Very low density lipoproteins stimulate surfactant lipid synthesis in vitro.

Surfactant synthesis is critically dependent on the availability of fatty acids. One fatty acid source may be circulating triglycerides that are transported in VLDL, and hydrolyzed to free fatty acids by lipoprotein lipase (LPL). To evaluate this hypothesis, we incubated immortalized or primary rat alveolar pre-type II epithelial cells with VLDL. The cells were observed to surface bind, internalize, and degrade VLDL, a process that was induced by exogenous LPL. LPL induction of lipoprotein uptake significantly increased the rates of choline incorporation into phosphatidylcholine (PC) and disaturated PC, and these effects were associated with a three-fold increase in the activity of the rate-regulatory enzyme for PC synthesis, cytidylyltransferase. Compared with native LPL, a fusion protein of glutathione S-transferase with the catalytically inactive carboxy-terminal domain of LPL did not activate CT despite inducing VLDL uptake. A variant of the fusion protein of glutathione S-transferase with the catalytically inactive carboxy-terminal domain of LPL that partially blocked LPL-induced catabolism of VLDL via LDL receptors also partially blocked the induction of surfactant synthesis by VLDL. Taken together, these observations suggest that both the lipolytic actions of LPL and LPL-induced VLDL catabolism via lipoprotein receptors might play an integral role in providing the fatty acid substrates used in surfactant phospholipid synthesis.

NDPK-D (NM23-H4)-mediated externalization of cardiolipin enables elimination of depolarized mitochondria by mitophagy.

Mitophagy is critical for cell homeostasis. Externalization of the inner mitochondrial membrane phospholipid, cardiolipin (CL), to the surface of the outer mitochondrial membrane (OMM) was identified as a mitophageal signal recognized by the microtubule-associated protein 1 light chain 3. However, the CL-translocating machinery remains unknown. Here we demonstrate that a hexameric intermembrane space protein, NDPK-D (or NM23-H4), binds CL and facilitates its redistribution to the OMM. We found that mitophagy induced by a protonophoric uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), caused externalization of CL to the surface of mitochondria in murine lung epithelial MLE-12 cells and human cervical adenocarcinoma HeLa cells. RNAi knockdown of endogenous NDPK-D decreased CCCP-induced CL externalization and mitochondrial degradation. A R90D NDPK-D mutant that does not bind CL was inactive in promoting mitophagy. Similarly, rotenone and 6-hydroxydopamine triggered mitophagy in SH-SY5Y cells was also suppressed by knocking down of NDPK-D. In situ proximity ligation assay (PLA) showed that mitophagy-inducing CL-transfer activity of NDPK-D is closely associated with the dynamin-like GTPase OPA1, implicating fission-fusion dynamics in mitophagy regulation.

F-box protein Fbxl18 mediates polyubiquitylation and proteasomal degradation of the pro-apoptotic SCF subunit Fbxl7.

Fbxl7, a subunit of the SCF (Skp-Cul1-F-box protein) complex induces mitotic arrest in cells; however, molecular factors that control its cellular abundance remain largely unknown. Here, we identified that an orphan F-box protein, Fbxl18, targets Fbxl7 for its polyubiquitylation and proteasomal degradation. Lys 109 within Fbxl7 is an essential acceptor site for ubiquitin conjugation by Fbxl18. An FQ motif within Fbxl7 serves as a molecular recognition site for Fbxl18 interaction. Ectopically expressed Fbxl7 induces apoptosis in Hela cells, an effect profoundly accentuated after cellular depletion of Fbxl18 protein or expression of Fbxl7 plasmids encoding mutations at either Lys 109 or within the FQ motif. Ectopic expression of Fbxl18 plasmid-limited apoptosis caused by overexpressed Fbxl7 plasmid. Thus, Fbxl18 regulates apoptosis by mediating ubiquitin-dependent proteasomal degradation of the pro-apoptotic protein Fbxl7 that may impact cellular processes involved in cell cycle progression.

Thrombospondin-1 restrains neutrophil granule serine protease function and regulates the innate immune response during Klebsiella pneumoniae infection.

Neutrophil elastase (NE) and cathepsin G (CG) contribute to intracellular microbial killing but, if left unchecked and released extracellularly, promote tissue damage. Conversely, mechanisms that constrain neutrophil serine protease activity protect against tissue damage but may have the untoward effect of disabling the microbial killing arsenal. The host elaborates thrombospondin-1 (TSP-1), a matricellular protein released during inflammation, but its role during neutrophil activation following microbial pathogen challenge remains uncertain. Mice deficient in TSP-1 (thbs1(-/-)) showed enhanced lung bacterial clearance, reduced splenic dissemination, and increased survival compared with wild-type (WT) controls during intrapulmonary Klebsiella pneumoniae infection. More effective pathogen containment was associated with reduced burden of inflammation in thbs1(-/-) mouse lungs compared with WT controls. Lung NE activity was increased in thbs1(-/-) mice following K. pneumoniae challenge, and thbs1(-/-) neutrophils showed enhanced intracellular microbial killing that was abrogated with recombinant TSP-1 administration or WT serum. Thbs1(-/-) neutrophils exhibited enhanced NE and CG enzymatic activity, and a peptide corresponding to amino-acid residues 793-801 within the type-III repeat domain of TSP-1 bridled neutrophil proteolytic function and microbial killing in vitro. Thus, TSP-1 restrains proteolytic action during neutrophilic inflammation elicited by K. pneumoniae, providing a mechanism that may regulate the microbial killing arsenal.

Effects of intratracheal instillation of TNF-alpha on surfactant metabolism.

Tumor necrosis factor-alpha (TNF-alpha) has been shown to play an integral role in the pathogenesis of the acute respiratory distress syndrome. This disorder is characterized by a deficiency of alveolar surfactant, a surface-active material that is composed of key hydrophobic proteins and the major lipid disaturated phosphatidylcholine (DSPC). We investigated how TNF-alpha might alter DSPC content in rat lungs by instilling the cytokine (2.5 microg) intratracheally for 10 min and then assaying parameters of DSPC synthesis and degradation in alveolar type II epithelial cells, which produce surfactant. Cells isolated from rats given TNF-alpha had 26% lower levels of phosphatidylcholine compared with control. TNF-alpha treatment also decreased the ability of these cells to incorporate [(3)H]choline into DSPC by 45% compared with control isolates. There were no significant differences in the levels of choline substrate or choline transport between the groups. However, TNF-alpha produced a 64% decrease in the activity of cytidylyltransferase, the rate-regulatory enzyme required for DSPC synthesis. TNF-alpha administration in vivo also tended to stimulate phospholipase A(2) activity, but it did not alter other parameters for DSPC degradation such as activities for phosphatidylcholine-specific phospholipase C or phospholipase D. These observations indicate that TNF-alpha decreases the levels of surfactant lipid by decreasing the activity of a key enzyme involved in surfactant lipid synthesis. The results do not exclude stimulatory effects of the cytokine on phosphatidylcholine breakdown.

Skp-cullin-F box E3 ligase component FBXL2 ubiquitinates Aurora B to inhibit tumorigenesis.

Aurora B kinase is an integral regulator of cytokinesis, as it stabilizes the intercellular canal within the midbody to ensure proper chromosomal segregation during cell division. Here we identified that the ubiquitin E3 ligase complex SCF(FBXL2) mediates Aurora B ubiquitination and degradation within the midbody, which is sufficient to induce mitotic arrest and apoptosis. Three molecular acceptor sites (K¹°², K¹°³ and K²°7) within Aurora B protein were identified as important sites for its ubiquitination. A triple Lys mutant of Aurora B (K¹°²/¹°³/(²°7R)) exhibited optimal resistance to SCF(FBXL2)-directed polyubiquitination, and overexpression of this variant resulted in a significant delay in anaphase onset, resulting in apoptosis. A unique small molecule F-box/LRR-repeat protein 2 (FBXL2) activator, BC-1258, stabilized and increased levels of FBXL2 protein that promoted Aurora B degradation, resulting in tetraploidy, mitotic arrest and apoptosis of tumorigenic cells, and profoundly inhibiting tumor formation in athymic nude mice. These findings uncover a new proteolytic mechanism targeting a key regulator of cell replication that may serve as a basis for chemotherapeutic intervention in neoplasia.

STAT1-regulated lung MDSC-like cells produce IL-10 and efferocytose apoptotic neutrophils with relevance in resolution of bacterial pneumonia.

Bacterial pneumonia remains a significant burden worldwide. Although an inflammatory response in the lung is required to fight the causative agent, persistent tissue-resident neutrophils in non-resolving pneumonia can induce collateral tissue damage and precipitate acute lung injury. However, little is known about mechanisms orchestrated in the lung tissue that remove apoptotic neutrophils to restore tissue homeostasis. In mice infected with Klebsiella pneumoniae, a bacterium commonly associated with hospital-acquired pneumonia, we show that interleukin (IL)-10 is essential for resolution of lung inflammation and recovery of mice after infection. Although IL-10(-/-) mice cleared bacteria, they displayed increased morbidity with progressive weight loss and persistent lung inflammation in the later phase after infection. A source of tissue IL-10 was found to be resident CD11b(+)Gr1(int)F4/80(+) cells resembling myeloid-derived suppressor cells (MDSCs) that appeared with a delayed kinetics after infection. These cells efficiently efferocytosed apoptotic neutrophils, which was aided by IL-10. The lung neutrophil burden was attenuated in infected signal transducer and activator of transcription 1 (STAT1)(-/-) mice with concomitant increase in the frequency of the MDSC-like cells and lung IL-10 levels. Thus, inhibiting STAT1 in combination with antibiotics may be a novel therapeutic strategy to address inefficient resolution of bacterial pneumonia.

F-box protein FBXL2 exerts human lung tumor suppressor-like activity by ubiquitin-mediated degradation of cyclin D3 resulting in cell cycle arrest.

Dysregulated behavior of cell cycle proteins and their control by ubiquitin E3 ligases is an emerging theme in human lung cancer. Here, we identified and characterized the activity of a novel F-box protein, termed FBXL2, belonging to the SCF (Skip-Cullin1-F-box protein) E3 ligase family. Ectopically expressed FBXL2 triggered G2/M-phase arrest, induced chromosomal anomalies and increased apoptosis of transformed lung epithelia by mediating polyubiquitination and degradation of the mitotic regulator, cyclin D3. Unlike other F-box proteins that target phosphodegrons within substrates, FBXL2 uniquely recognizes a canonical calmodulin (CaM)-binding motif within cyclin D3 to facilitate its polyubiquitination. CaM bound and protected cyclin D3 from FBXL2 by direct intermolecular competition with the F-box protein for access within this motif. The chemotherapeutic agent vinorelbine increased apoptosis of human lung carcinoma cells by inducing FBXL2 expression and cyclin D3 degradation, an effect accentuated by CaM knockdown. Depletion of endogenous FBXL2 stabilized cyclin D3 levels, accelerated cancer cell growth and increased cell viability after vinorelbine treatment. Last, ectopic expression of FBXL2 significantly inhibited the growth and migration of tumorogenic cells and tumor formation in athymic nude mice. These observations implicate SCF(FBXL2) as an indispensible regulator of mitosis that serves as a tumor suppressor.

Adenoviral gene transfer of a mutant surfactant enzyme ameliorates pseudomonas-induced lung injury.

Surfactant deficiency is an important contributor to the acute respiratory distress syndrome, a disorder that commonly occurs after bacterial sepsis. CTP:phosphocholine cytidylyltransferase (CCTalpha) is the rate-limiting enzyme required for the biosynthesis of dipalmitoylphosphatidylcholine (DPPC), the major phospholipid of surfactant. In this study, a cDNA encoding a novel, calpain-resistant mutant CCTalpha enzyme was delivered intratracheally in mice using a replication-deficient adenovirus 5 CTP:phosphocholine cytidylyltransferase construct (Ad5-CCT(Penta)) in models of bacterial sepsis. Ad5-CCT(Penta) gene transfer produced high-level CCTalpha gene expression, increased alveolar surfactant (DPPC) levels and improved lung surface tension and pressure-volume relationships relative to control mice. Pseudomonas aeruginosa (PA103) decreased DPPC synthesis, in part, via calpain-mediated degradation of CCTalpha. Deleterious effects of Pseudomonas on surfactant were lessened after infection with a mutant strain lacking the type III exotoxin, Exo U. Replication-deficient adenovirus 5 CTP:phosphocholine cytidylyltransferase gene delivery improved lung biophysical properties by optimizing surface activity in this Pseudomonas model of proteinase-mediated lung injury. The studies are the first demonstration of in vivo gene transfer of a lipogenic enzyme resulting in improved lung mechanics. The studies suggest that augmentation of DPPC synthesis via gene delivery of CCTalpha can attenuate impaired lung function in surfactant-deficient states such as bacterial sepsis.

Expression of immunoreactive cytidine 5'-triphosphate: cholinephosphate cytidylyltransferase in developing rat lung.

The principle rate-limiting enzyme required for phosphatidylcholine production is cytidine 5'-triphosphate:cholinephosphate cytidylyltransferase. Two functional forms of cytosolic cytidylyltransferase have been previously identified: an active high-molecular weight multimer (H-form) and a relatively inactive low-molecular-weight species (L-form). In the present study, we examined the maturational changes in enzyme mass in the subcellular fractions of fetal, neonatal, and adult rat lungs. Total enzyme mass, measured by immunoblotting of total cellular lung homogenates, revealed a large amount of immunoreactive enzyme during the fetal and neonatal periods and relatively low levels of enzyme in the adult lung. A similar developmental profile for enzyme mass was noted in the cytosolic and microsomal fractions. Further, in the fetus, the majority of cytosolic enzyme mass was expressed as an inactive form (L-form). Stimulation of fetal cytosol with phosphatidylglycerol converted the enzyme mass from an inactive form (L-form) to an active form (H-form). In the adult, a substantial portion of the cytosolic enzyme mass was expressed as the active species (H-form). These observations suggest that cytidylyltransferase activity early in lung development is accompanied by an increase in enzyme mass, the majority of which exists as an inactive low-molecular-weight species. In contrast, high levels of enzyme activity are maintained in the adult lung, despite relatively low levels of enzyme mass, because a significantly greater portion of the enzyme mass is expressed as an active high-molecular-weight multimer.

Lung CTP:choline-phosphate cytidylyltransferase: activation of cytosolic species by unsaturated fatty acid.

CTP:choline-phosphate cytidylyltransferase is the principal rate-limiting enzyme required for surfactant phosphatidylcholine synthesis. We examined the in vitro effect of unsaturated fatty acids on the expression of the two cytosolic forms of this enzyme in fetal and adult rat lung. In the adult, a substantial portion of cytidylyltransferase is expressed as the active form (H form). By contrast, the majority of enzyme mass in the fetus is in an inactive form (L form). Oleic acid, or its esterified derivative, oleoyl-CoA, each stimulated the inactive form (L form) in vitro. However, the addition of oleoyl-CoA directly to the active form (H form) resulted in a dose-dependent decrease in H-form activity, suggesting feedback inhibition. Further, exposure of the enzyme in fetal lung cytosol to either fatty acid increased the mass of the enzyme, consistent with a shift from the inactive form (L form) to the active species (H form). These observations support a key role for unsaturated fatty acids in the developmental regulation of this enzyme.

Isolation and immortalization of rat pre-type II cell lines.

The fetal respiratory distress syndrome is due, in part, to the presence of abundant pre-type II alveolar epithelial cells that have not yet differentiated into mature type II cells. Studies of this syndrome have been limited somewhat by the lack of an adequate in vitro model. In the present study we immortalized pre-type II cells by infecting primary isolates obtained from fetal rat lung with a retroviral construct expressing the adenoviral 12S E1A gene product. The immortalized pre-type II cells retained many of the ultrastructural features typical of pre-type II cells in primary culture, most notably lamellar bodies were not detected and the cells contained abundant stores of glycogen, expressed cytokeratin filaments, and bound the lectin Maclura pomifera. Karyotyping revealed that the cells are diploid. Growth studies demonstrate log phase growth in the presence of serum with a markedly decreased growth rate shortly after the cells reach confluence. Exposure of the immortalized pre-type II cells to hydrocortisone and dibutyryl cAMP resulted in the induction of lamellar bodylike organelles; however, these cells did not secrete surfactant or express surfactant protein A. These cells may serve as useful models for some in vitro studies of fetal type II cell maturation or the fetal respiratory distress syndrome, or both.

Regulation of CTP:choline-phosphate cytidylyltransferase by polyunsaturated n-3 fatty acids.

Disaturated phosphatidylcholine (DSPC) is the most distinctive surface-active lipid in pulmonary surfactant. The feeding of docosahexanoic acid (DHA) 22:6 n-3 has recently been described to elevate the levels of DSPC in rodent lung. The purpose of the present study was to determine the mechanisms by which this n-3 fatty acid might regulate CTP:choline-phosphate cytidylyltransferase, a key enzyme required for phosphatidylcholine (PC) synthesis. Cytidylyltransferase exists in lung cytosol as a large lipid-associated aggregate (H form) which is active, and as an inactive, low-molecular-weight species (L form). Fatty acids in vitro stimulate and aggregate the inactive L form to the active H form. Short-term (2-h) and long-term (24-h) exposure of fetal lung explants to DHA (150 microM) stimulated choline incorporation into PC by 54 and 64%, respectively. The fatty acid also enhanced DSPC synthesis by 88%. These changes were associated with an increase in the activity of cytidylyltransferase by 63% after addition of DHA to the explant medium. In vitro, DHA (50 microM) stimulated L form nearly 15-fold and appeared to be a more potent activator and aggregator of the enzyme than either linoleic 18:2 n-6 or arachidonic 20:4 n-6 acids. The effect of DHA on L-form activation was comparable, however, with other members of the n-3 family. Kinetic studies revealed that DHA increased the maximum velocity of enzyme reaction for cytidylyltransferase, although it did not alter the Michaelis constant of the enzyme for CTP. These observations provide in vitro evidence that n-3 fatty acids may play an important role in the regulation of surfactant PC biosynthesis.

Epidermal growth factor is a positive in vivo regulator of CTP:cholinephosphate cytidylyltransferase.

CTP:cholinephosphate cytidylyltransferase (CT) is a key enzyme required for surfactant phosphatidylcholine synthesis, and its activity is regulated by lung lipids. This study evaluated the effect of epidermal growth factor (EGF) on the phospholipid content and the expression of CT in the lung following direct in vivo administration to the newborn rat. EGF caused an increase in cytidylyltransferase activity by 58% in lung cytosol. The increase in cytosolic activity was not mediated by a corresponding increase in enzyme mass. Further, these changes in cytidylyltransferase activity were associated with a significant increase in total lung phospholipid and phosphatidylcholine content. The results suggest that EGF may have important maturational effects on lung surfactant metabolism.

Oxygen modulates surfactant protein mRNA expression and phospholipid production in human fetal lung in vitro.

We studied the effect of 20-95% O2 on mRNA levels for the surfactant-associated proteins (SP)-A, SP-B, and SP-C and [3H]choline incorporation into total phosphatidylcholine and type II cell-specific disaturated phosphatidylcholine (DPPC) in human fetal lung in culture. SP-A mRNA levels were increased by 25 and 39% in lung explants incubated in 70 and 95% O2, respectively, compared with levels in tissues incubated in 20% O2. SP-B mRNA levels were unaffected by O2, whereas SP-C mRNA levels were increased by 85, 102, and 115% in atmospheres of 35, 50, and 70% O2, respectively. [3H]choline incorporation into total phosphatidylcholine and DPPC were both increased in human fetal lung explants incubated in increased O2 concentrations compared with tissues incubated in 20% O2. Tissue levels of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) activity were not affected by O2 concentration, implying that the changes observed in SP mRNA levels and [3H]choline incorporation may not be mediated through alterations in PKA enzyme activity. These findings demonstrate that O2 regulates SP mRNA expression and phospholipid production in human fetal lung in vitro. We speculate that surfactant composition and possibly function may be regulated by O2 in human lung.

Sphingomyelin metabolism is developmentally regulated in rat lung.

We investigated several indices involved in sphingomyelin metabolism in developing rat lung. The levels of sphingomyelin gradually increased during lung maturation, with highest levels observed postnatally. The content of sphingosine and ceramide, biologically active sphingomyelin degradation products, did not significantly change in microsomes during the prenatal period, but increased to peak levels in neonatal and adult lung, respectively. Sphingosine content increased 6-fold between the fetal (Day 21) and neonatal period. The developmental profiles of two enzymes involved in sphingomyelin synthesis, serine palmitoyltransferase and sphingomyelin synthase, were similar. Serine palmitoyltransferase activity increased progressively from the fetal to neonatal period, and plateaued at high levels in the adult lung. The activity of serine palmitoyltransferase correlated with the levels of endogenous sphingolipid in lung tissue. Sphingomyelin synthase activity also increased during fetal lung development, but attained highest levels at Day 21 gestation; postnatally, enzyme activity was detected at lower levels. The activities of the sphingolipid hydrolases, acid and neutral sphingomyelinase and acid and alkaline ceramidase, were elevated in fetal lung, thereafter declining to low levels after birth. Studies conducted in alveolar macrophages, fibroblasts, and alveolar type II epithelial cells revealed that these developmental changes in enzyme activities in lung tissue were also occuring globally at the cellular level and were not restricted to any specific cell population. These studies suggest that the developmental increase in lung sphingomyelin content is due to coordinate regulation of enzymes involved in the biosynthesis and degradation of sphingomyelin. These observations also suggest a regulatory role for serine palmitoyltransferase in the generation of long chain sphingoid bases.

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.

Betamethasone activation of CTP:cholinephosphate cytidylyltransferase in vivo is lipid dependent.

Glucocorticoids increase surfactant phosphatidylcholine synthesis, in part, by stimulating the rate regulatory enzyme CTP:cholinephosphate cytidylyltransferase. This enzyme exists in mammalian lung cytosol as an active lipoprotein form (H-form) and an inactive apoprotein (L-form) species. We administered betamethasone to pregnant rats to examine the mechanisms for glucocorticoid stimulation of cytidylyltransferase activity in fetal lung. The hormone stimulated cytosolic activity threefold, and this effect was nearly abolished after lipid extraction. The addition of lipid extracts isolated from betamethasone-treated cytosolic preparations to L-form species increased enzyme activity to a greater extent than lipid extracts from control lungs. Further, the glucocorticoid increased the proportion of H-form activity from 34 to 55% of the total activity in the fetal lung cytosol. These changes were associated with a marked decrease in the activity of the L-form species. Analysis of the lipid composition of the H-form revealed that betamethasone increased the content of lipid activators, including phosphatidylglycerol and fatty acids. These observations provide evidence that glucocorticoid stimulation of CTP:cholinephosphate cytidylyltransferase in vivo is mediated by a conversion of the inactive form (L-form) to the active species (H-form). These studies further emphasize the critical role of lung lipids in mediating the glucocorticoid activation of this enzyme.

Betamethasone activation of CTP:cholinephosphate cytidylyltransferase is mediated by fatty acids.

The purpose of the present study was to determine the mechanisms by which glucocorticoids increase the activity of CTP:cholinephosphate cytidylyltransferase, a key enzyme required for the synthesis of surfactant phosphatidylcholine. Lung cytidylyltransferase exists as an inactive, light form low in lipids (L-form) and an active, heavy form high in lipid content (H-form). In vitro, fatty acids stimulate and aggregate the inactive L-form to the active H-form. In vivo, betamethasone increases the amount of H-form while decreasing the amount of L-form in fetal lung. There is also a coordinate increase in total free fatty acids in the H-form. In the present study, we used gas chromatography-mass spectrometry to measure the fatty acid species associated with the H-forms in fetal rat lung after the mothers were treated with betamethasone (1 mg/kg). In vivo, betamethasone increased the total amount of free fatty acids associated with the H-form by 62%. Further, the hormone selectively increased the mass of myristic and oleic acids in H-form by 52 and 82%, respectively. However, betamethasone produced the greatest increase in the amount of H-form linoleic acid, which increased fourfold relative to control. In vitro, each of the fatty acids increased L-form activity in a dose-dependent manner; however, linoleic acid was the most potent. Linoleic and oleic acids also effectively increased L-form aggregations. These observations suggest that in vivo glucocorticoids elevate the level of specific fatty acids which convert cytidylyltransferase to the active form.

TNF-alpha increases ceramide without inducing apoptosis in alveolar type II epithelial cells.

Ceramide is a bioactive lipid mediator that has been observed to induce apoptosis in vitro. The purpose of this study was to determine whether endogenous ceramide, generated in response to in vivo administration of tumor necrosis factor-alpha (TNF-alpha), increases apoptosis in primary rat alveolar type II epithelial cells. Intratracheal instillation of TNF-alpha (5 microgram) produced a decrease in sphingomyelin and activation of a neutral sphingomyelinase. These changes were associated with a significant increase in lung ceramide content. TNF-alpha concomitantly activated the p42/44 extracellular signal-related kinases and induced nuclear factor-kappaB activation in the lung. Hypodiploid nuclei studies revealed that intratracheal TNF-alpha did not increase type II cell apoptosis compared with that in control cells after isolation. A novel observation from separate in vitro studies demonstrated that type II cells undergo a gradual increase in apoptosis after time in culture, a process that was accelerated by exposure of cells to ultraviolet light. However, culture of cells with a cell-permeable ceramide, TNF-alpha, or a related ligand, anti-CD95, did not increase apoptosis above the control level. The results suggest that ceramide resulting from TNF-alpha activation of sphingomyelin hydrolysis might activate the mitogen-activated protein kinase and nuclear factor-kappaB pathways without increasing programmed cell death in type II cells.