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
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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- and -
but not PPAR-
.
Overexpression of cPLA2, but not group IIA
sPLA2 in the HepG2 cells, caused a significantly increased
PPAR-
/
-mediated reporter activity. Antisense inhibition of
cPLA2 resulted in a significantly decreased PPAR-
/
activity. The PPAR-
/
-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-
-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
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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-(12), and
15-deoxy-
12, 14 PGJ2
(15d-PGJ2), a cyclooxygenase pathway product, as a potent
ligand for PPAR-
(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 cPLA2 in most recent
literature in light of the cloning of 2 related isoforms
cPLA2
and
(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 (,
,
and
) that are often coexpressed in various tissues. PPAR-
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-/
-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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METHODS |
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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 -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-
, -
, and -
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.
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
-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
-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-
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 (-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- expression plasmid as
described above.
Immunoblotting analysis.
For immunoblotting analysis of cPLA2, group IIA
sPLA2, PPAR-, PPAR-
, and PPAR-
, 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-
, -
, and -
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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RESULTS |
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The HepG2 cells express both PPAR- and PPAR-
bot not
PPAR-
.
To determine the expression profile of PPARs in the HepG2 cells, we
first performed Western blot analysis for the three PPAR isoforms
(-
, -
, and and -
). Consistent with the well-documented expression of PPAR-
and -
in liver, the HepG2 cells express both
PPAR-
and PPAR-
but no detectable PPAR-
(Fig.
1). Two isoforms of PPAR-
(PPAR-
1
and PPAR-
2, which are produced by alternative splicing of the same
PPAR-
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-
and PPAR-
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-
and PPAR-
) upstream of the thymidine kinase minimal
promoter (12).
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Overexpression of cPLA2 but not group IIA
sPLA2 increases PPAR-/
-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-
/
-mediated gene transcription after calcium ionophore A23187 stimulation for 2 h. Similar results were
obtained when another reporter construct containing the PPAR-
/
response element was used (CAT reporter gene driven by the natural promoter of acyl CoA oxidase gene that contains the PPAR-
/
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-
/
-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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Antisense inhibition of cPLA2 decreases the
PPAR-/
-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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The effect of PLA2 protein levels on PPAR activation in
cells transfected with Gal4-PPAR- 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-
fused, in
frame, to the Gal4 DNA binding domain (Gal4-PPAR-
) 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-
-mediated
luciferase reporter activity when compared with control. In contrast,
overexpression of group IIA sPLA2 failed to increase the
PPAR-
-mediated luciferase activity. The PPAR-
-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.
|
Inhibitors of cPLA2, but not group IIA
sPLA2, reduce the PPAR-/
-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-
/
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-
/
-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.
|
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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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- (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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DISCUSSION |
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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- 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-
regulates genes involved in fatty acid uptake, activation to
acyl-CoA esters, degradation via the peroxisomal and mitochondrial
-oxidation pathways, and ketone body synthesis. All these genes
contain PPREs in their promoter regions, and induction of these genes
by PPAR-
activation results in a significant increase in fatty acid
oxidation in hepatocytes. The recognition of PPAR-
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-
in liver lipid metabolism has been further demonstrated in the PPAR-
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-
knockout mice failed to
show hepatic tumorigenesis in response to peroxisome proliferator
administration (18, 25), supporting the role of PPAR-
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-
include the
hypolipidemic drugs WY-14643 and fibrates, and the interaction of these
drugs with PPAR-
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-/
-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-. Although
15d-PGJ2 is a potent ligand of PPAR-
, 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-
/
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- expression plasmid was a
generous gift from Dr. F. Gonzalez at the National Institutes of Health (Bethesda, MD). The Gal4-PPAR-
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.
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