Proapoptotic effects of P. aeruginosa involve inhibition of surfactant phosphatidylcholine synthesis

Pseudomonas aeruginosa causes sepsis-induced acute lung injury, a disorder associated with deficiency of surfactant phosphatidylcholine (PtdCho). P. aeruginosa (PA103) utilizes a type III secretion system (TTSS) to induce programmed cell death. Herein, we observed that PA103 reduced alveolar PtdCho levels, resulting in impaired lung biophysical activity, an effect partly attributed to caspase-dependent cleavage of the key PtdCho biosynthetic enzyme, CTP:phosphocholine cytidylyltransferase-alpha (CCTalpha). Expression of recombinant CCTalpha variants harboring point mutations at putative caspase cleavage sites in murine lung epithelia resulted in partial proteolytic resistance of CCTalpha to PA103. Further, caspase-directed CCTalpha degradation, decreased PtdCho levels, and cell death in murine lung epithelia were lessened after exposure of cells to bacterial strains lacking the TTSS gene product, exotoxin U (ExoU), but not ExoT. These observations suggest that during the proapoptotic program driven by P. aeruginosa, deleterious effects on phospholipid metabolism are mediated by a TTSS in concert with caspase activation, resulting in proteolysis of a key surfactant biosynthetic enzyme.     

Tumor necrosis factor-alpha inhibits expression of CTP:phosphocholine cytidylyltransferase

We investigated the effects of tumor necrosis factor alpha (TNFalpha), a key cytokine involved in inflammatory lung disease, on phosphatidylcholine (PtdCho) biosynthesis in a murine alveolar type II epithelial cell line (MLE-12). TNFalpha significantly inhibited [(3)H]choline incorporation into PtdCho after 24 h of exposure. TNFalpha reduced the activity of CTP:phosphocholine cytidylyltransferase (CCT), the rate-regulatory enzyme within the CDP-choline pathway, by 40% compared with control, but it did not alter activities of choline kinase or cholinephosphotransferase. Immunoblotting revealed that TNFalpha inhibition of CCT activity was associated with a uniform decrease in the mass of CCTalpha in total cell lysates, cytosolic, microsomal, and nuclear subfractions of MLE cells. Northern blotting revealed no effects of the cytokine on steady-state levels of CCTalpha mRNA, and CCTbeta mRNA was not detected. Incorporation of [(35)S]methionine into immunoprecipitable CCTalpha protein in pulse and pulse-chase studies revealed that TNFalpha did not alter de novo synthesis of enzyme, but it substantially accelerated turnover of CCTalpha. Addition of N-acetyl-Leu-Leu-Nle-CHO (ALLN), the calpain I inhibitor, or lactacystin, the 20 S proteasome inhibitor, blocked the inhibition of PtdCho biosynthesis mediated by TNFalpha. TNFalpha-induced degradation of CCTalpha protein was partially blocked by ALLN or lactacystin. CCT was ubiquitinated, and ubiquitination increased after TNFalpha exposure. m-Calpain degraded both purified CCT and CCT in cellular extracts. Thus, TNFalpha inhibits PtdCho synthesis by modulating CCT protein stability via the ubiquitin-proteasome and calpain-mediated proteolytic pathways.     

Oxidized lipoproteins inhibit surfactant phosphatidylcholine synthesis via calpain-mediated cleavage of CTP:phosphocholine cytidylyltransferase

We investigated effects of pro-atherogenic oxidized lipoproteins on phosphatidylcholine (PtdCho) biosynthesis in murine lung epithelial cells (MLE-12). Cells surface-bound, internalized, and degraded oxidized low density lipoproteins (Ox-LDL). Ox-LDL significantly reduced [3H]choline incorporation into PtdCho in cells by selectively inhibiting the activity of the rate-regulatory enzyme, CTP:phosphocholine cytdylyltransferase (CCT). Ox-LDL coordinately increased the cellular turnover of CCTalpha protein as determined by [35S]methionine pulse-chase studies by inducing the calcium-activated proteinase, calpain. Forced expression of calpain or exposure of cells to the calcium ionophore, A23187, increased CCTalpha degradation, whereas overexpression of the endogenous calpain inhibitor, calpastatin, attenuated Ox-LDL-induced CCTalpha degradation. The effects of Ox-LDL on CCTalpha breakdown were attenuated in calpain-deficient cells. In vitro calpain digestion of CCTalpha isolated from cells transfected with truncated or internal deletion mutants indicated multiple cleavage sites within the CCTalpha primary structure, leading to the generation of a 26-kDa (p26) fragment. Calpain hydrolysis of purified CCTalpha generated p26, which upon NH2-terminal sequencing localized a calpain attack site within the CCTalpha amino terminus. Expression of a CCTalpha mutant where the amino-terminal cleavage site and a putative carboxyl-terminal hydrolysis region were modified resulted in an enzyme that was significantly less sensitive to proteolytic cleavage and restored the ability of cells to synthesize surfactant PtdCho after Ox-LDL treatment. Thus, these results provide a critical link between proatherogenic lipoproteins and their metabolic target, CCTalpha, resulting in impaired surfactant metabolism.     

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.     

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.     

The rate-limiting enzyme in phosphatidylcholine synthesis regulates proliferation of the nucleoplasmic reticulum

The nucleus contains a network of tubular invaginations of the nuclear envelope (NE), termed the nucleoplasmic reticulum (NR), implicated in transport, gene expression, and calcium homeostasis. Here, we show that proliferation of the NR, measured by the frequency of NE invaginations and tubules, is regulated by CTP:phosphocholine cytidylyltransferase-alpha (CCTalpha), the nuclear and rate-limiting enzyme in the CDP-choline pathway for phosphatidylcholine (PtdCho) synthesis. In Chinese hamster ovary (CHO)-K1 cells, fatty acids triggered activation and translocation of CCTalpha onto intranuclear tubules characteristic of the NR. This was accompanied by a twofold increase in NR tubules quantified by immunostaining for lamin A/C or the NE. CHO MT58 cells expressing a temperature-sensitive CCTalpha allele displayed reduced PtdCho synthesis and CCTalpha expression and minimal proliferation of the NR in response to oleate compared with CHO MT58 cells stably expressing CCTalpha. Expression of CCTalpha mutants in CHO58 cells revealed that both enzyme activity and membrane binding promoted NR proliferation. In support of a direct role for membrane binding in NR tubule formation, recombinant CCTalpha caused the deformation of liposomes into tubules in vitro. This demonstrates that a key nuclear enzyme in PtdCho synthesis coordinates lipid synthesis and membrane deformation to promote formation of a dynamic nuclear-cytoplasmic interface.     

LASS5 is the predominant ceramide synthase isoform involved in de novo sphingolipid synthesis in lung epithelia

Ceramide is a key bioactive mediator that inhibits surfactant phosphatidylcholine (PtdCho) synthesis in lung epithelia. Ceramide availability is governed by sphingomyelin (SM) hydrolysis, but less is known regarding its de novo synthesis. In this study, we observed that ceramide synthesis within murine lung epithelia was associated with high-level ceramide synthase (dihydroceramide synthase) activity. Longevity assurance homolog 5 (LASS5) was the predominant ceramide synthase isoform detected in lung epithelia, whereas relatively lower level expression was detected for the other five mammalian homologs. Pulmonary LASS5 was developmentally regulated, but its expression was spatially and gender nonspecific. Exogenously expressed LASS5 in lung epithelia was membrane-associated, triggering increased ceramide synthesis, whereas knockdown studies using fumonisin B1 or LASS5 small, interfering RNA reduced ceramide synthase activity by 78% or 45%, respectively. Overexpression of LASS5 also reduced PtdCho synthesis, but maximal inhibition was achieved when LASS5 was coexpressed with a plasmid encoding a neutral sphingomyelinase involved in SM hydrolysis. These results demonstrate that LASS5 is the major ceramide synthase gene product involved in sphingolipid production that may also regulate PtdCho metabolism in pulmonary epithelia.     

Pulmonary-specific expression of tumor necrosis factor-alpha alters surfactant lipid metabolism

Tumor necrosis factor (TNF)-alpha is a major cytokine implicated in inducing acute and chronic lung injury, conditions associated with surfactant phosphatidylcholine (PtdCho) deficiency. Acutely, TNF-alpha decreases PtdCho synthesis but stimulates surfactant secretion. To investigate chronic effects of TNF-alpha, we investigated PtdCho metabolism in a murine transgenic model exhibiting lung-specific TNF-alpha overexpression. Compared with controls, TNF-alpha transgenic mice exhibited a discordant pattern of PtdCho metabolism, with a decrease in PtdCho and disaturated PtdCho (DSPtdCho) content in the lung, but increased levels in alveolar lavage. Transgenics had lower activities and increased immunoreactive levels of cytidylyltransferase (CCT), a key PtdCho biosynthetic enzyme. Ceramide, a CCT inhibitor, was elevated, and linoleic acid, a CCT activator, was decreased in transgenics. Radiolabeling studies revealed that alveolar reuptake of DSPtdCho was significantly decreased in transgenic mice. These observations suggest that chronic expression of TNF-alpha results in a complex pattern of PtdCho metabolism where elevated lavage PtdCho may originate from alveolar inflammatory cells, decreased surfactant reuptake, or altered surfactant secretion. Reduced parenchymal PtdCho synthesis appears to be attributed to CCT enzyme that is physiologically inactivated by ceramide or by diminished availability of activating lipids.     

Oxysterol activation of phosphatidylcholine synthesis involves CTP:phosphocholine cytidylyltransferase alpha translocation to the nuclear envelope

In addition to suppressing cholesterol synthesis and uptake, oxysterols also activate glycerophospholipid and SM (sphingomyelin) synthesis, possibly to buffer cells from excess sterol accumulation. In the present study, we investigated the effects of oxysterols on the CDP-choline pathway for PtdCho (phosphatidylcholine) synthesis using wild-type and sterol-resistant CHO (Chinese-hamster ovary) cells expressing a mutant of SCAP [SREBP (sterol-regulatory-element-binding protein) cleavage-activating protein] (CHO-SCAP D443N). [(3)H]Choline-labelling experiments showed that 25OH (25-hydroxycholesterol), 22OH (22-hydroxycholesterol) and 27OH (27-hydroxycholesterol) increased PtdCho synthesis in CHO cells as a result of CCTalpha (CTP:phosphocholine cytidylyltransferase alpha) translocation and activation at the NE (nuclear envelope). These oxysterols also activate PtdCho synthesis in J774 macrophages. in vitro, CCTalpha activity was stimulated 2- to 2.5-fold by liposomes containing 5 mol% 25OH, 22OH or 27OH. Inclusion of up to 5 mol% cholesterol did not further activate CCTalpha. 25OH activated CCTalpha in CHO-SCAP D443N cells leading to a transient increase in PtdCho synthesis and accumulation of CDP-choline. CCTalpha translocation to the NE and intranuclear tubules in CHO-SCAP D443N cells was complete after 1 h exposure to 25OH compared with only partial translocation by 4-6 h in CHO-Mock cells. These enhanced responses in CHO-D443N cells were sterol-dependent since depletion with cyclodextrin or lovastatin resulted in reduced sensitivity to 25OH. However, the lack of effect of cholesterol on in vitro CCT activity indicates an indirect relationship or involvement of other sterols or oxysterol. We conclude that translocation and activation of CCTalpha at nuclear membranes by side-chain hydroxylated sterols are regulated by the cholesterol status of the cell.     

Oxysterols inhibit phosphatidylcholine synthesis via ERK docking and phosphorylation of CTP:phosphocholine cytidylyltransferase

Surfactant deficiency contributes to acute lung injury and may result from the elaboration of bioactive lipids such as oxysterols. We observed that the oxysterol 22-hydroxycholesterol (22-HC) in combination with its obligate partner, 9-cis-retinoic acid (9-cis-RA), decreased surfactant phosphatidylcholine (PtdCho) synthesis by increasing phosphorylation of the regulatory enzyme CTP:phosphocholine cytidylyltransferase-alpha (CCTalpha). Phosphorylation of CCTalpha decreased its activity. 22-HC/9-cis-RA inhibition of PtdCho synthesis was blocked by PD98059 or dominant-negative ERK (p42 kinase). Overexpression of constitutively active MEK1, the kinase upstream of p42 kinase, increased CCTalpha phosphorylation. Expression of truncated CCTalpha mutants lacking proline-directed sites within the C-terminal phosphorylation domain partially blocked oxysterol-mediated inhibition of PtdCho synthesis. Mutagenesis of Ser315 within CCTalpha was both required and sufficient to confer significant resistance to 22-HC/9-cis-RA inhibition of PtdCho synthesis. A novel putative ERK-docking domain N-terminal to this phosphoacceptor site was mapped within the CCTalpha membrane-binding domain (residues 287-300). The results are the first demonstration of a physiologically relevant phosphorylation site and docking domain within CCTalpha that serve as targets for ERKs, resulting in inhibition of surfactant synthesis.     

Lipid activation of CTP: phosphocholine cytidylyltransferase alpha: characterization and identification of a second activation domain

The CTP:phosphocholine cytidylyltransferase (CCT) governs the rate of phosphatidylcholine (PtdCho) biosynthesis, and its activity is governed by interaction with membrane lipids. The carboxy-terminus was dissected to delineate the minimum sequences required for lipid responsiveness. The helical domain is recognized as a site of lipid interaction, and all three tandem alpha-helical repeats from residues 257 through 290 were found to be required for regulation of enzymatic activity by this domain. Truncation of the carboxy-terminus to remove one or more of the alpha-helical repeats yielded catalytically compromised proteins that were not responsive to lipids but retained sufficient activity to accelerate PtdCho biosynthesis when overexpressed in vivo. The role of the helical region in lipid-activation was tested further by excising residues 257 through 309 to yield a protein that retained a 57-residue carboxy terminal domain fused to the catalytic core. This construct tested the hypothesis that the helical region inhibits activity in the absence of lipid rather than activates the enzyme in the presence of lipid. This hypothesis predicts constitutive activity for CCTalpha[Delta257-309]; however, this protein was tightly regulated by lipid with activities comparable to the full-length CCTalpha, in both the absence and presence of lipid. Activation of CCTalpha[Delta257-309] was dependent exclusively on anionic lipids, whereas full-length CCTalpha responded to either anionic or neutral lipids. Phosphatidic acid delivered in Triton X-100 micelles was the preferred activator of the second lipid-activation domain. These data demonstrate that CCTalpha can be regulated by lipids by two independent domains: (i) the three amphipathic alpha-helical repeats that interact with both neutral and anionic lipid mixtures and (ii) the last 57 residues that interact with anionic lipids. The results show that both domains are inhibitory in the absence of lipid and activating in the presence of lipid. Removal of both domains results in a nonresponsive, dysregulated enzyme with reduced activity. The data also demonstrate for the first time that the 57-residue carboxy-terminal domain in CCTalpha participates in lipid-mediated regulation and is sufficient for maximum activation of enzyme activity.     

15-deoxy-Delta12,14-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-Delta(12,14)-PGJ(2) (15d-PGJ(2)) 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-PGJ(2)-treated cells. 15d-PGJ(2) modified critical cysteine residues within CCTalpha as determined by mutagenesis studies and by incorporation of biotin-15d-PGJ(2) into CCTalpha. These effects of 15d-PGJ(2) 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.     

Activity of the phosphatidylcholine biosynthetic pathway modulates the distribution of fatty acids into glycerolipids in proliferating cells

PtdCho accumulation is a periodic, S phase-specific event that is modulated in part by cell cycle-dependent fluctuations in CTP:phosphocholine cytidylyltransferase (CCT) activity. A supply of fatty acids is essential to generate the diacylglycerol (DG) precursors for phosphatidylcholine (PtdCho) biosynthesis but it is not known whether the DG supply is also coupled to the cell cycle. Although the rate of fatty acid synthesis in a macrophage cell line was dramatically stimulated in response to the growth factor, CSF-1, it was not regulated by the cell cycle. Increased fatty acid synthesis correlated with elevated acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) steady-state mRNA levels. Cellular fatty acid synthesis was essential for membrane PL synthesis. Cerulenin inhibition of endogenous fatty acid synthesis also inhibited PtdCho synthesis, which was not relieved by exogenous fatty acids. Inhibition of CCT activity by the addition of lysophosphatidylcholine (lysoPtdCho) or temperature-shift of a conditionally defective CCT diverted newly synthesized DG to the TG pool where it accumulated. Enforced expression of CCT stimulated PtdCho biosynthesis and reduced TG synthesis. Thus, the cellular DG supply did not regulate PtdCho biosynthesis and CCT activity governs the partitioning of DG into either the PL or TG pools, thereby controlling both PtdCho and TG biosynthesis.     

Caspase processing and nuclear export of CTP:phosphocholine cytidylyltransferase alpha during farnesol-induced apoptosis

CTP:phosphocholine cytidylyltransferase alpha (CCT alpha) is a nuclear enzyme that catalyzes the rate-limiting step in the CDP-choline pathway, the primary route for synthesis of phosphatidylcholine (PtdCho) in eukaryotic cells. Induction of apoptosis by farnesol (FOH) and other cytotoxic drugs has been shown to alter PtdCho synthesis via the CDP-choline pathway. Here we report that FOH-induced apoptosis in CHO cells caused a dose-dependent activation of CCT alpha and inhibition of the final step in the pathway, resulting in a biphasic effect on PtdCho synthesis. Activation of CCT alpha was accompanied by enzyme translocation to the nuclear envelope within 30 min of FOH addition to cells. Following translocation to membranes, CCT alpha was exported from the nucleus and underwent caspase-mediated proteolysis that coincided with poly(ADP-ribose) polymerase cleavage. Site-directed mutagenesis and in vivo and in vitro expression studies mapped a caspase 6 and/or 8 cleavage site to TEED(28 downward arrow)G, the final residue in the CCT alpha nuclear localization signal. Nuclear export of CCT alpha appeared to be an active process in FOH-treated CHO cells that was independent of caspase removal of the nuclear localization signal. Caspase cleavage of CCT alpha occurred during UV or chelerythrine-induced apoptosis; however, nuclear membrane translocation and nuclear export were not evident under these conditions. Thus, caspase cleavage of CCT alpha was a late feature of several apoptotic programs that occurred in the nucleus or at the nuclear envelope. Activation and nuclear export of CCT alpha were early events in FOH-induced apoptosis that contributed to altered PtdCho synthesis and, in conjunction with caspase cleavage, excluded CCT alpha from the nucleus.     

Cellular responses to excess phospholipid

Phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells, and its synthesis is controlled by the activity of CDP:phosphocholine cytidylyltransferase (CCT). Enforced CCT expression accelerated the rate of PtdCho synthesis. However, the amount of cellular PtdCho did not increase as a result of the turnover of both the choline and glycerol components of PtdCho. Metabolic labeling experiments demonstrated that cells compensated for elevated CCT activity by the degradation of PtdCho to glycerophosphocholine (GPC). Phospholipase D-mediated PtdCho hydrolysis and phosphocholine formation were unaffected. Most of the GPC produced in response to excess phospholipid production was secreted into the medium. Cells also degraded the excess membrane PtdCho to GPC when phospholipid formation was increased by exposure to exogenous lysophosphatidylcholine or lysophosphatidylethanolamine. The replacement of the acyl moiety at the 1-position of PtdCho with a non-hydrolyzable alkyl moiety prevented degradation to GPC. Accumulation of alkylacyl-PtdCho was associated with the inhibition of cell proliferation, demonstrating that alternative pathways of degradation will not substitute. GPC formation was blocked by bromoenol lactone, implicating the calcium-independent phospholipase A2 as a key participant in the response to excess phospholipid. Owing to the fact that PtdCho is biosynthetically converted to PtdEtn, excess PtdCho resulted in overproduction and exit of GPE as well as GPC. Thus, general membrane phospholipid homeostasis is achieved by a balance between the opposing activities of CCT and phospholipase A2.