Participation of prostaglandin D2 in the mobilization of the nuclear-localized CTP:phosphocholine cytidylyltransferase alpha in renal epithelial cells.

Phosphatidylcholine (PC) is the main constituent of mammalian cell membranes. Consequently, preservation of membrane PC content and composition - PC homeostasis - is crucial to maintain cellular life. PC biosynthetic pathway is generally controlled by CTP:phosphocholine cytidylyltransferase (CCT), which is considered the rate-limiting enzyme. CCTα is an amphitropic protein, whose enzymatic activity is commonly associated with endoplasmic reticulum redistribution. However, most of the enzyme is located inside the nuclei. Here, we demonstrate that CCTα is the most abundant isoform in renal collecting duct cells, and its redistribution is dependent on endogenous prostaglandins. Previously we have demonstrated that PC synthesis was inhibited by indomethacin (Indo) treatment, and this effect was reverted by exogenous PGD(2). In this work we found that Indo induced CCTα distribution into intranuclear Lamin A/C foci. Exogenous PGD(2) reverted this effect by inducing CCTα redistribution to nuclear envelope, suggesting that PGD(2) maintains PC synthesis by CCTα mobilization. Interestingly, we found that the effect of PGD(2) was dependent on ERK1/2 activation. In conclusion, our previous observations and the present results lead us to suggest that papillary cells possess the ability to maintain their structural integrity through the synthesis of their own survival molecule, PGD(2), by modulating CCTα intracellular location.

Expression of phosphatidylcholine biosynthetic enzymes during early embryogenesis in the amphibian Bufo arenarum.

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.

Choline kinase alpha expression during RA-induced neuronal differentiation: role of C/EBPß.

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.

Mutations in PCYT1A cause spondylometaphyseal dysplasia with cone-rod dystrophy.

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.

Mechanistic insights into the nongenomic regulation of phospholipid synthesizing enzymes.

Lipid synthesis is a complex process regulated at multiple levels. Here, we will discuss nongenomic regulatory mechanisms, particularly the activation and/or recruitment of key enzymes to membranes. The phospholipid synthesis enzymes Lipin and CTP:phosphocholine cytidylyltransferase are taken as examples of these mechanisms that are mediated by posttranslational modifications or by an intrinsic property of the enzyme that senses lipid composition. In addition, special emphasis will be put on another relevant non genomic lipid synthesis regulation mechanism that is dependent on c-Fos, a protein that has deserved less attention so far. This latter regulatory mechanism is emerging as an important determinant for processes that require high rates of lipid synthesis such as those of growth and proliferation.

Effects of miltefosine on the proliferation, ultrastructure, and phospholipid composition of Angomonas deanei, a trypanosomatid protozoan that harbors a symbiotic bacterium.

Some trypanosomatids, such as Angomonas deanei formerly named as Crithidia deanei, present an obligatory intracellular bacterium, which maintains a mutualistic relationship with the host. Phosphatidylcholine (PC) is the major phospholipid in eukaryotes and an essential component of cell membranes playing structural, biochemical, and physiological roles. However, in prokaryotes, PC is present only in those species closely associated with eukaryotes, either in symbiotic or pathogenic interactions. In trypanosomatids, the endosymbiont envelope is composed by a reduced cell wall and by two membrane units that lack sterols and present cardiolipin (CL) and PC as the major phospholipids. In this study, we tested the effects of miltefosine in A. deanei proliferation, as well as, on the ultrastrucuture and phospholipid composition considering that this drug inhibits the CTP-phosphocholine cytidyltransferase (CCT), a key enzyme in the PC biosynthesis. Besides the low effect of miltefosine in cellular proliferation, treated protozoa presented ultrastructural alterations such as plasma membrane shedding and blebbing, mitochondrial swelling, and convolutions of the endosymbiont envelope. The use of (32) Pi as a tracer revealed that the production of PC, CL, and phosphatidylethanolamine decreased while phosphatidylinositol production remained stable. Mitochondrion and symbiont fractions obtained from protozoa treated with miltefosine also presented a decrease in phospholipid production, reinforcing the idea that an intensive metabolic exchange occurs between the host trypanosomatid and structures of symbiotic origin.

Role of phosphatidylcholine during neuronal differentiation.

Neuronal differentiation is characterized by neuritogenesis and neurite outgrowth, processes, which are critically dependent on membrane biosynthesis, and therefore, on the expression and regulation of enzymes involved in phospholipid biosynthesis. During the last decade a great effort was made to clarify where membrane lipids are synthesized, how the newly synthesized membrane components reach the membrane and are inserted during neuritogenesis and to elucidate the mechanism by which the supply of new membrane components is coordinated with the demand for growth. Phosphatidylcholine is the principal and essential component for mammalian membranes. This review updates the mechanism by which phosphatidylcholine biosynthesis takes place and how it is coordinately regulated during neuronal differentiation.

Specific interaction between E2F1 and Sp1 regulates the expression of murine CTP:phosphocholine cytidylyltransferase alpha during the S phase.

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.

Characterization of the murine CTP:phosphocholine cytidylyltransferase beta gene promoter.

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.

Zn-limited diet modifies the expression of the rate-regulatory enzymes involved in phosphatidylcholine and cholesterol synthesis.

Suboptimal intake of Zn is one of the most common nutritional worldwide problems. Previously, we showed that Zn deficiency produces alterations in lung lipid metabolism in rats. We studied the effect of a Zn-limited (ZL) diet on the expression of the enzymes involved in phosphatidylcholine and cholesterol synthesis. After 2 months of treatment with a ZL diet we found important variations in the lipid content of Wistar male rats: triacylglycerol (TG) decreased 60% (P<0.001) while esterified cholesterol (EC), free cholesterol and phospholipids (PL) increased 66%, 24 % and 25% respectively. We also observed a decrease of 40 % in the amount of (3)H incorporated into TG and an increase of 47% and 28% in the (3)H incorporated to PL and EC respectively. Fatty acid synthase and glucose-6-phosphate dehydrogenase activity was increased (P<0.01 and P<0.05 respectively). Glycerol-3-phosphate acyltransferase, lipoprotein lipase, diacyl glycerol acyl transferase and 3-hydroxy-3-methylglutaryl CoA reductase expression decreased (P<0.01 in all cases), while acetyl CoA carboxylase and cholinephosphate cytidylyltransferase increased (P<0.01 and P<0.005 respectively). These results suggest that ZL alters the expression of enzymes involved in phosphatidylcholine and cholesterol synthesis, which could lead to increased PL and cholesterol and decreased TG. This study suggests that major changes in the lipid composition of lung are induced by a ZL condition. Therefore, Zn deficiency must be taken into account in order to design therapies and public health interventions, such as Zn supplementation for high-risk subjects or certain diseases, such as asthma.

Phosphatidylcholine deficiency upregulates enzymes of triacylglycerol metabolism in CHO cells.

We studied the regulation of triacylglycerol (TAG) metabolism by phosphatidylcholine (PC) in CHO MT58 cells, which are deficient in PC synthesis because of a temperature-sensitive CTP:phosphocholine cytidylyltransferase. At the permissive growth temperature (34 degrees C), these cells contained 49% less TAG and 30% less PC than wild-type CHO K1 cells. Treatment with dipalmitoylphosphatidylcholine normalized both the PC and TAG levels. Despite low TAG levels, the incorporation of [14C]oleate into TAG was increased in CHO MT58 cells. The in vitro de novo synthesis of TAG and the activity of diacylglycerol acyltransferase were 90% and 34% higher, respectively. Two other key enzyme activities in TAG synthesis, acyl-CoA synthetase and mitochondrial glycerol-3-phosphate acyltransferase (GPAT), increased by 48% and 2-fold, respectively, and mitochondrial GPAT mRNA increased by approximately 4-fold. Additionally, TAG hydrolysis was accelerated in CHO MT58 cells, and in vitro lipolytic activity increased by 68%. These studies suggest that a homeostatic mechanism increases TAG synthesis and recycling in response to PC deficiency. TAG recycling produces diacylglycerol and fatty acids that can be substrates for de novo PC synthesis and for lysophosphatidylcholine (lysoPC) acylation. In CHO MT58 cells, in which de novo PC synthesis is blocked, lysoPC acylation with fatty acid originating from TAG may represent the main pathway for generating PC.

COX-2-mediated PGD2 synthesis regulates phosphatidylcholine biosynthesis in rat renal papillary tissue.

Phosphatidylcholine (PC) is the major membrane phospholipid in mammalian cells. Previous works from our laboratory demonstrated a close metabolic relationship between the maintenance of PC biosynthesis and the prostaglandins endogenously synthesized by cyclooxygenase (COX) in rat renal papilla. In the present work, we studied the COX isoform involved in papillary PC biosynthesis regulation. The incorporation of [methyl-3H]choline and [32P]orthophosphate to PC was determined in the absence and presence of SC-560 and NS-398, COX-1 and COX-2 specific inhibitors. PC synthesis was highly sensitive to COX-2 inhibition, while COX-1 inhibition only reduced PC synthesis at high SC-560 concentration. The analysis of choline-containing metabolites showed that COX-2 inhibition affected the formation of CDP-choline intermediary. The evaluation of PC biosynthetic enzymes revealed that microsomal, as well as nuclear, CTP:phosphocholine cytidylyltransferase (CCT), and nuclear-CDP-choline:1,2-diacylglycerol cholinephosphotransferase (CTP) activities were affected by COX-2 inhibition. The addition of exogenous prostaglandin D(2) (PGD(2)) restored nuclear-CCT and -CPT activities but not microsomal CCT. Papillary synthesis of PGD(2) was only detected in nuclear fraction where it was blocked by COX-2 inhibitor NS-398, but not by COX-1 inhibitor. All together, the present results demonstrated that COX-2-mediated PGD(2) synthesis is a PC biosynthesis regulator in rat renal papilla. Considering the importance of the maintenance of PC biosynthesis for the preservation of cell membrane homeostasis to ensure cell viability, and the extensive use of COX-2 inhibitors in therapeutics, the present results could have great pharmacological implications, and can constitute a biochemical explanation for the nephrotoxic effect of non-steroidal anti-inflammatory drugs.

Phospholipase C inhibitors and prostaglandins differentially regulate phosphatidylcholine synthesis in rat renal papilla. Evidence of compartmental regulation of CTP:phosphocholine cytidylyltransferase and CDP-choline:1,2-diacylglycerol cholinephosphotransferase.

Phosphatidylcholine (PC) is the most abundant phospholipid in mammalian cell membranes. Several lines of evidence support that PC homeostasis is preserved by the equilibrium between PC biosynthetic enzymes and phospholipases catabolic activities. We have previously shown that papillary synthesis of PC depends on prostaglandins (PGs) that modulate biosynthetic enzymes. In papillary tissue, under bradikynin stimulus, arachidonic acid (AA) mobilization (the substrate for PG synthesis) requires a previous phospholipase C (PLC) activation. Thus, in the present work, we study the possible involvement of PLC in PC biosynthesis and its relationship with PG biosynthetic pathway on the maintenance of phospholipid renewal in papillary membranes; we also evaluated the relevance of CDP-choline pathway enzymes compartmentalization. To this end, neomycin, U-73122 and dibutiryl cyclic AMP, reported as PLC inhibitors, were used to study PC synthesis in rat renal papilla. All the PLC inhibitors assayed impaired PC synthesis. PG synthesis was also blocked by PLC inhibitors without affecting cyclooxygenase activity, indicating a metabolic connection between both pathways. However, we found that PC biosynthesis decrease in the presence of PLC inhibitors was not a consequence of PG decreased synthesis, suggesting that basal PLC activity and PGs exert their effect on different targets of PC biosynthetic pathway. The study of PC biosynthetic enzymes showed that PLC inhibitors affect CTP:phosphocholine cytidylyltransferase (CCT) activity while PGD(2) operates on CDP-choline:1,2-diacylglycerol cholinephosphotransferase (CPT), both activities associated to papillary enriched-nuclei fraction. The present results suggest that renal papillary PC synthesis is a highly regulated process under basal conditions. Such regulation might occur at least at two different levels of the CDP-choline pathway: on the one hand, PLC operates on CCT activity; on the other, while PGs regulate CPT activity.