A Novel Positive Feedback Loop between Peroxisome Proliferator-activated Receptor-δ and Prostaglandin E2 Signaling Pathways for Human Cholangiocarcinoma Cell Growth
2006; Elsevier BV; Volume: 281; Issue: 45 Linguagem: Inglês
10.1074/jbc.m600135200
ISSN1083-351X
AutoresLihong Xu, Chang Han, Tong Wu,
Tópico(s)Cancer, Lipids, and Metabolism
ResumoPeroxisome proliferator-activated receptor-δ (PPARδ) is a nuclear receptor implicated in lipid oxidation and the pathogenesis of obesity and diabetes. This study was designed to examine the potential effect of PPARδ on human cholangiocarcinoma cell growth and its mechanism of actions. Overexpression of PPARδ or activation of PPARδ by its pharmacological ligand, GW501516, at low doses (0.5–50 nm) promoted the growth of three human cholangiocarcinoma cell lines (CCLP1, HuCCT1, and SG231). This effect was mediated by induction of cyclooxygenase-2 (COX-2) gene expression and production of prostaglandin E2 (PGE2) that in turn transactivated epidermal growth factor receptor (EGFR) and Akt. In support of this, inhibition of COX-2, EGFR, and Akt prevented the PPARδ-induced cell growth. Furthermore, PPARδ activation or PGE2 treatment induced the phosphorylation of cytosolic phospholipase A2α (cPLA2α), a key enzyme that releases arachidonic acid (AA) substrate for PG production via COX. Overexpression or activation of cPLA2α enhanced PPARδ binding to PPARδ response element (DRE) and increased PPARδ reporter activity, indicating a novel role of cPLA2α for PPARδ activation. Consistent with this, AA enhanced the binding of PPARδ to DRE, in vitro, suggesting a direct role of AA for PPARδ activation. In contrast, although PGE2 treatment increased the DRE reporter activity in intact cells, it failed to induce PPARδ binding to DRE in cell-free system, suggesting that cPLA2α-mediated AA release is required for PGE2-induced PPARδ activation. Taken together, these observations reveal that PPARδ induces COX-2 expression in human cholangiocarcinoma cells and that the COX-2-derived PGE2 further activates PPARδ through phosphorylation of cPLA2α. This positive feedback loop plays an important role for cholangiocarcinoma cell growth and may be targeted for chemoprevention and treatment. Peroxisome proliferator-activated receptor-δ (PPARδ) is a nuclear receptor implicated in lipid oxidation and the pathogenesis of obesity and diabetes. This study was designed to examine the potential effect of PPARδ on human cholangiocarcinoma cell growth and its mechanism of actions. Overexpression of PPARδ or activation of PPARδ by its pharmacological ligand, GW501516, at low doses (0.5–50 nm) promoted the growth of three human cholangiocarcinoma cell lines (CCLP1, HuCCT1, and SG231). This effect was mediated by induction of cyclooxygenase-2 (COX-2) gene expression and production of prostaglandin E2 (PGE2) that in turn transactivated epidermal growth factor receptor (EGFR) and Akt. In support of this, inhibition of COX-2, EGFR, and Akt prevented the PPARδ-induced cell growth. Furthermore, PPARδ activation or PGE2 treatment induced the phosphorylation of cytosolic phospholipase A2α (cPLA2α), a key enzyme that releases arachidonic acid (AA) substrate for PG production via COX. Overexpression or activation of cPLA2α enhanced PPARδ binding to PPARδ response element (DRE) and increased PPARδ reporter activity, indicating a novel role of cPLA2α for PPARδ activation. Consistent with this, AA enhanced the binding of PPARδ to DRE, in vitro, suggesting a direct role of AA for PPARδ activation. In contrast, although PGE2 treatment increased the DRE reporter activity in intact cells, it failed to induce PPARδ binding to DRE in cell-free system, suggesting that cPLA2α-mediated AA release is required for PGE2-induced PPARδ activation. Taken together, these observations reveal that PPARδ induces COX-2 expression in human cholangiocarcinoma cells and that the COX-2-derived PGE2 further activates PPARδ through phosphorylation of cPLA2α. This positive feedback loop plays an important role for cholangiocarcinoma cell growth and may be targeted for chemoprevention and treatment. Cholangiocarcinoma is a highly malignant neoplasm of the biliary tree, accounting for about 10–15% of the primary liver cancers. It often arises from background conditions that cause long standing inflammation, injury, and reparative biliary epithelial cell proliferation, such as primary sclerosing cholangitis (PSC), clonorchiasis, hepatolithiasis, or complicated fibropolycystic diseases (1Gores G.J. Hepatology. 2003; 37: 961-969Crossref PubMed Scopus (248) Google Scholar, 2Sirica A.E. Hepatology. 2004; 41: 5-15Crossref Scopus (264) Google Scholar, 3Berthiaume E.P. Wands J. Semin. Liver Dis. 2004; 24: 127-137Crossref PubMed Scopus (85) Google Scholar, 4Wu T. Biochim. Biophys. Acta. 2005; 1755: 135-150PubMed Google Scholar). Although chronic inflammation and cellular injury within bile ducts, together with partial obstruction of bile flow, appear to be relevant predisposing factors in the pathogenesis of cholangiocarcinoma (1Gores G.J. Hepatology. 2003; 37: 961-969Crossref PubMed Scopus (248) Google Scholar, 2Sirica A.E. Hepatology. 2004; 41: 5-15Crossref Scopus (264) Google Scholar, 3Berthiaume E.P. Wands J. Semin. Liver Dis. 2004; 24: 127-137Crossref PubMed Scopus (85) Google Scholar, 4Wu T. Biochim. Biophys. Acta. 2005; 1755: 135-150PubMed Google Scholar), the molecular mechanisms linking bile duct inflammation and cholangiocarcinogenesis remain to be further defined.Recent studies suggest that the cyclooxygenase-2 (COX-2) 2The abbreviations used are: COX-2, cyclooxygenase-2; AA, arachidonic acid; AACOCF3, arachidonyltrifluoromethyl ketone; cPLA2α, cytosolic phospholipase A2α; DRE, PPARδ response element; EGFR, epidermal growth factor receptor; GPCR, G protein-coupled receptor; PG, prostaglandin; PGE2, prostaglandin E2; PI3-K, phosphatidylinositol 3-kinase; PPARδ, peroxisome proliferator-activated receptor-δ; siRNA, small interfering RNA; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; MAPK, mitogen-activated protein kinase; ELISA, enzyme-linked immunosorbent assay. 2The abbreviations used are: COX-2, cyclooxygenase-2; AA, arachidonic acid; AACOCF3, arachidonyltrifluoromethyl ketone; cPLA2α, cytosolic phospholipase A2α; DRE, PPARδ response element; EGFR, epidermal growth factor receptor; GPCR, G protein-coupled receptor; PG, prostaglandin; PGE2, prostaglandin E2; PI3-K, phosphatidylinositol 3-kinase; PPARδ, peroxisome proliferator-activated receptor-δ; siRNA, small interfering RNA; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; MAPK, mitogen-activated protein kinase; ELISA, enzyme-linked immunosorbent assay.-derived prostaglandin E2 (PGE2), a potent lipid inflammatory mediator, may play an important role in cholangiocarcinogenesis (4Wu T. 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Moreover, our data show that the COX-2-derived PGE2 further activates PPARδ through a novel cPLA2α-dependent mechanism, thus forming a positive feedback loop that coordinately promotes tumor cell growth.EXPERIMENTAL PROCEDURESMaterials—Cell culture medium and Lipofectamine Plus™ reagent were purchased from Invitrogen. Cell proliferation reagent WST-1 was purchased from Roche Applied Science. [3H]Thymidine was from PerkinElmer Life Sciences. Luciferase Assay System and reporter lysis buffer were from Promega Corporation. Antibody providers are as follows: anti-COX-2 (Cayman Chemical Co.); anti-cPLA2α, anti-PPARδ, and anti-EGFR (Santa Cruz Biotechnology); anti-phospho-cPLA2α (Ser505) and Akt Kinase Assay kit (Cell Signaling Technology); antiphospho-EGFR (BD Biosciences), and anti-β-actin (Sigma). Chemiluminescence detection reagent was from Amersham Biosciences. PPARδ agonist, GW501516, was purchased from Cayman Chmical Co. (Ann Arbor, MI). Prostaglandin E2, indomethacin, arachidonic acid, stearic acid, oleic acid, α-linolenic acid, A23187, the cPLA2 inhibitors AACOCF3 and pyrrolidine derivative (cat. 525143), the EGFR tyrosine kinase inhibitor AG1478, the p38 kinase inhibitor SB203580, the protein kinase C inhibitor bisindolylmaleimide I, the phosphatidylinositol 3-kinase inhibitor LY294002 and the p44/42 MAPK inhibitor PD98059 were purchased from Calbiochem. The PGE2 enzyme immunoassay system was purchased from Amersham Biosciences. The PPARδ Transcription Factor Assay kit and recombinant human PPARδ were from Cayman Chemical Co. The nuclear extraction kit was purchased from Sigma. siRNA-PPARδ, siRNA-COX-2, and siRNA-control were from Dharmacon, Inc. The 5′-biotinylated DRE oligonucleotides were synthesized by Sigma-Genosys and the unlabeled DRE oligonucleotides were from Integrated DNA Technologies, Inc. (Coralville, IA). The immobilized streptavidin beads were purchased from Pierce. Poly(dI-dC) was from Amersham Biosciences.Cell Culture and WST-1 Assay—Three cholangiocarcinoma cell lines, CCLP1, HuCCT1, and SG231 were cultured respectively in medium DMEM, RPMI 1640, and MEMα as previously described (8Han C. Leng J. Demetris A.J. Wu T. Cancer Res. 2004; 64: 1369-1376Crossref PubMed Scopus (125) Google Scholar, 10Wu T. Han C. Lunz 3rd, J.G. Michalopoulos G. Shelhamer J.H. Demetris A.J. Hepatology. 2002; 36: 363-373Crossref PubMed Scopus (64) Google Scholar, 15Han C. Wu T. J. Biol. Chem. 2005; 280: 24053-24063Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Cell growth was determined using the cell proliferation reagent WST-1, which is a tetrazolium salt cleaved by mitochondrial dehydrogenases in viable cells. Briefly, the cells (3000/well) were seeded on 96-well plate and incubated at 37 °C overnight. The cells were then treated with GW501516 for indicated time periods. WST-1 (10 μl) was subsequently added to each well, and the culture continued for 30 min to 4 h prior to measurement of OD450 nm using an automatic enzyme-linked immunosorbent assay plate reader.[3H]Thymidine Incorporation—The cells cultured in 24-well plates were incubated with different concentrations of GW501516 for 48 h. [3H]Thymidine (1 μCi/ml) was added to the medium during the last 4 h of culture. The cells were then washed twice with cold PBS and incubated with 5% trichloroacetic acid at 4 °C for 30 min to precipitate macromolecules. The precipitant was washed once with cold PBS and incubated with 2% SDS. The radioactivity was quantitated in a liquid scintillation counter.Transient Transfection and Luciferase Reporter Assay—Cells were seeded in 6-well plate in culture medium containing 10% FBS the day before transfection. On the following day, the cells in each well (80–90% confluence) were transfected with 1 μgof plasmid using Lipofectamine Plus reagent (Plus reagent 6 μl, Lipofectamine 4 μl) in serum-free medium. For co-transfection with two plasmids, double volume of Lipofectamine Plus reagent was used. After 4 h of transfection, the transfection medium was replaced with culture medium containing 10% fetal bovine serum. After 16 h of incubation, the cells were washed three times in ice-cold PBS and lysed by reporter lysis buffer on ice for 20 min. The cells were then scraped down and spun at 14,000 rpm for 10 min in cold room. The supernatant was collected for luciferase activity assay using a Berthold AutoLumat LB 953 luminometer (Nashua, NH).Preparation of Whole Cell Lysate and Immunoblotting—CCLP1 and HuCCT1 cells were grown on 6-well plates and treated with different concentration of GW501516 for different time in 0.5% fetal bovine serum medium. The vehicle, Me2SO, was added to the control culture. Following treatment for indicated time periods, the cells were washed twice with cold PBS and scraped down. The cell pellets were washed two more times with cold PBS and then resuspended in homogenization buffer containing 50 mm Hepes (pH 7.55), 1 mm EDTA, 1 mm dithiothreitol, and 1 mm mammalian protease inhibitor mixture (Sigma). The cell suspension was placed on ice and sonicated for 15 s × 4. The samples were then centrifuged at 14,000 rpm for 10 min at 4 °C, and the supernatants were collected as whole cell lysate. The total protein concentration was measured by BCA reagent (Pierce). The cell lysate was aliquoted and frozen at –80 °C until use. For immunoblotting, 30 μg of protein was separated on 4–20% Tris-glycine gels and the separated proteins were electrophoretically transferred onto the nitrocellulose membrane (Bio-Rad). Nonspecific binding was blocked with 5% nonfat milk dissolved in buffer PBS-T(0.5% Tween 20 in buffer PBS) for 1 h at room temperature. The membrane was then incubated overnight with primary antibodies (1:1000 dilution for COX-2, EGFR, p-EGFR, Akt, p-Akt, and β-actin; 1:2000 dilution for PPARδ) in 5% milk PBS-T. Following repeated washing with PBS-T the next day, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibody (1:10,000 dilution) for 1 h at room temperature. After washing the blots were developed using the ECL Western blotting detection system and exposed to Eastman Kodak MR radiographic films.Immunoprecipitation and Western Blotting for cPLA2α Phosphorylation—To immunoprecipitate cPLA2α, 500 μl of whole CCLP1 cell lysate (about 40 μg protein) in a 1.5-ml Eppendorf tube was precleared with 20 μl of protein A/G-agarose (Santa Cruz Biotechnology) for 1 h at 4 °C. The cleared cell lysate was then incubated with 5 μl of mouse anti-human cPLA2α monoclonal antibody at 4 °C for 3 h, with gentle agitation. 20 μl of protein A/G-agarose was then added, and the sample was kept at 4 °C for 16 h, with gentle agitation, to precipitate cPLA2α-antibody complex. The protein A/G-agarose pellet was collected by centrifuge and washed four times with cold homogenization buffer at 4 °C. 20 μl of SDS sample loading buffer was then added to the pellet, and the mixture was boiled for 5 min prior to SDS-PAGE using 4–20% Tris-glycine gels. After blocking non-specific binding, the blot was incubated overnight with rabbit anti-phospho-cPLA2α (Ser505) antibody (1:1000 dilution) in 5% milk PBS-T at 4 °C. The HRP-conjugated donkey anti-rabbit antibody (1:10,000 dilution) was used as the second antibody. Specific cPLA2α band was visualized by ECL Western blotting detection system.Measurement of PGE2 Production—CCLP1 cells cultured in serum-free medium in 6-well plates were treated as indicated in the text. The supernatant was collected and centrifuged to remove floating cells. 100 μl of each sample was used to measure PGE2 level using the PGE2 enzyme immunoassay system as previously described (54Leng J. Han C. Demetris A.J. Michalopoulos G.K. Wu T. Hepatology. 2003; 38: 756-768Crossref PubMed Scopus (253) Google Scholar, 55Han C. Michalopoulos G.K. Wu T. J. Cell Physiol. 2005; 207: 261-270Crossref Scopus (1) Google Scholar).Purification of Nuclear Extract—CCLP1 cells cultured in 100-mm dishes at 80–90% confluence were treated as described in the text. Following treatment, the cells were washed twice with ice-cold PBS and scraped with a rubber policeman. The cell pellet was then swelled in 5-fold volume of hypotonic buffer for 20 min on ice. Following homogenization using 27-gauge sterile needle on ice, the nuclei were pelleted by centrifugation at 600 × g for 10 min. The nuclei were then washed three times in the isotonic buffer and resuspended in HKMG buffer (10 mm HEPES, pH 7.9, 100 mm KCl, 5 mm MgCl2, 10% glycerol, 1 mm dithiothreitol, and 0.5% of Nonidet P-40) containing protease inhibitors and phosphatase inhibitors. The nuclei suspension was then subjected to sonication, and the cellular debris was removed by centrifugation at 14,000 rpm for 20 min at 4 °C. The supernatant was collected as nuclear extract and frozen at –80 °C until use. Aliquots of the nuclear extracts were used to quantitate the protein concentration using the BCA reagent.ELISA-based PPARδ Binding to Its DNA Response Element—The experiments were carried out using the 96-well enzyme-linked immunosorbent assay (ELISA) kit purchased from Cayman (Ann Arbor, MI). Briefly, the oligonucleotide containing the PPARδ binding consensus sequence was immobilized onto the bottom of wells. 50 μg of nuclear extract from treated cells or control cells were added to the dsDNA-coated well and incubated at 4 °C overnight. After complete washing, PPARδ antibody was added, and the samples were incubated at room temperature for 1 h. The HRP-conjugated secondary antibody and developing solution were sequentially added and the OD655 nm value was determined.Biotinylated DRE Oligonucleotide Precipitation Assay—The assay was performed as previous reported with modification (56Hata A. Seoane J. Lagna G. Montalvo E. Hemmati-Brivanlou A. Massague J. Cell. 2000; 100: 229-240Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). The nucleotide sequences of biotinylated PPARδ response element (DRE) were 5′-GCGTGAGCGCTCACAGGTCAATTCG-3′ and 5′-CCGAATTGACCTGTGAGCGCTCACG-3′ (45He T.C. Chan T.A. Vogelstein B. Kinzler K.W. Cell. 1999; 99: 335-345Abstract Full Text Full Text PDF PubMed Scopus (1029) Google Scholar). These two complementary strands were an
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