RESUMO
In the principal route of phosphatidylcholine (PC) synthesis the regulatory steps are catalysed by CTP:phosphocholine cytidylyltransferase (CCT) and choline kinase (CK). Knock-out mice in Pcyt1a (CCT gene) and Chka1 (CK gene) resulted in preimplantation embryonic lethality, demonstrating the essential role of this pathway. However, there is still a lack of detailed CCT and CK expression analysis during development. The aim of the current work was to study the expression during early development of both enzymes in the external-fertilization vertebrate Bufo arenarum. Reverse transcription polymerase chain reaction (RT-PCR) and western blot confirmed their presence in unfertilized eggs. Analysis performed in total extracts from staged embryos showed constant protein levels of both enzymes until the 32-cell stage: then they decreased, reaching a minimum in the gastrula before starting to recover. CTP:phosphocholine cytidylyltransferase is an amphitropic enzyme that inter-converts between cytosolic inactive and membrane-bound active forms. Immunoblot analysis demonstrated that the cytosolic:total CCT protein ratio does not change throughout embryogenesis, suggesting a progressive decline of CCT activity in early development. However, PC (and phosphatidylethanolamine) content per egg/embryo remained constant throughout the stages analysed. In conclusion, the current data for B. arenarum suggest that net synthesis of PC mediated by CCT and CK is not required in early development and that supplies for membrane biosynthesis are fulfilled by lipids already present in the egg/embryo reservoirs.
Assuntos
Bufo arenarum/embriologia , Colina Quinase/metabolismo , Colina-Fosfato Citidililtransferase/metabolismo , Embrião não Mamífero/enzimologia , Fosfatidilcolinas/metabolismo , Proteínas de Anfíbios/genética , Proteínas de Anfíbios/metabolismo , Animais , Bufo arenarum/metabolismo , Colina Quinase/genética , Colina-Fosfato Citidililtransferase/genética , Citosol/enzimologia , Feminino , Masculino , Óvulo/enzimologia , Fosfatidilcolinas/biossíntese , Fosfatidiletanolaminas/metabolismoRESUMO
Neuronal differentiation is a complex process characterized by a halt in proliferation and extension of neurites from the cell body. This process is accompanied by changes in gene expression that mediate the redirection leading to neurite formation and function. Acceleration of membrane phospholipids synthesis is associated with neurite elongation, and phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells. The transcription of two genes in particular encoding key enzymes in the CDP-choline pathway for PtdCho biosynthesis are stimulated; the Chka gene for choline kinase (CK) alpha isoform and the Pcyt1a gene for the CTP:phosphocholine cytidylyltransferase (CCT) alpha isoform. We report that the stimulation of CKα expression during retinoic acid (RA) induced differentiation depends on a promoter region that contains two CCAAT/Enhancer-binding Protein-ß (C/EBPß) sites. We demonstrate that during neuronal differentiation of Neuro-2a cells, RA induces Chka expression by a mechanism that involves ERK1/2 activation which triggers C/EBPß expression. Elevated levels of C/EBPß bind to the Chka proximal promoter (Box1) inducing CKα expression. In addition we identified a downstream sequence named Box2 which together with Box1 is required for the promoter to reach the full induction. This is the first elucidation of the mechanism by which the expression of Chka is coordinately regulated during neuronal differentiation.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/genética , Diferenciação Celular/efeitos dos fármacos , Neurônios/metabolismo , Fosfolipídeos/biossíntese , Animais , Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Proliferação de Células , Colina Quinase/biossíntese , Colina Quinase/metabolismo , Colina-Fosfato Citidililtransferase/genética , Colina-Fosfato Citidililtransferase/metabolismo , Humanos , Camundongos , Neuritos/metabolismo , Neurônios/citologia , Fosfatidilcolinas/metabolismo , Fosfolipídeos/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Tretinoína/farmacologiaRESUMO
Spondylometaphyseal dysplasia with cone-rod dystrophy is a rare autosomal-recessive disorder characterized by severe short stature, progressive lower-limb bowing, flattened vertebral bodies, metaphyseal involvement, and visual impairment caused by cone-rod dystrophy. Whole-exome sequencing of four individuals affected by this disorder from two Brazilian families identified two previously unreported homozygous mutations in PCYT1A. This gene encodes the alpha isoform of the phosphate cytidylyltransferase 1 choline enzyme, which is responsible for converting phosphocholine into cytidine diphosphate-choline, a key intermediate step in the phosphatidylcholine biosynthesis pathway. A different enzymatic defect in this pathway has been previously associated with a muscular dystrophy with mitochondrial structural abnormalities that does not have cartilage and/or bone or retinal involvement. Thus, the deregulation of the phosphatidylcholine pathway may play a role in multiple genetic diseases in humans, and further studies are necessary to uncover its precise pathogenic mechanisms and the entirety of its phenotypic spectrum.
Assuntos
Colina-Fosfato Citidililtransferase/genética , Osteocondrodisplasias/genética , Retinose Pigmentar/genética , Adolescente , Brasil , Criança , Pré-Escolar , Colina-Fosfato Citidililtransferase/metabolismo , Feminino , Genes Recessivos , Homozigoto , Humanos , Lactente , Masculino , Oftalmologia/métodos , LinhagemRESUMO
Neuronal differentiation is characterized by neuritogenesis and neurite outgrowth, processes that are dependent on membrane biosynthesis. Thus, the production of phosphatidylcholine (PtdCho), the major membrane phospholipid, should be stimulated during neuronal differentiation. We demonstrate that during retinoic acid (RA)-induced differentiation of Neuro-2a cells, PtdCho synthesis was promoted by an ordered and sequential activation of choline kinase alpha (CK(alpha)) and choline cytidylyltransferase alpha (CCT(alpha)). Early after RA stimulation, the increase in PtdCho synthesis is mainly governed by the biochemical activation of CCT(alpha). Later, the transcription of CK(alpha)- and CCT(alpha)-encoding genes was induced. Both PtdCho biosynthesis and neuronal differentiation are dependent on ERK activation. A novel mechanism is proposed by which PtdCho biosynthesis is coordinated during neuronal differentiation. Enforced expression of either CK(alpha) or CCTalpha increased the rate of synthesis and the amount of PtdCho, and these cells initiated differentiation without RA stimulation, as evidenced by cell morphology and the expression of genes associated with neuritogenesis. The differentiation resulting from enforced expression of CCT(alpha) or CK(alpha) was dependent on persistent ERK activation. These results indicate that elevated PtdCho synthesis could mimic the RA signals and thus determine neuronal cell fate. Moreover, they could explain the key role that PtdCho plays during neuronal regeneration.
Assuntos
Neurônios/citologia , Neurônios/metabolismo , Fosfatidilcolinas/biossíntese , Animais , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Colina Quinase/genética , Colina-Fosfato Citidililtransferase/genética , Imunofluorescência , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Análise de Sequência com Séries de Oligonucleotídeos , Fosfatidilcolinas/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Tretinoína/farmacologiaRESUMO
CTP:phosphocholine cytidylyltransferase alpha (CCTalpha) is a key enzyme for phosphatidylcholine biosynthesis in mammalian cells. This enzyme plays an essential role in all processes that require membrane biosynthesis such as cell proliferation and viability. Thus, CCTalpha activity and expression fluctuate during the cell cycle to achieve PtdCho requirements. We demonstrated, for the first time, that CCTalpha is localized in the nucleus in cells transiting the S phase, whereas it is localized in the cytoplasm of G(0)-arrested cells, suggesting a specific role of nuclear CCTalpha during the S phase. We also investigated how E2F1 influences the regulation of the CCTalpha-promoter during the S phase; we demonstrated that E2F1 is necessary, but not sufficient, to activate CCTalpha expression when this factor is over-expressed. However, when E2F1 and Sp1 were over-expressed, the transcription from the CCTalpha-promoter reporter construct was super-activated. Transient transfection studies demonstrated that E2F1 could super-activate Sp1-dependent transcription in a promoter containing only the Sp1 binding sites "B" or "C", and that Sp1 could activate Sp1-dependent transcription in a promoter containing the E2F site, thus, further demonstrating a functional interaction of these factors. In conclusion, the present results allowed us to portray the clearest picture of the CCTalpha-gene expression in proliferating cells, and understand the mechanism by which cells coordinate cell cycle progression with the requirement for phosphatidylcholine.
Assuntos
Colina-Fosfato Citidililtransferase/metabolismo , Fator de Transcrição E2F1/metabolismo , Fase S , Fator de Transcrição Sp1/metabolismo , Animais , Sítios de Ligação , Colina-Fosfato Citidililtransferase/genética , Imunoprecipitação da Cromatina , Citidina Trifosfato/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Enzimológica da Expressão Gênica , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos C3H , Regiões Promotoras GenéticasRESUMO
CTP:phosphocholine cytidylyltransferase (CCT) is a key regulatory enzyme in phosphatidylcholine (PtdCho) biosynthesis by the Kennedy pathway. In mammals, there are two genes that encode the enzyme isoforms that catalyze this reaction: Pcyt1a for CCTalpha and Pcyt1b for CCTbeta. In mouse tissues two different CCTbeta variants named CCTbeta2 and CCTbeta3 have been identified. Although little is known about Pcyt1b gene expression, recent data from cell lines propose a distinct role for CCTbeta2 in neuronal differentiation. Also, gonadal dysfunction in the CCTbeta2 knockout mouse suggests a role for this protein in ovary maturation and the maintenance of sperm production. This work defines and characterizes two alternative promoters that drive the expression of the two murine CCTbeta isoforms. The promoter activities were measured in Neuro-2a (mouse neuroblastoma), TM4 (mouse Sertoli) and C3H10T1/2 (mouse embryo fibroblast) cell lines. The transcriptional start points of each transcript and the promoter regions essential for the expression of each isoform were determined. Analysis of the CCTbeta2 promoter sequence suggested the transcription factor AP-1 as a potential regulator of CCTbeta2 expression in neuronal cells. However, CCTbeta3 was not detected in this cell line suggesting a different role or regulation. The activities of alternative promoters provide for greater flexibility in the control of CCTbeta isoform expression.
Assuntos
Colina-Fosfato Citidililtransferase/genética , Isoenzimas/genética , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Encéfalo/enzimologia , Linhagem Celular , Colina-Fosfato Citidililtransferase/metabolismo , Biologia Computacional , Deleção de Genes , Isoenzimas/metabolismo , Camundongos , Dados de Sequência MolecularRESUMO
Antioxidants are known to reduce cardiovascular disease by reducing the concentration of free radicals in the vessel wall and by preventing the oxidative modification of low-density lipoproteins. The prooxidative effect of a vitamin-A-deficient diet on the aorta has previously been demonstrated by us. In this study, the lipid metabolism in the aorta of rats fed on a vitamin-A-deficient diet was evaluated. Vitamin A deficiency induced a hypolipidemic effect (lower serum triglyceride and cholesterol levels) and a decreased serum paraoxonase 1/arylesterase activity. The concentrations of triglycerides, total cholesterol, free and esterified cholesterol, and phospholipids were increased in the aorta of vitamin-A-deficient rats. The phospholipid compositions showed an increase in phosphatidylcholine (PC), phosphatidylinositol plus phosphatidylserine and phosphatidylethanolamine, a decrease in sphingomyelin, and no change in phosphatidylglycerol. In the aorta, the increase in triglycerides was associated with an increased fatty acid synthesis and mRNA expression of diacylglycerol acyltransferase 1. The increased PC content was attributed to an increased synthesis, as measured by [methyl-(14)C]choline incorporation into PC and high CTP:phosphocholine cytidylyltransferase-alpha mRNA expression. The cholesterol synthesis, evaluated by [1-(14)C]acetate incorporated into cholesterol and mRNA expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase, did not change. The lipoprotein lipase and lectin-like oxidized low-density lipoprotein receptor 1 mRNA expression levels increased in the aorta of vitamin-A-deficient animals. The incorporation of vitamin A into the diet of vitamin-A-deficient rats reverted all the changes observed. These results indicate that a vitamin-A-deficient diet,in addition to having a prooxidative effect, alters the aorta lipid metabolism.