. LIGHT-induced pathophysiology can be abrogated by the use of neutralizing antibodies directed at its receptors, HVEM and LTßR. These results indicate that LIGHT may have a previously unrecognized role in the pathophysiology of PE and that its detrimental effects are pregnancy-dependent. These findings immediately suggest novel therapeutic possibilities.

RESUMEN

The formation of the trophoblast cell lineage of the placenta is one of the first developmental events to occur in mammalian embryogenesis. To understand the mechanisms of gene regulation in the trophoblast cell lineage we have used the murine adenosine deaminase gene (Ada) as a model. Ada is highly expressed in trophoblast cells of the placenta and is critical for embryo development. A 770bp fragment of the mouse Ada 5' flanking region is capable of directing trophoblast cell-specific expression in a transgenic model system. Earlier studies identified several critical portions of this fragment, including three footprinting regions that are necessary for correct gene expression in the placenta. Using electromobility shift assays (EMSA), we identified a 5bp sequence within footprint 3 that computer databases predicted bound to the transcription factor RUNX1 (also known as acute myeloid leukemia 1). This prediction was confirmed by supershift analysis using antibodies specific for RUNX1. The functional importance of this binding was demonstrated by both transient transfections and transgenic approaches. A significant reduction in expression of the reporter gene in the placenta was seen when the 5bp RUNX1 binding site was mutated. The findings reported here indicate that the RUNX1 transcription factor plays a significant role in regulating Ada gene expression in the trophoblast cell lineage.

Asunto(s)

Adenosina Desaminasa/genética , Linaje de la Célula/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Placenta/metabolismo , Trofoblastos/metabolismo , Región de Flanqueo 5' , Animales , Secuencia de Bases , Sitios de Unión , Subunidad alfa 2 del Factor de Unión al Sitio Principal/análisis , Huella de ADN , Femenino , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Placenta/química , Placenta/citología , Embarazo

RESUMEN

The proliferation and differentiation of trophoblast cells is under the control of a variety of hormones and growth factors and is influenced by nutrient availability. The intracellular signaling pathways acting downstream of these mitogenic factors and nutrients to regulate trophoblast proliferation and placental development are poorly understood. Immortalized human trophoblast cells were used (HTR-8/SVneo) to investigate trophoblast proliferation in response to angiopoietin-2 (Ang-2), a major angiogenic factor and glucose (a major nutrient). Trophoblast cell proliferation was induced through activation of the phosphatidylinositol-3 (PI-3) kinase and the mammalian target of rapamycin (mTOR) signaling pathways, following Tie-2 receptor activation. Glucose also stimulated trophoblast cell proliferation through mTOR signaling. Ang-2 activated mTOR via PI-3 kinase-dependent signaling; whereas glucose-mediated mTOR activation was PI-3 kinase-independent and involved a novel nutrient sensor, glutamine fructose-6-phosphate amidotransferase (GFAT). Metabolites of the GFAT reaction acted upstream of mTOR and functioned as a nutrient sensor to regulate trophoblast cell proliferation in response to glucose. Overall, the results show that growth factor and nutrient signaling converge at tuberin, an upstream regulator of mTOR and that mTOR functions as an important placental growth signaling sensor. These results are the first to link mTOR with GFAT metabolites as nutrient sensors for trophoblast cell proliferation.

Asunto(s)

Placenta/fisiología , Placentación , Proteínas Quinasas/fisiología , Angiopoyetina 2/farmacología , Línea Celular , Proliferación Celular/efectos de los fármacos , Femenino , Glucosa/farmacología , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Humanos , Modelos Biológicos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Embarazo , Proteínas Represoras/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR , Trofoblastos/citología , Trofoblastos/efectos de los fármacos , Trofoblastos/fisiología , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/metabolismo

RESUMEN

Adenylosuccinate synthetase 1 (ADSS1) functions as an important component in adenine nucleotide biosynthesis and is abundant in the heart. Here we report that the Adss1 gene is up-regulated in two in vivo rodent models of surgically induced cardiac hypertrophy. In addition, we examined an in vitro hypertrophy system of rat neonatal cardiomyocytes treated with angiotensin II to study Adss1 gene regulation. We show that this stimulus triggers a signaling cascade that results in the activation of the Adss1 gene. The induction of Adss1 gene expression was blocked by cyclosporin A in vitro, suggesting that calcineurin, a calmodulin activated phosphatase, is involved in this signaling pathway. Consistent with this view we provide evidence that the induction of Adss1 by angiotension II requires the presence of an NFAT binding site located 556 base pairs upstream of the Adss1 transcription start site. We propose that ADSS1 plays a role in the development of cardiac hypertrophy through its function in adenine nucleotide biosynthesis.

Asunto(s)

Adenilosuccinato Sintasa/genética , Cardiomegalia , Regulación Enzimológica de la Expresión Génica , Proteínas Nucleares , Angiotensina II/farmacología , Animales , Animales Recién Nacidos , Células Cultivadas , Ciclosporina/farmacología , Proteínas de Unión al ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Ratones , Miocardio/citología , Miocardio/enzimología , Miocardio/metabolismo , Factores de Transcripción NFATC , Ratas , Factores de Transcripción/metabolismo , Regulación hacia Arriba

RESUMEN

Adenosine deaminase (ADA) deficiency in humans results in a severe combined immunodeficiency (SCID). This immunodeficiency is associated with severe disturbances in purine metabolism that are thought to mediate lymphotoxicity. The recent generation of ADA-deficient (ADA(-/-)) mice has enabled the in vivo examination of mechanisms that may underlie the SCID resulting from ADA deficiency. We demonstrate severe depletion of T and B lymphocytes and defects in T and B cell development in ADA(-/-) mice. T cell apoptosis was abundant in thymi of ADA(-/-) mice, but no increase in apoptosis was detected in the spleen and lymph nodes of these animals, suggesting that the defect is specific to developing thymocytes. Studies of mature T cells recovered from spleens of ADA(-/-) mice revealed that ADA deficiency is accompanied by TCR activation defects of T cells in vivo. Furthermore, ex vivo experiments on ADA(-/-) T cells demonstrated that elevated adenosine is responsible for this abnormal TCR signaling. These findings suggest that the metabolic disturbances seen in ADA(-/-) mice affect various signaling pathways that regulate thymocyte survival and function. Experiments with thymocytes ex vivo confirmed that ADA deficiency reduces tyrosine phosphorylation of TCR-associated signaling molecules and blocks TCR-triggered calcium increases.

Asunto(s)

Adenosina Desaminasa/deficiencia , Complejo Receptor-CD3 del Antígeno de Linfocito T/inmunología , Inmunodeficiencia Combinada Grave/patología , Transducción de Señal , Linfocitos T/patología , Timo/patología , Adenosina/farmacología , Animales , Antígenos CD/biosíntesis , Antígenos CD/genética , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Antígenos de Diferenciación de Linfocitos T/genética , Apoptosis , Linfocitos B/patología , Señalización del Calcio , Diferenciación Celular , Células Cultivadas , Nucleótidos de Desoxiadenina/metabolismo , Desoxiadenosinas/farmacología , Regulación de la Expresión Génica , Lectinas Tipo C , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Activación de Linfocitos , Ratones , Ratones Noqueados , Ratones SCID , Especificidad de Órganos , Fosforilación , Procesamiento Proteico-Postraduccional , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Receptores de Interleucina-2/biosíntesis , Receptores de Interleucina-2/genética , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/inmunología , Organismos Libres de Patógenos Específicos , Bazo/inmunología , Bazo/patología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Timo/efectos de los fármacos

RESUMEN

Murine fetal thymic organ culture was used to investigate the mechanism by which adenosine deaminase (ADA) deficiency causes T-cell immunodeficiency. C57BL/6 fetal thymuses treated with the specific ADA inhibitor 2'-deoxycoformycin exhibited features of the human disease, including accumulation of dATP and inhibition of S-adenosylhomocysteine hydrolase enzyme activity. Although T-cell receptor (TCR) Vbeta gene rearrangements and pre-TCR-alpha expression were normal in ADA-deficient cultures, the production of alphabeta TCR(+) thymocytes was inhibited by 95%, and differentiation was blocked beginning at the time of beta selection. In contrast, the production of gammadelta TCR(+) thymocytes was unaffected. Similar results were obtained using fetal thymuses from ADA gene-targeted mice. Differentiation and proliferation were preserved by the introduction of a bcl-2 transgene or disruption of the gene encoding apoptotic protease activating factor-1. The pan-caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone also significantly lessened the effects of ADA deficiency and prevented the accumulation of dATP. Thus, ADA substrates accumulate and disrupt thymocyte development in ADA deficiency. These substrates derive from thymocytes that undergo apoptosis as a consequence of failing to pass developmental checkpoints, such as beta selection.

Asunto(s)

Adenosina Desaminasa/deficiencia , Linfocitos T/citología , Linfocitos T/metabolismo , Adenosina Desaminasa/genética , Animales , Apoptosis , Secuencia de Bases , Cartilla de ADN/genética , Feto/citología , Feto/metabolismo , Hematopoyesis , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Técnicas de Cultivo de Órganos , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Linfocitos T/inmunología , Timo/citología , Timo/inmunología , Timo/metabolismo

RESUMEN

Adenosine deaminase (ADA) deficiency results in a combined immunodeficiency brought about by the immunotoxic properties of elevated ADA substrates. Additional non-lymphoid abnormalities are associated with ADA deficiency, however, little is known about how these relate to the metabolic consequences of ADA deficiency. ADA-deficient mice develop a combined immunodeficiency as well as severe pulmonary insufficiency. ADA enzyme therapy was used to examine the relative impact of ADA substrate elevations on these phenotypes. A "low-dose" enzyme therapy protocol prevented the pulmonary phenotype seen in ADA-deficient mice, but did little to improve their immune status. This treatment protocol reduced metabolic disturbances in the circulation and lung, but not in the thymus and spleen. A "high-dose" enzyme therapy protocol resulted in decreased metabolic disturbances in the thymus and spleen and was associated with improvement in immune status. These findings suggest that the pulmonary and immune phenotypes are separable and are related to the severity of metabolic disturbances in these tissues. This model will be useful in examining the efficacy of ADA enzyme therapy and studying the mechanisms underlying the immunodeficiency and pulmonary phenotypes associated with ADA deficiency.

Asunto(s)

Adenosina Desaminasa/deficiencia , Adenosina Desaminasa/uso terapéutico , Pulmón/fisiopatología , Inmunodeficiencia Combinada Grave/fisiopatología , Inmunodeficiencia Combinada Grave/terapia , Bazo/inmunología , Timo/inmunología , Adenosina/metabolismo , Adenosina Desaminasa/administración & dosificación , Adenosina Desaminasa/sangre , Adenosina Desaminasa/metabolismo , Adenosilhomocisteinasa , Animales , Nucleótidos de Desoxiadenina/análisis , Desoxiadenosinas/metabolismo , Modelos Animales de Enfermedad , Genotipo , Histocitoquímica , Hidrolasas/metabolismo , Células Asesinas Naturales/inmunología , Pulmón/metabolismo , Recuento de Linfocitos , Linfocitos/inmunología , Linfopenia/enzimología , Linfopenia/inmunología , Linfopenia/metabolismo , Linfopenia/terapia , Ratones , Ratones Transgénicos , Fenotipo , S-Adenosilhomocisteína/análisis , Inmunodeficiencia Combinada Grave/enzimología , Inmunodeficiencia Combinada Grave/inmunología , Bazo/fisiopatología , Timo/fisiopatología

RESUMEN

Adenosine deaminase (ADA) is a purine catabolic enzyme that manages levels of the biologically active purines adenosine and 2'-deoxyadenosine in tissues and cells. ADA-deficient mice die at 3 wk of age from severe respiratory distress. This phenotype is progressive and is linked to perturbations in pulmonary purine metabolism. The inflammatory changes found in the lungs of ADA-deficient mice included an accumulation of activated alveolar macrophages and eosinophils. These changes were accompanied by a pronounced enlargement of alveolar spaces and increases in mucus production in the bronchial airways. The alveolar enlargement was found to be due in part to abnormal alveogenesis. Lowering adenosine and 2'-deoxyadenosine levels using ADA enzyme therapy decreased the pulmonary eosinophilia and resolved many of the lung histopathologies. In addition, genetically restoring ADA to the forestomach of otherwise ADA-deficient mice prevented adenine metabolic disturbances as well as lung inflammation and damage. These data suggest that disturbances in purinergic signaling mediate the lung inflammation and damage seen in ADA-deficient mice.

Asunto(s)

Adenosina Desaminasa/deficiencia , Obstrucción de las Vías Aéreas/etiología , Pulmón/patología , Neumonía/etiología , Alveolos Pulmonares/crecimiento & desarrollo , Adenosina/metabolismo , Adenosina Desaminasa/genética , Adenosina Desaminasa/uso terapéutico , Animales , Líquido del Lavado Bronquioalveolar/química , Eosinófilos/fisiología , Inmunoglobulina E/sangre , Interleucina-5/análisis , Ratones , Ratones Transgénicos , Moco/fisiología

RESUMEN

Deficiency in the enzyme adenosine deaminase (ADA) in humans manifests primarily as severe lymphopenia and immunodeficiency, resulting in death by 6 months of age, if untreated. In this review, we discuss phenotypical, biochemical, and metabolic hallmarks of the disease, and describe a mouse model in which levels of ADA can be biochemically and genetically manipulated. This model provides exciting possibilities for uncovering the mechanisms by which this purine catabolic enzyme affects lymphopoiesis.

Asunto(s)

Adenosina Desaminasa/deficiencia , Adenosina Desaminasa/metabolismo , Linfocitos/enzimología , Linfocitos/inmunología , Inmunodeficiencia Combinada Grave/enzimología , Inmunodeficiencia Combinada Grave/inmunología , Adenosina/metabolismo , Adenosina Desaminasa/genética , Adenosina Desaminasa/farmacología , Animales , Desoxiadenosinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Ratones , Inmunodeficiencia Combinada Grave/fisiopatología , Inmunodeficiencia Combinada Grave/terapia

RESUMEN

Adenosine deaminase (ADA) deficiency in humans leads to a combined immunodeficiency characterized by severe T and B cell lymphopenia. ADA-deficient humans also display defective development of gut-associated lymphoid tissues (GALT). They lack lymphoid cells, and the Peyer's patches are without germinal centers. In mice, ADA-deficient fetuses die perinatally due to liver damage, but they also exhibit pathology in the thymus, spleen, and the small intestine. The GI phenotype associated with ADA-deficient humans prompted us to examine the effect of ADA-deficiency on mouse small intestine tissue. The work presented here focuses on understanding the physiological role of ADA in the GI tract, using ADA-deficient mice rescued from perinatal lethality by restoring Ada expression to trophoblast cells. Histologically and immunologically, the GALT was compromised at all sites in ADA-/- mice, with the most dramatic changes seen in the Peyer's patches. Profound disturbances in purine metabolism were detected in all the gastrointestinal tissues. In particular, adenosine and deoxyadenosine, the ADA substrates, increased markedly while the product inosine decreased. The activity of S-adenosylhomocysteine hydrolase decreased throughout the GI tract, indicating a possible disruption of cellular transmethylation and activation of apoptotic pathways. There were also disturbances in the purine metabolic pathway with a decrease in the production of downstream nucleosides hypoxanthine and xanthine.

Asunto(s)

Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Ganglios Linfáticos Agregados/enzimología , Inmunodeficiencia Combinada Grave/enzimología , Adenosina Desaminasa/deficiencia , Adenosilhomocisteinasa , Animales , Cruzamientos Genéticos , Heterocigoto , Homocigoto , Humanos , Hidrolasas/metabolismo , Mucosa Intestinal/enzimología , Mucosa Intestinal/patología , Intestino Delgado/enzimología , Intestino Delgado/patología , Ratones , Ratones Noqueados , Ganglios Linfáticos Agregados/patología , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/patología

RESUMEN

Electrically stimulated pacing of cultured cardiomyocytes serves as an experimentally convenient and physiologically relevant in vitro model of cardiac hypertrophy. Electrical pacing triggers a signaling cascade that results in the activation of the muscle-specific Adss1 gene and the repression of the nonmuscle Adss2 isoform. Activation of the Adss1 gene involves the calcineurin-mediated dephosphorylation of NFAT3, allowing its translocation to the nucleus, where it can directly participate in Adss1 gene activation. Mutational studies show that an NFAT binding site located in the Adss1 5'-flanking region is essential for this activation. Electrical pacing also results in the increased synthesis of GATA4, another critical cardiac transcription factor required for Adss1 gene expression. MEF2C also produces transactivation of the Adss1 gene reporter in control and paced cardiac myocytes. Using the Adss1 gene as a model, these studies are the first to demonstrate that electrical pacing activates the calcineurin/NFAT3 and GATA4 pathways as a means of regulating cardiac gene expression.

Asunto(s)

Adenilosuccinato Sintasa/genética , Proteínas de Unión al ADN/metabolismo , Estimulación Eléctrica , Regulación Enzimológica de la Expresión Génica , Miocardio/metabolismo , Proteínas Nucleares , Factores de Transcripción/metabolismo , Animales , Animales Recién Nacidos , Northern Blotting , Calcineurina/metabolismo , Estimulación Cardíaca Artificial , Células Cultivadas , Cloranfenicol O-Acetiltransferasa/metabolismo , Ciclosporina/farmacología , Proteínas de Unión al ADN/genética , Inhibidores Enzimáticos/farmacología , Factor de Transcripción GATA4 , Regulación de la Expresión Génica , Factores de Transcripción MEF2 , Factores Reguladores Miogénicos/metabolismo , Factores de Transcripción NFATC , Isoformas de Proteínas/metabolismo , Ratas , Factores de Tiempo , Factores de Transcripción/genética , Activación Transcripcional , Transfección , Regulación hacia Arriba

Asunto(s)

Adenosina Desaminasa/deficiencia , Adenosina/metabolismo , Adenosina Desaminasa/uso terapéutico , Adenosilhomocisteinasa , Animales , Desoxiadenosinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Hidrolasas/metabolismo , Linfocitos , Ratones , Ratones Noqueados , Receptor de Adenosina A3 , Receptores Purinérgicos P1/genética , Inmunodeficiencia Combinada Grave/terapia , Transducción de Señal

Asunto(s)

Adenosina Desaminasa/deficiencia , Inhibidores de Caspasas , Timo/enzimología , Clorometilcetonas de Aminoácidos/farmacología , Animales , Linfocitos B/citología , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/farmacología , Nucleótidos de Desoxiadenina/metabolismo , Ratones , Ratones Endogámicos C57BL , Técnicas de Cultivo de Órganos , Receptores de Antígenos de Linfocitos T alfa-beta , Linfocitos T/citología , Timo/embriología

RESUMEN

The mammalian heart begins contracting at the linear tube stage during embryogenesis and continuously pumps, nonstop, throughout the entire lifetime of the animal. Therefore, the cardiac energy metabolizing pathways must be properly established and efficiently functioning. While the biochemistry of these pathways is well defined, limited information regarding the regulation of cardiac metabolic genes is available. Previously, we reported that 1.9 kilobase pairs of murine adenylosuccinate synthetase 1 gene (Adss1) 5'-flanking DNA directs high levels of reporter expression to the adult transgenic heart. In this report, we define the 1.9-kilobase pair fragment as a cardiac-specific enhancer that controls correct spatiotemporal expression of a reporter similar to the endogenous Adss1 gene. A 700-base pair fragment within this region activates a heterologous promoter specifically in adult transgenic hearts. Proteins present in a cardiac nuclear extract interact with potential transcription factor binding sites of this region and these cis-acting sites play important regulatory roles in the cardiac expression of this reporter. Finally, we report that several different cardiac transcription factors trans-activate the 1.9HSCAT construct through these sites and that combinations result in enhanced reporter expression. Adss1 appears to be one of the first target genes identified for the bHLH factors Hand1 and Hand2.

Asunto(s)

Adenilosuccinato Sintasa/genética , Miocardio/enzimología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Análisis Mutacional de ADN , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasa HindIII/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Regulación Enzimológica de la Expresión Génica , Genes Reporteros , Corazón/embriología , Secuencias Hélice-Asa-Hélice , Hibridación in Situ , Ratones , Ratones Transgénicos , Contracción Miocárdica , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Activación Transcripcional

RESUMEN

The maturation of stratified squamous epithelium of the upper gastrointestinal tract is a highly ordered process of development and differentiation. Information on the molecular basis of this process is, however, limited. Here we report the identification of the first murine forestomach regulatory element using the murine adenosine deaminase (Ada) gene as a model. In the adult mouse, Ada is highly expressed in the terminally differentiated epithelial layer of upper gastrointestinal tract tissues. The data reported here represent the identification and detailed analysis of a 1. 1-kilobase (kb) sequence located 3.4-kb upstream of the transcription initiation site of the murine Ada gene, which is sufficient to target cat reporter gene expression to the forestomach in transgenic mice. This 1.1-kb fragment is capable of directing cat reporter gene expression mainly to the forestomach of transgenic mice, with a level comparable to the endogenous Ada gene. This expression is localized to the appropriate cell types, confers copy number dependence, and shows the same developmental regulation. Mutational analysis revealed the functional importance of multiple transcription factor-binding sites.

Asunto(s)

Adenosina Desaminasa/genética , Regulación Enzimológica de la Expresión Génica , Genes Reguladores/genética , Estómago/enzimología , Adenosina Desaminasa/biosíntesis , Animales , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa/genética , Técnicas de Cultivo , Genes Reporteros , Hibridación in Situ , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Análisis de Secuencia de ADN

RESUMEN

Trophoblast cells are specialized extra-embryonic cells present only in eutherian mammals. They play a major role in the implantation and placentation processes. To understand better the molecular mechanisms that control the development and function of trophoblast cells, we sought to identify the transcription factors that regulate murine adenosine deaminase (ADA) gene expression in the placenta. Here we report a detailed characterization of a placenta-specific footprinting region (FP1) in the Ada placental regulatory element. The sequence of FP1 was mapped by DNase I footprinting and was found to match a consensus AP-2 transcription factor-binding site. Electrophoretic mobility shift assays demonstrated that FP1 interacted with AP-2-like proteins. Further analysis using AP-2 antibody confirmed that AP-2 protein was indeed present in the placenta and bound to FP1. Mutation at the AP-2 site in FP1 abolished the ability of the Ada placental regulatory element to bind AP-2 proteins and failed to target chloramphenicol acetyltransferase reporter gene expression to placentas in transgenic mice, indicating that AP-2 is required for Ada expression in the placenta. In addition, RNase protection assays demonstrated that AP-2gamma was the predominant AP-2 family member expressed in the placenta. In situ hybridization analysis revealed that AP-2gamma expression was enriched in the trophoblast lineage throughout development, suggesting that AP-2gamma may be critical for trophoblast development and differentiation.

Asunto(s)

Adenosina Desaminasa/biosíntesis , Proteínas de Unión al ADN/metabolismo , Placenta/enzimología , Factores de Transcripción/metabolismo , Adenosina Desaminasa/genética , Alantoides/enzimología , Animales , Sitios de Unión , Corion/enzimología , Huella de ADN , Proteínas de Unión al ADN/genética , Embrión de Mamíferos/enzimología , Femenino , Regulación Enzimológica de la Expresión Génica , Genes Reporteros , Ratones , Ratones Endogámicos ICR , Placentación , Embarazo , Unión Proteica , Secuencias Reguladoras de Ácidos Nucleicos , Distribución Tisular , Factor de Transcripción AP-2 , Factores de Transcripción/genética , Transgenes , Trofoblastos/citología , Trofoblastos/enzimología

RESUMEN

Adenosine deaminase (ADA) deficiency in humans leads to a combined immunodeficiency. The mechanisms involved in the lymphoid specificity of the disease are not fully understood due to the inaccessibility of human tissues for detailed analysis and the absence of an adequate animal model for the disease. We report the use of a two-stage genetic engineering strategy to generate ADA-deficient mice that retain many features associated with ADA deficiency in humans, including a combined immunodeficiency. Severe T and B cell lymphopenia was accompanied by a pronounced accumulation of 2'-deoxyadenosine and dATP in the thymus and spleen, and a marked inhibition of S-adenosylhomocysteine hydrolase in these organs. Accumulation of adenosine was widespread among all tissues examined. ADA-deficient mice also exhibited severe pulmonary insufficiency, bone abnormalities, and kidney pathogenesis. These mice have provided in vivo information into the metabolic basis for the immune phenotype associated with ADA deficiency.

Asunto(s)

Adenosina Desaminasa/deficiencia , Errores Innatos del Metabolismo de la Purina-Pirimidina/enzimología , Inmunodeficiencia Combinada Grave/genética , Adenosina/metabolismo , Adenosina Desaminasa/genética , Adenosilhomocisteinasa , Animales , Antígenos CD/análisis , Antígenos de Diferenciación , Linfocitos B/citología , Huesos/anomalías , Desoxiadenosinas/metabolismo , Ingeniería Genética/métodos , Hidrolasas/análisis , Riñón/anomalías , Pulmón/patología , Recuento de Linfocitos , Linfopenia , Ratones , Insuficiencia de la Válvula Pulmonar , Errores Innatos del Metabolismo de la Purina-Pirimidina/genética , Errores Innatos del Metabolismo de la Purina-Pirimidina/patología , Proyectos de Investigación , Inmunodeficiencia Combinada Grave/enzimología , Bazo/patología , Linfocitos T/citología , Timo/patología , Distribución Tisular

RESUMEN

Adenosine deaminase (ADA) is an essential enzyme of purine metabolism that is enriched at the maternal-fetal interface of mice throughout postimplantation development. During early postimplantation stages Ada is highly expressed in both maternally derived decidual cells and zygotically derived trophoblast cells. For the current study we utilized genetically modified mice to delineate the relative contribution and importance of decidual and trophoblast ADA at the maternal-fetal interface. In females genetically engineered to lack decidual ADA a striking pattern of expression was revealed in giant trophoblast cells that surround the early postimplantation embryo. Embryos within gestation sites lacking both decidual and trophoblast ADA died during the early postimplantation period, whereas expression in trophoblast cells alone was sufficient for survival through this period. Severe disturbances in purine metabolism were observed in gestation sites lacking decidual ADA, including the accumulation of the potentially toxic ADA substrates adenosine and 2'-deoxyadenosine. These experiments provide genetic evidence that Ada expression at the maternal-fetal interface is essential for early postimplantation development in mice.

Asunto(s)

Adenosina Desaminasa/fisiología , Decidua/enzimología , Desarrollo Embrionario/fisiología , Trofoblastos/enzimología , Adenosina/metabolismo , Adenosina Desaminasa/análisis , Adenosina Desaminasa/genética , Animales , Desoxiadenosinas/metabolismo , Femenino , Inosina/metabolismo , Ratones , Ratones Transgénicos , Embarazo , Cigoto/enzimología , Cigoto/crecimiento & desarrollo

RESUMEN

Murine adenosine deaminase (ADA) is a ubiquitous purine catabolic enzyme whose expression is subject to developmental and tissue-specific regulation. ADA is enriched in trophoblast cells of the chorioallantoic placenta and is essential for embryonic and fetal development. To begin to understand the genetic pathway controlling Ada gene expression in the placenta, we have identified and characterized a 770-base pair fragment located 5.4 kilobase pairs upstream of the Ada transcription initiation site, which directs reporter gene expression to the placenta of transgenic mice. The expression pattern of the reporter gene reflected that of the endogenous Ada gene in the placenta. Sequence analysis revealed potential binding sites for bHLH and GATA transcription factors. DNase I footprinting defined three protein binding regions, one of which was placenta-specific. Mutations in the potential protein binding sites and footprinting regions resulted in loss of placental expression in transgenic mice. These findings indicate that multiple protein binding motifs are necessary for Ada expression in the placenta.

Asunto(s)

Adenosina Desaminasa/genética , Regulación Enzimológica de la Expresión Génica , Variación Genética , Placenta/enzimología , Animales , Secuencia de Bases , Genes Reguladores , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Mapeo Restrictivo