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Originally published In Press as doi:10.1074/jbc.M600135200 on September 11, 2006

J. Biol. Chem., Vol. 281, Issue 45, 33982-33996, November 10, 2006
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A Novel Positive Feedback Loop between Peroxisome Proliferator-activated Receptor-{delta} and Prostaglandin E2 Signaling Pathways for Human Cholangiocarcinoma Cell Growth*

Lihong Xu, Chang Han, and Tong Wu1

From the Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213

Received for publication, January 5, 2006 , and in revised form, August 24, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Peroxisome proliferator-activated receptor-{delta} (PPAR{delta}) 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 PPAR{delta} on human cholangiocarcinoma cell growth and its mechanism of actions. Overexpression of PPAR{delta} or activation of PPAR{delta} 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 PPAR{delta}-induced cell growth. Furthermore, PPAR{delta} activation or PGE2 treatment induced the phosphorylation of cytosolic phospholipase A2{alpha} (cPLA2{alpha}), a key enzyme that releases arachidonic acid (AA) substrate for PG production via COX. Overexpression or activation of cPLA2{alpha} enhanced PPAR{delta} binding to PPAR{delta} response element (DRE) and increased PPAR{delta} reporter activity, indicating a novel role of cPLA2{alpha} for PPAR{delta} activation. Consistent with this, AA enhanced the binding of PPAR{delta} to DRE, in vitro, suggesting a direct role of AA for PPAR{delta} activation. In contrast, although PGE2 treatment increased the DRE reporter activity in intact cells, it failed to induce PPAR{delta} binding to DRE in cell-free system, suggesting that cPLA2{alpha}-mediated AA release is required for PGE2-induced PPAR{delta} activation. Taken together, these observations reveal that PPAR{delta} induces COX-2 expression in human cholangiocarcinoma cells and that the COX-2-derived PGE2 further activates PPAR{delta} through phosphorylation of cPLA2{alpha}. This positive feedback loop plays an important role for cholangiocarcinoma cell growth and may be targeted for chemoprevention and treatment.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (1–4). 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 (1–4), the molecular mechanisms linking bile duct inflammation and cholangiocarcinogenesis remain to be further defined.

Recent studies suggest that the cyclooxygenase-2 (COX-2)2-derived prostaglandin E2 (PGE2), a potent lipid inflammatory mediator, may play an important role in cholangiocarcinogenesis (4). For example, increased COX-2 expression has been documented in cholangiocarcinoma cells and precancerous bile duct lesions but not in normal BECs (5–7). Overexpression of COX-2 in cultured human cholangiocarcinoma cells enhances PGE2 production and promotes tumor growth, whereas antisense depletion of COX-2 attenuates growth (8, 9). Treatment of cholangiocarcinoma cells with exogenous PGE2 increases tumor cell growth and prevents apoptosis (8–13). Consistent with these findings, selective COX-2 inhibitors prevent cholangiocarcinoma cell growth and invasion, in vitro and in nude mice (8, 9, 12–14), although their effect may be mediated through COX-2-dependent and -independent mechanisms. Transactivation of EGFR and Akt has recently been proposed as one of the important mechanisms for COX-2 and PGE2-mediated cholangiocarcinoma cell growth (15).

COX, including COX-1 and COX-2, is the rate-limited enzyme catalyzing the conversion of arachidonic acid (AA) into endoperoxide intermediates that are ultimately converted by specific synthases to prostanoids, including PGE2, the most abundant PG in human cholangiocarcinoma cells (16–19). Whereas COX-1 is constitutively expressed in most cells, COX-2 is highly induced by inflammatory cytokines/chemokines, growth factors, oncogene activation, and tumor promoters, thus contributing to the enhanced PG production when these signaling pathways are activated in inflammatory and neoplastic diseases (16–19). PGs transduce signals mainly through binding to their specific G protein-coupled receptors (GPCRs) along the plasma membrane. Although certain PGs including 15d-PGJ2 and PGI2 are known to activate peroxisome proliferators-activated receptors (PPARs) (20–22), the physiological implication of endogenous AA metabolism for PPAR activation in cells remains largely unknown.

PPARs belong to the nuclear hormone receptor superfamily and comprise of three subtypes: PPAR{alpha}, PPAR{gamma}, and PPAR{delta}/beta. As ligand-activated transcription factors, they form heterodimers with the retinoid X receptor (RXR) and bind to their response elements (PPREs) in the promoters of target genes upon activation (23, 24). A large body of evidence has documented an important role of PPARs in various cellular functions and in the pathogenesis of several human diseases including diabetes, obesity, and hyperlipidemia. PPAR{alpha} is highly expressed in hepatocytes and implicated in lipid catabolism (25–29), whereas PPAR{gamma} is predominantly expressed in adipose tissue and plays an important role in adipocyte differentiation, insulin sensitization, and glucose homeostasis (30–34). In contrast, PPAR{delta}/beta is ubiquitously expressed in most cells (26) and is implicated in fatty acid oxidation, cell differentiation, inflammation, cell motility, and cell growth (22, 35–44). More recently, emerging studies suggest a potential role of PPAR{delta} in carcinogenesis. For example, the expression of PPAR{delta} is elevated in human and rat colorectal cancer cells when compared with normal colon epithelial cells (45, 46). Exposure of Apcmin mice to the PPAR{delta} ligand, GW501516, increased the number and size of intestinal polyps (47). Conversely, disruption of PPAR{delta} in human colon cancer cells by targeted homologous recombination decreased tumor growth when the PPAR{delta}–/– cells were inoculated as xenografts in nude mice (48). These findings suggest a tumor-promoting role of PPAR{delta} during intestinal carcinogenesis. In addition, PPAR{delta} has also been implicated in the growth of other human cancers, including hepatocellular carcinoma, breast cancer, and prostate cancer (49, 50). PPAR{delta} is a downstream gene of Wnt-beta-catenin signal pathway and the target of nonsteroidal anti-inflammatory drugs (NSAIDs), which are COX inhibitors with anti-tumor effect (45, 51). Moreover, PPAR{delta} has also been shown to mediate the PGE2-induced intestinal adenoma growth (52). However, despite the documented tumor-promoting effect of PPAR{delta}, there is also evidence suggesting that PPAR{delta} might inhibit intestine tumor development (53). Therefore, the precise role of PPAR{delta} in tumorigenesis remains to be further defined.

This study was designed to evaluate the effect and mechanisms of PPAR{delta} in cholangiocarcinoma cell growth control. Our results demonstrate that overexpression of PPAR{delta} or activation of PPAR{delta} by its pharmacological ligand, GW501516, significantly enhances cholangiocarcinoma cell growth and this effect is mediated, at least in part, through induction of COX-2 expression and PGE2 production. Moreover, our data show that the COX-2-derived PGE2 further activates PPAR{delta} through a novel cPLA2{alpha}-dependent mechanism, thus forming a positive feedback loop that coordinately promotes tumor cell growth.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Cell culture medium and Lipofectamine PlusTM 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-cPLA2{alpha}, anti-PPAR{delta}, and anti-EGFR (Santa Cruz Biotechnology); anti-phospho-cPLA2{alpha} (Ser505) and Akt Kinase Assay kit (Cell Signaling Technology); antiphospho-EGFR (BD Biosciences), and anti-beta-actin (Sigma). Chemiluminescence detection reagent was from Amersham Biosciences. PPAR{delta} agonist, GW501516, was purchased from Cayman Chmical Co. (Ann Arbor, MI). Prostaglandin E2, indomethacin, arachidonic acid, stearic acid, oleic acid, {alpha}-linolenic acid, A23187 [GenBank] , 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 PPAR{delta} Transcription Factor Assay kit and recombinant human PPAR{delta} were from Cayman Chemical Co. The nuclear extraction kit was purchased from Sigma. siRNA-PPAR{delta}, 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 MEM{alpha} as previously described (8, 10, 15). 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).


Figure 1
Figure 1
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  		FIGURE 1.
Activation or overexpression of PPAR{delta} promotes human cholangiocarcinoma cell growth. A, WST-1 assay showing the effect of GW501516 on human cholangiocarcinoma cell growth. CCLP1, HuCCT1, and SG231 cells were seeded onto 96-well plate (3000 cells/well) in 10% fetal bovine serum medium overnight. After 24 h of serum deprivation, increasing concentrations of GW501516 were added to the cells (0.5–50 nM). After treatment for 24–72 h, WST-1 (10 µl) reagent was added to each well, and the cells were incubated for 4 h to determine OD value at 450 nM. B, [3H]thymidine incorporation assay showing the effect of GW501516 on human cholangiocarcinoma cell growth. The cells were treated with GW501516 for 48 h. [3H]Thymidine (1 µCi/ml) was added 4 h before the end of treatment. C, protein level of PPAR{delta} in human cholangiocarcinoma cell lines. 30 µg of whole cell lysate from untreated CCLP1, HuCCT1, and SG231 was used for SDS-PAGE and Western blot. Same amount of cell lystate from HepG2, a human hepatocellular carcinoma cell line, was used as the control. D, effect of PPAR{delta} overexpression on cholangiocarcinoma cell growth. CCLP1 and HuCCT1 cells (5000 cells/well) were attached to 96-well plate overnight. On the following day the cells in each well were transfected with 0.1 µg of the human PPAR{delta} expression plasmid or the control vector (SG5) using Lipofectamine Plus reagent for 4 h. The transfection medium was then removed and replaced with fresh medium and the cells were incubated overnight. Cell growth was determined by the WST-1 assay (panels a and b) and [3H]thymidine incorporation assay (panel c). *, p < 0.05 compared with vector control SG5. The protein level of PPAR{delta} was detected from the cells cultured on 6-well plate with the same transfection procedure. E, effect of PPAR{delta} overexpression and activation on DRE reporter activity. CCLP1 cells cultured on 6-well plates (80% confluence) were cotransfected with 1 µg of human DRE (PPAR{delta} response element) reporter construct and 1 µg of human PPAR{delta} expression plasmid or control vector SG5 for 4 h. Medium containing transfection reagent was then replaced by serum-free DMEM containing GW501516 (5 nM) or vehicle Me2SO (1: 10,000 dilution). The cells were then incubated overnight, and the cell lysates were obtained for the luciferase reporter activity assay. The values are expressed as mean ± S.D. **, p < 0.01, n = 3.

 
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 x 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 beta-actin; 1:2000 dilution for PPAR{delta}) 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 cPLA2{alpha} Phosphorylation—To immunoprecipitate cPLA2{alpha}, 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 cPLA2{alpha} 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 cPLA2{alpha}-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-cPLA2{alpha} (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 cPLA2{alpha} band was visualized by ECL Western blotting detection system.


Figure 2
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  		FIGURE 2.
PPAR{delta} enhances COX-2 expression and PGE2 production. A, influence of GW501516 on COX-2 expression in CCLP1 cells. CCLP1 cells (80–90% confluence) were treated with increasing concentrations of GW501516 (0.5–100 nM) for indicated time points. (The cells were serum-starved for 24 h before treatment.) The cell lysate was collected. 30 µg of protein were loaded onto a Tris-glycine gel for the Western blotting assay. B, influence of GW501516 on COX-2 expression in HuCCT1 cells. HuCCT1 cells were treated with GW501516 (0.5–500 nM) for indicated time points. 30 µg of protein from each sample were subjected to SDS-PAGE and Western blot analysis to determine COX-2 protein level. C, effect of GW501516 on PGE2 production in CCLP1 cells. CCLP1 cells were treated with GW501516 (1, 2, 5, 20 nM) for 8 h, and the medium was collected to determine PGE2 production. The values are expressed as mean ± S.D. (*, p < 0.05). D, effect of PPAR{delta} overexpression on COX-2 protein level. CCLP1 cells (80% confluence) cultured in 6-well plates were transfected with 1 µg of human PPAR{delta} expression plasmid or empty vector SG5. 30 µg of protein from each sample were subjected to SDS-PAGE and Western blot analysis to determine the COX-2 protein level. beta-Actin was used as loading control. Each experiment was repeated three times.

 
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 (54, 55).

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 x 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 PPAR{delta} 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 PPAR{delta} 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, PPAR{delta} 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 (56). The nucleotide sequences of biotinylated PPAR{delta} response element (DRE) were 5'-GCGTGAGCGCTCACAGGTCAATTCG-3' and 5'-CCGAATTGACCTGTGAGCGCTCACG-3' (45). These two complementary strands were annealed in TEN buffer. After transfection of CCLP1 cells (cultured in 6-well plate) with the cPLA2{alpha} expression plasmid or treatment with different reagents, the cells were lysed by sonication in 200 µl of HKMG buffer containing protease inhibitors and phosphatase inhibitors. The cellular debris was removed by centrifugation. The cell extracts (40 µg) were precleared with 20 µl of immobilized streptavidin-agarose beads for 1 h at 4 °C, with gentle agitation. The cleared nuclear extracts were then incubated with 1 µg of biotinylated double-strand DRE and 10 µg of poly(dI-dC)·poly(dI-dC) for 16 h. DRE-bound protein was pulled down by incubating the samples with 25 µl of streptavidin-agarose beads for 1 h at 4 °C, with gentle agitation. The agarose mixture was collected by centrifugation and washed four times with cold HKMG buffer. SDS sample buffer was then added to the pellet. The samples were boiled for 5 min and subjected to SDS-PAGE and Western blotting for PPAR{delta}.


Figure 3
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  		FIGURE 3.
PPAR{delta} activates EGFR and Akt in CCLP1 cells. A, GW501516 induces EGFR and Akt phosphorylation. CCLP1 cells were treated with GW501516 (10 nM) for 6–8 h, and the cell lysates were obtained. 30 µgof protein from each sample were subjected to SDS-PAGE and Western blotting for phospho-Akt(Thr308), total Akt, phospho-EGFR, total EGFR, COX-2, and beta-actin. B, siRNA inhibition of COX-2 prevents GW501516-induced phosphorylation of EGFR and Akt. CCLP1 cells were transfected with COX-2 siRNA or control siRNA using Lipofectamine 2000. On the following day the cells were incubated with GW501516 (10 nM) for 6 h, and the cell lysates were obtained for SDS-PAGE and Western blotting to determine the levels of phosphor-Akt(Thr308), total Akt, phospho-EGFR, total EGFR, COX-2, and beta-actin. C, siRNA inhibition of COX-2 prevents PPAR{delta} overexpression-induced phosphorylation of Akt or EGFR. CCLP1 cells (80% confluence in 6-well plate) were transfected with 1 µg of PPAR{delta} expression plasmid or the empty control vector (SG5), with cotransfection of human COX-2 siRNA or non-target control siRNA using Lipofectamine 2000 reagent. After 4 h of incubation, transfection medium was replaced by serum-free DMEM. The cells were cultured overnight, and the cell lyastes were obtained for SDS-PAGE and Western blot to determine the levels of phospho-EGFR, total EGFR, phospho-Akt, total Akt, COX-2, PPAR{delta}, and beta-actin. The experiments were repeated three times.

 
The Effect of Fatty Acids and PGE2 on the Binding of Recombinant Human PPAR{delta} (rhPPAR{delta}) to DRE—Biotinylated DRE oligonucleotide (0.2 µg) was preincubated with immobilized streptavidin-agarose beads (20 ul) for 1 h at room temperature in buffer A (4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, 50 mM NaCl, 10 mM Tris-HCl, pH 7.9). 500 nM of fatty acids (AA, oleic acid, {alpha}-linolenic acid, and stearic acid) or PGE2 (10 µM) was incubated with human recombinant PPAR{delta} (Cayman Chemical) (0.12 µg/sample) in Buffer A for 20 min at room temperature. Biotin-labeled DRE beads were added to the rhPPAR{delta}-fatty acid mixture, and the samples were incubated for an additional 20 min at room temperature. The beads were then washed four times using Buffer A. SDS sample buffer was then added to the pellet, and the samples were subjected to SDS-PAGE and Western blotting to detect PPAR{delta}.

Fatty Acid-Protein Overlay Assay—This assay was performed as previous report with modification (57). Briefly, various amounts of arachidonic acid were spotted onto Hybond C membrane (Amersham Biosciences) and completely dried. The blot was re-wet in deionized water and then blocked in 3% fatty acid-free bovine serum albumin (FAF-BSA)/PBS-T (0.05% Tween 20) for 1 h at room temperature. The blot was then incubated overnight with 0.24 µg/ml human recombinant PPAR{delta} (Cayman Chemical) in 1.5% FAF-BSA/PBS-T at 4 °C. The blot was washed gently and incubated with anti-PPAR{delta} antibody (1:1000) for 1 h followed by incubation with second antibody for additional 1 h at room temperature and developed using ECL.

Statistical Analysis—Statistical analysis was performed using Microsoft Excel 2003 software. Comparisons were performed using two-tailed unpaired Student's t test. Values of p < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
PPAR{delta} Promotes Cholangiocarcinoma Cell Growth—The effect of PPAR{delta} on human cholangiocarcinoma cell growth was evaluated by PPAR{delta} overexpression or treatment with GW501516, a selective PPAR{delta} ligand. As shown in Fig. 1A, GW501516 treatment significantly increased the growth of three human cholangiocarcinoma cell lines (CCLP1, HuCCT1, and SG231), as determined by the WST-1 assay. This effect was dose-dependent (0.5–50 nM) and was observed at different treatment periods (24–72 h). The dose-dependent effect of GW501516 on cell growth was also confirmed by the [3H]thymidine incorporation assay (Fig. 1B). The PPAR{delta} protein level was similar among the three cholangiocarcinoma cell lines utilized in this study (Fig. 1C). Consistent with the effect of GW501516, overexpression of PPAR{delta} also significantly increased the growth of human cholangiocarcinoma cells, as determined by both WST-1 and [3H]thymidine incorporation assays (Fig. 1D). The transcriptional activity of PPAR{delta} in these cells was verified by determining the reporter activity of a luciferase promoter construct containing the PPAR{delta} response element (DRE) (45). As shown in Fig. 1E, treatment of the PPAR{delta} ligand, GW501516, significantly increased the DRE-driven luciferase reporter activity (~2-fold, p < 0.01). Overexpression of PPAR{delta} alone or in combination with GW501516 further enhanced the DRE reporter activity (5.8 and 6.9-fold, respectively, p < 0.01). These observations reveal a growth-stimulatory effect of PPAR{delta} in human cholangiocarcinoma cells.


Figure 4
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  		FIGURE 4.
Inhibition of COX-2 and its downstream signaling prevents PPAR{delta}-induced cholangiocarcinoma cell growth. A and C, siRNA inhibition of COX-2 prevents PPAR{delta}-induced human cholangiocarcinoma cell growth. CCLP1 cells (A) and HuCCT1 cells (C) transfected with COX-2 siRNA or control siRNA were cotransfected with human PPAR{delta} expression plasmid or treated with GW501516 (10 nM) overnight to determine cell growth. The values represent mean ± S.D. of percentage change from control (*, p < 0.01 compared with control siRNA, n = 3). The COX-2 protein levels in the cells transfected with COX-2 siRNA, and control siRNA are shown at the lower panels. B and D, inhibition of Akt and EGFR prevents PPAR{delta}-induced human cholangiocarcinoma cell growth. CCLP1 cells (B) and HuCCT1 cells (D) were transfected with PPAR{delta} plasmid or treated with GW501516 (10 nM) in the presence or absence of the inhibitor of EGFR (AG1478, 10 µM) or Akt (LY294002, 10 µM) for overnight to determine cell growth. All values are expressed as mean ± S.D. of percentage change from control (*, p < 0.01 compared with vehicle treatment, n = 3).

 
PPAR{delta} Activation Induces COX-2 Expression and PGE2 Production—Further experiments were performed to determine the mechanisms by which PPAR{delta} promotes human cholangiocarcinoma growth. Because COX-2-derived PGE2 has been implicated in cholangiocarcinogenesis, we reasoned COX-2 and PGE2 signaling might play a role in PPAR{delta}-induced cholangiocarcinoma cell growth. Indeed, activation of PPAR{delta} by GW510516 significantly increased the expression of COX-2 protein in human cholangiocarcinoma cells. As shown in Fig. 2A, 1–50 nM GW501516 enhanced the expression of COX-2 in CCLP1 cells (especially at 1–10 nM); this effect occurred 4 h after treatment and persisted at 24 h. A similar effect was also seen in the HuCCT1 cells (Fig. 2B). Consistent with its effect on COX-2 expression, GW501516 also enhanced the production of PGE2 (Fig. 2C). Furthermore, overexpression of PPAR{delta} also enhanced the expression of COX-2 (Fig. 2D). These findings demonstrate a direct effect of PPAR{delta} on COX-2 expression and PGE2 production in human cholangiocarcinoma cells.


Figure 5
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  		FIGURE 5.
PGE2 activates cPLA2{alpha} through PI3-K or MAPK pathway in CCLP1 cells. A, PGE2 treatment increases cPLA2{alpha} phosphorylation. CCLP1 cells cultured in 6-well plates were serum-starved overnight followed by incubation with PGE2 (10 µM) for indicated time periods. The cell lysates were then collected to determine cPLA2{alpha} phosphorylation by immunoprecipitation and Western blot analysis as described under "Experimental Procedures." B, inhibition of p38 MAPK, p44/42 MAPK, or PI3-K prevents PGE2-induced cPLA2{alpha} phosphorylation. Serum-starved CCLP1 cells cultured in 6-well plates were preincubated for 30 min with the PKC inhibitor (bisindolylmaleimide I, 20 µM), PI3-K inhibitor (LY294002, 20 µM), p44/42 inhibitor (PD98059, 20 µM), or p38 inhibitor (SB203580, 10 µM) and co-incubated with PGE2 (10 µM) for 30 min. The cell lysates were collected and subjected to immunoprecipitation and Western blot analysis to determine cPLA2{alpha} phosphorylation. The experiments were repeated three times.

 
Involvement of COX-2/PGE2-mediated Transactivation of EGFR and Akt in PPAR{delta}-induced Cholangiocarcinoma Cell Growth—Given that COX-2-derived PGE2 has been show to promote cholangiocarcinoma cell growth through activation of EGFR and Akt (15), we next determined the potential effect of PPAR{delta} on EGFR and Akt phosphorylation. As shown in Fig. 3A, treatment of CCLP1 cells with 10 nM GW501516 enhanced the phosphorylation of both EGFR and Akt, whereas the levels of total Akt and EGFR were not altered. The GW501516-induced phosphorylation of Akt and EGFR was blocked by siRNA inhibition of COX-2 (Fig. 3B). Furthermore, overexpression of PPAR{delta} in CCLP1 cells also increased the phosphorylation of EGFR/Akt, and this effect was blocked by siRNA inhibition of COX-2 (Fig. 3C). These findings suggest that PPAR{delta} activates EGFR and Akt in human cholangiocarcinoma cells, and this effect is mediated, at least in part, through COX-2.

The role of COX-2/EGFR/Akt signaling in PPAR{delta}-induced cholangiocarcinoma cell growth was further documented. As shown in Fig. 4, A and C, siRNA inhibition of COX-2 prevented the growth of CCLP1 and HuCCT1 cells induced by PPAR{delta} overexpression and GW501516 treatment. Furthermore, the EGFR tyrosine kinase inhibitor, AG1478, and the PI 3-kinase inhibitor, LY294002, both blocked the PPAR{delta} overexpression or GW501516-induced cell growth (Fig. 4, B and D). These observations suggest the involvement of COX-2, EGFR, and Akt signaling in PPAR{delta}-mediated cholangiocarcinoma cell growth.

PPAR{delta} Induces cPLA2{alpha} Phosphorylation through COX-2-mediated PGE2 Production—cPLA2{alpha} is the rate-limiting enzyme that releases arachidonic acid from membrane phospholipids and thus provides substrate for COX enzymes. The cPLA2{alpha} and COX-2 controlled PG synthesis has been implicated in cholangiocarcinoma cell growth (4). Whereas coupled activation of cPLA2{alpha} and COX-2 plays an important role for PG production (58–60), there is also evidence indicating that PGE2 can further activates cPLA2{alpha} in prostate carcinoma cells (61). Therefore, we sought to further determine whether PPAR{delta}-induced PGE2 synthesis might affect cPLA2{alpha} activation in human cholangiocarcinoma cells. As shown in Fig. 5A, treatment of CCLP1 cells with 10 µM PGE2 induced a rapid phosphorylation of cPLA2{alpha}; this effect was observed at 5 min, peaked at 30 min and sustained at 2 h. The PGE2-induced cPLA2{alpha} phosphorylation was completely blocked by pretreatment with the inhibitor of Akt (LY294002, 20 µM), p44/42 MAPK (PD98059, 20 µM), or p38 MAPK (SB203580, 10 µM), and partially inhibited by the PKC inhibitor (bisindolylmaleimide I, 20 µM) (Fig. 5B), suggesting the involvement of p38, p42/44 MAPKs, Akt, or possibly PKC in PGE2-induced cPLA2{alpha} phosphorylation. Consistent with the effect of PPAR{delta} on COX-2 expression and PGE2 synthesis, activation of PPAR{delta} by GW501516 also increased cPLA2{alpha} phosphorylation, and this effect was blocked by siRNA inhibition of COX-2 (Fig. 6). Similarly, overexpression of PPAR{delta} also enhanced phosphorylation of cPLA2{alpha} (Fig. 6). Collectively, these data suggest that PPAR{delta} induces COX-2 expression and PGE2 production that in turn enhances cPLA2{alpha} phosphorylation, which further amplifies PGE2 signaling.

cPLA2{alpha} Enhances DRE Reporter Activity—Although recent evidence suggests the involvement of cPLA2{alpha} in the activation of PPAR{alpha} and PPAR{gamma} in primary and transformed hepatocytes and lung epithelial cells (62, 63), the potential role of cPLA2{alpha} in PPAR{delta} activation has not been investigated. In this study, the direct effect of cPLA2{alpha} on PPAR{delta} activation was investigated in human cholangiocarcinoma cells. For these experiments, CCLP1 cells were cotransfected with cPLA2{alpha} expression plasmid or control vector pMT-2 and DRE luciferase reporter construct. As shown in Fig. 7A, overexpression of cPLA2{alpha} significantly increased the DRE reporter activity (3.2-fold of control, p < 0.01). Consistent with this, activation of cPLA2{alpha} by the calcium ionophore A23187 [GenBank] (1 µM) also significantly increased the PPAR{delta} transcription activity in CCLP1 cells (4.5-fold of control, p < 0.01), which was inhibited by the cPLA2 inhibitor AACOCF3 (20 µM) (Fig. 7B). These findings demonstrate a direct role of cPLA2{alpha} for PPAR{delta} activation.

cPLA2{alpha} Enhances PPAR{delta} Binding to DRE in CCLP1 Cells—The role of cPLA2{alpha} in PPAR{delta} activation was further examined by assessing the binding of PPAR{delta} to DRE, in vitro. For this purpose, two complementary approaches were utilized, including the biotinylated oligonucleotide precipitation assay to characterize the specific binding phenomenon and the ELISA-based nuclear transcription factor assay to quantitate the amount of PPAR{delta} bound to its response element. As shown in Fig. 8A, overexpression of cPLA2{alpha} or activation of cPLA2{alpha} by the calcium ionophore, A23187 [GenBank] , significantly increased the binding of PPAR{delta} to its response element, as determined by the ELISA-based nuclear transcription factor assay. The effect of cPLA2{alpha} transfection or A23187 [GenBank] treatment appeared slightly less than that induced by the synthetic PPAR{delta} ligand, GW501516. Furthermore, the A23187 [GenBank] -induced PPAR{delta} binding to its response element was completely blocked by the selective cPLA2 inhibitor, AACOCF3 (Fig. 8A). These observations further support the role of cPLA2{alpha} in PPAR{delta} activation. The fact that AACOCF3 also inhibited PPAR{delta} binding in cells without cPLA2{alpha} overexpression or A23187 [GenBank] treatment (Fig. 8A) suggests the presence of endogenous cPLA2{alpha} for PPAR{delta} activation.


Figure 6
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  		FIGURE 6.
PPAR{delta} activates cPLA2{alpha} via PGE2. A, activation of PPAR{delta} by GW501516 induces cPLA2{alpha} phosphorylation. CCLP1 cells at 80% confluence were serum-starved overnight and then treated with GW501516 (10 nM) for indicated time periods. The cell lysates were collected and subjected to immunoprecipitation and Western blot analysis to determine cPLA2{alpha} phosphorylation. B, overexpression of PPAR{delta} induces cPLA2{alpha} phosphorylation. CCLP1 cells at 80% confluence were transfected with the PPAR{delta} expression plasmid or the control vector (SG5) (exposure to Lipofectamine Plus reagent for 4 h). After transfection, the cells were incubated in serum-free medium for 24 h. The whole cell lysate was subjected to immunoprecipitation and Western blot analysis to determine cPLA2{alpha} phosphorylation. C, siRNA inhibition of COX-2 prevents GW501516-induced cPLA2{alpha} phosphorylation. CCLP1 cells were transfected with COX-2 siRNA or non-target control siRNA for 4 h using Lipofectamine 2000 reagent. The transfection medium was then replaced by serum-free DMEM containing 10 nM of GW501516 or vehicle Me2SO, and the cells were incubated for 24 h. The cell lysate was then collected for immunoprecipitation and Western blot analysis to determine cPLA2{alpha} phosphorylation. The experiments were repeated three times.

 


Figure 7
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  		FIGURE 7.
cPLA2{alpha} enhances PPAR{delta} reporter activity in CCLP1 cells. A, cPLA2{alpha} overexpression enhances PPAR{delta} reporter activity. CCLP1 cells at 80% confluence were cotransfected with the human cPLA2{alpha} expression plasmid or the control vector pMT2 plus the DRE reporter construct for 4 h using Lipofectamine Plus reagent. The transfection medium was then replaced by fresh serum-free DMEM and the cells were incubated overnight. On the following day, the cells were washed with cold PBS and the cell lysate was obtained for luciferase activity assay to determine the DRE reporter activity. The values represent mean ± S.D. from three experiments (*, p < 0.01 compared with vector). The level of cPLA2{alpha} in these cells was determined by Western blot analysis. B, activation of cPLA2{alpha} by the calcium ionophore A23187 enhances DRE reporter activity. CCLP1 cells transfected with DRE reporter gene were incubated in serum-free medium overnight. On the following day, A23187 (10 µM), the cPLA2{alpha} inhibitor (AACOCF3, 20 µM) or vehicle was added to the cells and the culture was continued for 4 h. The cell lysate was then collected to determine the luciferase reporter activity. The values represent mean ± S.D. from three experiments (*, p < 0.01 compared with vehicle).

 
The effect of cPLA2{alpha} on PPAR{delta} binding to DRE was also confirmed by the biotinylated DRE oligonucleotide immunoprecipitation assay. Under this assay system, transfection of cPLA2{alpha} expression plasmid in CCLP1 cells also increased the binding of PPAR{delta} to DRE (Fig. 8B). The specificity of the assay was confirmed by the complete elimination of binding with the unlabeled DRE oligonucleotides. Similarly, the data from the biotinylated DRE oligonucleotide precipitation assay also confirmed that activation of cPLA2{alpha} by A23187 [GenBank] enhanced the binding of PPAR{delta} to DRE and that two structurally unrelated cPLA2{alpha} inhibitors, AACOCF3 and the pyrrolidine derivative, prevented PPAR{delta}-DRE binding (Fig. 8, C–E).

cPLA2{alpha} Induces COX-2 Gene Expression—The results presented in the above sections indicate that cPLA2{alpha}-mediated AA metabolites can activate PPAR{delta}. This finding, along with the observation that PPAR{delta} activation increases COX-2 expression, prompted us to evaluate the effect of cPLA2{alpha} on COX-2 gene expression. For this approach, the CCLP1 cells transfected with the cPLA2{alpha} expression plasmid or control vector were cotransfected with a luciferase reporter construct under the control of the COX-2 gene promoter, and the cell lysates were obtained to determine luciferase reporter activity. As shown in Fig. 9, overexpression of cPLA2{alpha} significantly increased COX-2 gene transcription activity (4.5-fold of control, p < 0.01) as well as COX-2 protein level and PGE2 production (0.34 versus 0.18 ng/ml, p < 0.05). These findings are consistent with the activation of PPAR{delta} by cPLA2{alpha} and the induction of COX-2 expression by PPAR{delta}, as described in the above sections.

PGE2 Activates PPAR{delta} through cPLA2{alpha} in CCLP1 Cells—Given that PGE2 can phosphorylate and activate cPLA2{alpha} and that cPLA2{alpha} is implicated in PPAR{delta} activity, we reasoned that treatment of human cholangiocarcinoma cells with PGE2 should also induce PPAR{delta} activation. This was examined by measuring the effect of PGE2 on DRE luciferase activity in CCLP1 cells. Indeed, as shown in Fig. 10A, PGE2 treatment significantly increased the DRE reporter activity in intact cells; this effect was blocked by the PI3-K/Akt inhibitor (LY294002), the p44/42 MAPK inhibitor (PD98059), the p38 MAPK inhibitor (SB203580), as well as by the cPLA2{alpha} inhibitor (AACOCF3). These data further support the role of PGE2-induced cPLA2{alpha} phosphorylation for PPAR{delta} activation in human cholangiocarcinoma cells.

To further delineate the effect of AA and PGE2 on PPAR{delta} activation, an in vitro system was employed, in which recombinant human PPAR{delta} was incubated with different fatty acids or PGE2 in the presence of biotinylated DRE oligonucleotide to determine the effect of fatty acids and PGE2 on PPAR{delta}-DRE binding. As shown in Fig. 10B, addition of 500 nM AA induced the binding of PPAR{delta} to DRE. In contrast, three other fatty acids, including {alpha}-linolenic acid, oleic acid, and stearic acid, failed to induce PPAR{delta} binding. PGE2 also failed to induce PPAR{delta} binding to DRE under similar conditions (up to 10 µM). Consistent with these results, fatty acid-protein overlay assay showed that AA directly bound PPAR{delta} in a dose-dependent manner (Fig. 10C). These findings suggest that PGE2 lacks the ability to directly activate PPAR{delta}, although AA itself can bind PPAR{delta} and alter PPAR{delta} transcription activity. The latter assertion is further supported by the observation that the COX-2 inhibitor, indomethacin, had no apparent influence on A23187 [GenBank] -induced PPAR{delta} binding to DRE (Fig. 10D). Thus, given that PGE2 activates PPAR{delta} only in intact cells, its effect is most likely mediated through cPLA2{alpha} phosphorylation-induced AA release rather than direct PPAR{delta} binding.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This study reveals an important role of PPAR{delta} in human cholangiocarcinoma cell growth. Our data show that PPAR{delta} overexpression or activation enhances human cholangiocarcinoma cell growth and this effect is mediated, at least in part, through induction of COX-2 gene expression and PGE2 synthesis. Moreover, the COX-2-derived PGE2 further activates PPAR{delta} through phosphorylation of cPLA2{alpha}. The interactions between PPAR{delta} and PG signaling pathways form a positive feedback loop that likely plays an important role in cholangiocarcinoma cell growth (Fig. 11). The most novel mechanistic aspect of this study is, perhaps, the identification of cPLA2{alpha}-controlled AA metabolism for endogenous PPAR{delta} activation.

Activation of PPAR involves ligand-induced conformational change which alters the binding of PPAR with other nuclear proteins and the basal transcriptional machinery. Although AA metabolites represent the natural ligands for PPAR activation, the individual enzymes involved in the control of eicosanoid production for PPAR activation remain to be further defined. This study provides the first evidence for the activation of PPAR{delta} by cPLA2{alpha} in human cells, which includes: 1) cPLA2{alpha} overexpression enhanced PPAR{delta} reporter activity in CCLP1 cells; 2) activation of cPLA2{alpha} by the ionophore A23187 [GenBank] enhanced PPAR{delta} reporter activity; and 3) PPAR{delta} reporter activity was blocked by the specific cPLA2 inhibitor, AACOCF3, and pyrrolidine. In addition to the data from the PPAR{delta} reporter activity assay, our results also demonstrate that cPLA2{alpha} overexpression or activation enhanced the association of PPAR{delta} to its specific DNA response element and this binding was blocked by inhibition of cPLA2. Thus, cPLA2{alpha} activity is involved in PPAR{delta} trans-activation, which underscores the importance of cPLA2{alpha} in PPAR{delta}-mediated gene transcription.

The importance of cPLA2{alpha} in PPAR activation can be explained by its unique characteristic of nuclear localization. The cPLA2{alpha} protein translocates from cytoplasm to nuclear envelope (7, 38–41) in response to calcium influx and this effect is mediated by its N-terminal Ca2+-dependent lipid binding domain (CaLB or C2 domain) (42, 64). As cPLA2{alpha} protein requires Ca2+ for its nuclear translocation, calcium ionophore A23187 [GenBank] was used in this study for maximal enzyme activation. Our data indicate that ionophore A23187 [GenBank] increased PPAR{delta} reporter activity and DNA binding in CCLP1 cells, which was blocked by the cPLA2 inhibitor, AACOCF3. These observations further support the involvement of calcium-mediated cPLA2{alpha} translocation in PPAR{delta}-mediated gene transcription.

In this study, the role of PPAR{delta} in cholangiocarcinoma growth was documented by utilization of the pharmacological PPAR{delta} ligand (GW501516) and PPAR{delta} overexpression. GW501516 is a synthetic pharmacological ligand that is selective for PPAR{delta} with no effect on PPAR{alpha} or PPAR{gamma} (even at dose as high as 10 µM) (35, 47, 64). In our system, we found that GW501516 was able to induce PPAR{delta} activation and enhance cholangiocarcinoma cell growth at relatively low doses (0.5–50 nM). These findings are consistent with the observations that GW501516 enhances the growth of other tumor cells in vitro (49, 50) and accelerate tumorigenesis in an animal model of intestinal adenoma (47). The direct effect of PPAR{delta} was further supported by the observation that overexpression of PPAR{delta} enhanced cholangiocarcinoma cell growth.


Figure 8
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  		FIGURE 8.
cPLA2{alpha} enhances PPAR{delta} binding to its response element in CCLP1 cells. A, effect of cPLA2{alpha} overexpression, A23187, and AACOCF3 on PPAR{delta} binding (ELISA-based PPAR{delta} transcription factor assay). Nuclear extracts (50 µg/each sample) from CCLP1 cells treated with GW501516 (5 nM) for 4 h, A23187 (1 µM) alone or in combination with the cPLA2{alpha} inhibitor (AACOCF3, 20 µM) for 4 h, or transfected with the cPLA2{alpha} expression plasmid, were added into PPAR response element-coated 96-well plate and incubated overnight to allow protein-DNA binding. After complete wash, anti-PPAR{delta} antibody was added into sample wells and incubated for 1 h. Subsequently, HRP-conjugated secondary antibody and color development solution were added and OD655 nm was measured (*, p < 0.05 compared with control; **, p < 0.01 compared with control). B, overexpression of cPLA2{alpha} enhances PPAR{delta} binding to DRE (biotinylated DRE oligonucleotide precipitation assay). Whole cell lysate from CCLP1 cells transfected with the cPLA2{alpha} expression plasmid or the control vector pMT-2 was precleared and then incubated with biotinylated DRE (1 µg) alone or with cold DRE oligonucleotide (no biotin modulation) (10 µg) overnight to allow PPAR{delta} binding to DRE. Immobilized streptavidin-agarose beads were then added to pull-down the protein-DNA complex for Western blot detection of bound PPAR{delta} as described under "Experimental Procedures." C, A23187 treatment enhances PPAR{delta} binding to DRE (biotinylated DRE oligonucleotide precipitation assay). This effect was blocked by pretreatment with the cPLA2{alpha} inhibitor, AACOCF3. Whole cell lysates from CCLP1 cells treated with A23187 (1 µM, 20 min) in the presence or absence of AACOCF3 (30 µM, 4 h) were precleared and then incubated overnight with biotinylated DRE (1 µg) in the presence or absence of cold DRE oligonucleotide (10 µg). Immobilized streptavidin-agarose beads were then added, and the samples were processed for biotinylated DRE oligonucleotide precipitation assay to detect bound PPAR{delta}. D, cPLA2{alpha} inhibitor, AACOCF3, prevents PPAR{delta} binding to DRE in CCLP1 cells under basal culture conditions (biotinylated DRE oligonucleotide precipitation assay). Whole cell lysates from CCLP1 cells treated with AACOCF3 (30 µM) at indicated time points were precleared and then incubated overnight with 1 µg of biotinylated DRE in the presence or absence of 10 µg of cold DRE oligonucleotide. Immobilized streptavidin-agarose beads were then added, and the samples were processed for biotinylated DRE oligonucleotide precipitation assay to detect bound PPAR{delta}. E, effect of a separate structurally unrelated cPLA2{alpha} inhibitor, pyrrolidine derivative, on PPAR{delta} binding to DRE (biotinylated DRE oligonucleotide precipitation assay). Whole cell lysate from CCLP1 cells treated with pyrrolidine derivative (2 µM) alone or in combination with A23187 (1 µM) was precleared and then incubated overnight with 1 µg of biotinylated DRE in the presence or absence of 10 µg of cold DRE oligonucleotide. Following addition of streptavidin-agarose beads, the samples were processed for biotinylated DRE oligonucleotide precipitation assay to detect bound PPAR{delta}. All the experiments were repeated three times.

 


Figure 9
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  		FIGURE 9.
Overexpression of cPLA2{alpha} enhances COX-2 gene transcription, COX-2 protein level, and PGE2 production. A, effect of cPLA2{alpha} overexpression on COX-2 gene transcription. CCLP1 cells were cotransfected with the cPLA2{alpha} expression plasmid or the pMT2 empty vector plus the COX-2 promoter construct (luciferase coding gene under the control of COX-2 promoter). The cell lysates were obtained for luciferase activity assay to determine the COX-2 reporter activity. The values represent mean ± S.D. from three experiments (*, p < 0.01 compared with control vector). B, effect of cPLA2{alpha} overexpression on COX-2 protein level. Cell lysates from CCLP1 cells transfected with the cPLA2{alpha} expression plasmid or pMT2 control vector were obtained for Western blot to determine the expression of cPLA2{alpha} and COX-2, with beta-actin as the loading control. C, level of PGE2 production in cells with cPLA2{alpha} overexpression. CCLP1 cells were transfected with the cPLA2{alpha} expression plasmid or pMT2 vector for 4 h using Lipofectamine Plus reagent. The transfection medium was then replaced by serum-free DMEM, and the culture continued overnight. 100 µl of supernatant was used to detect PGE2 level as described under "Experimental Procedures." The values are expressed as mean ± S.D. (*, p < 0.05 compared with control vector, n = 3).

 
Our data indicate that overexpression or activation of PPAR{delta} induces COX-2 expression in human cholangiocarcinoma cells. The role of COX-2 in PPAR{delta}-induced cholangiocarcinoma cell growth was supported by the observation that siRNA inhibition of COX-2 prevented PPAR{delta}-induced cell growth. The exact mechanism for PPAR{delta}-induced COX-2 expression, however, remains to be further defined. Because homologous alignment analysis revealed no DRE site in the human COX-2 gene promoter, the possibility of PPAR{delta} effect independent of DRE-binding cannot be excluded, giving that PPAR{alpha} and PPAR{gamma} are known to mediate their effect through interaction with other transcription factors (21, 65, 66), in addition to binding PPRE.

PGE2 has been shown to promote tumor cell growth through mechanisms including cell proliferation, anti-apoptosis, invasion, and angiogenesis. Certain prostanoids, including PGE2, have been shown to feed-forwardly increase COX-2 expression and PG production (67, 68); however, the molecular mechanism for this phenomenon is not fully understood. Our data presented in this study reveal a novel cPLA2{alpha}-mediated PPAR{delta} activation in PGE2-induced COX-2 expression in human cholangiocarcinoma cells. We found that PGE2 treatment induces cPLA2{alpha} phosphorylation at Ser505 in human cholangiocarcinoma cells and that this effect is likely mediated by activation of p38 MAPK, ERK1/2, and PI3-K. Our data suggest that activation of cPLA2{alpha} generates arachidonic acid for PPAR{delta} activation in the nucleus and this mechanism is likely implicated in COX-2 gene expression. This assertion is supported by the observations that PPAR{delta} activation induces both COX-2 expression and cPLA2{alpha} phosphorylation; that the PPAR{delta}-induced cPLA2{alpha} phosphorylation coincided with the expression of COX-2 (starting 4–6 h of GW501516 treatment or overnight following PPAR{delta} transfection); and that siRNA inhibition of COX-2 expression blocked the GW501516-induced cPLA2{alpha} phosphorylation. Notably, although PGE2 treatment increased the DRE reporter activity in intact cells, it failed to induce PPAR{delta} binding to DRE in cell-free system, further suggesting that the cPLA2{alpha}-mediated AA production is required for PGE2-induced PPAR{delta} activation and COX-2 expression. It is worth mentioning that the importance of cPLA2{alpha} for COX-2 expression documented in this study is consistent with the fact that the cPLA2{alpha}-null mice have less COX-2 expression than wild-type mice during inflammation (69).


Figure 10
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  		FIGURE 10.
PGE2 activates PPAR{delta} through cPLA2{alpha} in CCLP1 cells. A, PGE2 treatment increases DRE reporter activity; this effect is blocked by inhibition of cPLA2{alpha} activation. CCLP1 cells (80% confluence in 6-well plate) were transfected with human DRE reporter gene (1 µg) for 4 h using Lipofectamine Plus reagent. The transfection solution was then replaced by serum-free medium, and the cells were incubated overnight. On the following day, the cells were pre-incubated with the inhibitors of PI3-K (LY294002, 20µM), p44/42 (PD98059, 20µM), p38 (SB203580, 10µM) or cPLA2{alpha} (AACOCF3, 20µM) for 30 min, followed by PGE2 treatment (10µM) for additional 30 min. The cell lysates were obtained for luciferase activity assay to determine DRE reporter activity. The values are expressed as mean ± S.D. from three experiments (*, p < 0.05 compared with control; **, p < 0.01 compared with PGE2 treatment). B, AA induces PPAR{delta} binding to its response element DRE. Human recombinant PPAR{delta} (hrPPAR{delta}) (0.12µg) was incubated with AA (500 nM), stearic acid (500 nM), oleic acid (500 nM), {alpha}-linolenic acid (500 nM), PGE2 (10µM), or vehicle in Buffer A for 20 min. 0.2µg of biotin-labeled DRE-streptavidin beads (with or without 10-fold cold competitive DRE) were then added, and the samples were incubated for an additional 20 min. After washing four times with buffer A, SDS sample buffer was added to the pellet, and the samples were subjected to SDS-PAGE and Western blotting to detect PPAR{delta}. C, AA directly binds PPAR{delta} in vitro (fatty acid-protein overlay assay). Different amounts of AA in 5 µl of volume were spotted onto the Hybond-C membrane. The blot was dried, re-wet, and blocked in 3% fatty acid-free bovine serum albumin (FAF-BSA)/PBS-T (0.05% Tween 20). The blot was then incubated with 0.24 µg/ml human recombinant PPAR{delta}, followed by sequential incubation with anti-PPAR{delta} antibody (1:1000) and second antibody prior to development using ECL. D, inhibition of COX by indomethacin had no effect on A23187-induced PPAR{delta} binding to DRE in CCLP1 cells. Serum-starved CCLP1 cells were incubated with indomethacin (30 µM, overnight) prior to A23187 treatment (1 µM, 20 min). The whole cell lysates were obtained and incubated overnight with biotinylated DRE oligonucleotide (1µg) (with or without 10µg of cold competitive DRE). Streptavidin-agarose beads were then added to pull-down the protein-DNA complex for PPAR{delta} Western blot. All the experiments were repeated three times.

 


Figure 11
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  		FIGURE 11.
Mechanisms for PPAR{delta} and prostaglandin signaling in the regulation of cholangiocarcinoma cell growth. PPAR{delta} promotes cholangiocarcinoma cell growth through induction of COX-2 expression and PGE2 synthesis. The produced PGE2 mediates cell growth through mechanisms involving transactivation of EGFR and Akt. Moreover, PGE2 further amplifies growth through MAPK and PI3-K-mediated phosphorylation and activation of cPLA2{alpha}, which in turn releases arachidonic acid for PPAR{delta} activation and for PGE2 synthesis via COX-2. This positive feedback loop between PPAR{delta} and prostaglandin pathways likely plays an important role in cholangiocarcinogenesis.

 
Taken together, these studies reveal a novel cross-talk between PPAR{delta} and PG signaling pathways that coordinately regulate cholangiocarcinoma cell growth. Thus, interruption of this feed-forward loop may provide a novel therapeutic strategy for future chemoprevention and treatment.


   FOOTNOTES
 
* This work was supported by National Institutes of Health R01 Grants CA102325 and CA106280 (to T. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Pathology, University of Pittsburgh School of Medicine, MUH E-740, 200 Lothrop St., Pittsburgh, PA 15213. Tel.: 412-647-9504; Fax: 412-647-5237; E-mail: wut{at}upmc.edu.

2 The abbreviations used are: COX-2, cyclooxygenase-2; AA, arachidonic acid; AACOCF3, arachidonyltrifluoromethyl ketone; cPLA2{alpha}, cytosolic phospholipase A2{alpha}; DRE, PPAR{delta} response element; EGFR, epidermal growth factor receptor; GPCR, G protein-coupled receptor; PG, prostaglandin; PGE2, prostaglandin E2; PI3-K, phosphatidylinositol 3-kinase; PPAR{delta}, peroxisome proliferator-activated receptor-{delta}; 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. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Vogelstein and Kinzler at The Johns Hopkins Oncology Center, Baltimore, Maryland for providing the DRE-reporter plasmid, Dr. Liliane Michalik at University of Lausanne, Lausanne, Switzerland for the PPAR{delta} expression plasmid, and Drs. J. D. Clark and J. L. Knopf at the Genetics Institute, Boston, MA for the cPLA2 expression plasmid.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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