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Am J Physiol Gastrointest Liver Physiol 297: G559-G566, 2009. First published July 16, 2009; doi:10.1152/ajpgi.00133.2009 Free Article
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INFLAMMATION/IMMUNITY/MEDIATORS

Bile acids inhibit NAD+-dependent 15-hydroxyprostaglandin dehydrogenase transcription in colonocytes

Akira Miyaki,1 Peiying Yang,2 Hsin-Hsiung Tai,3 Kotha Subbaramaiah,1 and Andrew J. Dannenberg1

1Department of Medicine, Weill Cornell Medical College, New York, New York; 2Department of General Oncology, The University of Texas, M. D. Anderson Cancer Center, Houston, Texas; and 3Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky

Submitted 6 April 2009 ; accepted in final form 12 July 2009


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Multiple lines of evidence have suggested a role for both bile acids and prostaglandins (PG) in gastrointestinal carcinogenesis. Levels of PGE2 are determined by both synthesis and catabolism. Previously, bile acid-mediated induction of cyclooxygenase-2 (COX-2) was found to stimulate PGE2 synthesis. NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), the key enzyme responsible for the catabolism of PGE2, has been linked to colorectal carcinogenesis. In this study, we determined whether bile acids altered the expression of 15-PGDH in human colon cancer cell lines. Treatment with unconjugated bile acids (chenodeoxycholate and deoxycholate) suppressed the transcription of 15-PGDH, resulting in reduced amounts of 15-PGDH mRNA, protein, and enzyme activity. Conjugated bile acids were less potent suppressors of 15-PGDH expression than unconjugated bile acids. Treatment with chenodeoxycholate activated protein kinase C (PKC), leading in turn to increased extracellular signal-regulated kinase (ERK) 1/2 activity. Small molecules that inhibited bile acid-mediated activation of PKC and ERK1/2 also blocked the downregulation of 15-PGDH. Bile acids induced early growth response factor-1 (Egr-1) and Snail, a repressive transcription factor that bound to the 15-PGDH promoter. Silencing Egr-1 or Snail blocked chenodeoxycholate-mediated downregulation of 15-PGDH. Together, these data indicate that bile acids activate the signal transduction pathway PKC -> ERK1/2 -> Egr-1 -> Snail and thereby suppress 15-PGDH transcription. Bile acids appear to increase the release of PGs from cells by downregulating catabolism in addition to stimulating synthesis. These results provide new mechanistic insights into the link between bile acids and gastrointestinal carcinogenesis.

prostaglandin E2; early growth response factor-1; Snail; mitogen-activated protein kinase

A SUBSTANTIAL BODY OF EVIDENCE suggests that bile acids play a role in colorectal carcinogenesis. Epidemiological studies indicate that the risk of developing colon cancer is increased by consumption of a high-fat, low-fiber diet (37, 39, 45) and, following cholecystectomy (17, 41), conditions that are associated with bile acid excess. Secondary bile acids are also increased in patients with colorectal adenomas (4). Animal model studies have provided compelling evidence for a causal link between certain bile acids such as deoxycholate (DC) and colon carcinogenesis (31, 38, 43, 49). A variety of mechanisms including activation of protein kinase C and stimulation of prostaglandin E2 (PGE2) production are believed to contribute to the procarcinogenic effects of bile acids (8, 16, 29, 64).

Multiple findings suggest a significant role for PGs in colorectal carcinogenesis. Increased levels of PGE2 are found in colorectal adenomas and cancers (35, 40). Use of nonsteroidal anti-inflammatory drugs, prototypic inhibitors of cyclooxygenase (COX)-mediated synthesis of PGs, is associated with about a 50% reduction in risk of developing colon cancer (28, 51, 52). Selective COX-2 inhibitors are effective inhibitors of sporadic colorectal adenoma formation (5). An important role for PGE2 also has been suggested by experimental studies. Mice engineered to be deficient in EP2, a receptor for PGE2, were protected against the development of intestinal tumors (42, 47). In contrast, treatment of ApcMin/+ mice with exogenous PGE2 increased the size and number of intestinal adenomas, especially in the colon (57). Together, these findings strongly suggest that PGE2 plays a significant role in the development of colorectal cancer. It is important, therefore, to define the mechanisms that account for increased amounts of PGE2 in colorectal carcinogenesis. The mechanism(s) by which procarcinogenic bile acids induce PGE2 production are incompletely understood.

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 colon (46). COX-2 is an immediate-early response gene that is induced by a variety of inflammatory and mitogenic stimuli, including bile acids (14, 25, 63, 64). Microsomal prostaglandin E synthase-1 (mPGES-1) converts COX-derived PGH2 to PGE2 (26). Both COX-2 and mPGES-1 are commonly overexpressed in colorectal adenomas and cancers (15, 33, 62). Hence, increased synthesis of PGE2 can explain, at least in part, the elevated levels of PGE2 found in colorectal tumors. Tissue levels of PGE2 depend on catabolism in addition to synthesis (24). The key enzyme responsible for the metabolic inactivation of PGs, including PGE2, is nicotinamide adenine dinucleotide (NAD+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) (24, 50). This 29-kDa protein catalyzes the rate-limiting step of PG catabolism to yield 15-keto metabolites, which possess greatly reduced biological activities (50). Markedly reduced amounts of 15-PGDH occur in multiple tumor types, including colorectal cancers, and contribute to elevated levels of intratumoral PGE2 (2, 24, 30, 58, 59). Importantly, the formation of colon tumors was markedly increased in 15-PGDH knockout mice, highlighting the significance of PG catabolism in tumorigenesis (32).

In the present study, we showed that bile acids suppressed 15-PGDH transcription in colonocytes, which led, in turn, to increased production of PGE2. The reduction in 15-PGDH gene expression reflected activation of a signal transduction pathway comprising protein kinase C (PKC) -> extracellular signal-regulated kinase (ERK1/2) -> early growth response factor-1 (Egr-1) -> Snail. Given the importance of both 15-PGDH and PGE2 in colorectal carcinogenesis, our findings provide important new insights into the link between bile acids and colorectal carcinogenesis.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. McCoy's 5A medium and fetal bovine serum (FBS) were purchased from Invitrogen (Grand Island, NY). RPMI 1640, Leibovitz's L-15 medium, and FBS were purchased from American Type Culture Collection (Manassas, VA). Anti-human polyclonal antiserum to 15-PGDH was purchased from Novus Biologicals (Littleton, CO). Anti-human polyclonal antiserum to Egr-1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-human polyclonal antiserum to Snail was purchased from Abcam (Cambridge, MA). Antisera to ERK1/2 and phospho-ERK1/2 were purchased from Cell Signaling Technology (Beverly, MA). Antibody to β-actin, secondary antibody conjugated to horseradish peroxidase, bile acids, sodium arachidonate, calphostin C, PD-98059, L-glutamic dehydrogenase, {alpha}-ketoglutamic acid, and NAD+ were obtained from Sigma Chemical (St. Louis, MO). Western blot analysis detection reagents (ECL) and [32P]ATP were obtained from PerkinElmer Life Sciences (Boston, MA). Nitrocellulose membranes were obtained from Schleicher and Schuell (Keene, NH). Charcoal-activated powder was obtained from EM Science (Gibbstown, NJ). Bicinchoninic acid (BCA) protein assay reagent was obtained from Pierce (Rockford, IL). The RNeasy mini kit was obtained from Qiagen (Valencia, CA). MuLV reverse transcriptase, RNase inhibitor, oligo (dT)16, and SYBR green PCR master mix were obtained from Applied Biosystems (Foster City, CA). DharmaFECT and all high-performance liquid chromatography-grade solvents were purchased from Thermo Fisher Scientific (Waltham, MA). The PKC assay kit was purchased from Millipore (Lake Placid, NY). The chromatin immunoprecipitation (ChIP) assay kit was purchased from SA Bioscience (Frederick, MD).

Cell culture. The human colon cancer cell lines HT29, HCT15, and SW480 were obtained from American Type Culture Collection. HT29 cells were maintained in McCoy's 5A medium supplemented with 10% FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin. HCT15 cells were maintained in RPMI 1640 medium supplemented with 10% FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin. SW480 cells were maintained in Leibovitz's L-15 medium supplemented with 10% FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Cellular cytotoxicity was assessed by measurements of cell number, lactate dehydrogenase release, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. No evidence of cellular toxicity was detected in any of the experiments described below (data not shown).

Western blot analysis. Cell lysates were prepared by harvesting the cells using 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 µg/ml aprotinin, 10 µg/ml trypsin inhibitor, and 10 µg/ml leupeptin). 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 lysates was measured using the BCA method. SDS-PAGE was performed under reducing conditions on 10% polyacrylamide gels. The resolved proteins were transferred onto nitrocellulose sheets and then probed for 15-PGDH, ERK1/2, phospho-ERK1/2, Egr-1, Snail, and β-actin using previously described methods (48).

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 (53).

Real-time PCR. Total RNA was isolated using the RNeasy mini kit. One µg RNA was reversed transcribed using murine leukemia virus reverse transcriptase (Roche Applied Science, Indianapolis, IN) and oligo (dT)16 primer. The resulting cDNA was then used for amplification. The volume of the PCR was 20 µl and contained 5 µl of cDNA with the following primers: 15-PGDH (forward, 5'-TCTGTTCATCCAGTGCGATGT-3' and reverse, 5'-ATAATGATGCCGCCTTCACCT-3'), Egr-1 (forward, 5'-GCGACATCTGTGGAAGAAAGTTTGCC-3' and reverse, 5'-TTTCTTGTCCTTCTGCCGCAAGTG-3'), and Snail (forward, 5'-TCCCTCTTCCTCTCCATACCTG-3' and reverse, 5'-ATGGCAGTGAGAAGGATGTGG-3'). Real-time PCR was performed using 2x SYBR green PCR master mix on a 7900 HT real-time PCR system (Applied Biosystems) with β-actin (forward 5'-AGAAAATCTGGCACCACACC-3' and reverse 5'-AGAGGCGTACAGGGATAGCA-3') serving as an endogenous normalization control. Relative fold induction was determined using the {Delta}{Delta}CT (relative quantification) analysis protocol.

Transient transfections. Egr-1 small interfering RNA (siRNA), Snail siRNA, and nonspecific siRNA were obtained from Dharmacon. HCT15 cells were transfected with 100 nM of siRNA using DharmaFECT transfection reagent according to the manufacturer's instructions. After transfection for 36 h, the cells were collected for analysis or treatment.

Determination of PG levels. Levels of PGs were quantified by liquid chromatography/tandem mass spectroscopic analyses using previously described methods (24).

PKC activity. The activity of PKC was measured according to instructions from Millipore. Briefly, cells were plated in 10-cm dishes at 106 cells/dish and grown to 60% confluence. Cells were then treated with 0–100 µM chenodeoxycholate (CD) for 10 min. Total PKC activity was measured in cell lysates. To determine cytosolic and membrane-bound PKC activity, we centrifuged cell lysates at 100,000 g for 30 min. The resulting supernatant contains cytosolic PKC; membrane-bound PKC activity is present in the pellet. The system is based on PKC-catalyzed transfer of [{gamma}-32P]ATP to a PKC-specific peptide.

ChIP assay. ChIP assay was performed with a kit according to the manufacturer's instructions. Briefly, 4 x 106 cells were cross-linked in a 1% formaldehyde solution at 37°C for 10 min. Cells were then lysed and sonicated to generate 200- to 1,000-bp DNA fragments. After centrifugation, the cleared supernatant was incubated with 4 µg of the indicated antibody at 4°C overnight. Immune complexes were precipitated, washed, and eluted as recommended. DNA-protein cross-links were reversed by heating at 65°C for 4 h, and the DNA fragments were purified and used as a template for PCR amplification. Quantitative real-time PCR was carried out. 15-PGDH promoter oligonucleotide sequences for PCR primers were forward, 5'-CTCCGCTCTCCTTCTATCCA-3' and reverse, 5'-AACCCACGACTGTGTCACCT-3'. This primer set encompasses the 15-PGDH promoter sequence from nucleotide –366 to –155. PCR was performed at 94°C for 30 s, 62°C for 30 s, and 72°C for 45 s for 35 cycles, and real-time PCR was performed at 95°C for 15 s and 60°C for 60 s for 40 cycles. The PCR product generated from the ChIP template was sequenced, and the identity of the 15-PGDH promoter was confirmed.

Statistics. Each experiment was performed at least three times. Comparisons between groups were made using 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
 
Unconjugated bile acids inhibit the expression of 15-PGDH. Initially, we investigated whether treatment with CD or DC altered the expression of 15-PGDH. As shown in Fig. 1A, both CD and DC caused dose-dependent suppression of levels of 15-PGDH protein in several human colon cancer cell lines. Consistent with these findings, treatment with CD or DC also led to a dose-dependent decrease in 15-PGDH activity (Fig. 1B). Quantitative real-time PCR was performed to determine the effects of CD and DC on amounts of 15-PGDH mRNA. Treatment with either unconjugated bile acid led to a marked reduction in levels of 15-PGDH mRNA (Fig. 1C). 15-PGDH catalyzes the conversion of PGE2 to 15-keto-PGE2. To evaluate whether bile acid-mediated downregulation of 15-PGDH had functional consequences, we next evaluated whether treatment with CD altered the production of either PGE2 or 15-keto-PGE2. Treatment with CD led to a marked increase in the amount of PGE2 produced with a reciprocal decrease in the formation of 15-keto-PGE2 (Fig. 1D).


Figure 1
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  		Fig. 1. Unconjugated bile acids inhibit the expression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) in human colon cancer cell lines. A: HT29, HCT15, and SW480 cells were treated with indicated concentrations of chenodeoxycholate (CD) and deoxycholate (DC) for 24 h. Cellular protein (100 µg/lane) was loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose membranes. Immunoblots were probed with antibodies specific for 15-PGDH and β-actin. B: HT29 and HCT15 cells were treated with indicated concentrations of CD and DC for 24 h. Cellular levels of 15-PGDH enzyme activity were determined as described in MATERIALS AND METHODS. Enzyme activity is expressed as pmol·min–1·mg protein–1. Columns, means (n = 4); bars, SD. *P < 0.05; **P < 0.01; ***P < 0.001. C: HT29 and HCT15 cells were treated with indicated concentrations of CD and DC for 20 h. Total RNA was isolated, and quantitative real-time PCR was performed. Values of 15-PGDH mRNA were normalized to values obtained for β-actin. Columns, means (n = 4); bars, SD. *P < 0.05; **P < 0.01; ***P < 0.001. D: HT29 cells were treated with vehicle or 100 µM CD. Twenty-four hours later, the medium was replaced with fresh medium containing 10 µM sodium arachidonate for 30 min. The medium was then collected and analyzed for amounts of PGE2 and 15-keto-PGE2, respectively. Columns, means (n = 6); bars, SD. *P < 0.05; **P < 0.01.

 
Previously, conjugated bile acids were found to be less potent inducers of COX-2 than unconjugated bile acids (56, 63, 64). Hence, it was of interest to evaluate the effects of conjugated bile acids [glycochenodeoxycholate (GCD), taurochenodeoxycholate (TCD), glycodeoxycholate (GDC), and taurodeoxycholate (TDC)] on the expression and activity of 15-PGDH. As shown in Fig. 2A, high concentrations of conjugated bile acids were required to downregulate 15-PGDH. We also compared the effects of unconjugated and conjugated bile acids. Unconjugated bile acids (CD and DC) were much more potent inhibitors of 15-PGDH expression and activity than related conjugated bile acids (Fig. 2, B and C). Treatment with unconjugated bile acids also caused a marked reduction of 15-PGDH mRNA compared with conjugated bile acids (data not shown).


Figure 2
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  		Fig. 2. Conjugated bile acids are less potent suppressors of 15-PGDH expression than unconjugated bile acids. A: HT29 and HCT15 cells were treated with 0–300 µM glycochenodeoxycholate (GCD), taurochenodeoxycholate (TCD), glycodeoxycholate (GDC), or taurodeoxycholate (TDC) for 24 h. Std, standard. B: HCT15 cells were treated with vehicle (C) or 100 µM CD, GCD, TCD, DC, GDC, or TDC for 24 h. Cellular protein (100 µg/lane) was loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose membranes. Immunoblots were probed with antibodies specific for 15-PGDH and β-actin. C: HT29 and HCT15 cells were treated with vehicle (C) or 100 µM CD, GCD, TCD, DC, GDC, or TDC for 24 h. 15-PGDH enzyme activity is expressed as pmol·min–1·mg protein–1. Columns, means (n = 4); bars, SD. *P < 0.05; **P < 0.01; ***P < 0.001.

 
Bile acids activate the PKC -> ERK1/2 pathway, resulting in suppression of 15-PGDH transcription. Bile acids have been reported to activate cell signaling by stimulating the redistribution of PKC activity from cytosol to membrane (21, 27). As shown in Fig. 3A, treatment with CD led to a concentration-dependent translocation of PKC activity from cytosol to membrane. Next, we investigated whether increased membrane PKC activity was causally linked to CD-mediated downregulation of 15-PGDH. Treatment with calphostin C, an inhibitor of PKC activity, attenuated the suppressive effects of CD on 15-PGDH expression (Fig. 3B). Together, these results suggest that CD-mediated activation of PKC signaling is linked to the suppression of 15-PGDH levels. Activation of PKC signaling can stimulate ERK1/2 MAPK. As shown in Fig. 3C, treatment with CD caused a rapid concentration-dependent increase in ERK1/2 activity. In contrast, similar concentrations of conjugated bile acids (GCD and TCD) did not stimulate ERK1/2 phosphorylation (data not shown). Importantly, treatment with PD-98059, a compound that blocks the activation of ERK1/2, suppressed CD-mediated downregulation of 15-PGDH (Fig. 3D). This implies that the observed increase in ERK1/2 activity following bile acid treatment contributes to the suppression of 15-PGDH expression. To confirm that CD-mediated activation of PKC led to the observed increase in ERK1/2 activity, we determined the effects of calphostin C. As shown in Fig. 3E, treatment with calphostin C blocked CD-mediated stimulation of ERK1/2 activity. The transcription factor Snail was recently found to repress the transcription of 15-PGDH (3, 30). The expression of Snail is positively regulated by Egr-1, which, in turn, can be induced by ERK1/2 (20). To investigate whether bile acids induce Egr-1 and Snail, we carried out immunoblot analyses. Treatment with CD induced levels of both Egr-1 and Snail protein in a dose- and time-dependent manner (Fig. 4, A and B). The induction of Egr-1 occurred before the increase in levels of Snail. To determine whether regulation was pretranslational, we quantified levels of Egr-1 and Snail mRNAs. Consistent with the observed increase in Egr-1 and Snail protein levels, treatment with CD induced Egr-1 and Snail mRNAs (Fig. 4, C and D). Once again, the increase in Egr-1 message preceded the induction of Snail mRNA. We next investigated whether bile acid-mediated activation of the PKC -> ERK1/2 pathway was causally linked to the induction of Egr-1 and Snail. Treatment with calphostin C or PD-98059 blocked CD-mediated induction of Egr-1 and Snail (Fig. 4, E and F). Next, it was important to determine whether the induction of Egr-1 by CD was responsible for the increased levels of Snail and reduced amounts of 15-PGDH. Silencing of Egr-1 (Fig. 5A) blocked CD-mediated induction of Snail (Fig. 5B). Importantly, silencing of Egr-1 also blocked CD-mediated downregulation of 15-PGDH (Fig. 5C).


Figure 3
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  		Fig. 3. CD mediated activation of PKC -> ERK1/2 leads to reduced levels of 15-PGDH. A: HCT15 cells were treated with 0–100 µM CD for 10 min. Total PKC activity, cytosolic PKC activity, and membrane PKC activity were measured. Columns, means (n = 6); bars, SD. **P < 0.01; ***P < 0.001. B: HCT15 cells were pretreated with calphostin C (0–2 µM, 2 h), and then vehicle or CD (50 µM) was added for 12 h. Cellular protein (100 µg/lane) was loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose membranes. Immunoblot was probed with antibodies specific for 15-PGDH and β-actin. C: HCT15 cells were treated with 0–100 µM CD for 15 or 30 min. Cellular lysate protein (100 µg/lane) was loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose membranes. Immunoblots were probed with antibodies specific for phospho-ERK1/2 and ERK1/2. D: HCT15 cells were pretreated with PD-98059 (0–100 µM, 2 h), and then vehicle or CD (50 µM) was added for 12 h. Cellular protein (100 µg/lane) was loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose membranes. Immunoblot was probed with antibodies specific for 15-PGDH and β-actin. E: HCT15 cells were pretreated with vehicle or 2 µM calphostin C for 2 h, and then vehicle or 100 µM CD was added for 15 or 30 min. Cellular protein (100 µg/lane) was loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose membranes. The immunoblot was probed with antibodies specific for phospho-ERK1/2 and ERK1/2.

 

Figure 4
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  		Fig. 4. CD induces early growth response factor-1 (Egr-1) and Snail. A: HCT15 cells were treated with indicated concentrations of CD for 3 h. B: HCT15 cells were treated with vehicle or 100 µM CD for 0–3 h. In A and B, cellular protein (100 µg/lane) was loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose membranes. Immunoblots were probed with antibodies specific for Egr-1, Snail, and β-actin. C and D: HCT15 cells were treated with vehicle or 100 µM CD for 0.5–3 h. Total RNA was isolated, and quantitative real-time PCR was performed. Values for Egr-1 (C) and Snail mRNA (D) were normalized to values obtained for β-actin. Columns, means (n = 4); bars, SD. *P < 0.05; **P < 0.01. E: HCT15 cells were pretreated with vehicle or 2 µM calphostin C for 2 h. Subsequently, vehicle or 50 µM CD was added for 3 h. Cellular protein (100 µg/lane) was loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose membranes. Immunoblots were probed with antibodies specific for Egr-1, Snail, and β-actin. F: HCT15 cells were pretreated with vehicle or 100 µM PD-98059 for 2 h, and then vehicle or 50 µM CD was added for 3 h. Cellular protein (100 µg/lane) was loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose membranes. Immunoblots were probed with antibodies specific for Egr-1, Snail, and β-actin.

 

Figure 5
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  		Fig. 5. Silencing of Egr-1 blocks CD-mediated suppression of 15-PGDH expression. A: HCT15 cells were transfected with 100 nM Egr-1 small interfering RNA (siRNA) or nonspecific (NS) control siRNA. After transfection, cells were treated with vehicle or 50 µM CD for 3 (B) or 12 h (C). Cellular protein (100 µg/lane) was loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose membranes. Immunoblots were probed as indicated with antibodies specific for Egr-1, Snail, 15-PGDH, and β-actin.

 
To further evaluate the potential role of Snail in regulating 15-PGDH expression, it was important to determine whether CD stimulated the binding of Snail to the 15-PGDH promoter. Snail previously has been reported to bind to a region of the 15-PGDH promoter that contains E-boxes (30). ChIP assays were carried out using a primer set that included the 15-PGDH promoter segment containing E-boxes. As shown in Fig. 6A, treatment with CD caused a 1.5-fold increase in the binding of Snail to the 15-PGDH promoter. It was next important to evaluate whether Snail was important for CD-mediated downregulation of 15-PGDH expression. Silencing of Snail (Fig. 6B) blocked CD-mediated downregulation of 15-PGDH (Fig. 6C). Together, these results suggest that bile acid-mediated activation of the PKC -> ERK1/2 -> Egr-1 -> Snail pathway is responsible for the downregulation of 15-PGDH transcription.


Figure 6
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  		Fig. 6. Downregulation of 15-PGDH transcription induced by chenodeoxycholate is mediated by Snail. A: chromatin immunoprecipitation (ChIP) assays were performed. HCT15 cells were treated with vehicle or 100 µM CD for 3 h. Chromatin fragments were immunoprecipitated with antibodies against Snail, and the 15-PGDH promoter was amplified by PCR (top) or real-time PCR (bottom). DNA sequencing was carried out, and the PCR product was confirmed to be the 15-PGDH promoter. The 15-PGDH promoter was not detected when normal IgG was used or when antibody was omitted from the immunoprecipitation step (data not shown). Values are means ± SD; n = 3. *P < 0.05. B and C: HCT15 cells were transfected with 100 nM Snail siRNA or nonspecific control siRNA. After transfection, cells were treated with vehicle or 50 µM CD for 12 h. Cellular protein (100 µg/lane) was loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose membranes. Immunoblots were probed as indicated with antibodies specific for Snail, 15-PGDH, and β-actin.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Multiple lines of evidence suggest that PGs play a key role in gastrointestinal mucosal homeostasis, colitis, and colon carcinogenesis (30, 34, 35, 40, 48, 57). It is important, therefore, to understand the endogenous factors that regulate PG production. In humans, intracolonic release of PGs is stimulated by exposure to bile acid (7, 36). Previously, we found that unconjugated bile acids were potent inducers of COX-2 and PG synthesis (56, 63, 64). In the current study, we have shown that unconjugated bile acids suppress the transcription of 15-PGDH, resulting in increased production of PGE2 and reduced synthesis of 15-keto-PGE2.

Recent studies have suggested that 15-PGDH, the key enzyme responsible for metabolic inactivation of PGs, behaves as a tumor suppressor for several tumor types, including colon cancer (11, 32, 58, 59). Bile acid-mediated suppression of 15-PGDH, which results in increased levels of PGs, could be important for both the formation and progression of gastrointestinal cancers. In support of this notion, PGE2 stimulates cell proliferation, promotes angiogenesis, and inhibits apoptosis and immune surveillance (9, 18, 23, 44). PGE2 also can induce cell invasion and an epithelial mesenchymal transition (12, 19, 54, 55, 61), processes linked to metastasis. The potential significance of bile acid-mediated downregulation of 15-PGDH is underscored by extensive evidence that either inhibiting the synthesis of PGE2 or antagonizing its actions can suppress carcinogenesis (5, 28, 42, 47, 51, 52).

Bile acid-mediated suppression of 15-PGDH transcription reflected activation of the PKC -> ERK1/2 -> Egr-1 -> Snail signal transduction pathway. The involvement of PKC is suggested by two findings. Consistent with prior reports (21, 27), membranous PKC activity was stimulated by bile acid treatment. Importantly, bile acid-mediated suppression of 15-PGDH was attenuated by treatment with an inhibitor of PKC activity. Additional studies are needed to determine which isoforms of PKC are involved. The involvement of ERK1/2 is supported by two findings. First, treatment with bile acid led to rapid activation of ERK1/2. Second, an inhibitor of MAPK kinase blocked bile acid-mediated downregulation of 15-PGDH. An inhibitor of PKC activity blocked bile acid-mediated activation of ERK1/2, suggesting that PKC is upstream of ERK1/2.

We also report that suppression of 15-PGDH reflects bile acid-mediated induction of Egr-1 and Snail. Recently, activation of MAPK was found to induce Egr-1 leading, in turn, to increased Snail expression (20). Moreover, bile acids have been reported to induce Egr-1 and Snail, a known repressor of 15-PGDH transcription in colonocytes (6, 30). Treatment with bile acid led to increased levels of Egr-1 and Snail message and protein. Importantly, the increase in Egr-1 preceded the change in Snail expression. The induction of Egr-1 and Snail by bile acid was suppressed by treatment with inhibitors of PKC or MAPK kinase. Bile acid-mediated induction of Snail and suppression of 15-PGDH expression was blocked by silencing of Egr-1. Previous studies have reported that the transcriptional repressor Snail binds to E-boxes in the 15-PGDH promoter and thereby inhibits transcription (30). There are three E-boxes located in the 15-PGDH 5'-flanking region. ChIP analysis indicated that treatment with bile acid stimulated the binding of Snail to a region of the 15-PGDH promoter containing the three E-boxes. To evaluate the functional significance of increased Snail binding, we used siRNA. Silencing of Snail blocked bile acid-mediated downregulation of 15-PGDH.

Both conjugated and unconjugated bile acids caused a reduction in 15-PGDH expression and activity. However, unconjugated bile acids were much more potent than conjugated bile acids. This finding is consistent with our previous results for COX-2 (56, 63, 64). Notably, the concentrations of unconjugated bile acids that suppressed levels of 15-PGDH are found in the aqueous phase of the fecal stream (10, 22). The observed difference in suppressive activity is likely to reflect differences in the hydrophobicity of unconjugated and conjugated bile acids. CD and DC are hydrophobic bile acids. Conjugation of these bile acids with either glycine or taurine makes them more hydrophilic and less potent inhibitors of 15-PGDH expression. These findings are consistent with recent evidence that the ability to bile acids to activate PKC{alpha} and ERK1/2 is hydrophobicity dependent (1).

Levels of 15-PGDH are markedly reduced in both inflammatory bowel disease and colorectal cancer (2, 34, 59). A number of different mechanisms can downregulate 15-PGDH transcription. Hypermethylation of the 15-PGDH promoter, changes in histone acetylation, and increased binding of repressive transcription factors, including Snail, lead to reduced transcription (3, 30, 58, 60). Future studies are needed to determine whether bile acids induce epigenetic changes such as hypermethylation of the 15-PGDH promoter that also contribute to reduced expression. Our data suggest that bile acids can suppress levels of 15-PGDH, which could, in turn, promote the development of colorectal cancer. Recently, inducers of 15-PGDH have been proposed as potential chemopreventive agents (13). On the basis of this study, it would be worthwhile to determine whether agents that block bile acid-mediated downregulation of 15-PGDH possess anticancer properties.


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This work was supported by National Cancer Institute Grant R01 CA111469, the New York Crohn's Foundation, and the Center for Cancer Prevention Research.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. J. Dannenberg, Dept. of Medicine and Weill Cornell Cancer Center, 525 East 68th St., Rm. F-206, New York, NY 10065 (e-mail: ajdannen{at}med.cornell.edu)


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