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Originally published In Press as doi:10.1074/jbc.M404852200 on August 4, 2004

J. Biol. Chem., Vol. 279, Issue 43, 44344-44354, October 22, 2004
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Transforming Growth Factor-{beta} (TGF-{beta}) Activates Cytosolic Phospholipase A2{alpha} (cPLA2{alpha})-mediated Prostaglandin E2 (PGE)2/EP1 and Peroxisome Proliferator-activated Receptor-{gamma} (PPAR-{gamma})/Smad Signaling Pathways in Human Liver Cancer Cells

A NOVEL MECHANISM FOR SUBVERSION OF TGF-{beta}-INDUCED MITOINHIBITION*

Chang Han{ddagger}, A. Jake Demetris{ddagger}, Youhua Liu{ddagger}, James H. Shelhamer§, and Tong Wu{ddagger}

From the {ddagger}Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213 and the §Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, April 30, 2004 , and in revised form, August 2, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Transforming growth factor-{beta} (TGF-{beta}) potently inhibits the growth of human epithelial cells. However, neoplastic epithelial cells become resistant to TGF-{beta}-mediated mitoinhibition, and the mechanisms for this alteration during tumorigenesis are not fully understood. This study was designed to determine whether there is an association between the cytosolic phospholipase A2{alpha} (cPLA2{alpha})-controlled eicosanoid metabolism and the growth response to TGF-{beta} in human liver cancer cells. TGF-{beta} treatment induced simultaneous Smad-mediated gene transcription and phosphorylation of cPLA2{alpha}. Whereas Smad activation inhibited tumor cell growth, phosphorylation of cPLA2 {alpha} promoted growth and counteracted Smad-mediated mitoinhibition. TGF-{beta}1 failed to prevent the growth of cells with high basal expression of cPLA2{alpha}, but inhibition of cPLA2 {alpha}, cyclooxygenase-2 (COX-2), or EP1 receptor restored mi-toinhibition by TGF-{beta}1 in these cells. These results suggest that resistance of tumor cells to TGF-{beta}-mediated mitoinhibition involves activation of cPLA2 {alpha}/COX-2/EP1 signaling. Furthermore, the TGF-{beta}1-induced Smad transcriptional activity and mitoinhibition were blocked by overexpression of cPLA2{alpha} or peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) but enhanced by depletion of cPLA2{alpha} or PPAR-{gamma}. These findings, along with the observations that cPLA2{alpha} activates PPAR-{gamma} and that PPAR-{gamma} binds Smad3, illustrate novel cPLA2{alpha}/COX-2/EP1 and cPLA2{alpha}/PPAR-{gamma}/Smad signaling pathways that counteract the mitoinhibition by TGF-{beta} in human cancer cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Transforming growth factor-{beta}s (TGF-{beta}s)1 are multifunctional cytokines that regulate cell proliferation, differentiation, apoptosis, migration, and extracellular matrix production (1–6). A large number of experimental and clinical studies have established that the TGF-{beta} system can function as a tumor suppressor pathway. For example, transgenic mice overexpressing active TGF-{beta}1 in mammary and skin epithelium are resistant to carcinogenesis (7, 8). Expression of dominant negative T{beta}RII enhances carcinogenesis in several animal models (9, 10). Mice with disruption of the Tgfb1, Smad3, or Smad4 gene exhibit enhanced tumorigenesis (11–14). Consistent with these observations, mutations or deletions in the genes for Smad4, T{beta}RII, or Smad2 are observed in some human tumors (1, 2, 4–6). On the other hand, there is also abundant evidence indicating that TGF-{beta}s can promote tumor growth (1, 2, 4–6). Cancer cells often resist TGF-{beta}-mediated mitoinhibition, and late stage human tumors frequently show increased production of TGF-{beta} that is associated with increased tumor progression. This phenomenon is exemplified in hepatobiliary neoplasia, with TGF-{beta} potently inhibiting primary hepatobiliary epithelial cell proliferation (15–17) but not liver cancer cell growth (16, 18). However, the mechanisms for the paradoxical effects of TGF-{beta} on tumor cell growth are not fully understood.

There are three mammalian TGF-{beta} isoforms, TGF-{beta}1, TGF-{beta}2, and TGF-{beta}3, which bind to the same receptor complex and signal predominantly through the Smad pathway (3). The TGF-{beta} receptor is composed of a heteromeric complex of transmembrane serine/threonine kinases, the type II and type I receptors (T{beta}RII and T{beta}RI). Following ligand binding to T{beta}RII, T{beta}RI is recruited to the complex, allowing for the constitutively active T{beta}RII kinase to transphosphorylate and activate the T{beta}RI kinase, which in turn phosphorylates Smad2 and Smad3. The phosphorylated Smad2/3 then associate with Smad4 and translocate to the nucleus, where they regulate gene transcription. The mitoinhibition by TGF-{beta} is predominantly mediated through activation of the Smad pathway. In addition, the activated T{beta}RII and T{beta}RI receptor complex can also signal independently of Smads via extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K) (1).

The current rationalization for the paradoxical effects of TGF-{beta}s on tumor growth is that TGF-{beta}s function as tumor suppressors early in tumorigenesis when epithelial cell responsiveness to TGF-{beta} is still relatively normal. Later during the process, genetic or epigenetic alterations in multiple pathways compromise the tumor suppressor activity, and the TGF-{beta}s then function predominantly as oncogenes to promote tumor growth. It is generally believed that during multistage tumorigenesis, the mitoinhibitory effect of TGF-{beta}s becomes lost, either through mutation of the TGF-{beta} signaling molecules or by subversion of the normal signaling pathway because of activation of other molecules (1, 2, 4–6). Because mutation of TGF-{beta} signaling molecules occurs only in a minority of human tumors, disruption of TGF-{beta}-mediated mitoinhibition by other signaling pathways appears to be a more generalized mechanism, although the exact nature of such pathways remains to be further defined.

Arachidonic acid (AA) metabolites, eicosanoids, have been implicated in the growth control of various human cells and cancers (19–28). Eicosanoid biosynthesis is tightly controlled by a series of enzymes including the group IV{alpha} cytosolic phospholipase A2 (cPLA2{alpha}), which selectively cleaves AA from membrane phospholipids, and cyclooxygenase-2 (COX-2), which converts AA substrate to prostaglandins (PGs). The expression of COX-2 and the production of PGs are increased in various human cancers including carcinomas of the colon, liver, stomach, breast, and lung. Abundant evidence has shown that prostaglandins promote tumor cell proliferation, survival, and angiogenesis (19, 23–28). Knockout of the COX-2 gene suppresses intestinal tumorigenesis (29); targeted expression of COX-2 in mammary gland (30) and skin (31) enhances tumorigenesis. Mutation or deletion of cPLA2{alpha} suppresses intestine and lung tumorigenesis in mice (32–34). Consistent with these experimental observations, large epidemiological studies have documented a strong correlation between the use of non-steroidal anti-inflammatory drugs (COX inhibitor) and a decreased incidence of human cancers (24–27). These findings strongly suggest the importance of eicosanoid metabolism in the development and progression of human cancers.

Eicosanoids regulate cell functions through activation of specific G-protein-coupled receptors (GPCRs). For example, there are four receptor subtypes that can bind PGE2 (EP1, EP2, EP3, and EP4), with EP1 signaling through a Gq-mediated increase in intracellular calcium, EP2 and EP4 signaling through a Gs-mediated increase in intracellular cAMP, and EP3 signaling through a Gi-mediated decrease of cAMP formation. Recently, evidence points toward the possibility that eicosanoids may also modulate cellular pathways by acting directly at the nucleus. For example, certain AA metabolites can activate the nuclear eicosanoid receptor, the peroxisome proliferator-activated receptor (PPAR) (35–43). cPLA2{alpha} is translocated to the nuclear envelope upon activation (22, 44, 45). However, the biological implications of PPAR activation by endogenous eicosanoid metabolism are currently unknown.

This study was designed to evaluate whether there is an association between the cPLA2{alpha}-controlled eicosanoid metabolism and the growth response to TGF-{beta} in human liver cancer cells. We report that TGF-{beta} regulates tumor cell growth through simultaneous activation of Smad-mediated gene transcription and phosphorylation of cPLA2{alpha}. Whereas Smad activation inhibits tumor cell growth, phosphorylation of cPLA2{alpha} initiates two signaling pathways that counteract Smad-mediated mitoinhibition, including production of PGE2 for activation of its G-protein-coupled plasma membrane receptor EP1 and activation of nuclear eicosanoid receptor PPAR-{gamma}. Our findings establish a novel link between two individual signaling cascades that are important for control of tumor growth.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Minimum essential medium {alpha}, Dulbecco's modified minimum essential medium, RPMI 1640, fetal bovine serum, glutamine, antibiotics, and the LipofectAMINE PlusTM reagent and LipofectAMINETM 2000 reagent were purchased from Invitrogen. The recombinant human TGF-{beta}1 was purchased from R&D Systems (Minneapolis, MN). The cPLA2{alpha} inhibitor arachidonyltrifluoromethyl ketone (AACOCF3) and the p38 MAPK inhibitor SB203580 (purchased from Calbiochem, San Diego, CA) were dissolved at 50 mM in dimethyl sulfoxide as stock. The [{gamma}-32P]ATP was purchased from PerkinElmer Life Sciences. The luciferase assay system was purchased from Promega (Madison, WI). The cell proliferation assay reagent WST-1 was purchased from Roche Applied Science. The antibodies against human cPLA2{alpha}, Smad2/3, p38 MAP kinase, ERK2, and PPAR-{gamma} were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibody against phospho-specific Smad2 was obtained from Upstate Biotechnology (Lake Placid, NY). The antibodies against phospho-cPLA2{alpha} (Ser505), phospho-p38 MAP kinase (Thr180/Tyr182), and phospho-ERK1/2(Thr202/Tyr204) were purchased from Cell Signaling (Beverly, MA). The antibodies against EP1 receptor and COX-2 were purchased from Cayman Chemical (Ann Arbor, MI). The antibody against {beta}-actin was purchased from Sigma. [3H]Arachidonic acid, horseradish peroxidase-linked streptavidin, and chemiluminescence detection reagents were purchased from Amersham Biosciences. The EP1 receptor agonist ONO-004 and antagonist ONO-8711 were obtained from the Ono Pharmaceutical Co., Ltd., Japan. The p3TP-Lux reporter plasmid containing TGF-{beta}-responsive elements (46) was kindly provided by Dr. J. Massague (Memorial Sloan-Kettering Cancer Center, New York). The Ski expression vector (47) was provided by Dr. R. Weinberg, Massachusetts Institute of Technology, and the cPLA2{alpha} expression plasmid (48) was provided by Drs. J. D. Clark and J. L. Knopf at the Genetics Institute, Boston, MA.

Cell Culture—Human liver cancer cells including the cholangiocarcinoma cell lines SG231, CCLP1, and HuCCT1 and the hepatocellular carcinoma cell lines HepG2 and Hep3B were cultured according to our previously described methods (49–52). Briefly, SG231 cells (53) were cultured in serum-supplemented medium (SSM) (minimum essential medium {alpha} with 2 mM L-glutamine, 50 µg/ml gentamycin, 10 mM HEPES, and 10% fetal bovine serum). CCLP1 cells (54) were cultured in Dulbecco's modified minimum essential medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 50 µg/ml gentamycin. HuCCT1 cells (55) (obtained from the Japanese Health Science Research Resources Bank) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 50 µg/ml gentamycin. Hep3B cells (obtained from ATCC) were cultured in minimum essential medium (with Eagle's salt and glutamine) with 10% (v/v) fetal bovine serum. HepG2 cells (obtained from ATCC) were cultured in modified minimum essential medium with 2 mM L-glutamine, 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids, 1.5 g/liter sodium bicarbonate supplemented with 10% fetal bovine serum and antibiotics. The cells were cultured at 37 °C in a humidified CO2 incubator. Experiments were performed when cells reached ~80% confluence. The cells were subjected to serum starvation for 24 h prior to TGF-{beta}1 stimulation; all the experiments were performed in serum-free medium.

Transient Transfections and Reporter Activities—Cultured cells were seeded at a concentration achieving 80% confluence in 6-well plates 18 h before transfection. The cells were transfected with 1 µg of p3TP-Lux reporter vector with 1 µg of cPLA2{alpha} expression plasmid or 1 µg of Ski expression plasmid using LipofectAMINE PlusTM reagent. After transfection, the cells were washed twice with phosphate-buffered saline and lysed with 200 µl of 1x reporter lysis buffer/well (Promega). Luciferase activity was assayed with 20 µl of the cell extract in a Berthold AutoLumat LB 953 luminometer (Nashua, NH) by using a luciferase assay system from Promega as described by the manufacturer. All values are expressed as -fold induction relative to basal activity.

Stable Transfection of Antisense cPLA2{alpha} Plasmid in SG231 Cells— The SG231 cells were exposed to the mixture of LipofectAMINE PlusTM reagents and antisense cPLA2{alpha} plasmid (56) or pcDNA3 control vector for 4 h. Following removal of the transfection mixtures, fresh minimum essential medium {alpha} with 10% fetal bovine serum was added. On the second day, the medium was changed, and the cells were incubated with medium containing 600 µg/ml G418 sulfate (Calbiochem). Subsequent cultures of selected SG231 cells were routinely grown in the presence of selective pressure. Western blotting analysis for cPLA2{alpha} was performed in the selected cells permanently transfected with antisense cPLA2{alpha} or control plasmids. The selected cells with successful reduction of cPLA2{alpha} expression were subsequently used for further experiments.

Cell Proliferation—Cell growth was determined by using the cell proliferation reagent WST-1, a tetrazolium salt that is cleaved by mitochondrial dehydrogenases in viable cells. Briefly, 100 µl of cell suspension (containing 0.5–2 x 104 cells) were plated in each well of 96-well plates. Cells were cultured overnight to allow reattachment. The cells were then incubated with different treatments at indicated concentrations and time periods. Cell proliferation reagent WST-1 (10 µl) was subsequently added to each well. The incubation was continued from 30 min to 4 h at 37 °C, and A450 nm was measured using an automatic enzyme-linked immunosorbent assay plate reader.

Preparation of Whole Cell Lysate—At the end of each treatment, cells were scraped off the plates and centrifuged, washed twice with cold phosphate-buffered saline (PBS) containing 0.5 mM phenylmethylsulfonyl fluoride and 10 µg/ml leupeptin, and resuspended in 5-fold volume of hypotonic buffer consisting of 50 mM HEPES, pH 7.55, 1 mM EDTA, 1 mM dithiothreitol, and protease inhibitor mixture tablets (Roche Diagnostics). After sonication, the whole cell lysate was collected by centrifugation at 14,000 rpm at 4 °C for 10 min using a microcentrifuge to remove cell debris and stored in aliquots at –20 °C until use. The protein concentrations in the cell extracts were determined by the Bio-Rad protein assay.

Immunoblotting—20 µg of cellular protein were separated on 4–20% Tris-glycine gels (Invitrogen) for immunoblotting detection with cPLA2{alpha}, COX-2, EP1, p38 MAPK, phospho-p38 MAPK, ERK2, phospho-ERK1/2, Smad2/3, phospho-Smad2, and PPAR-{gamma} using Tris-glycine SDS running buffer. The separated proteins were electrophoretically transferred onto the nitrocellulose membrane (Bio-Rad). Nonspecific binding was blocked with PBS-T (0.5% Tween 20 in PBS) containing 5% nonfat milk for 1 h at room temperature. The membranes were then incubated overnight at 4 °C with specific primary antibodies in PBS-T containing 1% nonfat milk at the dilutions specified by the manufacturers. Following three washes with PBS-T, the membranes were then incubated with the horseradish peroxidase-conjugated secondary antibodies at a 1:10,000 dilution in PBS-T containing 1% nonfat milk for 1 h at room temperature. The membranes were then washed three times with PBS-T, and the protein bands were visualized with the ECL Western blotting detection system (Amersham Biosciences) according to the manufacturer's instructions.

Phosphorylation of cPLA2{alpha}—Analysis for cPLA2{alpha} phosphorylation was performed as we described previously (49, 57). Equal amounts of cell lysate were preincubated with 5 µg/ml mouse anti-human cPLA2{alpha} monoclonal antibody at 4 °C, followed by addition of 20 µl of protein A/G-agarose (Santa Cruz Biotechnology). The cell lysate preincubated with mouse IgG was used as negative control. The mixtures were incubated overnight at 4 °C. After three washes with the same hypotonic buffer, the pellet was used for immunoblotting using rabbit antiphospho-cPLA2{alpha} (Ser505) antibody.

AA Release and PGE2 Production—To measure arachidonic acid release, the cells grown in 6-well plates were labeled for 18 h with [3H]arachidonic acid (218 Ci/mmol) in serum-supplemented medium. After washing three times with 2 ml of serum-free medium, 2 ml of fresh medium were added to each well. Subsequently, some cells were exposed to 10 ng/ml TGF-{beta}1 for 30 min, whereas others were maintained as controls. In some experiments, the cPLA2{alpha} inhibitor AACOCF3, the p38 MAP kinase inhibitor SB203580, or the MEK1/2 inhibitor PD98059 was added 30 min prior to the addition of TGF-{beta}1. After incubation with TGF-{beta}1 and/or inhibitors, 0.5 ml of medium was removed from each well for scintillation counting.

To measure PGE2 production, the cells cultured in 6-well plates were serum-starved overnight. The cells were exposed to TGF-{beta}1 in the presence or absence of inhibitors for cPLA2{alpha}, COX-2, p38 MAPK, or MEK1/2. After a 30-min incubation, the spent medium was collected, and a 100-µl centrifuged sample was analyzed for PGE2 production using the PGE2 enzyme immunoassay system (Amersham Biosciences) according to the manufacturer's protocol.

RNA Interference—The sequences of PPAR-{gamma} siRNA and EP1 siRNA were selected based on a method described previously (58). The targeted sequences that effectively mediate the silencing of PPAR-{gamma} expression are 5'-GUUGACACAGGAUGCCAUUTT-3' for PPAR-{gamma}1 and 5'-GGUGAAACUCUGGGAGAUUCTT-3' for PPAR-{gamma}2 (sense sequence); the 21-nucleotide synthetic siRNA duplexes were prepared by annealing the single-stranded siRNAs obtained from IDT (Coralville, IA) in the annealing buffer (100 mM potassium acetate, 30 mM HEPES, pH 7.4, 2 mM magnesium acetate) for 2 min at 95 °C and slowly cooled to room temperature. The targeted sequence that effectively mediates the silencing of EP1 expression is 5'-AGCUUGUCGGUAUCAUGGUTT-3' (sense sequence); the 21-nucleotide synthetic siRNA duplex was prepared by Dharmacon Research. Cells were transfected with either PPAR-{gamma} siRNA or EP1 siRNA or with a 21-nucleotide irrelevant RNA duplex as a control using LipofectAMINETM 2000 (Invitrogen). Depletion of PPAR-{gamma} or EP1 was confirmed by Western blotting.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The Effect of TGF-{beta}1 on Liver Cancer Cell Growth—Human liver cancer cell lines were examined for their response to TGF-{beta}1 treatment. As shown in Fig. 1A, inhibition of growth was observed in CCLP1 cells, which express a low level of cPLA2{alpha} protein (Fig. 1B). Treatment of CCLP1 cells with 10 ng/ml TGF-{beta}1 for 24–48 h induced 24–32% inhibition of cell growth. In contrast, TGF-{beta}1 did not inhibit the growth of SG231 and HuCCT1 cells, which express a high level of cPLA2{alpha}. TGF-{beta}1 treatment induced ~20–30% of inhibition of growth in Hep3B and HepG2 cells, which also contain a low level of cPLA2{alpha} protein (data not shown). These observations suggest that the level of cPLA2{alpha} expression may influence cellular response to TGF-{beta}.



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  		FIG. 1.
The effect of TGF-{beta}1 on human liver cancer cell growth and cPLA2{alpha} phosphorylation. A, effect of TGF-{beta}1 on cell growth. Human liver cancer cells were seeded on 96-well plates in serum-supplemented medium for 24 h to allow attachment. After 24 h of serum starvation, the cells were treated with 10 ng/ml TGF-{beta}1 for 24–48 h, and cell growth was assessed by using the cell proliferation reagent WST-1 as described under "Experimental Procedures." The data were obtained from six individual experiments and are expressed as mean ± S.D. of percentage of the respective control (p < 0.01). B, a representative Western blot showing the level of cPLA2{alpha} protein in human liver cancer cells. Equal amounts of cellular protein (20 µg) were subjected to SDS-PAGE followed by Western blot analysis using specific antibodies for cPLA2{alpha} and EP1 receptor. The same blot was then stripped and reprobed for {beta}-actin as loading control. C, TGF-{beta}1 induces phosphorylation of cPLA2{alpha} in human liver cancer cells. SG231, CCLP1, and HuCCT1 cells were treated with 10 ng/ml TGF-{beta}1 for 1, 5, 15, 30, and 60 min, and the cell lysates were obtained for immunoprecipitation with mouse anti-human cPLA2{alpha} monoclonal antibody. The precipitated pellets were then separated by gel electrophoresis on 10% Tris-glycine gels, followed by Western blot analysis with polyclonal antibody against phospho-cPLA2{alpha} (Ser505). The assay specificity was verified by the absence of signal when the control cell lysate was immunoprecipitated with mouse IgG. D, TGF-{beta}1 had no effect on cPLA2{alpha} and COX-2 protein levels in human liver cancer cells. The cells were treated with 10 ng/ml TGF-{beta}1 for 3–48 h, and the cell lysates (20 µg) were obtained for SDS-PAGE and Western blot for cPLA2{alpha} and COX-2. The same blot was then stripped and reprobed for {beta}-actin as loading control.

 
TGF-{beta}1 Induces cPLA2{alpha} Phosphorylation in Human Liver Cancer Cells—We then examined the effect of TGF-{beta}1 on cPLA2{alpha} phosphorylation and protein expression. Fig. 1C shows that treatment of human liver cancer cells with 10 ng/ml TGF{beta}1 induced a rapid phosphorylation of cPLA2{alpha}. In SG231 cells, cPLA2{alpha} phosphorylation occurred within 1 min (3-fold increase), peaked at 15 min (3.5-fold increase), and gradually declined over 60 min. A similar pattern of cPLA2{alpha} phosphorylation was observed in HuCCT1 cells, with a 3-fold increase at 1 min and 7-fold increase at 15–30 min. Phosphorylation of cPLA2{alpha} in CCLP1 cells occurred at 15 min (7-fold increase) and declined at 60 min. In contrast, treatment with 10 ng/ml TGF-{beta}1 for up to 48 h had no effect on the protein expression of cPLA2{alpha} and COX-2 (Fig. 1D). These results demonstrate a direct effect of TGF-{beta}1 on cPLA2{alpha} phosphorylation. Consistent with this, TGF-{beta}1 treatment significantly increased AA release and PGE2 production in these cells (see below).

TGF-{beta}1 Activates p38 MAPK, ERK1/2, and Smad in Human Liver Cancer Cells—Because cPLA2{alpha} is phosphorylated by protein kinases including p38 MAPK and ERK1/2 (p44/42 MAPK), we then examined the effect of TGF-{beta} on p38 MAPK and ERK1/2 activation in our system. Fig. 2 shows that TGF-{beta}1 treatment induced a rapid phosphorylation of p38 MAPK and ERK1/2 (occurred within 1–5 min). In addition, TGF-{beta}1 treatment also induced a rapid Smad2 phosphorylation. These findings, along with the significant increase of Smad reporter activity by TGF-{beta}1 (see below), indicate intact TGF-{beta}-initiated signaling in these cells and support the notion that resistance to TGF-{beta}-mediated mitoinhibition is unlikely because of mutation of TGF-{beta} downstream signaling molecules. This assertion is further supported by Western blot analysis showing expression of both T{beta}RII and T{beta}RI in these cells (data not shown).



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  		FIG. 2.
TGF-{beta}1 activates Smad2, p38 MAPK, and ERK1/2 in human liver cancer cells. CCLP1 cells were treated with 10 ng/ml TGF-{beta}1 for 1–60 min, and the cell lysates were obtained for SDS-PAGE and Western blot using antibodies against phosphorylated Smad2 (A), phosphorylated p38 MAPK (B), phosphorylated ERK1/2 (C), total Smad2/3 (A), total p38 MAP kinase (B), and ERK2 (C). Similar results were also obtained in SG231 and HuCCT1 cells.

 
TGF-{beta}1 Induces AA Release and PGE2 Production via p38 MAPK- and ERK1/2-mediated cPLA2{alpha} Phosphorylation—We next investigated whether inhibition of p38 MAPK or ERK1/2 would block TGF-{beta}1-induced cPLA2{alpha} phosphorylation. Fig. 3A shows that TGF-{beta}1-induced cPLA2{alpha} phosphorylation in SG231 and CCLP1 cells was inhibited by the p38 MAPK inhibitor SB203580 or MEK1/2 inhibitor PD98059. Consistent with this, the TGF-{beta}1-induced [3H]AA release and PGE2 production were also inhibited by pretreatment for 30 min with SB203580, PD98059, and the cPLA2{alpha} inhibitor AACOCF3 (Fig. 3, B and C). These findings further demonstrate the role of p38 MAPK- and ERK1/2-mediated cPLA2{alpha} activation in TGF-{beta}1-induced AA release and PGE2 synthesis. In addition, the TGF-{beta}1-induced PGE2 production was also inhibited by the selective COX-2 inhibitor NS-398 (Fig. 3C), although the level of COX-2 expression was not altered. Time course analysis revealed that TGF-{beta}1-induced [3H]AA release and PGE2 production peaked within 30 min without delayed elevation (up to 48 h, data not shown), which is consistent with the observation that the levels of cPLA2{alpha} and COX-2 proteins were not altered by TGF-{beta}1 treatment (Fig. 1D).



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  		FIG. 3.
TGF-{beta}1 induces AA release and PGE2 production via p38 MAPK- and ERK1/2-mediated cPLA2{alpha} phosphorylation in human liver cancer cells. A, the effect of the p38 MAPK inhibitor SB203580 and the MEK1/2 inhibitor PD98059 on TGF-{beta}1-induced cPLA2{alpha} phosphorylation. SG231 and CCLP1 cells were treated with either 25 µM SB203580 or 25 µM PD98059 for 30 min prior to stimulation with 10 ng/ml TGF-{beta}1 for 15 min. The cell lysates were obtained for immunoprecipitation with mouse anti-human cPLA2{alpha} monoclonal antibody. The precipitated pellets were then separated by gel electrophoresis on 10% Tris-glycine gels, followed by Western blot analysis with polyclonal antibody against phospho-cPLA2{alpha} (Ser505). The data represent an average of three independent experiments (*, p < 0.01 compared with TGF-{beta}1 treatment alone). B, the effect of the cPLA2{alpha} inhibitor AACOCF3, the p38 MAPK inhibitor SB203580, and the MEK1/2 inhibitor PD98059 on TGF-{beta}1-induced [3H]AA release. SG231 and CCLP1 cells grown on 6-well plates and prelabeled with 1 µCi/ml [3H]AA were treated with 10 ng/ml TGF-{beta}1 for 30 min in the presence or absence of AACOCF3 (25 µM), SB203580 (25 µM), or PD98059 (25 µM). The media were then collected for measurement of [3H]AA release. The results are presented as mean ± S.D. of four experiments (*, p < 0.01 compared with control; **, p < 0.05 compared with TGF-{beta}1 treatment). C, the effect of a p38 MAPK inhibitor, a MEK1/2 inhibitor, a cPLA2{alpha} inhibitor, or a COX-2 inhibitor on TGF-{beta}1-induced PGE2 production. SG231 and CCLP1 cells were treated with 10 ng/ml TGF-{beta}1 for 30 min in the presence or absence of SB203580 (25 µM), PD98059 (25 µM), AACOCF3 (25 µM), and the COX-2 inhibitor NS-398 (50 µM). The media were then collected from each well to measure PGE2 production. The results are presented as mean ± S.D. of four experiments (*, p < 0.01 compared with control; **, p < 0.05 compared with TGF-{beta}1 treatment).

 
Blocking cPLA2{alpha}-mediated PGE2 Signaling Restores the Mitoinhibitory Effect of TGF-{beta}1 in SG231 Cells—The findings presented in the above sections suggest that activation of cPLA2{alpha}-controlled arachidonic acid metabolism may represent an important growth-promoting pathway that counteracts TGF-{beta}1-mediated mitoinhibition. To further evaluate this hypothesis, we examined whether inhibition of cPLA2{alpha} would alter the cellular response to TGF-{beta}. SG231 cells were treated with 10 ng/ml TGF-{beta}1 in the presence of the p38 MAPK inhibitor SB203580 (25 µM), the MEK1/2 inhibitor PD98059 (25 µM), the cPLA2{alpha} inhibitor AACOCF3 (25 µM), or the COX-2 inhibitor NS-398 (50 µM) to determine cell growth. As shown in Fig. 4A, although treatment with TGF-{beta}1 alone did not alter the growth of SG231 cells, it induced 36% of mitoinhibition when the cells were cotreated with the cPLA2{alpha} inhibitor AACOCF3; this result is statistically significant when compared with cells treated with AACOCF3 alone (p < 0.01). Consistent with this, TGF-{beta}1 also significantly inhibited cell growth in the presence of the p38 MAPK inhibitor SB203580, the MEK1/2 inhibitor PD98059 (25 µM), and the COX-2 inhibitor NS-398. Therefore, inhibition of cPLA2{alpha} activation and PGE2 synthesis can partially restore TGF-{beta}-induced mitoinhibition. The observation that blocking cPLA2{alpha} activation and PGE2 production also prevents cell growth in the absence of TGF-{beta}1 indicates the involvement of cPLA2{alpha}/COX-2/PGE2 signaling in the spontaneous liver cancer cell growth.



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  		FIG. 4.
Blocking cPLA2{alpha}-mediated PGE2 signaling restores mitoinhibition by TGF-{beta}1 in SG231 cells. A, inhibition of PGE2 synthesis restores TGF-{beta}1-mediated growth inhibition. SG231 cells were treated with 10 ng/ml TGF-{beta}1 in the presence or absence of the p38 MAPK inhibitor SB203580 (25 µM), the MEK1/2 inhibitor PD98059 (25 µM), the cPLA2{alpha} inhibitor AACOCF3 (25 µM), or the COX-2 inhibitor NS-398 (50 µM). The cell proliferation was assessed after 48 h of treatment. The data were obtained from six individual experiments and are expressed as mean ± S.D. of percentage of control. *, p < 0.01 compared with cells without TGF-{beta}1 treatment. B, the effect of selective EP1 antagonist on TGF-{beta}1-mediated growth inhibition. SG231 cells were treated with 10 µM ONO-004, 50 µM PGE2, or 10 ng/ml TGF-{beta}1 in the absence or presence of 10 µM ONO-8711; cell proliferation was assessed after 48 h. The data were obtained from six individual experiments and are expressed as mean ± S.D. of percentage of control. *, p < 0.05, significant increase of cell growth compared with the control; **, p < 0.01, significant decrease of cell growth compared with cells without ONO-8711 treatment. C, the effect of siRNA suppression of the EP1 receptor on TGF-{beta}1-mediated growth inhibition. SG231 cells were transfected with EP1 siRNA for 24 h. The cells were then treated with 50 µM PGE2 or 10 ng/ml TGF-{beta}1 for 48 h to assess cell proliferation (top). The data were obtained from six individual experiments and are expressed as mean ± S.D. of percentage of control. *, p < 0.01 compared with cells transfected with control RNA. Approximately 50% reduction of EP1 protein was observed in cells transfected with EP1 siRNA when compared with cells transfected with control RNA or without transfection (bottom).

 
We then examined the direct effect of PGE2 and an EP1 receptor agonist on human liver cancer cell growth. Treatment with PGE2 or ONO-004 (selective EP1 receptor agonist) resulted in 31–42% increase of cell growth, which was inhibited by the EP1 receptor antagonist ONO-8711 (Fig. 4B). Consistent with this, the PGE2-induced cell growth was also blocked by siRNA suppression of the EP1 receptor (Fig. 4C). Furthermore, the mitoinhibitory effect of TGF-{beta}1 was partially restored by the EP1 receptor antagonist ONO-8711 (Fig. 4B) and EP1 siRNA (Fig. 4C). These findings demonstrate a key role of the EP1 receptor for attenuation of TGF-{beta}1-induced mitoinhibition.

Suppression of Smad Activation Prevents TGF-{beta}1-mediated Inhibition of CCLP1 Cell Growth—The role of Smad in human liver cancer cell growth was examined by overexpressing Ski, a transcriptional co-repressor of Smads. CCLP cells were selected because the growth of these cells is inhibited by TGF-{beta}1 treatment alone (Fig. 1A). The cells transfected with Ski expression plasmid or control vector were treated with vehicle or 10 ng/ml TGF-{beta}1 for 48 h to determine cell growth. As shown in Fig. 5, overexpression of Ski inhibited Smad-mediated transcriptional activity and partially reversed TGF-{beta}1-induced mitoinhibition. These results suggest a role of Smad activation in TGF-{beta}1-induced inhibition of liver cancer cell growth.



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  		FIG. 5.
Blocking Smad activation prevents TGF-{beta}1-mediated inhibition of CCLP1 cell growth. A, the effect of Ski (Smad transcriptional co-repressor) on TGF-{beta}1-mediated growth inhibition. Equal numbers of CCLP1 cells stably transfected with Ski expression plasmid or control vector were treated with vehicle or 10 ng/ml TGF-{beta}1 for 48 h to determine cell growth. The data were obtained from six individual experiments and are expressed as mean ± S.D. of percentage of the control. *, p < 0.01 compared with vector control cells without TGF-{beta}1 treatment. B, the effect of Ski overexpression on TGF-{beta}1-mediated transcriptional activity. CCLP1 cells were transiently transfected with the Ski expression plasmid or the control vector pcDNA3 with cotransfection of the p3TP-Lux reporter construct containing the TGF-{beta}-responsive elements. After transfection, the cells were treated with vehicle or 10 ng/ml TGF-{beta}1 for 24 h, and the cell lysates were obtained to determine the luciferase reporter activity. The relative luciferase activities (arbitrary units) were calculated after normalization of cellular protein. The data are presented as mean ± S.D. of six independent experiments. *, p < 0.01 compared with vector control cells in the absence of TGF-{beta}1. **, p < 0.01 compared with vector control cells in the presence of TGF-{beta}1. C, a representative Western blot showing the level of Ski protein in cells transfected with the Ski expression plasmid or control vector pcDNA3. Equal amounts of cellular protein (20 µg) were used, and the same blot was then stripped and reprobed for {beta}-actin as loading control.

 
Overexpression of cPLA2{alpha} Blocks TGF-{beta}1-mediated Smad Activation and Mitoinhibition—We then investigated whether cPLA2{alpha}-mediated AA release might influence TGF-{beta}-mediated Smad transcriptional activity. CCLP1 cells (with a low basal level of cPLA2{alpha} expression) were transiently transfected with the cPLA2{alpha} expression plasmid or the control plasmid MT-2 with cotransfection of the p3TP-Lux reporter construct (containing the TGF-{beta}-responsive element) (46); the cells were then treated with TGF-{beta}1 to determine the luciferase reporter activity. Overexpression of cPLA2{alpha} significantly inhibited TGF-{beta}1-mediated Smad transcriptional activity (Fig. 6), indicating a role of cPLA2{alpha} for inhibition of Smad activation. Consistent with this, overexpression of cPLA2{alpha} partially prevented TGF-{beta}1-induced mitoinhibition.



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  		FIG. 6.
Overexpression of cPLA2{alpha} blocks TGF-{beta}1-mediated Smad activation and growth inhibition. A, the effect of cPLA2{alpha} overexpression on TGF-{beta}1-mediated reporter activity. CCLP1 cells were transiently transfected with the cPLA2{alpha} expression plasmid or control plasmid MT-2 with cotransfection of the p3TP-Lux reporter construct containing the TGF-{beta}-responsive element. After transfection, the cells were treated with vehicle or TGF-{beta}1 (10 ng/ml) for 24 h, and the cell lysates were obtained to determine the luciferase reporter activity. The data are presented as mean ± S.D. of six independent experiments. *, p < 0.05 compared with cells transfected with MT-2 in the absence of TGF-{beta}1. **, p < 0.01 compared with cells transfected with MT-2 in the presence of TGF-{beta}1. B, the effect of cPLA2{alpha} overexpression on TGF-{beta}1-mediated growth inhibition. CCLP1 cells were transiently transfected with the cPLA2{alpha} expression plasmid or MT-2 control vector. After transfection, the cells were treated with vehicle or 10 ng/ml TGF-{beta}1 for 48 h to determine cell growth. The data are presented as mean ± S.D. of six independent experiments. *, p < 0.05 compared with cells transfected with MT-2 in the absence of TGF-{beta}1. **, p < 0.01 compared with cells transfected with MT-2 in the presence of TGF-{beta}1. C, a representative Western blot showing the levels of cPLA2{alpha} protein in cells transfected with the cPLA2{alpha} expression plasmid or MT-2 control vector (with or without 10 ng/ml TGF-{beta}1 treatment). Equal amounts of cellular protein (20 µg) were used, and the same blot was then stripped and reprobed for {beta}-actin as loading control.

 
Antisense Inhibition of cPLA2{alpha} Enhances TGF-{beta}1-mediated Smad Transcriptional Activity—The effect of cPLA2{alpha} on Smad activation was further investigated by using antisense inhibition of cPLA2{alpha}. SG231 cells (with a relatively high level of cPLA2{alpha} expression) were stably transfected with the antisense cPLA2{alpha} plasmid (56) or control vector pcDNA3. The selected cells with optimal depletion of cPLA2{alpha} protein were then transiently transfected with the TGF{beta}-responsive p3TP-Lux reporter plasmid to determine Smad activation. Whereas Smad-mediated gene transcription was not altered by TGF-{beta}1 in control vector cells, a significant increase of Smad transcriptional activity was observed when cPLA2{alpha} antisense cells were treated with 10 ng/ml TGF-{beta}1 for 24 h (Fig. 7). These observations further demonstrate the involvement of cPLA2{alpha} in TGF-{beta}1-induced Smad activation.



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  		FIG. 7.
Antisense inhibition of cPLA2{alpha} enhances TGF-{beta}1-mediated Smad activation. SG231 cells stably transfected with the antisense (AS) cPLA2{alpha} plasmid or control vector pcDNA3 were transiently transfected with the TGF-{beta}-responsive p3TP-Lux reporter plasmid, and the cells were treated with or without TGF-{beta}1 (10 ng/ml) for 24 h. The cell lysates were then obtained to determine the luciferase reporter activity, and the data were normalized by the amount of cellular proteins. The results are presented as mean ± S.D. of six independent experiments. *, p < 0.05 compared with pcDNA3 in the presence of TGF-{beta}1.

 
The Effect of PPAR-{gamma} on TGF-{beta}1-mediated Smad Activation—Given that PPAR-{gamma} is the nuclear eicosanoid receptor known to bind and inhibit Smad3 in vitro (59) and that cPLA2{alpha} is able to activate PPAR-{gamma} (60), we postulated that cPLA2{alpha} might inhibit Smad3 through activation of PPAR-{gamma}. We examined the expression and function of PPAR-{gamma} in different liver cancer cell lines, all of which expressed a comparable level of PPAR-{gamma}. Our previous transfection experiments with a reporter construct containing peroxisome proliferator-response element (PPRE) revealed ~2.5-fold increase of PPRE reporter activity in response to PPAR-{gamma} ligand treatment (5 µM 15d-PGJ2 or 10 µM troglitazone), indicating that the endogenous PPAR-{gamma} protein in these cells is functional (50). Furthermore, the PPRE reporter activity was significantly increased in cPLA2{alpha}-overexpressed cells but reduced in cells with antisense depletion of cPLA2{alpha}, suggesting activation of PPAR by cPLA2{alpha} in human liver cancer cells (61).

To further document the role of PPAR-{gamma} in TGF-{beta}-mediated Smad transcriptional activity, CCLP1 cells were transiently cotransfected with a PPAR-{gamma} expression plasmid and the TGF-{beta}-responsive p3TP-Lux reporter plasmid. As shown in Fig. 8, overexpression of PPAR-{gamma} partially inhibited TGF-{beta}1-mediated Smad transcriptional activity and prevented TGF-{beta}1-induced mitoinhibition. In contrast, siRNA inhibition of PPAR-{gamma} increased TGF-{beta}1-mediated Smad transcriptional activity and partially restored TGF-{beta}1-mediated mitoinhibition in SG231 cells (Fig. 9). These results demonstrate a direct role of PPAR-{gamma} in TGF-{beta}1-mediated Smad activation and growth regulation of human liver cancer cells.



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  		FIG. 8.
Overexpression of PPAR-{gamma} blocks TGF-{beta}1-induced Smad activation and mitoinhibition. A, CCLP1 cells were transiently cotransfected with a PPAR-{gamma} expression plasmid and the TGF-{beta}-responsive p3TP-Lux reporter plasmid. After transfection, the cells were treated with vehicle or 10 ng/ml TGF-{beta}1 for 24 h, and the cell lysates were obtained to determine luciferase reporter activity. The obtained data were normalized by the amount of cellular proteins, and the results are presented as mean ± S.D. of six independent experiments. *, p < 0.01 compared with pcDNA3 in the absence of TGF-{beta}1. **, p < 0.05 compared with the pcDNA3 group in the presence of TGF-{beta}1. B, overexpression of PPAR-{gamma} partially prevents TGF-{beta}1-induced mitoinhibition. CCLP1 cells were seeded on 96-well plates in serum-supplemented medium for 24 h to allow attachment. After transient transfection with a PPAR-{gamma} expression plasmid or pcDNA control vector, the cells were treated with 10 ng/ml TGF-{beta}1 for 48 h to determine cell growth. The data were obtained from six individual experiments and are expressed as mean ± S.D. of percentage of the control (*, p < 0.05 compared with TGF-{beta}1-treated vector control cells). C, a representative Western blot showing the level of PPAR-{gamma} protein in cells transfected with the PPAR-{gamma} expression plasmid or pcDNA3 control vector.

 



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  		FIG. 9.
siRNA suppression of PPAR-{gamma} enhances TGF-{beta}1-induced Smad activation. A, SG231 cells were transiently cotransfected with PPAR-{gamma} siRNA and the TGF-{beta}-responsive p3TP-Lux reporter plasmid. After transfection, the cells were treated with vehicle or 10 ng/ml TGF-{beta}1 for 24 h, and the cell lysates were obtained to determine luciferase reporter activity. The obtained data were normalized by the amount of cellular protein, and the results are presented as mean ± S.D. of six independent experiments. *, p < 0.05 compared with PPAR-{gamma} siRNA cells without TGF-{beta}1 or control siRNA cells with TGF-{beta}1. B, siRNA suppression of PPAR-{gamma} restores mitoinhibition by TGF-{beta}1. SG231 cells were seeded on 96-well plates in serum-supplemented medium for 24 h to allow attachment. After transfection with PPAR-{gamma} siRNA, the cells were treated with 10 ng/ml TGF-{beta}1 for 48 h to determine cell growth. The data were obtained from six individual experiments and are expressed as mean ± S.D. of percentage of the control (*, p < 0.01 compared with PPAR-{gamma}-transfected cells without TGF-{beta}1 treatment or control vector cells with TGF-{beta}1 treatment). C, a representative Western blot showing the level of PPAR-{gamma} protein in cells transfected with the PPAR-{gamma} siRNA or control siRNA.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
As a result of oncogenic stimuli or other unknown mechanisms, human cancers attenuate TGF-{beta}-induced suppression but exploit TGF-{beta}-mediated mechanisms conducive to tumor progression. Here we provide novel evidence that links eicosanoid metabolism to TGF-{beta}-mediated Smad activation and tumor growth control. Our findings suggest that the cPLA2{alpha}-mediated eicosanoid cascade counteracts Smad-induced mitoinhibition through two parallel mechanisms, i.e. PGE2/EP1 and PPAR-{gamma}/Smad signaling pathways, as outlined in Fig. 10. These conclusions are based on several lines of evidence. 1) TGF-{beta} treatment of human liver cancer cells results in simultaneous Smad-mediated gene transcription (hence inhibiting growth) and phosphorylation of cPLA2{alpha} (hence promoting growth). 2) Tumor cells with high cPLA2{alpha} expression are more resistant to TGF-{beta}1-mediated mitoinhibition. 3) TGF-{beta}1 treatment increases AA release and PGE2 production in tumor cells. 4) Inhibition of cPLA2{alpha}- and COX-2-mediated PGE2 synthesis and EP1 receptor restores the mitoinhibition by TGF-{beta}1. 5) Overexpression of cPLA2{alpha} increases PPRE reporter activity and blocks TGF-{beta}1-mediated Smad activation, whereas the opposite phenomenon is seen with antisense inhibition of cPLA2{alpha}. 6) Overexpression of PPAR-{gamma} partially prevented TGF-{beta}1-mediated Smad activation and mitoinhibition. 7) siRNA depletion of PPAR-{gamma} enhanced TGF-{beta}1-mediated Smad activation and partially restored the mitoinhibition by TGF-{beta}1.



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  		FIG. 10.
Proposed mechanisms for the cPLA2{alpha}-controlled AA cascade in subversion of TGF-{beta}-mediated mitoinhibition. TGF-{beta} regulates tumor cell growth through simultaneous activation of Smad-mediated gene transcription and phosphorylation of cPLA2{alpha}. Whereas Smad activation inhibits tumor cell growth, phosphorylation of cPLA2{alpha} initiates two signaling pathways that counteract Smad-mediated mitoinhibition, including production of PGE2 for activation of its G-protein-coupled plasma membrane receptor, EP1, and activation of the nuclear eicosanoid receptor, PPAR-{gamma}.

 
cPLA2{alpha} is a cytoplasmic enzyme that selectively cleaves AA from membrane phospholipids, which provides substrate for eicosanoid synthesis by downstream enzymes including cyclooxygenase, lipoxygenase, and monooxygenase (20, 62–64). The cPLA2{alpha} activity is tightly regulated by mechanisms including enzyme phosphorylation, calcium influx, and induction of gene expression. In this study, we show that TGF-{beta} treatment enhances PGE2 production in human liver cancer cells, which in turn promotes cell growth through activation of the EP1 receptor. The observations that TGF-{beta} treatment enhances cPLA2{alpha} phosphorylation and that inhibition of cPLA2{alpha} blocks TGF-{beta}1-induced AA release and PGE2 production document an important role of cPLA2{alpha} phosphorylation in TGF-{beta}-induced PGE2 synthesis. Our data demonstrate the role of p38 MAPK and ERK1/2 in TGF-{beta}-induced cPLA2{alpha} phosphorylation. Induction of cPLA2{alpha} or COX-2 enzyme expression does not appear to be involved because the levels of these proteins were not altered by TGF-{beta} treatment. It is worth noting that although COX-2 protein expression was not altered by TGF-{beta}, pretreatment with the selective COX-2 inhibitor NS-398 significantly blocked TGF-{beta}1-induced PGE2 production and restored TGF-{beta}-mediated mitoinhibition. The latter effect appears similar to that observed with cPLA2{alpha} inhibition, suggesting coupling of cPLA2{alpha} and COX-2 for PGE2 synthesis and liver cancer cell growth. The importance of PGE2 and the EP1 receptor in TGF-{beta}-mediated growth regulation is further supported by the observations that TGF-{beta}-mediated mitoinhibition is restored by pretreatment with the EP1 receptor antagonist ONO-8711 or siRNA.

Upon activation through phosphorylation and intracellular calcium influx, cPLA2{alpha} is translocated to cellular membranes releasing arachidonic acid from membrane phospholipids. Based on the key role of cPLA2{alpha} in AA release (20, 62–64), the activation of PPAR by AA in cultured cells (65, 66), and the association of cPLA2{alpha} with the nuclear envelope (22, 44, 45), we hypothesized that translocation of cPLA2{alpha} to the nuclear envelope upon activation may lead to increased production of AA in the nuclei for PPAR activation. This hypothesis was first presented elsewhere by using a reporter construct containing PPRE (60). The results in this study lead us to speculate that cPLA2{alpha} may activate PPAR-{gamma} and regulate cell growth through mechanisms independent of the PPRE cis-element.

PPAR-{gamma} is one of the low affinity dietary lipid receptors (67). It is widely expressed in various tissues including adipose tissue and in cells of hepatobiliary origin (50, 68, 69). After binding a ligand (e.g. thiazolidinediones (TZDs), arachidonate, or others), it forms a heterodimer with the cis-retinoid acid receptor RXR (retinoic X receptor), binds to a PPRE, and activates transcription of selected genes. In this study, we provide the first evidence that activation of PPAR-{gamma} by cPLA2{alpha} alters Smad-mediated transcriptional activity. The effect is likely mediated through interaction between PPAR-{gamma} and R-Smad. This contention is supported by the experimental findings in the current study, the established role of cPLA2{alpha}-mediated AA for PPAR activation (60), and the direct binding between PPAR-{gamma} and Smad3 in vitro (59).

The exact role of PPAR-{gamma} in tumor development remains the subject of intense debate; whereas extensive studies have documented the antiproliferative effects of PPAR-{gamma} activation, equally convincing experimental evidence describes its oncogenic actions (70). Studies using tumor cell lines including hepatobiliary carcinoma cells have shown growth arrest, differentiation, and apoptosis upon ligand-induced PPAR-{gamma} activation, supporting a role for PPAR-{gamma} in tumor suppression (50, 71, 72). This assertion is also supported by the identification of hemizygous loss-of-function mutations in the PPAR-{gamma} gene in certain human cancers (73). However, such mutations are rare overall, and the loss of both alleles has not been found in any tumor (74). On the other hand, recent evidence has begun to challenge the antitumorigenic functions of PPAR-{gamma}. For example, ligands such as thiazolidinediones, often used to study PPAR-{gamma} functions, are known to affect cell growth and survival through mechanisms independent of PPAR-{gamma} activation (70). Differential and seemingly opposite dose-dependent responses of PPAR-{gamma} ligands have been reported in neoplastic cells (75). In a recent genetic study, transgenic mice expressing a constitutively active form of PPAR-{gamma} in mammary gland showed enhanced polyoma virus middle T antigen-induced tumor development, indicating that PPAR-{gamma} may serve as a tumor promoter after an initiating event (76). Such a view is also supported by the observations that PPAR-{gamma} ligand treatment increased the frequency of colon tumors in the adenomatous polyposis coli (APCMin) mice (77, 78), an animal model with ablation in the APC tumor suppressor gene predisposing it to intestinal neoplasia. Further, genetic and pharmacological studies suggest that PPAR-{gamma} activation in colon neoplasia may be bifunctional, prooncogenic after tumor initiation caused by APC loss but otherwise protective, presumably because of its effects on promoting differentiation (79). The above observations in mammary gland and colon tumor models indicate that PPAR-{gamma} may be involved in tumor maintenance and growth rather than initiation. In this study, by altering the expression level of PPAR-{gamma} in human liver cancer cells, we provide evidence that PPAR-{gamma} prevents TGF-{beta}1-mediated Smad activation and mitoinhibition in vitro. The fact that PPAR-{gamma} signaling seems to exacerbate tumor growth in cancer-prone backgrounds underscores that contextual variance may determine PPAR-{gamma} signaling outcomes in neoplasms. Further studies are needed to determine whether similar conclusions can be drawn in animal models of hepatobiliary carcinogenesis and to determine the validity of these observations in human cancers.

In summary, the results in this study establish a novel link between two separate signaling cascades that are crucial for control of tumor growth. Given that the cPLA2{alpha}- and COX-2-mediated eicosanoid metabolism is up-regulated in a variety of human cancers, the signaling pathways depicted in this paper may represent a common mechanism by which cancer cells escape mitoinhibition and acquire signaling leading to tumor progression during carcinogenesis.


   FOOTNOTES
 
* This work was supported by grants from the American Liver Foundation and the Cancer Research Foundation of America and by Grant DK49615 from the National Institutes of Health. 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

To whom correspondence should be addressed: Dept. of Pathology, University of Pittsburgh School of Medicine, Presbyterian University Hospital C902, 200 Lothrop St., Pittsburgh, PA 15213. Tel.: 412-647-9504; Fax: 412-647-5237; E-mail: wut{at}upmc.edu.

1 The abbreviations used are: TGF-{beta}, transforming growth factor-{beta}; T{beta}RI and -II, TGF-{beta} receptor type I and II, respectively; AA, arachidonic acid; cPLA2{alpha}, cytosolic phospholipase A2{alpha}; COX-2, cyclooxygenase-2; PGE2, prostaglandin E2; PPAR-{gamma}, peroxisome proliferator-activated receptor-{gamma}; AACOCF3, arachidonyltrifluoromethyl ketone; MAP, mitogen-activated protein; MAPK, MAP kinase; ERK, extracellular signal-regulated kinase; PBS, phosphate-buffered saline; PBS-T, 0.5% Tween 20 in PBS; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; siRNA, small interfering RNA; PPRE, peroxisome proliferator-response element. Back


   ACKNOWLEDGMENTS
 
We thank Dr. J. Massague at the Memorial Sloan-Kettering Cancer Center, New York, for providing the p3TP-Lux reporter plasmid, Dr. R. Weinberg at the Massachusetts Institute of Technology, Boston, for providing the Ski expression plasmid, and Drs. J. D. Clark and J. L. Knopf at the Genetics Institute, Boston, for the cPLA2 expression plasmid. The EP1 receptor agonist ONO-004 and antagonist ONO-8711 were kindly provided by the Ono Pharmaceutical Co., Ltd., Japan.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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