RESUMEN
Cell growth and differentiation are opposite events in the myogenic lineage. Growth factors block the muscle differentiation program by inducing the expression of transcription factors that negatively regulate the expression of muscle regulatory genes like MyoD. In contrast, extracellular clues that induce cell cycle arrest promote MyoD expression and muscle differentiation. Thus, the regulation of MyoD expression is critical for muscle differentiation. Here we show that estrogen induces MyoD expression in mouse skeletal muscle in vivo and in dividing myoblasts in vitro by relieving the MyoD promoter from AP-1 negative regulation through a mechanism involving estrogen receptor/AP-1 protein-protein interactions but independent of the estrogen receptor DNA binding activity.
Asunto(s)
Regulación de la Expresión Génica , Desarrollo de Músculos/genética , Proteína MioD/genética , Receptores de Estrógenos/metabolismo , Factor de Transcripción AP-1/metabolismo , Animales , Estrógenos/metabolismo , Estrógenos/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Mioblastos Esqueléticos/efectos de los fármacos , Mioblastos Esqueléticos/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
We have developed a simple and reproducible fingerprinting method for screening the genome for regions of DNA that have altered patterns of DNA methylation associated with oncogenic transformation. Restriction enzymes with different sensitivities to cytosine methylation in their recognition sites were used to digest genomic DNAs from primary tumors, cell lines, and normal tissues prior to arbitrarily primed PCR amplification. Fragments that showed differential methylation were cloned and sequenced after resolving the PCR products on high-resolution polyacrylamide gels. The cloned fragments were then used as probes for Southern analysis to confirm differential methylation of these regions in colon tissues and cell lines. Forty-four DNA fragments associated with a total of five different regions of genomic DNA containing methylation sites were detected in 10 matched sets of normal and tumor colon DNAs and 7 colon cancer cell lines. A novel CpG island was also isolated that was found to be frequently hypermethylated in bladder and colon tumors. We have demonstrated that this technique is a rapid and efficient method that can be used to screen for altered methylation patterns in genomic DNA and to isolate specific sequences associated with these changes.
Asunto(s)
Neoplasias del Colon/genética , Dermatoglifia del ADN/métodos , Metilación de ADN , ADN de Neoplasias/genética , Reacción en Cadena de la Polimerasa/métodos , Neoplasias de la Vejiga Urinaria/genética , Southern Blotting , Humanos , Datos de Secuencia Molecular , Células Tumorales CultivadasRESUMEN
BACKGROUND: Vitamin E is well known as an antioxidant, and numerous studies suggest that it has a preventive role in atherosclerosis, although the mechanism of action still remains unclear. METHODS AND RESULTS: The original aim of this study was to establish whether alpha-tocopherol (the most active form of vitamin E) acts at the earliest events on the cascade of atherosclerosis progression, that of oxidized LDL (oxLDL) uptake and foam-cell formation. We show here that the CD36 scavenger receptor (a specific receptor for oxLDL) is expressed in cultured human aortic smooth muscle cells (SMCs). Treatment of SMCs and HL-60 macrophages with alpha-tocopherol (50 micromol/L, a physiological concentration) downregulates CD36 expression by reducing its promoter activity. Furthermore, we find that alpha-tocopherol treatment of SMCs leads to a reduction of oxLDL uptake. CONCLUSIONS: This study indicates that CD36 is expressed in cultured human SMCs. In these cells, CD36 transports oxLDL into the cytosol. alpha-Tocopherol inhibits oxLDL uptake by a mechanism involving downregulation of CD36 mRNA and protein expression. Therefore, the beneficial effect of alpha-tocopherol against atherosclerosis can be explained, at least in part, by its effect of lowering the uptake of oxidized lipoproteins, with consequent reduction of foam cell formation.
Asunto(s)
Antígenos CD36/genética , Lipoproteínas LDL/farmacocinética , Músculo Liso Vascular/metabolismo , Vitamina E/farmacología , Aorta/citología , Arteriosclerosis/metabolismo , Antígenos CD36/metabolismo , Células Cultivadas , Citometría de Flujo , Colorantes Fluorescentes/farmacocinética , Regulación de la Expresión Génica/efectos de los fármacos , Células HL-60 , Humanos , Microscopía Confocal , Músculo Liso Vascular/citología , Regiones Promotoras Genéticas/fisiología , ARN Mensajero/análisis , TransfecciónRESUMEN
Two plasmid systems, containing the easily assayable galK and lacZ functions, were employed to study the regulation of the bacteriophage P1 tail-fibre and dar operons. Various P1 DNA fragments carrying either the 5' end of lydA (the 1st gene in the dar operon) or the tail-fibre gene 19 precede the promoterless coding region of galK or were fused, in-frame, to the lacZ gene. In the presence of an induced P1 prophage, GalK and LacZ activities were both detected after a 20 to 30 minute lag period, indicating that the dar and tail-fibre operons are expressed from positively regulated, late promoters. The corresponding DNA operons are expressed from positively regulated, late promoters. The corresponding DNA region of the closely related p15B plasmid exhibits comparable promoter properties. Deletion analysis mapped the promoter of a gene 19-lacZ fusion to a DNA region upstream from gene R, an open reading frame that precedes the coding frame of gene 19. The tail-fibre gene thus forms the second gene in a three gene operon (genes R, 19 (S) and U). Sequence comparison between this promoter region, upstream sequences of the lydA gene and the corresponding portions of the p15B genome allowed the identification of a highly conserved 38 base-pair sequence, which most likely represents a P1-specific late promoter. This was confirmed by 5' mapping of P1 mRNA. Transcription of both the tail-fibre and dar operons is initiated at sites five and six base-pairs, respectively, downstream from the first conserved nucleotide of this sequence. The conserved motif consists of a standard Escherichia coli -10 region followed by a nine base-pair palindromic sequence located centrally about position -22.
Asunto(s)
Colifagos/genética , Operón , Regiones Promotoras Genéticas , Secuencia de Bases , Colifagos/ultraestructura , Modelos Genéticos , Datos de Secuencia Molecular , Plásmidos , Homología de Secuencia de Ácido NucleicoRESUMEN
Cytosine to thymine transition mutations at the CpG dinucleotide are the most common point mutations in cancer and genetic disease. We calculated the in vivo rate of CpG mutation in the primate germline by deriving a primordial consensus sequence for an Alu repetitive element which inserted into intron 6 of the primate p53 gene 35 to 55 million years ago. Comparison of this primordial sequence to the Alu sequence in intron 6 of present-day primates was used to determine the nature and rate of mutations which occurred during evolution. We estimate the half-life of a CpG nucleotide to be 24 to 60 million years, and the rate constant for mutation at this dinucleotide to be 1.2 x 1O(-8) to 2.9 x 1O(-8) years(-1). These results were confirmed by the analysis of a second Alu sequence in intron 10 of the p53 gene. The in vivo mutation rate is at least 1250-fold slower than the in vitro chemical rate of 5-methylcytosine deamination in double-stranded DNA, showing that current estimates of CpG mutation repair have been significantly underestimated. Furthermore, the mutability of the CpG dinucleotide has led to the depletion of this dinucleotide from the vertebrate genome, and calculations in this study suggest that current levels of the CpG dinucleotide in the primate genome are very close to a steady state equilibrium in which the rate of CpG mutation is equal to the rate of CpG formation by random mutation.
Asunto(s)
Fosfatos de Dinucleósidos/genética , Genes p53 , Mutación de Línea Germinal , Primates/genética , Secuencias Repetitivas de Ácidos Nucleicos , Animales , Secuencia de Bases , Evolución Biológica , Línea Celular , ADN , ADN-Citosina Metilasas/metabolismo , Humanos , Intrones , Metilación , Datos de Secuencia Molecular , Polimorfismo Conformacional Retorcido-Simple , Homología de Secuencia de Ácido Nucleico , Células Tumorales CultivadasRESUMEN
Since the discovery of vitamin E in 1922, its deficiency has been associated with various disorders, particularly atherosclerosis, ischemic heart disease, and the development of different types of cancer. A neurological syndrome associated with vitamin E deficiency resembling Friedreich ataxia has also been described. Whereas epidemiological studies have indicated the role of vitamin E in preventing the progression of atherosclerosis and cancer, intervention trials have produced contradictory results, indicating strong protection in some cases and no significant effect in others. Although it is commonly believed that phenolic compounds like vitamin E exert only a protective role against free radical damage, antioxidant molecules can exert other biological functions. For instance, the antioxidant activity of 17-beta-estradiol is not related to its role in determining secondary sexual characters, and the antioxidant capacity of all-trans-retinal is distinguished from its role in rhodopsin and vision. Thus, it is not unusual that alpha-tocopherol (the most active form of vitamin E) has properties independent of its antioxidant/radical scavenging ability. The Roman god Janus, shown in ancient coins as having two faces in one body, inspired the designation of 'Janus molecules' for these substances. The new biochemical face of vitamin E was first described in 1991, with an inhibitory effect on cell proliferation and protein kinase C activity. After a decade, this nonantioxidant role of vitamin E is well established, as confirmed by authoritative studies of signal transduction and gene regulation. More recently, a tocopherol binding protein with possible receptor function has been discovered. Despite such important developments in understanding the molecular mechanism and the targets of vitamin E, its new Janus face is not fully elucidated. Greater knowledge of the molecular events related to vitamin E will help in selecting the parameters for clinical intervention studies such as population type, dose response effects, and possible synergism with other compounds.
Asunto(s)
Antioxidantes/farmacología , División Celular/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Vitamina E/farmacología , Animales , Arteriosclerosis/prevención & control , Ensayos Clínicos como Asunto , HumanosRESUMEN
INTRODUCTION: During the oil frying process lipid peroxidation compounds are formed. These products can modulate gene expression and alter cellular behaviour. The cellular uptake of oxidized LDL, a key step in the development of atherosclerosis, is mediated by the CD36 scavenger receptor, whose expression is down-regulated by alpha-tocopherol. OBJECTIVE: To determine the effects of water-soluble aldehydes, obtained from thermally oxidized sunflower oil on the expression of CD36 scavenger receptor in human monocytes (THP-1 cells). We also wanted to study the effects of alpha-tocopherol on CD36 expression in the presence of water-soluble aldehydes. MATERIALS AND METHODS: Sunflower oil was heated in a frying pan, at 180--200 degrees C for 40 min, water-soluble aldehydes were isolated, and the content of thiobarbituric acid reacting substances (TBARS) was determined. THP-1 monocytes were cultured in RPMI medium during 24 h and incubated with increasing concentrations of the water-soluble aldehydes (ranging from 0.05 to 1 microM) and with or without 50 microM of alpha-tocopherol. In parallel, THP-1 cells were cultured with the same volume of an extract obtained from non-oxidized oil or distilled water. The CD36 expression at the cell surface was studied with fluorescence-activated cell sorting (FACS). RESULTS: Monocytes incubated in a medium containing water-soluble aldehydes, showed a dose dependent increase in the expression of the CD36 protein on the cell surface, compared to with the control groups. When the cells were treated simultaneously with 50 microM of alpha-tocopherol a significant reduction in the expression of the CD36 protein was observed. CONCLUSION: Water-soluble aldehydes, extracted from thermally oxidized culinary oil, increase the expression of CD36. This effect is partially decreased by the presence of alpha-tocopherol.
Asunto(s)
Aldehídos/farmacología , Antígenos CD36/biosíntesis , Monocitos/efectos de los fármacos , Aldehídos/aislamiento & purificación , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Calor , Humanos , Peroxidación de Lípido , Monocitos/inmunología , Aceites de Plantas/química , Aceite de Girasol , alfa-Tocoferol/farmacologíaRESUMEN
The revised sequence of a bacteriophage P1 DNA fragment containing the 5' end of the tail-fibre gene, gene 19, revealed that this gene is closely preceded by another open reading frame (ORF) of 432 bp. We have designated this ORF as gene R. The tail-fibre gene and gene R are transcriptionally and translationally coupled. Thus, the tail-fibre operon of bacteriophage P1 consists of three genes: gene R, gene 19 (or gene S) and gene U.
Asunto(s)
Bacteriófagos/genética , Desoxirribonucleasa BamHI/genética , Genes Virales , Operón , Proteínas Virales/genética , Secuencia de Bases , ADN Viral/genética , Regulación de la Expresión Génica , Datos de Secuencia Molecular , Mutación , Plásmidos , Biosíntesis de Proteínas , Transcripción Genética , Proteínas de la Cola de los VirusRESUMEN
The function of vitamin E has been attributed to its capacity to protect the organism against the attack of free radicals by acting as a lipid based radical chain breaking molecule. More recently, alternative non-antioxidant functions of vitamin E have been proposed and in particular that of a "gene regulator". Effects of vitamin E have been observed at the level of mRNA or protein and could be consequent to regulation of gene transcription, mRNA stability, protein translation, protein stability and post-translational events. Given the high priority functions assigned to vitamin E, it can be speculated that it would be inefficient to consume it as a radical scavenger. Rather, it would be important to protect vitamin E through a network of cellular antioxidant defences, similarly to what occurs with proteins, nucleic acids and lipids.
Asunto(s)
Antioxidantes/metabolismo , alfa-Tocoferol/metabolismo , Enfermedades de los Animales/prevención & control , Animales , Arteriosclerosis/prevención & control , Ataxia/prevención & control , Enfermedades Cardiovasculares/epidemiología , Enfermedades Cardiovasculares/prevención & control , Radicales Libres/metabolismo , Regulación de la Expresión Génica , Humanos , Masculino , Neoplasias de la Próstata/prevención & control , Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , ARN Mensajero/metabolismo , Transducción de Señal , Transcripción Genética , gamma-Tocoferol/metabolismoRESUMEN
Total protein kinase C (PKC) activity in human skin fibroblasts increases during in vivo aging as a function of the donor's age. During in vitro aging protein kinase C activity is also increased, as a function of cell passage number. Using PKC isoform specific antibodies, we demonstrate that the increase in total PKC activity is mainly due to the PKC a isoform. PKC alpha protein expression increased up to 8 fold during in vivo aging. Collagenase (MMP-1) gene transcription and protein expression also increased with age, concomitant with the increase in protein kinase C alpha. Furthermore, alpha-tocopherol, which inhibits protein kinase C activity, is able to diminish collagenase gene transcription without altering the level of its natural inhibitor, tissue inhibitor of metalloproteinase, TIMP-1. We propose that an aging program leads to increased protein kinase C alpha expression and activity. This event would induce collagenase overexpression followed by increased collagen degradation. Our in vitro experiments with skin fibroblasts suggest that alpha-tocopherol may protect against skin aging by decreasing the level of collagenase expression, which is induced by environmental insults and by aging.
Asunto(s)
Colagenasas/metabolismo , Isoenzimas/metabolismo , Metaloproteinasa 1 de la Matriz/metabolismo , Proteína Quinasa C/metabolismo , Vitamina E/farmacología , Adulto , Factores de Edad , Anciano , Células Cultivadas , Colágeno/metabolismo , Colagenasas/genética , Inhibidores Enzimáticos/farmacología , Femenino , Fibroblastos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Recién Nacido , Metaloproteinasa 1 de la Matriz/genética , Persona de Mediana Edad , Proteína Quinasa C-alfa , ARN Mensajero/metabolismo , Envejecimiento de la Piel/efectos de los fármacos , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Atherosclerosis and its complications, such as coronary heart disease, heart infarction and stroke, are the leading causes of death in the developed world. High blood pressure, diabetes, smoking and a diet high in cholesterol and lipids clearly increase the likelihood of premature atherosclerosis, albeit other factors, such as the individual genetic makeup, may play an additional role. During atherosclerosis, uncontrolled cholesterol and lipid accumulation in macrophages and smooth muscle cells leads to foam cell formation and to the progression of the atherosclerotic plaque. This review will focus on foam cell formation within the atherosclerotic lesion, the involvement of the scavenger receptor genes in this process, and the possibility to interfere with scavenger receptor function to reduce the progression of atherosclerosis. To date, the regulatory mechanisms for the expression of scavenger receptor genes and their role in atherosclerosis are not well characterized. Knowledge on this subject could lead to a better understanding of the process, prevention and therapy of this disease.
Asunto(s)
Arteriosclerosis/fisiopatología , Macrófagos/fisiología , Proteínas de la Membrana , Receptores Inmunológicos/fisiología , Receptores de Lipoproteína , Animales , Colesterol/metabolismo , Células Espumosas/fisiología , Regulación de la Expresión Génica , Humanos , Metabolismo de los Lípidos , Receptores Inmunológicos/genética , Receptores Depuradores , Receptores Depuradores de Clase BRESUMEN
OBJECTIVE: Consuming curcumin may benefit health by modulating lipid metabolism and suppressing atherogenesis. Fatty acid binding proteins (FABP-4/aP2) and CD36 expression are key factors in lipid accumulation in macrophages and foam cell formation in atherogenesis. Our earlier observations suggest that curcumin's suppression of atherogenesis might be mediated through changes in aP2 and CD36 expression in macrophages. Thus, this study aimed to further elucidate the impact of increasing doses of curcumin on modulation of these molecular mediators on high fat diet-induced atherogenesis, inflammation, and steatohepatosis in Ldlr(-/-) mice. METHODS: Ldlr(-/-) mice were fed low fat (LF) or high fat (HF) diet supplemented with curcumin (500 HF + LC; 1000 HF + MC; 1500 HF + HC mg/kg diet) for 16 wks. Fecal samples were analyzed for total lipid content. Lipids accumulation in THP-1 cells and expression of aP2, CD36 and lipid accumulation in peritoneal macrophages were measured. Fatty streak lesions and expression of IL-6 and MCP-1 in descending aortas were quantified. Aortic root was stained for fatty and fibrotic deposits and for the expression of aP2 and VCAM-1. Total free fatty acids, insulin, glucose, triglycerides, and cholesterol as well as several inflammatory cytokines were measured in plasma. The liver's total lipids, cholesterol, triglycerides, and HDL content were measured, and the presence of fat droplets, peri-portal fibrosis and glycogen was examined histologically. RESULTS: Curcumin dose-dependently reduced uptake of oxLDL in THP-1 cells. Curcumin also reduced body weight gain and body fat without affecting fat distribution. During early intervention, curcumin decreased fecal fat, but at later stages, it increased fat excretion. Curcumin at medium doses of 500-1000 mg/kg diet was effective at reducing fatty streak formation and suppressing aortic expression of IL-6 in the descending aorta and blood levels of several inflammatory cytokines, but at a higher dose (HF + HC, 1500 mg/kg diet), it had adverse effects on some of these parameters. This U-shape like trend was also present when aortic root sections were examined histologically. However, at a high dose, curcumin suppressed development of steatohepatosis, reduced fibrotic tissue, and preserved glycogen levels in liver. CONCLUSION: Curcumin through a series of complex mechanisms, alleviated the adverse effects of high fat diet on weight gain, fatty liver development, dyslipidemia, expression of inflammatory cytokines and atherosclerosis in Ldlr(-/-) mouse model of human atherosclerosis. One of the mechanisms by which low dose curcumin modulates atherogenesis is through suppression of aP2 and CD36 expression in macrophages, which are the key players in atherogenesis. Overall, these effects of curcumin are dose-dependent; specifically, a medium dose of curcumin in HF diet appears to be more effective than a higher dose of curcumin.
Asunto(s)
Aterosclerosis/tratamiento farmacológico , Aterosclerosis/genética , Curcumina/administración & dosificación , Hígado Graso/genética , Receptores de LDL/genética , Tejido Adiposo , Animales , Aterosclerosis/metabolismo , Peso Corporal , Antígenos CD36/metabolismo , Línea Celular , Células Cultivadas , Citocinas/metabolismo , Dieta Alta en Grasa , Proteínas de Unión a Ácidos Grasos/metabolismo , Femenino , Humanos , Inflamación , Metabolismo de los Lípidos , Lipoproteínas LDL/metabolismo , Hígado/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxígeno/química , Triglicéridos/metabolismoRESUMEN
Recent research on alpha-tocopherol has revealed specific cellular functions of this compound belonging to the vitamin E family. Alpha-tocopherol can act as a radical scavenger, as a pro-oxidant, as an anti-alkylation agent and, most important, by mechanisms that are independent of the above properties. To the last group belong protein kinase C and 5-lipoxygenase inhibition at post-translational level, as well as alpha-tocopherol activation of protein phosphatase 2A and diacylglycerol kinase. Furthermore, at transcriptional level, several genes (CD36, alpha-TTP, alpha-tropomyosin, and collagenase) are modulated by alpha-tocopherol. These effects result in inhibition of smooth muscle cell proliferation, platelet aggregation, and monocyte adhesion and may be related to the alleged protection of atherosclerosis by vitamin E. On the other side, epidemiological and intervention studies have shown some inconsistent results. Rather than disregarding vitamin E as a means to protect against atherosclerosis progression, it would be wiser to better design clinical trials based on current knowledge of the biological properties of the molecule.
Asunto(s)
Depuradores de Radicales Libres/metabolismo , Radicales Libres/metabolismo , Vitamina E/metabolismo , Animales , Arteriosclerosis/complicaciones , Arteriosclerosis/metabolismo , Progresión de la Enfermedad , Regulación de la Expresión Génica , Humanos , Enfermedades del Sistema Nervioso/complicaciones , Enfermedades del Sistema Nervioso/metabolismo , Transcripción Genética , Deficiencia de Vitamina E/complicaciones , Deficiencia de Vitamina E/metabolismo , alfa-Tocoferol/metabolismoRESUMEN
We have isolated the mouse MyoD1 gene flanked by its promoter region by screening a genomic library with synthetic oligonucleotides. The structural gene is interrupted by two G + C rich introns. Transfection of the cloned gene inserted into an expression vector converts fibroblasts to myoblasts. Sequence analysis of about 650 bp of the 5' upstream region revealed the presence of several potential regulatory elements such as a TATA-box, an AP2-box, two SP1-boxes and a CAAT-box. In addition, there are three half palindromic estrogen response elements, a potential cAMP response element and various muscle specific elements such as a muscle-specific CAAT-box (MCAT) and four potential binding sites for MyoD1. Using S1 protection analysis the major start site of transcription in muscle and myoblast cells was mapped 3 bp upstream of the published cDNA 5' end. Promoter activity of the 650 bp upstream fragment was tested by in vitro transcription and by transfection analysis of myoblasts and fibroblasts. In all promoter test systems used, MyoD1 promoter activity was detected in myoblasts as well as in fibroblasts. Furthermore, DNA methylation was found to turn off MyoD1 promoter activity both in myoblasts and in fibroblasts.
Asunto(s)
Genes , Proteína MioD , Proteínas Nucleares/genética , Fosfoproteínas/genética , Regiones Promotoras Genéticas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN , Fosfatos de Dinucleósidos/metabolismo , Fibroblastos/citología , Regulación de la Expresión Génica , Biblioteca Genómica , Metilación , Ratones , Datos de Secuencia Molecular , Músculos/citología , Sondas de Oligonucleótidos , Especificidad de Órganos/genética , Mapeo Restrictivo , Transcripción Genética , TransfecciónRESUMEN
We show that in mouse myoblasts the MyoD1 promoter is highly stimulated by MyoD1 expression, suggesting that it is controlled by a positive feedback loop. Using deletion and mutation analyses, we identified the targets for MyoD1 promoter autoregulation as the two proximal E-boxes located close to the MyoD1 core promoter. Gel mobility shift competition assays with MyoD1 antibodies as competitor suggest that the MyoD1 protein is binding directly to these E-boxes. Autoregulation did not occur in fibroblasts cotransfected with the expression vector of MyoD1. It is assumed that autoregulation is controlled by the stoichiometry between the MyoD1 protein and negatively regulatory proteins like Id, which is known to be highly expressed in fibroblasts. When the MyoD1 promoter was methylated, autoregulation only occurred when the density of methylated sites was low. The density of DNA methylation, therefore, can determine the accessibility of the MyoD1 promoter to transcription factors and interfere with the auto- and crossregulatory loop. The MyoD1 promoter in vivo was found to be only partially methylated in all tissues tested except in skeletal muscle where it was demethylated. We propose that high level expression of the MyoD1 gene is a result of release from constraints such as negative regulatory factors and/or DNA methylation interfering with MyoD1 autoregulation.
Asunto(s)
Regulación de la Expresión Génica , Proteína MioD/genética , Regiones Promotoras Genéticas , Transactivadores/genética , Animales , Secuencia de Bases , Línea Celular , ADN , Elementos de Facilitación Genéticos , Retroalimentación , Fibroblastos/citología , Metilación , Ratones , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Músculos/citología , Proteína MioD/metabolismo , Unión Proteica , Transactivadores/metabolismoRESUMEN
We have studied the transcriptional activity of the mouse MyoD1 gene promoter in vivo and in vitro using mouse G8 myoblasts and muscle cell nuclear extracts. 5' deletion analysis of the promoter and transcription-competition analysis using oligonucleotides corresponding to several cis-acting elements revealed that the basal activity of the MyoD1 promoter is conferred by two SP1 boxes, an AP-2 box, and a CAAT box. We have identified a negative regulatory sequence located between nucleotide position -342 to -322 with respect to the cap site. The negative regulatory element shows sequence homology with cAMP-responsive element (CRE) and AP-1 binding site (5'-GAGCACTGAGGTCAGTACAG-3'). As determined by gel mobility shift competition analysis, oligonucleotides containing AP-1 binding sites inhibit protein interactions with the MyoD1 CRE-like element. We also show that binding to this element is down-regulated during myogenic differentiation and can be reinduced by the addition of serum. Furthermore, mutation of the CRE-like element induces MyoD promoter activity in diving myoblasts. By using anti-c-Fos antibodies we show that AP-1 is binding to the MyoD1 CRE-like element. Our results indicate that AP-1 negatively modulates MyoD1 expression in growing myoblasts and strongly suggest that c-Fos and c-Jun inhibit myogenesis and MyoD1 expression by direct binding to a negative cis-acting element in the MyoD1 promoter.
Asunto(s)
AMP Cíclico/metabolismo , Expresión Génica , Músculos/citología , Músculos/metabolismo , Proteína MioD/biosíntesis , Proteína MioD/genética , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-jun/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , División Celular , Línea Celular , Cartilla de ADN , Genes fos , Genes jun , Ratones , Datos de Secuencia Molecular , Proteínas Musculares/biosíntesis , Proteínas Musculares/genética , Oligodesoxirribonucleótidos/síntesis química , Oligodesoxirribonucleótidos/farmacología , Plásmidos , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-jun/biosíntesis , Ratas , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Transcripción Genética/efectos de los fármacosRESUMEN
Most prokaryotic (cytosine-5)-DNA methyltransferases increase the frequency of deamination at the cytosine targeted for methylation in vitro in the absence of the cofactor S-adenosylmethionine (AdoMet) or the reaction product S-adenosylhomocysteine (AdoHcy). We show here that, under the same in vitro conditions, the prokaryotic methyltransferase, M.MspI (from Moraxella sp.), causes very few cytosine deaminations, suggesting a mechanism in which M.MspI may avoid enzyme-mediated cytosine deamination. Two analogues of AdoMet, sinefungin and 5'-amino-5'-deoxyadenosine, greatly increased the frequency of cytosine deamination mediated by M.MspI presumably by introducing a proton-donating amino group into the catalytic centre, thus facilitating the formation of an unstable enzyme-dihydrocytosine intermediate and hydrolytic deamination. Interestingly, two naturally occurring analogues, adenosine and 5'-methylthio-5'-deoxyadenosine, which do not contain a proton-donating amino group, also weakly increased the deamination frequency by M.MspI, even in the presence of AdoMet or AdoHcy. These analogues may trigger a conformational change in the enzyme without completely inhibiting the access of solvent water to the catalytic centre, thus allowing hydrolytic deamination of the enzyme-dihydrocytosine intermediate. Under normal physiological conditions the enzymes M.HpaII (from Haemophilus parainfluenzae), M. HhaI (from Haemophilus hemolytica) and M.MspI all increased the in vivo deamination frequency at the target cytosines with comparable efficiency.
Asunto(s)
Proteínas Bacterianas/metabolismo , Citosina/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Moraxella/enzimología , Adenosina/análogos & derivados , Adenosina/metabolismo , Proteínas de Unión al ADN/análisis , Desaminación , Desoxiadenosinas/metabolismo , Estructura Molecular , Mutación/genética , Neomicina/química , S-Adenosilhomocisteína/análogos & derivados , S-Adenosilhomocisteína/metabolismo , S-Adenosilmetionina/análogos & derivados , S-Adenosilmetionina/metabolismoRESUMEN
DNA methylation is essential for normal embryonic development. Distinctive genomic methylation patterns must be formed and maintained with high fidelity to ensure the inactivities of specific promoters during development. The mutagenic and epigenetic aspects of DNA methylation are especially interesting because they may lead to the inactivation of genes which are involved in human carcinogenesis. The mutagenicity of 5-Methylcytosine (5mC) and the role of promoter hypermethylation in gene silencing, particularly in cancer, suggest a clinical significance for the design of novel DNA methylation inhibitors which may be utilized to reverse the effects of DNA methylation.
Asunto(s)
Metilación de ADN/efectos de los fármacos , Diseño de Fármacos , Animales , Humanos , Neoplasias/genéticaRESUMEN
Lipoproteins modified by oxidation, glycation, alkylation, and nitration are generated by oxidative stress during inflammation, diabetes, and inadequate supply of dietary antioxidants. A family of genes, the scavenger receptors, recognizes and internalizes modified lipoproteins, making them susceptible to degradation. Clearance of modified lipoproteins by scavenger receptors occurs mainly in macrophages, dendritic cells, and Kupffer cells of the liver. However, scavenger receptor expression also occurs in other cells, such as endothelial cells, aortic smooth muscle cells, neuronal cells, and keratinocytes. Thus, the local clearance of oxidized low-density lipoprotein and the resolution of inflammatory processes may rely in part on the expression of scavenger receptors in "nonprofessional" phagocytes. Uptake of oxidized low-density lipoprotein, without an efficient machinery to degrade them and uncontrolled expression of scavenger receptors, may lead to cellular deregulation, apoptosis, and formation of foam cells. Diseases accompanied by oxidation of lipoproteins, such as atherosclerosis, Alzheimer disease, glomerulosclerosis, ataxia with vitamin E deficiency, and possibly age-dependent lipofuscin deposition, may share a common pathogenetic feature. This review will focus on foam cell formation, mainly within the atherosclerotic lesion, and the possible involvement of aberrant regulation of the scavenger receptor genes. To date, the regulatory mechanisms at the basis of scavenger receptor gene expression and their roles in atherosclerosis and other diseases are not well established. Knowledge on this subject could lead to a better understanding of the pathogenesis, prevention, and therapy of these diseases.
Asunto(s)
Lipoproteínas LDL/metabolismo , Proteínas de la Membrana , Receptores Inmunológicos/metabolismo , Receptores de Lipoproteína , Animales , Aorta/metabolismo , Apoptosis , Arteriosclerosis/metabolismo , Arteriosclerosis/patología , Adhesión Celular , Células Dendríticas/metabolismo , Endotelio Vascular/metabolismo , Humanos , Queratinocitos/metabolismo , Ligandos , Macrófagos/metabolismo , Modelos Biológicos , Músculo Liso Vascular/metabolismo , Neuronas/metabolismo , Estrés Oxidativo , Oxígeno/metabolismo , Fagocitosis , Receptores Depuradores , Receptores Depuradores de Clase B , Transducción de SeñalRESUMEN
Atherosclerosis and its complications such as coronary heart disease, myocardial infarction and stroke are the leading causes of death in the developed world. High blood pressure, diabetes, smoking and a diet high in cholesterol and lipids clearly increase the likelihood of premature atherosclerosis, albeit other factors, such as the individual genetic makeup, may play an additional role. Several epidemiological studies and intervention trials have been performed with vitamin E, and some of them showed that it prevents atherosclerosis. For a long time, vitamin E was assumed to act by decreasing the oxidation of LDL, a key step in atherosclerosis initiation. However, at the cellular level, vitamin E acts by inhibition of smooth muscle cell proliferation, platelet aggregation, monocyte adhesion, oxLDL uptake and cytokine production, all reactions implied in the progression of atherosclerosis. Recent research revealed that these effects are not the result of the antioxidant activity of vitamin E, but rather of precise molecular actions of this compound. It is assumed that specific interactions of vitamin E with enzymes and proteins are at the basis of its non-antioxidant effects. Vitamin E influences the activity of several enzymes (e.g. PKC, PP2A, COX-2, 5-lipooxygenase, nitric oxide synthase, NADPH-oxidase, superoxide dismutase, phopholipase A2) and modulates the expression of genes that are involved in atherosclerosis (e.g. scavenger receptors, integrins, selectins, cytokines, cyclins). These interactions promise to reveal the biological properties of vitamin E and allow designing better strategies for the protection against atherosclerosis progression.