Microsomal Prostaglandin E Synthase-1 Is Overexpressed in Inflammatory Bowel Disease

Microsomal prostaglandin E synthase-1 (mPGES-1) catalyzes the conversion of cyclooxygenase-derived prostaglandin (PG) H2 to PGE2. Increased amounts of mPGES-1 were detected in inflamed intestinal mucosa from patients with inflammatory bowel disease (IBD). Treatment with tumor necrosis factor (TNF)-α stimulated mPGES-1 transcription in human colonocytes, resulting in increased amounts of mPGES-1 mRNA and protein. The inductive effect of TNF-α localized to the GC box region of the mPGES-1 promoter. Binding of Egr-1 to the GC box region of the mPGES-1 promoter was enhanced by treatment with TNF-α. Notably, increased Egr-1 expression and binding activity were also detected in inflamed mucosa from IBD patients. Treatment with TNF-α induced the activities of phosphatidylcholine-phospholipase C (PC-PLC) and protein kinase (PK) C and enhanced NO production. A pharmacological approach was used to implicate PC-PLC → PKC → NO signaling as being important for the induction of mPGES-1 by TNF-α. TNF-α also enhanced guanylate cyclase activity and inhibitors of guanylate cyclase activity blocked the induction of mPGES-1 by TNF-α. YC-1, an activator of guanylate cyclase, induced mPGES-1. Overexpressing a dominant negative form of PKG blocked TNF-α-mediated stimulation of the mPGES-1 promoter. Taken together, these results suggest that overexpression of mPGES-1 in IBD is the result of Egr-1-mediated activation of transcription. Moreover, TNF-α induced mPGES-1 by stimulating PC-PLC → PKC → NO → cGMP → PKG signal transduction pathway.

Inflammatory bowel disease (IBD) 1 including Crohn's disease and ulcerative colitis, is characterized by chronic, relaps-ing inflammation of the gastrointestinal tract. This disease is multifactorial in origin and affects at least 1 in 1000 people in Western countries (1)(2)(3). Multiple lines of evidence suggest an important role for prostaglandin E 2 (PGE 2 ) in IBD. For example, increased amounts of PGE 2 are detected at sites of intestinal inflammation and correlate with disease activity (4 -6). Moreover, nonsteroidal anti-inflammatory drugs (NSAIDs), prototypic inhibitors of cyclooxygenase (COX) activity and PGE 2 production, can trigger or worsen the disease (7,8). Inhibiting the expression of either of the two isoforms of COX, COX-1 or COX-2, exacerbated the extent of colitis in experimental animals (9). Mice engineered to be deficient in the PGE 2 receptor EP4 were sensitized to developing experimental colitis (10). Conversely, treatment with an EP4-selective agonist ameliorated colitis in wild-type mice (10). Taken together, these results strongly suggest that PGE 2 is important for maintaining mucosal homeostasis. It is important, therefore, to define the pathways that are dysregulated in IBD resulting in increased amounts of mucosal PGE 2 .
The synthesis of PGE 2 from arachidonic acid requires two enzymes that act in sequence. COX catalyzes the synthesis of PGH 2 from arachidonic acid. COX-1 is constitutively expressed in most tissues, including the intestine (11,12). In contrast, COX-2 is not expressed in the normal gut but is induced by cytokines, growth factors, and tumor promoters (13)(14)(15)(16)(17)(18)(19). Increased amounts of COX-2 have been detected in inflamed mucosa in IBD (20). Recent studies suggest that both cytosolic and microsomal enzymes can convert COX-derived PGH 2 to PGE 2 . cPGES/p23 has been reported to be constitutively expressed in many tissues and functionally coupled to COX-1 (21). mPGES-1, an inducible enzyme, is overexpressed in inflamed joints, atherosclerotic plaques, and neoplastic tissues (22)(23)(24)(25)(26)(27). It is functionally coupled with COX-2 in preference to COX-1 (28). The expression of mPGES-1 is positively regulated by proinflammatory cytokines including TNF-␣ and IL-1␣ (29,30). Several findings suggest that mPGES-1 is extremely important for the synthesis of PGE 2 . It has a high catalytic * This work was supported by the New York Crohn's Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
In this study, we evaluated whether mPGES-1 was overexpressed in inflamed intestinal mucosa from patients with IBD. TNF-␣ plays a central role in mucosal inflammation and is postulated to be at the apex of an inflammatory cascade in IBD (1,33). Hence, we have defined the signal transduction pathway by which TNF-␣ stimulated mPGES-1 transcription in human colonocytes. We show that levels of mPGES-1 are increased in inflamed intestinal mucosa. Moreover, TNF-␣ induced mPGES-1 by stimulating a signal transduction pathway comprising PC-PLC 3 PKC 3 NO 3 cGMP 3 PKG, resulting in Egr-1-mediated activation of transcription.

EXPERIMENTAL PROCEDURES
Materials-Dulbecco's modified Eagle's medium, fetal bovine serum, PKC assay kits, and LipofectAMINE were purchased from Invitrogen. Rabbit polyclonal anti-human mPGES-1 serum, blocking peptide against human mPGES-1, cDNA probe for mPGES-1, and spermine NONOate were from Cayman Chemical Co. (Ann Arbor, MI). Human TNF-␣, antibody to ␤-actin, methylene blue, phosphatidylcholine-phospholipase C from Bacillus cereus, Lowry protein assay kits, and secondary antibody conjugated to horseradish peroxidase were from Sigma. Western blotting detection reagents (ECL) were from Amersham Biosciences. Nitrocellulose membranes were from Schleicher & Schuell. Calphostin C, YC-1, S-nitroso-N-acetylpenicillamine (SNAP), and diphenyleneiodonium were from Biomol (Plymouth Meeting, PA). D609 and LY83583 were from Calbiochem (La Jolla, CA). Griess reagent kit (nitrite assay) was from Molecular Probes (Eugene, OR). Monoclonal anti-human cPGES/p23 antibody was from Affinity Bioreagents (Golden, CO Patient Samples-Specimens were obtained at the time of colonoscopic examination from patients with ulcerative colitis or Crohn's disease. Biopsies were taken from mucosa that was macroscopically inflamed or normal. Tissue samples were immediately snap-frozen in liquid nitrogen and stored at Ϫ80°C until analysis. The study was approved by the Committee on Human Rights in Research at the participating institutions. Immunohistochemistry-mPGES-1 immunostaining was performed as previously described (25,26). Neutral buffered formalin-fixed tissue was embedded in paraffin. Tissue sections (4 m) were prepared using a microtome and mounted on Superfrost/Plus slides. Immunohistochemical analysis was performed within 24 h of the sections being cut. Sections were deparaffinized in xylene, rehydrated in graded alcohols, and washed in distilled water. Antigen retrieval was performed by steaming the sections in 10 mM citric acid (pH 6.0) for 30 min. Subsequently, endogenous peroxidase activity was blocked with 3% hydrogen peroxide. The slides were washed three times in PBS and blocked for 20 min with 5% normal goat serum. Tissue sections were then incubated with antiserum to mPGES-1 at a 1:750 dilution (2% bovine serum albumin in PBS) for 18 h at 4°C. Control sections were incubated with mPGES-1 antiserum preabsorbed with a 100-fold excess of mPGES-1 blocking peptide or with preimmune serum. After washing three times with PBS, the sections were incubated with biotinylated anti-rabbit antibody at a 1:500 dilution for 1 h at room temperature. The slides were then washed three times in PBS and labeled using 1:500 streptavidin-horseradish peroxidase for 1 h at room temperature. The reaction was visualized using 3,3Ј-diaminobenzidine. Subsequently, the slides were rinsed in tap water and counterstained with hematoxylin. The slides were then dehydrated with ethanol, rinsed with xylene, and mounted.
Cell Culture-Human colon cancer cell line HCA7 was established from moderately differentiated adenocarcinoma of the colon (34) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 g/ml strepto-mycin. DLD1 and HCT116 colon cell lines were maintained in McCoy's 5A medium supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 g/ml streptomycin. All treatments were carried out in serum-free medium.
Western Blotting-Frozen tissue was thawed in ice-cold lysis buffer (150 mM NaCl, 100 mM Tris (pH 8.0), 1% Tween 20, 50 mM diethyldithiocarbamate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml trypsin inhibitor, and 10 g/ml leupeptin). Tissues were sonicated for 20 s on ice and centrifuged at 10,000 ϫ g for 10 min at 4°C to remove the particulate material. The protein concentration of the supernatant was measured using the method of Lowry et al. (35). Cell lysates were prepared as described previously (24 -26). Immunoblot analysis of mPGES-1 was performed using methods described in previous studies (24 -26).
Northern Blotting-Total cellular RNA was isolated from cell monolayers using an RNA isolation kit from Qiagen Inc. 15 g of total cellular RNA/lane were electrophoresed in a formaldehyde-containing 1.0% agarose gel and transferred to nylon-supported membranes. mPGES-1 and 18 S rRNA probes were labeled with [ 32 P]CTP by random priming. The blot was probed using previously described methods (24).
Nuclear Run-off Assay-Cells (2.5 ϫ 10 5 ) were plated in three 10-cm dishes for each condition. Cells were grown in growth medium until they were ϳ60% confluent. Nuclei were isolated and stored in liquid nitrogen. For the transcription assay, nuclei (1.0 ϫ 10 7 ) were thawed and incubated in reaction buffer (10 mM Tris, pH 8.5, 5 mM MgCl 2 , and 0.3 M KCl) containing 100 Ci of uridine 5Ј-[ 32 P]triphosphate and 1 mM unlabeled nucleotides. After 30 min, labeled nascent RNA transcripts were isolated. The human mPGES-1 and 18 S rRNA cDNAs were immobilized onto nitrocellulose and prehybridized overnight in hybridization buffer. Hybridization was carried out at 42°C for 24 h using equal cpm/ml of labeled nascent RNA transcripts for each treatment group. The membranes were washed twice with 2ϫ SSC buffer for 1 h at 55°C and then treated with 10 mg/ml RNase A in 2ϫ SSC at 37°C for 30 min, dried, and autoradiographed.
Plasmids-The mPGES-1 promoter constructs have been described previously (36). Wild type and dominant negative PKG expression vectors were a generous gift of Dr. Renate Pilz (University of California, San Diego, CA) (37).
Transient Transfection-HCA7 cells were seeded at a density of 5 ϫ 10 4 cells/well in 6-well dishes and grown to 50% confluence. For each well, 2 g of plasmid DNA were introduced into cells using 4 g of LipofectAMINE as per the instructions from the manufacturer. After 16 h of incubation, the medium was replaced with culture medium. The activities of luciferase and ␤-galactosidase were measured in cellular extracts as described previously (24).
Electrophoretic Mobility Shift Assay-Nuclear extracts were prepared from cells or tissues (40). For binding studies, oligonucleotides containing the GC box region of mPGES-1 promoter (5Ј-CGTGGGGC-GGGGCGTGGGCGGTGCTG-3Ј (sense) or Egr-1 consensus site (5Ј-CG-CCCTCGCCCCCGCGCCGGG-3Ј (sense) (41) were used. The complementary oligonucleotides were annealed in 20 mM Tris (pH 7.6), 50 mM NaCl, 10 mM MgCl 2 , and 1 mM dithiothreitol. The annealed oligonucleotide was phosphorylated at the 5Ј end with [␥-32 P]ATP and T4 polynucleotide kinase. The binding reaction was performed by incubating 5 g of nuclear protein in 20 mM HEPES (pH 7.9), 10% glycerol, 300 g of bovine serum albumin, and 1 g of poly(dI⅐dC) in a final volume of 10 l for 10 min at 25°C. The labeled oligonucleotides were added to the reaction mixture and allowed to incubate for an additional 20 min at 25°C. The samples were electrophoresed on a 4% nondenaturing polyacrylamide gel. The gel was then dried and subjected to autoradiography at Ϫ80°C.
Measurement of PC-PLC Activity-PC-PLC activity was measured following a method described previously (42). Cells were plated in 10-cm dishes at 2 ϫ 10 5 cells/dish and grown to 60% confluence. Cells were then treated with fresh basal medium containing vehicle or TNF-␣ (10 ng/ml) for 30 and 60 min. Pellets were dissolved in 300 l of 0.01% Triton X-100 and then were sonicated. 16 l of lysate was incubated at 37°C for 2 h in buffer (50 mM Tris-HCl, pH 7.3, 6.3 mM CaCl 2 , 0.13 g/ml bovine serum albumin, 0.15 M ammonium sulfate, and L-3-phosphatidyl[N-methyl- 14 C]choline, 50 mCi) in a total volume of 80 l. The reaction was stopped and the lipids were extracted by adding CHCl 3 : CH 3 OH (1:2, v/v; 180 l), 0.9% NaCl (60 l), and CHCl 3 (60 l). The aqueous and organic phases containing [ 14 C]phosphocholine and [ 14 C]phosphatidylcholine, respectively, were separated and quantified by liquid scintillation. PC-PLC activation was expressed as cpm incorporated/g of protein.
Measurement of PKC Activity-PKC activity was determined in both cytosolic and membranous fractions as well as in crude whole cell fractions of treated cells. The activity of PKC was measured according to directions from Invitrogen (43). Briefly, cells were plated in 10-cm dishes at 2 ϫ 10 5 cells/dish and grown to 60% confluence. Cells were then treated with fresh basal medium containing vehicle or TNF-␣ (10 ng/ml) for 30 or 60 min. Total PKC activity was measured in cell lysates.
To determine cytosolic and membrane-bound PKC activity, cell lysates were centrifuged at 100,000 ϫ g for 30 min. The resulting supernatant contains cytosolic PKC; membrane-bound PKC activity is present in the pellet. Subsequently, DEAE cellulose columns were used to partially purify PKC enzymes. PKC activity was then measured by incubating partially purified PKC with [ 32 P]ATP (3000 -6000 Ci/mmol) and the substrate myelin basic protein for 20 min at room temperature. The activity of PKC is expressed as cpm incorporated/g of protein.
Measurement of Nitrite Concentration in the Culture Cell Supernatant-HCA7 cells were seeded at a density of 5 ϫ 10 4 cells/well in 6-well dishes. When the cells became 60% confluent, the medium was changed to serum-free for 24 h. Following treatment, the supernatant was collected to assess nitrite concentration using Griess reagent as per the instructions from the manufacturer.
Statistics-Comparisons between groups were made by Student's t test. A difference between groups of p Ͻ 0.05 was considered significant.

mPGES-1 Expression Is Enhanced in Inflammatory Bowel
Disease-We compared amounts of mPGES-1 protein in inflamed and non-inflamed colonic mucosa from patients with Crohn's disease and ulcerative colitis. Elevated levels of mPGES-1 were detected in inflamed mucosa in 10 of 10 patients with Crohn's disease. Similarly, increased amounts of mPGES-1 were observed in 10 of 12 patients with ulcerative colitis. Representative immunoblots are shown in Fig. 1. Immunohistochemistry was performed to identify the cell types that expressed mPGES-1 in IBD. In inflamed colonic mucosa, mPGES-1 was detected in both epithelial cells and inflammatory cells (Fig. 2).
TNF-␣ Stimulated the Transcription of mPGES-1-TNF-␣ is widely recognized to contribute to inflammation in IBD (1,33). It was relevant, therefore, to determine whether TNF-␣ regulated the expression of mPGES-1 in human colonocytes. As shown in Fig. 3A, TNF-␣ caused dose-dependent induction of mPGES-1 in HCA7 cells. To exclude the possibility that the inductive effects of TNF-␣ were unique to HCA7 cells, we also determined whether TNF-␣ induced mPGES-1 in HCT116 and DLD1 human colonocytes. mPGES-1 was induced by TNF-␣ in these two cell lines as well (Fig. 3B). In contrast, to mPGES-1, amounts of cPGES were unaffected by treatment with TNF-␣ (Fig. 3A). Given the marked increase in amounts of mPGES-1 following treatment with TNF-␣, it was important to determine whether mPGES-1 was important for TNF-␣-mediated stimulation of PGE 2 production. Treatment of HCA7 cells with TNF-␣ led to an ϳ3-fold increase in synthesis of PGE 2 (Fig.  3C). Importantly, overexpressing antisense to mPGES-1 essentially abrogated the increase in mPGES-1 (data not shown) and PGE 2 production induced by TNF-␣ (Fig. 3C). To determine whether regulation of mPGES-1 was pretranslational, Northern blot analysis was carried out. TNF-␣ caused a marked increase in amounts of mPGES-1 mRNA (Fig. 3D).  , ϫ200). B, mPGES-1 expression is marked in ulcerative colitis. In this photograph both colonic epithelium and rare lamina propria inflammatory cells express mPGES-1 (right of center) (original magnification, ϫ200). C and D, mPGES-1 expression is also marked in Crohn's disease. Panel C is an example of mild chronic, active colitis with mPGES-1 expression that is more prominent in epithelial cells. Panel D shows moderate chronic active colitis, including a crypt abscess. mPGES-1 expression is seen primarily in epithelial cells and also in rare inflammatory cells that surround the damaged crypt (center) (original magnification, ϫ200).
TNF-␣ (Fig. 3E). Consistent with this result, TNF-␣ caused approximately a 1.5-fold increase in mPGES-1 promoter activity (Fig. 4B). To define the region of mPGES-1 promoter (Fig. 4A) that responded to TNF-␣, transient transfections were performed utilizing different deletions of the human mPGES-1 promoter (Fig. 4B). The inductive effects of TNF-␣ were lost when the cells were transfected with the deletion construct pGL3/99. There are two GC boxes (Ϫ112 to Ϫ119 (GC box 2) and Ϫ101 to Ϫ108 (GC box 1) in the human mPGES-1 promoter. We postulated that either one or both of these GC boxes might be crucial for TNF-␣-mediated induc-tion of mPGES-1 promoter activity. As shown in Fig. 4C, mutagenesis of either GC box 1, GC box 2, or both GC boxes (double mutant) abrogated TNF-␣-mediated induction of mPGES-1 promoter activity.
Egr-1 has been observed to bind to the GC box of the mPGES-1 promoter and thereby regulate transcription (36). Hence, electrophoretic mobility shift assays were performed to evaluate whether Egr-1 was important for TNF-␣-mediated induction of mPGES-1 transcription. As shown in Fig.  5A, treatment with TNF-␣ led to increased binding of nuclear protein to the GC box region of mPGES-1 promoter. Super-FIG. 3. TNF-␣ induces mPGES-1 transcription and stimulates production of PGE 2 . A, HCA7 cells were treated with vehicle or TNF-␣ (0.1, 1.0, and 10 ng/ml) for 24 h. Cellular lysate protein (100 g/lane) was loaded onto a 12.5% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose. The immunoblot was sequentially probed with antibodies specific for mPGES-1, cPGES, and ␤-actin. B, cellular lysate protein (100 g/lane) from HCA7, HCT116, and DLD1 cells treated with vehicle (C) or TNF-␣ (10 ng/ml) for 24 h was loaded onto a 12.5% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose. The immunoblot was sequentially probed with antibodies specific for mPGES-1 and ␤-actin. C, bars labeled control and TNF-␣ represent HCA7 cells treated with vehicle and TNF-␣ (10 ng/ml), respectively. Bars labeled TNF-␣ ϩ mPGES-1-S and TNF-␣ ϩ mPGES-1-AS represent cells treated with TNF-␣ (10 ng/ml) that were transfected with 1.8 g of scrambled (S) and antisense (AS) oligonucleotides for mPGES-1, respectively. Treatments were for 24 h. The medium was then replaced with fresh medium containing 10 M sodium arachidonate. 30 min later, the medium was collected to determine amounts of PGE 2 . Production of PGE 2 was determined by enzyme immunoassay. Columns, means; bars, S.D.; n ϭ 6. D, total cellular RNA was isolated from HCA7 cells treated with vehicle or 10 ng/ml TNF-␣ for 16 h. 10 g of RNA was added to each lane. The blot was hybridized with probes that recognized mPGES-1 mRNA and 18 S rRNA. E, nuclei were isolated from HCA7 cells treated with vehicle (C) or TNF-␣ (10 ng/ml) for 16 h. Nuclear run-offs were performed as described under "Experimental Procedures." mPGES-1 and ␤-actin cDNAs were immobilized onto nitrocellulose membranes and hybridized with labeled nascent RNA transcripts.

mPGES-1 and Inflammatory Bowel Disease
shift analysis identified Egr-1 in the binding complex. In contrast, c-Jun, CEBP-␣, c-Fos, p65, and PEA3 were not identified in the binding complex. Consistent with this finding, binding was prevented by incubating nuclear extract with an excess of GC box cold probe or a consensus Egr-1 oligonucleotide (Fig. 5A). To confirm the involvement of Egr-1 in TNF-␣-mediated induction of mPGES-1, we utilized siRNA as well as antisense technologies. Transfection of HCA7 cells with siRNA to Egr-1 inhibited the expression of Egr-1 (data not shown). As shown in Fig. 5B, when cells were transfected with siRNA to Egr-1, TNF-␣-mediated induction of mPGES-1 was blocked. In contrast, overexpressing siRNA to GFP did not suppress the induction of mPGES-1 by TNF-␣. Additionally, transfection of antisense to Egr-1 suppressed TNF-␣mediated stimulation of mPGES-1 promoter activity (Fig.  5C). Neither antisense nor scrambled Egr-1 oligonucleotides affected basal transcription (data not shown). Taken together, these data clearly show that Egr-1 is crucial for TNF-␣-mediated induction of mPGES-1.
Deregulated TNF-␣ signaling plays a central role in IBD. Because mPGES-1 is overexpressed in IBD, we next investigated the expression and binding activity of Egr-1 in IBD. As shown in Fig. 6A, levels of Egr-1 were increased in inflamed mucosa from patients with Crohn's disease (lanes 1-4) and ulcerative colitis (lanes 5-7) compared with non-inflamed mucosa (lanes 8 -10). Notably, enhanced binding of nuclear protein to the GC box region of the mPGES-1 promoter was detected in extracts prepared from inflamed mucosa from patients with Crohn's disease and ulcerative colitis (Fig. 6B, lanes 2 and 4,  respectively). Egr-1 was detected in the binding complex (Fig.  6C). Incubating nuclear extract with an excess of consensus Egr-1 oligonucleotide prevented binding.

FIG. 5. TNF-␣ induces mPGES-1 transcription by enhancing the binding of Egr-1 to the GC box region of the mPGES-1 promoter.
A, 5 g of nuclear protein from HCA7 cells was incubated with a 32 P-labeled oligonucleotide containing the GC box region of mPGES-1. Cells were treated with vehicle (lane 1) or TNF-␣ (10 ng/ml; lane 2) for 30 min. Lanes 3, 4, and 5 represent nuclear extract from TNF-␣-treated cells incubated with a 32 P-labeled mPGES-1 GC box oligonucleotide and a 100-, 10-, and 50-fold excess of unlabeled oligonucleotide, respectively. Lane 7 represents nuclear extract from TNF-␣-treated cells incubated with a 32 P-labeled mPGES-1 GC box oligonucleotide and a 100-fold excess of unlabeled Egr-1 consensus oligonucleotide. Lanes 6, 8, and 9 represent nuclear extract from TNF-␣-treated cells incubated with antibodies to Egr-1 (lane 6), c-Jun (lane 8), and p65 (lane 9), respectively. The protein-DNA complex that formed was separated on a 4% polyacrylamide gel. B, HCA7 cells were transfected with Egr-1 siRNA or GFP siRNA. Subsequently, cells were treated with vehicle or TNF-␣ (10 ng/ml) for 24 h. Cellular lysate protein (100 g/lane) was then loaded onto a 12.5% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose. The immunoblot was sequentially probed with antibodies specific for mPGES-1 and ␤-actin. C, cells were transfected with 0.9 g of human mPGES-1 promoter construct (pGL3/650) and 0.2 g of pSV␤gal. Bars labeled Egr-1 scrambled and Egr-1 antisense also received 0.9 g of scrambled or antisense Egr-1 oligonucleotides. The total amount of DNA in each reaction was kept constant at 2 g by using empty vector. After transfection, cells were treated with vehicle or 10 ng/ml TNF-␣. Reporter activities were measured in cellular extracts 24 h later. Luciferase Activity represents data that have been normalized with ␤-galactosidase. Columns, means; bars, S.D.; n ϭ 6.

Signal Transduction Pathway Responsible for TNF-␣mediated Induction of mPGES-1-TNF-␣ receptor activation
stimulates PC-PLC and PKC signaling. Hence, we were interested in the possibility that PC-PLC and PKC were involved in TNF-␣-mediated induction of mPGES-1. As shown in Fig. 7A, treatment with TNF-␣ for 30 min led to a 2-fold induction of PC-PLC activity. To determine whether this increase in PC-PLC activity was linked to the induction of mPGES-1, cells were treated with purified PC-PLC. Notably, treatment with PC-PLC caused a significant increase in amounts of mPGES-1 (Fig. 7B). Moreover, D609, an inhibitor of PC-PLC, blocked TNF-␣-mediated induction of mPGES-1 (Fig. 7C), providing further evidence of the involvement in PC-PLC in TNF-␣mediated induction of mPGES-1. TNF-␣ also activated PKC, a downstream target of PC-PLC activation (Fig. 7D). The fact that calphostin C, an inhibitor of PKC activity, abrogated TNF-␣-mediated induction of mPGES-1 (Fig. 7E) indicates that PKC contributes to the induction of mPGES-1. Both TNF-␣ and PKC can stimulate mitogen-activated protein kinase (MAPK) activity. Neither PD98059 nor SB202190, compounds that inhibit MAPK kinase and p38, blocked TNF-␣-mediated induction of mPGES-1 (data not shown).
TNF-␣ can stimulate the production of NO and cGMP in a PC-PLC and PKC-dependent fashion. Treatment of cells with TNF-␣ led to ϳ2-fold increase in NO production (Fig. 8A). As shown in Fig. 8 (B and C), the increase in production of NO mediated by TNF-␣ was attenuated by D609 and calphostin C, respectively. These findings strongly suggest that TNF-␣-mediated increases in NO production occur downstream of activation of PC-PLC and PKC. We next investigated whether NO was involved in TNF-␣-mediated induction of mPGES-1. Treatment with SNAP or NONOate, two different NO donors, induced mPGES-1 implying a positive role for NO in TNF-␣mediated induction of mPGES-1 (Fig. 8, D and E). Notably, diphenyleneiodonium chloride, an inhibitor of NO production, caused dose-dependent suppression of TNF-␣-mediated induction of mPGES-1 (Fig. 8F). NO activates guanylate cyclase resulting in enhanced synthesis of cGMP. Accordingly, we investigated whether cGMP was involved in the induction of mPGES-1 by TNF-␣. Treatment with YC-1, a compound that stimulates cGMP production, induced mPGES-1 (Fig. 9A). cGMP production can be blocked by inhibiting guanylate cyclase activity. Methylene blue, an inhibitor of guanylate cyclase, suppressed the induction of mPGES-1 by TNF-␣ (Fig.  9B). LY83583, another inhibitor of guanylate cyclase, also blocked TNF-␣-mediated induction of mPGES-1 (Fig. 9C). cGMP activated PKG (data not shown). To determine whether PKG was important for regulating mPGES-1 expression, transient transfections were performed. Overexpressing wild-type PKG caused nearly a 2-fold increase in mPGES-1 promoter activity (data not shown). Importantly, TNF-␣-mediated activation of the mPGES-1 promoter was blocked by overexpressing a dominant negative form of PKG (Fig. 9D).

DISCUSSION
In this study, we found increased levels of mPGES-1, an inducible form of PGES, in inflamed mucosa from patients with IBD. Treatment with TNF-␣ a cytokine implicated in mucosal inflammation in IBD (1), induced mPGES-1 transcription in human colonocytes. Stimulation of mPGES-1 promoter activity by TNF-␣ was mediated by tandem GC boxes (Ϫ101 to Ϫ119). Another recent report indicated that these GC boxes play a significant role in regulating the transcription of both human and mouse mPGES-1 genes (36). Several findings support a role for Egr-1 in mediating the induction of mPGES-1 by TNF-␣. Increased binding of Egr-1 to the GC boxes of the mPGES-1 promoter was detected in TNF-␣-treated cells. The functional importance of Egr-1 was established because TNF-␣-mediated induction of mPGES-1 was suppressed with siRNA to Egr-1. Moreover, antisense to Egr-1 inhibited TNF-␣-mediated activation of the mPGES-1 promoter. Taken together, these results indicate that Egr-1 is a key transcription factor in regulating the inducible expression of mPGES-1.
Based on the above findings, we also evaluated whether either the amounts or binding activity of Egr-1 were deregulated in IBD. Notably, both an increase in amounts and binding activity of Egr-1 were detected in inflamed mucosa. Although Egr-1 is rapidly activated by tissue injury (44), to the best of our knowledge, neither of these findings has been reported previously in IBD. Recently, NO was reported to enhance Egr-1 expression (45). Increased levels of NO synthesizing enzymes occur in IBD (46). It is reasonable to postulate, therefore, that NO played a role in stimulating the expression of Egr-1, which contributed, in turn, to the elevated levels of mPGES-1 in inflamed mucosa.
FIG. 6. Egr-1 expression and binding activity are enhanced in inflammatory bowel disease. A, immunoblot analysis was performed using tissue lysates from inflamed (lanes 1-4, Crohn's disease; lanes 5-7, ulcerative colitis) and non-inflamed (lanes 8 -10) mucosa. Tissue lysate protein (100 g/lane) was loaded onto a 12.5% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose. Lane 11 represents a Egr-1 standard. The immunoblot was probed sequentially with antibodies specific for Egr-1 and ␤-actin. In B and C, 5 g of nuclear protein from the colonic mucosa of patients with inflammatory bowel disease was incubated with a 32 P-labeled oligonucleotide containing the GC box region of mPGES-1. In B, lanes 1 and 3 represent binding with nuclear protein from non-inflamed mucosa from Crohn's disease and ulcerative colitis patients, respectively. Lanes 2 and 4 represent binding observed with nuclear protein from inflamed mucosa from patients with Crohn's disease and ulcerative colitis, respectively. In C, lanes 1-4 represent binding of nuclear protein from inflamed Crohn's mucosa to a 32 P-labeled mPGES-1 GC box oligonucleotide. Lanes 2 and 4 represent nuclear protein incubated with normal IgG (lane 2) or antibodies to Egr-1 (lane 4). In lane 3 nuclear protein was incubated with a 32 P-labeled mPGES-1 GC box oligonucleotide and a 100-fold excess of unlabeled Egr-1 consensus oligonucleotide. The protein-DNA complex that formed was separated on a 4% polyacrylamide gel.  Egr-1 can also induce TNF-␣ (47). PGE 2 , the catalytic product of mPGES-1, can induce Egr-1 (48). Hence, a complex feedback loop appears to exist contributing to the observed increase in levels of Egr-1 and mPGES-1 in inflamed mucosa.
In contrast to COX-2 (49), very little is known about the signal transduction pathway(s)that regulate the expression of mPGES-1. We show that TNF-␣ induced mPGES-1 by a signaling pathway comprising PC-PLC 3 PKC 3 NO 3 cGMP and PKG (Fig. 10). TNF-␣ can stimulate the hydrolysis of phospholipids (50 -52). TNF-␣ induced the activity of PC-PLC but not phosphatidylinositol-phospholipase C (data not shown). Treating cells with purified PC-PLC induced mPGES-1 whereas an inhibitor of PC-PLC activity suppressed TNF-␣mediated induction of mPGES-1. These results indicate that TNF-␣ induced mPGES-1 by a signaling pathway that includes PC-PLC. Notably, PC-PLC has already been reported to play a role in TNF-␣-mediated induction of COX-2 (52). Diacylglycerol, a product of PC-PLC-mediated hydrolysis of phospholipids, activates PKC (50). We showed that TNF-␣ stimulated PKC activity, whereas a prototypic inhibitor of PKC blocked TNF-␣-mediated induction of mPGES-1. Presumably, diacylglycerol was responsible at least, in part, for activating PKC. Activation of PKC can stimulate p38 and ERK1/2 MAPK activity (53). Others have suggested that MAPKs can mediate the FIG. 9. Guanylate cyclase/PKG signal pathway is important in mPGES-1 induction by TNF-␣ in HCA7 cells. A, cells were treated with vehicle or the indicated concentration of YC-1 for 24 h. B, cells were treated with vehicle, TNF-␣ (10 ng/ml), or TNF-␣ plus the indicated concentration of methylene blue for 24 h. C, cells were pretreated with vehicle or 10 -100 M LY83583 for 1 h prior to being treated with vehicle (C) or TNF-␣ for 24 h. In A-C, cellular lysate protein (100 g/lane) was loaded onto a 12.5% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose. The immunoblots were sequentially probed with antibodies specific for mPGES-1 and ␤-actin. D, cells were transfected with 0.9 g of human mPGES-1 promoter construct ligated to luciferase (Ϫ651/Ϫ20) and 0.2 g of pSV␤gal. The bar labeled TNF-␣ ϩ PKG DN represents cells that also received 0.9 g of expression vector for PKG dominant negative. The total amount of DNA in each reaction was kept constant at 2 g by using a corresponding empty expression vector. Following transfection, cells were treated with vehicle or TNF-␣ (10 ng/ml) for 24 h at which time reporter activities were measured. Luciferase activity represents data that have been normalized with ␤-galactosidase. Columns, means; bars, S.D.; n ϭ 6.
induction of mPGES-1 (54). Although TNF-␣ activated MAPK activity in colonocytes, treatment with pharmacological inhibitors of MAPK kinase and p38 did not suppress the induction of mPGES-1. To further elucidate the mechanism by which TNF-␣ induced mPGES-1, it was necessary to investigate other signaling mechanisms that can be activated by PKC.
TNF-␣, PC-PLC, and PKC can induce the production of NO (55)(56)(57). Treatment with TNF-␣ stimulated the production of NO in colonocytes. Inhibitors of PC-PLC and PKC activity suppressed the increase in NO production mediated by treatment with TNF-␣. Additional experiments were carried out to establish a causal link between the observed increase in NO production mediated by TNF-␣ and the induction of mPGES-1. In support of this mechanism, treatment with NO donors induced mPGES-1, whereas an inhibitor of NO synthase blocked TNF-␣-mediated induction of mPGES-1. Nitric oxide is a potent transcriptional regulator influencing a variety of genes either by cGMP-dependent or -independent mechanisms (58,59). cGMP appears to be important for TNF-␣-mediated induction of mPGES-1. This conclusion was supported by evidence that an inducer of cGMP production caused an increase in amounts of mPGES-1 whereas two inhibitors of guanylate cyclase activity blocked TNF-␣-mediated induction of mPGES-1. PKG can mediate the inductive effects of cGMP (58). A dominant negative form of PKG blocked TNF-␣-mediated induction of mPGES-1 promoter activity. Taken together, these results suggest that a signal transduction pathway comprising PC-PLC 3 PKC 3 NO 3 cGMP and PKG is responsible for the induction of mPGES-1 by TNF-␣. TNF-␣ regulates mucin production by a similar mechanism (60). However, this pathway has not been implicated in previous studies that have attempted to elucidate the regulation of mPGES-1 expression. It is important to acknowledge that TNF-␣ can stimulate a variety of other signaling mechanisms. Pharmacological inhibitors, e.g. D609, of components of the proposed signaling pathway did not completely block the induction of mPGES-1. This suggests that additional pathways may also contribute to TNF-␣-mediated induction of mPGES-1. Based on these findings, future studies will be needed to determine whether this signal transduction pathway can explain the increase in levels of mPGES-1 detected in other inflammatory (22,23,30) and neoplastic (24 -27) conditions.
The results of this study provide other important insights. We showed that overexpressing antisense to mPGES-1 blocked TNF-␣-mediated stimulation of PGE 2 production. This implies that mPGES-1, in addition to COX-2, is likely to contribute to the elevated levels of PGE 2 detected in inflamed mucosa in IBD (4 -6). In the short term, enhanced production of PGE 2 in inflamed mucosa appears to be an adaptive mechanism that promotes wound healing. For example, PGE 2 can induce mitogenesis by activating epidermal growth factor receptor signaling (61). Increased production of PGE 2 has also been linked to enhanced production of vascular endothelial growth factor and angiogenesis (62), another important component of wound healing. NSAIDs inhibit PGE 2 production and Egr-1 expression (63), effects that are predicted to inhibit wound healing and exacerbate IBD. Thus, our finding that Egr-1 plays a key role in regulating the expression of mPGES-1 and thereby the production of PGE 2 provides new insights into the mechanism by which NSAIDs trigger or worsen IBD .
Patients with longstanding IBD are at increased risk for developing colon cancer (64). Neoplasia develops in the context of increased epithelial cell turnover and regeneration induced by the chronic inflammatory state (65). Elevated levels of COX-2 are commonly observed in colorectal cancers and IBD (12,20). This study suggests that mPGES-1 is frequently overexpressed in IBD in addition to colorectal cancer (25). Recently, cells overexpressing mPGES-1 and COX-2 were observed to produce more PGE 2, grow faster, and exhibit abnormal morphology compared with cells in which either mPGES-1 or COX-2 were overexpressed (66). Cotransfection of COX-2 and mPGES-1 into HEK293 cells resulted in cellular transformation manifested by colony formation in soft agar culture and tumor formation when injected subcutaneously into nude mice (66). cDNA array analyses revealed that mPGES-1-directed cellular transformation was accompanied by changes in the expression of a variety of genes related to morphology, proliferation, cell cycle, and adhesion (66). In the short term, it seems likely that increased production of PGE 2 will contribute to the healing of inflamed mucosa. Chronic elevation of COX-2, mPGES-1, and PGE 2 synthesis in intestinal mucosa should stimulate mitogenesis and increase the risk of developing colorectal cancer. In support of these ideas, treatment with an EP4 receptor antagonist exacerbated experimental colitis (10), but protected against the development of colon cancer (67). Based on the findings in this study, it will be important to determine whether targeting mPGES-1 impacts on either intestinal inflammation or carcinogenesis.