85-kDa cPLA2 plays a critical role in PPAR-mediated gene transcription in human hepatoma cells

Chang Han1, A. Jake Demetris1, George Michalopoulos1, James H. Shelhamer2, and Tong Wu1

1 Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213; and 2 Critical Care Medicine Department, National Institutes of Health, Bethesda, Maryland 20892


    ABSTRACT
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In an effort to understand the role of key eicosanoid-forming enzymes in the activation of peroxisome proliferator-activated receptor (PPAR), this study was designed to evaluate the possible contributions of cytosolic phospholipase A2 (cPLA2) and group IIA secretory phospholipase A2 (sPLA2) in the regulation of PPAR-mediated gene transcription in a human hepatoma cell line (HepG2). The HepG2 cells express both PPAR-alpha and -gamma but not PPAR-beta . Overexpression of cPLA2, but not group IIA sPLA2 in the HepG2 cells, caused a significantly increased PPAR-alpha /gamma -mediated reporter activity. Antisense inhibition of cPLA2 resulted in a significantly decreased PPAR-alpha /gamma activity. The PPAR-alpha /gamma -induced gene transcription in the HepG2 cells was inhibited by the cPLA2 inhibitors methyl arachidonyl fluorophosphonate and arachidonyltrifluoromethyl ketone, but not by the sPLA2 inhibitor LY311727. The expression of PPAR-alpha -mediated endogenous gene apolipoprotein A-II was increased in cells with overexpression of cPLA2, decreased in cells with antisense inhibition of cPLA2, but unaltered in cells with overexpression of group IIA sPLA2. The above results demonstrated an important role of cPLA2, but not group IIA sPLA2 in the control of PPAR activation. The cPLA2-mediated PPAR activation was likely mediated by arachidonic acid and prostaglandin E2. This study reveals a novel intracellular function of cPLA2 in PPAR activation in HepG2 cells. The cPLA2 thus may represent a potential therapeutic target for the control of PPAR-related liver and metabolic disorders such as obesity, lipid metabolic disorders, diabetes mellitus, and atherosclerosis.

HepG2 cell; liver; arachidonic acid; prostaglandin; peroxisome proliferator response element


    INTRODUCTION
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INTRODUCTION
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ARACHIDONIC ACID (AA) metabolites leukotrienes (LTs) and prostaglandins (PGs), termed eicosanoids, are biologically active lipid molecules that can act as potent inflammatory mediators or participate in intracellular signal transduction (1, 5, 8-10, 27, 30, 32, 42). In addition to the well-documented effect of AA metabolites in inflammation, numerous studies have established that AA metabolites play important roles in a variety of cellular functions including cell growth and differentiation, regulation of ion transport, neurotransmitter uptake and release, blood vessel tone, and cytoskeleton and membrane remodeling. Despite the documented effects of AA metabolites in inflammation and intracellular signal transduction, the detailed mechanisms of eicosanoids in intracellular signal transduction are poorly understood. Recent studies have shown that eicosanoids are the natural ligands for peroxisome proliferator-activated receptors (PPARs) with leukotriene B4 (LTB4), a lipoxygenase product of AA, as a potent natural ligand for PPAR-alpha (12), and 15-deoxy-Delta 12, 14 PGJ2 (15d-PGJ2), a cyclooxygenase pathway product, as a potent ligand for PPAR-gamma (15, 23). These findings demonstrate a direct role of eicosanoids in the regulation of gene expression in the nucleus and also suggest the importance of eicosanoids in mediating the diverse functions of PPARs.

The biosynthesis of eicosanoids is tightly controlled by a series of enzymes including phospholipase A2s (PLA2; EC 3.1.1.4), cyclooxygenases, and lipoxygenases. PLA2s are a group of enzymes that catalyze the hydrolysis of the sn-2-ester bond of phospholipids, resulting in the production of free fatty acid and lysophospholipids, which can then be further metabolized to produce eicosanoids and platelet-activating factor (1, 5, 8-10, 27, 30, 32, 42). These lipid molecules are crucial for various cellular responses such as inflammation, signal transduction, and membrane remodeling. The group IV 85-kDa cytosolic PLA2 [cPLA2, also termed as cPLA2alpha in most recent literature in light of the cloning of 2 related isoforms cPLA2beta and gamma  (40, 47)] is a rate-limiting key enzyme in the liberation of AA from membrane phospholipids for subsequent production of bioactive eicosanoids in activated cells. The cPLA2 selectively cleaves AAs in the sn-2 position of substrate phospholipids and is regulated by phosphorylation, physiologically relevant concentrations of Ca2+, induction of gene expression, G proteins, and the S-100 protein p11 (3, 6, 9, 19, 20, 22, 24, 27, 28, 34, 35, 52, 54, 55, 57). Various Ca2+-mobilizing agonists and proinflammatory cytokines induce cPLA2 activation for immediate and delayed eicosanoid biosynthesis through mechanisms involving intracellular Ca2+ elevation, phosphorylation by mitogen-activated protein kinase, and induction of cPLA2 enzyme synthesis. In addition to cPLA2, accumulating evidence also shows that other forms of PLA2s, including type II secretory PLA2 (sPLA2), may also participate in the release of AA release in various types of cells. Numerous studies have documented the involvement of AA metabolites, PGs, and leukotrienes in various liver physiological and pathophysiological processes including liver regeneration, growth regulation of hepatocytes, inflammation, cirrhosis, and hepatocytic ischemic/hypoxic injuries (31, 36, 37, 49, 51, 53, 59). However, the detailed mechanisms for their actions and the regulation of the key eicosanoid-forming enzymes in liver tissue or liver cells are not well understood. Although hepatocytes are the major source for group IIA sPLA2 (7, 14), they also express cPLA2. The physiological functions of individual PLA2s in the hepatocytes are currently unknown.

PPARs belong to the superfamily of ligand-activated nuclear transcription factors (11, 16, 48), which, on heterodimerization with the retinoid X receptor (RXR), bind to specific peroxisome proliferator response elements (PPRE) to regulate the expression of target genes. There are three PPAR subtypes (alpha , beta , and gamma ) that are often coexpressed in various tissues. PPAR-alpha is highly expressed in tissues with elevated rates of fatty acid metabolism such as liver, where it plays a critical role in liver lipid metabolism and hepatocarcinogenesis (11, 16, 48). The PPAR ligands (including natural ligands AA metabolites and pharmacological ligands) directly bind to PPARs and thus regulate the transcription activities of PPARs in the nucleus. Although AA metabolites have been identified as the natural ligands for PPARs, the potential role of eicosanoid-forming enzymes in production of eicosanoids for PPAR activation in the nucleus has not been studied. Because several different forms of PLA2s may coexist within the same cell or tissue and various PLA2s carry out essentially the same enzymatic reaction, it has been very difficult to correlate the various PLA2 activities with the various physiological functions, such as AA release and eicosanoid production for modulation of inflammation or signal transduction. Accumulating evidence has shown that both cPLA2 and group IIA sPLA2 may participate in hormone-induced release of AA for intracellular signal transduction. The role of cPLA2 in intracellular signal transduction is supported by the characteristics of this enzyme, including its selectivity for substrate AA and its regulation by phosphorylation, physiologically relevant concentration of calcium, and G proteins. One of the intriguing characteristics of cPLA2 is its preferential translocation from cytoplasm to nuclear envelope in response to intracellular calcium increase (17, 27, 38, 39, 41). However, the physiological significance of this phenomenon and the potential functions of AA metabolites in the nucleus remain largely unknown.

On the basis of the documented nuclear targeting of cPLA2 and the finding that AA metabolites represent the natural PPAR ligands, we reasoned that translocation of cPLA2 to nuclear envelope on activation would likely result in production of eicosanoids in the nuclei that might modulate the transcriptional activity of PPARs. This study was thus designed to document the role of cPLA2 in PPAR-mediated gene transcription in a human liver cell line (HepG2), and this effect was compared with the group IIA sPLA2. Our results demonstrate that cPLA2, but not group IIA sPLA2, plays an important role in the regulation of PPAR-alpha /gamma -mediated gene transcription in HepG2 cells. These findings suggest that cPLA2 may represent a potential therapeutic target for the control of PPAR-related liver and metabolic disorders such as obesity, lipid metabolic disorders, diabetes mellitus, and atherosclerosis.


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Materials. MEM, fetal bovine serum, glutamine, antibiotics, and the Lipofectamine plus reagent were purchased from Life Technologies (Rockville, MD). Chloramphenicol acetyltransferase (CAT) enzyme assay system was purchased from Promega (Madison, WI). Chemiluminescent reporter assay for beta -galactosidase was purchased from Tropix (Bedford, MA). The sPLA2 inhibitor LY311727 was a generous gift from Dr. E. Mihelich at Lilly Research Laboratories (Indianapolis, IN). Other PLA2 inhibitors including methyl arachidonyl fluorophosphonate (MAFP) and arachidonyltrifluoromethyl ketone (AACOCF3) as well as ionophore A23187 and PGE2 were obtained from Calbiochem (San Diego, CA). [3H]chloramphenicol and [3H-]AA and sn-2-[14C]arachidonyl phosphatidylcholine were purchased from DuPont New England Nuclear (Boston, MA). Plasmid purification reagents were from Qiagene (Valencia, CA). The antibodies for human cPLA2 and PPAR-alpha , -beta , and -gamma were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibody for human group IIA sPLA2 was obtained from Cayman Chemical (Ann Arbor, MI). PGE2 enzymeimmunoassay system, horseradish peroxidase (HRP)-linked streptavidin, chemiluminescence detection reagents, and the sn-2-[14C]linoleoyl phosphatidylethanolamine were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Unlabeled phospholipids were purchased from Avanti Polar Lipids. Unless indicated otherwise, all other chemicals were from Sigma.

The cPLA2 expression plasmid was kindly provided by Drs. J. Clark and J. Knopf at the Genetics Institute (Cambridge, MA) (6). The PPRE reporter construct was kindly provided by Dr. W. Wahli (Lausanne, Switzerland). The antisense cPLA2 plasmid was constructed by us as reported previously (56). The PPAR-alpha expression plasmid was a generous gift from Dr. F. Gonzalez at the National Institutes of Health (Bethesda, MD). The Gal4-PPAR-alpha and the Gal4DBD expression plasmids were kindly provided by Dr. D. Kelly at Washington University (St. Louis, MO). The reporter plasmid pG5Luc was obtained from Promega.

Cell culture. The HepG2 cells (human hepatoma cell line) were cultured in MEM supplemented with 10% fetal bovine serum, 2 mM glutamine, and antibiotics. All experiments were performed when cells reached ~80% confluence and conducted in serum-free medium with serum deprivation for 12 h before experiments.

Plasmid construction. To construct group IIA sPLA2 expression plasmid, we first obtained full-length human group IIA sPLA2 cDNA using RT-PCR of human lung RNA. The primer pair was constructed according to the cDNA sequence (43). It amplified an 823-bp product and was composed of the following sequences: 5' primer-CAACTCTGGAGTCCTCTGAGAGAGCC (8-33); 3' primer-GCTAATTGCTTTATTCAGAAGAGAC (830-806). The amplified full-length human group IIA sPLA2 cDNA was then cloned in natural orientation into the mammalian expression vector pcDNA3.1 (Invitrogen, San Diego, CA). The identity and orientation of this construct were confirmed by DNA sequence.

Reporter activity assessment. The HepG2 cells grown in 75-cm2 flasks were seeded in six-well culture dishes. Transfection experiments were performed 24 h after cells were plated in six-well dishes (~80% confluence). The cells were transfected with the PPRE reporter plasmid expressing the CAT gene using Lipofectamine plus reagent (1.5 µg in each transfection). An internal control reporter plasmid expressing the beta -galactosidase gene (pIGP LacZ) was used to normalize the transfection efficiency (0.5 µg in each transfection). After exposure to the transfection reagents and plasmids for 4 h, the media containing transfection reagents were replaced with serum-free medium and the cultures were continued for 24 h. The cells were then incubated with or without 10-6 M A23187 for 2 h, washed twice with PBS, and collected with 1× reporter lysis buffer (obtained from Promega). The CAT reporter activity in cell extract was determined using the CAT enzyme assay system (Promega) according to the manufacturer's protocol. The beta -galactosidase activity was measured with the chemiluminescent reporter assay (Tropix) according to the manufacturer's recommendation. In some experiments, the pG5Luc reporter construct and the Gal4-PPAR-alpha expression vector were transfected to the cells with cotransfection of cPLA2 and group IIA sPLA2 or to the cells with antisense inhibition of cPLA2. The cells were cultured in serum-free medium after transfection for 24 h, and the cell extracts were then obtained to measure the luciferase reporter activity.

Transient transfection of cPLA2 and group IIA sPLA2 expression plasmids in HepG2 cells. The HepG2 cells grown in six-well culture dishes were transfected with 1.5 µg each of PLA2 expression plasmids (cPLA2 in pMT-2 and group IIA sPLA2 in pcDNA3.1) or vectors (pMT-2 and pcDNA3.1) as well as 1.5 µg PPRE-CAT reporter plasmid and 0.5 µg pIGP LacZ (beta -galactosidase expression vector) using Lipofectamine plus reagent as described above. The cultures were continued in serum-free medium for 24 h after exposure to the mixture of transfection reagents and plasmids for 4 h, and the cell lysates were collected to measure the CAT reporter activity. Western blot for cPLA2 and group IIA sPLA2 was also performed to determine the overexpression efficiency.

Stable transfection of cPLA2 antisense plasmids in HepG2 cells. The HepG2 cells were exposed to the mixture of Lipofectamine plus reagents and plasmids (antisense cPLA2 and pcDNA3.1 control vector) for 4 h. After removal of the transfection mixtures, fresh MEM with 10% fetal bovine serum was added. On the second day, fresh MEM with 10% fetal bovine serum and 800 µg/ml G418 sulfate (Calbiochem) was added. Colonies of resistant cells appeared after ~14 days and were subcultured at ~18 days. Subsequent cultures of selected HepG2 cells were routinely grown in the presence of selective pressure. Western blotting analysis for cPLA2 was then performed in the selected cells permanently transfected with the antisense or control plasmids. The selected cells with successfully reduced cPLA2 protein level were subsequently used for transfection with the PPRE reporter plasmid as well as the pG5Luc reporter construct and the GAL4-PPAR-alpha expression plasmid as described above.

Immunoblotting analysis. For immunoblotting analysis of cPLA2, group IIA sPLA2, PPAR-alpha , PPAR-beta , and PPAR-gamma , the cell lysates from HepG2 cells were prepared using lysis buffer containing protease inhibitor cocktail tablets (Roche Diagnostics). Samples containing 10 µg of cellular protein were separated on 4-20% or 16% Tris-glycine gels (Novex, San Diego, CA) using Tris-glycine SDS running buffer. The separated proteins were then electrophoretically transferred onto nitrocellulose membranes (Novex). Nonspecific binding was blocked with 3% nonfat milk in PBS containing 0.05% Tween 20 (PBS-T) at room temperature for 1 h. The membranes were then incubated with primary antibodies (1:200 dilution of mouse anti-human cPLA2 monoclonal antibody, 1:200 dilution of rabbit anti-human group IIA sPLA2 polyclonal antiserum, and 1:500 dilutions of rabbit anti-human PPAR-alpha , -beta , and -gamma polyclonal antibodies) in PBS-T containing 3% nonfat milk. After overnight incubation at 4°C, the membranes were washed three times with PBS-T and then incubated at room temperature for 1 h with 1:5,000 dilution of the corresponding HRP-conjugated secondary antibodies in PBS-T containing 3% nonfat milk. After being washed three times with PBS-T, the protein bands were visualized with the enhanced chemiluminescence Western blotting detection system (Amersham) according to the manufacturer's instructions.

Assay of PLA2 activity. In vitro cPLA2 and group IIA sPLA2 activity assays were performed as previously described (55, 57, 58). The HepG2 cells grown in 100-mm culture dishes (~80% confluence) were transfected with the cPLA2 or sPLA2 expression plasmid for 4 h using Lipofectamine plus reagent, and the cultures were continued in serum-free medium for 24 h. The cells were scraped in medium, centrifuged, and then washed once with PBS containing protease inhibitor cocktail tablets (Roche Diagnostics). The cells from each culture dish were lysed by sonication in 0.5 ml of 100 mM HEPES buffer (pH 7.5, containing protease inhibitor cocktail). The cell lysates were then centrifuged at 100,000 g at 4°C for 1 h, and the supernatants were then collected for subsequent PLA2 activity assays. For cPLA2 activity assay, sn-2-[14C]arachidonyl phosphatidylcholine was used as substrate, and the reaction was performed in 100 µl of reaction mixture containing 100 µM arachidonyl phosphatidylcholine/phosphatidylinositol 4,5-bisphosphate (97:3) [containing 100,000 cycles/min (cpm) of 14C arachidonyl phosphatidylcholine] in 100 mM HEPES (pH 7.5), 1 mM Ca2+, 1 mM EDTA, 2 mM 1,4-dithiothreitol, and 0.1 mg/ml bovine serum albumin. For group II sPLA2 assay, sn-2-[14C]linoleoyl phosphatidylethanolamine was used as substrate, and the reaction was performed in 100 µl of reaction mixture containing 100 µM linoleoyl phosphatidylethanolamine/sn-2 oleoyl phosphatidylserine (1:1; containing 100,000 cpm of [14C]linoleoyl phosphatidylethanolamine) in 100 mM HEPES (pH 7.5), 1 mM Ca2+, and 1 mg/ml bovine serum albumin. The reaction mixtures were incubated at 37°C for 1 h and were terminated by the addition of 2:1 chloroform/methanol containing 1% acetic acid and 1 mg/ml free AA (for cPLA2 assay) or 1 mg/ml free linoleic acid (for group IIA sPLA2 assay). The release of free fatty acids were analyzed using silica gel H thin layer chromatography plates (Analtech, Newark, DE), and the plates were developed with heptane/isopropyl ether/acetic acid (60:40:4). Free AA or linoleic acid was then scraped and quantified by scintillation counting. The protein concentrations in the cell fractions were determined by the Bio-Rad protein assay.

Analysis of AA metabolites by reverse phase high performance liquid chromatography. Equal numbers of HepG2 cells were grown on 100-mm culture dishes, and experiments were performed when the cells reached ~80% of confluence. The cells were labeled for 18 h with 0.3 µCi/ml [5,6,8,9,11,12,14,15-3H]AA (180-240 Ci/mmol, New England Nuclear) in 8 ml MEM media. After repeated washing with media, 8 ml of fresh media were added to each dish and the supernatants were harvested after 8 h of incubation. The samples were extracted by Sep-Pak C18 cartridges and chromatographed by reverse-phase HPLC as we previously described (54-57). Individual octadecyl-silane C18 cartridges (Sep-Pak C18; Waters Associates, Milford, MA) were prepared with 15 ml of methanol followed by 5 ml of 5 mM ethylenediaminetetra-acetic acid and 10 ml of water. Samples were loaded onto the cartridges, washed with 10 ml of water, and eluted with 4 ml of methanol. The methanol fraction was collected, evaporated to dryness under steady-flow nitrogen gas, and resuspended in 200 µl of mobile phase A for analysis by HPLC. An Ultrasphere C18 (Beckman Instruments, Fullerton, CA) column (4.7 × 250 mm) with 5-µm particle size was used. A gradient program was used with mobile phase A: water/acetonitrile/phosphoric acid (75:25:0.025) and mobile phase B: methanol/acetonitrile/trifluoroacetic acid (60:40:0.0016) at a flow rate of 1.5 ml/min. One-minute fractions of the HPLC elution were collected and counted for radioactivity over 80 min.

Measurement of PGE2 by enzyme immunoassays. Nonradiolabeled HepG2 cells cultured in 12-well plates were incubated with 1 ml of serum-free medium in the presence or absence of PLA2 inhibitors (LY311727, AACOCF3, and MAFP) for 6 h. The supernatants were then collected and assayed for PGE2 production by enzymeimmunoassay as recommended by the manufacture's instruction.


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The HepG2 cells express both PPAR-alpha and PPAR-gamma bot not PPAR-beta . To determine the expression profile of PPARs in the HepG2 cells, we first performed Western blot analysis for the three PPAR isoforms (-alpha , -beta , and and -gamma ). Consistent with the well-documented expression of PPAR-alpha and -gamma in liver, the HepG2 cells express both PPAR-alpha and PPAR-gamma but no detectable PPAR-beta (Fig. 1). Two isoforms of PPAR-gamma (PPAR-gamma 1 and PPAR-gamma 2, which are produced by alternative splicing of the same PPAR-gamma gene) are present in the HepG2 cells. On the basis of this finding, we elected to use the PPRE reporter construct that can be activated by both PPAR-alpha and PPAR-gamma in the subsequent experiments. The PPRE reporter construct used in this study has optimal reporter activity in hepatocytes and contains the CAT coding sequence driven by a promoter consisting of two copies of the CYP4A6 PPRE (binding element for PPAR-alpha and PPAR-gamma ) upstream of the thymidine kinase minimal promoter (12).


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Fig. 1.   Protein expression of peroxisome proliferator-activated receptor (PPAR)-alpha , -beta , and -gamma in HepG2 cells. An equal amounts of cellular proteins (10 µg) isolated from HepG2 cells and from various positive control cells was used for Western blot analysis for PPAR isoforms. A: immunoblot of PPAR-alpha with cell lysate isolated from primary human hepatocytes (containing high levels of PPAR-alpha ) as positive control. B: immunoblot of PPAR-beta with cell lysate isolated from primary human bronchial epithelial cells (containing high level of PPAR-beta ) as positive control. C: immunoblot of PPAR-gamma with cell lysate isolated from a human cholangiocarcinoma cell line (SG231 cells, containing high levels of PPAR-gamma ) as positive control.

Overexpression of cPLA2 but not group IIA sPLA2 increases PPAR-alpha /gamma -mediated gene transcription. To study the direct contribution of cPLA2 and sPLA2-induced AA metabolism in the regulation of PPAR-mediated gene transcription, we first examined the possible effect of transient overexpression of cPLA2 and group IIA sPLA2 on PPAR activation. The HepG2 cells were cotransfected with the cPLA2 or group IIA sPLA2 expression plasmid as well as the PPRE reporter plasmid. Western blot analysis of the cell lysate collected 24 h after transfection showed that the cells transfected with the cPLA2 or group IIA sPLA2 expression plasmids exhibited significantly increased expression of the two PLA2 proteins when compared with the cells transfected with vectors alone (Figs. 2 and 3). Because the activities of both cPLA2 and group IIA sPLA2 are increased in the presence of calcium, the cells with cotransfection of PLA2 expression plasmid and the reporter plasmid for 24 h were then stimulated with calcium ionophore A23187 for maximal AA release. On the basis of the previously documented calcium-induced cPLA2 translocation to nuclear envelope (17, 27, 38, 39, 41), we hypothesized that cells with cPLA2 overexpression would likely have increased AA and eicosanoid production in the nuclei for PPAR activation in response to the calcium mobilizing agents. As shown in Fig. 2, cells cotransfected with the cPLA2 expression plasmid and the PPRE reporter plasmid for 24 h showed significantly increased PPAR-alpha /gamma -mediated gene transcription after calcium ionophore A23187 stimulation for 2 h. Similar results were obtained when another reporter construct containing the PPAR-alpha /gamma response element was used (CAT reporter gene driven by the natural promoter of acyl CoA oxidase gene that contains the PPAR-alpha /gamma response element; data not shown). The PPRE reporter activity increase in cells with cPLA2 overexpression was less prominent in the absence of ionophore A23187 stimulation. These results indicate that calcium-mediated cPLA2 activation plays an important role for PPAR-alpha /gamma -mediated gene transcription in the HepG2 cells. Overexpression of cPLA2 failed to increase the reporter activity when the negative control reporter construct (CAT reporter plasmid with deletion of PPRE) was used.


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Fig. 2.   Overexpression of cPLA2 increases PPAR-alpha /gamma activation in HepG2 cells. A: structure of the PPRE reporter construct used in this study. It contains the chloramphenicol acetyl transferase (CAT) coding sequence driven by a promoter consisting of 2 copies of the CYP4A6 peroxisome proliferator-response element (PPRE; binding element for PPAR-alpha and PPAR-gamma ) upstream of the thymidine kinase (TK) minimal promoter (12). B: PPAR-alpha /gamma activity in cells with or without overexpression of cytosolic phospholipase A (cPLA2). The PPAR-alpha /gamma activity was assessed by measuring the PPRE-CAT reporter activity. The HepG2 cells were transfected with the cPLA2 expression vector (cPLA2 in pMT-2) or the control vector (pMT-2) with cotransfection of the PPRE reporter plasmid. The cells were cultured in medium without serum for 24 h and then stimulated with 10-6 M A23187 for an additional 2 h after transfection. The cell extracts were then prepared and processed to measure the CAT reporter activity as described in METHODS. The cells with cPLA2 overexpression showed significantly increased CAT reporter activity when compared with the cells transfected with control vector (P < 0.01, n = 6). C: immunoblot analysis for cPLA2 protein expression. An equal amount of cellular proteins isolated from the above cells transfected with the cPLA2 expression vector or control vector was used for Western blot analysis for cPLA2. The blot was reprobed using a primary antibody to beta -actin as a control for protein loading. The cells transfected with the cPLA2 expression vector showed significantly increased cPLA2 protein expression. D: the effect of PPAR-alpha ligand WY-14643 on PPRE reporter activity. The HepG2 cells transfected with the PPRE reporter plasmid (without cPLA2 overexpression) were stimulated with 10-6 M WY-14643 for 24 h, and the cell extracts were obtained to measure the CAT reporter activity. The cells treated with WY-14643 showed significantly increased CAT reporter activity when compared with control (P < 0.01, n = 3).



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Fig. 3.   Overexpression of group IIA secretory phospholipase A (sPLA2) failed to increase PPAR-alpha /gamma activation in HepG2 cells. A: the PPAR-alpha /gamma activity in cells with or without group IIA sPLA2 overexpression. The HepG2 cells were transfected with the group IIA sPLA2 expression vector (sPLA2 in pcDNA3.1) or the control vector (pcDNA3.1) with cotransfection of the peroxisome proliferator response element (PPRE) reporter plasmid. The cells were cultured in medium without serum for 24 h and then stimulated with 10-6 M A23187 for an additional 2 h after transfection. The cell extracts were then prepared and processed to measure the CAT reporter activity as described in METHODS. The cells with group IIA sPLA2 overexpression failed to show increased CAT reporter activity when compared with the control cells. Instead, the CAT reporter activity in cells with group IIA sPLA2 was slightly lower than that in the control cells (n = 6). B: immunoblot analysis for group IIA sPLA2 protein expression. An equal amount of cellular proteins isolated from the above cells transfected with the group IIA sPLA2 expression vector or control vector was used for Western blot analysis for group IIA sPLA2. The blot was reprobed using a primary antibody to beta -actin as a control for protein loading. The cells transfected with the group IIA sPLA2 expression vector showed significantly increased group IIA sPLA2 protein expression.

To compare the magnitude of PPAR activation by cPLA2 overexpression with that induced by the known PPAR ligand, we examined the PPRE reporter activity in HepG2 cells stimulated with WY14643, a PPAR-alpha ligand known to activate this reporter construct. The cells transfected with the PPRE reporter construct were stimulated with 50 µM of WY14643 for 24 h, and the cell lysates were obtained to measure the CAT reporter activity. As shown in Fig. 2D, WY14643 induced an increased PPRE-CAT reporter activity (3.5-fold of control). The PPRE reporter activity in cells with cPLA2 overexpression is ~70% of that induced by WY14643 in the HepG2 cells.

In contrast to cPLA2, group IIA sPLA2 overexpression failed to show PPRE reporter activity increase in response to calcium ionophore stimulation (Fig. 3A). Similar results were obtained when experiments were performed in the absence of calcium ionophore A23187 stimulation (data not shown). In fact, the PPRE reporter activity in cells with group IIA sPLA2 overexpression was slightly lower than in control cells. Therefore, the above experiments with overexpression of PLA2s demonstrated an important role of cPLA2 but not group IIA sPLA2 in the production of AA metabolites in nuclei for PPAR-alpha /gamma activation in the HepG2 cells.

To assess the PLA2 activities in cells with overexpression of cPLA2 and group IIA sPLA2, the cell lysates were obtained to measure specific cPLA2 and group IIA sPLA2 activities. As shown in Fig. 4A, overexpression of cPLA2 and group IIA sPLA2 resulted in a similar degree of specific PLA2 activity increase. To exclude the possibility that the difference in PPRE reporter activity might relate to altered PPAR protein, Western blot for PPAR was performed. The levels of PPAR-alpha and PPAR-gamma proteins in HepG2 cells were not altered by transfection of either cPLA2 or group IIA sPLA2. Figure 4B shows a representative immunoblot for PPAR-alpha with recombinant PPAR-alpha as the positive control. The above results indicate that the observed difference in PPRE reporter activity in cells with overexpression of cPLA2 and group IIA sPLA2 is not due to different PLA2 activity or PPAR protein expression.


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Fig. 4.   Effect of cPLA2 and group IIA sPLA2 overexpression on PLA2 activity and PPAR protein level in HepG2 cells. A: overexpression of cPLA2 and group IIA sPLA2 results in comparable increase of PLA2 activity. The HepG2 cells were transfected with the cPLA2 expression plasmid or group IIA sPLA2 expression plasmid and the control plasmids pMT-2 or pcDNA3. The cells were cultured in serum-free medium for 24 h, and the in vitro activities of cPLA2 or group IIA sPLA2 were measured as described in the METHODS. The results represent the average of 3 experiments. B: PPAR-alpha protein levels in cells with or without transient PLA2 overexpression. An equal amount of cell lysates (10 µg) from HepG2 cells transiently transfected with the cPLA2 expression plasmid, group IIA sPLA2 expression plasmid, and the control plasmids pMT-2 and pcDNA3 were used for Western blot analysis for PPAR-alpha . The blot was reprobed using a primary antibody to beta -actin as a control for protein loading. The PPAR-alpha protein level was not altered by transfection of cPLA2 or group IIA sPLA2. Recombinant PPAR-alpha standard was used as the positive control.

Antisense inhibition of cPLA2 decreases the PPAR-alpha /gamma -mediated gene transcription. To further study the contribution of cPLA2 in the PPAR-mediated gene transcription, we then used antisense inhibition of cPLA2 in the HepG2 cells. Cells stably transfected with the cPLA2 antisense plasmid were selected using the method described in METHODS. Western blot analysis showed that the cells stably expressing antisense plasmids showed successfully decreased cPLA2 protein levels (Fig. 5B). The cells stably expressing the cPLA2 antisense plasmid and control plasmid were then transfected with the PPRE reporter plasmid for 24 h, and the PPRE reporter activity was measured following 2 h ionophore A23187 stimulation. As shown in Fig. 5A, the cells with decreased cPLA2 protein levels showed significantly decreased PPRE reporter activity. This result again demonstrated the importance of cPLA2 in the PPAR-mediated gene transcription in the HepG2 cells.


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Fig. 5.   Antisense inhibition of cPLA2 downregulates PPAR-alpha /gamma activation in HepG2 cells. A: PPAR-alpha /gamma activity in cells with or without antisense inhibition of cPLA2. The HepG2 cells stably transfected with the antisense cPLA2 plasmid (As-cPLA2 in pcDNA3.1) or control plasmid (pcDNA3.1) were selected as described in METHODS. The cells were then transfected with the PPRE reporter plasmid and the beta -galactosidase expression vector. The cells were cultured in medium without serum for 24 h and then stimulated with 10-6 M A23187 for an additional 2 h after transfection. The cell extracts were then prepared and processed to measure the CAT reporter activity. The cells with antisense inhibition of cPLA2 showed significantly decreased CAT reporter activity when compared with the control cells (P < 0.01, n = 6). B: immunoblot analysis for cPLA2 protein expression. An equal amount of cellular protein isolated from the above cells stably transfected with the cPLA2 antisense plasmid or control vector was used for Western blot analysis for cPLA2. The blot was reprobed using a primary antibody to beta -actin as a control for protein loading. The cells stably transfected with the cPLA2 antisense plasmid showed significantly decreased cPLA2 protein expression.

The effect of PLA2 protein levels on PPAR activation in cells transfected with Gal4-PPAR-alpha expression plasmid. To further examine the contribution of cPLA2 and group IIA sPLA2 on PPAR activation, we used a different reporter construct containing the luciferase coding gene under the control of Gal4 upstream activating sequence (pG5Luc). This reporter construct was transfected with an expression vector encoding PPAR-alpha fused, in frame, to the Gal4 DNA binding domain (Gal4-PPAR-alpha ) or the Gal4 DNA binding domain alone (Gal4DBD) in cells with transient overexpression of cPLA2 and group IIA sPLA2 or in cells with antisense inhibition of cPLA2. As shown in Fig. 6, transient transfection of cPLA2 significantly increased the PPAR-alpha -mediated luciferase reporter activity when compared with control. In contrast, overexpression of group IIA sPLA2 failed to increase the PPAR-alpha -mediated luciferase activity. The PPAR-alpha -mediated luciferase reporter activity was significantly decreased in cells with antisense inhibition of cPLA2. These results again demonstrated the important role of cPLA2 but not group IIA sPLA2 in PPAR activation in the HepG2 cells.


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Fig. 6.   The effect of altered PLA2 expression on PPAR-alpha activation. A: structure of the reporter construct pG5Luc. It contains the luciferase coding gene under the control of Gal4 upstream activating sequence (UAS; pG5Luc). B: PPAR-alpha activity in cells with altered PLA2 expression. The pG5Luc reporter construct was transfected with the expression vector encoding PPAR-alpha fused, in frame, to the Gal4 DNA binding domain (Gal4-PPAR-alpha ) or the expression vector encoding the Gal4 DNA binding domain alone (Gal4DBD) to the HepG2 cells with transient overexpression of cPLA2 and group IIA sPLA2 or to the cells with antisense inhibition of cPLA2. The cells were cultured in serum-free medium for 24 h after transfection. The cell extracts were then prepared and processed to measure the luciferase reporter activity as described in the METHODS. The results represent the average of 3 experiments.

Inhibitors of cPLA2, but not group IIA sPLA2, reduce the PPAR-alpha /gamma -mediated gene transcription. In addition to overexpression and antisense inhibition approaches, we tested the potential effect of cPLA2 and sPLA2 inhibitors on the PPRE reporter activities in HepG2 cells. The cPLA2 inhibitors AACOCF3 and MAFP and the sPLA2 inhibitor LY311727 were used in the experiments. The HepG2 cells pretransfected with the PPRE reporter plasmid were incubated with various PLA2 inhibitors for 24 h before reporter activity measurement. We found that although the sPLA2 inhibitor LY311727 exhibited no inhibitory effect on PPAR-alpha /gamma activation, the cPLA2 inhibitors AACOCF3 and MAFP significantly inhibited the PPRE reporter activity (Fig.7). Therefore, the above data with the PLA2 inhibitors also demonstrated the essential role of cPLA2 but not sPLA2 in the activation of PPAR-alpha /gamma -mediated gene transcription in HepG2 cells. Because AACOCF3 and MAFP may also have inhibitory effect on the intracellular calcium-independent PLA2 (iPLA2), the above result does not exclude the possibility of iPLA2 involvement.


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Fig. 7.   The effect of PLA2 inhibitor on PPAR-alpha /gamma activity. The HepG2 cells were cotransfected with the PPRE reporter plasmid and the beta -galactosidase expression vector. The cells were cultured in serum-free medium in the presence of different inhibitors of PLA2s as indicated for 24 h after transfection. The cells were then lysed, and the cell extracts were obtained for measurement of CAT reporter activity. Although there was no significant difference in the measured CAT reporter activity between control cells and cells treated with the sPLA2 inhibitor LY311727, the CAT reporter activity in cells treated with the cPLA2 inhibitors arachidonyltrifluoromethyl ketone (AACOCF3) and methyl arachidonyl fluorophosphate (MAFP) was significantly decreased when compared with that in control cells (P < 0.01, n = 3).

AA metabolites in HepG2 cells. In an effort to identify the potential AA derivatives mediating PPAR activation in HepG2 cells, we used HPLC to identify the AA metabolism profile in these cells. The HPLC method used in this study allows separation of a wide spectrum of AA metabolites including PGs, leukotrienes, and hydroxyeicosatetraenoic acids. The cells were prelabeled with [3H]AA for 18 h, and the release of AA metabolites was analyzed as described in the METHODS. As shown in Fig. 8, the predominant radioactive peak in HepG2 cells was AA, and the major AA metabolite was PGE2. Other AA metabolites such as PGD2, 15d-PGJ2, leukotriene B4, and hydroxyeicosatetraenoic acids were not identified. The presence of PGE2 in the HepG2 cells was further confirmed by the enzymeimmunoassay, which showed that 4,103 pg/ml of PGE2 were released into the culture medium during the 6-h culture period (Fig. 9). Furthermore, the release of PGE2 was significantly decreased by the cPLA2 inhibitors AACOCF3 and MAFP but not by the sPLA2 inhibitor LY311727, indicating that PGE2 is the major AA metabolite controlled by cPLA2 in HepG2 cells.


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Fig. 8.   Analysis of arachidonic acid (AA) metabolites in HepG2 cells by HPLC. The HepG2 cells grown on 100-mm dishes were labeled for 18 h with 0.3 µCi/ml [3H]AA in 8 ml of medium. The cells were incubated with 8 ml of medium for 8 h after repeated washing, and the supernatants were collected to examine the release of AA metabolites. The samples were extracted by Sep-Pak C18 cartridges and chromatographed by HPLC as described in METHODS. As indicated in the chromatogram, the major radioactive peak is AA, and the predominant AA metabolite produced in HepG2 cells is PGE2. The eluted peaks of phospholipid (PL) is also indicated in the chromatogram. No radioactive peaks corresponding to the retention times of other AA metabolites [such as thromboxane B2 (TXB2) PGD2, 15d-PGJ2, leukotriene B4 (LTB4), leukotriene C4(LTC4) or hydroxyeicosatetraenoic acids (HETE)] are detected. The chromatogram shown is a representative of 6 experiments. The retention times of 3H-labeled eicosanoid standards are indicated by arrows.



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Fig. 9.   The effect of PLA2 inhibitors on PGE2 production. The HepG2 cells cultured in 12-well plates were incubated in serum-free medium for 6 h in the absence or presence of sPLA2 inhibitor LY311727 and cPLA2 inhibitors AACOCF3 and MAFP. The supernatants were collected and processed for the measurement of PGE2 release using the enzymeimmunoassay. The amount of PGE2 is expressed as picograms/milliliters. The results were obtained from 4 experiments. * P < 0.01.

Although LTB4 and 15d-PGJ2 are the known natural ligands for PPAR-alpha and -gamma , these two AA metabolites were not identified in the HepG2 cells (Fig. 8). Therefore, the cPLA2-induced PPAR activation in HepG2 cells is likely mediated by other AA metabolites in light of the known PPAR activation by fatty acids and eicosanoids in various cells (11, 16, 29, 46, 48, 60). Because AA represents the predominant peak in an HPLC chromatogram (Fig. 8) and AA is known to activate PPAR in HepG2 cells (60), the production of AA likely contributes to the cPLA2-mediated PPAR activation. Because the potential effect of PGE2 on PPAR activation has not been well documented in hepatocytes, further experiments were performed to study the direct effect of PGE2 on PPAR-alpha /gamma activation in HepG2 cells. Figure 10A showed that treatment of cells with 100 µM PGE2 increased the PPAR-alpha /gamma -mediated reporter activity (~2.7-fold of control). Figure 10B showed that the cPLA2 inhibitor MAFP-induced decrease of PPRE reporter activity was partially reversed by cotreatment with 100 µM PGE2. Although the PPAR-alpha /gamma -mediated reporter activity in cells treated with MAFP was 47% of control cells, the reporter activities in cells treated with MAFP plus PGE2 was 76% of control cells (P < 0.01). Treatment with lower concentrations of PGE2 (1 and 10 µM) also increased the PPRE reporter activity, although the effect was slightly less prominent. The above results demonstrate that in addition to AA, PGE2 also partially contributes to the cPLA2-mediated PPAR-alpha /gamma activation in HepG2 cells.


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Fig. 10.   The effect of PGE2 on PPAR-alpha /gamma activation in HepG2 cells. A: the effect of PGE2 on the PPRE reporter activity. The HepG2 cells were transfected with the PPRE reporter plasmid and the beta -galactosidase expression vector. The cells were incubated with PGE2 in serum-free medium for 24 h after transfection. The cells were then lysed, and the cell extracts were obtained for measurement of CAT reporter activity. The PPRE reporter activity was significantly increased after 24 h treatment with 100 µM PGE2 (P < 0.01, n = 3). B: the effect of PGE2 on MAFP-induced PPAR-alpha /gamma inhibition. The HepG2 cells were cotransfected with the PPRE reporter plasmid and the beta -galactosidase expression plasmid. The cells were treated with the cPLA2 inhibitor MAFP (10 µM) in the absence or presence of PGE2 (100 µM) for 24 h after transfection. The cells were then lysed, and the cell extracts were obtained for measurement of CAT reporter activity. The data shown were obtained from 4 individual experiments. The MAFP-induced inhibition of CAT reporter activity was partially reversed by cotreatment with PGE2 (P < 0.01).

The role of cPLA2 in the expression of apolipoprotein A-II in HepG2 cells. To further examine the roles of cPLA2 and group IIA sPLA2 in PPAR activation, additional experiments were performed to document the expression of PPAR/PPRE-controlled endogenous gene in HepG2 cells. Because apolipoprotein A-II gene contains PPRE in its 5'-flanking region and its expression in HepG2 cells is upregulated by activation of PPAR-alpha (50), the expression of apolipoprotein A-II was examined in HepG2 cells with overexpression of cPLA2 and group IIA sPLA2 as well as antisense inhibition of cPLA2. We found that overexpression of cPLA2 significantly increased the production of apolipoprotein A-II when compared with control (Fig. 11). In contrast, overexpression of group IIA sPLA2 did not alter the apolipoprotein A-II level. The production of apolipoprotein A-II was significantly decreased in cells with antisense inhibition of cPLA2. These results demonstrate an important role of cPLA2 but not group IIA sPLA2 in the regulation of PPAR-mediated endogenous gene in HepG2 cells.


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Fig. 11.   The effect of altered PLA2 expression on apolipoprotein A-II production in HepG2 cells. The expression of apolipoprotein A-II, a PPAR-alpha -mediated endogenous gene, was examined in cells with altered PLA2 expression. The cells with overexpression of cPLA2 and group IIA sPLA2 or antisense inhibition of cPLA2 were incubated in serum-free medium for 24 h, and the supernatants were collected to measure the apolipoprotein A-II level by ELISA using antibody against human apolipoprotein A-II (Biogenesis, Kingston, NH) as described previously (50). The results represent the average of 3 experiments. Overexpression of cPLA2 significantly increased the apolipoprotein A-II production when compared with control. In contrast, overexpression of group IIA sPLA2 failed to increase apolipoprotein A-II production. The production of apolipoprotein A-II was significantly reduced in cells with antisense inhibition of cPLA2.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PPARs belong to the superfamily of ligand-activated nuclear transcription factors (11, 16, 48), which, on heterodimerization with the 9-cis-retinoic acid receptor RXR, bind to specific PPRE to regulate the expression of target genes. PPAR-alpha is highly expressed in tissues with elevated rates of fatty acid metabolism such as liver, where it plays a critical role in liver lipid metabolism and hepatocarcinogenesis (11, 16, 48). PPAR-alpha regulates genes involved in fatty acid uptake, activation to acyl-CoA esters, degradation via the peroxisomal and mitochondrial beta -oxidation pathways, and ketone body synthesis. All these genes contain PPREs in their promoter regions, and induction of these genes by PPAR-alpha activation results in a significant increase in fatty acid oxidation in hepatocytes. The recognition of PPAR-alpha in hepatocyte lipid metabolism may have important implications in understanding the pathogenesis and in the treatment of hepatic steatosis, hyperlipidemia, obesity, diabetes, and atherosclerosis. The critical role of PPAR-alpha in liver lipid metabolism has been further demonstrated in the PPAR-alpha knockout mice models, which showed prominent hepatic fatty change in response to fasting (26) or perturbation of mitochondrial fatty acid import by carnitine palmitoyltransferase I inhibitors (13). Furthermore, these PPAR-alpha knockout mice failed to show hepatic tumorigenesis in response to peroxisome proliferator administration (18, 25), supporting the role of PPAR-alpha in hepatic carcinogenesis. The PPAR ligands (including natural ligands AA metabolites and pharmacological ligands) directly bind to PPARs and thus regulate the transcription activities of PPARs in the nucleus. Many pharmacological ligands were known to act as peroxisome proliferators in rodents before their PPAR binding was recognized. For example, the major pharmacological ligands for PPAR-alpha include the hypolipidemic drugs WY-14643 and fibrates, and the interaction of these drugs with PPAR-alpha in hepatocytes accounts for their therapeutic value in hyperlipidemia and cardiovascular disorders. The recognition of AA metabolites as the natural ligands for PPARs suggests a possible link between the eicosanoid-mediated inflammation and the diverse PPAR-mediated noninflammatory pathophysiological processes including various hepatic and metabolic disorders.

Despite of the findings that eicosanoids are the natural ligands for the activation of PPARs (11, 12, 15, 16, 23, 48), the involvement of key eicosanoid-forming enzymes in PPAR regulation was not known before this study. Although it has been documented that cPLA2 is preferentially translocated from cytoplasm to nucleus in response to calcium elevation (17, 27, 38, 39, 41), the functional importance of this intriguing phenomenon was not well understood. We hypothesized that the translocation of cPLA2 from cytoplasm to nuclei would result in the release of AA from nuclear envelope for eicosanoid production, and the AA metabolites in the nuclei might directly bind to and activate nuclear transcription factors. This study was thus designed to study the possible contributions of cPLA2 and group IIA sPLA2, the two most important PLA2s of AA metabolism cascade, in the regulation of PPAR-mediated gene transcription in the human hepatoma cell line HepG2 cells. By overexpression or antisense inhibition of cPLA2 and group IIA sPLA2 in the HepG2 cells, as well as the use of chemical inhibitors of PLA2s, we have demonstrated that cPLA2 but not the group IIA sPLA2 plays an important role in the regulation of PPAR-alpha /gamma -mediated gene transcription in these cells. These results reveal previously unrecognized functions of cPLA2: the cPLA2-regulated production of eicosanoids and the activation of PPARs in nuclei for gene transcription.

Coexistence of different forms of PLA2, including cPLA2 and sPLA2, have been found within the same cell or tissue (1, 8, 9). Because a number of these cells produces its own unique lipid mediator profile and exhibits different biological characteristics, it seems possible that various PLA2 isoforms may play different roles in cell lipid metabolism and in the regulation of cell functions in various cell types. Experiments using several cell models have revealed that in general, more than one type of PLA2s is involved in cellular regulation and lipid messenger formation. It has been proposed that both the cPLA2 and sPLA2 (types IIA and V) may contribute to hormone and cytokine-induced AA release and eicosanoid production and thus may potentially play important roles in intracellular signal transduction (1, 8, 9). Results in this study documented a clear distinction between cPLA2 and group IIA sPLA2 in the PPAR-mediated gene transcription in HepG2 cells. Experiments with overexpression and antisense inhibition of cPLA2 as well as the use of chemical inhibitors of cPLA2 have established that the cPLA2-mediated production of AA metabolites plays an important role in the activation of PPAR in HepG2 cells (Figs. 2, 5, 6, and 7). In contrast, the group IIA sPLA2 failed to activate the PPAR-mediated reporter activity, as documented by overexpression of group IIA sPLA2 (Figs. 3 and 6) and by using the sPLA2 inhibitor LY311727 (Fig. 7). Furthermore, experiments with overexpression of cPLA2 and group IIA sPLA2 and antisense inhibition of cPLA2 have also demonstrated an important role of cPLA2 but not group IIA sPLA2 in the expression of the endogenous PPAR-mediated gene apolipoprotein A-II (Fig. 11). These results clearly demonstrated that it is the cPLA2 but not group IIA sPLA2 that plays a critical role in the control of PPAR-mediated gene transcription.

The different effects of cPLA2 and group IIA sPLA2 on PPAR activation can be explained by their different enzyme characteristics. One of the most important characteristics of cPLA2 regulation is its calcium-dependent translocation from cytoplasm to membrane [preferentially nuclear envelope (17, 27, 38, 39, 41)], which is mediated by its NH2-terminal Ca2+-dependent lipid-binding domain (CaLB or C2 domain) (6, 34). This is in contrast with the group IIA sPLA2, which exists either as soluble form (located in extracellular space) or cell-associated form (1, 9, 33, 45). Although the group IIA sPLA2 requires Ca2+ for catalytic activity, it lacks the Ca2+-dependent membrane association. Therefore, the above unique enzyme characteristics of cPLA2 and group IIA sPLA2 likely explain the different regulatory roles of these two enzymes in PPAR activation. Because cPLA2 protein requires Ca2+ for its nuclear translocation and group IIA sPLA2 requires Ca2+ for its catalysis, calcium ionophore A23187 was used in this study for maximal enzyme activation. When experiments with cPLA2 overexpression were performed in the absence of ionophore A23187, the cPLA2-induced increase of PPRE reporter activity was less prominent. For experiments with overexpression of group IIA sPLA2, a similar degree of PPAR activation was observed in the presence or absence of ionophore A23187. These observations again highlight the importance of calcium-mediated translocation of cPLA2 in PPAR-mediated gene transcription.

LTB4 is the natural ligand of PPAR-alpha . Although 15d-PGJ2 is a potent ligand of PPAR-gamma , its concentration in human cells is not high enough to be physiologically important. Other natural ligands such as unsaturated fatty acids and other AA metabolites have also been shown to activate PPARs (11, 16, 29, 46, 48, 60). Results in this study suggest that the cPLA2-induced PPAR activation in HepG2 cells is unlikely to be mediated by LTB4 and 15d-PGJ2, because these two metabolites were not detectable in these cells (Fig. 8). Instead, our findings have pointed to the role of AA and PGE2 in cPLA2-induced PPAR activation in HepG2 cells. The contribution of AA to cPLA2-mediated PPAR activation is supported by the following evidences: 1) AA represents the predominant peak in HPLC chromatogram; 2) AA binds to PPRE in several in vitro binding assays and increases PPRE reporter activities (11, 29, 46, 60); 3) AA activates PPAR in HepG2 cells (60); and 4) cPLA2 is the key enzyme for AA release. The contribution of PGE2 to cPLA2-mediated PPAR-alpha /gamma activation in HepG2 cells is supported by the following observations: 1) PGE2 represents the predominant AA metabolite; 2) PGE2 increases the PPRE reporter activity; 3) the PPRE reporter activity inhibited by the cPLA2 inhibitor is partially reversed by cotreatment with PGE2; and 4) PGE2 has been shown to activate PPAR in other cell types (21, 29). Although the concentration of PGE2 required for induction of PPRE reporter activity is higher than the amount of PGE2 released into the medium, the nuclear localization of eicosanoid-forming enzymes suggests that local eicosanoids and AA in the nucleus can reach high levels and that intranuclear action of endogenous eicosanoids may be feasible (2, 4, 27, 39, 44). In addition to cPLA2, it is interesting to note that some other key enzymes in the AA cascade, including cyclooxygenase-2, 5-lipoxygenase, and 5-lipoxygenase activating protein, are also associated with nuclear membrane in response to calcium elevation (2, 4, 27, 44). The finding that cPLA2 plays an important role in the regulation of PPAR-mediated gene transcription provides novel evidence for the potential functions of nuclear targeting of key eicosanoid-forming enzymes in human cells.

In summary, this study provides the first evidence for the critical role of cPLA2 but not group IIA sPLA2 in the regulation of PPAR-mediated gene transcription in human cells. We have documented a novel intracellular function of cPLA2 in PPAR activation in human hepatoma cells. Because AA metabolites and PPARs have been implicated in several important pathophysiological processes in the liver, recognition of the cPLA2-PPAR signal-transduction pathway will provide insight into the mechanisms and biological functions of eicosanoids and PPARs in liver cells and in liver disorders. This knowledge may provide future therapeutic implications for the treatment of liver and metabolic diseases involving eicosanoids and PPARs, such as lipid metabolic disorders, diabetes mellitus, obesity, atherosclerosis, hepatitis, and cirrhosis.


    ACKNOWLEDGEMENTS

We thank Drs. J. D. Clark and J. L. Knopf at the Genetics Institute (Boston, MA) for providing the cPLA2 expression plasmid and Dr. W. Wahli at Universite de Lausanne (Switzerland) for the PPRE reporter plasmid. The PPAR-alpha expression plasmid was a generous gift from Dr. F. Gonzalez at the National Institutes of Health (Bethesda, MD). The Gal4-PPAR-alpha and the Gal4DBD expression plasmids were kindly provided by Dr. D. Kelly at Washington University (St. Louis, MO). The sPLA2 inhibitor LY311727 was a generous gift from Dr. E. Mihelich at Lilly Research Laboratories (Indianapolis, IN).


    FOOTNOTES

This work was partially supported by the American Liver Foundation Liver Scholar Award (to T. Wu).

Address for reprint requests and other correspondence: T. Wu, Dept. of Pathology, Univ. of Pittsburgh School of Medicine, Presbyterian Univ. Hospital C902, 200 Lothrop St., Pittsburgh, PA 15213 (E-mail: wut{at}msx.upmc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpgi.00305.2001

Received 13 July 2001; accepted in final form 6 December 2001.


    REFERENCES
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METHODS
RESULTS
DISCUSSION
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