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• W1974876622 abstract "The growth of any solid tumor depends on angiogenesis. Vascular endothelial growth factor (VEGF) plays a prominent role in vesical tumor angiogenesis regulation. Previous studies have shown that the peroxisome proliferator-activated receptor γ (PPARγ) was involved in the angiogenesis process. Here, we report for the first time that in two different human bladder cancer cell lines, RT4 (derived from grade I tumor) and T24 (derived from grade III tumor), VEGF (mRNA and protein) is differentially up-regulated by the three PPAR isotypes. Its expression is increased by PPARα, β, and γ in RT4 cells and only by PPARβ in T24 cells via a transcriptional activation of theVEGF promoter through an indirect mechanism. This effect is potentiated by an RXR (retinoid-X-receptor), selective retinoid LG10068 providing support for a PPAR agonist-specific action on VEGF expression. While investigating the downstream signaling pathways involved in PPAR agonist-mediated up-regulation of VEGF, we found that only the MEK inhibitor PD98059 reduced PPAR ligand-induced expression of VEGF. These data contribute to a better understanding of the mechanisms by which PPARs regulate VEGF expression. They may lead to a new therapeutic approach to human bladder cancer in which excessive angiogenesis is a negative prognostic factor. The growth of any solid tumor depends on angiogenesis. Vascular endothelial growth factor (VEGF) plays a prominent role in vesical tumor angiogenesis regulation. Previous studies have shown that the peroxisome proliferator-activated receptor γ (PPARγ) was involved in the angiogenesis process. Here, we report for the first time that in two different human bladder cancer cell lines, RT4 (derived from grade I tumor) and T24 (derived from grade III tumor), VEGF (mRNA and protein) is differentially up-regulated by the three PPAR isotypes. Its expression is increased by PPARα, β, and γ in RT4 cells and only by PPARβ in T24 cells via a transcriptional activation of theVEGF promoter through an indirect mechanism. This effect is potentiated by an RXR (retinoid-X-receptor), selective retinoid LG10068 providing support for a PPAR agonist-specific action on VEGF expression. While investigating the downstream signaling pathways involved in PPAR agonist-mediated up-regulation of VEGF, we found that only the MEK inhibitor PD98059 reduced PPAR ligand-induced expression of VEGF. These data contribute to a better understanding of the mechanisms by which PPARs regulate VEGF expression. They may lead to a new therapeutic approach to human bladder cancer in which excessive angiogenesis is a negative prognostic factor. transitional cell carcinoma vascular endothelial growth factor peroxisome proliferator-activated receptor retinoid-X-receptor mitogen-activated protein peroxisome proliferator response element extracellular signal-regulated kinase phosphatidylinositol (MAP kinase)/ERK kinase fatty acid-binding protein oxidized low-density lipoprotein reverse transcriptase enzyme-linked immunosorbent assay nuclear localization signal Bladder cancer comprises a wide range of tumors including transitional cell carcinoma (TCC)1 (1Friedel G.H. Nagy G.K. Cohen S.M. Bryan G.T. Cohen S.M. The Pathology of Bladder Cancer. I. CRC Press, Inc., Boca Raton, FL1983: 11-42Google Scholar, 2Pauli B.U. Alroy J. Weinstein R.S. Bryan G.T. Cohen S.M. The Pathology of Bladder Cancer. II. CRC Press, Inc., Boca Raton, FL1983: 41-140Google Scholar). This cancer represents the second cancer of the urinary tract in men. TCC is classified histopathologically into three types: superficial (papillary tumors), confined to the bladder wall (pT1, pTa tumors), and invasive (stages T2–T4). Superficial bladder cancers represent a heterogeneous group of tumors, and about 60% of them will recur after transurethral resection (3Heney N.M. Ahmed S. Flanagar M.J. Frable W. Corder M.P. Hafermann M.D. Hawkins I.R. J. Urol. 1983; 130: 1083-1086Crossref PubMed Scopus (822) Google Scholar). Some of them will progress to invasive and/or metastatic tumors and are therefore potentially lethal (4Levi F., La Vecchia C. Randimbison L. Franceschi S. Int. J. Cancer. 1993; 55: 419-554Crossref PubMed Scopus (24) Google Scholar). Angiogenesis, the process by which new vascular networks are formed from preexistent capillaries, is an essential component of the tumor growth and the metastatic pathway (5Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7209) Google Scholar). Tumor angiogenesis is regulated by the production of angiogenic stimulators (6Folkman J. Shing Y. J. Biol. Chem. 1992; 267: 10931-10934Abstract Full Text PDF PubMed Google Scholar) including the vascular endothelial growth factor (VEGF), which has emerged as a key regulatory factor of the angiogenic process in either physiological or pathological conditions (7Risau W. Nature. 1997; 386: 671-674Crossref PubMed Scopus (4834) Google Scholar, 8Korpelainen E.I. Alitalo K. Curr. Opin. Cell Biol. 1998; 10: 159-164Crossref PubMed Scopus (174) Google Scholar, 9Neufeld G. Cohen T. Gengrinovitch S. Poltorak Z. FASEB J. 1999; 13: 9-22Crossref PubMed Scopus (3142) Google Scholar). VEGF is overexpressed in most human tumors such as kidney and bladder cancers (10Brown L.F. Berse B. Jackman R.W. Tognazzi K. Manseau E.J. Dvorak H.F. Senger D.R. Am. J. Pathol. 1993; 143: 1255-1262PubMed Google Scholar). Elevated expression of VEGF in human tumor biopsies as well as the rise of VEGF levels in urine or serum have been reported to be independent prognostic and predictive factors of recurrence and disease progression in patients with superficial urothelial cancer (11O' Brien T. Cranston D. Fuggle S. Bicknell R. Harris A.L. Cancer Res. 1995; 55: 510-513PubMed Google Scholar, 12Crew J.P. O'Brien T. Bradburn M. Fuggle S. Bicknell R. Cranston D. Harris A.L. Cancer Res. 1997; 57: 5281-5285PubMed Google Scholar, 13Crew J.P. O'Brien T. Bicknell R. Fuggle S. Cranston D. Harris A.L. J. Urol. 1999; 161: 799-804Crossref PubMed Scopus (123) Google Scholar, 14Miyake H. Hara I. Yamanaka K. Gohji K. Arakawa S. Kaminodo S. Urology. 1999; 53: 302-307Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 15Bernardini S. Fauconnet S. Chabannes E. Adessi G.L. Bittard H. J. Urol. 2001; 166: 1275-1279Crossref PubMed Scopus (101) Google Scholar). Peroxisome proliferator-activated receptors (PPAR) belong to the steroid receptor superfamily and as such are ligand-activated transcription factors (16Issemann I. Green S. Nature. 1990; 347: 645-650Crossref PubMed Scopus (3037) Google Scholar, 17Dreyer C. Krey G. Keller H. Givel F. Helftenbein G. Wahli W. Cell. 1992; 68: 879-887Abstract Full Text PDF PubMed Scopus (1199) Google Scholar, 18Schoonjans K. Martin G. Staels B. Auwerx J. Curr. Opin. Lipidol. 1997; 8: 159-166Crossref PubMed Scopus (468) Google Scholar, 19Desvergne B. Wahli W. Endocr. Rev. 2000; 20: 649-688Google Scholar). They control gene expression by binding with their heterodimeric partner retinoid-X-receptor (RXR) (20Kliewer S.A. Umesomo K. Noonan D.J. Heyman R.A. Evans R.M. Nature. 1992; 358: 771-774Crossref PubMed Scopus (1520) Google Scholar) to peroxisome proliferator responsive elements (PPREs) (17Dreyer C. Krey G. Keller H. Givel F. Helftenbein G. Wahli W. Cell. 1992; 68: 879-887Abstract Full Text PDF PubMed Scopus (1199) Google Scholar, 20Kliewer S.A. Umesomo K. Noonan D.J. Heyman R.A. Evans R.M. Nature. 1992; 358: 771-774Crossref PubMed Scopus (1520) Google Scholar, 21Palmer C.N.A. Hsu M.H. Griffin K.J. Johnson E.F. J. Biol. Chem. 1995; 270: 16114-16121Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Three PPAR isotypes, PPARα (NR1C1), PPARβ (NR1C2), and PPARγ (NR1C3) (22Nuclear Receptors Nomenclature Committee Cell. 1999; 97: 161-163Abstract Full Text Full Text PDF PubMed Scopus (940) Google Scholar) have been cloned and identified (17Dreyer C. Krey G. Keller H. Givel F. Helftenbein G. Wahli W. Cell. 1992; 68: 879-887Abstract Full Text PDF PubMed Scopus (1199) Google Scholar). PPARα is predominantly found in the liver, heart, kidney, brown adipose tissue, and stomach mucosa; PPARγ is primarily found in adipose tissue; PPARβ is ubiquitously expressed (23Kliewer S.A. Forman B.M. Blumberg B. Ong E.S. Borgmeyer U. Mangelsdorf D.J. Umesomo K. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7355-7359Crossref PubMed Scopus (1278) Google Scholar, 24Mukherjee R. Jow L. Croston G.E. Paterniti J.R. J. Biol. Chem. 1997; 272: 8071-8076Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar). Fatty acid derivatives and eicosanoids were identified as natural ligands for PPARs. Furthermore, fibrates, including WY 14,643, are synthetic ligands for PPARα that mediates the lipid-lowering activity of these drugs (25Devchand P.R. Keller H. Peters J.M. Vazquez M. Gonzalez F.J. Wahli W. Nature. 1996; 384: 39-43Crossref PubMed Scopus (1205) Google Scholar, 26Krey G. Braissant O. L'Horset F. Kalkhoven E. Perroud M. Parker M.G. Wahli W. Mol. Endocrinol. 1997; 11: 779-791Crossref PubMed Scopus (908) Google Scholar, 27Kliewer S.A. Sundseth S.S. Jones S.A. Brown P.J. Wisely G.B. Koble C.S. Devchand P. Wahli W. Willson T.M. Lenhard J.M. Lehmann J.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4318-4323Crossref PubMed Scopus (1885) Google Scholar, 28Forman B.M. Chen J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4312-4317Crossref PubMed Scopus (1861) Google Scholar). The synthetic antidiabetic thiazolidinedione (TZD) agents are specific PPARγ agonists (29Tontonoz P., Hu, E. Graves R.A. Budavari A.I. Spiegelman B.M. Genes Dev. 1994; 8: 1224-1234Crossref PubMed Scopus (1996) Google Scholar, 30Lehmann J.M. Moore L.B. Smith-Olivier T.A. Wilkison W.O. Willson T.M. Kliewer S.A. J. Biol. Chem. 1995; 270: 12953-12956Abstract Full Text Full Text PDF PubMed Scopus (3456) Google Scholar, 31Berger J. Bailey P. Biswas C. Cullinan C.A. Doebber T.W. Hayes N.S. Saperstein R. Smith R.G. Leibowitz M.D. Endocrinology. 1996; 137: 4189-4195Crossref PubMed Scopus (346) Google Scholar). Recently, the L-165041 compound has been identified as being the first PPARβ-selective synthetic agonist (32Berger J. Leibowitz M.D. Doebber T.W. Elbrecht A. Zhang B. Zhou G. Biswas C. Cullinan C.A. Hayes N.S., Li, Y. Tanen M. Ventre J., Wu, M.S. Berger G.D. Mosley R. Marquis R. Santini C. Sahoo S.P. Tolman R.L. Smith R.G. Moller D.E. J. Biol. Chem. 1999; 274: 6718-6725Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). PPARα plays an important role in fatty acid catabolism (33Lee S.S. Pineau T. Drago J. Lee E.J. Owens J.W. Kroetz D.L. Fernandez-Salguero P.M. Westphal H. Gonzales F.J. Mol. Cell. Biol. 1995; 15: 3012-3022Crossref PubMed Scopus (1498) Google Scholar) and homeostasis in the liver as well as in the control of inflammatory response (25Devchand P.R. Keller H. Peters J.M. Vazquez M. Gonzalez F.J. Wahli W. Nature. 1996; 384: 39-43Crossref PubMed Scopus (1205) Google Scholar,34Staels B. Koenig W. Habib A. Merval R. Lebret M. Torra I.P. Delerive P. Fadel A. Chinetti G. Fruchart J.C. Najib J. Maclouf J. Tedgui A. Nature. 1998; 393: 790-793Crossref PubMed Scopus (1053) Google Scholar). PPARγ is involved in lipid metabolism, glucose metabolism, preadipocyte differentiation, inflammatory response, and macrophage differentiation (18Schoonjans K. Martin G. Staels B. Auwerx J. Curr. Opin. Lipidol. 1997; 8: 159-166Crossref PubMed Scopus (468) Google Scholar, 35Jiang C. Ting A.T. Seed B. Nature. 1998; 391: 82-86Crossref PubMed Scopus (538) Google Scholar, 36Tontonoz P. Nagy L. Alvarez J.G. Thomazy V.A. Evans R.M. Cell. 1998; 93: 241-252Abstract Full Text Full Text PDF PubMed Scopus (1610) Google Scholar, 37Ricote M. Huang J. Fajas L., Li, A. Welch J. Najib J. Witztum J.L. Auwerx J. Palinski W. Glass C.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7614-7619Crossref PubMed Scopus (682) Google Scholar, 38Spiegelman B.M. Cell. 1998; 93: 153-155Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). The PPARβ function is poorly known. However, this receptor might be linked to colorectal cancer (39Nagy L. Tontonoz P. Alvarez J.G. Chen H. Evans R.M. Cell. 1998; 93: 229-240Abstract Full Text Full Text PDF PubMed Scopus (1586) Google Scholar) and skin wound healing (40Michalik L. Desvergne B. Dreyer C. Gavillet M. Laurini R. Wahli W. Int. J. Dev. Biol. 2002; 46: 105-114PubMed Google Scholar). VEGF expression is regulated by many growth factors, environmental factors, and cytokines. A PPARγ-mediated up-regulation of VEGF (mRNA and protein secretion) has been established in human vascular smooth muscle cells (41Yamakawa K. Hosoi M. Koyama H. Tanaka S. Fukumoto S. Morii H. Nishizawa Y. Biochem. Biophys. Res. Commun. 2000; 271: 571-574Crossref PubMed Scopus (140) Google Scholar). In addition, oxidized low-density lipoproteins (Ox-LDL) up-regulate VEGF expression in macrophages and endothelial cells, at least in part, through the activation of PPARγ (42Inoue M. Itoh H. Tanaka T. Chun T.H. Doi K. Fukunaga Y. Sawada N. Yamshita J. Masatsugu K. Saito T. Sakagushi S. Sone M. Yamahara K.I. Yurugi T. Nakao K. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 560-566Crossref PubMed Scopus (154) Google Scholar). Two of the major oxidized lipid components of Ox-LDL, 9-hydroxy-(S)-10,12-octadecadienoic acid (9-HODE), and 13-hydroxy-(S)-10,12-octadecadienoic acid (13-HODE) have been identified as endogenous activators and ligands of PPARγ (39Nagy L. Tontonoz P. Alvarez J.G. Chen H. Evans R.M. Cell. 1998; 93: 229-240Abstract Full Text Full Text PDF PubMed Scopus (1586) Google Scholar). All of these studies suggest that PPARγ may be an important molecular target for the development of therapeutic inhibitors of angiogenesis in the treatment of cancer. No effect on VEGF expression has been observed in the presence of PPARα and PPARβ agonists. So far, in human cancers, a PPAR-mediated regulation of VEGF expression has never been described. Taking into account the importance of VEGF in the angiogenic process and its prognostic significance in the fate of TCC, the present investigation aimed to study VEGF gene regulation by the three PPAR isotypes (α, β, and γ) in RT4 cells (derived from grade I tumor) and T24 cells (derived from grade III tumor). Both groups of cells were derived from human bladder cancer and were used to clarify the intracellular signaling mechanisms involved. In this study, we uncovered a differential regulation of VEGF expression by PPARs according to the differentiated state of the cells. This regulated VEGF expression occurs through a transcriptional activation of the VEGF promoter via an indirect mechanism requiring an intermediary protein factor. In addition, the MAP kinase ERK 1/2 pathway modulates this regulation because an inhibition of PPAR-induced VEGF expression was observed only in the presence of PD98059 (MAP kinase/ERK 1/2 inhibitor). The hypolipidemic drug WY 14,643 came from Chemsyn Science Laboratories (Campro Scientific, Veenendaal, The Netherlands). L-165041, LG10068, and BRL 49653 compounds were a kind gift from Parke Davis. The MAP kinase/ERK 1/2 inhibitor PD98059 and the p38 MAP kinase-specific inhibitor SB203580 were purchased from Calbiochem(France Biochem, Meudon, France). Cycloheximide, actinomycin D, and wortmannin (specific PI 3-kinase inhibitor activity) were purchased from Sigma (La Verpillère, France). Ligands were dissolved in 100% Me2SO or ethanol and added to cell cultures at a concentration of less than 0.1%. The RT4 and T24 cell lines were purchased from the American Type Culture Collection (Biovalley, Conches, France). The cells were maintained at 37 °C in a 5% CO2 atmosphere in phenol red-free Mc COY's 5a medium (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen), 1% antibiotic antimycotic mixture (10 mg/ml streptomycin, 10,000 units/ml penicillin, 25 μg/ml amphotericin B), 2 mm glutamine, and 15 mm Hepes (Sigma). The cells were tested for the absence of mycoplasma before the experiments were started. For the VEGF expression studies, cells were grown to 100% confluence to avoid any variation in VEGF expression in Mc COY's 5a medium supplemented with 5% decomplemented fetal calf serum, 2 mm glutamine, and 15 mm Hepes. Before stimulation, cells were washed three times for 24 h with serum-free Mc COY's 5a medium in order to prevent any remaining serum effect. For stimulation with the PPAR ligands (WY 14,643 or L-165041 or BRL 49653) and RXR ligand LG10068, cells were incubated for 24 h in serum-free Mc COY's 5a medium. In the inhibitory experiments of protein synthesis and cellular signaling pathways, confluent cells were incubated for 24 h with 10 μg/ml cycloheximide, 1 or 20 μm PD98059, 100 nmwortmannin, or 10 μm SB203580 alone or in the presence of PPAR agonists. The VEGF mRNA expression analysis was then measured by Northern blotting as described below. The pSG5 hPPARα, pBS hPPARβ, and pBS hPPARγ plasmids were a kind gift from L. Michalik (IBA, Lausanne, Switzerland). They were used as positive controls in RT-PCR assays, generating fragments of 125-bp, 100-bp, and 130-bp lengths, respectively, corresponding to the coding region from the A/B domain of each nuclear receptor. The reporter plasmid Cyp2XPal-LUC (26Krey G. Braissant O. L'Horset F. Kalkhoven E. Perroud M. Parker M.G. Wahli W. Mol. Endocrinol. 1997; 11: 779-791Crossref PubMed Scopus (908) Google Scholar) was also a kind gift from L. Michalik. The VEGF promoter-luciferase reporter construct was a kind gift from A. Weisz (Instituto di Patologia generale e Oncologia, Facultà di Medicina e chirurgica, seconda Università di Napoli, Naples, Italy). This pGL2 basic vector contains the human VEGF promoter from −2279 to +56, linked to the firefly luciferase reporter gene (43Minchenko A. Salceda S. Bauer T. Caro J. Cell Mol. Biol. Res. 1994; 40: 35-39PubMed Google Scholar). The eukaryotic expression vector pSG▵2 containing the NLS LacZ gene from pMMuLV NLS LacZ (NLS LacZ construct) (44Ambrosino C. Cicatiello L. Cobellis G. Addeo R. Sica V. Bresciani F. Weisz A. Mol. Endocrinol. 1993; 7: 1472-1483PubMed Google Scholar) was used as an internal control of transfection efficiency and was called hereafter the β-gal plasmid. Total RNA from RT4 and T24 cells was isolated using TRIzolR reagent purchased from Invitrogen. Contaminating genomic DNA was removed with RNase-free DNase I (Invitrogen) according to the manufacturer's instructions. Total RNA from human tissues was used as a positive control and was provided by CLONTECH (Saint Quentin Yvelines, France). The synthesis of cDNA was performed in a total volume of 20 μl using 6 μg of total RNA extracted from human liver (positive control for PPARα and PPARγ) and human kidney (positive control for PPARβ) or 1 μg of total RNA extracted from RT4 or T24 cells. The reaction was performed in the presence of 200 units of Moloney murine leukemia virus reverse transcriptase (M-MLV RT) (Invitrogen) and 0.5 μg of oligo(dT)12–18 (Invitrogen). Subsequent amplifications of the partial cDNA encoding hPPARα, hPPARβ, and hPPARγ were performed using 6 μl of reverse-transcribed mixture, which was one-third diluted as a template with specific oligonucleotide primers, as follows: hPPARα sense, 5′-ACTCTGCCCCCTCTCGCCACTC-3′ and antisense, 5′-GCCAAAGCTTCCAGAACTATCCTC-3′; hPPARβ sense, 5′-GAGCAGCCACAGGAGGAAGCC-3′ and antisense, 5′-GCTGTGGTCCCCCAT-3′; hPPARγ sense, 5′-AGAGATGCCATTCTGGCCCAC-3′ and antisense, 5′-GTGGAGTAGAAATGCTGGAGA-3′. PCR reactions were performed in a total volume of 20 μl in the presence of 100 pmol of each oligonucleotide primer, 20 mmTris-HCl (pH 8.4), 50 mm KCl, 200 μm dNTP, 1.5 mm MgCl2, and 5 units of Taq DNA recombinant polymerase (Invitrogen). The expected sizes of PCR products for hPPARα, β, and γ were 125, 100, and 130 base pairs, respectively. Negative controls for reverse transcription and PCR amplifications were included. For the plasmid controls, 0.5 μg of plasmid was used. The PCR mixtures were subjected to 30 cycles of amplifications by denaturation (30 s at 94 °C), hybridization (30 s at 60 °C), and elongation (20 s at 72 °C). The PCR products were analyzed by 1.5% agarose gel electrophoresis with ethidium bromide. Total RNA from confluent cells was isolated using a commercially available kit TRI reagent (Molecular Research Center, Euromedex, Souffelwyersheim, France) according to the manufacturer's recommendations. The RNA (30 μg) was size-fractionated by electrophoresis on a 1.2% agarose gel and transferred to a nylon membrane (Zeta-Probe GT Genomic (Bio-Rad) using a vacuum blotting system. The filters were prehybridized for 5 min at 42 °C in a solution containing 50% formamide, 0.25m NaCl, 7% SDS, and 0.12 mNa2HPO4 (pH 7.2). The hybridizations were performed for 48 h in the same solution at 42 °C with the VEGF cDNA probe (45Chin K. Kurashima Y. Ogura T. Tajiri H. Yoshida S. Esumi H. Oncogene. 1997; 15: 437-442Crossref PubMed Scopus (157) Google Scholar) labeled with [α-32P]dCTP (PerkinElmer Life Sciences) using the random hexamer labeling method (Prime-a-gene labeling system, Promega, Lyon, France). After a rapid wash in 2× SSC solution at room temperature, two washes were performed for 15 min at room temperature in 2× SSC, 0.1% SDS and 0.5× SSC, 0.1% SDS, respectively. The final wash was performed for 15 min at 55 °C in 0.1× SSC, 0.1% SDS. To check the loading of equivalent amounts of total RNA and to normalize the experiments, the filters were hybridized with a 1200-bp mouse β-actin probe labeled with [α-32P]dCTP by the random hexamer labeling method. The VEGF and β-actin mRNA were quantitated using PhosphorImager analysis (Molecular ImagerR System, GS-505, Bio-Rad). After a serum-free period of 24 h, confluent cells were stimulated for 24 h in the presence of 50 μm WY 14,643 or 25 μm L-165041 or 10 μm BRL 49653 or vehicle. VEGF protein levels in cell-conditioned medium were determined by ELISA, using a human VEGF immunoassay (R&D Systems, Minneapolis, MN) according to the manufacturer's protocol. Data are expressed in ng/mg of total cellular proteins and are the mean values of three independent experiments in quadruplicate. The total cellular protein concentration was determined using a protein assay according to the Bradford method (Bio-Rad). To evaluate VEGF mRNA stability in RT4 and T24 cells, we measured the half-life of VEGF mRNA in the cells after 24 h of incubation in the presence of PPAR ligands. The transcription inhibitor actinomycin D (5 μg/ml) (Sigma) was added to the culture to block further gene transcription. Cells were harvested at 30 min, and 1, 2, 3, 4, and 6 h after the addition of actinomycin D. The amount of VEGF and β-actin at each time point was quantified after Northern blotting using phosphorimager analysis; the amount of VEGF mRNA was corrected for loading differences using the amount of β-actin mRNA. For functional studies, T24 cells were seeded in 6-well plates at a concentration of 1.5 × 105 cells per well in Mc COY's 5a medium supplemented with 5% delipidated serum. All transient transfections were performed using LipofectinRReagent (Invitrogen) according to the manufacturer's recommended protocol. A total amount of 4 μg of DNA (2 μg of β-gal plasmid and 2 μg of reporter plasmid) was transfected with Lipofectin reagent (2 μg/μg plasmid DNA). After 24 h, cells were incubated for 12 h in Mc COY's 5a medium supplemented with 5% delipidated serum and then stimulated with test drugs in the absence of serum for 24 h more. Cells were harvested using reporter lysis buffer purchased from Promega. Luciferase activity was measured using the luciferase assay system (Promega) according to the manufacturer's recommendations. The β-galactosidase activity was spectrophotometrically measured using orthonitrophenyl β-d-galactopyranoside as substrate. Luciferase activity values were normalized to a β-galactosidase activity content, and -fold activation was calculated. Each experiment, subjected to a statistical analysis, was performed independently at least three times with similar results. The significance of the data was determined using Student's t test (two-tailed). p < 0.05 was deemed significant. The data presented consist of mean ±S.D. RT-PCR was performed to demonstrate the expression of all three hPPAR (α, β, and γ) mRNAs in RT4 and T24 cells culturedin vitro. Based on the primers used in this study to amplify the cDNA of hPPARα, hPPARβ, and hPPARγ, fragments were expected to be 125, 100, and 130 base pairs in length, respectively. RNA samples from human liver and kidney were used as positive controls as well as plasmids containing the fragments of 125, 100, and 130 bp of hPPARα, hPPARβ, and hPPARγ, respectively. As shown in Fig.1, all three PPAR mRNAs were expressed in both cell lines. Negative controls, performed in the absence of mRNA or directly on mRNA, yielded no detectable band (data not shown). Although the expression of hPPARγ mRNA and protein in T24 cells has been reported previously (46Guan Y.F. Zhang Y.H. Breyer R.M. Davis L. Breyer M.D. Neoplasia. 1999; 1: 330-339Crossref PubMed Google Scholar), this study demonstrates for the first time the expression of hPPARα and hPPARβ mRNAs in RT4 and T24 cells and that of hPPARγ mRNA in RT4 cells. To examine the regulation of VEGF expression in RT4 (derived from grade I tumor) and T24 (derived from grade III tumor) bladder cancer cells, we first investigated the ability of these tumor cells to express the VEGF gene constitutively. Total RNA was extracted from these cells and was subjected to Northern blot analysis. On the Northern blots (Fig. 2,upper panels), three bands at ∼5.2, 4.5, and 1.7 kb were observed with the VEGF-A cDNA probe. Thus, RT4 and T24 cell lines express VEGF-A. The basal VEGF mRNA levels were lower in T24 cells than in RT4 cells. In RT4 cells after the ligand-dependent activation of the three PPAR isotypes (WY 14,643 for PPARα, L-165041 for PPARβ, and BRL 49653 for PPARγ) for 24 h, we observed a significant induced VEGF mRNA expression in each case for each of the VEGF transcripts (Fig. 2). The 5.2-kb transcript level was increased 5.5- and 5.3-fold with WY 14,643 (50 μm) and L-165041 (25 μm), respectively. In the case of the 4.5-kb transcript, PPARα and β agonists increased its expression 5.6- and 6.2-fold, respectively. For the 1.7-kb transcript, PPARα and β ligands stimulated this transcript expression to the same extent with 4.8- and 4.6-fold increases, respectively. The thiazolidinedione BRL 49653, a PPARγ ligand, induced the three VEGF transcripts to a lower extent than the other two ligands, WY 14,643 and L-165041, with 2.7-, 2.7-, and 2.5-fold inductions for the 5.2-, 4.5-, and 1.7-kb transcripts, respectively. These results contrast with the expression of VEGF observed in T24 cells. Indeed, in these cells no effect of PPARα and γ ligands on VEGF expression was observed; only PPARβ regulated the VEGF gene with 2-, 3.6-, and 2.9-fold inductions for the 5.2-, 4.5-, and 1.7-kb transcripts, respectively. In conclusion, in cells derived from grade I bladder cancer, VEGF expression is regulated by PPARα, β, and γ. In contrast, in cells derived from grade III bladder cancer, PPARα and PPARγ-mediated up-regulation of VEGF expression cannot be found despite the presence of receptors in these cells. VEGF expression is induced only by PPARβ. Thus, for the first time we demonstrate a differential up-regulation of the expression of VEGF mRNA by PPAR agonists in bladder cancer cells according to the differentiation state of the cells. To determine whether the up-regulation of VEGF mRNA levels by PPAR ligands correlates with higher VEGF protein levels in RT4- and T24 cell-conditioned media, we treated cells with vehicle alone or with 50 μm WY 14,643, or 25 μm L-165041, or 10 μm BRL 49653 for 24 h. Then, we performed an enzyme-linked immunosorbent assay analysis of RT4 and T24 cell-conditioned media (Fig.3). The amount of VEGF proteins was greater in RT4 cell-conditioned medium than that measured in T24 cell-conditioned medium. We found that the conditioned media of RT4 and T24 control cells (in the presence of vehicle alone) contained 3.7 ± 0.6 and 1.1 ± 0.2 ng/mg total cellular proteins, respectively. The PPAR activators WY 14,643, L-165041, and BRL 49653 significantly increased VEGF protein levels by 2.6-, 3-, and 1.7-fold, respectively, after 24 h stimulation of RT4 cells. In T24 cell-conditioned medium, only the PPARβ activator L-165041 increased VEGF protein levels by 4.4-fold. Thus, the PPAR agonist-dependent increase in VEGF gene expression correlates with increased levels of VEGF protein in the culture medium. To examine the efficacy of a retinoid in potentiating the PPAR ligand effect on VEGF mRNA expression, and thus to confirm the specificity of the effect of PPAR agonists WY 14,643, L-165041, and BRL 49653, we treated cells with 1 μm LG10068, a RXR-selective ligand, alone or in the presence of PPAR ligands. PPARs are known to activate cis-acting elements in the promoters of target genes as heterodimers with RXR (20Kliewer S.A. Umesomo K. Noonan D.J. Heyman R.A. Evans R.M. Nature. 1992; 358: 771-774Crossref PubMed Scopus (1520) Google Scholar). As shown in Fig. 4, no VEGF transcript was induced by LG10068 alone after 24 h of stimulation. The RXR agonist potentiated the effects of WY 14,643, L-165041, and BRL 49653 for the three VEGF transcripts. This result indicates the involvement of the RXR/PPAR heterodimer complex in the regulation of VEGF expression in RT4 cells. To determine whether PPAR synthetic ligands can increase the stability of VEGF mRNA, cells were left untreated or treated with 50 μm WY 14,643 or 25 μm L-165041 or 10 μm BRL 49653 for 24 h prior to the addition of the transcriptional inhibitor actinomycin D (5 μg/ml). Then, the VEGF mRNA half-life was estimated with quantitative Northern blot analysis. As shown in Fig. 5, in the control cells there was a rapid decay of VE" @default.
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• W1974876622 date "2002-06-01" @default.
• W1974876622 modified "2023-09-29" @default.
• W1974876622 title "Differential Regulation of Vascular Endothelial Growth Factor Expression by Peroxisome Proliferator-activated Receptors in Bladder Cancer Cells" @default.
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• W1974876622 doi "https://doi.org/10.1074/jbc.m200172200" @default.
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