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• W2095495874 abstract "Oncogenic ras induces the expression of cyclooxygenase-2 (COX-2) in a variety of cells. Here we investigated the role of transforming growth factor-β (TGF-β) in the Ras-mediated induction of COX-2 in intestinal epithelial cells (RIE-1). RIE-1 cells were transfected with an inducible Ha-RasVal12 cDNA and are referred as RIE-iRas cells. the addition of 5 mmisopropyl-1-thio-β-d-galactopyranoside (IPTG) induced the expression of Ha-RasVal12, closely followed by an increase in the expression of COX-2. Neutralizing anti-TGF-β antibody partially blocked the Ras-induced increase in COX-2. Combined treatment with IPTG and TGF-β1 resulted in a 20–50-fold increase in the levels of COX-2 mRNA. The t 12 of COX-2 mRNA was increased from 13 to 24 min by Ha-Ras induction alone. The addition of TGF-β1 further stabilized the COX-2 mRNA (t 12 > 50 min). Stable transfection of a luciferase reporter construct containing the COX-2 3′-untranslated region (3′-UTR) revealed that TGF-β1 treatment and Ras induction each stabilized the COX-2 3′-UTR. Combined treatment with IPTG and TGF-β1 synergistically increased the luciferase activity. Furthermore, a conserved AU-rich region located in the proximal COX-2 3′-UTR is required for maximal stabilization of COX-2 3′-UTR by Ras or TGF-β1 and is necessary for the synergistic stabilization of COX-2 3′-UTR by oncogenic Ras and TGF-β1. Oncogenic ras induces the expression of cyclooxygenase-2 (COX-2) in a variety of cells. Here we investigated the role of transforming growth factor-β (TGF-β) in the Ras-mediated induction of COX-2 in intestinal epithelial cells (RIE-1). RIE-1 cells were transfected with an inducible Ha-RasVal12 cDNA and are referred as RIE-iRas cells. the addition of 5 mmisopropyl-1-thio-β-d-galactopyranoside (IPTG) induced the expression of Ha-RasVal12, closely followed by an increase in the expression of COX-2. Neutralizing anti-TGF-β antibody partially blocked the Ras-induced increase in COX-2. Combined treatment with IPTG and TGF-β1 resulted in a 20–50-fold increase in the levels of COX-2 mRNA. The t 12 of COX-2 mRNA was increased from 13 to 24 min by Ha-Ras induction alone. The addition of TGF-β1 further stabilized the COX-2 mRNA (t 12 > 50 min). Stable transfection of a luciferase reporter construct containing the COX-2 3′-untranslated region (3′-UTR) revealed that TGF-β1 treatment and Ras induction each stabilized the COX-2 3′-UTR. Combined treatment with IPTG and TGF-β1 synergistically increased the luciferase activity. Furthermore, a conserved AU-rich region located in the proximal COX-2 3′-UTR is required for maximal stabilization of COX-2 3′-UTR by Ras or TGF-β1 and is necessary for the synergistic stabilization of COX-2 3′-UTR by oncogenic Ras and TGF-β1. cyclooxygenase-2 transforming growth factor-β1 isopropyl-1-thio-β-d-galactopyranoside rat intestinal epithelial cells that conditionally express Ha-RasVal12 5,6-dichlorobenzimidazole riboside 3′-untranslated region AU-rich element base pair(s) Although increased expression of COX-21 in human and rodent intestinal tumors has been widely observed (1.Eberhart C.E. Coffey R.J. Radhika A. Giardiello F.M. Ferrenbach S. DuBois R.N. Gastroenterology. 1994; 107: 1183-1188Abstract Full Text PDF PubMed Google Scholar, 2.Kargman S. O'Neill G. Vickers P. Evans J. Mancini J. Jothy S. Cancer Res. 1995; 55: 2556-2559PubMed Google Scholar, 3.Sano H. Kawahito Y. Wilder R.L. Hashiramoto A. Mukai S. Asai K. Kimura S. Kato H. Kondo M. Hla T. Cancer Res. 1995; 55: 3785-3789PubMed Google Scholar, 4.Shao J. Sheng H. Aramandla R. Pereira A. Lubet R.A. Hawk E. Grogan L. Kirsch I.R. Washington M.K. Beauchamp R.D. DuBois R.N. Carcinogenesis. 1999; 20: 185-191Crossref PubMed Scopus (84) Google Scholar), the mechanisms that regulate the expression of COX-2 in colonic tumors are not completely understood. COX-2 expression is induced by cytokines, growth factors, and tumor promoters (reviewed in Ref. 5.Williams C.S. DuBois R.N. Am. J. Physiol. 1996; 270: G393-G400PubMed Google Scholar). Up-regulation of COX-2 is a downstream effect of Ras-mediated transformation in intestinal epithelial cells (RIE-1) (6.Sheng G.G. Shao J. Sheng H. Hooton E.B. Isakson P.C. Morrow J.D. Coffey R.J. DuBois R.N. Beauchamp R.D. Gastroenterology. 1997; 113: 1883-1891Abstract Full Text PDF PubMed Scopus (180) Google Scholar), fibroblasts (7.Sheng H. Williams C.S. Shao J. Liang P. DuBois R.N. Beauchamp R.D. J. Biol. Chem. 1998; 273: 22120-22127Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar), mammary epithelial cells (8.Subbaramaiah K. Telang N. Ramonetti J.T. Araki R. DeVito B. Weksler B.B. Dannenberg A.J. Cancer Res. 1996; 56: 4424-4429PubMed Google Scholar), and non-small cell lung cancer cells (9.Heasley L.E. Thaler S. Nicks M. Price B. Skorecki K. Nemenoff R.A. J. Biol. Chem. 1997; 272: 14501-14504Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). The mechanisms underlying the regulation of COX-2 expression are complex. Previously, it was reported that COX-2 expression was regulated at the transcriptional level by activated Ha-ras in mammary cells (8.Subbaramaiah K. Telang N. Ramonetti J.T. Araki R. DeVito B. Weksler B.B. Dannenberg A.J. Cancer Res. 1996; 56: 4424-4429PubMed Google Scholar, 10.Mestre 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) and by oncogene v-src (11.Xie W. Herschman H.R. J. Biol. Chem. 1995; 270: 27622-27628Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar) and growth factors (12.Xie W. Herschman H.R. J. Biol. Chem. 1996; 271: 31742-31748Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar) in NIH 3T3 cells. Interleukin-1α induced rapid but transient activation of COX-2 transcription and also prolonged the half-life of the COX-2 mRNA (13.Ristimaki A. Garfinkel S. Wessendorf J. Maciag T. Hla T. J. Biol. Chem. 1994; 269: 11769-11775Abstract Full Text PDF PubMed Google Scholar). Post-transcriptional regulation of cytokine-induced cyclooxygenase-2 transcript isoforms by dexamethasone has also been reported (14.Ristimaki A. Narko K. Hla T. Biochem. J. 1996; 318: 325-331Crossref PubMed Scopus (210) Google Scholar). We have found that the induction of COX-2 in conditionally Ha-RasVal12-transformed Rat-1 cells occurs via a modest increase (∼50%) in COX-2 transcription and a 3-fold increase in the half-life of COX-2 mRNA (7.Sheng H. Williams C.S. Shao J. Liang P. DuBois R.N. Beauchamp R.D. J. Biol. Chem. 1998; 273: 22120-22127Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). The TGF-βs are 25-kDa homodimeric polypeptides belonging to a superfamily of growth regulatory molecules. TGF-β has previously been characterized as a potent growth inhibitor for cultured rat intestinal crypt cells (15.Kurokowa M. Lynch K. Podolsky D.K. Biochem. Biophys. Res. Commun. 1987; 142: 775-782Crossref PubMed Scopus (262) Google Scholar, 16.Barnard J.A. Beauchamp R.D. Coffey R.J. Moses H.L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1578-1582Crossref PubMed Scopus (319) Google Scholar, 17.Filmus J. Zhao J. Buick R.N. Oncogene. 1992; 7: 521-526PubMed Google Scholar, 18.Ko T.C. Beauchamp R.D. Townsend Jr., C.M. Thompson J.C. Surgery. 1993; 114: 147-154PubMed Google Scholar). Through the activation of specific receptors, the TGF-β ligands activate the Smad signal transduction pathway, which appears to serve an important tumor suppressor function (19.Markowitz S. Wang J. Myeroff L. Parsons R. Sun L. Lutterbaugh J. Fan R.S. Zborowska E. Kinzler K. Vogelstein B. Brattain M. Willson J.K.V. Science. 1995; 268: 1336-1338Crossref PubMed Scopus (2145) Google Scholar, 20.Hahn S.A. Schutte M. Hoque A.T.M.S. Moskaluk C.A. da Costa L.T. Rozenblum E. Weinstein C.L. Fischer A. Yeo C.J. Hruban R.H. Kern S.E. Science. 1996; 271: 350-353Crossref PubMed Scopus (2171) Google Scholar, 21.Takagi Y. Kohmura H. Futamura M. Kida H. Tanemura H. Shimokawa K. Saji S. Gastroenterology. 1996; 111: 1369-1372Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 22.Eppert K. Scherer S.W. Ozcelik H. Pirone R. Hoodless P. Kim H. Tsui L.C. Bapat B. Gallinger S. Andrulis I.L. Thomsen G.H. Wrana J.L. Attisano L. Cell. 1996; 86: 543-552Abstract Full Text Full Text PDF PubMed Scopus (779) Google Scholar). However, there is mounting evidence that TGF-β may enhance malignant transformation and tumor progression for several different epithelial tumors under certain circumstances (23.Torre-Amione G. Beauchamp R.D. Koeppen H. Park B.H. Schreiber H. Moses H.L. Rowley D.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1486-1490Crossref PubMed Scopus (383) Google Scholar, 24.Sieweke M.H. Thompson N.L. Sporn M.B. Bissell M.J. Science. 1990; 248: 1656-1660Crossref PubMed Scopus (183) Google Scholar, 25.Sieweke M.H. Bissell M.J. Crit. Rev. Oncog. 1994; 5: 297-311Crossref PubMed Scopus (96) Google Scholar, 26.Zhang X. Wang T. Batist G. Tsao M.S. Cancer Res. 1994; 54: 6122-6128PubMed Google Scholar, 27.Cui W. Fowlis D.J. Bryson S. Duffie E. Ireland H. Balmain A. Akhurst R.J. Cell. 1996; 86: 531-542Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar, 28.Oft M. Peli J. Rudaz C. Schwarz H. Beug H. Reichmann E. Genes Dev. 1996; 10: 2462-2477Crossref PubMed Scopus (565) Google Scholar, 29.Sheng H. Shao J. O'Mahony C.A. Lamps L. Albo D. Isakson P.C. Berger D.H. DuBois R.N. Beauchamp R.D. Oncogene. 1999; 18: 855-867Crossref PubMed Scopus (66) Google Scholar). One of the remarkable effects of TGF-β on intestinal epithelial cells and other cell types is the induction or augmentation of COX-2 expression (4.Shao J. Sheng H. Aramandla R. Pereira A. Lubet R.A. Hawk E. Grogan L. Kirsch I.R. Washington M.K. Beauchamp R.D. DuBois R.N. Carcinogenesis. 1999; 20: 185-191Crossref PubMed Scopus (84) Google Scholar, 29.Sheng H. Shao J. O'Mahony C.A. Lamps L. Albo D. Isakson P.C. Berger D.H. DuBois R.N. Beauchamp R.D. Oncogene. 1999; 18: 855-867Crossref PubMed Scopus (66) Google Scholar, 30.Gilbert R.S. Reddy S.T. Kujubu D.A. Xie W. Luner S. Herschman H.R. J. Cell. Physiol. 1994; 159: 67-75Crossref PubMed Scopus (47) Google Scholar, 31.Gilbert R.S. Reddy S.T. Targan S. Herschman H.R. Cell. Mol. Biol. Res. 1994; 40: 653-660PubMed Google Scholar, 32.Li J. Simmons D.L. Tsang B.K. Endocrinology. 1996; 137: 2522-2529Crossref PubMed Scopus (19) Google Scholar, 33.Pilbeam C. Rao Y. Voznesensky O. Kawaguchi H. Alander C. Raisz L. Herschman H. Endocrinology. 1997; 138: 4672-4682Crossref PubMed Scopus (34) Google Scholar, 34.Sheng H. Shao J. Hooton E.B. Tsujii M. DuBois R.N. Beauchamp R.D. Cell Growth Differ. 1997; 8: 463-470PubMed Google Scholar). Expression of either activated Ras or Src proteins activates transcription of the TGF-β1 gene (35.Birchenall-Roberts M.C. Ruscetti F.W. Kasper J. Lee H.D. Friedman R. Geiser A. Sporn M.B. Roberts A.B. Kim S.J. Mol. Cell. Biol. 1990; 10: 4978-4983Crossref PubMed Scopus (103) Google Scholar, 36.Geiser A.G. Kim S. Roberts A.B. Sporn M.B. Mol. Cell. Biol. 1991; 11: 84-92Crossref PubMed Google Scholar). Furthermore, TGF-β collaborates with oncogenic Ras in the transformation of mammary epithelial cells (28.Oft M. Peli J. Rudaz C. Schwarz H. Beug H. Reichmann E. Genes Dev. 1996; 10: 2462-2477Crossref PubMed Scopus (565) Google Scholar). Based upon the observations that COX-2 was overexpressed in 85–90% of human colon cancers (1.Eberhart C.E. Coffey R.J. Radhika A. Giardiello F.M. Ferrenbach S. DuBois R.N. Gastroenterology. 1994; 107: 1183-1188Abstract Full Text PDF PubMed Google Scholar) and that TGF-β was abnormally expressed in over 90% of human colon cancers (37.Avery A. Paraskeva C. Hall P. Flanders K.C. Sporn M. Moorghen M. Br. J. Cancer. 1993; 68: 137-139Crossref PubMed Scopus (114) Google Scholar), we hypothesized that TGF-β may play a role in the regulation of COX-2 expression during the adenoma to carcinoma sequence of events that are involved in the neoplastic transformation of colonic epithelial cells (4.Shao J. Sheng H. Aramandla R. Pereira A. Lubet R.A. Hawk E. Grogan L. Kirsch I.R. Washington M.K. Beauchamp R.D. DuBois R.N. Carcinogenesis. 1999; 20: 185-191Crossref PubMed Scopus (84) Google Scholar). This study describes the observation that TGF-β synergistically enhances the expression of COX-2 in conditionally Ha-Ras-transformed intestinal epithelial cells. We have also evaluated the mechanisms by which Ras and TGF-β mediate the induction of COX-2. TGF-β-neutralizing antibody partially inhibits the increased expression of COX-2 after induction of Ha-Ras, suggesting an important autocrine effect of Ras-induced TGF-β1 expression. Both Ha-RasVal12 and TGF-β1-mediated induction of COX-2 involve stabilization of COX-2 mRNA, and the combined effects of Ha-RasVal12 and TGF-β treatment cause a synergistic increase in COX-2 mRNA by prolonging the half-life of the mRNA. Using chimeric reporter constructs containing the COX-2 3′-untranslated region (3′-UTR) linked to the luciferase reporter gene, we provide the evidence that this region contains thecis-acting elements necessary to confer both Ha-Ras and TGF-β responsiveness. RIE-iRas cell line with an inducible activated Ha-RasVal12 cDNA was generated by using LacSwitch eukaryotic expression system (Stratagene, La Jolla, CA) and was maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 400 μg/ml G418 (Life Technologies, Inc.), and 150 μg/ml hygromycin B (Calbiochem). The Ha-RasVal12 cDNA is under the transcriptional control of the Lac operon in rat intestinal epithelial (RIE-1) cells. Isopropyl-1-thio-β-d-galactopyranoside (IPTG; Life Technologies, Inc.) at a concentration of 5 mm was used to induce the expression of mutated Ha-Ras. Anti-TGF-β antibody (R&D Systems, Inc. Minneapolis MN) was used to block the endogenous TGF-β activity. The extraction of total cellular RNA was performed as described previously (7.Sheng H. Williams C.S. Shao J. Liang P. DuBois R.N. Beauchamp R.D. J. Biol. Chem. 1998; 273: 22120-22127Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). RNA samples (20 μg/lane) were separated on formaldehyde-agarose gels and blotted onto nitrocellulose membranes. The blots were hybridized with cDNA probes labeled with [α-32P]dCTP by random primer extension (Stratagene). After hybridization and washes, the blots were subjected to autoradiography. 18 S rRNA signals were used as controls to determine integrity of RNA and equality of the loading. For determination of mRNA stability, RIE-iRas cells were treated with IPTG, TGF-β1, or both IPTG and TGF-β1 for 24 h, then the transcription was stopped by the addition of 100 μm DRB (5,6-dichlorobenzimidazole riboside; Sigma). The RNA samples were isolated at 0, 10, 20, 30, 40, and 50 min following the DRB treatment and analyzed for mRNA levels by Northern blotting. Immunoblot analysis was performed as described previously (38.Ko T.C. Sheng H.M. Reisman D. Thompson E.A. Beauchamp R.D. Oncogene. 1995; 10: 177-184PubMed Google Scholar). Briefly, the cells were lysed for 30 min in radio immunoprecipitation assay buffer (1× phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mg/ml phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 1 mmsodium orthovanadate), then clarified cell lysates were denatured and fractionated by SDS-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred to nitrocellulose membrane. The filters were then probed with the indicated antibodies, developed by the enhanced chemiluminescence system (ECL, Amersham Pharmacia Biotech), and exposed to X-AR5 film (Eastman Kodak Co.). Quantitation was by densitometry. The anti-COX-2 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-pan Ras antibody was purchased from Calbiochem. Reporter construct phPES2(−327/+59) containing the 5′-flanking region of the human COX-2 gene (nucleotides −327 to +59) was a kind gift of Dr. H. Inoue (National Cardiovascular Center Research Institute, Osaka, Japan) and described previously (39.Inoue 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). phPES2(−327/+59) was co-transfected with pcDNA3/zeo into RIE-iRas cells. Transfected cells were selected by neomycin (600 μg/ml), hygromycin (150 μg/ml), and zeocin (250 μg/ml). Pooled clones were used for determine the luciferase activity. The construction of reporter expression vectors pLuc+3′-UTR, pLuc+3′-UTRΔARE, and pLuc+ARE was described elsewhere. 2Dixon, D. A., Kaplan, C. D., McIntyre, T. M., Zimmerman, G. A., and Prescott, S. M. (2000) J. Biol. Chem. 275, in press. Briefly, a reporter vector, pLuc, was generated by inserting the luciferase cDNA into pcDNA3/zeo (Invitrogene, Carlsbad, CA). The addition of the COX-2 3′-UTR (1451 bp) was accomplished by polymerase chain reaction amplification of the COX-2 3′-UTR using XbaI-tailed primers and inserting them adjacent to the luciferase coding region to yield pLuc+3′-UTR. pLuc+3′-UTRΔARE was generated by digesting pLuc+3′-UTR with ApaI and ScaI to release a 1036-bp region of the COX-2 3′-UTR, and this 3′-UTR region was cloned into the ApaI and filled-in XbaI sites of pLuc. For pLuc+ARE, pLuc+3′-UTR was digested with PmeI andScaI to generate a 1818-bp Luc+ARE fragment that was inserted into pcDNA3/zeo vector. RIE-iRas cells were transfected with pLuc (RIE-iRas/luc), pLuc+3′-UTR (RIE-iRas/Luc+3′-UTR), pLuc+3′-UTRΔARE (RIE-iRas/Luc+3′-UTRΔARE), or pLuc+ARE (RIE-iRas/Luc+ARE). The stable transfected clones were selected with neomycin (600 μg/ml), hygromycin (150 μg/ml), and zeocin (250 μg/ml). For the luciferase activity assay, cells were treated for the indicated hours and lysed with passive lysis buffer (Promega, Madison WI). Twenty μl of lysate was used for the firefly luciferase reading by using a luciferase reporter assay system (Promega) and a model TD-20/20 luminometer. Firefly luciferase values were standardized to the protein contents and presented as mean ± S.E. of assays performed in triplicate. We previously reported that COX-2 expression is significantly increased in a rat intestinal epithelial cell line stably transformed by Ha-Ras (6.Sheng G.G. Shao J. Sheng H. Hooton E.B. Isakson P.C. Morrow J.D. Coffey R.J. DuBois R.N. Beauchamp R.D. Gastroenterology. 1997; 113: 1883-1891Abstract Full Text PDF PubMed Scopus (180) Google Scholar) and conditional expression of Ha-Ras rapidly induced the expression of COX-2 in rat fibroblasts (7.Sheng H. Williams C.S. Shao J. Liang P. DuBois R.N. Beauchamp R.D. J. Biol. Chem. 1998; 273: 22120-22127Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). To determine whether COX-2 is an early target of oncogenic Ha-Ras in RIE cells, we have introduced the inducible Ha-RasVal12cDNA vectors into the RIE-1 cells (RIE-iRas). Noninduced RIE-iRas cells displayed the same nontransformed morphology as the parental RIE-1 cells. Morphological transformation of the RIE-iRas cells was observed between 24–48 h after IPTG treatment. During this interval, cell-cell contact inhibition was lost. The cells acquired a spindly appearance and grew in overlapping clusters (Fig.1 A). The morphological transformation of RIE-iRas cells could be completely reversed upon withdrawal of IPTG for 72 h (−72 h). As shown in Fig.1 B, the addition of IPTG into the culture medium induced activated Ha-Ras protein by 4 h. Thereafter, the level of Ras protein was continuously elevated for the duration of IPTG treatment. COX-2 was expressed at very low levels in RIE-iRas cells before IPTG treatment. The elevation of COX-2 protein was detected by 8 h after the addition of IPTG. The induction of COX-2 temporally coincided with the induction of Ha-Ras protein. We next investigated whether Ras-induced expression of COX-2 involves signaling through TGF-β. Northern analysis revealed that induction of Ras significantly increased the levels of TGF-β1 mRNA (Fig.2 A). In the absence of Ras induction, the addition of exogenous TGF-β1 transiently increased the levels of COX-2 in RIE-iRas cells, with a peak between 12–24 h following TGF-β treatment (Fig. 2 B). To determine whether endogenous TGF-β mediates a component of the Ras-induced COX-2 expression, TGF-β-neutralizing antibody was added before the IPTG treatment. Indeed, induction of COX-2 by activated Ha-Ras was partially blocked by TGF-β-neutralizing antibody (Fig. 2 C), implying a functional autocrine role for TGF-β in Ras-mediated induction of COX-2. Although TGF-β expression is normally restricted to the lumenal one-third of the intestinal epithelium (16.Barnard J.A. Beauchamp R.D. Coffey R.J. Moses H.L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1578-1582Crossref PubMed Scopus (319) Google Scholar, 40.Barnard J.A. Warwick G.J. Gold L.I. Gastroenterology. 1993; 105: 67-73Abstract Full Text PDF PubMed Google Scholar), the interstitial cells may produce large amounts of paracrine TGF-β. It was of interest to determine whether there was a cooperative effect of exogenous TGF-β1 and oncogenic Ras on the expression of COX-2. The RIE-iRas cells were treated with 3 ng/ml TGF-β1 without IPTG, and the levels of COX-2 mRNA were analyzed by Northern blotting. As shown in Fig.3 A, in the absence of Ras induction, treatment with TGF-β1 increased the level of COX-2 mRNA, which reached a peak (3.5-fold) by 8 h after the treatment. Induction of Ras by IPTG treatment increased the levels of COX-2 mRNA by 8 h, and the levels remained at an elevated plateau between 12–72 h. Interestingly, combined treatment with TGF-β1 and IPTG resulted in a marked induction of COX-2. The levels of COX-2 mRNA were synergistically elevated by the combination of TGF-β1 and Ras by 24 h after the combined treatment. A 20–50-fold increase in the levels of COX-2 mRNA was observed between 24–72 h. Western analysis further confirmed that the increased levels of COX-2 mRNA level also reflected an increase in the level of COX-2 protein (Fig. 3 B). We have previously reported that induction of Ha-Ras stabilizes COX-2 mRNA in Rat-1 fibroblasts (7.Sheng H. Williams C.S. Shao J. Liang P. DuBois R.N. Beauchamp R.D. J. Biol. Chem. 1998; 273: 22120-22127Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). To determine the mechanism of synergistic induction of COX-2 that results from oncogenic Ras and TGF-β1, we examined the stability of COX-2 mRNA in RIE-iRas cells after TGF-β1 and IPTG treatment. RIE-iRas cells were treated with IPTG, TGF-β1, or both IPTG and TGF-β1 for 24 h, then transcription was stopped by the addition of 100 μmDRB. The RNA samples were isolated at 0, 10, 20, 30, 40, and 50 min following the DRB treatment and analyzed for mRNA levels by Northern blotting. As demonstrated in Fig.4, A and B, COX-2 mRNA was rapidly degraded in noninduced RIE-iRas cells (t 12 ∼ 13 min). Individually, either TGF-β1 or IPTG treatment increased the stability of COX-2 mRNA to a similar extent (t 12 ∼ 24–30 min). The COX-2 mRNA from the RIE-iRas cells after combined TGF-β1 and IPTG treatment was extremely stable, and the t 12 was greater than 50 min. To further study transcriptional and post-transcriptional regulation of COX-2 in RIE-iRas cells, we transfected the luciferase reporter gene linked with either COX-2 promoter region or 3′-UTR into RIE-iRas cells. The 5′-flanking region of the human COX-2 gene (nucleotides −327 to +59) includes the nuclear factor responsible for Interleukin-6 expression (NF-IL6) site and the cyclic AMP response element (CRE) (39.Inoue 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). This reporter gene exhibited promoter activity that was modestly increased by induction of oncogenic Ras or by the addition of exogenous epidermal growth factor (EGF) (Fig.5 A). Treatment with TGF-β1 for 6 and 24 h did not alter the activity of this promoter region. We next engineered the RIE-iRas cells to express the cytomegalovirus promoter-driven luciferase reporter gene alone (RIE-Ras/luc) or linked with 1.5 kilobases of COX-2 3′-UTR (RIE-iRas/luc+3′-UTR). Stably transfected clones were selected and analyzed. Treatment with IPTG for 24 h increased the luciferase activity by 100% in RIE-iRas/luc+3′-UTR cells but did not alter the luciferase activity in RIE-iRas/luc control cells (Fig. 5 B). These results suggested that induction of Ras stabilized luciferase mRNA via the linked COX-2 3′-UTR and not through a transcriptional induction of the luciferase gene alone. Northern blot analysis confirmed that the increased luciferase activity resulted from an increase in the levels of luciferase mRNA (Fig. 5 C). The level of luciferase mRNA was clearly elevated by 8 h after the induction of Ha-Ras. A 2–3-fold increase in the level of luciferase mRNA was observed between 24–48 h after Ha-RasVal12 was induced by the addition of IPTG (Fig. 5 C). There was no significant induction of luciferase activity in control RIE-iRas/luc cells that were treated with IPTG, TGF-β1, or both IPTG and TGF-β1 for 6, 24, and 72 h (Fig. 5 D). However, the luciferase activity in RIE-iRas/luc+3′-UTR cells was induced by either IPTG or TGF-β1 treatment. The luciferase activity was additively increased by 24 h after combined treatment with TGF-β1 and IPTG. A synergistic increase in luciferase activity was observed by 72 h after the RIE-iRas/luc+3′-UTR cells were treated with both TGF-β1 and IPTG. These results are consistent with the findings of Northern analysis described in Fig. 3 A. The 3′-untranslated region of the COX-2 transcript is extremely AU-rich and contains 14 copies of the Shaw-Kamens sequence (AUUUA), otherwise known as AU-rich elements (AREs) (41.Shaw G. Kamen R. Cell. 1986; 46: 659-667Abstract Full Text PDF PubMed Scopus (3123) Google Scholar). To determine whether the stabilization of COX-2 mRNA by oncogenic Ras and TGF-β1 treatment was dependent upon highly conserved AU-rich elements located in the proximal COX-2 3′-UTR (14.Ristimaki A. Narko K. Hla T. Biochem. J. 1996; 318: 325-331Crossref PubMed Scopus (210) Google Scholar), we constructed two additional reporter vectors. The pLuc+3′-UTRΔARE construct was derived from pLuc+3′-UTR by removal of a fragment of 415 bp of nucleotide including 8 conserved AU-rich elements from the proximal COX-2 3′-UTR. For the pLuc+ARE, the 415 bp of conserved AU-rich fragment was linked to the downstream of luciferase cDNA in pLuc vector (see “Experimental Procedures”). RIE-iRas cells were transfected with either pLuc+3′-UTRΔARE or pLuc+ARE. Stably transfected clones were selected by the treatment with zeocin, pooled, and referred to as RIE-iRas/Luc+3′-UTRΔARE or RIE-iRas/Luc+ARE. IPTG treatment (Ha-Ras induction) of the RIE-iRas/Luc+3′-UTRΔARE cells resulted in a 44% increase in luciferase activity after 24 h (Fig. 6 A) and a 3-fold increase after 72 h (Fig. 6 B). Treatment of the RIE-iRas/Luc+3′-UTRΔARE cells with TGF-β1 alone altered luciferase activity by no more than 27%, and the combination of TGF-β1 and IPTG did not increase the levels over that observed with IPTG alone (Fig. 6,A and B). In contrast, in the RIE-iRas/Luc+ARE cells, IPTG treatment increased luciferase activity 5.4-fold by 24 h (Fig. 6 A) and 7.2-fold by 72 h (Fig. 6 B). TGF-β1 treatment of the RIE-iRas/Luc+ARE cells increased luciferase activity 4.7-fold by 24 h (Fig. 6 A) and 2.9-fold by 72 h (Fig. 6 B) after treatment. Treatment of RIE-iRas/Luc+ARE cells with the combination of IPTG and TGF-β1 increased the luciferase activity 15.7-fold by 24 h (Fig.6 A) and 23-fold by 72 h (Fig. 6 B). Thus, the conserved 415-bp ARE region of the COX-2 3′-UTR appears to be necessary for the synergistic increase in expression caused by the combination of activated Ras and TGF-β1. Numerous studies have suggested that cyclooxygenase activity and prostaglandin synthesis may be involved in intestinal carcinogenesis. COX-2 expression is increased in human colorectal adenocarcinomas when compared with normal adjacent colonic mucosa (1.Eberhart C.E. Coffey R.J. Radhika A. Giardiello F.M. Ferrenbach S. DuBois R.N. Gastroenterology. 1994; 107: 1183-1188Abstract Full Text PDF PubMed Google Scholar, 2.Kargman S. O'Neill G. Vickers P. Evans J. Mancini J. Jothy S. Cancer Res. 1995; 55: 2556-2559PubMed Google Scholar, 3.Sano H. Kawahito Y. Wilder R.L. Hashiramoto A. Mukai S. Asai K. Kimura S. Kato H. Kondo M. Hla T. Cancer Res. 1995; 55: 3785-3789PubMed Google Scholar). Furthermore there is mounting evidence that COX-2 expression in colorectal cancer cells provides a growth and survival advantage (42.Sheng H. Shao J. Morrow J.D. Beauchamp R.D. DuBois R.N. Cancer Res. 1998; 58: 362-366PubMed Google Scholar), increases tumor cell invasiveness (43.Tsujii M. Sunao K. DuBois R.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3336-3340Crossref PubMed Scopus (1328) Google Scholar), and enhances tumor angiogenesis (44.Tsujii M. Kawano S. Tsuji S. Sawaoka H. Hori M. DuBois R.N. Cell. 1998; 93: 705-716Abstract Full Text Full Text PDF PubMed Scopus (2214) Google Scholar). The importance of cyclooxygenase function in colorectal tumorigenesis is further supported by the observations that chronic ingestion of nonsteroidal anti-inflammatory drugs is associated with a reduced incidence of colorectal cancer in humans (45.Thun M.J. Namboodiri M.M. Calle E.E. Flanders W.D. Heath C.W.J. Cancer Res. 1993; 53: 1322-1327PubMed Google Scholar). Nonsteroidal anti-inflammatory drugs can also significantly inhibit colorectal tumorigenesis in animal models (46.Rao C.V. Rivenson A. Simi B. Zang E. Kelloff G. Steele V. Reddy B.S. Cancer Res. 1995; 55: 1464-1472PubMed Google Scholar, 47.Jacoby R.F. Marshall D.J. Newton M.A. Novakovic K. Tutsch K. Cole C.E. Lubet R.A. Kelloff G.J. Verma A. Moser A.R. Dove W.F. Cancer Res. 1996; 56: 710-714PubMed Google Scholar). Sel" @default.
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