CTP:phosphocholine Cytidylyltransferase Activity Research Articles

Interdomain communication in the phosphatidylcholine regulatory enzyme, CCTα, relies on a modular αE helix

CTP:phosphocholine cytidylyltransferase (CCT), the rate-limiting enzyme in phosphatidylcholine (PC) synthesis, is an amphitropic enzyme that regulates PC homeostasis. Recent work has suggested that CCTα activation by binding to a PC-deficient membrane involves conformational transitions in a helix pair (αE) that, along with a short linker of unknown structure (J segment), bridges the catalytic domains of the CCTα dimer to the membrane-binding (M) domains. In the soluble, inactive form, the αE helices are constrained into unbroken helices by contacts with two auto-inhibitory (AI) helices from domain M. In the active, membrane-bound form, the AI helices are displaced and engage the membrane. Molecular dynamics simulations have suggested that AI displacement is associated with hinge-like bending in the middle of the αE, positioning its C terminus closer to the active site. Here, we show that CCTα activation by membrane binding is sensitive to mutations in the αE and J segments, especially within or proximal to the αE hinge. Substituting Tyr-213 within this hinge with smaller uncharged amino acids that could destabilize interactions between the αE helices increased both constitutive and lipid-dependent activities, supporting a link between αE helix bending and stimulation of CCT activity. The solvent accessibilities of Tyr-213 and Tyr-216 suggested that these tyrosines move to new partially buried environments upon membrane binding of CCT, consistent with a folded αE/J structure. These data suggest that signal transduction through the modular αE helix pair relies on shifts in its conformational ensemble that are controlled by the AI helices and their displacement upon membrane binding.

Metformin Decouples Phospholipid Metabolism in Breast Cancer Cells.

IntroductionThe antidiabetic drug metformin, currently undergoing trials for cancer treatment, modulates lipid and glucose metabolism both crucial in phospholipid synthesis. Here the effect of treatment of breast tumour cells with metformin on phosphatidylcholine (PtdCho) metabolism which plays a key role in membrane synthesis and intracellular signalling has been examined.MethodsMDA-MB-468, BT474 and SKBr3 breast cancer cell lines were treated with metformin and [3H-methyl]choline and [14C(U)]glucose incorporation and lipid accumulation determined in the presence and absence of lipase inhibitors. Activities of choline kinase (CK), CTP:phosphocholine cytidylyl transferase (CCT) and PtdCho-phospholipase C (PLC) were also measured. [3H] Radiolabelled metabolites were determined using thin layer chromatography.ResultsMetformin-treated cells exhibited decreased formation of [3H]phosphocholine but increased accumulation of [3H]choline by PtdCho. CK and PLC activities were decreased and CCT activity increased by metformin-treatment. [14C] incorporation into fatty acids was decreased and into glycerol was increased in breast cancer cells treated with metformin incubated with [14C(U)]glucose.ConclusionThis is the first study to show that treatment of breast cancer cells with metformin induces profound changes in phospholipid metabolism.

Molecular Mechanism for the Thermo-Sensitive Phenotype of CHO-MT58 Cell Line Harbouring a Mutant CTP:Phosphocholine Cytidylyltransferase.

Control and elimination of malaria still represents a major public health challenge. Emerging parasite resistance to current therapies urges development of antimalarials with novel mechanism of action. Phospholipid biosynthesis of the Plasmodium parasite has been validated as promising candidate antimalarial target. The most prevalent de novo pathway for synthesis of phosphatidylcholine is the Kennedy pathway. Its regulatory and often also rate limiting step is catalyzed by CTP:phosphocholine cytidylyltransferase (CCT). The CHO-MT58 cell line expresses a mutant variant of CCT, and displays a thermo-sensitive phenotype. At non-permissive temperature (40°C), the endogenous CCT activity decreases dramatically, blocking membrane synthesis and ultimately leading to apoptosis. In the present study we investigated the impact of the analogous mutation in a catalytic domain construct of Plasmodium falciparum CCT in order to explore the underlying molecular mechanism that explains this phenotype. We used temperature dependent enzyme activity measurements and modeling to investigate the functionality of the mutant enzyme. Furthermore, MS measurements were performed to determine the oligomerization state of the protein, and MD simulations to assess the inter-subunit interactions in the dimer. Our results demonstrate that the R681H mutation does not directly influence enzyme catalytic activity. Instead, it provokes increased heat-sensitivity by destabilizing the CCT dimer. This can possibly explain the significance of the PfCCT pseudoheterodimer organization in ensuring proper enzymatic function. This also provide an explanation for the observed thermo-sensitive phenotype of CHO-MT58 cell line.

Expression of phosphatidylcholine biosynthetic enzymes during early embryogenesis in the amphibianBufo 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.

Abstract 1874: Phosphatidylcholine synthesis as a potential therapeutic target in multiple myeloma.

Abstract A metabolics screen with 8226 cells treated with AICAr showed that de novo pyrimidine synthesis was inhibited. AICAr -induced apoptosis in 5 different myeloma cell lines. This screen showed a significant decrease in CDP-choline, an intermediate of the Kennedy pathway involved in de novo synthesis of phosphatidylcholine (PC). Phosphatidylcholine is the most predominant phospholipid in mammalian ER membranes. The rate limiting step in this pathway is the conversion of phosphocholine and CTP to CDP-choline via the enzyme CTP:phosphocholine cytidylyltransferase (CCT). Although it is likely that the decrease in CDP-choline is a consequence of pyrimidine starvation, the fact that this pathway showed a significant decrease within 8 hours of AICAr treatment suggested to us that this pathway may have a high flux in 8226 cells and that phosphatidylcholine synthesis may be a relevant target in myeloma cells. Multiple myeloma (MM) cells may be particularly dependent on this biosynthetic reaction because of their high consistent level of ER stress and requirement to continuously replenish ER membranes. Indeed, CCT-null mice have a defect in differentiation of B lymphocytes to plasma cells and deficiencies in Ig synthesis. To test whether this pathway remains critical in survival of malignant MM cells, we exposed MM cell lines to an inhibitor shown to inhibit CCT activity, HexPC. HexPC induced apoptosis in all MM cell lines in a concentration- and time-dependent manner. The addition of lysophosphatidylcholine (LPC), presumably converted to PC independently of the Kennedy pathway, completely rescued MM cell apoptosis. Knocking down of CCT with shRNA in myeloma cell lines also resulted in apoptosis. Apoptosis of MM cells induced by HexPC was associated with induction of ER stress as shown by enhanced phosphorylation of IRE1 and eIF-2alpha. This ER stress was also prevented when LPC was added to HexPC although LPC could not prevent similar ER stress induced by bortezomib. These results underscore the importance of this phosphatidylcholine synthesis pathway in MM cells and provide new targets for future therapy. Citation Format: Carolyne Bardeleben, Alan Lichtenstein. Phosphatidylcholine synthesis as a potential therapeutic target in multiple myeloma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1874. doi:10.1158/1538-7445.AM2013-1874

Ras-induced Up-regulation of CTP:Phosphocholine Cytidylyltransferase α Contributes to Malignant Transformation of Intestinal Epithelial Cells

Cancer cells have enhanced lipogenic capacity characterized by increased synthesis of fatty acids and complex lipids, including phosphatidylcholine (PC). As the rate-limiting enzyme in the CDP-choline pathway for PC synthesis, CTP:phosphocholine cytidylyltransferase α (CCTα) is implicated in the provision of membranes and bioactive lipids necessary of cell proliferation. In this study, we assessed the role of CCTα in malignant intestinal epithelial cells transformed with activated H-ras (IEC-ras). Three IEC-ras clones had significant up-regulation CCTα expression, but PC synthesis and in vitro activity of CCTα were similar to control IEC. RNA interference of CCTα in adherent IEC-ras did not affect PC synthesis, confirming that the enzyme was relatively inactive. However, CCTα silencing in ras-transformed IEC reduced anchorage-independent growth, a criterion for malignant transformation, as well as tumorigenicity in mice. Relative to their adherent counterparts, detached IEC-ras had increased PC synthesis that was attenuated by inducible CCTα silencing. Detachment of IEC-ras was accompanied by increased CCTα phosphorylation and cytosolic enzyme activity. We conclude that the expanded pool of CCTα in IEC-ras is activated by detachment. This provides the increased PC biosynthetic capacity that contributes to malignant transformation of intestinal epithelial cells when detached from the extracellular matrix.

The Intrinsically Disordered Nuclear Localization Signal and Phosphorylation Segments Distinguish the Membrane Affinity of Two Cytidylyltransferase Isoforms

Membrane phosphatidylcholine homeostasis is maintained in part by a sensing device in the key regulatory enzyme, CTP:phosphocholine cytidylyltransferase (CCT). CCT responds to decreases in membrane phosphatidylcholine content by reversible membrane binding and activation. Two prominent isoforms, CCTα and -β2, have nearly identical catalytic domains and very similar membrane binding amphipathic helical (M) domains but have divergent and structurally disordered N-terminal (N) and C-terminal phosphorylation (P) regions. We found that the binding affinity of purified CCTβ2 for anionic membranes was weaker than CCTα by more than an order of magnitude. Using chimeric CCTs, insertion/deletion mutants, and truncated CCTs, we show that the stronger affinity of CCTα can be attributed in large part to the electrostatic membrane binding function of the polybasic nuclear localization signal (NLS) motif, present in the unstructured N-terminal segment of CCTα but lacking in CCTβ2. The membrane partitioning of CCTβ2 in cells enriched with the lipid activator, oleic acid, was also weaker than that of CCTα and was elevated by incorporation of the NLS motif. Thus, the polybasic NLS can function as a secondary membrane binding motif not only in vitro but in the context of cell membranes. A comparison of phosphorylated, dephosphorylated, and region P-truncated forms showed that the in vitro membrane affinity of CCTβ2 is more sensitive than CCTα to phosphorylation status, which antagonizes membrane binding of both isoforms. These data provide a model wherein the primary membrane binding motif, an amphipathic helical domain, works in collaboration with other intrinsically disordered segments that modulate membrane binding strength. The NLS reinforces, whereas the phosphorylated tail antagonizes the attraction of domain M for anionic membranes.

Nuclear export of the rate-limiting enzyme in phosphatidylcholine synthesis is mediated by its membrane binding domain

CTP:phosphocholine cytidylyltransferase alpha (CCTalpha), the rate-limiting enzyme in the CDP-choline pathway for phosphatidylcholine (PtdCho) synthesis, is activated by translocation to nuclear membranes. However, CCTalpha is cytoplasmic in cells with increased capacity for PtdCho synthesis and following acute activation, suggesting that nuclear export is linked to activation. The objective of this study was to identify which CCTalpha domains were involved in nuclear export in response to the lipid activators farnesol (FOH) and oleate. Imaging of CCT-green fluorescent protein (GFP) mutants expressed in CCTalpha-deficient CHO58 cells showed that FOH-mediated translocation to nuclear membranes and export to the cytoplasm required the membrane binding amphipathic helix (domain M). Nuclear export was reduced by a mutation that mimics constitutive phosphorylation of the CCT phosphorylation (P) domain. However, domain M alone was sufficient to promote translocation to the nuclear envelope and export of a nuclear-localized GFP construct in FOH- or oleate-treated CHO58 cells. In the context of acute activation with lipid mediators, nuclear export of CCT-GFP mutants correlated with in vitro activity but not PtdCho synthesis. This study describes a nuclear export pathway that is dependent on membrane interaction of an amphipathic helix, thus linking lipid-dependent activation to the nuclear/cytoplasmic distribution of CCTalpha.

Contribution of Each Membrane Binding Domain of the CTP:Phosphocholine Cytidylyltransferase-α Dimer to Its Activation, Membrane Binding, and Membrane Cross-bridging

CTP:phosphocholine cytidylyltransferase (CCT), a rate-limiting enzyme in phosphatidylcholine synthesis, is regulated by reversible membrane interactions mediated by an amphipathic helical domain (M) that binds selectively to anionic lipids. CCT is a dimer; thus the functional unit has two M domains. To probe the functional contribution of each domain M we prepared a CCT heterodimer composed of one full-length subunit paired with a CCT subunit truncated before domain M that was also catalytically dead. We compared this heterodimer to the full-length homodimer with respect to activation by anionic vesicles, vesicle binding affinities, and promotion of vesicle aggregation. Surprisingly for all three functions the dimer with just one domain M behaved similarly to the dimer with two M domains. Full activation of the wild-type subunit was not impaired by loss of one domain M in its partner. Membrane binding affinities were the same for dimers with one versus two M domains, suggesting that the two M domains of the dimer do not engage a single bilayer simultaneously. Vesicle cross-bridging was also unhindered by loss of one domain M, suggesting that another motif couples with domain M for cross-bridging anionic membranes. Mutagenesis revealed that the positively charged nuclear localization signal sequence constitutes that second motif for membrane cross-bridging. We propose that the two M domains of the CCT dimer engage a single bilayer via an alternating binding mechanism. The tethering function involves the cooperation of domain M and the nuclear localization signal sequence, each engaging separate membranes. Membrane binding of a single M domain is sufficient to fully activate the enzymatic activity of the CCT dimer while sustaining the low affinity, reversible membrane interaction required for regulation of CCT activity.

15-Deoxy-Δ12,14-prostaglandin J2 Impairs Phosphatidylcholine Synthesis and Induces Nuclear Accumulation of Thiol-modified Cytidylyltransferase

Synthesis of phosphatidylcholine, the major phospholipid of animal cell membranes, requires the key enzyme cytidylyltransferase (CCTalpha). Cysteine sulfhydryls within CCTalpha are needed for full catalytic activity. Here we show that prostaglandin 15-deoxy-Delta12,14-PGJ2 (15d-PGJ2) inactivates CCTalpha by inducing generation of reactive oxidant species and the appearance of a cross-linked CCTalpha dimer in cells. N-Acetyl-l-cysteine reduced oxidative stress, prevented CCTalpha cross-linking, and restored CCT function in 15d-PGJ2-treated cells. 15d-PGJ2 modified critical cysteine residues within CCTalpha as determined by mutagenesis studies and by incorporation of biotin-15d-PGJ2 into CCTalpha. These effects of 15d-PGJ2 were associated with CCTalpha accumulation within the nucleus. The data indicate that bioactive prostanoids significantly impair membrane phospholipid production by promoting cysteine cross-bridging within CCTalpha.

Probucol therapy overcomes the reproductive defect in CTP: phosphocholine cytidylyltransferase β2 knockout mice

The synthesis of phosphatidylcholine (PtdCho), the major phospholipid in mammalian cells, is regulated by the CTP:phosphocholine cytidylyltransferase (CCT). Loss of the CCTβ2 isoform expression in mice results in gonadal dysfunction. CCTβ2−/− females exhibit ovarian tissue disorganization with progressive loss of follicle formation and oocyte maturation. Ultrastructure revealed a disrupted association between ova and granulosa cells and disorganized Golgi apparati in oocytes of CCTβ2−/− mice. Probucol is a cholesterol-lowering agent that stimulates the uptake and retention of lipids carried by lipoproteins in peripheral tissues. Probucol therapy significantly lowered both serum cholesterol and PtdCho levels. Probucol therapy increased fertility in the CCTβ2−/− females 100%, although it did not completely correct the phenotype, the morphological abnormalities in the knockout ovaries or itself stimulate CCT activity directly. These data indicated that a deficiency in de novo PtdCho synthesis could be complemented by altering the metabolism of serum lipoproteins, an alternative source for cellular phospholipid.

Differential Targets and Subcellular Localization of Antitumor Alkyl-lysophospholipid in Leukemic Versus Solid Tumor Cells

Synthetic alkyl-lysophospholipids represent a family of promising anticancer drugs that induce apoptosis in a variety of tumor cells. Here we have found a differential subcellular distribution of the alkyl-lysophospholipid edelfosine in leukemic and solid tumor cells that leads to distinct anticancer responses. Edelfosine induced rapid apoptosis in human leukemic cells, including acute T-cell leukemia Jurkat and Peer cells, but promoted a late apoptotic response, preceded by G(2)/M arrest, in human solid tumor cells such as cervix epitheloid carcinoma HeLa cells and lung carcinoma A549 cells. c-Jun amino-terminal kinase (JNK) and caspase-3 were accordingly activated at earlier times in edelfosine-treated Jurkat cells as compared with drug-treated HeLa cells. Both leukemic and solid tumor cells took up this alkyl-lysophospholipid and expressed the two putative edelfosine targets, namely cell surface Fas death receptor (also known as APO-1 or CD95) and endoplasmic reticulum CTP: phosphocholine cytidylyltransferase. However, edelfosine was mainly located to plasma membrane lipid rafts in Jurkat and Peer leukemic cells and to endoplasmic reticulum in solid tumor HeLa and A549 cells. Edelfosine induced translocation of Fas, Fas-associated death domain-containing protein, and JNK into membrane rafts in Jurkat cells, but not in HeLa cells. In contrast, edelfosine inhibited phosphatidylcholine biosynthesis in both HeLa and A549 cells, but not in Jurkat or Peer leukemic cells, before the triggering of apoptosis. These data indicate that edelfosine targets two different subcellular structures in a cell type-dependent manner, namely cell surface lipid rafts in leukemic cells and endoplasmic reticulum in solid tumor cells.

CDP-choline Significantly Restores Phosphatidylcholine Levels by Differentially Affecting Phospholipase A2 and CTP: Phosphocholine Cytidylyltransferase after Stroke

Phosphatidylcholine (PtdCho) is a major membrane phospholipid, and its loss is sufficient in itself to induce cell death. PtdCho homeostasis is regulated by the balance between hydrolysis and synthesis. PtdCho is hydrolyzed by phospholipase A2 (PLA2), PtdChospecific phospholipase C (PtdCho-PLC), and phospholipase D (PLD). PtdCho synthesis is rate-limited by CTP:phosphocholine cytidylyltransferase (CCT), which makes CDP-choline. The final step of PtdCho synthesis is catalyzed by CDP-choline:1,2-diacylglycerol cholinephosphotransferase. PtdCho synthesis in the brain is predominantly through the CDP-choline pathway. Transient middle cerebral artery occlusion (tMCAO) significantly increased PLA2 activity, secretory PLA2 (sPLA2)-IIA mRNA and protein levels, PtdCho-PLC activity, and PLD2 protein expression following reperfusion. CDP-choline treatment significantly attenuated PLA2 activity, sPLA2-IIA mRNA and protein levels, and PtdCho-PLC activity, but did not affect PLD2 protein expression. tMCAO also resulted in loss of CCT activity and CCTalpha protein, which were partially restored by CDP-choline. No changes were observed in cytosolic PLA2 or calcium-independent PLA2 tMCAO. protein levels after Up-regulation of PLA2, PtdCho-PLC, and PLD and regulation of CCT collectively down-resulted in loss of PtdCho, which was significantly restored by CDP-choline treatment. CDP-choline treatment significantly attenuated the infarction volume by 55 +/- 5% after 1 h of tMCAO and 1 day of reperfusion. Taken together, these results suggest that CDP-choline significantly restores Ptd-Cho levels by differentially affecting sPLA2-IIA, PtdCho-PLC, and CCTalpha after transient focal cerebral ischemia. A hypothetical scheme is proposed integrating results from this study and from other reports in the literature.

CDP‐CHOLINE PARTLY RESTORES THE PHOSPHATIDYLCHOLINE LEVELS BY DIFFERENTIALLY AFFECTING SECRETORY PHOSPHOLIPASE A 2 IIA AND CTP‐PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE AFTER STROKE

Phosphatidylcholine (PtdCho) is a major membrane phospholipid and its loss is sufficient in itself to induce cell death. PtdCho homeostasis is regulated by a balance between hydrolysis and synthesis. PtdCho is hydrolyzed by phospholipase A2 (PLA2), PtdCho-phospholipase C (PtdCho-PLC) and phospholipase D (PLD). PtdCho synthesis is rate-limited by CTP:phosphocholine cytidylyltransferase (CCT) that makes CDP-choline. The final step of PtdCho synthesis is catalyzed by CDP-choline: 1,2-diacylglycerol choline phosphotransferase (CPT). PtdCho synthesis in the brain is predominantly through the CDP-choline pathway. Transient middle cerebral artery occlusion (tMCAO) significantly increased secretory PLA2 (sPLA2) IIA mRNA and protein levels, PtdCho-PLC activity and PLD2 expression at 1 d reperfusion. CDP-choline treatment significantly attenuated sPLA2 mRNA and protein levels and PtdCho-PLC activity, but did not affect PLD2 expression. tMCAO also resulted in loss of CCT activity and protein that were partially restored by CDP-choline. No changes were observed in the cytosolic PLA2 or calcium-independent PLA2 protein levels after tMCAO. The up-regulation of phospholipases and loss of CCT collectively resulted in the loss of PtdCho and total phospholipids, which were partially restored by CDP-choline. CDP-choline treatment significantly attenuated the infarction volume by 55% ± 5 after stroke. TNF-α and IL-1α/β are up-regulated in the brain after stroke. TNF-α/IL-1 activate phospholipases and down-regulate CCT, thus increasing PtdCho hydrolysis and inhibiting PtdCho synthesis. Our studies integrate CDP-choline effects with cytokine biology and lipid metabolism in stroke. Support: NIH UW VA.

Contribution of lipid mediators to the regulation of phosphatidylcholine synthesis by angiotensin

Angiotensin stimulates a cellular mitogenic response via the AT1 receptor. We have examined the effect of angiotensin on the rate of phosphatidylcholine (PC) synthesis and have begun to dissect the pathway linking the AT1 receptor to the rate-limiting enzyme in PC synthesis, CTP: phosphocholine cytidylyltransferase (CCT), using CHO cells engineered to express the AT1a receptor. Since CCT can be directly activated by lipid mediators, we probed for their involvement in the PC synthesis response to angiotensin. Angiotensin stimulated CCT activity and PC synthesis two- to threefold after a 30-min delay. The kinetics of this stimulation most closely paralleled an increase in diacylglycerol (DAG) derived from myristic acid-enriched phospholipids. The production of arachidonic acid, phosphatidic acid, or reactive oxygen species either peaked much earlier or not at all. Moreover, manipulation of the intracellular supply of oxygen free radicals, arachidonic acid, HETEs, or phosphatidic acid (using inhibitors and/or exogenous addition) did not generate parallel effects on the rate of PC synthesis. Restricting the production of DAG by inhibition of PLCβ with U73122 reduced both basal and angiotensin-stimulated PC synthesis. The U73122 inhibition of PC synthesis was accompanied by a similar inhibition of ERK1/2 phosphorylation. Addition of exogenous DAG stimulated basal and angiotensin-dependent PC synthesis, and partially reversed the effect of the PLC inhibitor on PC synthesis. These results do not provide support for lipid mediators as direct stimulators of CCT and PC synthesis downstream of angiotensin, but give rise to the idea that angiotensin effects might be mediated via ERK1/2.

Effects of hypoxia, glucose deprivation and acidosis on phosphatidylcholine synthesis in HL-1 cardiomyocytes. CTP:phosphocholine cytidylyltransferase activity correlates with sarcolemmal disruption

A decrease in [3H]Cho (choline) incorporation in to PtdCho (phos-phatidylcholine) preceded the onset of LDH (lactate dehydrogenase) release in HL-1 cardiomyocytes submitted to simulated ischaemia. This observation led us to examine the role of PtdCho synthesis in sarcolemmal disruption in HL-1 cardiomyocytes. To address this objective we analysed the individual effects of hypoxia, glucose deprivation and acidosis, three prominent components of ischaemia, on the different steps of the Kennedy pathway for the synthesis of PtdCho. Pulse and pulse-chase experiments with [3H]Cho, performed in whole HL-1 cells submitted to hypoxia or normoxia, in the presence or absence of glucose at different pHs indicated first, that CK (choline kinase) was inhibited by hypoxia and acidosis, whereas glucose deprivation exacerbated the inhibition caused by hypoxia. Second, the rate-limiting reaction in PtdCho synthesis, catalysed by CCT (CTP:phosphocholine cytidylyltransferase), was inhibited by hypoxia and glucose deprivation, but unexpectedly activated by acidosis. In cellfree system assays, acidosis inhibited both CK and CCT. In experiments performed in whole cells, the effect of acidosis was likely to be direct on CK, but indirect or intact-cell-dependent on CCT. Since hypoxia and glucose deprivation favoured membrane disruption, but acidosis prevented it, we hypothesized that the modulation of CCT could be an important determinant of cell survival. Supporting this hypothesis, we show that CCT activity in whole-cell experiments clearly correlated with LDH release, but not with ATP concentration. Altogether our results suggest a significant role for CCT activity in sarcolemmal disruption during ischaemia.

Cytidine 5'-diphosphocholine (CDP-choline) in stroke and other CNS disorders.

Brain phosphatidylcholine (PC) levels are regulated by a balance between synthesis and hydrolysis. Pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1alpha/beta) activate phospholipase A(2) (PLA(2)) and PC-phospholipase C (PC-PLC) to hydrolyze PC. PC hydrolysis by PLA(2) releases free fatty acids including arachidonic acid, and lyso-PC, an inhibitor of CTP-phosphocholine cytidylyltransferase (CCT). Arachidonic acid metabolism by cyclooxygenases/lipoxygenases is a significant source of reactive oxygen species. CDP-choline might increase the PC levels by attenuating PLA(2) stimulation and loss of CCT activity. TNF-alpha also stimulates proteolysis of CCT. TNF-alpha and IL-1beta are induced in brain ischemia and may disrupt PC homeostasis by increasing its hydrolysis (increase PLA(2) and PC-PLC activities) and inhibiting its synthesis (decrease CCT activity). The beneficial effects of CDP-choline may result by counteracting TNF-alpha and/or IL-1 mediated events, integrating cytokine biology and lipid metabolism. Re-evaluation of CDP-choline phase III stroke clinical trial data is encouraging and future trails are warranted. CDP-choline is non-xenobiotic, safe, well tolerated, and can be considered as one of the agents in multi-drug treatment of stroke.

Increased Phosphatidylcholine Production but Disrupted Glycogen Metabolism in Fetal Type II Cells of Mice That Overexpress CTP:Phosphocholine Cytidylyltransferase

CTP:phosphocholine cytidylyltransferase (CCT) is a rate-determining enzyme in the de novo synthesis of phosphatidylcholine (PtdCho). Alveolar type II cells synthesize large quantities of disaturated PtdCho, the surface-active agent of pulmonary surfactant, particularly at late gestation when the lung prepares itself for postnatal air breathing. To clarify the role of CCTalpha in lung surfactant maturation, we overexpressed CCTalpha(1-367) using the surfactant protein-C promoter. Lungs of transgenic mice were analyzed at day 18 of gestation (term = 19 days). Overexpression of CCTalpha(1-367) increased the synthesis and content of PtdCho in fetal type II cells isolated from the transgenic mice. Also, PtdCho content of fetal lung fluid was increased. No changes in surfactant protein content were detected. Interestingly, fetal type II cells of transgenic mice contained more glycogen than control cells. Incorporation studies with [U-(14)C]glucose demonstrated that overexpression of CCTalpha(1-367) in fetal type II cells increased glycogen synthesis without affecting glycogen breakdown. To determine which domain contributes to this glycogen phenotype, two additional transgenes were created overexpressing either CCTalpha(1-239) or CCTalpha(239-367). Glycogen synthesis and content were increased in fetal type II cells expressing CCTalpha(239-367) but not CCTalpha(1-239)(.) We conclude that overexpression of CCTalpha increases surfactant PtdCho synthesis without affecting surfactant protein levels but that it disrupts glycogen metabolism in differentiating type II cells via its regulatory domain.

Contribution of lipid second messengers to the regulation of phosphatidylcholine synthesis during cell cycle re-entry

During entry into the cell cycle a phosphatidylcholine (PC) metabolic cycle is activated. We have examined the hypothesis that PC synthesis during the G 0 to G 1 transition is controlled by one or more lipid products of PC turnover acting directly on the rate-limiting enzyme in the synthesis pathway, CTP: phosphocholine cytidylyltransferase (CCT). The acceleration of PC synthesis was two- to threefold during the first hour after addition of serum to quiescent IIC9 fibroblasts. The rate increased to ∼15-fold above the basal rate during the second hour. The production of arachidonic acid, diacylglycerol (DAG), and phosphatidic acid (PA) preceded the second, rapid phase of PC synthesis. However, an increase in the cellular content of these lipid mediators was detected only for DAG. CCT activation and translocation to membranes accompanied the second phase of the PC synthesis acceleration. Bromoenol lactone (BEL), an inhibitor of calcium-independent phospholipase A 2 and PA phosphatase, blocked production of fatty acids and DAG, inhibited both phases of the PC synthesis response to serum, and reduced CCT activity and membrane affinity. The effect of BEL on PC synthesis was partially reversed by in situ generation of DAG via exogenous PC-specific phospholipase C to generate ∼2-fold elevation in PC-derived DAG. Exogenous arachidonic acid also partially reversed the inhibition by BEL, but only at a concentration that generated a supra-physiological cellular content of free fatty acid. 1-Butanol, which blocks PA production, had no effect on DAG generation, or on PC synthesis. We conclude that fatty acids and DAG could contribute to the initial slow phase of the PC synthesis response. DAG is the most likely lipid regulator of CCT activity and the rapid phase of PC synthesis. However, processes other than direct activation of CCT by lipid mediators likely contribute to the highly accelerated phase during entry into the cell cycle.

Phosphatidylcholine synthesis is elevated in neuronal models of Gaucher disease due to direct activation of CTP:phosphocholine cytidylyltransferase by glucosylceramide.

Glucosylceramide (GlcCer) accumulates in the inherited metabolic disorder, Gaucher disease, because of the defective activity of lysosomal glucocerebrosidase. We previously demonstrated that upon GlcCer accumulation, cultured hippocampal neurons exhibit modified growth patterns, altered endoplasmic reticulum density, and altered calcium release from intracellular stores. We here examined the relationship between GlcCer accumulation and phospholipid synthesis. After treatment of neurons with an active site-directed inhibitor of glucocerebrosidase, or in neurons obtained from a mouse model of Gaucher disease, [14C]methyl choline incorporation into [14C]phosphatidylcholine ([14C]PC) and [14C]sphingomyelin was elevated, as were [14C]CDP-choline levels, suggesting that CTP:phosphocholine cytidylyltransferase (CCT) is activated. Indeed, CCT activity was elevated in neurons that had accumulated GlcCer. GlcCer, but not galactosylceramide (GalCer), stimulated CCT activity in rat brain homogenates, and significantly higher levels of CCT were membrane associated in cortical homogenates from a mouse model of Gaucher disease compared with wild-type mice. Because CCT mRNA and protein levels were unaltered in either neurons or brain tissue that had accumulated GlcCer, it appeared likely that GlcCer activates CCT by a post-translational mechanism. This was verified by examination of the effect of GlcCer on CCT purified about 1200-fold from rat brain. GlcCer stimulated CCT activity, with stimulation observed at levels as low as 2.5 mol% and with maximal activation reached at 10 mol%. In contrast, GalCer had no effect. Together, these data demonstrate that GlcCer directly activates CCT, which results in elevated PC synthesis, which may account for some of the changes in growth rates observed upon neuronal GlcCer accumulation.