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.

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.

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.

Identification of sex-specific differences in surfactant synthesis in rat lung.

Delayed lung maturation and lower levels of surfactant phosphatidylcholine have been previously identified in male fetuses compared with female fetuses in several species. We investigated the mechanisms for sex differences in surfactant content by examining parameters of phosphatidylcholine turnover and biosynthesis; the latter was evaluated by measuring metabolic steps within the biosynthetic pathway. Compared with male lung cells, freshly isolated lung cells from female fetuses contained higher levels of disaturated phosphatidylcholine, a marker of surfactant lipid. Female mixed monolayer cultures exhibited a 71% increase in choline incorporation into disaturated phosphatidylcholine compared with male cultures. Male cultures exhibited significantly greater release of [3H]-arachidonic acid into the medium compared with females, suggesting sex differences in phospholipase activity. However, pulse-chase studies showed no sex differences in degradation of disaturated phosphatidylcholine, which was confirmed by assays of phospholipase A2, phosphatidylcholine-specific phospholipase C, and phospholipase D. Female mixed lung cells, however, had greater rates of cellular choline transport and activity of cytidylyltransferase, the rate-regulatory enzyme for phosphatidylcholine synthesis. Separate studies showed that exposure of sex-specific pretype II cell cultures to cortisol-stimulated fibroblast-conditioned medium plus transforming growth factor-beta-neutralizing antibody stimulated cytidylyltransferase activity to a greater extent in male cells compared with female cells. These studies indicate that sex differences in surfactant phospholipid content are not due to differences in phospholipid turnover, but rather differential regulation of specific metabolic steps within the surfactant biosynthetic pathway. The data also support a role for transforming growth factor-beta as a negative regulator of a key surfactant biosynthetic enzyme within male lungs.

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.

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.

Betamethasone modulation of sphingomyelin hydrolysis up-regulates CTP:cholinephosphate cytidylyltransferase activity in adult rat lung.

Glucocorticoids appear to play an integral role in stimulating surfactant synthesis by activating the rate-regulatory enzyme for phosphatidylcholine synthesis, CTP:cholinephosphate cytidylyltransferase (CT). The activity of liver CT, in vitro, has been shown to be inhibited by the sphingomyelin hydrolysis product, sphingosine. In order to investigate the mechanisms by which glucocorticoids alter CT activity, in vivo, we administered betamethasone (1 mg/kg intraperitoneally) sequentially to adult male rats for 5 days. Betamethasone increased CT activity 2-fold relative to control in whole lung. The hormone also increased membrane-bound activity, but did not affect cytosolic enzyme activity. Betamethasone modestly increased CT mRNA as determined by the reverse-transcription PCR and Southern analysis of PCR products, but did not alter the levels of immunoreactive enzyme in lung membranes as demonstrated by Western blotting. The hormone did, however, produce a nearly 3-fold increase in membrane-associated sphingomyelin, and co-ordinately a substantial decrease in the levels of sphingosine in lung membranes. Sphingosine, but not sphinganine, was a competitive, reversible inhibitor of lung CT with respect to the enzyme activator, phosphatidylglycerol. Betamethasone decreased the activities of the sphingomyelin hydrolases: acid sphingomyelinase by 33% and of alkaline ceramidase by 21%. The hormone also inhibited the generation of sphingosine from lysosphingomyelin in lung membranes. There was no significant effect of the hormone on serine palmitoyltransferase activity, the first committed enzyme for sphingolipid biosynthesis. Further, administration of L-cycloserine, an inhibitor of sphingosine formation, was shown to stimulate CT activity by 74% and increase disaturated phosphatidylcholine in alveolar lavage by 52% relative to control. These observations suggest that glucocorticoids up-regulate surfactant synthesis at the level of a key regulatory enzyme by significantly altering the availability of inhibitory metabolites resulting from sphingomyelin hydrolysis.

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.

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.

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.

Effect of dietary fat saturation, cholesterol and cholestyramine on acyl-CoA: cholesterol acyltransferase activity in rabbit intestinal microsomes.

The regulation of intestinal acyl-CoA: cholesterol acyltransferase was investigated by dietary manipulation. Rabbits were fed the following diets: normal rabbit chow, 10% safflower oil, safflower oil plus 1% cholesterol, coconut oil plus 1% cholesterol, or cholestyramine. Acyl-CoA: cholesterol acyltransferase activity was increased in intestinal microsomes from animals fed safflower oil but not from animals fed coconut oil. Both diets containing cholesterol increased acyl-CoA: cholesterol acyltransferase activity; however, the safflower oil plus cholesterol diet was a more potent stimulator than coconut oil plus cholesterol. Cholestyramine decreased microsomal acyl-CoA: cholesterol acyltransferase activity. The different diets significantly modified microsomal lipid content in these groups. The two cholesterol diets resulted in equal increments in microsomal cholesterol. Microsomal cholesterol was unchanged in animals on the safflower oil diet; however, coconut oil and cholestyramine decreased the cholesterol content. Linoleic acid content increased in microsomes from animals on both the safflower oil-containing diets. Myristic acid accumulated and linoleic acid was decreased in microsomes from animals on both diets containing coconut oil. Subcellular fractionation of the intestine yielded a 4-fold enhancement of acyl-CoA: cholesterol acyltransferase activity in the rough endoplasmic reticulum. The lipid modifications and the subsequent changes in acyl-CoA: cholesterol acyltransferase activity in the rough endoplasmic reticulum from animals on normal, safflower oil, and safflower oil plus cholesterol diets paralleled that observed in the crude microsomal preparations. The changes in acyl-CoA: cholesterol acyltransferase activity observed with the different diets were not due to changes in microsomal fatty acyl-CoA pool size. It is concluded that dietary manipulation can alter microsomal lipid content. Microsomal fat saturation, independent of microsomal cholesterol content, regulates intestinal acyl-CoA: cholesterol acyltransferase and modifies the stimulatory effect of exogenous cholesterol on this enzyme.

Collagenase activity in epidermoid carcinoma of the oral cavity and larynx.

Summary--Tumor invasion requires the breadkdown of the main structural protein, collagen. A series of fourteen epidermoid carcinomas of the larynx and oral cavity produced a collagen dissolving enzyme in vitro as demonstrated by the breakdown of 14C-labeled collagen. Oral cavity tumors showed greater activity than laryngeal carcinomas while both sites were more active than uninvolved mucosa from the same patients. Tumor associated collagenase activity, in common with previously described collagenases, can only be demonstrated in vitro and requires protein synthesis. Maximum tumor collagenase occurred at 24 hours in vitro and then declined as compared with the maximum collagenase at 72 hours in vitro produced by oral cavity mucosa. The 14 patients in our series were ranked in order of the collagenase activity of their tumors. At 18 months after the diagnosis, four of the six patients with the most active tumors were dead of cancer and one patient was alive with persistent cancer. High collagenase activity may be a factor in the clinical aggressiveness of epidermoid carcinomas of the head and neck.