Interleukin- 1 Rapidly Stimulates Lysophosphatidate Acyltransferase and Phosphatidate Phosphohydrolase Activities in Human Mesangial Cells*

Phosphatidic acid (PA) is a cytokine in a variety of cell types, and an intermediary in cell activation. It is produced from membrane phospholipids by either lysophosphatidate acyl-C0A:acyltransferase (lyso-PA AT) or phospholipase D. Interleukin-1 (IL-1) stimulation of human mesangial cells (HMC) induced activation of lyso-PA AT, and synthesis of new PA species with significant increase in PA mass. These PA species were enriched in long-chain unsaturated acyl side chains (ClS:l, C18:2, C20:5, and C22:6) in both the en-2 and sn-l positions, and stimulated the action of the lyso-PA AT as a positive feedback mechanism. Gas-liquid chromatography and mass spectrometry demonstrate that the acyl composition of phosphatidic acid does not resemble that of the major phospholipid fractions of this preparation and therefore is not the product of HPLC analysis was performed with a Waters p-Porasil silica column (0.45 25 of gradient of 1-9% hexane/ isopropyl ml/min. separation of DAG, PA, PE, PI, PS, PC, sphingomyelin. subfrac-tions, lipids microsomes, asolectin, egg phosphatidyl- may be blocked by preincubation with pertussis toxin, preventing activation of phosphatidate phosphohydrolase and conversion of PA to 1,2-DAG. In these circumstances, mass accumulates in phosphatidic acid, suggesting that lyso-PA AT activity is not mediated by a G-protein.

Interleukin-1 Rapidly Stimulates Lysophosphatidate Acyltransferase and Phosphatidate Phosphohydrolase Activities in Human Mesangial Cells* (Received for publication, March 12, 1991) Stuart L. BurstenSpY, Ward E. HarrisJI, Karol BomsztykS, and David Lovett** Phosphatidic acid (PA) is a cytokine in a variety of cell types, and an intermediary in cell activation. It is produced from membrane phospholipids by either lysophosphatidate acyl-C0A:acyltransferase (lyso-PA AT) or phospholipase D. Interleukin-1 (IL-1) stimulation of human mesangial cells (HMC) induced activation of lyso-PA AT, and synthesis of new PA species with significant increase in PA mass. These PA species were enriched in long-chain unsaturated acyl side chains (ClS:l, C18:2, C20:5, and C22:6) in both the en-2 and sn-l positions, and stimulated the action of the lyso-PA AT as a positive feedback mechanism. Gasliquid chromatography and mass spectrometry demonstrate that the acyl composition of phosphatidic acid does not resemble that of the major phospholipid fractions of this preparation and therefore is not the product of phospholipase D. The PA species were rapidly converted to 1,2-sn-diacylglycerols by phosphatidate phosphohydrolase, which also was activated by IL-1 via a separate mechanism involving a pertussis-sensitive G-protein. The activities of lyso-PA AT and phosphatidate phosphohydrolase were associated with plasma membrane enriched and refined microsomal fractions. IL-1 stimulation of a murine T cell (thymoma) line, EL-4, also caused stimulation of lyso-PA AT, resulting in PA formation. EL-4 mutants with defective IL-1 receptors did not demonstrate stimulation of lyso-PA AT, showing the necessity of intact IL-1 receptors for activation of this enzyme. We conclude that PA is a significant signaling intermediary for IL-1 via activation of lyso-PA AT and a G-protein, which activates phosphatidate phosphohydrolase. This system suggests a novel mechanism whereby a low intensity signal may be translated into cellular activation.
The cytokines interleukin-la and -1/3 (IL-la and IL-lp)' are central mediators of inflammation (Dinarello, 1985;Mizel, 1989). The cellular responses to IL-1 molecules are protean, and include activation of T and B lymphocytes, maturation of precursor thymocyte forms, and induction of lymphokine synthesis. IL-1 binding induces proliferation of T and B cells, fibroblasts, smooth muscle cells, and glomerular mesangial cells Mizel, 1988). In addition to glomerular mesangial cell (MC) proliferation, IL-1 induces production of prostaglandin EP and a type IV collagenase in rat and human MC (Lovett et al., ,1986(Lovett et al., ,1987. IL-1 may act in the glomerulus to amplify local tissue injury processes, such as those seen in acute immune complex-mediated glomerulonephritis (Werber et al., 1987).
We have recently suggested that PA is an important cellular activator of MC (Bursten et al., 1989) and that lipid A may derive its biological effect from its mimicry of PA structure. In this paper we show that IL-1 acts to stimulate rapid formation of PA by a pathway involving lyso-PA AT acylation of lyso-PA. IL-1 initially activates lyso-PA AT to produce transiently a subspecies of PA enriched in palmitate and C18:1, C182, (2205, C22:6 unsaturated fatty acids. The newly synthesized PA is converted within 15-30 s into 1,2-DAG by phosphatidate phospohydrolase. Lyso-PA AT activity is not regulated via a G-protein, whereas phosphatidate phosphoydrolase is dependent upon the initial activation of a pertussissensitive Gp. These pathways interact independently of phosphoinositide metabolism, which is quiescent throughout the first 5 min after IL-1 stimulation. These data indicate the presence of a unique signaling pathway activated by IL-1.

EXPERIMENTAL PROCEDURES
Materials-Proliferating human MC were maintained in RPMI 1640 containing 20% fetal bovine serum (Irvine Scientific, Irvine, CA) and supplemented with 300 pg/ml glutamine, 100 units/ml penicillin, 100 pg/ml streptomycin, 2.5 pg/ml vancomycin (Sigma), 5 pg/ml transferrin, M insulin, and 5 ng/ml selenous acid (ITS Pre-mix, Collaborative Research, Waltham, MA). All media components were screened for the presence of exogenous endotoxin using a Limulus amoebocyte lysate assay sensitive to 10-100 pg of endotoxin/ ml (E-toxate, Sigma). Human recombinant interleukin-la was provided by Dr. K. Bomsztyk (University of Washington Medical Center, Seattle, WA), as was a murine monoclonal IgG antibody ("15) directed against common sites in the T cell/fibroblast IL-1 receptors.
The anti-IL-1 receptor IgG mAb was generated and isolated as described (Bomsztyk et al., 1989). Rabbit polyclonal IgG antibody directed against human IL-1 was obtained from Endogen (Boston, MA). This antibody was prepared against purified natural human IL-1, and recognizes common epitopes on the secreted 17-kDa forms of both IL-la and IL-1p. cis-Parinaric acid (Molecular Probes, Eugene, OR) was prepared as a 1 mM stock solution in ethanol with butylated hydroxytoluene as an antioxidant. A molar extinction coefficient (E) of 78,000 M" cm" was used to establish concentration.
Stock solutions of phospholipids were prepared by dissolving the compounds above in phosphate-buffered saline without Ca2+ containing 0.5% (w/v) BSA, such that final concentrations of BSA in reaction volumes ranged from 0.002-0.04% (w/v). Lipids were dispersed in 500 nM stock solutions by active vortexing alternated with 15-s sonications at 4 "C, for a total time of sonication of 2-3 min. This resulted in suspension for all lipid solutions with BSA in the range described. All phospholipids were characterized for purity via thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) as described below. Gas-liquid chromatography (GLC) of methyl esters was used to confirm acyl chain composition. Dilinoleoyl-PC was converted to dilinoleoyl-PA with phospholipase D (cabbage) (Kates and Sastry, 1983). Reaction products were separated by normal phase HPLC, the dilinoleoyl-PA fraction isolated, and repurified. The identity of dilinoleoyl-PA was confirmed by TLC and mass spectrometry. Acyl chain characterization by GLC demonstrated that >99.5% of acyl ester side chains were linoleate.
Preparation of Human Mesangial Cells, EL-4 Murine Thymoma Cells, Crude Microsomes, and Plasma Membranes-Cultures of human mesangial cells (HMC) were prepared, cultured, and characterized as described in detail elsewhere (Bursten et al., 1988). MC were used between the 4th and 10th passages for preparation of microsomes or cell labeling experiments. Where indicated, cycling MC were treated with pertussis toxin, 0.1-100 ng/ml, for 4 h prior to harvesting and microsomal preparation. EL-4 wild-type cells and EL-4 mutants were maintained as previously described (Bomsztyk et al., 1989).
Microsomes (crude microsomal fraction containing plasma membranes) were prepared from HMC and EL-4 cells as reported (Lovett et al., 1988). Confluent human MC layers were washed in PBS (4 "C), scraped, and pelleted by centrifugation at 400 X g for 10 min. The pellets were suspended in 20 mM sodium borate, pH 10.2, 10 mM EDTA, 1 pg/ml pepstatin, and 2 mM phenylmethylsulfonyl fluoride, followed by disruption by 10 strokes (HMC) or 50 strokes (EL-4) with tight-fitting Dounce homogenizers. The broken cell suspension was centrifuged at 500 X g for 10 min and the supernatant recentrifuged at 15,200 X g for 25 min. The resultant crude microsomal preparation was washed in 20 mM HEPES, pH 7.4, followed by centrifugation immediately or stored in liquid nitrogen at a concentration of 2-6 pg proteinlpl. Microsomal protein was quantitated using the Bradford (1976) method. Plasma membrane-enriched and refined microsomal fractions were prepared by layering crude microsomal fractions on a 35% (w/w) sucrose cushion in 20 mM HEPES buffer, pH 7.4, followed by centrifugation at 75,000 X g for 30 min, to obtain a plasma membrane-enriched fraction at the interface. The plasma membrane-enriched fraction was verified in three ways: 1) a 30-fold increase in Na'/K+-ATPase activity as compared to crude microsomes, 2) transmission electron micrography after staining with 2% aqueous uranyl acetate and lead citrate demonstrated >90% open planar sheets of bilayers, (3) presence of IL-1 receptors by "'1-IL-1 photolabeling (described in detail in Lovett et al., 1988).
Whole Cell-labeling Studies-Human MC were grown to nearconfluence in 75-cm2 culture flasks in complete growth medium. The medium was removed, the cell layers washed three times in 37 "C PBS, and fresh serum-free medium containing 0.5% BSA added. To label the glycerol moiety of cellular lipids, the cultures were incubated for 4 h at 37 "C with 8 pCi/ml [3H]glycerol (diluted with unlabeled glycerol to a final specific activity of 400 mCi/mmol). The labeling medium was removed and the cell layers washed twice with 37 "C PBS. Fresh medium containing 0.5% BSA with or without lo-" M IL-la or -10 was added. Flasks were harvested at sequential times by washing twice with 4 "C PBS, followed by two washes with methanol. Precipitated cells were scraped from the dishes and the lipids extracted in 2:l ch1oroform:methanol (Folch et al., 1957). For [14C] linoleate labeling, washed cell layers were incubated for 3 h with 0.2 pCi/ml ['4C]linoleic acid in serum-free medium containing 0.5% BSA. The cell layers were washed three times, given fresh serum-free medium, exposed to lo-" M IL-la or -ID, and processed as above. In each case, control cells were maintained in serum-free medium and processed in parallel to experimental samples. Whole cells were also reacted in the presence of 1-palmitoyl lyso-PA, 1.5-500 nM (final concentration) in the presence and absence of 1 p~ oleate and linoleate. This experiment was repeated in the presence of 10"' M IL-1, and in the presence of the DAG kinase inhibitor, R50922 (100 pM). HMC incubated with R50922 alone did not differ from control HMC; R50922 was shown to be effective in DAG kinase inhibition in the BC3Hl myocyte assay.
Assay of Microsomal Acyltransferase Actiuity-HMC microsomes or EL-4 microsomes, 100-300 pg, were suspended in 500 pl reaction buffer (125 mM Tris HCl, pH 7.4, 5 mM Na2ATP, 5 mM MgCl,, and 0.1 mM CoA). Polarization of fluorescence was measured in an SLM 4800 sub-nanosecond fluorometer. After a 5-min equilibration period, 4 p M cis-PnA was added to the microsomal preparation and mixed rapidly. Where indicated, IL-1 was added (1-10 p1) to a final concentration of 10-"-10-9 M. In specified experiments, microsomes were preincubated with anti-IL-1 at a concentration of 5 pg/ml for 5 min prior to addition of IL-1, or "15 (mAb against IL-1 receptor) at 5 pg/ml for 1 h prior to addition of IL-1. Light scattering by the microsomal preparation contributed between 5 and 8% of the total signal and remained constant throughout the period of study.
HPLC Analysis of Acyl Transferase Products-Preparations of HMC microsomes containing 100-300 pg of protein were preincubated for 5 min at 37 "C in assay buffer. cis-PnA, 4 p~, was added to initiate the reaction. Experimental conditions included 1) IL-la, lo-" M; 2) GTPyS, 100 pM; 3) lyso-PA, 1.5-500 nM; 4) linoleate, 100 nM to 10 pM; 5 ) dilinoleoyl-PA, 100 nM to 4 pM; and 6) dilinoleoyl-PC, 4 pM. In specified experiments, microsomes were preincubated with "15 mAb for 1 h prior to addition of IL-1. Experiments were repeated in the presence of 100 p~ R50922. At sequential times, aliquots were removed and lipids extracted. HPLC analysis was performed with a Waters p-Porasil silica column (0.45 X 25 cm). The mobile phase consisted of a gradient of 1-9% water in hexane/ isopropyl alcohol (3:4, v/v) run at a flow rate of 1 ml/min. This was found to give adequate separation of DAG, PA, PE, PI, PS, PC, and sphingomyelin. To permit better separation of PA and PE subfractions, a different gradient was used, as follows: 2.5-5.5% water from 0 to 10 min, 5.5% water from 10-14 min, and 5.5-10% water from 14 to 18 min (carrier organic solvents were hexane/propanol 3:4, v/v). Lipids in column effluent were monitored at 206 nm; fluorescence was monitored sequentially with excitation at 325 nm and emission at >390 nm. HPLC fractions were analyzed for phosphorus content, amines, and acyl esters according to established methods (Keenan et al., 1968;Kornberg and Pricer, 1953). Extracted lipids from sheep, rat, and human kidney microsomes, asolectin, and egg phosphatidylcholine hydrolyzed with commercial phospholipases C and D were also run on HPLC and then analyzed to confirm phospholipase identity. Fast-atom bombardment mass spectrometry was used to confirm fraction identities for similar lipid species. By this method, PA1 was determined to be largely l-palmitoyl/l-myristoyl2-saturated PA, PA2 was determined to be 1-palmitoyl 2-unsaturated PA, and PA3 was found to be 1-unsaturated 2-unsaturated PA (largely 1oleoyl-2-oleoyl, 1-oleoyl-2-linoleoyl, and 1-linoleoyl-2-linoleoyl PAS) (cf. Fig. 4; Bursten and Harris, 1991).
["C]Linoleoyl-CoA (4 PM) was also used as a substrate in place of cis-PnA. Lipids were extracted, and separated on HPLC, where 0.5min fractions from HPLC separation were collected. Fractions were either counted in a Packard 460 scintillation counter or analyzed via TLC, GLC, and mass spectrometry. Experiments measuring ["C] linoleoyl-CoA uptake were also repeated using 50-100-pg HMC plasma membrane fractions.
TLC-Extracts of cellular, microsomal, or HPLC-derived phospholipids were analyzed by TLC on Whatman K-6 Silica G plates developed at 4 "C in chloroform, methanol, 0.9% NaCl, 502k2.5 v/v, visualized by brief exposure to iodine vapor, and compared with purified standards (Goppelt and Resch, 1984). This system provides adequate resolution of PC, PS, PI, and PE in one dimension. Phosphatidic acid was analyzed on Silica Gel G plates using the upper phase from a mixture of ethyl acetate:2,2,4-trimethylpentane:acetic acidwater, 95:2:10, v/v (Billah et al., 1981). Neutral lipids were separated from phospholipids by TLC on Silica Gel G plates using petroleum ether:diethyl ether:acetic acid, 50501, v/v. Quantitation of radiolabel incorporation was performed by liquid scintillation counting of identified, scraped lipid spots. Assay of Phosphatidate Phosphohydrohe Actiuity-For direct quantitation of phosphatidate phospohydrolase activity, 300-600 pg of HMC microsomes or 50-100 pg of HMC plasma membrane were placed in acylation buffer with cis-PnA and allowed to come to equilibrium, synthesizing sn-2-labeled parinoyl-PA. These PA fractions were then isolated by HPLC as above, repurified by HPLC, dried down with argon, and dispersed as previously described. Labeling of the sn-2 position by the parinoyl group was verified by formation of methyl esters and comparison of fatty acid methyl ester concentrations by GLC (cf. below). Due to formation of PA in acyltransferase solution, no Ca2+ was present in the final solution; a small amount of Mgl+ was present. PA concentration was calculated by using phospholipid phosphorus determinations and a stock solution of parinoyl PA made at a concentration of 500 nM. 2-sn-Parinoyl-PA was then added to HMC microsomes or plasma membranes in constant amount (5-100 nM, final concentration) in the presence and absence of to 10"' M IL-1, and 100 pM GTPrS. The conversion rate to 1,2-sn-diacylglycerol was calculated by using the extinction coefficient of cis-parinaric acid. These methods of determining 1,2sn-DAG formation were then compared to rates determined in conventional fashion using [2-sn-"C]oleoyl PA (Jamal et al., 1991;Cascales et al., 1984), and were found to correspond closely in measuring rates of phosphatidate phospohydrolase activity (also cf. Bursten and Harris, 1991).
Methyl Esterification of Lipids and GLC-The acyl content of free fatty acid fractions, neutral lipids, or phospholipids was determined by GLC after esterification with BFa/methanol (Johnson et al., 1990). GLC was performed on a Hewlett-Packard model 5790 A GLC using a 6-ft X %-inch column packed with GP 3%, SP-23 1/2% on 100/120 Chromasorb WAW using nitrogen as a carrier gas. The oven temperature was programmed for initial 2 min at 190 "C, followed by a gradient to 220 "C at the rate of 2 "C/min.
Fast-Atom Bombardment Mass Spectrometry (FABIMS) of HPLC Isolated Fractions-FABIMS spectra were acquired using a VG 70 SEQ tandem hybrid instrument of EBqQ geometry (VG Analytical, Altrincham, U.K.). The instrument was equipped with a standard unheated VG FAB ion source and a standard saddle-field gun (Ion Tech Ltd., Middlesex, U.K.) producing a beam of xenon atoms at 8 keV and 1 mA. The mass spectrometer was adjusted to a resolving power of 1000 and spectra were obtained at 8 kV and at a scan speed of 10 s/decade. In this study, all samples were applied to the FAB target as solutions of known concentrations. 2-Hydroxyethyl disulfide was used as matrix in the positive ion FAB/MS and triethanolamine was used as a matrix in the negative FAB/MS. Statistics-Representative experiments performed independently two to three times are given for the whole cell labeling studies. In each case, the phospholipid analyses were performed in groups of two to four, and the data are presented as the means * S.E. For analysis of lipid samples at AZffinm, repetition of experiments two to six times showed that control AZffinm for a given lipid fraction varied by no more than 1-2% of total unsaturated acyl mass. Other data were analyzed by analysis of variance and multiple comparison techniques, with p values of <0.05 considered significant (Tukey, 1949;Dunnett, 1964). For polarization of fluorescence studies, samples from 5-10 individual batches of HMC were run three to five times each in the acyltransferase assay system and the data are given as the means of these determinations (S.E. <lo% in all cases). Linear regression analyses of polarization of fluorescence kinetics, statistical analyses including chi-square test, and curve fitting of data were performed with STATview I1 (Abacus Concepts, Berkeley, CA).

RESULTS AND DISCUSSION
Evidence for Rapid IL-1-stimulated PA Formation from 1-Acylglycerol Phosphate Pools-Human MC in culture were labeled with [3H]glycerol to study interconversion between lipid species in vitro. Lipid species were separated by HPLC, and quantitated using UV absorption at 206 nm, a wavelength measuring unsaturated acyl chains. By comparing the distribution of radioactivity with the distribution of UV-absorbing phospholipid mass, it was possible to follow alterations in lipid species mass by [3H]glycerol content and also detect changes in relative mass of unsaturated acyl side chains due to lysophospholipid acylation or lipase activity. Data shown were obtained in the absence of NaF, and are identical to those obtained in the presence of 25 mM NaF', used to inhibit phospholipase C activity. PA and PE were singled out as the phospholipid fractions changing during the first 5 min after IL-1 stimulation.
Change thereafter, while a decrease in PA was observed. Increases in relative PA mass in IL-1-stimulated HMC observed within 5 s to 1 min ( Fig. 1B) therefore appeared to be correlated with incorporation of unsaturated acyl chains into PA rather than shift of phospholipid mass. Subsequent increases in PA mass (2 min to 4 min) reflect parallel changes in the glycerol backbone and acyl chains (Fig. 1) and are the inverse of PE changes. Whole HMC were then studied for changes in absolute phospholipid species mass during the first min of IL-1 stimulation (Table I). During this time, corresponding to the period when unsaturated acyl absorption in PA was increasing Whole HMC, grown as described, were placed in rest medium for 4 h, followed by addition of either rest medium as control or IL-1, lo-" M, at time 0. The reaction was stopped at the indicated time by pouring off the medium and adding 4 ml of ice-cold methanol, followed by lipid extraction in chloroform:methanol, 2:l. HMC lipids were separated by HPLC, and fractions collected using a fraction collector. Masses were then measured as phospholipid phosphorus (Harris and Stahl, 1983  Confluent HMC layers were prelabeled with [3H]glycerol in serumfree media (see "Experimental Procedures"). Following removal of the labeling media, the washed cell layers were incubated for 0-5 min with serum-free media containing 10"' M IL-la. Lipids from unstimdated cultures were analyzed in parallel to stimulated cultures. At the indicated times, cells were washed with 4 ml of 4 "C methanol, then extracted into 2:l chlorofomxmethanol and lipids analyzed via HPLC. Lipid species were detected and relative mass determined by UV absorption spectroscopy at 206 nm (Amax for C16-C22 unsaturated acyl side chains). Fractions of eluent were collected, and analyzed by @-scintillation counting. ( Fig. lB), there was a parallel 3-4-fold increase in PA mass in stimulated cells compared to control cells ( p < 0.01 by ANOVA). The mass in other phospholipid species, including PE, did not change during this time. We hypothesized from these data that IL-1 stimulated immediate increases in PA mass in HMC (5-60 s), resulting from rapid incorporation of unsaturated acyl groups into both preexisting stable pools of 1-acylglycerol phosphate (lysophosphatidic acid), and pools formed from saturated PA. These latter pools did not appear to originate from other phospholipid species, as there was no increase in glycerol incorporation in PA, but rather from possible phospholipase Az action on saturated PA species (Lands cycle).
[3H]Glycerol levels did not decrease in other phospholipid species during the first min after IL-1 addition (cf. Fig. lA and legend) in either the presence or absence of 25 mM NaF, which suggests that phospholipase C does not play a role in these lipid changes. The stability in phospholipid glycerol pools and phospholipid mass over 60 s in PC, PE, PS, and PI also makes it unlikely that the increase in PA results from the activity of phospholipase D. The PA events in the first min contrast with the PAand PE-related events after 2 min of IL-1 stimulation, in which glycerol decreases in PE and reciprocally increases in PA. The PA content of HMC membranes could be increased by the action of the enzyme DG kinase, which phosphorylates DAG to PA, and is activated in stimulation of other cell systems (Soeling et al., 1989). Since the PA acyl content and mass increase observed do not parallel an increase in PA glycerol content, it would appear that this pathway of PA synthesis is not significant within this time frame. To confirm that PA did not result from DAG kinase activity, HMC were stimulated with IL-1 in the presence of the DAG kinase inhibitor R50922. In these circumstances, a 4-fold increase in PA mass and a parallel increase in unsaturated acyl content of PA was observed ( p < 0.01 by ANOVA; data not shown in figure), eliminating the possibility that PA resulted from DAG kinase activity. Lyso-PA:Acyl-CoA Acyltransferase Activity-The constant amount of [3H]glycerol in PA for 60 s after IL-1 addition, while an increase in unsaturated acyl-chain incorporation was observed, suggested that an acyltransferase activity was re- linoleic acid into PA in HMC. Confluent HMC layers were labeled with [3H]linoleic acid (see "Experimental Procedures"). Following removal of the labeling media, the washed cell layers were incubated for 0-5 min with serum-free medium with or without 10"' M IL-1. At the indicated times, washed cell layers were fixed and scraped into 4 ml of methanol and the lipids extracted with 2:l chloroform:methanol. The lipids were separated by HPLC, monitoring effluent by UV absorption spectroscopy and @-scintillation counting.
During the 5-min time period analyzed, [3H]linoleate in the following phospholipid fractions did not change after stimulation with IL-1: PC (92,500 k 9,600 dpm/100 pg protein), PS (1145 k 150 dpm/100 pg protein), and PI (700 k 125 dpm/100 pg protein). During the first 2 min, the radioactivity in PE did not vary significantly (33,000 k 1,400 dpm/100 pg protein), but began to decrease over the next 3 min (to 16-17,000 dpm/100 pg protein). Free fatty acid fractions (FFA) were labeled to 29,000 k 1,075 dpm/100 pg protein at zero time, but decreased to ~16,350 within 2 min after IL-1 stimulation. Radioactivity in PA of control cells (O), PA in cells stimulated with IL-1 (a).
Panel B, uptake of ["C]linoleoyl-CoA into HMC microsomes used as a lyso-PA acyltransferase assay. ["C]Linoleoyl CoA (4 p M ) and 1palmitoyl-lyso-PA (50 nM) were added to 100-300 pg HMC microsomes in the presence of absence of 10"' M IL-1. At indicated times, ch1oroform:methanol (2:l) was added to stop the acylation reaction, the lipids extracted, separated by HPLC, and the radioactivity in individual fractions determined. >85% of radioactivity was recovered within the PA fractions at all time points examined for controls. The remaining radioactivity was either in the solvent front (10-12%) or PE (3-5%). In HMC microsomes treated with IL-1, 15-20% of radioactivity was incorporated into 1,a-sn-DAG fractions after the first 5 s. Recalculating enzyme activity as nanomoles of fatty acid incorporated/mg protein/min gave a range of 17-75 nMol/mg/min for control microsomes, and a range of 36-168 nmol/mg/min for IL-sponsible for increasing unsaturated acyl groups in PA. To examine this possibility, intact HMC were labeled with [3H] linoleate in medium without serum, washed, and placed in medium with or without lo-" M IL-la. Control cells (Fig. 2 A ) showed uptake of labeled linoleate into PA, consistent with previous findings (Yamashita et al., 1975). Stimulation of HMC with IL-1 caused an 8-fold increase in uptake of [3H] linoleate into PA in the first minute ( p < 0.05 for each time point by ANOVA), followed by a decrease in the enhanced rate to approximately unstimulated levels of incorporation after 1-2 min. During the first min of stimulation, there was no change in linoleate levels in PC, PE, PI, or PS (cf. Fig. 2 legend) which argued against the possibility that phospholipase C or D was hydrolyzing these lipids during this time. However, a decrease of 43% in [14C]linoleate content of the free fatty acid pool was detected in the first min after IL-1 stimulation (cf. Fig. 2 legend), arguing for acyltransferase activity.
HMC microsomes were used to examine the possibility that the IL-1-mediated increase in C182 incorporation into PA resulted from activation of lyso-PA AT. The kinetics of uptake of the lyso-PA AT substrate, ['4C]linoleoyl CoA, into PA were measured, using crude HMC microsomes as a source of this enzyme. Fig. 2B shows that control microsomes demonstrated trans-acylation of ['4C]C182 CoA consistent with unstimulated specific activity described previously (24-46 nmol fatty acid/mg protein/min) (Soeling et al., 1989). Stimulation of HMC microsomes with IL-1 resulted in enhanced uptake of labeled linoleoyl-CoA into PA. Analysis of enzyme activity (percent increase over control) demonstrates that after IL-1 stimulation, there was an increase in acylation at 5 s comparing stimulated to control (132%; p 0.05 by ANOVA) consistent with the observed IL-1 effect in whole HMC (cf. Fig. 2 A ) . This was followed by a sustained increase in enzyme activity between 30 and 60 s ( p < 0.01 for each point).
A separate assessment of IL-1-modulated lyso-PA AT activity was obtained by examining rates of PA acylation with cis-parinaric acid (9,11,13,15-all-cis-octadecaenoic acid), a naturally occurring fluorescent congener of linoleic acid (Sklar et al., 1977). Covalent uptake of cis-PnA into the membrane (i.e. lysophospholipid acylation) may be followed by monitoring the rate of change of fluorescence polarization (Harris and Stahl, 1983). Incorporation of cis-PnA into lysophospholipids is shown in Fig. 2C; cis-PnA incorporation was most rapid during the first minute, followed by establishment of equilibrium rate. Addition of lo-" M IL-la or -1p with cis-PnA resulted in enhanced uptake during the first minute compared to uptake in unstimulated microsomes. Regression analysis of the initial kinetic curve revealed an increase of rate induced by IL-1 of 60-70% (cf . Table 11). In the times acyltransferase activity (Soeling et al., 1989). Radioactivity of PA 1-stimulated microsomes. This is comparable to measured lyso-PA  in human MC microsomes The relative activity of the acyl-CoA:acyltransferase in human mesangial cell microsomes (100-300 pg) under the indicated conditions with and without interleukin-la (lo-" M) is presented. Enzyme activity was measured by the change in polarization of fluorescence of cis-PnA as this fatty acid was acylated to endogenous lysophospholipids in HMC microsomes (see "Experimental Procedures"). The additions were made to microsomes suspended in acylation buffer except as follows: for the analysis of effect of pertussis toxin, intact HMC were incubated with pertussis toxin (0.1-100 ng/ml) for 4 h, followed by washing in cold PBS X 4 and isolation of the crude microsomal fraction as described under "Experimental Procedures." A control curve for initial kinetics of cis-PnA uptake (0-60 s) was established using linear regression analysis (n = 3 experiments). The control curve was assigned the value of unity, and other curves were compared to this value.
(preincubated) Significant change, p < 0.05, comparing conditions to the initial control curve, using the chi-squared test.
Significant change, p < 0.05, comparing conditions to the initial curve generated by control HMC microsomes in the presence of IL-1. IL-1 significantly enhances acyltransferase activity. At concentrations below those known to damage biological membranes (>1 p~) 1-palmitoyl-lyso-PA had a stimulating effect on lyso-PA AT, while 1-palmitoyl-lyso-PC had no equivalent effect. 1,2-sn-Dilinoleoyl-PA stimulated enzyme activity, and was synergistic with IL-1, whereas 1,2-sn-dilinoleoyl-PC had no effect. NaF and GTPyS had small stimulating effects on enzyme activity. Pretreatment with pertussis toxin did not reduce baseline acyltransferase activity, nor reduce the stimulatory effect of IL-1. studied, no incorporation of cis-PnA into phospholipids of the microsomal membrane occurred without AT cofactors ATP, CoA, and M P . Sonication of microsomes for 30 min also resulted in loss of cis-PnA incorporation (data not shown). This was evidence that a specific enzyme was responsible for incorporation of cis-PnA into HMC microsomal PA. Table I1 presents other properties of lyso-PA AT activity in HMC. Linear regression analysis was used to compare initial rates of cis-PnA incorporation into control microsomes uersus incorporation following additions to the reaction media. Exogenous increase in the second substrate of the lyso-P A AT-mediated acyltransferase reaction, l-palmitoyl-lyso-PA, caused an augmentation of cis-PnA incorporation, and also augments IL-1 stimulation of this reaction. Addition of 1-palmitoyl-lyso-PC, which contains a different phospholipid head group, had no stimulatory action on this enzyme. Addition of the end product congener, 1,2-sn-dilinoleoyl-PA, resulted in strong augmentation of lyso-PA AT activity. This appears to be specific to PA, as addition of dilinoleoyl-PC did not change basal or stimulated kinetics. These data suggested that initial accumulation of PA further stimulates lyso-PA AT in a positive feedback manner. This is in agreement with earlier reports of lysophospholipid acyltransferase activity including oleoyl CoA:lysolectithin acyltransferase, oleoyl CoA:lyso-PE acyltransferase, and linoleoyl-CoA:lyso-PA acyltransferase (rat MC) (Ferber and Resch, 1977;Szamel and Resch, 1981;Bursten and Harris, 1991). This provides an explanation for delay in fully enhanced uptake observed in Fig. 2B (ie. accumulation of PA is required before full stimulation is observed Szamel and Resch, 1981).
Calcium salts inhibit the activity of lysophospholipid AT (Lands and Hart, 1965). 5 mM CaCL in the acylation solution results in significant diminution in initial rate of incorporation and IL-1-stimulated uptake. 5 mM NaF, which stimulates G-proteins in the presence of aluminum (Birnbaumer et al., 1987), and GTP+, a nonhydrolyzable activator of G-proteins, caused a small enhancement in cis-PnA uptake, but did not duplicate the action of IL-1. Preincubation of HMC with pertussis toxin prior to preparation of microsomes had no effect on lyso-PA AT activity.
The specificity of uptake of unsaturated acyl groups into PA was confirmed by chemical analysis of labeled phospholipid species. Separation of lipid species by HPLC after labeling with ['4C]linoleoyl-CoA demonstrated that basal uptake of label into HMC microsomes was directed into PA fractions (Fig. 3A), and that IL-1 stimulation of microsomes resulted in enhancement of PA fraction unsaturated mass and radioactive labeling (Fig. 3B). There was no labeling of or change in unsaturated mass in other phospholipid species, consistent with Table I. This specificity of uptake of unsaturated fatty acids into PA was confirmed by fluorescent labeling of microsomal PA fractions by C18:4 cis-PnA (Fig. 3C), and significant enhancement of PA unsaturated mass and fluorescence labeling after IL-la stimulation (Fig. 3 0 ) . We also observed that, with basal (unstimulated) uptake of linoleoyl-CoA or cis-PnA, there was no labeling of DAG fractions. However, following stimulation with IL-1, a significant percentage of label was found in DAG fractions (cf. Fig. 3, B and D). This suggested that an IL-1-stimulated DAG fraction found at 5-60 s originated from PA, but could not completely exclude activation of monoacylglycerol AT.
Gas-Liquid Chromatography and Mass Spectrometry of PA Species Produced by Lyso-PA AT-Experiments were performed to determine the acyl chain composition of the PA first synthesized by lyso-PA AT after IL-1 stimulation and compare it to other phospholipids (Table 111). HMC whole cells were stimulated with IL-1, followed by extraction of lipids and separation by HPLC. Lipid fractions were then divided into two aliquots. The first aliquot was treated with BF3-methanol to hydrolyze and methylate acyl side chains for analysis via GLC; the other aliquot was analyzed by fast atom bombardment mass spectrometry. Results in Table TI1 from GLC support the hypothesis that increases in PA mass result from acylation of lyso-PA. The composition of PA found in control HMC is heavily saturated (15% myristate, 33% palmitate, 20% stearate) and contains little to no linoleate (C182). After 15 s of stimulation by IL-1, the resulting PA species is significantly enriched in unsaturated side chains with an increase in oleate and significant increases in linoleate, arachidonate, docosapentaenoate, and docosahexaenoate (22:5, 22:6). There is a decrease in unsaturation in the PE species after IL-1 stimulation, suggesting that PE + PA tram-acylation may occur, but this cannot solely account for the amount of mass change in PA fractions (cf. Table I), nor does it account for the decrease in unsaturated free fatty acids ( Fig. 2A legend). There is no change in acyl distribution in PC or PS after stimulation with IL-1, arguing that deacylation of the sn-2 position of these phospholipids cannot account for observed changes in PA fractions. After IL-1 stimulation, the free fatty acid pool shows an increase in saturated acyl content

FIG. 3. ['4C]Linoleoyl CoA and cis-parinaric acid incorporation
into PA. HPLC analysis of lipids from HMC microsomes incubated with 4 p~ ["C]linoleoyl CoA or cis-parinaric acid in the presence or absence of 10"O M ILl a is shown. 50 nM 1-oleoyl-lyso-PA and 50 nM 1-palmitoyl-lyso-PA were present. The microsomal phospholipids were extracted and separated by HPLC as detailed under "Experimental Procedures." The upper tracing represents relative phospholipid mass as measured at 206 nm; the lower tracing represents either @-scintillation counts due to incorporation of ["C]linoleoyl-CoA (panelsA and B ) or fluorescence due to incorporation of cis-PnA (panels C and D). Labeled PA fractions represent HPLC separation of 1-palmitoyl 2-unsaturated PAS from 1-oleoyl-2-oleoyl and 1-oleoyl-2-linoleoyl PAS. Panel A, HPLC profile of lipids from control HMC microsomes incubated for 5 s at 37 "C; panel E , HPLC profile of HMC microsomal lipids after treatment with 10"' M IL-1 for 5 s at 37 "C; panel C, HPLC profile of lipids from control HMC microsomes incubated for 5 s, +cis-PnA; panel D, HPLC profile of microsomal lipids after treatment with IL-1, 10"O M, 5 s, +cis-PnA.  (myristate increased from 1.4 to 8%; palmitate increased from 12 to 18.4%; and stearate increased from 15 to 24.6%). This is further evidence that one source of 1-acylglycerol phosphate (lyso-PA) is the Lands cycle. It was assumed that saturated acyl chains were found in the C-1 position and unsaturated acyl chains in the C-2 position. From these GLC data, most probable configurations were predicted and matched with mass spectrometric data (see Fig. 4 legend for identification of individual PA species) Control PA, found in amounts consistent with Table I, is highly saturated as predicted from Table I11 (Fig. 4A). This PA contains large amounts of myristate, palmitate, and stearate, as well as 1-alkyl 2-acyl PA species. The PA first synthesized by lyso-PA AT, in contrast, was greatly enriched in oleate, linoleate, arachidonate, docosapentaenoate, and docosahexaenoate as determined by GLC determination (Fig.   4B). m/z ratios from Fig. 4B show that the fraction contains large concentrations of 1-palmitoyl %unsaturated and 1oleoyl 2-unsaturated PAS. This PA species was found to be enriched in palmitate relative to the composition of PE, PI, PS, and PC (21% compared with 3-12% in other fractions). In addition, this PA was found to have less stearate compared with other phospholipids (17% compared with 24-34% in other fractions). Mass spectrometry of other phospholipid fractions (m/z spectra not shown) demonstrates that these species conform with predictions from acyl chain composition (Table 111). Predominant PE species include l-palmitoyl-2-oleoyl (m/z 718, 719), 1-stearoyl-2-oleoyl (m/z 744, 745), and 1-stearoyl-2-arachidonoyl ( n / z 767-769) PES. Predominant PC species include 1-palmitoyl-2-oleoyl (m/z 761) and 1palmitoyl-2-stearoyl (m/z 763) PC. The predominant PS species include 1-stearoyl-2-oleoyl-PS (m/z 901, 902) and 1-24:0 2-stearoyl-PS (m/z 986-988). The only PI species detected during the first min after IL-1 stimulation is 1-stearoyl 2arachidonoyl inositol 1,4,5-diphosphate (m/z 1092, with strong m/z 465-471 inositol 1,4,5-trisphosphate spectrum components). There is no change in m/z determination of PC, PE, PS, or PI species after stimulation of HMC with IL-1 over 1 min. The GLC data and m/z configurations supported our hypothesis that the PA generated by lyso-PA AT would be rich in 18:1, 182, 205, and 22:6 unsaturated acyl side chains; the lack of similarity in acyl composition to other phospholipids supported our contention that this PA was not derived from other phospholipids by action of a phospholipase D.
IL-1 and Lyso-PA Stimulation of Lyso-PA A T and Formation of DAG-To examine the specificity of IL-1 stimulation of lyso-PA AT, HMC microsomes were equilibrated with cis-PnA and required cofactors, followed by addition of IL-la (Fig. 5A). The addition of IL-la to microsomes equilibrated with cis-PnA resulted in enhanced cis-PnA incorporation and establishment of a new reaction rate. The rapidity of the reaction and the similarity in change of kinetics with different times of IL-1 addition suggested that IL-1 activates already Acyl side chain composition of HMC phospholipid fractions f interleukin-1 Whole resting HMC were reacted in the presence and absence of IL-1, lo-" M, for 15 s as previously described, and the reaction was terminated with 4 ml of ice-cold methanol. HMC were scraped into 2:1 chloroform:methanol, lipids extracted, and individual lipid fractions isolated using HPLC (see "Experimental Procedures"). Lipid fractions were then split into two aliquots: one aliquot was used for mass spectrometry for fraction identity, and the other was reacted with BF3/methanol to form methyl esters of acyl side chains. The fatty acid methyl esters were then identified and quantitated against standards using gas-liquid chromatography. The results above are representative for significant HMC phospholipid fractions from three to five separate experiments. extant lyso-PA AT. The specificity of IL-1 stimulation of the acylation reaction was shown by addition of excess a-IL-la antibody prior to IL-la. In the presence of a-IL-1 antibody, IL-1-stimulated uptake of cis-PnA into HMC microsomes was abolished, and original reaction rate maintained (Fig. 5A).
The dose response of cis-PnA incorporation into lyso-PA after IL-1 stimulation is shown in Fig. 5B. Addition of 10-fold increments of IL-1 resulted in continued enhancement of cis-PnA incorporation to 5 X 10"' M IL-1, the maximum concentration used. The enhancement of acyltransferase activity by the lyso-P A AT substrate, 1-palmitoyl-lyso-PA (Table 11), also caused increased amounts of unsaturated PA to be produced (Fig.  5 C ) . In further experiments, we monitored formation of 1,2-DAG, as addition of lyso-PA to cells may stimulate G-proteins (VanCorven et al., 1989) and synthesis of DAG. Lyso-PA from 1.5 to 500 nM was added to whole HMC and caused no cell disruption that could be detected using trypan blue exclusion or increases in 51Cr leakage (Bursten and Harris, 1991). Representative results were taken from HMC incubated with 50 nM 1-palmitoyl-lyso-PA. Incubation of control HMC at 37 "C for 5 and 15 s (Fig. 5C) did not change the proportion of unsaturation in PA, or the level of DAG unsaturation. Addition of lyso-PA alone resulted in a 3-6-fold increase of unsaturation in both PA and DAG, which occurred between 30 and 60 s ( p < 0.01). Within 5 s after addition of lyso-PA and IL-1 together, 10-20-fold stimulation of PA and DAG unsaturation was observed (Fig. 5 C ) ( p < 0.001).
These data suggested that both lyso-PA and IL-1 might stimulate initial formation of DAG and reinforced our hypothesis that PA was the precursor of DAG. Studies with lyso-PA in HMC microsomes (Fig. 5 0 ) also supported this hypothesis. Lyso-PA alone stimulated the increase of unsat- urated acyl mass by a factor of 10 in both PA and DAG within 30 s (p < 0.01). Lyso-PA added with IL-1 to HMC microsomes caused 3-fold enhancement in both PA and DAG at 15 s (p < 0.01) but was associated with a fall in PA acyl unsaturation and a marked %fold increase in DAG unsaturation at 30 s (p < 0.01). During this time period, there was no change in masses of other lipid fractions (Table I). Lyso-PA and IL-1 appeared to stimulate formation of DAG from PA synergistically, as would be expected if they are operating to accelerate different but complementary aspects of DAG formation.
IL-1 Stimulation of Plasma Membrane Lyso-PA A T and Phosphatidate Phosphohydrolase Via a G-protein-Phosphatidate phospholydrolase is the enzyme which hydrolyzes PA to DAG. Lyso-PA and IL-1 might operate synergistically on a Gp which regulates this enzyme. Phosphatidate phosphohydrolase is tightly regulated in intact cell systems, but may be relatively unregulated in microsomes (Cascales et al., 1988;Bursten and Harris, 1991). This difference in regulation between whole cells and microsomes could account for the constant level of PA during DAG synthesis in whole HMC (Fig. 5C) and the large decrease in PA coupled with the large increase in DAG that was seen in HMC microsomes (Fig.  50). To dissect the possible effects of IL-1 and lyso-PA on a Gp-mediating phosphatidate phosphohydrolase, whole HMC Whole HMC were preincubated with pertussis toxin (0.1-100 ng/ml) for 4 h, followed by isolation of microsomal fractions (see "Experimental Procedures"). HMC microsomes were incubated in the presence and absence of 5 pg/mlM-15 for 1 h prior to reaction in acylation buffer in the presence or absence of 10"' M IL-1 for 5 and 15 s. The reaction was stopped with 2:l chloroform:methanol, and lipids were extracted and separated by HPLC, followed by determination A2% nm of 1,2-sn-DAG and PA fractions. Results are shown for HMC micro- were preincubated for 4 h with pertussis toxin (0.1-100 ng/ ml). Microsomes were prepared from these cells and assayed. To examine the relation of IL-1 effect on lyso-PA AT and phosphatidate phosphohydrolase to IL-1-[IL-1-receptor] binding, aliquots of microsomes were preincubated with a mAb ("15) directed against fibroblast/T cell type IL-1 receptors.

Lyso-PA Acyltransferase
After 5 and 15 s, at 37 "C, control microsomes showed an increase in PA but no increase in 1,2-DAG (Fig. 6). Preincubation of microsomes with "15 mAb had no detectable agonistic effect on either PA or DAG concentrations. Stimulation of microsomes with IL-1 resulted in 4-fold increase in PA unsaturation at 5 s and a &fold increase after 15 s of stimulation compared with controls ( p < 0.01 by ANOVA).
After stimulation of microsomes with IL-1 for 5 s, DAG demonstrated a 15-fold increase in unsaturation, which continued for 15 s ( p < 0.001 by ANOVA). Preincubation of microsomes with "15 mAb abolished the increase in PA and DAG induced by stimulation with IL-1.
Microsomes from cells incubated with pertussis toxin demonstrated the same increase in unsaturation in PA as control microsomes. In contrast, stimulation of pertussis pretreated microsomes with IL-1 resulted in enhancement of PA unsaturation at 5 s, and an even greater (50%; p < 0.05) enhancement at 15 s. However, unsaturation of acyl chains in DAG reaction was stopped with 2:l chloroform:methanol, and lipids were extracted and separated by normal phase HPLC. Absorption at 206 nm of 1,2-sn-DAG and PA fractions were determined. A2mnm of PE and PC was constant under these conditions during the times studied.

IL-1 Stimulates Lyso-PA Acyltransferase 20741
in these microsomes was not increased compared with M-15or pertussis toxin-treated microsomes at either 5 or 15 s. These data established that IL-1 occupation of the IL-1 receptor was necessary for stimulation of lyso-PA AT and phosphatidate phosphohydrolase. Pertussis toxin prevented activation of phosphatidate phosphohydrolase.
Blockade of lyso-PA AT activation through mAb occupation of the IL-1 receptor suggested that lyso-PA AT activity may be found in the plasma membrane. To examine this possibility further, HMC plasma membranes were isolated from crude microsomal fractions. Assay of lyso-PA AT activity indicates its presence in the plasma membrane (Table IV), that it is stimulated by IL-1, and this stimulation is blocked by "15 mAb. We conclude that IL-1 stimulates a plasma membrane-associated lyso-PA AT, through association with its receptor. The data in Fig. 6 also indicate that IL-1 stimulation of lyso-PA AT is not linked to a pertussis toxin-sensitive Gp. The accumulation of unsaturated mass in PA after IL-1 stimulation in pertussis toxin-treated HMC microsomes suggested that increases in DAG unsaturated mass occurring with IL-1 result from conversion of PA to DAG via phosphatidate phosphohydrolase. This latter enzyme is regulated by a pertussis-toxin sensitive Gp, and hence the blockage of phosphatidate phosphohydrolase results in PA mass accumulation. This hypothesis would also explain the stimulation of cis-PnA uptake by NaF and GTPyS (Table 11) as the result of a change in equilibrium induced by shift of mass from PA to DAG due to the G-protein-induced action of phosphatidate phosphohydrolase.
To further examine Gp activation of phosphatidate phosphohydrolase, microsomes from HMC were reexamined in the presence and absence of IL-1 and GTPyS following labeling of phosphatidic acid with cis-parinaric acid or [14C]oleate allowing estimation of phosphatidate phosphohydrolase reaction rates. Control microsomes incubated at 37 "C demonstrated formation rates of <0.1 nmol DAG/mg/min ( Table  V). In contrast, microsomes stimulated with IO-" M IL-1 showed formation rates of DAG from cis-PnA-labeled PA of 6.2 f 2.0 nmol DAG/mg/min within 15 s after stimulation. Microsomes stimulated with 100 ~L M GTPrS showed formation rates of 5.3 * 1.5 nmol DAG/mg/min within 15 s after stimulation (Table V). NaF (5 and 10 mM) also induced  V Phosphatidate phosphohydrolase activity in HMC cellular fractions HMC microsomes and plasma membrane enriched fractions were isolated as described under "Experimental Procedures." 100-300 pg of microsomes or 50-100 pg of plasma membranes were reacted with reagents as described, or following equilibration for 1 h with "15 mAb, in the presence of 4 p~ cis-PnA-labeled PA, or ['4C]oleoyl-PA. The reaction was quenched by addition of 2 ml of ice-cold methanol, and lipids were then extracted in 2:l ch1oroform:methanol and separated by HPLC. Formation of DAG was assayed using fluorescence intensity of DAG or scintillation counting. 9.6 f 2.6' 6.1 f 1.8' <0.lC Activity is expressed as nmoles of DAG/mg/min f S.E.
Significantly decreased phosphatidate phosphohydrolase activity, p < 0.01, compared to IL-1-stimulated microsomes or plasma membranes. moderate increases in phosphatidate phosphohydrolase activity compared to control (3.1 and 2.4 nmol DAG/mg/min), but did not increase rates to the same extent as IL-1 or GTP+. These data are consistent with an inhibitory effect of NaF on phosphatidate phosphohydrolase itself (Jamal et al., 1991) and with previous data concerning phosphatidate phosphohydrolase activity in rat MC (Bursten and Harris, 1991); they confirm significant activation of phosphatidate phosphohydrolase after IL-1 stimulation, via a Gp-related mechanism. Phosphatidate phosphohydrolase is present in eukaryotic cells in hormone-sensitive forms and may be tightly regulated by phosphorylation and/or translocation (Hosaka et al., 1975;Berglund et al., 1981;Cascales et al., 1984Cascales et al., , 1988. In addition, a specific plasma membrane-associated form of phosphatidate phosphohydrolase has recently been described which may be present in association with signaling (Jamal et al., 1991) consistent with our findings (Table V). Evidence linking IL-1 to Gp activation that may mediate enzymes other than adenyl cyclase or phospholipase A2 has been reported (O'Neill et al., 1990).

IL-1 Effect on Murine Thymoma (EL-4) Cell
Lipids-A close interaction between the IL-1 receptor and the lyso-PA acyltransferase was suggested, since the monoclonal antibody against the IL-1 receptor could prevent stimulation of this enzyme in mesangial cells. A murine T cell line, EL-4, was used to determine if this interaction was limited to specific cell types or was a general phenomenon. One minute after EL-4 cells were stimulated with lo-" M IL-1, a %fold increase in the unsaturation of acyl groups in P A was observed ( p < 0.01, ANOVA) which increased to a 4-fold enhancement 2 min poststimulation ( p < 0.01, ANOVA) (Fig. 7A). The PAunsaturated acyl content did not change in unstimulated EL-4 cells. Acyltransferase activity was measured in microsomes prepared from EL-4 cells using the change in polarization of fluorescence of cis-PNA as this fatty acid was incorporated into phospholipids (Harris and Stahl, 1983). Addition of IL-1 to EL-4 microsomes stimulated the endogenous AT activity (Fig. 7 B ) , similar to IL-1 effect of lyso-PA A T activity in IL-1 Stimulates Lyso-PA  8. Proposed mechanism of IL-1 action in human mesangial cells. IL-1, by binding to its receptor, activates lysophosphatidic acid acyltransferase, which synthesizes a phosphatidic acid fraction enriched in sn-2 unsaturated acyl chains, (218, C20, and C22. This PA fraction further stimulates the activity of the acyltransferase, promoting further PA synthesis. Binding of IL-1 to its receptor also stimulates a pertussis-sensitive G-protein which activates phosphatidate phosphohydrolase, resulting in hydrolysis of the newly synthesized PA into 1,2-diacylglycerols. This second activity may be blocked by preincubation with pertussis toxin, preventing activation of phosphatidate phosphohydrolase and conversion of PA to 1,2-DAG. In these circumstances, mass accumulates in phosphatidic acid, suggesting that lyso-PA AT activity is not mediated by a G-protein. microsomes from mesangial cells (cf. Fig. 2C). Microsomes were prepared from an EL-4 mutant that contained nonfunctional IL-1 receptors. Addition of IL-1 to these microsomes had no effect on the AT activity measured by polarization of fluorescence (Fig. 7C). This suggested that a functional IL-1 receptor was required for lyso-PA AT stimulation. A dose response of EL-4 cells to IL-1 (Fig. 7 0 ) demonstrated that the generation of unsaturated PA occurred in the same physscribed under "Experimental Procedures." Microsomes were then incubated in acylation buffer, and change in polarization of fluorescence of cis-parinaric acid was observed in the presence and absence of lo-" M IL-1, added with cis-PnA at time 0. Results shown are representative of five to seven experiments. Panel C, EL-4 microsomes from wild-type cells responsive to IL-1, and microsomes from mutant EL-4 cells with greatly diminished response to IL-1 and recently characterized as having atypical IL-1 receptors were isolated as described under "Experimental Procedures." Microsomes were incubated in acylation buffer, and change in polarization of fluorescence of cis-parinanic acid was observed in the presence and absence of lo-" M IL-1. In the first series of experiments, IL-1 was added at time 0 with cis-PnA. IL-1 was also added (after equilibration of cis-PnA with microsomes) at t = 10 min in the next series of experiments. Mutant EL-4 microsomes were then incubated in acylation buffer, and uptake of cis-PnA observed as previously described. Following equilibration at 10 min, mutant microsomes were stimulated with IO-" M IL-1. Panel D, dose response curve of EL-4 whole cell PA unsaturated acyl group incorporation to IL-1. EL-4 whole cells in suspension were stimulated with 2 X to 5 X 10"O M IL-1 for 30 s or 1 min. Equal aliquots of cells (2 X 10') were pipetted into 2 ml of ice-cold methanol. Lipids were then extracted in 2:l chloroform:methanol, separated by HPLC, and analyzed at A206 ", , , . Results shown are representive of two to three experiments. iologic range as the response of HMC to IL-1 and was maximal at to lo-'' M.
In summary, we have demonstrated activation of two novel mechanisms produced by IL-1 stimulation of HMC (and EL-4 murine T lymphoblasts) which occurs within 5-15 s after addition of the cytokine: 1) a lyso-PA-specific acyltransferase activity that used lysophosphatidic acid as a substrate to form PA, and caused an increase in PA mass, and 2) a G-proteindependent phosphatidate phosphohydrolase activity that hydrolyzes PA to DAG (Fig. 8). The increase in PA mass observed appears to come from incorporation of stable pools of lyso-PA (1-acylglycerol phosphate), and production from the Lands cycle, i.e. phospholipase A2-mediated conversion of PA to lyso-PA. In addition, there is evidence that lyso-PA may be produced form other sources such as lyso-PE, but this remains Both activities are associated with the plasma membrane, and the presence of intact IL-1 receptors. Abolition of response with IL-1-receptor antibodies demonstrates that this is a specific IL-1 interaction with the cell.
The importance of PA as a cytokine and signaling intermediate has been described (Moolenaar et aL, 1986;Altin and Bygrave, 1987;Murayama and Ui, 1987). Other evidence implicates lyso-PA separately as a mediator of early cell signaling pathways, and as a possible activator of G-proteins. The stimulation of lyso-PA AT activity by IL-1 provides several explanations for the effects observed with lyso-PA (VanCorven et al., 1989). In our system, lyso-PA acted synergistically with IL-1 in formation of both PA and 1,2-sn-DAG. This synergism may derive both from lyso-PA as a substrate for AT, and as an activator of phosphatidate phospohydrolase via Gp. A second effect of lyso-PA on cell dynamics may be as the rate-limiting substrate for lyso-PA AT. Production of sn-2-unsaturated PA species from 1-palmitoyl and 1-unsaturated lyso-PA substrates by the acyltransferase may be sufficient as a cell activator. The importance of unsaturated acyl chains in the sn-2 position of PA in cell activation of mouse mammary epithelium has been reported (Imagawa et a/., 1989).
This study provides evidence that PA is a cellular signaling intermediate; however, it also emphasizes that there are variant pathways that may be utilized to give PA and lyso-PA their relevance to cell signaling. In addition, our data elucidate the mechanisms by which IL-1 elicits changes in cell membrane metabolism. The complexities of rapid membrane responses to cytokines are evident, and data suggest how Gprotein and non-G-protein-mediated mechanisms may interact to amplify cell responses (Fig. 8). The various interactions of both PA and lyso-PA in these mechanisms is worthy of further investigation.