Epidermal Growth Factor Stimulation of Prostaglandin Ea ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 263, No. 16, Issue of June 5, pp. 7846-7854,1988 Printed in U S. A.

Epidermal Growth Factor Stimulationof Prostaglandin Ea Biosynthesis inAmnion Cells INDUCTIONOFPROSTAGLANDINHzSYNTHASE* (Received for publication, November 3, 1987)

M. Linette CaseyS, Klaus Korte, and Paul C. MacDonald From the The Cecil H.and Ida Green Center for Reproductive BiologySciences and the Departments of Biochemistry and Obstetrics and Gynecology, The University of Texas Southwestern Medical School, Dalles, Texas75235 Amnion is believed to be a tissue of signal importance, anatomically and functionally, in the maintenance of pregnancy and during the initiation of parturition. Epidermal growth factor (EGF)-like agents cause a striking increase in the secretion of prostaglandin E, (PGE2) in human amnion cells but only if arachidonic acid is present in the culture medium. To investigate the regulation of arachidonic acid metabolism by EGF-like agentsin amnion, weused mEGF and human amnion cells in primary monolayer culture as a model system. The amountof PGE, secreted into the culture medium was quantified by radioimmunoassay and the rate of conversion of [14C]arachidonic acidto [14C]PGE2(PGH, synthase activity) in cell sonicates in vitro conditions. wasdeterminedunderoptimal Treatment of amnion cells with mEGF led to a marked increase in the rate of production of PGE,. The specific activity of PGHz synthase (uiz.the combined activities of prostaglandin endoperoxide (PGH2) synthase and PGH,-PGE isomerase) was increased by 2-5-fold in cells treated with mEGF. Treatment of amnion cells with mEGF for 4 h didnot affect the specific activities of phospholipase A2 or phosphatidylinositol-specific phospholipase C. By immunoisolationof newly synthesized, [36S]methionine-labeled PGH, synthase, we found that mEGF stimulated de nouo synthesis of the enzyme. Thus, mEGF acts in human amnion cells in primary monolayer culture to increase rate the of PGEz biosynthesis by a mechanism that involves inductionof PGH, synthase; the manifestation of EGF action on PGEzbiosynthesis is dependent on the presence of nonesterified arachidonic acid.

affecting the rate of prostaglandin biosynthesis in various tissues. Evidence now is accumulating,however, that de novo synthesis of prostaglandin (PG)’endoperoxide synthase (PGH, synthase) (EC 1.14.99.1) also is instrumental in governing the rate of prostaglandin production (1-3); and, in some tissues, thismay be the principal mechanism operative to regulate prostaglandin formation. For example, increased PGH, synthase activity leads to increased prostaglandin biosynthesis in mouse osteoblastic cells treated with epidermal growth factor (EGF) (2) and in 3T3 cells treated with plateletderived growth factor (1). Human amnion cells are useful as a model system for the study of the regulation of prostaglandin biosynthesis (4) because in amnion cells (5), like amnion tissue (6), only one prostaglandin, viz. PGE,, is biosynthesized, and the PGE, formed is notmetabolized in this tissue. Previously, we found that EGF acts to stimulate PGE, production by 2-150-fold in human amnion cells maintained in primary monolayer culture; but, the effectiveness of mEGF in stimulating PGE, production was dependent on the presence of arachidonic acid in the culture medium (4). The regulation of arachidonic acid metabolism by way of phospholipase A, and phosphatidylinositol-specific phospholipase C is well-characterized in amnion tissue (8-11). The regulation of prostaglandin formation in the extraembryonic fetal membranes and uterine decidua is believed to be fundamental to the maintenance of pregnancy and important during parturition(cf. 12). In this study we evaluated the effect of mEGF on the conversion of arachidonic acid to PGE,, viz. the combined activities of PGH, synthase and PGH,-PGE isomerase (EC 5.3.99.3), inamnion cells. WefoundthatmEGFactsto stimulate PGEz production in intact amnion cells by a mechanism(s) that involves an increase in de novo synthesis of Until recently, it has been generally assumed that the rate PGH, synthase. of biosynthesis of prostaglandins of the 2-series is regulated EXPERIMENTALPROCEDURES by the availabilityof nonesterified arachidonic acid. Because intracellular arachidonic acid is esterified principally in the Materi~k-[l-’~C]Arachidonicacid (59.6 mCi/mmol), [1-14C]PGE2 sn-2 position of glycerophospholipids, alterations in the rate (58.4 mCi/mmol), L-3-phosphatidyl[2-3H]inositol(17.1 Ci/mmol), of hydrolysis of arachidonic acid from these compounds by and 1-acyl 2-[l-’4C]arachidonoy1phosphatidylethanolamine (59 mCi/ the actions of phospholipase A, and by the combined actions mmol) were purchased from Amersham Corp. Nonradiolabeled PGE, of phosphatidylinositol-specific phospholipase C, diacylglyc- was obtained from Cayman Chemical Company, Ann Arbor, MI. Nonradiolabeled arachidonicacid and glycerophospholipids were purerol lipase, and monoacylglycerol lipase areimportantin Canada. Mouse chased from Serdary Research Laboratories, London, epidermal growth factor (culture grade) was purchased from Collab* This investigation was supported, in part, by National Institutes orative Research, Inc., Lexington, MA.PGHzsynthase, purified from of Health Grant 5-P50-HD11149. The costs of publication of this ram seminal vesicles, was purchased from Oxford Biochemical Co., article were defrayed in part by the payment of page charges. This Oxford, MI. [35S]Methioninewas purchased from ICN Radiochemiarticle must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The abbreviations used are: PG, prostaglandin; EGF, epidermal $ To whom correspondence should be addressed Cecil H. and Ida sulfate; EGTA, [ethyleneGreen Center for Reproductive Biology Sciences,University of Texas growth factor; SDS, sodiumdodecyl Southwestern Medical School, 5323 Harry Hines Blvd., Dallas, TX bis(oxyethylenenitrilo)]tetraacetic acid Temed, N,N,N’,N’-tetramethylethylenediamine. 75235. ~~~

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Induction of PGHz Synthase by EGF in AmnionCells cals, Irvine, CA. Essentially fatty acid-free bovine serum albumin and cycloheximide were obtained from Sigma. [3H]Acetylsalicylic acid was synthesized by incubation of [3H] acetic anhydride (42.6 Ci/mol, Amersham Corp.) with salicylic acid (Sigma) in the presence of pyridine a t 40 "C for 2 h. The [3H] acetylsalicylic acid formed was purified byliquidliquidpartition column chromatography on celite with a solvent system of isooctane/ tert-butanol/methanol/water(25:1087, by volume). The purity of the [3H]acetylsalicylicacid was confirmed by thin-layer chromatography in a solvent system of benzene/ethanol/acetic acid (85:15:0.1, by volume). Preparation and Maintenance of Amnion Cells in Primary Monolnyer Culture and Determination of PGE, Production-Human amnion tissue was obtained aseptically from normal pregnancies at the time of elective cesarean section conducted before the onset of labor. Amnion cells were dispersed enzymatically (4), placed inculture dishes (60-mm diameter), and allowed to replicate to confluence in Ham F-l2/Dulbecco's minimal essential medium (l:l, v/v) that contained heat-inactivated fetal calf serum (lo%, v/v), penicillin (200 unitslml), streptomycin (200 pg/ml), fungizone (0.5 pglml), kanamycin (200 pglml), and gentamicin (200 pglml). Confluent cells were incubated with mEGF (or appropriate test agents). Thereafter, the culture media were collected for quantification of PGE, by specific radioimmunoassay (5), andthe cells were collected for assay of enzyme activities. Assay of PGH, Synthase Activity-The rate of conversion of arachidonic acid to PGE, was determined by use of the method described previously (6) with modifications. The assay of the conversion of arachidonic acid to PGE, was optimized with respect to substrate concentration, incubation time,and cell protein concentration. Thus, in these studies, we determined the combined activities of PGH, synthase and PGH,-PGE isomerase. Intact amnion cells in monolayer culture were incubated with mEGF, in various concentrations, for various times. The culture media were removed and aliquots were assayed for PGE, by radioimmunoassay. The cells were scraped from the dishes and sonicated in potassium phosphate (50 mM, pH 7.4) buffer that contained EDTA (2 mM). The sonicates were centrifuged a t 750 X g and 4 "C for 10 min, and the supernatantwas used as the enzyme source. Assays were conducted by incubation of aliquots of the cell preparation with [1-"Clarachidonic acid (10 p ~ ) L-trypto, phan (4.2 mM), reduced glutathione (5.1 mM), and hematin (1.75 p M ) a t 37 'C for 10 min in 1ml (total volume). Reactions were terminated by the addition of acetic acid, nonradiolabeled PGE, (15 pg) was added, and PGE, was extracted into ethyl acetate. The solvent was removed by evaporation under nitrogenand radiolabeled arachidonic acid was separated from radiolabeled PGE, by silicic acid column chromatography as described (6). PGE, was purified further by thinlayer chromatography (131, and radioactivity was quantified by liquid scintillation spectrometry. To account for losses during the extraction and purification procedure, ["CIPGE, was used as a recovery marker. On average the recovery ranged from 70-80%. All experimental values were corrected for incorporation of radioactivity into PGE, in incubations conducted in the absence of cell protein ( 4 % of total radioactivity) or with preparations of cell sonicates that previously were heated a t 100 "C for 10 min ( 4 % of total radioactivity). Assays were conducted in triplicate orquadruplicate. Protein was quantified by the method of Lowry et al. (14) with bovine serum albumin as the standard. Statistical analyses were conducted by use of the Student's t test. Assays of Phospholipase Actiuities-Phospholipase A, activity was assayed by use of a method described previously (8). Confluent amnion cells were scraped from the culture dishes and sonicated in a solution of sucrose (0.25 M) and dithiothreitol (1 mM). The sonicates were centrifuged at 750 X g for 10 min at 2 "C; the supernatant fraction was used as the enzyme source. Incubation mixtures consisted of Tris-HC1 (77 mM, p H 8.5), CaCl, (5 mM), EGTA (1 mM), 1-oleoyl 2-[l-'4C]arachidonoy1 sn-glycero-3-phosphoethanolamine (80 pM, 59 mCi/mmol) as substrate, and aliquots of the cell preparation (0.1-0.2 mg ofprotein). The substrate was prepared by sonication in a solution of bovine serum albumin (fatty acid-free; 1%,w/v). The reaction mixtures were incubated a t 37 "C for 30 min and were terminated by the addition of acetic acid. Lipids were extracted by use of the method of Folch et al. (15) and arachidonic acid was separated from radiolabeled glycerophospholipids and lysoglycerophospholipids by thin-layer chromatography as described (16). Radioactivity that comigrated with authentic arachidonic acid was quantified by liquid scintillation spectrometry. The specific activity of phosphatidylinositol-specific phospholipase

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C was determined by use of the method described previously (10). The assay conditions were optimized for amnion cells in primary monolayer culture with respect to linearity with time and concentration of cytosolic protein and saturation of the enzyme with phosphatidylinositol. Confluent amnioncells were scraped from the dishes in a solution of sucrose (0.32 M) and were sonicated. The sonicates were centrifuged successively at 750 X g, 10,000 X g, and 105,000 X g;the 105,000 x g supernatant (cytosolic) fraction was used as the enzyme source. Reaction mixtures consisted of Tris-HC1 (20 mM, pH 7.0) buffer, CaC1, (4 mM), P-mercaptoethanol (4 mM), phosphatidyl [23H]inositol (2 mM,0.04 pCi/mol), and an aliquot of the cytosolic fraction (60-100 pg of protein) in a total volume of 0.25 ml. Incubations were conducted at 37 'C for 30 min, the reaction was terminated by the addition of chloroform/methanol (L2, v/v), and the glycerophospholipids were extracted by the method of Bligh and Dyer (17). Thereafter, [3H]inositolin the aqueous phase was quantified by liquid scintillation spectrometry. Radwlubeling and Analysis of Glycerophospholipids of Amnion Cells-Amnion cells in monolayer culture (confluent cells in 60-mm dishes) were preincubated for 24 h with [I4C]arachidonicacid (2.3 p ~1.7 , X 10'dpm) in serum-free culture medium that contained fatty acid-free bovine serum albumin (2.5 mg/ml). At t = 0, the culture medium wasremoved and replaced with serum-free medium that contained fatty acid-free bovine serum albumin (2.5 mg/ml) with or without mEGF (15 ng/ml). At 4 h the mediumwas collected for analysis of radiolabeled arachidonic acid and PGE,; the cells were collected for determination of [14C]arachidonicacid in glycerophospholipids. Lipids were extracted by the method of Bligh and Dyer (17), and glycerophospholipidswere separated by thin-layer chromatography as described (16). Arachidonic acid and PGE, were extracted from the culture medium (after acidification to pH 4.0) with ethyl acetate and separated by thin-layer chromatography as described (13). The glycerophospholipid, PGE,, or arachidonic acid that comigrated with authentic standards was eluted from the silica gel, and radioactivity was quantified by liquid scintillation spectrometry. In another study, glycerophospholipids of amnion cells were radiolabeled with [14C]arachidonicacid in the same manner. After preincubation for 24 h, theculture medium that contained ["Clarachidonic acid was removed and replaced with serum-free culture medium that contained [3H]arachidonic acid (2.3 pM, 1.7 X lo6 dpm) with or without mEGF (15 ng/ml). Thereafter, a t 1, 2, and 4 h, the culture medium was removed and thecells were collected. PGE, and arachidonic acid were extracted from the medium and arachidonic acid was isolated from the lipid extract of the cells. The tritium/carbon-14 ratio in each of these substanceswas determined by liquid scintillation spectrometry. Each of these studies was conducted with replicates of three dishes of amnion cells. Preparation and Characterization of a Rabbit Polyclonal Antibody toPGH, Synthase-The homogeneity of apreparation of PGH, synthase purified from ram seminal vesicles, purchased from Oxford Biochemicals Co., Oxford, MI, was assessed by SDS-polyacrylamide gel electrophoresis on 7.5% gels and on 7.5-15% gradient gels. The preparation was comprised of a single protein (M, -72,000), and purity was judged to be greater than 99%. A young male rabbit was immunized initially by subcutaneous injection of PGH, synthase (150 pg) in complete Freunds' adjuvant. Four times thereafter, at intervals of 3 weeks, the rabbit was immunized with PGH, synthase (100-150 pg) in incomplete Freunds' adjuvant. Six months after the initial injection, the rabbit was bled, serum was collected, and the IgG was purified partially by precipitation with ammonium sulfate (40%, w/ VI.

The antibody was characterized by Ouchterlony double diffusion analysis and by Western blot analysis of microsomal preparations of ram, bovine, and human seminal vesicles. Ram and bovine seminal vesicles (frozen) were purchased from Pel Freeze Biologicals (Rogers, AR). Human seminal vesicles were obtained at the time of surgery from a 74-year-old man with carcinoma of the testes; the tissue was kept a t 4 'C prior to homogenization. Homogenates of these tissues were prepared in a solution of sucrose (0.25 M) and phenylmethylsulfonyl fluoride (0.1 mM)by use of a Polytron homogenizer. The homogenates were centrifuged at 800 X g for 10 min at 4 "C, and then the supernatants were centrifuged sequentially at 15,000 X g for 10 min at 4 "C and 105,000 X g at 4 "C for 1 h. The 105,000 X g pellet (microsomes) was used for Western blot analysis. Microsomal pellets were suspended in electrophoresis sample buffer, and the solubilized proteins were separated by SDS-polyacrylamide gel electrophoresis on 7.5-15% gels. To evaluate further the specificity of the antibody for PGH,

7848

Induction of PGH, Synthase by EGF in AmnionCells

synthase, ram seminal vesicle PGH, synthase (2 pg) was incubated at 37 “C for 10 min with [3H]acetylsalicylicacid (1.4 nmol). At the end of the incubation period, the [3H]acetyl-PGH2synthase was isolated by immunoprecipitation with anti-PGH, synthase IgG and SDS-polyacrylamide gel electrophoresis (of the immunoisolate). The gelwas sliced into 1-mm pieces; each gel slice was solubilized in Protosol (Du Pont-NewEngland Nuclear), and radioactivity was quantified by liquid scintillation spectrometry. The inhibition of PGH, synthase activity by anti-PGH, synthase antibody was evaluated. Sonicates of mEGF-treated amnion cells were preincubated for 30 min a t 0 “C with anti-PGH, synthase IgG or preimmune IgG (1or 10 pg) prior to the addition of cofactors and [“Clarachidonic acid for assay of PGH, synthase activity. SDS-PolyacrylnmideGel Electrophoresis-SDS-polyacrylamide gel electrophoresis was conducted by the method of Laemmli (18) on 7.5% slab gels or 7.5-15% gradient slabgels. The gels werecomprised of appropriate concentrations of bis-acrylamide and acrylamide to achieve the desired cross-linking, Tris (1.5 M,pH 8.8), EDTA (8 mM), and SDS (0.4%, w/v). Temed and ammonium persulfate were added to facilitate polymerization. Electrode buffer consisted of Tris (0.025 M),glycine (0.192 M), SDS (0.1%, w/v), and EDTA (0.002M),pH 8.3. Samples were applied to the gels in electrophoresis sample buffer that consisted of Tris (10 mM, pH 8.0), SDS (l%, w/v), 0-mercaptoethanol ( l % , v/v), EDTA (1 mM), glycerol (5%, v/v), and bromphenol blue (0.02%, w/v). Electrophoresis was conducted at 2 “C and40 mA for 15-16 h. Western Blot Analysis-For Western blot analysis, proteins were transferred electrophoretically (4“C, 20 V, 15-16 h) from the polyacrylamide gel to nitrocellulose paper in buffer that contained Tris (0.02 M), glycine (0.15 M), and methanol (20%, v/v). The blot was blocked by incubation at 37 “C for 45 min in a solution of Tris-Cl(l0 mM, pH 7.5), NaCl (0.15 M), bovine serum albumin (5%, w/v), Nonidet P-40 (0.2%, v/v). Thereafter, the blot was incubated for 3 h in the same buffer solution that contained anti-PGH, synthase IgG (2 pg/ml). Finally, the blot was incubated a t 25 ‘C for 2 h in asolution of the blocking buffer that contained horseradish peroxidase-conjugated goat anti-rabbit IgG (diluted 1:1000, Bio-Rad). The proteins that bound the first and thence the second antibodies were visualized by color development with the horseradish peroxidase color reagent (Bio-Rad). The relative molecular mass of the proteins was estimated from the migration distances of “molecular weight markers” purchased from Bio-Rad. The proteins used were phosphorylase b (Mr = 92,500), bovine serum albumin (M,= 66,200), ovalbumin (M, = 45,000), carbonic anhydrase (M, = 31,000), soybean trypsin inhibitor (M.= 21,500), and lysozyme (M.= 14,400).In addition, ram seminal vesicle PGH, synthase (Oxford Biochemical) was used as a standard. Immunoisolntion of r5S]Methionine-labeled, Newly Synthesized PGH, Synthase-To evaluate the effect of mEGF onthe induction of synthesis of PGH, synthase, confluent amnion cells in primarymonolayer culture (plated in 60-mm diameter culture dishes) were maintained in methionine-free culture medium for 2 h prior to the commencement of the experiment. Thereafter, the cells were incubated in the absence or presence of mEGF (15 ng/ml) in methionine-free medium that contained [36S]methionine (40 pCi/dish). At 0.5, 1, 2, and 4 h thereafter, the medium was removed and discarded, and the cells were scraped from the dishes in a solution of NaCl(O.15 M) that contained sodium phosphate (10 mM, pH 7.4) buffer and phenylmethylsulfonyl fluoride (0.1 mM). The cells were pelleted by centrifugation a t 4 “C and lysed by freeze-thawing and sonication in a solution of sodium phosphate (10 mM, pH 7.4) buffer that contained NaCl(O.15 M), Triton X-100 (lo%,v/v), deoxycholic acid (0.55%, w/ v), SDS (0.1%, w/v), L-methionine (10 mM), and bovine serum albumin (5 ng/ml). The samples were clarified by centrifugation (800 X g, 20 min, 4 “C). Theamount of [36S]methionineincorporated into total trichloroacetic acid-precipitable protein was determined in triplicate aliquots of each sample. Newly synthesized, radiolabeled PGH, synthase was immunoisolated from aliquots that contained equal amounts of [36S]methionine-labeledtotal protein. The samples were preabsorbed with Pansorbin (Behring Diagnostics) and preimmune IgG. Pansorbin and associated IgG waspelleted by centrifugation for 5 min in a microcentrifuge a t room temperature. Thereafter, PGH, synthase was immunoprecipitated by the addition of anti-PGH, synthase IgG and Pansorbin. Immunoprecipitated proteins were evaluated by SDS-polyacrylamide gel electrophoresis and autoradiography as described. Autoradiography-For autoradiography of gels that contained immunoisolated radiolabeled protein(s), the separated proteins were fixed in an aqueous solution of glacial acetic acid (lo%, v/v) and

isopropyl alcohol (25%, v/v). The gels were stained with Coomassie Blue R-250 (0.05%, w/v) in glacial acetic acid/isopropyl alcohol/ water (2:5:13, by volume) and were destained in glacial acetic acid/ isopropyl alcohol/water (1:1:8,by volume) to visualize molecular weight and PGH, synthase markers. The gels were dried and placed on Kodak X-Omat film for 4-14 days a t 70 “C. Radiolabeled proteins were visualized after development of the exposed film. Estimates of relative molecular mass were based on migration distances of molecular weight markers purchased from Bio-Rad as described. RESULTS

Effect ofmEGF on the SpecificActivity ofPGH, S y n t h e Optimal conditions were established for the determination of the rate of conversion of arachidonic acid to PGE, in sonicated preparations of cultured human amnioncells. The conversion of [“Clarachidonic acid (10 p M ) to [l4C]PGEZat 37 “C was linear with incubation times up to 10 min (Fig. lA). In incubations conducted for 10 min, the reaction was linear with protein concentrationsbetween 0.08 and 0.9 mg/ml (Fig. 1B).By Lineweaver-Bulk analysis of the data obtained in incubations conducted with [14C]arachidonicacid, in various concentrations (1-19 pM), the apparent K,,, was computed to be 1 p~ (Fig. 1C).All subsequent assays were conducted for 10 min at 37 “C with protein concentrations of 0.5-0.7 mg/ml . and arachidonic acid at a concentration of 10 p ~ Similarly, studies were conducted to define optimal conditions for the assay of phospholipase Az and phospholipase C. The specific activities of phospholipase A, and phosphatidylinositol-specific phospholipase C as well as the rates of conversion of arachidonic acid to PGEz, i.e. PGH, synthase (determined under optimal conditions), were determined in

n

e

w“ -3 E

0

5

15

25

45 35

TIME (min 1 0

-

0 PROTEIN (pgl

ARACHIDONIC ACID (pM1

FIG. 1. The conversion of arachidonic acid to PGE, as a function of time (A), as a function of protein ( B ) , and as a function of the concentration of arachidonic acid (C, mean 2 S.E.).Assays wereconducted with sonicated preparations of cultured human amnion cells as described under “Experimental Procedures.”

Induction of PGHz Synthase by sonicated preparations of amnion cells (Table I). Treatment of confluent amnion cells in monolayer culture with mEGF (15 ng/ml, 4 h) did not affect the specific activities of either phospholipase A, or phospholipase C. On the other hand, the rate of conversion of arachidonic acid to PGE, was increased by 2.7-foldin EGF-treatedcells. In 10 experiments the specific activity of PGH, synthase was increased by 2-5-fold in cells treated with mEGF (15 ng/ml) for 4 h compared with that in nontreated cells. In another study, PGE, production (as evaluated by radioimmunoassay of PGE, in the culture medium, panel A) and PGH, synthase activity (panel B) were determined in amnion cells incubated with mEGF in various concentrations (0.1-25 ng/ml). In nontreated cells, the amount of PGE, that accumulated in the medium during a 4-h incubation period was 4.2 f 0.5 pmol/mg of protein/4 h (mean f S.E., n = 3); and, maximal accumulation of PGE,, 175 f 28 pmol/mg of protein/ 4h ( n = 3), was effected by treatment with mEGF at a concentration of 5 ng/ml and was maintained at thislevel in cells treated with mEGF in higher concentrations (Fig. 2 A ) . In this same experiment, the specific activity of PGH, synthase was stimulated maximally (4.4-fold) by mEGF at a concentration of 12.5 ng/ml (Fig. 2B). As a function of time of treatment with mEGF (15 ng/ml), the accumulation of PGE, in the culture medium reached maximal levels after 4 h (Fig. 3A). From these data the rate of PGE, production of nontreated cells was computed. The rate of production of PGE, (mean f S.E.) was maximal at 2 h, 55.9 f 13 pmol/mg of protein/h (n = 3), and declined thereafter to 6.1 f 3.0 and 6.7 f 0.8 pmol/mg of protein/h ( n = 3) by 4 and 8 h, respectively (Fig. 3B). The rate of production of PGE, in cells that were treated with mEGF increased with time up to 4 h (maximum: 219 k 39 pmol/mg of protein/ h, n = 3), and decreased precipitously to 20 f 10 pmol/mg of protein/h (n = 3) by 8 h (Fig. 3B). As .a function of time of treatment with mEGF (15 ng/ml), the specific activity of PGH, synthase (mean f S.E.) was increased maximally by 4 h (Fig. 3C). Importantly, the increase in specific activity of PGH, synthase in amnioncells that were treated with mEGF was maintained for 8 h (Fig. 3C). To investigate the possible effect of mEGF treatment on the apparent K,,, of cyclooxygenase for arachidonic acid, the rate of conversion of [14C]arachidonicacid to [“C]PGE, was evaluated in assaysthat were conducted with arachidonic acid in various concentrations (1-19 WM)and sonicates of amnion

EGF in Amnion Cells

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ann

_I

0

5

10

15

20

25

mEGF (ng I m l 1

FIG. 2. A, the accumulation of PGE, in the culture medium (as determined by radioimmunoassay),and, B, the rate of conversion of arachidonic acid to PGE, (PGH, synthase) as a function of the concentration of mEGF in the culture medium. Amnioncell cultures were treated with mEGF in various concentrations for 4 h. The specific activity of PGH, synthase is expressed as % increase over that in cells that were not treated with mEGF. Assayswere conducted as describedunder“ExperimentalProcedures”and the data are presented as the mean f S.E. (n = 3).

cells that were maintained for 4 h inthe absence or presence of mEGF (15 ng/ml). By Lineweaver-Burk analysis of the data obtained, the Vmaxin control cells was 0.51 pmol/min/ mg of protein and that in mEGF-treated cells was 1.83pmol/ min/mg of protein (3.6-fold increase). The apparent K,,, of the enzyme for arachidonic acid (1.0 WM) was notaltered by treatment of the cells with mEGF. We found that in the presence of cycloheximide (35 WM),treatment of amnion cells with mEGF (15 ng/ml) for 4 h was ineffective in stimulating PGH,synthase activity and PGE, production (Table 11). Similar results were obtained in studies conducted with puTABLE I romycin as an inhibitor of protein synthesis. Effect of treatment of human amnion cells with mEGFon the specific The stimulation of PGE, production by mEGF was dependactivities of phospholipase A%phosphutidylinositol-specific phospholipase C, and PGH, synthase ent on the presence of arachidonic acid in the culture medium. Confluent human amnion cells in primary monolayer culture were Treatment of amnion cells with mEGF (15 ng/ml) in serumincubated in the absence or presence of mEGF (15 ng/ml) for 4 h. free culture medium (that contained fatty acid-free bovine Thereafter, the cells were scraped from the dishes; preparations of serum albumin (2.5 mg/ml)) caused an increase in thespecific cells and assays were conducted as described under “Experimental activity of PGH, synthase but not PGE, production (Table Procedures.” 111).When arachidonic acid (5 p ~ was ) added to the serumSpecific activity, free culture medium, mEGF was effective in stimulating both mean % S.E. Enzyme PGH, synthase and PGE, production in a manner similar to Control mEGF that effected by mEGF in medium that contained fetal bovine Phospholipase A, (nmol/30 min/mg 1.1 f 0.15 1.0 f 0.09 serum(Table 111). Similar findings were obtained in cells protein; n = 4) maintained in the absence of serum for 24-48 h; namely, Phosphatidylinositol-specific phospho1.2 f 0.06 1.1 f 0.07 PGE, production was dependent on the presence of arachilipase C (pmol/h/mg protein; n = 4) donic acid in the culture medium (data not shown). PGHz synthase” (pmol/min/mg protein; 1.9 f 0.2 5.2 k 0.5* Metabolic Fate of Radiolabeled Arachidonic Acid in mEGFn = 3) PGH, synthase refers to the specific rate of conversion of radio- treated Amnion Cells-To determine the origin of arachidonic labeled arachidonic acid to radiolabeled PGE, as determined under acid that is utilized as substrate for PGE, biosynthesis in mEGF-treated amnion cells, two studies were conducted. In optimal in vitro conditions. * p < 0.001 compared with Control. the first study, amnion cells were preincubated with [“C]

Induction of PGH, Synthase by EGF in Amnion Cells

7850

TABLE 111 Effect ofmEGF on PGE, productionand PGH, synthase activity in amnion cells Human amnion cells in primary monolayer culture (confluent) were incubated for 10 h in culture media that contained fetal bovine serum, fatty acid-free bovine serum albumin, arachidonic acid, or mEGF as indicated. Thereafter, PGE, in the culture media was quantified and thespecific activity of PGH, synthase was determined as described under "Experimental Procedures." The experiment was conducted with replicates of four dishes of amnion cells, and the data are expressed as mean f S.E. The abbreviation used is: DMEM, Dulbecco's Modified Eagles Medium. Composition of culturemediumPGEzproduction

PGH, synthase specific activity

pg/mg proteinll0 h pmol/min/mg protein

F12:DMEM + Fetal bovine serum (IO%, v/v) Control 16.6 f 3.81 mEGF (15 ng/ml) 162 f 17.0" Serum-free F12:DMEM + BSA (2.5 mg/ml) Control 3.6 f 1.27 mEGF 3.7 f 0.90 Serum-free F12:DMEM BSA arachidonic acid (5 pM) Control 8.4 f 1.04 224 f 18.3" mEGF " p c 0.001, compared with Control. b p < 0.05, compared with Control.

+

05

2

4

FIG. 3. The accumulation of PGE, in the culture medium (A),the rate of production of PGE, based on the quantity of PGE, in the culture medium as determined by radioimmunoassay ( B ) , and the specific activity of PGH, synthase (0. Amnion cell cultures were maintained in the absence (0)or presence (0)of mEGF (15 ng/ml) for various times. The dataare expressed as the mean f S.E. ( n = 3). The data from a single experiment are presented graphically in two ways in panels A and B.

TABLEI1 Effect of cycloheximide on mEGF-mediated induction of PGH, synthase activity and PGE, production in amnion cells Human amnioncells in primary monolayer culture were incubated for 4 h in the absence or presence of cycloheximide and mEGF. Thereafter, PGE, in the culture medium was quantified by specific radioimmunoassay; the specific activity PGH, synthase was determined in cell sonicates as described under "Experimental Procedures." Data are expressed as mean f S.E. for replicates of 3. PGE, production pg/mg proteinl4 h

30 f 5.0 Control 2.2 f 0.7 Cycloheximide (35 PM) 145 f 18" mEGF (15 ng/nl) 4.1 f 0.3' Cycloheximide + mEGF " p< 0.005, compared with Control. b p < 0.002, compared with Control. ' p c 0.02, compared with mEGF. d p c 0.05, compared with mEGF.

0.4 f 0.01 1.4 f 0.42b

+

0.9 f 0.20 1.70 f 0.25'

e

TIME (hl

Treatment

1.3 f 0.21 2.1 f 0.20b

PGHz synthase specific activity pmol/min/mg protein 1.8 f 0.08 1.0 f 0.09 3.7 f 0.50'

2.2 f 0.22d

arachidonic acid to effect radiolabeling of the lipids, in particular, the glycerophospholipids. Thereafter, the cells were incubated (4 h) in the absence or presence of mEGF (15 ng/ ml). At the end of the incubation period, the radioactivity in glycerophospholipids, i.e. phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine, was determined. Treatment of amnion cells with mEGF had no effect on the amount of radioactivity in these glycerophospholipids (Table IV). To address this issue further, we conducted a similar ex-

TABLEIV Effect of mEGF treatment on arachidonic acid content of glycerophosphlipids Human amnion cells in primary monolayer culture were incubated for 24 h in serum-free culture medium that contained fatty acid-free bovine serum albumin (2.5 mg/ml) and ['4C]arachidonic acid (2.3 pM, 1.7 X lo6 dpm). Thereafter, the culture medium was removed and replaced with serum-free culture medium that contained fatty acidfree bovine serum albumin. After incubation for 4 h in the absence or presence of mEGF (15 ng/ml), the cells were collected and radioactivity in glycerophospholipids was quantified as described under "Experimental Procedures." Data are presented as mean f S.E. with replicates of 3. ["C]Arachidonic acid Glycerophospholipids mEGF

Control dpmlmg protein

Phosphatidylcholine 810,409 f 55,060 960,402 f 51,023" 479,729 f 66,088" Phosphatidylethanol339,825 f 100,331 amine 484,036 f 28,404469,590 f 21,362" Phosphatidylinositol Phosphatidylserine 64,650 f 1,740 82,958 f 5,625" a Not significantly ( p > 0.05) different, compared with Control.

periment in which glycerophospholipids were radiolabeled with [14C]arachidonicacid (as in the first experiment). Then, the medium that contained [14C]arachidonic acidwas removed, and the cells were incubated with or without mEGF in serum-free medium that contained fatty acid free-bovine serum albumin and [3H]arachidonic acid. A t 1, 2, and 4 h thereafter, we determined the tritium/carbon-14 ratios of PGE, and arachidonic acid in the culture media and that of intracellular nonesterified arachidonic acid. For each incubation time, the tritium/carbon-14 ratio of PGE, and arachidonic acid in the culture medium were similar, irrespective of treatment with mEGF (Table V). In all cases, the tritium/ carbon-14 ratio of intracellular nonesterified arachidonic acid (