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Am J Physiol Gastrointest Liver Physiol 290: G361-G368, 2006. First published September 29, 2005; doi:10.1152/ajpgi.00348.2005
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INFLAMMATION/IMMUNITY/MEDIATORS

Levels of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase are reduced in inflammatory bowel disease: evidence for involvement of TNF-{alpha}

Taisuke Otani,1,2 Kentaro Yamaguchi,1,2 Ellen Scherl,1 Baoheng Du,1 Hsin-Hsiung Tai,3 Melanie Greifer,4 Lydia Petrovic,5 Takiko Daikoku,6 Sudhansu K. Dey,6 Kotha Subbaramaiah,1 and Andrew J. Dannenberg1

Departments of 1Medicine, 4Pediatrics, and 5Pathology, Weill Medical College of Cornell University, New York, New York; 2Department of Surgery, Tokyo Women's Medical University and Daini Hospital, Tokyo, Japan; 3Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky; and 6Departments of Pediatrics, Cell and Developmental Biology, and Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee

Submitted 26 July 2005 ; accepted in final form 26 September 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Increased amounts of PGE2 have been detected in the inflamed mucosa of patients with inflammatory bowel disease (IBD). This increase has been attributed to enhanced synthesis rather than reduced catabolism of PGE2. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) plays a major role in the catabolism of PGE2. In this study, we investigated whether amounts of 15-PGDH were altered in inflamed mucosa from patients with IBD. Amounts of 15-PGDH protein and mRNA were markedly reduced in inflamed mucosa from patients with Crohn's disease and ulcerative colitis. In situ hybridization demonstrated that 15-PGDH was expressed in normal colonic epithelium but was virtually absent in inflamed colonic mucosa from IBD patients. Because of the importance of TNF-{alpha} in IBD, we also determined the effects of TNF-{alpha} on the expression of 15-PGDH in vitro. Treatment with TNF-{alpha} suppressed the transcription of 15-PGDH in human colonocytes, resulting in reduced amounts of 15-PGDH mRNA and protein and enzyme activity. In contrast, TNF-{alpha} induced two enzymes (cyclooxygenase-2 and microsomal prostaglandin E synthase-1) that contribute to increased synthesis of PGE2. Overexpressing 15-PGDH blocked the increase in PGE2 production mediated by TNF-{alpha}. Taken together, these results suggest that reduced expression of 15-PGDH contributes to the elevated levels of PGE2 found in inflamed mucosa of IBD patients. The decrease in amounts of 15-PGDH in inflamed mucosa can be explained at least, in part, by TNF-{alpha}-mediated suppression of 15-PGDH transcription.

prostaglandin E2; inflammatory bowel disease; tumor necrosis factor-{alpha}; cyclooxygenase

INFLAMMATORY BOWEL DISEASE (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), is characterized by chronic, relapsing inflammation of the gastrointestinal tract. This disease affects at least 1 in 1,000 people in Western countries. Multiple lines of evidence suggest a significant role for PGE2 in IBD (14, 18). For example, increased levels of PGE2 are found at sites of intestinal inflammation and correlate with disease activity (6, 32, 43). Nonsteroidal anti-inflammatory drugs (NSAIDs), prototypic inhibitors of cyclooxygenase (COX) activity and thereby PGE2 synthesis, can exacerbate or trigger IBD (4, 17). An important role for PGE2 has also been suggested by experimental studies. Both COX-1–/– and COX-2–/– mice showed increased susceptibility to dextran sodium sulfate-induced colonic epithelial injury (24). In contrast, administration of 16,16-dimethyl PGE2 protected against trinitrobenzene sulfonic acid-induced colitis (1). Recently, mice engineered to be deficient in EP4, a receptor for PGE2, were found to be sensitized to developing experimental colitis; treatment with an EP4 receptor agonist ameliorated colitis in wild-type mice (16). Taken together, these findings strongly suggest that PGE2 is important for maintaining mucosal homeostasis. It is important, therefore, to define the mechanisms that account for increased amounts of mucosal PGE2 in IBD.

The synthesis of PGE2 from arachidonic acid requires two sequential enzymatic steps. COX catalyzes the synthesis of PGH2 from arachidonic acid. COX-1 is a housekeeping gene that is expressed constitutively in most tissues including the intestine (35). COX-2 is an immediate-early response gene that is induced by a variety of inflammatory and mitogenic stimuli (35). Increased amounts of COX-2 have been observed in IBD and undoubtedly contribute to elevated production of PGH2 (34). Several enzymes, including microsomal prostaglandin E synthase-1 (mPGES-1), convert COX-derived PGH2 to PGE2 (15). Similar to COX-2, levels of mPGES-1 are markedly elevated in inflamed mucosa in IBD (37). Thus far, the elevated levels of PGE2 found at sites of mucosal inflammation have been attributed to increased synthesis caused by overexpression of COX-2 and mPGES-1.

In theory, reduced catabolism of PGE2 might also contribute to increased mucosal levels of PGE2 in IBD. The key enzyme responsible for metabolic inactivation of prostaglandins, including PGE2, is NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) (38). The human gene is located on chromosome 4 and codes for a 29-kDa enzyme that catalyzes the formation of 15-ketoprostaglandins (30, 38). 15-Ketoprostaglandins possess greatly reduced biological activities. Genetic deletion of 15-PGDH leads to an increase in tissue levels of PGE2 (7). Although this enzyme is ubiquitously expressed in mammalian tissues, little, if anything, is known about its levels in inflamed human tissues. In this study, we investigated whether amounts of 15-PGDH were reduced in inflamed intestinal mucosa from patients with IBD. TNF-{alpha} plays a central role in inflammation and is believed to be at the apex of an inflammatory cascade in IBD (29, 42). Hence, we also evaluated the effects of TNF-{alpha} on the expression of the 15-PGDH gene in human colonocytes. We show that levels of 15-PGDH mRNA and protein are markedly reduced in inflamed mucosa in patients with CD and UC. Treatment with TNF-{alpha} suppressed the transcription of 15-PGDH resulting in reduced amounts of 15-PGDH mRNA and protein and enzyme activity in human colonocytes. The results of the present study suggest that the observed increase in mucosal levels of PGE2 in IBD is likely to be a consequence of reduced catabolism in addition to increased synthesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. McCoy's 5A medium, DMEM-F-12 medium, fetal bovine serum (FBS), and LipofectAMINE 2000 were purchased from Invitrogen (Carlsbad, CA). Rabbit anti-human polyclonal antibodies to mPGES-1, mPGES-2, and 15-PGDH, monoclonal anti-human cPGES/p23 antibody, and enzyme immunoassay reagents for PGE2 assays were from Cayman Chemical (Ann Arbor, MI). Human TNF-{alpha}, goat anti-human COX-2 antibody, antibody to {beta}-actin, Lowry protein assay kits, and secondary antibody conjugated to horseradish peroxidase, L-glutamic dehydrogenase, {alpha}-ketoglutamic acid, and nicotinamide adenine dinucleotide (NAD+) were from Sigma Chemical (St. Louis, MO). Western blot analysis detection reagents (ECL) were from Amersham Biosciences (Piscataway, NJ). Nitrocellulose membranes were from Schleicher and Schuell (Keene, NH). Plasmid DNA isolation kits were from Promega (Madison, WI). Taq polymerase was from Applied Biosystems (Foster City, CA). Reagents for the luciferase assay were from Analytical Luminescence (San Diego, CA). Charcoal-activated powder was from EM Science (Gibbstown, NJ). The RNeasy Mini kit was from Qiagen (Valencia, CA). 18 S rRNA cDNA was purchased from Ambion (Austin, TX).

Human tissue. Specimens were obtained at the time of colonoscopic examination from patients with UC or CD. Biopsies were taken from mucosa that was macroscopically inflamed or normal appearing. As a control, mucosal biopsies were also taken from patients without evidence of colonic disease. Tissue samples were immediately snap frozen in liquid nitrogen and stored at –80°C until analysis. The study was approved by the Committee on Human Rights in Research at Weill Medical College of Cornell University.

Cell culture. The human colon cancer cell line HT29 was maintained in McCoy's 5A medium supplemented with 10% FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin. The FHC cell line was grown in a DMEM-F-12 medium supplemented with 10% FBS, 100 IU/ml penicillin, 100 µg/ml streptomycin, 0.005 mg/ml insulin, 0.005 mg/ml transferrin, and 100 ng/ml hydrocortisone (33). All treatments were carried out in serum-free medium. Both cell lines were purchased from American Type Culture Collection (Manassas, VA).

PGE2 production. Cells (1 x 104 cells/well) were plated in six-well dishes and grown to 60% confluence in McCoy's 5A medium containing 10% FBS. The medium was replaced with serum-free McCoy's 5A medium for 16 h. Cells were then treated with vehicle or TNF-{alpha} (5 ng/ml) for 12–48 h. At the end of the treatment period, culture medium was collected for analysis of PGE2. Amounts of PGE2 were measured by enzyme immunoassay. Amounts of PGE2 were normalized to protein concentrations.

Western blot analysis. Tissue or cell lysates were prepared by treatments with lysis buffer as described previously (37). Lysates were sonicated for 20 s on ice and centrifuged at 10,000 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. (19). SDS-PAGE was performed under reducing conditions on 12.5% polyacrylamide gels. The resolved proteins were transferred onto nitrocellulose sheets and then probed for COX-2, mPGES-1, mPGES-2, cPGES, 15-PGDH, and {beta}-actin using previously described methods (8, 36, 37).

PCR analysis. To determine tissue or cellular levels of mRNA for 15-PGDH, total RNA was isolated from frozen specimens using the RNeasy Mini Kit according to the manufacturer's instructions. Reverse transcription was performed using 2 µg of RNA per 50 µl of reaction. The reaction mixture contained 1 x PCR buffer II, 2.5 mM MgCl2, 0.5 mM dNTPs, 2.5 µM oligo(dT)16 primer, 50 units RNase inhibitor, and 125 units MuLV. Samples were amplified in a thermocycler at 25°C for 10 min, 42°C for 15 min, 99°C for 5 min, and 5°C for 5 min. The resulting cDNA was used for amplification. The volume of the PCR was 25 µl and contained 2 µl cDNA, 1 x PCR buffer II, 2 mM MgCl2, 0.4 mM dNTPs, 400 nM forward primer, 400 nM reverse primer, and 2.5 units Taq polymerase (Applied Biosystems). Samples were denatured at 95°C for 2 min and then amplified for 30 cycles in a thermocycler under the following conditions: 95°C for 30 s, 62°C for 30 s, and 70°C for 45 s. Subsequently, the extension was carried out at 70°C for 10 min. Primer sequences were as follows: 15-PGDH, forward: 5'-GTAAAGCTGCCCTGGATGAG-3' and reverse: 5'-AACAAAGCCTGGACAAATGG-3'; and {beta}-actin, forward 5'-GGTCACCCACACTGTGCCCAT-3' and reverse: 5'-GGATGCCACAGGACTCCATGC-3'. Samples were subjected to electrophoresis on a 1% agarose gel with 0.5 µg/ml ethidium bromide. The identity of each PCR product was confirmed by DNA sequencing.

In situ hybridization. In situ hybridization was performed as previously described by us (12). In brief, frozen sections (12 µm) were mounted onto poly-L-lysine-coated slides and fixed in cold 4% paraformaldehyde in PBS. The sections were prehybridized and hybridized at 45°C for 4 h in 50% formamide hybridization buffer containing the 35S-labeled antisense or sense cRNA probes. Ribonuclease A-resistant hybrids were detected by autoradiography. Sections hybridized with the sense probes did not exhibit any positive signals and served as negative controls. Sense and antisense 35S-labeled cRNA probes were generated using Sp6 and T7 polymerases, respectively. Probes had specific activities of ~2 x 109 disintegrations·min–1·µg–1.

Transient transfection assays. Cells were seeded at a density of 5 x 104 cells/well in six-well dishes and grown to 50% confluence. The 15-PGDH-luciferase promoter construct contains 1.6 kb of the 5'-flanking region (–1640 to –1 relative to the ATG start codon) ligated to luciferase (21). For each well, 2 µg of plasmid DNA were introduced into cells using 8 µg of LipofectAMINE 2000 as per the manufacturer's instructions. Reporter activities were measured as described previously (22).

15-PGDH activity assay. 15-PGDH enzyme activity in cellular lysates was assayed by measuring the transfer of tritium from 15(S)-[15-3H]PGE2 to glutamate by coupling 15-PGDH with glutamate dehydrogenase as described previously (40).

Statistics. Comparisons between groups were made by Student's t-test. A difference between groups of P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Levels of 15-PGDH are reduced in inflamed mucosa of IBD patients. We compared amounts of 15-PGDH protein in colonic mucosa from IBD patients versus normal subjects. Reduced levels of 15-PGDH were consistently observed in inflamed mucosa (Fig. 1). RT-PCR was carried out to determine whether mucosal inflammation was also associated with reduced amounts of 15-PGDH mRNA. Consistent with the immunoblot findings, levels of 15-PGDH mRNA were markedly reduced in inflamed mucosa from patients with either CD or UC (Fig. 2).



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  		Fig. 1. Levels of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) protein are reduced in inflamed mucosa in inflammatory bowel disease (IBD). A: samples of normal appearing colonic mucosa were obtained from 6 patients without evidence of colonic disease. B and C: paired samples were obtained from inflamed or noninflamed colonic mucosa from patients with Crohn's disease (n = 8) or ulcerative colitis (n = 9). Equal concentrations of protein (100 µg/lane) were loaded onto a 12.5% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose membranes. The immunoblot was sequentially probed with antibodies specific for 15-PGDH and {beta}-actin. STD, standard.

 


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  		Fig. 2. Levels of 15-PGDH mRNA are reduced in inflamed colonic mucosa in IBD. A and B: normal (NL) mucosa was obtained from subjects without evidence of colonic disease, and paired noninflamed (N) and inflamed (I) mucosa was obtained from patients with Crohn's disease or ulcerative colitis. RT-PCR was carried out to assess the amounts of 15-PGDH mRNA. C: results of densitometry (expressed in arbitrary units). Values are means ± SD. *P < 0.05 and **P < 0.01 compared with normal mucosa.

 
To determine the location of 15-PGDH in normal human colonic mucosa, we performed in situ hybridization for 15-PGDH. In multiple normal colon samples, 15-PGDH mRNA was expressed by epithelial cells located at the upper reaches and luminal surface of colonic crypts (Fig. 3A). Consistent with the results obtained by Western blot analysis (Fig. 1) and RT-PCR (Fig. 2), the signal for 15-PGDH was virtually absent in samples of inflamed colonic mucosa from CD (Fig. 3B) and UC (Fig. 3C) patients.



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  		Fig. 3. In situ hybridization of 15-PGDH. Representative dark-field photomicrographs of human colonic sections showing 15-PGDH mRNA expression (bar = 400 µm) are shown. A: normal colonic mucosa. The 15-PGDH mRNA signal is strongly expressed in the surface epithelium, with most of the cells being absorptive, and without any significant inflammatory component. B: section from a patient with Crohn's disease. In contrast to the normal colonic mucosa, the 15-PGDH mRNA signal is significantly downregulated (virtually absent) from the surface mucosal epithelium. It is also absent from the crypt epithelium. C: section from a patient with ulcerative colitis. Similar to the findings in B, the 15-PDGH mRNA signal is virtually absent from the surface mucosal and crypt epithelium.

 
Treatment with TNF-{alpha} induced amounts of COX-2 and mPGES-1 while suppressing the expression of 15-PGDH. TNF-{alpha} is widely recognized to contribute to inflammation in IBD. Moreover, increased amounts of PGE2 are detected at sites of intestinal inflammation. Hence, experiments were carried out to determine the effect of TNF-{alpha} on the production of PGE2 in human colonocytes. As shown in Fig. 4, treatment with TNF-{alpha} led to a time-dependent increase in PGE2 production. Approximately, a eightfold increase in production of PGE2 was detected 48 h after treatment with TNF-{alpha}. Enhanced production of PGE2 could be a consequence of increased synthesis, reduced catabolism, or both. Immunoblot analysis was performed to determine the effects of TNF-{alpha} on amounts of enzymes that are important for the synthesis of PGE2. As shown in Fig. 5A, TNF-{alpha} was a potent inducer of COX-2 and mPGES-1. In contrast, TNF-{alpha} did not induce either cPGES or mPGES-2 (Fig. 5B).



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  		Fig. 4. Treatment with TNF-{alpha} induced PGE2 synthesis. HT29 cells were treated with vehicle (control) or TNF-{alpha} (5 ng/ml) for 12–48 h. Culture medium was assayed for spontaneous production of PGE2. Values are means ± SD; n = 6. *P < 0.001.

 


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  		Fig. 5. TNF-{alpha} induces cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase (mPGES)-1. HT29 cells were treated with vehicle (C) or TNF-{alpha} (5 ng/ml) for 12–48 h. Cellular lysate protein (100 µg/lane) was loaded onto a 12.5% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose membranes. Immunoblots were sequentially probed with antibodies specific for COX-2, mPGES-1, mPGES-2, cPGES, and {beta}-actin.

 
Because of its importance in the degradation of PGE2, we also evaluated the effects of TNF-{alpha} on amounts of 15-PGDH. Consistent with the findings in inflamed mucosa, treatment with TNF-{alpha} caused a marked decrease in amounts of 15-PGDH protein (Fig. 6A). A corresponding time-dependent decrease in cellular 15-PGDH activity was observed (Fig. 6B). To determine whether TNF-{alpha} suppressed amounts of 15-PGDH by a pretranslational mechanism, levels of 15-PGDH mRNA were assessed (Fig. 6C). Treatment with TNF-{alpha} caused a marked reduction in amounts of 15-PGDH mRNA consistent with the observed decrease in protein levels. Transient transfections were carried out to determine whether TNF-{alpha} suppressed the expression of 15-PGDH by altering transcription. TNF-{alpha} suppressed 15-PGDH promoter activity (Fig. 7A). This inhibitory effect was observed after treatment with TNF-{alpha} for 12–24 h. Importantly, the suppressive effect of TNF-{alpha} was not unique to HT-29 cells. TNF-{alpha} also inhibited 15-PGDH activity in FHC cells, a human fetal normal colon epithelial cell line (Fig. 7B). Gene transfection was used to confirm the significance of 15-PGDH as a determinant of PGE2 production. Notably, overexpressing 15-PGDH blocked TNF-{alpha}-mediated induction of PGE2 production (Fig. 7C).



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  		Fig. 6. TNF-{alpha} suppresses amounts of 15-PGDH protein, enzyme activity, and mRNA in HT29 cells. Cells were treated with vehicle (C) or TNF-{alpha} (5 ng/ml) for 12–48 h. A: cellular lysate protein (100 µg/lane) was loaded onto a 12.5% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose membranes. The immunoblot was sequentially probed with antibodies specific for 15-PGDH and {beta}-actin. B: cellular levels of 15-PGDH enzyme activity were determined by enzyme assay as described in MATERIALS AND METHODS. Values are means ± SD; n = 6. *P < 0.01; **P < 0.001. C: total cellular RNA was isolated. RT-PCR was carried out to assess amounts of 15-PGDH and {beta}-actin, respectively.

 


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  		Fig. 7. Treatment with TNF-{alpha} suppresses the expression of 15-PGDH, an enzyme that is important for the catabolism of PGE2. A: TNF-{alpha} reduces 15-PGDH promoter activity. HT29 cells were transfected with 1.8 µg 15-PGDH promoter (–1640 to –1) ligated to luciferase and 0.2 µg pSV{beta}gal. After transfection, cells were treated with vehicle (control) or TNF-{alpha} (5 ng/ml) for 12–24 h, at which time reporter activities were measured. Luciferase activity represents data that have been normalized with {beta}-galactosidase. Values are means ± SD; n = 6. *P < 0.001. B: FHC cells were treated with 0–1.0 ng/ml TNF-{alpha} for 24 h. Cellular levels of 15-PGDH enzyme activity were determined by enzyme assay as described in MATERIALS AND METHODS. Values are means ± SD; n = 6. *P < 0.01. C: HT29 cells were transfected with empty vector or a 15-PGDH expression vector. After transfection, cells were treated with vehicle or TNF-{alpha} (5 ng/ml) for 24 h. Culture medium was assayed for spontaneous production of PGE2. Values are means ± SD; n = 6. *P < 0.001.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we found markedly reduced levels of 15-PGDH in inflamed colonic mucosa from IBD patients. This is significant because 15-PGDH is the key enzyme responsible for metabolic inactivation of PGE2 (38). Previous studies have demonstrated increased amounts of COX-2 and mPGES-1 in inflamed mucosa in IBD (34, 37). COX-2 and mPGES-1 play pivotal roles in the synthesis of PGE2. Hence, it is likely that the elevated level of PGE2 in inflamed mucosa is a consequence of both reduced catabolism and increased synthesis (Fig. 8). On the basis of the present findings, it will be worthwhile to determine whether amounts of 15-PGDH are reduced in other inflammatory conditions including esophagitis and arthritis, in which elevated levels of PGE2 have been demonstrated (27).



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  		Fig. 8. Schematic illustration of the mechanism underlying increased amounts of PGE2 in IBD. Elevated levels of COX-2 and mPGES-1 result in enhanced synthesis of PGE2. Reduced amounts of 15-PGDH cause a reduction in the rate of PGE2 catabolism.

 
TNF-{alpha} plays a central role in mucosal inflammation in IBD (29, 42). It was important, therefore, to determine whether treatment with TNF-{alpha} altered amounts of COX-2, mPGES-1, and 15-PGDH in colonocytes in a manner that reproduced the aberrant expression profile observed in IBD. Remarkably, TNF-{alpha} was a potent inducer of COX-2 and mPGES-1 while causing a significant reduction in amounts of 15-PGDH. Importantly, the changes in amounts of enzymes mediated by TNF-{alpha} were associated with increased production of PGE2. TNF-{alpha}-mediated induction of PGE2 production was suppressed by overexpressing 15-PGDH, confirming the functional significance of this enzyme. These in vitro findings strongly suggest that deregulated TNF-{alpha} signaling contributes to the changes in enzyme and PGE2 levels that occur in inflamed mucosa. In the future, it will be of considerable interest to determine whether these in vitro findings translate in patients with IBD. The results of this study predict that treatment with monoclonal anti-TNF-{alpha} antibodies will cause a rapid normalization of deregulated enzyme expression in inflamed mucosa. However, it is important to consider the possibility that other proinflammatory cytokines will also suppress amounts of 15-PGDH in inflamed mucosa. IL-1{beta} has been reported to reduce levels of 15-PGDH in trophoblast cells in primary culture (23). If the same mechanism is operative in IBD, the observed reduction in amounts of 15-PGDH in inflamed mucosa could reflect the effects of multiple proinflammatory cytokines.

Previously, TNF-{alpha} has been found to induce both COX-2 and mPGES-1 (20, 37). To our knowledge, the present study represents the first evidence that TNF-{alpha} can suppress the expression of 15-PGDH in colonocytes. Consequently, additional experiments were carried out to further define the mechanism by which TNF-{alpha} suppressed levels of 15-PGDH. Our studies suggest that TNF-{alpha} suppressed the transcription of 15-PGDH, resulting in reduced levels of 15-PGDH mRNA and protein and enzyme activity. Recently, AP-1, Ets, and CREB proteins have been implicated in the transcriptional regulation of 15-PGDH (26). Future studies will be needed to determine whether any one of these trans-activating factors is involved in TNF-{alpha}-mediated suppression of 15-PGDH transcription. TNF-{alpha} has been reported to induce mPGES-1 in colonocytes by stimulating a signal transduction pathway comprised of PC-PLC->PKC->nitric oxide->GMP->PKG. (37). Additional studies are warranted to determine whether TNF-{alpha} suppressed the transcription of 15-PGDH by this pathway or a different signaling mechanism.

As mentioned above, elevated levels of PGE2 in inflamed mucosa appear to be a consequence of both increased synthesis and decreased catabolism (Fig. 8). The fact that changes in both synthetic and catabolic pathways occur in inflamed mucosa strongly suggests that elevated tissue levels of PGE2 must be important in some way. This point is underscored by the longstanding clinical observation that NSAIDs can trigger or exacerbate IBD (4, 17). In all likelihood, elevated levels of PGE2 in inflamed mucosa promote wound healing. In support of this idea, PGE2 has been observed to promote crypt stem cell survival and proliferation (9, 39). Although the precise mechanism by which PGE2 stimulates cell proliferation is uncertain, PGE2 can activate epidermal growth factor receptor (EGFR) signaling (28). PGE2 can activate EGFR either by increasing the production of ligands of EGFR or via an intracellular Src-mediated event independent of the release of an extracellular ligand of EGFR (5, 28, 31). Whether this mechanism is operative in IBD remains uncertain. Additionally, there is extensive evidence linking increased production of PGE2 to enhanced production of vascular endothelial growth factor and angiogenesis (3, 41), another important component of wound healing. Thus, in the short term, it seems likely that reduced degradation of PGE2 will contribute to the healing of inflamed mucosa.

Patients with longstanding IBD are at increased risk for developing colon cancer (13). Neoplasia develops in association with increased epithelial cell turnover and regeneration induced by the chronic inflammatory state (11). In fact, colitis markedly accelerated the formation of dysplasia and intestinal cancer in experimental animals (10). Chronic elevation of PGE2 in inflamed mucosa should stimulate mitogenesis and inhibit apoptosis, thereby reducing the threshold for the development of colorectal cancer. Consistent with this idea, reduced levels of 15-PGDH were recently found in colorectal cancer, supporting the possibility that this is a gene with tumor suppressor activity (2, 44). Taken together, it appears likely that short-term elevation of mucosal PGE2 in IBD is an adaptive mechanism that enhances wound healing, whereas chronic elevation of PGE2 promotes the formation of colorectal cancer. In support of these ideas, treatment with an EP4 receptor antagonist exacerbated experimental colitis (16) but protected against the development of colon cancer (25). On the basis of the findings in this study, future studies are needed to determine whether targeting 15-PGDH impacts on either intestinal inflammation or carcinogenesis.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by the New York Crohn's Foundation, the Mary Kay Ash Foundation, the Center for Cancer Prevention Research, and National Cancer Institute Grants P01 CA-77839 and R01 CA-111469.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. J. Dannenberg, Div. of Gastroenterology and Hepatology, New York Presbyterian-Cornell, 525 E. 68th St., Rm. F-206, New York, NY 10021 (e-mail: ajdannen{at}med.cornell.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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