Tumor Necrosis Factor-α Inhibits Expression of CTP:Phosphocholine Cytidylyltransferase

We investigated the effects of tumor necrosis factor α (TNFα), a key cytokine involved in inflammatory lung disease, on phosphatidylcholine (PtdCho) biosynthesis in a murine alveolar type II epithelial cell line (MLE-12). TNFα significantly inhibited [3H]choline incorporation into PtdCho after 24 h of exposure. TNFα 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 TNFα inhibition of CCT activity was associated with a uniform decrease in the mass of CCTα 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 CCTα mRNA, and CCTβ mRNA was not detected. Incorporation of [35S]methionine into immunoprecipitable CCTα protein in pulse and pulse-chase studies revealed that TNFα did not alterde novo synthesis of enzyme, but it substantially accelerated turnover of CCTα. Addition ofN-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 TNFα. TNFα-induced degradation of CCTα protein was partially blocked by ALLN or lactacystin. CCT was ubiquitinated, and ubiquitination increased after TNFα exposure. m-Calpain degraded both purified CCT and CCT in cellular extracts. Thus, TNFα inhibits PtdCho synthesis by modulating CCT protein stability via the ubiquitin-proteasome and calpain-mediated proteolytic pathways.

Phosphatidylcholine (PtdCho (1)), 1 the most abundant mammalian phospholipid, has several well recognized roles, including serving as an integral component of pulmonary alveolar surfactant and providing the cell with a reservoir for the generation of bioactive lipid second messengers. The biosynthesis of PtdCho is tightly regulated within the CDP-choline pathway (1). A key step in this pathway is the conversion of choline phosphate to CDP-choline, which is catalyzed by the ratelimiting enzyme CTP:phosphocholine cytidylyltransferase (CCT, EC 2.7.7.15 (1)).
CCT localizes primarily to the endoplasmic reticulum and nucleus (2), but it is also found associated with Golgi and transport vesicles (1,3). CCT activity in cells is controlled primarily by association with membrane lipids and by gene expression (1). Positive and negative regulatory lipids govern CCT activity when presented within the context of a PtdCho lamellar structure. Activating lipids include unsaturated fatty acids, anionic phospholipids, and diacylglycerol, whereas negative regulatory lipids include sphingosine and lyso-PtdCho (4 -7). Phosphorylation of CCT modulates activity by attenuating lipid activation, and an inverse correlation exists between CCT phosphorylation and PtdCho synthesis in some systems (8). Protein kinases that phosphorylate purified CCT in vitro include p42/44 mitogen-activated protein kinase, protein kinase C␣, and casein kinase II (9,10). Although there is substantial evidence supporting regulation of CCT by biochemical and protein modification mechanisms, changes in the level of mRNA and protein have also been identified (11)(12)(13)(14). In this regard, studies have shown that the developmental increase in CCT mRNA in alveolar epithelium is due to an increase in the stability of the CCT transcript (15).
Three isoforms of CCT exist in cells as follows: CCT␣, CCT␤1, and CCT␤2 (2,16). The 367-amino acid CCT␣ isoform contains four distinct functional domains including an aminoterminal nuclear targeting domain, a catalytic sequence, a regulatory domain consisting of amphipathic ␣-helices that interact with lipids, and a carboxyl-terminal phosphorylation domain. Unlike CCT␣, the CCT␤ isoforms lack a nuclear localization motif and differ in their carboxyl-terminal phosphorylation sites (2,16). However, the CCT␤ isoforms exhibit full catalytic activity and, like CCT␣, require the presence of lipid regulators for optimal activation. The control mechanisms underlying the differential expression of CCT␣ and CCT␤ are not known.
Several factors such as phorbol ester (17) colony-stimulating factor 1 (11), cholecystokinin (13), and exogenous lipoproteins (18,19) have been used to modulate CCT activity to investigate the physiological mechanisms that govern PtdCho synthesis. In the lung, TNF␣ is a key cytokine released by alveolar macrophages that has been implicated as a major factor for inducing acute lung injury associated with sepsis (20). Many of the manifestations of sepsis can be reproduced by the in vivo administration of this cytokine, and these deleterious effects can be attenuated by treatment with TNF␣ antibodies (21).
Sepsis-associated injury is characterized by elevated serum levels of TNF␣ and with reduced levels of PtdCho within critical organs (22,23). Specifically, one important feature of TNF␣-induced acute lung injury is depletion of alveolar surfactant PtdCho, which may occur as a result of accelerated PtdCho hydrolysis (22,24,25). TNF␣ stimulates PtdCho hydrolysis rapidly, often within minutes after cytokine exposure (26). However, there is limited information about long term effects of TNF␣ on phospholipid metabolism and whether the cytokine might regulate PtdCho biosynthesis. Thus, the primary objectives of this study were to investigate whether exposure to TNF␣ down-regulates PtdCho biosynthesis in alveolar epithelial cells and to characterize the mechanisms responsible for reduced cellular PtdCho levels after long term cytokine exposure. Recent studies demonstrating that TNF␣ suppresses [ 14 C]glucose incorporation into disaturated phosphatidylcholine (DSPtdCho (27)), a marker of surfactant phospholipid, led us to hypothesize that the inhibitory actions of TNF␣ on surfactant phospholipid metabolism might be mediated by inhibition of PtdCho synthesis at the CTP:phosphocholine cytidylyltransferase step.

EXPERIMENTAL PROCEDURES
Materials-Human tumor necrosis factor ␣ (1 g ϭ 1.1 ϫ 10 5 activity units) was obtained from Endogen (Minneapolis, MN). Silica LK5D (250 mm ϫ 20 ϫ 20 cm) TLC plates were purchased from Whatman. Hite's medium was obtained from the University of Iowa Tissue Culture and Hybridoma Facility (Iowa City, IA). All radiochemicals were purchased from NEN Life Science Products. The MLE-12 cell line was kindly provided by Dr. Jeffrey Whitsett (Cincinnati (28)). Lactacystin, N-Acetyl-Leu-Leu-Nle-CHO (ALLN), and calpastatin were purchased from Calbiochem. Immunoblotting membranes were obtained from Millipore (Bedford, MA), and Sepharose CL-B4 was from Sigma. The ECL Western blotting detection system and GammaBind® Plus Sepharose® were from Amersham Pharmacia Biotech. Protein A was purchased from Repligen (Cambridge, MA). Anti-ubiquitin rabbit polyclonal antibody was purchased from StressGen Biotechnologies Corp. (Victoria, Canada). Anti-CCT␣ and anti-CCT␤ rabbit polyclonal antiserum were prepared against synthetic peptides as described previously (2,16). The anti-CCT␣ rabbit polyclonal antiserum was raised against a synthetic peptide (MDAQSSAKVNSRKRRKE) corresponding to the first 17 amino acids of CCT␣. The anti-CCT␤ rabbit polyclonal antiserum was raised against a synthetic peptide (MEEIEHTCPQPRL) corresponding to amino acids 27-39 of CCT␤. A rabbit polyclonal antibody to a synthetic peptide corresponding to residues 164 -176 of CCT␣ and crossreactive with CCT␤ (29) was generated by Covance Research Products Inc. (Richmond, CA) and is termed pan-reactive. The CCT␣ and CCT␤ cDNA clones have been described (16). The random primed labeling kit used was Redprime II TM (Buckinghamshire, UK).
Cell Culture-Cells were cultured in Hite's medium with the inclusion of 2% fetal bovine serum for 48 h and then cultured in Hite's medium alone for 2-24 h in the presence or absence of TNF␣ (500 ng/ml (27)). In some studies, cells were preincubated with lactacystin (1-5 M) or ALLN (10 -40 g/ml) alone, or with both protease inhibitors for 1 h with or without subsequent exposure of cells to TNF␣. Cells lysates were isolated after brief sonication in Buffer A (150 mM NaCl, 50 mM Tris, 1.0 mM EDTA, 2 mM dithiothreitol, 0.025% sodium azide, 1 mM PMSF, pH 7.4) at 4°C prior to analysis. Cell cytosolic and microsomal subfractions were prepared using sequential centrifugation (30). Cell nuclei were prepared by nuclease treatment of cellular lysates as described (31).
Cell Viability-Effects of TNF␣ on cell lysis were measured by a standard chromium-51 release assay. Cells were pulsed with 1 Ci of 51 Cr-labeled sodium chromate overnight. After labeling, cells were rinsed 4 times with Hite's medium and subsequently incubated for an additional 24 h with or without TNF␣ (500 ng/ml). An aliquot of medium was collected for gamma counting. Cell-specific lysis was calculated as a ratio of medium cpm/total cpm as described (32). Cell cultures were also examined morphologically using an Olympus IX 70 inverted microscope (Leeds Precision Instruments, Inc., Minneapolis, MN).
Phosphatidylcholine (PtdCho) and Disaturated Phosphatidylcholine (DSPtdCho) Analysis-Lipids were extracted from equal amounts of cellular protein using the method of Bligh and Dyer (33). Lipids were dried under nitrogen gas, spotted on silica LK5D plates, and resolved in chloroform:methanol:petroleum ether:acetic acid:boric acid (40:20:30: (34)). Samples that comigrated with PtdCho standard as detected by exposure to iodine vapor were scraped from the silica gel and quantitatively assayed for phosphorus content (35). In other studies, cells were pulsed with 1 Ci of [methyl-3 H]choline chloride during the final 2 h of incubation. Cellular lipids were extracted; PtdCho was resolved using TLC and the lipid reacted with osmium tetroxide prior to a run in the second dimension (36). Incorporation of label into PtdCho and DSPtdCho were then quantitated using scintillation counting.
Enzyme Assays-The activity of choline kinase was assayed as described (37). The reaction mixture (0.1-ml volume) contained 100 mM Tris-HCl buffer, pH 8.0, 10 mM magnesium acetate, 0.016 mM [ 14 C]choline (specific activity ϳ7000 dpm/nmol), 10 mM ATP, and 50 -100 g of cell sample. After a 1-h incubation at 37°C, the reaction was terminated with 0.02 ml of cold 50% trichloroacetic acid. Aliquots (20 l) of the mixture were spotted on Whatman 3MM paper and choline metabolites resolved using paper chromatography as described (37). Spots that comigrated with the radiolabeled standard, choline phosphate, were cut and used for scintillation counting.
CCT activity was determined by measuring the rate of incorporation of [methyl-14 C]phosphocholine into CDP-choline using a charcoal extraction method (30). All assays were performed without the inclusion of a lipid activator in the reaction mixture unless otherwise stated.
The lipid substrate was prepared by combining appropriate amounts of 1,2 dioleoylglycerol (1 mM) and phosphatidylglycerol (0.8 mM) in a test tube, drying under nitrogen gas, and brief sonication prior to addition to the assay mixture to achieve the final desired concentration. The reaction proceeded for 1 h at 37°C and was terminated with 4 ml of methanol:chloroform:water (2:1:7, v/v). The remainder of the assay was performed exactly as described (38).
Immunoblot Analysis-For immunoblot analysis, equal amounts of protein from cell homogenates, cytosolic, microsomal, and nuclear extracts were used. Each sample was adjusted to give a final concentration of 60 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.1% bromphenol blue, and 5% ␤-mercaptoethanol and heated at 100°C for 5 min. Samples were then electrophoresed through a 10% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. CCT␣ and CCT␤ isoforms were detected by using the ECL Western blotting detection system as instructed by the manufacturer. The dilution factor for anti-CCT isoform-specific antibodies was 1:1000. The pan-reactive CCT rabbit polyclonal antibody was used for immunoprecipitation studies (29).
Detection of CCT␣ mRNA-Total cellular RNA was isolated from cells by cesium chloride gradient centrifugation following lysis with guanidine thiocyanate (39). Total RNA (30 g) containing ethidium bromide was separated electrophoretically using a 1% agarose gel containing 2.2 M formaldehyde with a circulating running buffer of 20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA, and 2.2 M formaldehyde. RNA was capillary-blotted to a nitrocellulose membrane using a Turboblot apparatus and hybridized at 42°C with 7.5 ϫ 10 6 cpm/15 ml of a 32 P-labeled probe using standard Northern blot hybridization protocols (16). Probes were prepared by random primed labeling of cDNA fragments isolated from agarose gels using a Geneclean kit. The probes were prepared for the following cDNAs inserted in pcDNA3: a 1.3-kb HindIII-BamHI fragment of the rodent CCT␣; a 1.3-kb BamHI-XhoI fragment from the human CCT␤. The blot was washed four times for 1 min at 22°C and then 42°C for 10 min, following hybridization with the CCT␣ probe, or four times for 1 min at 22°C only following hybridization with the CCT␤ probe.
Single Immunoprecipitation and Sequential Immunoprecipitation with Anti-CCT and Anti-ubiquitin Antibodies-Single immunoprecipitations were used to evaluate CCT␣ synthesis and degradation, whereas sequential immunoprecipitations were performed to determine if CCT␣ becomes ubiquitinated in MLE cells. CCT␣ synthesis was assessed in control and TNF␣-treated cells by labeling cells with [ 35 S]methionine (60 Ci/ml in methionine-deficient medium) during the last 4 h of culture at 37°C. After labeling, cells were scraped in RIPA buffer (10 mM Na 2 HPO 4 , 100 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1 mM dithiothreitol, 1 mM PMSF, 20 M leupeptin, 1% Triton X-100, 0.1% SDS, 20 mM L-methionine, and 2 mM L-cysteine; pH 7.4), sonicated briefly, centrifuged at 15,000 ϫ g for 60 s, and then precleared for 2 h at 4°C using Sepharose CL-4B and preimmune rabbit serum. Cleared supernatants, containing equal amounts of protein (500 g), were incubated overnight at 4°C with 1 g of rabbit anti-CCT antibody, which was previously bound to GammaBind® Plus Sepharose®. The following morning, the immunoprecipitates were washed with lysis buffer (50 mM HEPES, 150 mM NaCl, 0.5 mM EGTA, 1 mM PMSF, 50 mM NaF, 1 mM NaVO4, 1 mM PMSF, and 1 M aprotinin; pH 7.6), and the pellets were placed in SDS protein sample buffer and heated to 95°C for 5 min. Soluble proteins were separated using 10% SDS-PAGE, and the gels were stained with Coomassie Blue, destained, and then dried for both autoradiography and liquid scintillation counting of excised 42-kDa protein bands.
Turnover of CCT␣ was determined using pulse-chase procedures. MLE-12 cells were preincubated for 1 h in methionine-deficient medium and then pulsed with [ 35 S]methionine (60 Ci/ml) for 4 h at 37°C. Cells were rinsed twice in similar medium and chased with serum-free Hite's medium containing 10 mM methionine and 3 mM cysteine for 0 to 24 h in the presence or absence of TNF␣. Cells were scraped and lysed in RIPA buffer, and CCT␣ was immunoprecipitated prior to separation using SDS-PAGE as described above.
For detection of ubiquitinated CCT␣, MLE-12 cells were labeled with with [ 35 S]methionine as described above. The radiolabeled cells were washed with ice-cold phosphate-buffered saline and solubilized in RIPA buffer. CCT␣ was immunoprecipitated using 1 g of rabbit anti-CCT antibody as described above. The CCT immunoprecipitates were washed twice with lysis buffer, resuspended in 400 l of the same buffer, and boiled for 4 min to release proteins from the GammaBind® Plus Sepharose® beads. Samples were placed on ice for at least 10 min and allowed to cool. These samples were then used for a second round of immunoprecipitation, which was conducted overnight at 4°C using rabbit anti-ubiquitin antibody at a dilution of 1:100. The ubiquitin immunocomplexes were captured with protein A and Sepharose CL-B4 and subsequently analyzed by SDS-PAGE and autoradiography of dried gels.
Assay of Calpain and 20 S Proteasome Proteolytic Activity-Incubations for calpain activity were conducted for 16 h at 30°C in a reaction mixture (total volume, 150 l) containing calpain (0 -500 g; 0.00 -0.90 units/ml), 20 mM Tris, pH 7.5, 50 mM NaCl, 1 mM dithiothreitol, 0.5 mM EGTA, 0.5 mM EDTA, and 20% v/v glycerol. Calpastatin (50 g), a calpain protease inhibitor, was added to selected samples. Proteins from cellular lysates (300 g) and a CCT␣ preparation (30 g) purified from rat liver cytosol using octyl glucoside were used as substrates (40). The reaction was started by adding 1 l of CaCl 2 to a final concentration of 3 mM and was terminated by adding SDS protein sample buffer and then heating samples to 95°C. Degradation of CCT␣ protein was evaluated by immunoblot analysis.
For assay of 20 S proteasome, the reaction mixture (total volume, 100 l) contained 0 -40 nM of 20 S proteasome, 40 mM Tris-HCl, pH 8.0, 0.04% w/v SDS, and 2 mM ␤-mercaptoethanol. Rat liver CCT␣ protein (30 g) served as substrate. Reactions were carried out for 24 h at 37°C and then stopped by adding SDS protein sample buffer and heating samples to 95°C. In selected assays, proteasomes were pretreated with an inhibitor, 50 M lactacystin, for 10 min at 37°C before addition of protein substrate. Immunoblotting was then conducted to evaluate CCT␣ degradation in these samples.

RESULTS
Cell Viability-Cells cultured in the presence or absence of TNF␣ exhibited no significant differences in cell cytotoxicity as determined by 51 1A (inset)) and choline incorporation into DSPtdCho by 76% (p ϭ 0.001, Fig. 1B (inset)). These results show that TNF␣ substantially reduces the biosynthesis of PtdCho, an essential component of surfactant, in alveolar epithelial cells.
Enzymes Assays-To confirm whether TNF␣ inhibits PtdCho synthesis, we assayed the activities of enzymes within the CDP-choline pathway, the principal pathway for PtdCho synthesis. There were no significant effects of the cytokine on choline kinase activity, the first committed step in the pathway, or on cholinephosphotransferase, the terminal enzyme involved in PtdCho synthesis (Table I). However, TNF␣ significantly decreased CCT activity to approximately 40% of control values after 24 h of treatment (p Ͻ 0.001, Table I) and 50% of control after 30 h (data not shown). These negative effects of TNF␣ were not only observed in cellular homogenates but also in the soluble, microsomal, and nuclear fractions (data not shown). The presence of the lipid activator (PtdCho:oleic acid) in the reaction mixture had little effect, as enzyme activity decreased 47% after 24 h of TNF␣ treatment. These results indicate that the primary effect of TNF␣ is to down-regulate the conversion of choline phosphate to CDP-choline at the rate-limiting step within the PtdCho biosynthetic pathway.
Immunoblot Analysis-We performed immunoblot analysis to determine if TNF␣ inhibited CCT activity by decreasing the amount of enzyme. Although two distinct CCT isoforms have been described in mammalian tissues, CCT␣ appears to be the only isoform detectable in MLE-12 cells. TNF␣ significantly decreased the amount of steady-state CCT␣ after long term exposure to the cytokine. No effects on immunoreactive levels were seen after 4 h of TNF␣ treatment, and modest inhibitory effects of the cytokine were observed after 12 h of exposure ( Fig.  2A). However, densitometric analysis showed that 24 h of TNF␣ exposure overall produced a 50 -70% decrease in the amount of immunoreactive enzyme in these cells. Following TNF␣ treatment, the levels of immunoreactive CCT␣ uniformly decreased in total cell lysates and in the cytosolic and microsomal fractions compared with control (Fig. 2B). The most significant effect of TNF␣ was observed in the soluble fraction, where control cells contained 3-fold greater amounts of CCT␣ compared with TNF␣-treated cells. Unlike CCT␤, the CCT␣ isoform contains a nuclear targeting domain, and the nucleus appears to contain a large reservoir of enzyme (16). Consistent with its effects in whole cells and on nuclear CCT activity, TNF␣ decreased the amount of nuclear-associated enzyme. As a control, there was no demonstrable effect of TNF␣ on levels of ␤-actin (Fig. 2B). These results indicate that TNF␣ decreases PtdCho synthesis in MLE cells by decreasing the amount of CCT␣ protein.
Detection of CCT␣ mRNA-We next conducted studies to determine if TNF␣ decreased CCT␣ protein by decreasing the amount of CCT␣ mRNA. By using probes prepared from 1.3and 1.3-kb fragments of rodent CCT␣ and human CCT␤, respectively, Northern blotting was performed on total RNA isolated from control cells and cells exposed to TNF␣ for various times (Fig. 3). As shown in Fig. 3, TNF␣ did not significantly alter the amount of the 1.9-kb CCT␣ transcript. Consistent with our immunoblotting results, mRNA for CCT␤ was not detected in MLE cells. These results indicate that TNF␣ does not regulate CCT␣ at the level of gene transcription and that CCT␤ is not expressed in MLE cells.
De Novo Synthesis and Degradation of CCT␣-We next examined whether TNF␣ alters de novo synthesis or degradation of CCT␣. For synthesis studies, cells were pulsed the last 4 h of culture with [ 35 S]methionine, and CCT␣ was then immunoprecipitated using the panreactive antibody. These experiments did not reveal a significant effect of TNF␣ on newly synthesized CCT␣ between 4 and 24 h of analysis (Fig. 4A). The cytokine, however, substantially enhanced CCT␣ degradation (Fig. 4B). The turnover of newly synthesized CCT␣ was examined in pulse-chase experiments in which the amount of [ 35 S]methionine incorporated into immunoprecipitable CCT␣ was determined after a 4-h pulse, followed by a chase (0 -24 h) with unlabeled methionine conducted in the presence or absence of TNF␣. At 0 h (start of chase), equal amounts of CCT␣ were present in control and TNF␣-treated cells (Fig. 4B).
However, after only 4 h of TNF␣ treatment, CCT␣ degradation was detectable, and by 16 h, substantially less CCT␣ was observed in TNF␣ samples compared with control. TNF␣ treatment resulted in further CCT␣ breakdown at the 24-h time point (Fig. 4B). In contrast to effects of TNF␣ on CCT␣, the cytokine did not alter the turnover of ␤-actin (Fig. 4C). On the basis of these results, the half-life for CCT␣ protein was estimated to be ϳ8 h in control MLE cells and reduced by approximately 50% by TNF␣ (Fig. 4D). These results indicate that TNF␣ decreases PtdCho synthesis by selectively enhancing the degradation of CCT␣.
Effect of Protease Inhibition on CCT Expression-TNF␣ induces catabolism of cellular proteins by activating two principal pathways: cytosolic calcium-activated proteases (calpains) and the ubiquitin-proteasome pathway (42,43). To investigate the role of these proteolytic pathways in mediating TNF␣induced CCT degradation, control and cytokine-treated cells were preincubated with ALLN, an inhibitor of calpain I, and lactacystin, a 20 S proteasome inhibitor. Samples were then analyzed for CCT content and DSPtdCho mass (Figs. 5 and 6). Pretreatment with either lactacystin or ALLN partly blocked TNF␣-induced CCT proteolysis by 47 and 55%, respectively, as determined by densitometric analysis of immunoblots (Fig. 5, A  and B). When MLE cells were pretreated with a combination of ALLN and lactacystin, subsequent TNF␣ exposure did not result in a substantial decrease in CCT mass (Fig. 5C). In separate studies, each inhibitor alone totally blocked the negative effects of TNF␣ on DSPtdCho content within cells (Fig. 6). These observations implicate the calpain and proteasome pathways in mediating TNF␣-induced proteolysis of CCT and inhi-

FIG. 1. Effect of TNF␣ on PtdCho content and choline incorporation into PtdCho in MLE cells. Cells were incubated in
Hite's medium with the inclusion of 2% fetal bovine serum for 48 h prior to changing the medium to Hite's serum-free medium with or without the addition of TNF␣ (500 ng/ml) for the indicated times. A, lipids were extracted using a Bligh-Dyer method, PtdCho resolved using TLC, and the lipid reacted with osmium tetroxide prior to a run in the second dimension. The levels of PtdCho and DSPtdCho (inset) were quantitatively assayed for using the phosphorus assay. B, in other studies, cells were pulsed with 1 Ci of [methyl-3 H]choline chloride during the final 2 h of incubation. Cellular lipids were extracted, and the lipids were processed as described above. Incorporation of label into PtdCho and DSPtdCho (inset) were then quantitated using scintillation counting. The results represent the mean Ϯ S.E. of three independent experiments. *, p Ͻ 0.05 versus control; **, p Ͻ 0.005 versus control.

bition of surfactant PtdCho synthesis.
Effect of m-Calpain on CCT Proteolysis-To determine if CCT could be a potential substrate for TNF␣-activated proteases, in vitro assays were conducted using purified CCT␣ in the presence of catalytically active m-calpain. The products of the reaction were then analyzed by immunoblotting using the pan-reactive anti-CCT antibody. Incubation of purified CCT␣ with calpain produced a dose-dependent decrease in the levels of the 42-kDa enzyme yielding several lower molecular weight degradation products at high doses of calpain (Fig. 7A). CCT proteolysis by calpain was substantially blocked by inclusion of the endogenous inhibitor, calpastatin, in the reaction mixture. In addition to effects on purified CCT, incubation of calpain with cellular lysates under similar reaction conditions resulted in proteolysis of CCT. Proteolysis of CCT in MLE cell lysates was also inhibited by calpastatin (Fig. 7B). The degradation products observed on the immunoblots did not originate from m-calpain because m-calpain did not react with CCT antibody (Fig. 7C). Preliminary studies were also performed to assess the role of the proteasome on degradation of CCT, using the  Fig. 1 with or without TNF␣ (500 ng/ml) for the indicated times. Total cellular RNA was separated (30 g) on 1% agarose gels, transferred to nitrocellulose membranes, and probed with CCT␣ or CCT␤ cDNAs. Below, ethidium bromide staining of RNA gels was visualized to confirm equal RNA loading. 20 S proteasome which functions as the proteolytic core of the 26 S proteasome complex. The 20 S proteasome induced only partial degradation of the enzyme up to a concentration of 20 nmol in the reaction mixture (data not shown). The results may be attributed to the fact that unlike the 26 S proteasome, the 20 S proteasome lacks recognition sites for ubiquitinated proteins. Collectively, these results indicate that CCT serves as a substrate for calpain and possibly the proteasomal complex.
Sequential Immunoprecipitation with Anti-CCT and Antiubiquitin Antibodies-The data above support a role for the proteasome pathway in partially mediating TNF␣-induced degradation of CCT. To identify whether TNF␣-induced proteolysis of CCT involved a ubiquitin-dependent or ubiquitin-independent proteasome pathway, cells were first exposed to TNF␣, lactacystin, or a combination of lactacystin with TNF␣. Immunoblotting studies were conducted probing with an anti-ubiquitin antibody to determine if TNF␣ induces protein ubiquitination in MLE cells. As shown in Fig. 8A, in the presence of TNF␣, accumulation of several high molecular weight bands became discernible. Similar high molecular weight poly-ubiquitinated proteins were observed after cells were treated with lactacystin. Cells exposed to a combination of TNF␣ and the proteasome inhibitor expressed the highest levels of ubiquitinated proteins (Fig. 8A). Thus, these results indicate that TNF␣ stimulates protein ubiquitination within the alveolar epithelial cell line.
To determine if CCT is a direct substrate for ubiquitination, separate studies were conducted where unlabeled cells were lysed, and the enzyme was immunoprecipitated using the panreactive CCT antibody. The samples were then resolved by SDS-PAGE, immunoblotted using a rabbit polyclonal ubiquitin antibody, and ubiquitinated CCT detected by using the ECL detection system. By using these methods, only low levels of CCT were detected under control conditions, but after stimulation with TNF␣, greater amounts of ubiquitinated CCT were present (Fig. 8C). As a control, immunoblotting of these preparations with only the anti-CCT antibody is shown revealing that TNF␣ reduced the levels of immunoreactive CCT␣ in these cells (Fig. 8B). Thus, under conditions of TNF␣ exposure, the total CCT␣ pool decreases but CCT␣ ubiquitination increases. To investigate further ubiquitination of CCT, cells were metabolically labeled with [ 35 S]methionine. The enzyme was immunoprecipitated first using a polyclonal CCT antibody followed by a second immunoprecipitation using the anti-ubiquitin antibody (Fig. 8D). Under unstimulated conditions, no products were detectable on autoradiograms prepared from dried polyacrylamide gels. However, after exposure of cells to TNF␣, CCT␣ was found to be ubiquitinated (Fig. 8D). Together, these results suggest that under steadystate conditions low levels of the enzyme are targeted for ubiquitin-dependent proteolysis. These levels are increased in the setting of TNF␣ exposure.

FIG. 4. Effect of TNF␣ on de novo synthesis and degradation of CCT␣.
A, MLE cells were cultured as described in Fig. 1 and then pulsed with [ 35 S]methionine (60 Ci/ml, in methionine-deficient medium) during the last 4 h of culture with or without the addition of TNF␣ (500 ng/ml). Cells were lysed, and CCT␣ was immunoprecipitated, and immunoprecipitates were separated by SDS-PAGE prior to autoradiography to detect radiolabeled CCT. A pan-reactive antibody raised against CCT was used for immunoprecipitation. B, turnover of CCT␣ was determined by pulsing cells with the same label (60 Ci/ml in methionine-free medium) followed by various chase times (4 -24 h) in Hite's medium containing 10 mM L-methionine plus 3 mM L-cysteine with or without the addition of TNF␣. Immunoprecipitation, SDS-PAGE, and autoradiography were then performed as above. Right, overexposure of the 24-h samples was performed to visualize bands. C, turnover of ␤-actin was also determined. D, quantitation of CCT during the pulse-chase studies in B was performed by excising and counting the 42-kDa bands. Three independent experiments were performed in studies A and B above and experiments C and D were performed twice. C, control.

DISCUSSION
Our results show for the first time that TNF␣ modulates PtdCho synthesis by inhibiting CCT activity via proteolytic degradation of the enzyme. Based on the current results, it appears that specific degradative pathways exist in cells to control expression of CCT. These pathways include a constitutively active ubiquitin-proteasome pathway that may be important in regulating enzyme turnover under steady-state conditions. This system, together with an inducible calpain pathway, likely mediates TNF␣ effects on CCT proteolysis. The results should prompt investigations into the molecular mechanisms by which CCT is selected for degradation under normal or pro-inflammatory conditions. Results from such work could lead to maneuvers directed at modulation of these degradative pathways which in turn could serve as a means to control CCT lifespan within cells.
TNF␣ decreased the activity of CCT selectively. In contrast to effects of interleukin-1␤, there were no appreciable effects of TNF␣ on other enzymatic steps within the biosynthetic pathway (44). Immunoblotting of MLE-12 cells and primary alveolar type II cells in both control and TNF␣-treated extracts with antisera specific for the CCT␣ and CCT␤ isoforms indicated that CCT␣ was the only isoform detected in these cells. Thus, from these and other recent results (2), CCT␣ appears to be the predominant isoform involved in surfactant phospholipid synthesis in mature alveolar type II cell epithelium. Further analysis confirmed that inhibition of CCT activity was due to a reduction of total cellular enzyme mass. A global decrease in CCT protein was observed in all subcellular fractions that were analyzed (Fig. 2), arguing against the likelihood that a shift in cellular CCT content from a membrane-bound form to a cytosolic form accompanied the down-regulation of activity. It is unlikely that TNF␣ altered translational efficiency because metabolic labeling studies showed no changes in newly synthesized CCT␣ but clearly showed that the cytokine accelerated enzyme turnover. The native enzyme had a half-life (t1 ⁄2 ϭ ϳ8 h) consistent with half-lives of other regulatory enzymes (45), but it was much more labile after cytokine stimulation (Fig. 4).
There have been no prior studies that have investigated which proteolytic pathways control CCT turnover. Groblewski et al. (13) showed that CCT can be degraded in response to cholecystokinin and these effects could be influenced by en- FIG. 7. CCT proteolysis by purified m-calpain. Purified CCT was incubated with various amounts of m-calpain (A and B) after which the reactions were terminated and the samples run on SDS-PAGE followed by immunoblotting for CCT content. A pan-reactive antibody raised against CCT was used for immunoblotting. A, purified CCT (30 g) was incubated with m-calpain (0 -500 g, 0.00 -0.90 units/ml) or m-calpain incubated with calpastatin (50 g, far right two lanes) prior to immunoblotting. B, either purified CCT (30 g) or MLE cell lysates (300 g) were incubated in the absence (lane 1) or presence of m-calpain (500 g, lanes 2 and 3) with calpastatin (50 g, lane 3). C, gels were loaded with purified CCT (30 g) or 500 g of m-calpain, and immunoblotting was performed using the anti-CCT antibody as described above.  4) for 24 h. The levels of ubiquitinated proteins were assayed in cell lysates using immunoblotting. B, unlabeled cells exposed to diluent or TNF␣ were lysed and immunoblotted using a rabbit anti-CCT polyclonal antibody. The far left lane shows CCT␣ standard. The levels of CCT in control and TNF␣-treated cells are shown. C, in the same preparations, cell lysates (500 g) were immunoprecipitated using a rabbit anti-CCT polyclonal antibody. The immunoprecipitates were resolved using SDS-PAGE and immunoblotted using a rabbit anti-ubiquitin polyclonal antibody. CCT was detected using an ECL detection system. D, cells were pulse-labeled with [ 35 S]methionine (60 Ci/ml in methionine-deficient medium) for 4 h followed by a 24-h chase in the presence or absence of TNF␣ (500 ng/ml). Sequential immunoprecipitation was performed on MLE cell lysates (500 g) using anti-CCT polyclonal antibody for the first immunoprecipitation followed by anti-ubiquitin antibody for the second immunoprecipitation. Immunoprecipitates were separated by SDS-PAGE, and autoradiography (using an intensifier screen) was performed on dried gels. A pan-reactive anti-CCT antibody was used for immunoblotting or immunoprecipitation in (B-D) above. zyme phosphorylation. The primary mechanisms for protein catabolism in viable cells include lysosomal proteolysis, calcium-activated neutral proteolysis (calpains), and the ATP-dependent ubiquitin-proteasome pathway. Of these, the calpain and ubiquitin-proteasome systems have been most prominently identified in mediating TNF␣ proteolysis (42,43,46). For example, TNF␣-induced IB␣ proteolysis is mediated by both the calpain and ubiquitin-proteasome pathways (42), and IB␣ degradation in response to the cytokine was also observed in our cells (data not shown). However, the kinetics of CCT␣ degradation in alveolar epithelial cells and IB␣ degradation in HepG2 cells differ significantly (42). IB␣ is rapidly degraded within a few minutes with an 80% reduction in the half-life of the protein and is effectively eliminated during the acute phase response to TNF␣. On the other hand, CCT␣, a metabolic regulatory protein with an estimated half-life of 8 h, is degraded at a slower rate, and its half-life is reduced 56% in response to the cytokine (Fig. 4D). The differences between the kinetics of CCT␣ degradation and IB␣ degradation are probably due to multiple factors. First, the various cellular responses to TNF␣ are dose-and time-dependent (47) and also cell type-dependent. For example, unlike proteolysis of IB␣, p21WAF1 proteolysis in ME-180 cells is not detectable until 4 h after TNF addition (42,48). The kinetics of calpain-mediated proteolysis also differ with respect to different protein substrates due to the fact that there are multiple active forms of -calpain within cells that have unique substrate specificities and distinct functional roles (49).
Second, the molecular context of CCT can influence the rate of proteolysis. For example, the presence of DNA decreases the Ca 2ϩ requirement for degradation of nuclear matrix proteins (50), phosphorylation of connexin-32 prevents its proteolysis (51), interaction of calmodulin with brain spectrin results in a marked acceleration of the rate of spectrin degradation by calpains (52), and binding of polyamines to spermidine/spermine N 1 -acetyltransferase prevents proteasomal degradation (53). CCT␣ resistance to proteolytic degradation correlates with binding to membrane lipid (54), suggesting the possibility that nucleoplasmic CCT␣ might be more susceptible to digestion by calpain or the proteasome complex compared with CCT␣ located at membranous sites. In addition, the rate of CCT␣ modification by ubiquitination (Fig. 8) or phosphorylation in response to TNF␣ likely plays a role in determining the kinetics of proteolysis. CCT␣ phosphorylation stabilizes the protein from degradation in response to cholecystokinin (13), suggesting by analogy that dephosphorylation could reduce its half-life in response to TNF␣.
These effects of TNF␣-inducible CCT proteolysis are also partially blocked by inhibitors of either calpain or the 20 S proteasome. Interestingly, CCT␣ degradation was nearly completely blocked by the combination of both inhibitors. Perhaps more importantly, inhibition of the ubiquitin and calpain pathways totally abrogated the TNF␣-induced decrease in DSPtd-Cho mass (Fig. 6). Lactacystin is a selective, potent, inhibitor of the proteasome without effects on either serine or cysteine proteases, whereas ALLN is less specific for cysteine proteases, such as calpain, especially at higher doses (55). Thus, we performed direct in vitro studies examining whether purified CCT was a substrate for catalytically active m-calpain, and it degraded purified CCT in a dose-dependent manner resulting in the generation of low molecular weight degradation products (Fig. 7). Our inability to detect similar proteolytic fragments in cells following TNF␣ treatment is probably due to several factors. The data indicate that CCT␣ is targeted by two proteolytic systems, as is IB␣ where fragments also were not easily observed and Western blots required long gel exposures (42).
Whereas two major fragments of about 40 and 20 kDa resulted from calpain digestion of purified CCT␣ in vitro (Fig. 7), recent studies indicate that the sizes of products of the 20 S and 26 S proteasome are only 500 Da (56). Such small products are only detected in vitro by more sensitive methods and would run near the dye front of the PAGE. Thus, the proteolytic fragments resulting from digestion by the combination of calpain and/or the proteasome are probably very small, on the order of 5-6 residues. Another factor contributing to this result is the fact that the analyses were performed at 4-to 8-h intervals following cytokine addition (Figs. 2 and 4), and it has been shown that proteasome fragments are subject to further rapid hydrolysis by endopeptidases (57), thus probably eliminating the products of calpain/proteasome degradation.
Because both ubiquitin-dependent and ubiquitin-independent proteasome pathways have been described (58), we performed additional studies to determine if CCT and ubiquitin coimmunoprecipitated. Our pulse-chase and sequential immunoprecipitation studies indicate that radiolabeled CCT, which represents a relatively small pool of total cellular enzyme, apparently is not ubiquitinated in the absence of TNF␣ (Fig.  8D). However, it is likely that some CCT is targeted for ubiquitination under steady-state conditions perhaps as the enzyme becomes misfolded or modified post-synthetically (Fig.  8C). Thus, low levels of a constituently active ubiquitin-dependent proteolytic pathway appear to regulate enzyme turnover in the native state. The activity of this pathway is stimulated after TNF␣ exposure, and results in a greater amount of CCT that becomes ubiquitinated (Fig. 8). We suspect that both the ubiquitin-proteasome and calpain systems may be relevant to CCT stability because purified CCT was a substrate for m-calpain in vitro, and these proteolytic systems localize both in the cytoplasm and nucleus (59,60).
The mechanisms by which calpain and the proteasome are activated by TNF␣ and how CCT is targeted for degradation require further study. It appears that lipid mediators implicated in TNF␣ signaling can stimulate calpain activity (61). On the other hand, proteins that are abnormally synthesized or modified post-translationally are often targeted for ubiquitination (42,60). Since TNF␣ alters oxidant tone within cells (62), it is possible that oxidation of CCT methionine residues, or other post-synthetic modifications such as serine phosphorylation, or alterations in NH 2 -terminal protein folding could destabilize the enzyme facilitating incorporation into the ubiquitin pathway.
Unlike the present results showing alterations in CCT protein stability, most prior studies have shown that enzyme activity in cells is regulated by other post-translational events involving lipid regulation or changes in the degree of enzyme phosphorylation (1). These mechanisms might occur independently or act in concert with TNF␣-induced CCT proteolysis. For example, the cytokine stimulates phospholipase A 2 and neutral sphingomyelinase activities (24,25,63,64). One potential consequence of activation of these hydrolases is the generation of bioactive lipid intermediates such as lyso-PtdCho, ceramide, or potentially sphingosine which have been linked to inhibition of either CCT activity or PtdCho synthesis (4,6,30,65). Ceramide, resulting from TNF␣ activation of sphingomyelinase, has also been shown to activate calpain (61). Furthermore, TNF␣ has been shown to stimulate mitogen-activated protein kinases, such as p42/p44 extracellularly regulated kinase and protein kinase C␣, which may modulate CCT activity by stimulating enzyme phosphorylation (9,66). Preliminary studies in our laboratory reveal that the cytokine activates p42/p44 extracellular signal-regulated kinase within minutes in MLE cells (data not shown) and in primary alveolar type II epithe-lium (64) simultaneously with an increase in CCT phosphorylation suggesting that these pathways are initial upstream events in TNF␣ signaling. Whether these changes in enzyme phosphorylation or alterations in lipid association of CCT affect its vulnerability to proteolysis in the setting of TNF␣ exposure require further investigation. Results of such work might lead to novel therapies targeted at protease inhibition in the setting of sepsis-associated lung injury.