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Dihydroxy Bile Acids Activate the Transcription of Cyclooxygenase-2

1998; Elsevier BV; Volume: 273; Issue: 4 Linguagem: Inglês

10.1074/jbc.273.4.2424

ISSN

1083-351X

Autores

Fan Zhang, Kotha Subbaramaiah, Nasser K. Altorki, Andrew J. Dannenberg,

Tópico(s)

Estrogen and related hormone effects

Resumo

Bile acids, endogenous promoters of gastrointestinal cancer, activate protein kinase C (PKC) and the activator protein-1 (AP-1) transcription factor. Because other activators of PKC and AP-1 induce cyclooxygenase-2 (COX-2), we determined the effects of bile acids on the expression of COX-2 in human esophageal adenocarcinoma cells. Treatment with the dihydroxy bile acids chenodeoxycholate and deoxycholate resulted in an ∼10-fold increase in the production of prostaglandin E2(PGE2). Enhanced synthesis of PGE2 was associated with a marked increase in the levels of COX-2 mRNA and protein, with maximal effects at 8–12 and 12–24 h, respectively. In contrast, neither cholic acid nor conjugated bile acids affected the levels of COX-2 or the synthesis of PGE2. Nuclear run-off assays and transient transfections with a human COX-2promoter construct showed that induction of COX-2 mRNA by chenodeoxycholate and deoxycholate was due to increased transcription. Bile acid-mediated induction of COX-2 was blocked by inhibitors of PKC activity, including calphostin C and staurosporine. Treatment with bile acid enhanced the phosphorylation of c-Jun and increased binding of AP-1 to DNA. These data are important because dihydroxy bile acid-mediated induction of COX-2 may explain, at least in part, the tumor-promoting effects of bile acids. Bile acids, endogenous promoters of gastrointestinal cancer, activate protein kinase C (PKC) and the activator protein-1 (AP-1) transcription factor. Because other activators of PKC and AP-1 induce cyclooxygenase-2 (COX-2), we determined the effects of bile acids on the expression of COX-2 in human esophageal adenocarcinoma cells. Treatment with the dihydroxy bile acids chenodeoxycholate and deoxycholate resulted in an ∼10-fold increase in the production of prostaglandin E2(PGE2). Enhanced synthesis of PGE2 was associated with a marked increase in the levels of COX-2 mRNA and protein, with maximal effects at 8–12 and 12–24 h, respectively. In contrast, neither cholic acid nor conjugated bile acids affected the levels of COX-2 or the synthesis of PGE2. Nuclear run-off assays and transient transfections with a human COX-2promoter construct showed that induction of COX-2 mRNA by chenodeoxycholate and deoxycholate was due to increased transcription. Bile acid-mediated induction of COX-2 was blocked by inhibitors of PKC activity, including calphostin C and staurosporine. Treatment with bile acid enhanced the phosphorylation of c-Jun and increased binding of AP-1 to DNA. These data are important because dihydroxy bile acid-mediated induction of COX-2 may explain, at least in part, the tumor-promoting effects of bile acids. Recent studies have established that there are two distinct isoforms of cyclooxygenase (COX), 1The abbreviations used are: COX, cyclooxygenase; PKC, protein kinase C; PGE2, prostaglandin E2; AP-1, activator protein-1; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PMA, phorbol 12-myristate 13-acetate; CD, chenodeoxycholate; DC, deoxycholate. both of which catalyze the formation of prostaglandins from arachidonic acid.COX-1 is thought to be a housekeeping gene with essentially constant levels of expression (1Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1854) Google Scholar). In contrast, the gene forCOX-2 is an early-response gene that, like c-fosand c-jun, is induced rapidly following stimulation of quiescent cells. The expression of COX-2 is stimulated by growth factors, cytokines, oncogenes, phorbol esters, and carcinogens and by overexpression of PKC (2Kujubu D.A. Fletcher B.S. Varnum B.C. Lim R.W. Herschman H.R. J. Biol. Chem. 1991; 266: 12866-12872Abstract Full Text PDF PubMed Google Scholar, 3O'Banion M.K. Sadowsky H.B. Winn V. Young D.A. J. Biol. Chem. 1991; 266: 23261-23267Abstract Full Text PDF PubMed Google Scholar, 4Jones D.A. Carlton D.P. McIntyre T.M. Zimmerman G.A. Prescott S.M. J. Biol. Chem. 1993; 268: 9049-9054Abstract Full Text PDF PubMed Google Scholar, 5DuBois R.N. Awad J. Morrow J. Roberts L.J. Bishop P.R. J. Clin. Invest. 1994; 93: 493-498Crossref PubMed Scopus (378) Google Scholar, 6Xie W. Fletcher B.S. Andersen R.D. Herschman H.R. Mol. Cell. Biol. 1994; 14: 6531-6539Crossref PubMed Scopus (189) Google Scholar, 7Subbaramaiah K. Telang N. Ramonetti J.T. Araki R. DeVito B. Weksler B.B. Dannenberg A.J. Cancer Res. 1996; 56: 4424-4429PubMed Google Scholar, 8Kelley D.J. Mestre J.R. Subbaramaiah K. Sacks P.G. Schantz S.P. Tanabe T. Inoue H. Ramonetti J.T. Dannenberg A.J. Carcinogenesis (Lond.). 1997; 18: 795-799Crossref PubMed Scopus (148) Google Scholar, 9Miller B.W. Baier L.D. Morrison A.R. Am. J. Physiol. 1997; 273: C130-C136Crossref PubMed Google Scholar). The different responses of the genes forCOX-1 and COX-2 reflect, in part, differences in the regulatory elements in the 5′-flanking regions of the two genes (10Inoue H. Yokoyama C. Hara S. Tone Y. Tanabe T. J. Biol. Chem. 1995; 270: 24965-24971Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar). A large body of data from a variety of experimental systems suggest that COX-2 is important for tumor formation. Oshima et al.(11Oshima M. Dinchuk J.E. Kargman S.L. Oshima H. Hancock B. Kwong E. Trzaskos J.M. Evans J.F. Taketo M.M. Cell. 1996; 87: 803-809Abstract Full Text Full Text PDF PubMed Scopus (2284) Google Scholar) have reported, for example, that a null mutation of COX-2 is associated with a marked reduction in the number and size of intestinal polyps in ApcΔ716 (adenomatouspolyposis coli) knockout mice, which are a model for human familial adenomatous polyposis. Treatment with a selective inhibitor of COX-2 also reduced the number of polyps in ApcΔ716 mice (11Oshima M. Dinchuk J.E. Kargman S.L. Oshima H. Hancock B. Kwong E. Trzaskos J.M. Evans J.F. Taketo M.M. Cell. 1996; 87: 803-809Abstract Full Text Full Text PDF PubMed Scopus (2284) Google Scholar). COX-2 deficiency also appears to protect against the formation of tumors in other epithelia becauseCOX-2 knockout mice develop fewer chemically induced skin papillomas than control mice (12Tiano H. Chulada P. Spalding J. Lee C. Loftin C. Mahler J. Morham S. Langenbach R. Proc. Am. Assoc. Cancer Res. 1997; 38: 1727Google Scholar). Although the exact causal link(s) between the activity of COX-2 and carcinogenesis remain uncertain, there are several different mechanisms that can account for this linkage (13Prescott S.M. White R.L. Cell. 1996; 87: 783-786Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Prostaglandins, e.g. PGE2, affect cell proliferation and inhibit immune surveillance; thus, overproduction of prostaglandins could favor malignant growth (14Goodwin J.S. Ceuppens J. J. Clin. Immunol. 1983; 3: 295-314Crossref PubMed Scopus (652) Google Scholar). Overexpression of COX-2 inhibits apoptosis and increases the invasiveness of malignant cells (15Tsujii M. DuBois R.N. Cell. 1995; 83: 493-501Abstract Full Text PDF PubMed Scopus (2137) Google Scholar, 16Tsujii M. Kawano S. DuBois R.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3336-3340Crossref PubMed Scopus (1328) Google Scholar). Amounts of COX-2 are increased in transformed cells and tumors (7Subbaramaiah K. Telang N. Ramonetti J.T. Araki R. DeVito B. Weksler B.B. Dannenberg A.J. Cancer Res. 1996; 56: 4424-4429PubMed Google Scholar, 17Kutchera W. Jones D.A. Matsunami N. Groden J. McIntyre T.M. Zimmerman G.A. White R.L. Prescott S.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4816-4820Crossref PubMed Scopus (445) Google Scholar, 18Kargman S.L. O'Neill G.P. Vickers P.J. Evans J.F. Mancini J.A. Jothy S. Cancer Res. 1995; 55: 2556-2559PubMed Google Scholar, 19Ristimaki A. Honkanen N. Jankala H. Sipponen P. Harkonen M. Cancer Res. 1997; 57: 1276-1280PubMed Google Scholar), which results in enhanced synthesis of prostaglandins in malignant tissue (20Bennett A. Prog. Lipid Res. 1986; 25: 539-542Crossref PubMed Scopus (81) Google Scholar, 21Bennett A. Civier A. Hensby C.N. Melhuish P.B. Stamford I.F. Gut. 1987; 28: 315-318Crossref PubMed Scopus (111) Google Scholar). Given these data, a reasonable strategy for inhibiting carcinogenesis is to suppress the expression of COX-2 (22Mestre J.R. Subbaramaiah K. Sacks P.G. Schantz S.P. Tanabe T. Inoue H. Dannenberg A.J. Cancer Res. 1997; 57: 1081-1085PubMed Google Scholar, 23Mestre J.R. Subbaramaiah K. Sacks P.G. Schantz S.P. Tanabe T. Inoue H. Dannenberg A.J. Cancer Res. 1997; 57: 2890-2895PubMed Google Scholar). It therefore becomes important to identify the factors that modulate the expression of COX-2. In this regard, bile acids are known promoters of gastrointestinal cancer in vivo and enhance cell transformation in vitro (24Reddy B.S. Wynder E.L. J. Natl. Cancer Inst. 1973; 50: 1437-1442Crossref PubMed Scopus (364) Google Scholar, 25Narisawa T. Magdie N.E. Weisburger J.H. Wynder E.L. J. Natl. Cancer Inst. 1974; 53: 1093-1097Crossref PubMed Scopus (642) Google Scholar, 26Reddy B.S. Watanabe K. Weisburger J.H. Wynder E.L. Cancer Res. 1977; 37: 3238-3242PubMed Google Scholar, 27Hill M.J. Eur. J. Cancer Prevention. 1991; 1: 69-74Crossref PubMed Scopus (80) Google Scholar, 28Kaibara N. Yurugi E. Koga S. Cancer Res. 1984; 44: 5482-5485PubMed Google Scholar). The precise mechanism(s) by which bile acids promote tumor formation is not known. But like tumor-promoting phorbol esters (29Angel P. Karin M. Biochim. Biophys. Acta. 1991; 1072: 129-157Crossref PubMed Scopus (3270) Google Scholar), bile acids activate PKC (30Fitzer C.J. O'Brian C.A. Guillem J.G. Weinstein I.B. Carcinogenesis. 1987; 8: 217-220Crossref PubMed Scopus (103) Google Scholar, 31Guillem J.G. O'Brian C.A. Fitzer C.J. Johnson M.D. Forde K.A. LoGerfo P. Weinstein I.B. Arch. Surg. 1987; 122: 1475-1478Crossref PubMed Scopus (43) Google Scholar, 32Huang X.P. Fan X.T. Desjeux J.F. Castagna M. Int. J. Cancer. 1992; 52: 444-450Crossref PubMed Scopus (100) Google Scholar) and induce AP-1 activity (33Hirano F. Tanaka H. Makino Y. Okamoto K. Hiramoto M. Handa H. Makino I. Carcinogenesis (Lond.). 1996; 17: 427-433Crossref PubMed Scopus (65) Google Scholar). The latter effect may be especially important because inhibitors of AP-1-mediated transcription have anti-cancer properties (34Huang C. Ma W.-Y. Dawson M.I. Rincon M. Flavell R.A. Dong Z. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5826-5830Crossref PubMed Scopus (205) Google Scholar). To further examine the mechanism by which bile acids promote tumor formation, we have examined their effects as inducers of COX-2. The rationale for this approach to defining the bile acid-cancer connection is that phorbol esters are prototypic inducers of COX-2 (2Kujubu D.A. Fletcher B.S. Varnum B.C. Lim R.W. Herschman H.R. J. Biol. Chem. 1991; 266: 12866-12872Abstract Full Text PDF PubMed Google Scholar, 4Jones D.A. Carlton D.P. McIntyre T.M. Zimmerman G.A. Prescott S.M. J. Biol. Chem. 1993; 268: 9049-9054Abstract Full Text PDF PubMed Google Scholar, 5DuBois R.N. Awad J. Morrow J. Roberts L.J. Bishop P.R. J. Clin. Invest. 1994; 93: 493-498Crossref PubMed Scopus (378) Google Scholar). Our data show that selected bile acids enhance the transcription ofCOX-2 and that inhibitors of PKC activity block this effect. DMEM, Opti-MEM, FCS, and Lipofectin were from Life Technologies, Inc. Enzyme immunoassay reagents for PGE2 assays were from Cayman Chemical Co., Inc. (Ann Arbor, MI). Bile acids, sodium arachidonate, staurosporine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; thiazolyl blue) and phorbol 12-myristate 13-acetate (PMA) were from Sigma. Calphostin C was from BIOMOL Research Labs Inc. (Plymouth Meeting, PA). [32P]CTP and [32P]UTP were from NEN Life Science Products. The AP-1 consensus oligonucleotide was obtained from Promega (Madison, WI). Random-priming kits were from Boehringer Mannheim. Reagents for the luciferase assay were from Analytical Luminescence Laboratory (San Diego, CA). Nitrocellulose membranes were from Schleicher & Schuell. The 18 S rRNA cDNA was from Ambion Inc. (Austin, TX). Rabbit polyclonal anti-human COX-2 antiserum was from Oxford Biomedical Research, Inc. (Oxford, MI). Goat polyclonal anti-human COX-1 and monoclonal anti-c-Jun (specific for the phosphorylated form of c-Jun) antisera were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Western blot detection reagents (ECL) were from Amersham Corp. Plasmid DNA was prepared using a kit from QIAGEN Inc. (Chatsworth, CA). The SK-GT-4 cell line was established from a well differentiated adenocarcinoma arising in Barrett's epithelium of the distal esophagus (35Altorki N. Schwartz G.K. Blundell M. Davis B.M. Kelsen D.P. Albino A.P. Cancer (Phila.). 1993; 72: 649-657Crossref PubMed Scopus (49) Google Scholar). Cells were maintained in DMEM supplemented with 10% FCS, 100 units/ml penicillin, and 100 μg/ml streptomycin in a 5% CO2/water-saturated incubator at 37 °C. Treatments with vehicle (0.1% ethanol) or bile acid were carried out in 1% FCS. Cellular cytotoxicity was assessed by measurements of cell number, release of lactate dehydrogenase, trypan blue exclusion, and MTT assay. Levels of lactate dehydrogenase release were measured in the supernatants used for PGE2 analyses. MTT assay was performed according to the method of Denizot and Lang (36Denizot F. Lang R. J. Immunol. Methods. 1986; 89: 271-277Crossref PubMed Scopus (4322) Google Scholar). Lactate dehydrogenase assays were performed according to the manufacturer's instructions. For trypan blue analysis, following treatment with bile acid for 12 h, cells were combined 1:1 with 0.4% trypan blue and examined for dye exclusion. Cells (1 × 104/well) were plated in 24-well dishes and grown to 60% confluence in DMEM containing 10% FCS. The medium was then replaced with DMEM containing 1% FCS and vehicle (0.1% ethanol) or bile acids (400 μm) for 12 h. At the end of the treatment period, the culture medium was collected to determine the amounts of PGE2 secreted spontaneously by these cells. Fresh DMEM containing 1% FCS and 10 μm sodium arachidonate was then added. After 30 min, the medium was collected for analysis of PGE2. The levels of PGE2 released by the cells were measured by enzyme immunoassay (7Subbaramaiah K. Telang N. Ramonetti J.T. Araki R. DeVito B. Weksler B.B. Dannenberg A.J. Cancer Res. 1996; 56: 4424-4429PubMed Google Scholar). Rates of production of PGE2 were normalized to protein concentrations. Lysates for the detection of COX-2 and COX-1 were prepared by treating cells with lysis buffer (150 mm NaCl, 100 mm Tris (pH 8.0), 1% Tween 20, 50 mm diethyl dithiocarbamate, 1 mm EDTA, and 1 mm phenylmethylsulfonyl fluoride). Lysates for the detection of the phosphorylated form of c-Jun were prepared by treating cells in radioimmune precipitation assay buffer (150 mmNaCl, 50 mm Tris (pH 7.5), 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and 1 mm phenylmethylsulfonyl fluoride). Lysates were sonicated for 20 s on ice and centrifuged at 10,000 × g for 10 min to sediment the particulate material. The protein concentration of the supernatant was measured by the method of Lowry et al. (37Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). SDS-polyacrylamide gel electrophoresis was performed under reducing conditions on 10% polyacrylamide gels as described by Laemmli (38Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar). The resolved proteins were transferred onto nitrocellulose sheets as detailed by Towbinet al. (39Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44924) Google Scholar). The nitrocellulose membrane was then incubated with rabbit polyclonal anti-COX-2 antiserum, polyclonal anti-COX-1 antiserum, or a monoclonal antibody to phosphorylated c-Jun. The blots were probed with the corresponding secondary antibodies to IgG conjugated to horseradish peroxidase. Blots were probed with the ECL Western blot detection system according to the manufacturer's instructions. Total cellular RNA was isolated from cell monolayers using an RNA isolation kit from QIAGEN Inc. 10 μg of total cellular RNA/lane was electrophoresed on a formaldehyde-containing 1.2% agarose gel and transferred to nylon-supported membranes. After baking, membranes were prehybridized overnight in a solution containing 50% formamide, 5 × sodium chloride/sodium phosphate/EDTA buffer (SSPE), 5 × Denhardt's solution, 0.1% SDS, and 100 μg/ml single-stranded salmon sperm DNA and were then hybridized for 12 h at 42 °C with a radiolabeled human COX-2 cDNA probe. After hybridization, membranes were washed twice for 20 min in 2 × SSPE and 0.1% SDS at room temperature, twice for 20 min in the same solution at 55 °C, and twice for 20 min in 0.1 × SSPE and 0.1% SDS at 55 °C. Washed membranes were then subjected to autoradiography. To verify equivalency of RNA loading in the different lanes, the blot was stripped of radioactivity and rehybridized to determine the levels of 18 S rRNA. COX-2 and 18 S rRNA probes were labeled with [32P]CTP by random priming. Cells (1 × 105) were plated in 100-mm dishes. Cells were grown in DMEM containing 10% FCS until ∼60% confluent. The medium was then replaced with fresh DMEM containing 1% FCS and vehicle (0.1% ethanol), CD (400 μm), or DC (400 μm) for 8 h. Nuclei were isolated and stored in liquid nitrogen. For the transcription assay, nuclei (1.0 × 107) were thawed and incubated in reaction buffer (10 mm Tris (pH 8), 5 mmMgCl2, and 0.3 m KCl) containing 100 μCi of uridine 5′-[α-32P]triphosphate and 1 mmunlabeled nucleotides. After 30 min, labeled nascent RNA transcripts were isolated. The human COX-2 and 18 S rRNA cDNAs were immobilized onto nitrocellulose and prehybridized overnight in hybridization buffer (10 mm TES, 10 mm EDTA, 0.2% SDS, and 0.6 m NaCl). 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 2 × SSC for 1 h at 55 °C and then treated with 10 mg/ml RNase A in 2 × SSC for 30 min at 37 °C, dried, and autoradiographed. The COX-2 promoter construct (−327/+59) was a gift of Dr. Tadashi Tanabe (National Cardiovascular Center Research Institute, Osaka, Japan) (10Inoue H. Yokoyama C. Hara S. Tone Y. Tanabe T. J. Biol. Chem. 1995; 270: 24965-24971Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar). The humanCOX-2 cDNA was generously provided by Dr. Stephen M. Prescott (University of Utah, Salt Lake City, UT). pSV-βgal was obtained from Promega. SK-GT-4 cells were seeded at a density of 5 × 104/well in 6-well dishes and grown to 50–60% confluence in DMEM containing 10% FCS. For each well, 1.8 μg of COX-2-luciferase construct and 0.2 μg of pSV-βgal were cotransfected into SK-GT-4 cells at a 1:4 ratio of DNA to Lipofectin following the manufacturer's instructions. After transfection, the medium was replaced with DMEM containing 10% FCS for 24 h. The cells were then treated with DMEM containing 1% FCS and vehicle (0.1% ethanol), CD (400 μm), or DC (400 μm). The activities of luciferase and β-galactosidase were measured in cellular extract 8 h later (22Mestre J.R. Subbaramaiah K. Sacks P.G. Schantz S.P. Tanabe T. Inoue H. Dannenberg A.J. Cancer Res. 1997; 57: 1081-1085PubMed Google Scholar). To adjust for differences in transfection efficiencies, the luciferase values were normalized using β-galactosidase. SK-GT-4 cells were treated with DMEM containing 1% FCS and vehicle (0.1% ethanol) or CD for 2 or 6 h, respectively. Cells were harvested and nuclear extracts were prepared as described previously (40Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 19: 2499Crossref PubMed Scopus (2211) Google Scholar). For binding studies, an AP-1 consensus oligonucleotide was used: 5′-CGCTTGATGAGTCAGCCGGAA-3′ (sense) and 3′-GCGAACTACTCAGTCGGCCTT-5′ (antisense). The complementary oligonucleotides were annealed in 20 mm Tris (pH 7.6), 50 mm NaCl, 10 mmMgCl2, and 1 mm dithiothreitol. The annealed oligonucleotide was phosphorylated at the 5′-end with [γ-32P]ATP and T4 polynucleotide kinase. The binding reaction was performed by incubating 2 μg of nuclear protein in 20 mm HEPES (pH 7.9), 10% glycerol, 300 μg of bovine serum albumin, and 1 μg of poly(dI·dC) in a final volume of 10 μl for 10 min at 25 °C. The labeled AP-1 consensus oligonucleotide was 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 −80 °C. Comparisons between groups were made by Student's t test. A difference between groups ofp < 0.05 was considered significant. Fig. 1 shows the effects of several bile acids and bile acid conjugates on the production of PGE2. CD and DC caused ∼3- and 10-fold increases in the spontaneous production of PGE2, respectively. Cholic acid and all conjugated bile acids, however, had no effect on rates of spontaneous synthesis of PGE2. To examine the synthesis of PGE2 in more detail, we also measured the effect of bile acids on the production of PGE2when an excess of arachidonic acid was added to the system. This experiment was done because PGE2 production can be affected by changes in the activity of phospholipase A2, which provides the substrate for COX-catalyzed reactions. Adding excess arachidonate minimizes any contribution of phospholipase A2activity to the rate of production of PGE2. As for rates of spontaneous synthesis of PGE2, treatment with CD or DC induced an ∼10-fold increase in the synthesis of PGE2 in the presence of excess arachidonate (Fig. 1). Neither cholic acid nor the conjugated bile acids affected the production of PGE2in the presence of excess arachidonic acid. Cytotoxicity in these experiments was assessed by cell number, lactate dehydrogenase release, MTT assay, and trypan blue exclusion. No evidence of cellular toxicity was detected (data not shown). Western blotting was carried out to determine whether the above differences in PGE2 production were related to differences in amounts of COX-2. We found that treatment with CD caused a dose-dependent induction of COX-2 protein with a peak effect at 400 μm CD (Fig.2). Moreover, CD and DC were the only bile acids that induced COX-2 protein (Fig.3), which is consistent with their effects on the production of PGE2 (Fig. 1). We also compared the time course for induction of COX-2 by CD versusPMA. Maximal induction of COX-2 protein occurred after 12–24 h of treatment with CD (Fig. 4). In contrast, COX-2 was induced maximally by PMA after 8–12 h of treatment (Fig. 4). COX-1 was not detectable in this cell line by Western blotting under basal or stimulated conditions (data not shown).Figure 3Dihydroxy bile acids induce COX-2.SK-GT-4 cells were treated for 12 h with vehicle (0.1% ethanol) or with 400 μm cholic acid (CA), taurocholate (TC), CD, glycochenodeoxycholate (GCD), taurochenodeoxycholate (TCD), DC, or taurodeoxycholate (TDC). Cellular lysate protein (30 μg) was loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose. Immunoblots were probed with antibody specific for COX-2. Ovine Cox-2 was used as a standard.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4CD and PMA cause prolonged induction of COX-2. SK-GT-4 cells were treated for 4–36 h with vehicle (0.1% ethanol), CD (400 μm), or PMA (50 ng/ml). Cellular lysate protein (30 μg) was loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose. Immunoblots were probed with antiserum specific for COX-2. Ovine Cox-2 was used as a standard.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further elucidate the mechanism responsible for the changes in amounts of COX-2 protein, we measured the steady-state levels of COX-2 mRNA by Northern blotting. CD and DC stimulated the formation of COX-2 mRNA; maximal induction of COX-2 mRNA was observed after treatment for ∼8 h (Fig. 5). Bile acid-mediated induction of COX-2 mRNA was still apparent after 24 h of treatment, which is consistent with the effects detected by Western blotting. Differences in the levels of mRNA could reflect altered rates of transcription or stability of mRNA. Nuclear run-off assays were performed to distinguish between these possibilities. As shown in Fig. 6, we detected higher rates of synthesis of nascent COX-2 mRNA after treatment with CD or DC, consistent with the differences observed by Northern blotting. To further investigate the importance of bile acids in modulating the expression of COX-2, transient transfections were performed with a human COX-2 promoter-luciferase construct. Treatment with CD or DC led to approximately a doubling of COX-2promoter activity (Fig. 7).Figure 7Bile acids increase COX-2promoter activity. SK-GT-4 cells were cotransfected with 1.8 μg of human COX-2 promoter construct (−327/+59) and 0.2 μg of pSV-βgal. After transfection, cells were treated with 400 μm CD or DC or with vehicle (0.1% ethanol). Reporter activities were measured in cellular extract 8 h later. Luciferase activity represents data that have been normalized with β-galactosidase activity. Six wells were used for each of the three conditions. The columns are the means, and thebars are the S.D. *, p < 0.001 compared with control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Bile acids have been reported to activate PKC. Therefore, to elucidate the signaling mechanism by which bile acids activate the transcription ofCOX-2, we measured the induction of COX-2 by CD in the presence of known inhibitors of PKC. Staurosporine and calphostin C were used as inhibitors. Both agents blocked the induction of COX-2 by bile acids (Fig. 8, A andB). Dexamethasone also blocked the induction of COX-2 by CD and PMA (Fig. 8 C). Because activators of PKC modulate gene expression via AP-1, we determined the effects of CD on the phosphorylation state of c-Jun and AP-1 DNA-binding activity (Fig.9). Treatment with CD increased the phosphorylation of c-Jun (Fig. 9 A). Electrophoretic mobility shift assay revealed increased binding of AP-1 to DNA following treatment with CD (Fig. 9 B).Figure 9CD enhances the phosphorylation of c-Jun and induces AP-1 DNA-binding activity. Cells were treated with CD (400 μm) or vehicle (0.1% ethanol) for 2 or 6 h. InA, cellular lysate protein (30 μg/lane) was loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose. The blot was probed with a monoclonal antibody directed toward the phosphorylated form of c-Jun. InB, 2 μg of nuclear protein was incubated with a32P-labeled oligonucleotide containing an AP-1 consensus site. The protein-DNA complex that formed was separated on a 4% polyacrylamide gel as described under “Experimental Procedures.”View Large Image Figure ViewerDownload Hi-res image Download (PPT) Bile acids are known tumor promoters that activate PKC, which may be the mechanism of bile acid-induced carcinogenesis (31Guillem J.G. O'Brian C.A. Fitzer C.J. Johnson M.D. Forde K.A. LoGerfo P. Weinstein I.B. Arch. Surg. 1987; 122: 1475-1478Crossref PubMed Scopus (43) Google Scholar, 32Huang X.P. Fan X.T. Desjeux J.F. Castagna M. Int. J. Cancer. 1992; 52: 444-450Crossref PubMed Scopus (100) Google Scholar, 33Hirano F. Tanaka H. Makino Y. Okamoto K. Hiramoto M. Handa H. Makino I. Carcinogenesis (Lond.). 1996; 17: 427-433Crossref PubMed Scopus (65) Google Scholar). Dihydroxy bile acids are more effective in activating PKC than mono- and trihydroxy bile acids (32Huang X.P. Fan X.T. Desjeux J.F. Castagna M. Int. J. Cancer. 1992; 52: 444-450Crossref PubMed Scopus (100) Google Scholar). The exact mechanism by which bile acids change the activity of PKC is uncertain. However, bile acids increase the activity of PKC, in part, by inducing the translocation of selected isoforms from cytosol to membrane fractions (31Guillem J.G. O'Brian C.A. Fitzer C.J. Johnson M.D. Forde K.A. LoGerfo P. Weinstein I.B. Arch. Surg. 1987; 122: 1475-1478Crossref PubMed Scopus (43) Google Scholar). It is also known, in the context of the tumor-promoting effects of bile acids, that dihydroxy bile acids stimulate the release of PGE2 by colonic epithelium (41Rampton D.S. Breuer N.F. Vaja S.G. Sladen G.E. Dowling R.H. Clin. Sci. 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Bile acid-mediated induction of COX-2 can be important for tumorigenesis in the gastrointestinal tract because the products of COX-2 activity, e.g. prostaglandins, inhibit apoptosis, diminish immune surveillance, and increase the invasiveness of malignant cells (14Goodwin J.S. Ceuppens J. J. Clin. Immunol. 1983; 3: 295-314Crossref PubMed Scopus (652) Google Scholar, 15Tsujii M. DuBois R.N. Cell. 1995; 83: 493-501Abstract Full Text PDF PubMed Scopus (2137) Google Scholar, 16Tsujii M. Kawano S. DuBois R.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3336-3340Crossref PubMed Scopus (1328) Google Scholar). These conclusions are consistent with evidence that inhibitors of COX-2 are chemopreventive against cancers of the colon and upper gastrointestinal tract in experimental animals (45Rao C.V. Rivenson A. Simi B. Zang E. Kelloff G. Steele V. Reddy B.S. Cancer Res. 1995; 55: 1464-1472PubMed Google Scholar, 46Boolbol S.K. Dannenberg A.J. Chadburn A. Martucci C. Guo X. Ramonetti J.T. 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Suppression of bile acid-mediated induction of COX-2 by dexamethasone is consistent with prior reports that AP-1 activity can be regulated negatively by glucocorticoids (29Angel P. Karin M. Biochim. Biophys. Acta. 1991; 1072: 129-157Crossref PubMed Scopus (3270) Google Scholar). AP-1 sites are only one of several cis-elements that mediate the effects of PKC. Other PKC-responsive cis-elements include the AP-2 recognition site (54Imagawa M. Chiu R. Karin M. Cell. 1987; 51: 251-260Abstract Full Text PDF PubMed Scopus (1030) Google Scholar) and the NF-κB-binding site (55Lenardo M. Baltimore D. Cell. 1989; 58: 227-229Abstract Full Text PDF PubMed Scopus (1258) Google Scholar). Irrespective of the exact mechanism, however, the results reported above suggest that inhibitors of PKC could be useful for down-regulating COX-2 and thereby preventing and/or treating cancer. Although relatively high concentrations of bile acids were required to induce COX-2, similar concentrations of bile acids are found in bile (56Matoba N. Une M. Hoshita T. J. Lipid Res. 1986; 27: 1154-1162Abstract Full Text PDF PubMed Google Scholar) and the aqueous phase of the fecal stream (57McJunkin B. Fromm H. Sarva R.P. Amin P. Gastroenterology. 1981; 80: 1454-1464Abstract Full Text PDF PubMed Scopus (103) Google Scholar). It will therefore be worthwhile to investigate whether bile acids also induce COX-2in vivo. Further studies are also needed to confirm that induction of COX-2 by bile acids is mediated via AP-1 and to identify the responsible COX-2 promoter element(s). Given the already demonstrated effects of bile acids on the activity of PKC, it also becomes important to identify the specific isoforms of PKC that mediate the effects of bile acids on COX-2. A more detailed understanding of the precise mechanism(s) by which bile acids induce COX-2 should facilitate the development of chemopreventive strategies to diminish the risk of carcinogenesis within regions of the gastrointestinal tract exposed to bile acids. We thank Drs. Babette B. Weksler and David Zakim for reviewing this manuscript.

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