THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 280, No. 27, Issue of July 8, pp. 25830 –25839, 2005
Printed in U.S.A.
Search of Factors That Intermediate Cytokine-induced Group IIA
Phospholipase A2 Expression through the Cytosolic Phospholipase
A2- and 12/15-Lipoxygenase-dependent Pathway*
Received for publication, January 5, 2005, and in revised form, April 8, 2005
Published, JBC Papers in Press, May 6, 2005, DOI 10.1074/jbc.M500168200
Hiroshi Kuwata, Takuya Nonaka, Makoto Murakami‡, and Ichiro Kudo§
From the Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai,
Shinagawa-ku, Tokyo 142-8555, Japan
Inducible expression of group IIA secretory phospholipase A2 (sPLA2-IIA) by interleukin-1␤ (IL-1␤) and tumor
necrosis factor-␣ (TNF␣) is under the control of group IVA
cytosolic PLA2␣ and 12/15-lipoxygenase (12/15-LOX) in rat
fibroblastic 3Y1 cells. We show here that this cytokine
induction of sPLA2-IIA mRNA requires de novo protein
synthesis. By means of cDNA array analysis, we found
that the level of the CXC chemokine MIP-2 (macrophage
inflammatory protein-2) was significantly elevated in 12/
15-LOX-transfected cells compared with control cells. IL1␤/TNF␣-stimulated induction of endogenous MIP-2 preceded that of sPLA2-IIA, and exogenous MIP-2 induced
sPLA2-IIA dose-dependently. Moreover, a MIP-2-specific
antisense oligonucleotide and small interfering RNA attenuated the IL-1␤/TNF␣-induced expression of sPLA2IIA, suggesting that MIP-2 is an absolute intermediate
requirement for optimal induction of sPLA2-IIA. In addition, the expression of c-jun and fra-1, which are components of the transcription factor AP-1, was elevated in
12/15-LOX-transfected cells, in which cytokine-dependent
binding of AP-1 to the sPLA2-IIA promoter was increased
significantly. Conversely, the receptors for transforming
growth factor-␤ and platelet-derived growth factor,
which contributed to down-regulation of sPLA2-IIA expression, were decreased following 12/15-LOX overexpression. Taken together, 12/15-LOX-dependent up-regulation of sPLA2-IIA expression may result from the
interplay between accelerated MIP-2 signaling, AP-1 activation, and attenuated transforming growth factor-␤ and
platelet-derived growth factor signaling.
Phospholipase A2s (PLA2s)1 are a family of enzymes that
hydrolyze the sn-2 position of the ester bond in glycerophos* This work was supported by grants-in-aid for scientific research
from the Ministry of Education, Science, Culture, Sports, and Technology of Japan. The costs of publication of this article were defrayed in
part by the payment of page charges. This article must therefore be
hereby marked “advertisement” in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
‡ Present address: The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan.
§ To whom correspondence should be addressed: Dept. of Health
Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8
Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. Tel.: 81-3-37848196; Fax: 81-3-3784-8245; E-mail: [email protected].
1
The abbreviations used are: PLA2, phospholipase A2; AACOCF3,
arachidonyl trifluoromethyl ketone; CHX, cycloheximide; COX, cyclooxygenase; DMEM, Dulbecco’s modified Eagle’s medium; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; FABP, fatty acidbinding protein; A-FABP, adipocyte-FABP; FCS, fetal calf serum;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL, interleukin;
iNOS, inducible nitric-oxide synthase; L-NMMA, N(G)-monomethyl-Larginine; LOX, lipoxygenase; NDGA, nordihydroguaiaretic acid;
pholipids, releasing free fatty acids and lysophospholipids. In
turn, the released free fatty acids, especially arachidonic acid,
are converted to bioactive lipid mediators such as prostaglandins (PGs) and leukotrienes through the cyclooxygenase (COX)
and lipoxygenase (LOX) pathways, respectively (1–7). To date,
a number of mammalian intracellular and secretory PLA2s
(sPLA2) have been identified, and some of them, including
group IVA cytosolic PLA2 (cPLA2␣) and several sPLA2s, can be
functionally coupled with the COX or LOX pathways for the
stimulus-induced production of bioactive eicosanoids (1, 8 –10).
Among the sPLA2s, group IIA sPLA2 (sPLA2-IIA) is a prototypic proinflammatory sPLA2 that is found to be highly elevated both in the circulation and locally in the tissue in association with a variety of pathological conditions such as
rheumatoid arthritis, sepsis, and atherosclerosis (11–13). Consequently, it is thought that sPLA2-IIA participates in the
progression of proinflammatory reactions. The expression of
sPLA2-IIA is inducible in a wide variety of cells and tissues
following various stimuli, such as interleukin (IL)-1␤, IL-6,
tumor necrosis factor-␣ (TNF␣), lipopolysaccharide, interferon-␥, phorbol esters, and cAMP-elevating agents. Consistent
with this marked inducibility, the promoter region of the
sPLA2-IIA gene contains TATA and CAAT boxes as well as
several elements homologous with consensus sequences for
binding of stimulus-activated transcription factors, such as
C/EBPs, cAMP-response element-binding protein, NF-␬B,
STAT, and AP-1 (14 –16). In contrast, stimulus-induced sPLA2IIA expression has been shown to be negatively regulated by
anti-inflammatory cytokines or growth factors, such as transforming growth factor-␤ (TGF-␤), insulin-like growth factor-1,
and platelet-derived growth factor (PDGF) (17–19). Thus, the
molecular mechanisms underlying the regulation of sPLA2-IIA
expression appear to be diverse, and the mechanisms for the
induction or repression of sPLA2-IIA expression by particular
stimuli differ according to cell type and even animal species.
Our current studies have demonstrated that stimulation of
rat fibroblastic 3Y1 cells with IL-1␤ and TNF␣ synergistically
induces the expression of sPLA2-IIA, leading to delayed PGE2
biosynthesis in cooperation with inducible COX-2 (20). In this
system, the expression of sPLA2-IIA is sensitive to inhibitors of
cPLA2 and LOX and conversely is enhanced by overexpression
of 12/15-LOX, the only LOX isozyme that is endogenously detectable in 3Y1 cells (20 –22). Furthermore, suppression of
PAF-R, platelet-activating factor receptor; PDGF, platelet-derived
growth factor; PDGFR, PDGF receptor; PG, prostaglandin; PMSF,
phenylmethylsulfonyl fluoride; PPAR, peroxisome proliferator-activated receptor; RT, reverse transcriptase; siRNA, small interfering
RNA; sPLA2, secretory PLA2; TGF, transforming growth factor; TGF␤RII, TGF-␤ receptor II; TNF, tumor necrosis factor; cPLA2␣, cytosolic
PLA2; PPRE, peroxisome proliferator-response element.
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This paper is available on line at http://www.jbc.org
Regulation of Group IIA sPLA2 Induction by MIP-2
sPLA2-IIA expression by the cPLA2 inhibitor could be restored
by certain lipid products of the cPLA2␣-12/15-LOX pathway
(22). Although these observations suggest that the expression
of sPLA2-IIA depends on prior activation of the cPLA2-12/15LOX pathway in these cells, and the requirement for cPLA2␣
and/or 12/15-LOX for sPLA2 expression has also been demonstrated in several, even if not all, cellular systems (23), the
precise underlying machinery for this regulatory pathway and
its physiological relevance are not yet fully understood.
In the present study, we found that IL-1␤/TNF␣-stimulated
induction of sPLA2-IIA mRNA requires prior induction of certain regulatory proteins, expression of which depends on the
cPLA2-12/15-LOX pathway. In search of such factors, we identified macrophage inflammatory protein (MIP)-2, a CXC chemokine, as a critical intermediate regulator of IL-1␤/TNF␣-dependent, 12/15-LOX-regulated induction of sPLA2-IIA.
Additionally, we provide evidence that the transcription factor
AP-1 contributes to the positive regulation of sPLA2-IIA transcription and that signaling by TGF-␤ and PDGF contributes to
its negative regulation.
EXPERIMENTAL PROCEDURES
Materials—Mouse IL-1␤, human TNF␣, and human PDGF-BB were
purchased from Genzyme. Nordihydroguaiaretic acid (NDGA), a LOX
inhibitor, was purchased from BioMol. Cycloheximide (CHX) was purchased from Wako Chemicals. Arachidonyl trifluoromethyl ketone (AACOCF3), a cPLA2 inhibitor, and the enzyme immunoassay kit for PGE2
were obtained from Cayman Chemicals. Rat MIP-2, N(G)-monomethylL-arginine (L-NMMA), and polyclonal antibody against human
PDGF-BB were purchased from Sigma. Human TGF-␤1 and monoclonal antibody against TGF-␤1, -2, and -3 were from R & D Systems.
Monoclonal antibody against human cPLA2␣ and polyclonal antibodies
against mouse IL-8RA (CXCR-1) and mouse IL-8B (CXCR-2) were purchased from Santa Cruz Biotechnology. Polyclonal antibody against rat
MIP-2 was purchased from PeproTech. The cDNAs for rat sPLA2-IIA
(24) and mouse cPLA2␣-(1–522) (25) were prepared as described previously. Lipofectamine 2000, Oligofectamine, Opti-MEM medium, TRIzol
reagent, geneticin, hygromycin, and the pcDNA3.1 series of mammalian expression vectors were obtained from Invitrogen. 3Y1 cells were
generously given by Dr. Y. Uehara (National Institute of Infectious
Disease) and were maintained in culture medium composed of Dulbecco’s modified Eagle’s medium (DMEM; Nissui Pharmaceutical) supplemented with 10% (v/v) fetal calf serum (FCS), penicillin/streptomycin
(100 units/ml and 100 ␮g/ml, respectively), and 2 mM glutamine (Invitrogen) at 37 °C in a CO2 incubator flushed with 5% CO2 in humidified air. 3Y1 cells stably expressing 12/15-LOX were described previously (21).
Activation of 3Y1 Cells—The media of 3Y1 cells that had attained
80% confluence in 6-well plates (Iwaki Glass) were replaced with 2 ml
of DMEM supplemented with 2% FCS. After culture for 24 h, 1 ng/ml
mouse IL-1␤ and 2 ng/ml human TNF␣ or various concentrations of rat
MIP-2 were added to the cultures. In other sets of experiments, 3Y1
cells were pretreated with various concentrations of rat PDGF-BB, rat
TGF-␤1, or their antibodies for 3 h and then treated with IL-1␤/TNF␣
for 24 h. For RNA blotting, TRIzol reagent was added directly to the cell
monolayer.
cDNA Array Analysis—mRNAs isolated from mock- and 12/15-LOXtransfected 3Y1 cells (107 cells for each) were reverse-transcribed into
cDNA and 32P-labeled with an Atlas cDNA expression arrays kit (Clontech). Hybridization was performed on the Atlas Rat 1.2 array kit
(Clontech). After exposure to an image plate (BAS III; Fuji Photo Film),
signals were analyzed with AtlasImage 1.0 software (Clontech).
Reverse Transcriptase (RT)-PCR—Synthesis of cDNA was performed
with 1 ␮g of total RNA and avian myeloblastosis virus-reverse transcriptase according to the manufacturer’s instructions supplied with
the RNA PCR kit (avian myeloblastosis virus, version 2.1; Takara
Biomedicals). Subsequent amplifications of the partial cDNA were performed using 0.5 ␮l of the reverse-transcribed mixture as a template
with specific oligonucleotide primers (Sigma) as follows: rat MIP-2,
sense 5⬘-TTT GGT CCA GAG CCA TGG CC-3⬘ and antisense 5⬘-CCA
GGT CAG TTA GCC TTG CC-3⬘; rat c-JUN, sense 5⬘-CGC GTG AAG
TGA CCG ACT GT-3⬘ and antisense 5⬘-TGA GGT TGG CGT AGA CCG
GA-3⬘; rat FRA-1, sense 5⬘-CAT GTA CCG AGA CTT CGG GG-3⬘ and
antisense 5⬘-GCC TCA CAA AGC CAG GAG TG-3⬘; rat inducible nitric-
25831
oxide synthase (iNOS), sense 5⬘-GCA CAT GCA GAA TGA GTA CC-3⬘
and antisense 5⬘-GTC TGG CGA AGA ACA ATC C-3⬘; rat fatty acidbinding protein (FABP)-5, sense 5⬘-CAT GGC CAG CCT TAA GGA
CC-3⬘ and antisense 5⬘-CTG CTG TCC AGG ATG ACG AG-3⬘; rat
adipocyte FABP (A-FABP), sense 5⬘-ATG TGT GAT GCC TTT GTG
GGG-3⬘ and antisense 5⬘-TTA TGC TCT TTC ATA AAC TCT TGT A-3⬘;
rat IL-6, sense 5⬘-GCC CAC CAG GAA CGA AAG TC-3⬘ and antisense
5⬘-ACT AGG TTT GCC GAG TAG ACC-3⬘; rat platelet-activating factor
receptor (PAF-R), sense 5⬘-CTG CCC AGA GCA ATG GAG CA-3⬘ and
antisense 5⬘-GAA GGC TCT GGA CCT GGC TT-3⬘, rat PDGF receptor
␣ chain (PDGFR␣), sense 5⬘-CAG AAT ACT GCT TCT ATG GG-3⬘ and
antisense 5⬘-GAT GAA AGT GGA ACT ACT GG-3⬘; rat TGF-␤ receptor
type II (TGF-␤RII), sense 5⬘-ACG ACC CCA AGT TCA CCT AC-3⬘ and
antisense 5⬘-GGC CAT GTA TCT CGC TGT TC-3⬘; rat CXCR1, sense
5⬘-AGG GTT CCA ATG ACC AAC AGG3⬘ and antisense 5⬘-AGA CGT
GCG AAA GGA AGC AG-3⬘; rat CXCR2, sense 5⬘-ATC AAC AGG TAT
GCT GTG GTT G-3⬘ and antisense 5⬘-CAG GTC TCC TTG ATC AGC
TTG-3⬘; and rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH),
sense 5⬘-ATG GTG AAG GTC GGT GTG AAC G-3⬘ and antisense
5⬘-TTA CTC CTT GGA GGC CAT GTA G-3⬘. The PCR mixtures were
subjected to 35 cycles of amplification by denaturation (30 s at 94 °C),
annealing (30 s at 60 °C), and elongation (1 min at 72 °C). The PCR
products were analyzed by 1% agarose gel electrophoresis with
ethidium bromide.
RNA Blot Analysis—All of the blotting procedures were performed as
described previously (20). Briefly, equal amounts (10 ␮g) of total RNA
were applied to each lane of 1.2% (w/v) formaldehyde-agarose gels,
electrophoresed, and then transferred to Immobilon-N membranes
(Millipore). The resulting blots were then probed with cDNA inserts
and labeled with [32P]dCTP (PerkinElmer Life Sciences) by random
priming (Takara Biomedicals). Hybridization was carried out at 42 °C
overnight in a solution comprising 50% (v/v) formamide, 0.75 M NaCl,
75 mM sodium citrate, 0.1% (w/v) SDS, 1 mM EDTA, 10 mM sodium
phosphate, pH 6.8, 5⫻ Denhardt’s solution (Nakalai Tesque), 10% (w/v)
dextran sulfate (Sigma), and 100 ␮g/ml salmon sperm DNA (Wako).
The membranes were washed three times at room temperature with
150 mM NaCl, 15 mM sodium citrate, 1 mM EDTA, 0.1% SDS, and 10 mM
sodium phosphate, pH 6.8, for 5 min each, followed by two washes at
55 °C with 30 mM NaCl, 3 mM sodium citrate, 1 mM EDTA, 1% SDS, and
10 mM sodium phosphate, pH 6.8, for 15 min each. The blots were
visualized by autoradiography with Kodak X-Omat AR films and double-intensifying screens at ⫺80 °C.
Transfection of Antisense Oligonucleotide for MIP-2—The antisense
(5⬘-TGG CGA GTG GGA GGG GCC AT-3⬘) and sense (5⬘-ATG GCC CTC
CCA CTC GCC CA-3⬘) oligonucleotides, which correspond to the translation initiation site for rat MIP-2, were each incubated at 200 nM with
Oligofectamine reagent in 200 ␮l of Opti-MEM for 15 min at room
temperature and then added to cells that had attained 60 – 80% confluence in 6-well plates and had been supplemented with 800 ␮l of OptiMEM. After incubation for 6 h at 37 °C, the medium was replaced with
2 ml of DMEM supplemented with 2% FCS in the continued presence of
200 nM oligonucleotide. After culture for 24 h, 1 ng/ml IL-1␤ and 2 ng/ml
TNF␣ were added to the culture to activate the cells.
Establishment of MIP-2 Small Interfering RNA (siRNA)-expressing
Cells—The target sequences for MIP-2 siRNA were subcloned into
pRNA-U6.1/Hygro (GenScript Corporation) at the HindIII/BamHI
sites. The sense sequences of the synthesized oligonucleotides (BEX)
used were as follows: 5⬘-GAT CCC GAA CAT CCA GAG CTT GAC GTT
CAA GAG ACG TCA AGC TCT GGA TGT TCT TTT TTG GAA A-3⬘ and
5⬘-GAT CCC GCC CCC TTG GTT CAG AGG ATT CAA GAG ATC CTC
TGA ACC AAG GGG GCT TTT TTG GAA A-3⬘. The plasmids were
transfected into 3Y1 cells with Lipofectamine 2000 according to the
manufacturer’s instructions. Three days after transfection, the cells
were seeded into 96-well plates in the presence of 100 ␮g/ml hygromycin
in order to establish stable transfectants. The IL-1␤/TNF␣-dependent
expression levels of MIP-2 in the obtained transfectants were assessed
by RNA blotting and immunoblotting.
Establishment of cPLA2␣-(1–522)-expressing Cells—The cDNA for
cPLA2␣-(1–522), which had been subcloned into pcDNA3.1/Hygro, was
transfected into 3Y1 cells with Lipofectamine 2000 according to the
manufacturer’s instructions. Three days after transfection, the cells
were seeded into 96-well plates in the presence of 100 ␮g/ml hygromycin
in order to establish stable transformants expressing cPLA2␣-(1–522).
The expression levels of cPLA2␣-(1–522) in the obtained transfectants
were assessed by immunoblotting. To assess immediate and delayed
PGE2 biosynthesis in these transfectants, the cells were activated with
1 ␮M A23187 for 10 min and IL-1␤/TNF␣ for 24 h, respectively, and the
amounts of PGE2 released into the supernatant were measured.
25832
Regulation of Group IIA sPLA2 Induction by MIP-2
Immunoblot Analysis—Cell lysates (105 cells equivalent) were subjected to SDS-PAGE using 10 or 15% (w/v) gels under reducing conditions. The separated proteins were electroblotted onto nitrocellulose
membranes (Schleicher & Schuell) with a semidry blotter (Bio-Rad)
according to the manufacturer’s instructions. After blocking for 2 h with
3% (w/v) skimmed milk in 10 mM Tris-HCl, pH 7.4, containing 150 mM
NaCl and 0.05% Tween 20 (TBS-Tween), the membranes were probed
for 2 h with the respective antibodies (1:5,000 for MIP-2, CXCR-2, and
CXCR-1; 1:2,000 for cPLA2␣), followed by incubation with horseradish
peroxidase-conjugated anti-mouse (for cPLA2␣), anti-rabbit (for MIP-2
and CXCR2), or anti-goat (for CXCR1) IgG (1:10,000 dilution in TBSTween). After washing, the membranes were visualized with Western
Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences), as described previously (26).
Electrophoretic Mobility Shift Assay (EMSA)—Double-stranded oligonucleotides containing the consensus sequences for the binding sites
for AP-1 and peroxisome proliferator-response element (PPRE) in the
promoter region of the rat sPLA2-IIA gene were radiolabeled with
[␣-32P]dCTP at their 3⬘ ends with a Klenow fragment (Takara Biomedicals) and then purified with a Micro Bio-Spin 6 chromatography column
(Bio-Rad). The sense sequences of the synthesized oligonucleotides used
were as follows: IIA-AP-1, 5⬘-GAA TGA ATG ACT GAC ACG TG-3⬘;
IIA-AP-1 mutant, 5⬘-GAA TGA ATA GTC GAC ACG TG-3⬘ (mutated
sequence underlined); and IIA-PPRE (34), 5⬘-CAG CTG TTG GGG GGA
AAA GGG GA-3⬘. Nuclear extracts of the cells were prepared as described previously (27). Briefly, the cells were suspended in 10 mM
HEPES, pH 7.9, containing 10 mM KCl, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 2 ␮g/ml
leupeptin, 2 ␮g/ml pepstatin, and 2 ␮g/ml aprotinin and were incubated
for 10 min on ice. Nonidet P-40 was added to the suspension at a final
concentration of 0.4% (v/v), and the mixtures were vortex-mixed vigorously for 10 s; the nuclei were then pelleted by centrifugation at
15,000 ⫻ g for 1 min. The pellets were resuspended in 20 mM HEPES,
pH 7.9, containing 0.4 M NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT,
0.5 M PMSF, 2 ␮g/ml leupeptin, 2 ␮g/ml pepstatin, and 2 ␮g/ml aprotinin by vigorous mixing for 5 min at 4 °C. After centrifugation at
10,000 ⫻ g for 5 min, the supernatants were used as nuclear extracts.
The nuclear extracts were incubated with 1 ng of radiolabeled AP-1 or
PPRE oligonucleotide in binding buffer (10 mM HEPES, pH 7.9, containing 50 mM KCl, 5 mM MgCl2, 0.5 mM EDTA, 5 mM DTT, 0.7 mM
PMSF, 2 ␮g/ml leupeptin, 2 ␮g/ml pepstatin, 2 ␮g/ml aprotinin, 10%
(v/v) glycerol, and 1 ␮g/ml poly(dI-dC)䡠poly(dI-dC)) for 30 min at 25 °C.
For competition assay, a 50-fold excess of unlabeled oligonucleotide was
added to the binding reaction prior to the addition of the radiolabeled
probe. These incubation mixtures were electrophoresed in 4.5% native
polyacrylamide gels with Tris borate-EDTA buffer, pH 8.8. The gels
were dried and analyzed with a BAS2000 (Fuji Photo Film).
RESULTS
sPLA2-IIA mRNA Expression in IL-1␤/TNF␣-treated 3Y1
Cells Requires de Novo Protein Synthesis—We have shown
previously that delayed PGE2 generation induced by IL-1␤ and
TNF␣ in 3Y1 cells is regulated by functional coupling between
two inducible enzymes, sPLA2-IIA and COX-2, and that the
induction of sPLA2-IIA, but not COX-2, is suppressed by cPLA2
and LOX inhibitors at the transcriptional level (20). Conversely, when 3Y1 cells overexpressing 12/15-LOX (a LOX
isozyme expressed in 3Y1 cells (21)) were cultured with IL-1␤/
TNF␣ for 24 h, the expression levels of sPLA2-IIA, but not
COX-2, mRNA (Fig. 1, 2nd and 5th lanes) and protein (21) were
markedly up-regulated relative to that in replicate mock-transfected cells. In addition to these observations, we found herein
that de novo protein synthesis was required for full induction of
sPLA2-IIA, but not COX-2, mRNA, because adding CHX, a
protein synthesis inhibitor, to 3Y1 cells greatly decreased the
amount of IL-1␤/TNF␣-induced sPLA2-IIA mRNA in mock- and
12/15-LOX-transfected cells (Fig. 1, lanes 3 and 6). Appropriate
expression of 12/15-LOX in the transfectants was confirmed by
its enzymatic activity and mRNA expression, as described previously (21). Based on these results, we hypothesized that
stimulation by IL-1␤/TNF␣ induces certain regulatory factor(s), which in turn amplify the expression of sPLA2-IIA in a
manner dependent upon the product(s) of the cPLA2-12/15LOX pathway.
FIG. 1. IL-1␤/TNF␣-stimulated sPLA2-IIA mRNA expression is
sensitive to CHX. Mock- or 12/15-LOX-transfected 3Y1 cells grown in
6-well plates were incubated with (⫹) or without (⫺) 1 ng/ml IL-1␤ and
2 ng/ml TNF␣ in the presence (⫹) or absence of 1 ng/ml CHX. After 24 h
of incubation, the expression of sPLA2-IIA and COX-2 mRNAs was
assessed by RNA blotting. Equal loading of samples in each lane was
verified by staining of rRNA with ethidium bromide in agarose gels. A
representative result of three independent experiments is shown.
Identification of 12/15-LOX-regulated Genes—In an attempt
to identify such factor(s) that intermediate the cytokine- and
cPLA2-12/15-LOX-mediated induction of sPLA2-IIA, we performed cDNA array analysis to select a panel of genes in which
expression was significantly altered in IL-1␤/TNF␣-treated 12/
15-LOX-overexpressing 3Y1 cells relative to that in replicate
mock-transfected cells. As summarized in Table I, increased
genes included those required for the inflammatory response
(e.g. the CXC chemokine MIP-2, several proinflammatory cytokines such as IL-6, and iNOS), gene transcription (e.g. junD,
fra-1, and c-jun), and material transport (e.g. A-FABP and
FABP-5). Conversely, decreased genes included receptors for
several growth factors (e.g. PDGFR␣ and insulin-like growth
factor II receptor) and for anti-inflammatory cytokines (e.g.
TGF-␤RII) and annexins (which can inhibit the PLA2 reaction
through substrate depletion (28)).
To assess the IL-1␤/TNF␣-stimulated inducibility of the molecules identified by the cDNA array (Table I) and to ascertain
whether the expression levels of these molecules were indeed
altered following 12/15-LOX expression, we compared by using
high sensitivity RT-PCR and RNA blotting their expression
levels in mock-transfected and 12/15-LOX-expressing 3Y1 cells
after culture with or without IL-1␤/TNF␣ for 24 h. Examples of
RT-PCR for several genes are shown in Fig. 2, where the
expression levels of MIP-2, c-jun, fra-1, iNOS, PAF-R, FABP-5,
and A-FABP were significantly higher in 12/15-LOX-transfected cells than in control cells. Elevated expression of MIP-2
and FABPs in 12/15-LOX-expressing cells over control cells
was particularly evident (Fig. 2), consistent with the fact that
these genes showed the highest inducibility in the cDNA array
assay (Table I). Although the expressions of MIP-2 and iNOS
were undetectable without IL-1␤/TNF␣ stimulation, they were
markedly increased after IL-1␤/TNF␣ stimulation. Unlike
these molecules, the expressions of c-jun and fra-1 were already evident in unstimulated cells and were modestly increased after IL-1␤/TNF␣ stimulation, particularly in 12/15LOX-expressing cells (Fig. 2). A similar result was observed
with the expression of PAF-R, except that it was detectable in
unstimulated cells and was markedly decreased following IL1␤/TNF␣ stimulation. By contrast, the expression of FABP-5
and A-FABP was minimally affected by IL-1␤/TNF␣ stimulation. Collectively, these results indicate that MIP-2, iNOS,
c-jun, and fra-1, which display inducibility by 12/15-LOX and
IL-1␤/TNF␣, are potential candidates for the factor(s) that
intermediate the cytokine induction of sPLA2-IIA.
Cytokine Induction of sPLA2-IIA Is Intermediated by MIP2—To establish whether these inducible factors indeed intermediate the IL-1␤/TNF␣-induced expression of sPLA2-IIA, we
first investigated MIP-2 because, among the inducible genes
identified thus far, its expression was up-regulated most dra-
Regulation of Group IIA sPLA2 Induction by MIP-2
25833
TABLE I
Genes that were altered by overexpression of 12/15-LOX in
IL-1␤/TNF␣-stimulated rat fibroblastic 3Y1 cells
Numbers indicate the fold changes in the expression of genes in
IL-1␤/TNF␣-treated 12/15-LOX transfectants relative to that of mock
transfectants. Intensity ratios (12/15-LOX versus mock, ⱖ2.0 or ⱕ0.4)
are included. A representative result of three independent experiments
is shown.
Genes
Growth factors, cytokines, chemokines and their
receptors
MIP-2
Granulocyte colony-stimulating factor
Granulocyte-macrophage colony-stimulating factor
Thrombopoietin
Interferon ␣1
Fibroblast growth factor 5
Interleukin 18
Glial cell line-derived neurotrophic factor
IL-6
Interleukin 10
Vascular endothelial growth factor D
Vascular endothelial growth factor receptor 2
Insulin-like growth factor II receptor
PDGF receptor ␣
TGF-␤ receptor II
Signal transduction-related genes
Neural visinin-like protein 1
JunD proto-oncogene
Fos-related antigen 1 (FRA1)
Early growth response protein 1
Mitogen-activated protein kinase 10
PAF-R
Jun proto-oncogene
Casein kinase I␦
Glycogen synthase kinase 3␣
Clusterin
BCL2-associated X protein membrane isoform ␣
RalA
Transporter/carrier-related genes
A-FABP
Fatty acid-binding protein 5 (FABP5)
Excitatory amino acid transporter 4
Kidney oligopeptide transporter
Synaptic vesicle protein 2B
Cationic amino acid transporter 1
Multidrug resistance protein 2
Zinc transporter
Antigen peptide transporter 2
Sodium/phosphate cotransporter 1
Sodium/potassium-transporting ATPase ␣2 subunit
Synaptobrevin 1
Syntaxin 1A
Intrinsic factor
Syntaxin 4
Annexin IV
Metabolism-related genes
Glutathione S-transferase P subunit
Soluble superoxide dismutase 1
P450 1B1
Cytochrome P450 51
Corticosteroid 11␤-dehydrogenase isozyme 1
iNOS
Adenylate kinase 3
cAMP-dependent protein kinase type I␣ regulatory
subunit
Annexin III
Fructose-bisphosphate aldolase A
Annexin I
Sertoli cell cytochrome c oxidase polypeptide I
-Fold
4.5
3.8
3.4
3.0
2.9
2.4
2.4
2.1
2.1
2.0
2.0
0.31
0.31
0.18
0.21
FIG. 2. Altered expression of several genes following 12/15LOX overexpression. Mock- or 12/15-LOX-transfected 3Y1 cells
grown in 6-well plates were cultured for 24 h with (⫹) or without (⫺)
IL-1␤/TNF␣, and the expression levels of each mRNA were assessed by
RT-PCR. GAPDH was used as a control for equal sample loading on
each lane. The bands for each PCR product were detected by ethidium
bromide staining in agarose gels. The results are representative of
three independent experiments.
2.9
2.4
2.3
2.2
2.2
2.1
2.0
0.08
0.13
0.18
0.23
0.15
4.5
4.1
2.7
2.6
2.5
2.4
2.3
2.2
2.2
2.1
2.1
2.1
2.0
2.0
0.15
0.27
2.6
2.4
2.3
2.3
2.2
2.1
0.09
0.16
0.16
0.17
0.17
0.26
matically following cytokine stimulation and by 12/15-LOX
expression (Table I and Fig. 2). As shown in Fig. 3A, when
mock-transfected or 12/15-LOX-expressing 3Y1 cells were cultured with IL-1␤/TNF␣, the expression of sPLA2-IIA mRNA
started to increase after culture for 3 h, increased markedly
after 6 h, and reached its maximum at a plateau level after
12–24 h. This induction of sPLA2-IIA mRNA was accompanied
FIG. 3. Time courses of MIP-2 and sPLA2-IIA induction after
IL-1␤/TNF␣ stimulation. Mock- or 12/15-LOX-transfected (A) and
parental (B) 3Y1 cells grown in 35-mm dishes were cultured for the
indicated periods with IL-1␤/TNF␣. A, the expression levels of MIP-2
and sPLA2-IIA mRNAs were assessed by RNA blotting. Equal loading of
samples in each lane was verified by staining of rRNA with ethidium
bromide in agarose gels. B, the expression of MIP-2 protein was assessed by immunoblotting. 105 cell equivalents were applied to each
lane. Representative results of three independent experiments are
shown.
by induction of its protein expression (as assessed by immunoblotting) and enzymatic activity (as assessed by PLA2 enzyme
assay), as reported previously (20). In comparison, the expression of MIP-2 mRNA appeared as early as 30 min, reached a
maximum level by 3 h, and then decreased over 12–24 h.
Increased expression of MIP-2 mRNA was followed by that of
its protein, which was minimal before stimulation, increased
after culture with IL-1␤/TNF␣ for 3–18 h, and tended to decline
gradually thereafter (Fig. 3B). Thus, IL-1␤/TNF␣ stimulation
led to transient induction of MIP-2 expression, followed by
persistent induction of sPLA2-IIA expression. Furthermore,
MIP-2 expression was higher in 12/15-LOX-expressing cells
than in control cells at each time point, thus correlating with
subsequent increased expression of sPLA2-IIA in the former
cells (Fig. 3A).
The cytokine inducibility of endogenous MIP-2 prior to that
of sPLA2-IIA prompted us to examine whether exogenous
MIP-2 directly induces expression of sPLA2-IIA mRNA. When
3Y1 cells were stimulated with recombinant rat MIP-2 for 12 h,
the induction of sPLA2-IIA, as well as that of endogenous
25834
Regulation of Group IIA sPLA2 Induction by MIP-2
FIG. 4. Recombinant MIP-2 induces
sPLA2-IIA expression. A, 3Y1 cells
grown in 12-well plates were incubated
with various concentrations of recombinant rat MIP-2. B, cells were treated with
(⫹) or without (⫺) 1 ␮g/ml recombinant
MIP-2 for the indicated periods. C, 3Y1
cells were cultured with (⫹) or without
(⫺) IL-1␤/TNF␣ for 24 h, and the expression levels of CXCRs were assessed by
RT-PCR (upper panel) and immunoblotting (lower panel). The bands for each
CXCR were detected by ethidium bromide
staining in agarose gels. D, cells were
stimulated for 12 h with (⫹) or without
(⫺) 1 ␮g/ml MIP-2 (lanes 1–5) or IL-1␤/
TNF␣ (lanes 6 –9) in the presence or absence of 10 ␮M AACOCF3 (lanes 3 and 8),
20 ␮M NDGA (lanes 4 and 9), or 1 ng/ml
CHX (lane 5). The expression of sPLA2IIA (A, B, and D) and MIP-2 (A) mRNAs
was assessed by RNA blotting, and equal
loading of samples in each lane was verified by staining of rRNA with ethidium
bromide in agarose gels. Representative
results of two to three independent experiments are shown.
MIP-2, occurred in a dose-dependent manner (Fig. 4A). As
shown in Fig. 4B, the expression of sPLA2-IIA induced by
recombinant MIP-2 became obvious by 3 h, reached its maximum at a plateau level after 6 –12 h, and declined by 24 h.
Thus, induction of sPLA2-IIA by MIP-2 (⬃3– 6 h) occurs kinetically earlier than that by IL-1␤/TNF␣ (⬎6 h; see Fig. 3), in line
with the hypothesis that IL-1␤/TNF␣ induces MIP-2, which in
turn facilitates sPLA2-IIA expression. Because several members of the CXC chemokine family, to which MIP-2 belongs,
exert their bioactivities through the G protein-coupled receptors CXCR1 and CXCR2 (29 –31), we assessed by RT-PCR and
immunoblot analyses the expression of these receptors in 3Y1
cells. Both CXCR1 and CXCR2 mRNAs (Fig. 4C, upper panel)
and proteins (Fig. 4C, lower panel) were constitutively expressed in 3Y1 cells, with the former showing a modest increment following IL-1␤/TNF␣ stimulation.
To assess whether the MIP-2 signaling for sPLA2-IIA induction requires the cPLA2-12/15-LOX pathway, we examined the
effects of AACOCF3 and NDGA on exogenous MIP-2-dependent
sPLA2-IIA induction. As in the case of IL-1␤/TNF␣ stimulation
(Fig. 4D, lanes 6 –9), the induction of sPLA2-IIA mRNA by
recombinant MIP-2 was also suppressed by AACOCF3 or
NDGA (Fig. 4D, lanes 3 and 4), suggesting the involvement of
the cPLA2-12/15-LOX pathway in this MIP-2-mediated event.
By contrast, the addition of CHX, which reduced IL-1␤/TNF␣dependent sPLA2-IIA mRNA induction (Fig. 1), failed to suppress MIP-2-dependent sPLA2-IIA mRNA induction, but rather
tended to increase it (Fig. 4D, lane 5), implying that de novo
protein synthesis is not required for MIP-2 signaling leading to
sPLA2-IIA induction.
To verify further the involvement of cPLA2␣ in IL-1␤/TNF␣induced MIP-2 expression, cDNA for cPLA2␣-(1–522), a dominant-negative mutant for cPLA2␣ (25), was transfected into
3Y1 cells to establish transfectants stably expressing cPLA2␣(1–522). Fig. 5A depicts the expression levels of full-length
(endogenous) cPLA2␣ and cPLA2␣-(1–522) in cells stably transfected with cPLA2␣-(1–522) and in mock-transfected control
cells, as assessed by immunoblotting. The expression of
cPLA2␣-(1–522) protein was detected only in cPLA2␣-(1–522)
transfectants, whereas the expression levels of endogenous,
full-length cPLA2␣ protein were similar in both cell types (Fig.
5A). When these cells were incubated for 10 min with A23187,
which elicits immediate PGE2 biosynthesis via the cPLA2␣ and
COX-1 pathway (20), the expression of cPLA2␣-(1–522) led to
FIG. 5. cPLA2␣-(1–522), a dominant-negative form of cPLA2␣,
attenuates the induction of MIP-2 and sPLA2-IIA expression. A,
expression of cPLA2␣ and cPLA2␣-(1–522) proteins in cPLA2␣-(1–522)transfected cells (right lane) and control cells (left lane) was assessed by
immunoblotting with anti-cPLA2␣ antibody. B, effects of cPLA2␣-(1–
522) overexpression on immediate (left panel) and delayed (right panel)
PGE2 biosynthesis. Cells were stimulated for 10 min with A23187 or for
24 h with IL-1␤/TNF␣, and the supernatants were taken for PGE2
enzyme immunoassay. C, effects of cPLA2␣-(1–522) overexpression on
IL-1␤/TNF␣-induced MIP-2 and sPLA2-IIA mRNA induction were assessed by RNA blotting. Equal loading of samples in each lane was
verified by staining of rRNA with ethidium bromide in agarose gels.
Representative results of three independent experiments are shown.
⬃80% reduction of A23187-induced immediate PGE2 biosynthesis (Fig. 5B, left panel), confirming the dominant-negative
action of cPLA2␣-(1–522) in this situation. As shown in Fig. 4C,
when these cells were stimulated for 24 h with IL-1␤/TNF␣,
which elicits delayed COX-2-dependent PGE2 synthesis, significant (even though partial) reduction of sPLA2-IIA and MIP-2
expression was observed in cPLA2␣-(1–522) transfectants compared with mock transfectants. In accordance with the partial
reduction of sPLA2-IIA expression, delayed PGE2 release was
also attenuated by ⬃40% following introduction of cPLA2␣-(1–
522) (Fig. 5B, right panel). These results further support the
idea that cytokine induction of MIP-2 (and thereby that of
Regulation of Group IIA sPLA2 Induction by MIP-2
FIG. 6. Down-regulation of MIP-2 attenuates IL-1␤/TNF␣-mediated sPLA2-IIA expression. A, effects of MIP-2-directed antisense
and sense oligonucleotides on sPLA2-IIA mRNA expression. 3Y1 cells
pretreated with antisense (lane 2) or sense (lane 3) oligonucleotides for
MIP-2 or without treatment (lane 1) were stimulated with IL-1␤/TNF␣
for 24 h, as described under “Experimental Procedures,” and expression
of MIP-2, sPLA2-IIA, and COX-2 mRNAs was assessed by RNA blotting.
Equal loading of samples in each lane was verified by staining of rRNA
with ethidium bromide in agarose gels. B, effect of MIP-2 siRNA on
MIP-2 and sPLA2-IIA expression. Cells were stimulated with or without
0.1 ng/ml IL-1␤ and 0.2 ng/ml TNF␣ (lanes 2 and 6), 0.3 ng/ml IL-1␤ and
0.6 ng/ml TNF␣ (lanes 3 and 7), and 1 ng/ml IL-1␤ and 2 ng/ml TNF␣
(lanes 4 and 8) for 24 h. The expression levels of MIP-2, sPLA2-IIA, and
cPLA2␣ proteins in MIP-2 siRNA- (lanes 5– 8) and mock- (lanes 1– 4)
transfected cells were assessed by immunoblotting.
sPLA2-IIA, which lies downstream of MIP-2) is under the control of cPLA2␣. However, only partial suppression of the delayed response, relative to profound reduction of the immediate
response, by cPLA2␣-(1–522) might be indicative of the contribution of additional PLA2(s) (possibly other cPLA2 isozymes) to
the delayed response, a possibility that is under investigation.
To obtain more conclusive evidence for the participation of
MIP-2 in IL-1␤/TNF␣-induced sPLA2-IIA expression, we used
antisense and siRNA technologies to specifically reduce MIP-2
expression. As shown in Fig. 6A, transfection of the antisense,
but not sense, oligonucleotide for MIP-2 into 3Y1 cells reduced
IL-1␤/TNF␣-induced MIP-2 expression (⬃40% reduction compared with sense oligonucleotide-treated cells; Fig. 6A, lane 3),
with concomitant reduction of IL-1␤/TNF␣-induced sPLA2-IIA
expression (⬃20% reduction compared with sense oligonucleotide-treated cells; Fig. 6A, lane 3). Less sensitivity of sPLA2IIA than MIP-2 to the MIP-2 antisense may be because the
residual MIP-2 expression is still capable of inducing sPLA2IIA to some extent or because other proteinaceous factor(s) also
participate in sPLA2-IIA induction. The antisense oligonucleotide did not suppress the IL-1␤/TNF␣-induced expression of
COX-2, implying that the suppression of MIP-2 expression was
responsible for selective attenuation of sPLA2-IIA expression.
Next, we transfected MIP-2-specific siRNA into 3Y1 cells and
examined the expression levels of MIP-2, sPLA2-IIA, and
cPLA2␣ proteins by immunoblotting. As shown in Fig. 6B, the
cytokine-dependent induction of MIP-2 protein in MIP-2
siRNA-transfected cells was markedly reduced (⬎80% reduction) compared with that in mock-transfected cells. Accordingly, there was 50 – 60% reduction of sPLA2-IIA expression in
MIP-2 siRNA-treated cells relative to that in control cells at
each IL-1␤/TNF␣ concentration employed (Fig. 6B). Taken together, these results indicate that MIP-2 acts as a positive
intermediate regulator for IL-1␤/TNF␣-induced sPLA2-IIA
expression.
12/15-LOX Enhances Binding of AP-1 to Rat sPLA2-IIA Promoter—As already shown in Table I and Fig. 2, 12/15-LOX
enhances the expression of c-jun and fra-1, both of which are
components of the transcription factor AP-1 (32, 33). Detailed
time course studies of c-jun expression revealed that it was
increased only transiently at 30 min after IL-1␤/TNF␣ stimulation in control cells and that its expression levels were higher
25835
FIG. 7. AP-1 is involved in 12/15-LOX-dependent sPLA2-IIA
transcription. A, mock- or 12/15-LOX-transfected 3Y1 cells grown in
35-mm dishes were cultured for the indicated periods with IL-1␤/TNF␣,
and the expression levels of c-jun were assessed by RNA blotting. Equal
loading of samples in each lane was verified by staining of rRNA with
ethidium bromide in agarose gels. B and D, EMSA was carried out with
nuclear extracts isolated from mock- or 12/15-LOX-transfected 3Y1
cells that had been cultured with (⫹) or without (⫺) IL-1␤/TNF␣ for
18 h. IIA-AP-1 (B) and IIA-PPRE (D) were used as probes in the binding
reaction. To verify the specificity of the binding, a 50-fold excess of cold
specific and mutant (AP-1 mut.) oligonucleotides was added, as indicated. C, EMSA was carried out with nuclear extracts isolated from 3Y1
cells that had been cultured with (⫹) or without (⫺) MIP-2 for the
indicated periods. IIA-AP-1 was used as a probe in the binding reaction.
Similar results were obtained in three independent experiments.
in 12/15-LOX-transfected cells than in control cells at each time
point tested (Fig. 7A). Furthermore, following the first transient increase at 30 min, there was a second gradual increase in
c-jun over 6 –12 h in 12/15-LOX-transfected cells. To assess the
contribution of c-JUN to 12/15-LOX-dependent sPLA2-IIA expression, we next examined by EMSA the ability of AP-1 to
bind to the sPLA2-IIA promoter. 12/15-LOX- or mock-transfected 3Y1 cells (Fig. 7B, lanes 5– 8 and lanes 1– 4, respectively)
were treated with or without IL-1␤/TNF␣ for 18 h, and then the
nuclear extracts from these cells were subjected to EMSA. As
shown in Fig. 7B, constitutive binding of AP-1 to the AP-1binding motif in the sPLA2-IIA promoter (IIA-AP-1 (⫺470 to
⫺480)) was already evident in control cells regardless of the
stimulus, and was significantly increased in 12/15-LOX-transfected cells particularly under the IL-1␤/TNF␣-stimulated condition. The specificity of the AP-1 complex was verified by
inhibition with an excess of unlabeled IIA-AP-1, but not IIAAP-1 mutant, oligonucleotide (Fig. 7B, lanes 3, 4, 7, and 8). In
addition, when 3Y1 cells were treated with recombinant MIP-2,
the binding of AP-1 to IIA-AP-1 was significantly increased
over 3–18 h (Fig. 7C), indicating that MIP-2-mediated induction of sPLA2-IIA also involves AP-1.
Because PPAR␥ has been reported to play a critical role in
the regulation of sPLA2-IIA expression in IL-1␤-treated rat
vascular smooth muscle cells (34), and because potential ligands for this transcription factor include 12/15-LOX metabolites (35, 36), we also tested whether this transcription factor
could affect sPLA2-IIA transcription, by using the same
method. Although binding of PPAR␥ to the sPLA2-IIA promoter
(⫺160 to ⫺133; a region reported previously to be responsible
for the binding to PPAR␥ (34)) was indeed observed in our
EMSA, with a trend to increase after IL-1␤/TNF␣ stimulation,
it was minimally affected by 12/15-LOX overexpression (Fig.
7D). The specificity of the PPAR␥ complex was verified by
inhibition with an excess of unlabeled oligonucleotide (Fig. 7D,
lanes 3 and 6). These results suggest that 12/15-LOX increases
the basal and IL-1␤/TNF␣-induced expression of AP-1 compo-
25836
Regulation of Group IIA sPLA2 Induction by MIP-2
FIG. 8. IL-1␤/TNF␣-induced iNOS expression is up-regulated
by 12/15-LOX independently of sPLA2-IIA induction. A, mock- or
12/15-LOX-transfected 3Y1 cells grown in 35-mm dishes were cultured
for the indicated periods with IL-1␤/TNF␣, and the expression levels of
iNOS were assessed by RNA blotting. B, mock- or 12/15-LOX-transfected 3Y1 cells grown in 6-well plates were incubated with (⫹) or
without (⫺) IL-1␤/TNF␣ in the presence (⫹) or absence (⫺) of 5 mM
L-NMMA, an NOS inhibitor. After 24 h of incubation, the induction of
sPLA2-IIA was assessed by RNA blotting. Equal loading of samples in
each lane was verified by staining of rRNA with ethidium bromide in
agarose gels. The results are representative of two reproducible
experiments.
nents, which leads to enhanced sPLA2-IIA transcription,
whereas the contribution of PPAR␥ to this 12/15-LOX-regulated event through the reported PPRE site is negligible.
iNOS Is Not Involved in sPLA2-IIA Expression—Because
nitric oxide has a wide variety of biological functions and one of
its biosynthetic enzymes, iNOS, has been implicated in the
regulation of eicosanoid synthesis in certain situations (37, 38),
we next examined whether iNOS is also involved in IL-1␤/
TNF␣-induced sPLA2-IIA expression. When mock-transfected
cells were cultured with IL-1␤/TNF␣, the expression of iNOS
mRNA, which was undetectable in the basal state, reached a
maximum level after 3 h and decreased thereafter over 6 –24 h
(Fig. 8A, left panel). By contrast, the expression of iNOS mRNA
was enhanced and prolonged markedly over 3–24 h in replicate
12/15-LOX-transfected cells (Fig. 8A, right panel), suggesting
that 12/15-LOX affects IL-1␤/TNF␣-induced iNOS mRNA expression and/or its stability. However, when 3Y1 cells were
cultured with IL-1␤/TNF␣ for 24 h in the presence or absence
of L-NMMA, a NOS inhibitor, the induction of sPLA2-IIA expression was unaffected in both mock-transfected and 12/15LOX-expressing cells (Fig. 8B). No effect of L-NMMA on sPLA2IIA expression was found even at earlier time points (data not
shown). These results suggest that, even though augmented
induction of iNOS by 12/15-LOX occurs, this event is not functionally linked to the regulation of sPLA2-IIA expression.
PDGF and TGF-␤ Signalings Negatively Regulate IL-1␤/
TNF␣-induced sPLA2-IIA Expression—As shown in Table I,
our cDNA array assay identified the receptors for PDGF and
TGF-␤ as 12/15-LOX-down-regulated products. Therefore, we
examined by RNA blotting whether the expression levels of
these receptors were indeed altered in 12/15-LOX-expressing
cells. As shown in Fig. 9A, both PDGFR␣ and TGF-␤RII were
expressed constitutively and were modestly increased over
3–24 h of stimulation with IL-1␤/TNF␣ in control cells. The
IL-1␤/TNF␣-stimulated expression of PDGFR␣ and TGF-␤RII
FIG. 9. The PDGF and TGF-␤ signaling pathways negatively
regulate cytokine-dependent sPLA2-IIA induction. A, mock- or
12/15-LOX-transfected 3Y1 cells grown in 35-mm dishes were cultured
for the indicated periods with IL-1␤/TNF␣, and the expression levels of
PDGFR␣ and TGF-␤RII mRNAs were assessed by RNA blotting. B and
C, 3Y1 cells pretreated with the indicated concentrations of PDGF-BB
(B) or TGF-␤1 (C) for 3 h were incubated with (⫹) or without (⫺)
IL-1␤/TNF␣ for 24 h, and the expression of sPLA2-IIA mRNA was
assessed. D, 3Y1 cells grown in 12-well plates were incubated with or
without IL-1␤/TNF␣ in the presence or absence of 10 ng/ml anti-TGF-␤,
anti-PDGF-BB, or control antibody. After incubation for 24 h, the induction of sPLA2-IIA was assessed by RNA blotting. E, 3Y1 cells grown
in 35-mm dishes were cultured for the indicated periods with IL-1␤/
TNF␣, and the expression of TGF-␤1 mRNA was assessed by RNA
blotting. Equal loading of samples in each lane was verified by staining
of rRNA with ethidium bromide in agarose gels. The results are representative of three independent experiments.
was markedly decreased in 12/15-LOX-expressing cells compared with that in control cells, and the decreased expression of
the former receptor was already evident in unstimulated cells
(Fig. 9A).
When 3Y1 cells were pretreated with PDGF-BB (Fig. 9B) or
TGF-␤ (Fig. 9C) for 3 h and then incubated with IL-1␤/TNF␣
for 24 h in their continued presence, IL-1␤/TNF␣-induced
sPLA2-IIA mRNA expression was dose-dependently reduced by
each of them, with their minimum effects being observed at 10
and 0.1 ng/ml, respectively, suggesting that these factors negatively regulate sPLA2-IIA expression. To confirm the contribution of endogenous PDGF and/or TGF-␤ signalings on the
regulation of sPLA2-IIA expression, we examined the effects of
the neutralizing antibodies specific for TGF-␤1 and PDGF-BB
on IL-1␤/TNF␣-induced sPLA2-IIA expression. The IL-1␤/
TNF␣-mediated induction of sPLA2-IIA was significantly increased only by the addition of anti-TGF-␤ antibody but not by
that of anti-PDGF-BB and control antibodies (Fig. 9D). Consistent with this observation, when 3Y1 cells were treated with
IL-1␤/TNF␣, endogenous TGF-␤1 mRNA was modestly increased over 1–24 h (Fig. 9E), whereas no expression of endogenous PDGF-BB mRNA was observed (data not shown). These
results suggest that the endogenous TGF-␤ signaling regulates
sPLA2-IIA induction negatively in 3Y1 cells.
Regulation of Group IIA sPLA2 Induction by MIP-2
25837
FIG. 10. Schematic diagram of the
signaling pathway involved in the
sPLA2-IIA gene expression in rat fibroblastic 3Y1 cells. A, during the earliest CHX-sensitive stage, proinflammatory cytokines (IL-1␤ and TNF␣) generate
certain lipid metabolite(s) (diamond) via
the cPLA2 and 12/15-LOX pathway, and
the metabolite(s) in turn induce the expression of MIP-2 and AP-1. B, after induction of MIP-2, it binds to its receptors
CXCR1 and/or CXCR2 and stimulates the
expression of sPLA2-IIA via the cPLA2
and LOX pathway and the AP-1 pathway.
Because MIP-2-dependent sPLA2-IIA induction is largely insensitive to CHX, this
stage does not need de novo protein biosynthesis, even though MIP-2 induces its
own expression weakly. C, during the late
stage of cytokine stimulation, TGF-␤1, as
well as the receptors for TGF-␤ and
PDGF, which are negative regulators for
sPLA2-IIA expression, is induced, thereby
resulting in attenuation of sPLA2-IIA expression. Although PDGF is not produced
endogenously in 3Y1 cells, it could be supplied from tissue microenvironments in
certain in vivo circumstances.
DISCUSSION
We have shown previously that stimulation by IL-1␤ and
TNF␣ causes delayed PGE2 production in rat fibroblastic 3Y1
cells by promoting the de novo induction of sPLA2-IIA and
COX-2 at the transcriptional level, and that the expression of
sPLA2-IIA, but not COX-2, is dependent upon prior activation
of cPLA2␣ and its downstream enzyme in another arm of
arachidonate metabolism, 12/15-LOX (20 –22). The results presented in this paper have provided new insights into the regulation of this cPLA2␣-12/15-LOX-dependent expression of
sPLA2-IIA, as summarized in Fig. 10. We found that the protein biosynthesis inhibitor CHX suppresses IL-1␤/TNF␣-induced sPLA2-IIA mRNA expression, suggesting that de novo
induction of certain protein factor(s) is needed for cytokine- and
cPLA2␣-12/15-LOX-dependent sPLA2-IIA mRNA induction. To
identify such factor(s), we used cDNA array analysis to search
for genes for which the expression is up-regulated by cytokines
and 12/15-LOX overexpression. Of the inducible 12/15-LOXresponsive genes identified thus far, we identified the CXC
chemokine MIP-2 as one of the critical mediators of sPLA2-IIA
induction. Three lines of evidence support this conclusion.
First, treatment of 3Y1 cells with IL-1␤/TNF␣ increased MIP-2
expression prior to sPLA2-IIA induction, and the expressions of
both MIP-2 and sPLA2-IIA were markedly enhanced by overexpression of 12/15-LOX. Second, treatment of 3Y1 cells with
exogenous MIP-2 induced sPLA2-IIA in a time- and dose-dependent manner. Third, suppression of endogenous MIP-2 induction by MIP-2-directed antisense oligonucleotide and siRNA
markedly attenuated cytokine-dependent sPLA2-IIA expres-
sion. This again provides strong support for the notion that
MIP-2 functions as an intermediary for regulation of cytokineand cPLA2␣-12/15-LOX-dependent sPLA2-IIA expression. We
also found that overexpression of 12/15-LOX up-regulates the
transcription factor AP-1, which acts as a positive regulator for
sPLA2-IIA expression, and down-regulates the receptors for
TGF-␤ and PDGF, signaling from which negatively regulates
sPLA2-IIA expression. Thus, 12/15-LOX-dependent induction
of sPLA2-IIA expression results from the interplay between
accelerated MIP-2 expression, AP-1 activation, and attenuated
TGF-␤ and PDGF (particularly the former) signaling (Fig. 10).
Regulation of the Expression of Various Genes by 12/15LOX—12/15-LOX catalyzes the stereoselective oxidation of
arachidonic acid at C12 or C15 and can also directly oxidize
esterified fatty acids in lipoproteins and phospholipids membranes (39 – 42). We found that the overexpression of 12/15LOX affects the expression of various genes, implying that its
metabolic product(s) can control their transcription either directly or indirectly. The transcription factor AP-1 is involved in
the transcriptional activation of a wide variety of genes (33).
Because the components of AP-1 (c-jun and fra-1) are sensitive
to 12/15-LOX, it is possible that some of the 12/15-LOX-induced
genes are indirectly regulated by AP-1. Indeed, several 12/15LOX-responsive genes listed in Table I, including MIP-2 (58),
contain AP-1-binding elements in their promoter regions. It is
also possible that overproduction of oxidized lipid metabolites
following 12/15-LOX overexpression may produce oxidative
stress, which in turn leads to the activation of stress-sensitive
transcriptional factors.
25838
Regulation of Group IIA sPLA2 Induction by MIP-2
Because the products of 12/15-LOX can activate PPARs (35,
36), some of the 12/15-LOX-induced genes might be regulated
by this particular class of nuclear receptors. Indeed, the promoter regions of the genes for some FABPs, including A-FABP,
as well as that for sPLA2-IIA, contain PPRE elements to which
PPARs can bind and thereby transactivate their target genes
(34, 43– 45). Although our present EMSA analysis argues
against a requirement for the reported PPAR␥-binding element
(⫺160 to ⫺133) in the sPLA2-IIA promoter (34) for 12/15-LOXdependent induction of this enzyme, the promoter region may
contain additional PPAR-responsive element(s) that could account for PPAR-dependent induction of sPLA2-IIA. In fact, we
have shown recently that PPAR␣, rather than PPAR␥, may be
involved in sPLA2-IIA induction in 3Y1 cells (22). Similarly,
PPAR␣ appears to be involved in sPLA2-IIA expression in rat
mesangial cells (45).
Mechanisms for MIP-2 Induction of sPLA2-IIA—MIP-2,
which contains a glutamic acid-leucine-arginine (ELR) sequence immediately preceding the CXC chemokine motif, is a
potent neutrophil chemoattractant (46). In addition to this
chemotactic activity, MIP-2 can also activate NF-␬B (47), a key
transcription factor leading to the induction of various proinflammatory genes, which include cytokines (IL-1␤ and TNF␣),
iNOS, and COX-2 as well as sPLA2-IIA. The expression of
MIP-2 has been shown to be increased in macrophages, epithelial cells, and fibroblasts in response to lipopolysaccharide,
TNF␣, and oxidative stress (48 –51). We observed that MIP-2dependent sPLA2-IIA induction is still sensitive to inhibitors of
cPLA2 and LOX, implying that this chemokine-directed event
still requires the product(s) of the cPLA2␣-12/15-LOX pathway.
In support of this, IL-1␤/TNF␣ treatment increases the expression of MIP-2 mRNA in a time-dependent manner, an event
that is enhanced by the overexpression of 12/15-LOX. Moreover, the induction of MIP-2 is markedly attenuated by overexpression of a dominant-negative form of cPLA2␣. Most interestingly, Pawliczak et al. (52) have shown that in epidermal
growth factor- and A23187-treated human lung epithelial cell
line A549 cells, the stimulus-dependent induction of IL-8, another member of the ELR⫹ CXC chemokine family, is under the
control of certain product(s) of the cPLA2 pathway and that this
effect depends partly on PPAR␥ (52). Thus, even though the
precise targets through which cPLA2␣ and 12/15-LOX activate
MIP-2 transcription remain to be identified, a possible mechanism for this up-regulation might involve, in part, activation of
certain PPARs. Also, although it needs to be clarified which
specific intracellular signals produced by the G protein-coupled
MIP-2 receptor are able to trigger sPLA2-IIA transcription,
such signals appear to involve AP-1, because the binding of
AP-1 to the sPLA2-IIA promoter is significantly elevated following treatment with exogenous MIP-2. We have also shown
that stimulation of 3Y1 cells with exogenous MIP-2 induces the
expression of endogenous MIP-2. Thus, IL-1␤/TNF␣ signaling,
MIP-2 signaling, and the cPLA2␣-12/15-LOX pathway may
form a positive amplification relay leading to optimal sPLA2IIA expression (Fig. 10, A and B). Of note, unlike IL-1␤/TNF␣
stimulation, MIP-2-induced sPLA2-IIA mRNA expression
does not require de novo protein biosynthesis. This suggests
that MIP-2 is a main intermediate determinant that influences subsequent CHX-sensitive induction of sPLA2-IIA
transcription.
Down-regulation of sPLA2-IIA Expression by PDGF and
TGF-␤ Signaling—Several recent studies have shown that
stimulus-induced sPLA2-IIA expression is negatively regulated
by anti-inflammatory cytokines or growth factors, such as
TGF-␤ and PDGF (17, 19). Consistent with this, we found that
pretreatment of 3Y1 cells with TGF-␤1 or PDGF-BB dose-de-
pendently suppressed IL-1␤/TNF␣-dependent sPLA2-IIA induction. Furthermore, we also found that the expression of the
receptors for these factors, namely PDGFR-␣ and TGF-␤RII,
was markedly up-regulated after IL-1␤/TNF␣ stimulation and
that the expression of PDGFR-␣ (constitutive and inducible)
and TGF-␤RII (inducible) was markedly attenuated by 12/15LOX overexpression. These results suggest that certain 12/15LOX product(s) may down-regulate negative regulatory signals
for sPLA2-IIA induction, thereby eventually leading to increased expression of sPLA2-IIA in 12/15-LOX-expressing cells.
Notably, TGF-␤, but not PDGF, is endogenously expressed in
3Y1 cells, and the incubation of the cells with anti-TGF-␤, but
not with anti-PDGF, antibody resulted in enhanced IL-1␤/TNF␣dependent sPLA2-IIA induction, suggesting that endogenous
TGF-␤, rather than PDGF, signaling may significantly contribute to the regulation of sPLA2-IIA expression in our system
(Fig. 10C). Nevertheless, considering the in vivo circumstances,
attenuation of sPLA2-IIA expression by PDGF or TGF-␤, which
could be supplied endogenously or even exogenously from tissue microenvironments at the late stage of inflammation,
might contribute, at least in part, to sequestering inflammatory reactions promoted by this proinflammatory enzyme and
thus facilitating the tissue repair process promoted by these
growth factors.
PGE2 Generation in 3Y1 Cells—cPLA2␣ is the most important PLA2 enzyme involved in the production of various eicosanoids as well as lysophospholipid-derived mediators such as
PAF (8, 9, 53). Not only does cPLA2␣ directly supply arachidonic acid to COX and LOX enzymes for the production of
eicosanoids, but it also modifies eicosanoid production by
sPLA2s (54, 55). In some cases, cPLA2␣-derived lipid product(s)
appear to regulate the expression of sPLA2-IIA and sPLA2-V
(20, 23). Thus, cPLA2␣ often mediates the stimulus-dependent
induction of these sPLA2s and subsequent membrane hydrolysis in several cell types (20, 23), and conversely, sPLA2s often
induce eicosanoid production by activating cPLA2␣ (56). The
synergistic action of cPLA2␣ and sPLA2s has been supported
further by gene targeting studies as follows: macrophages derived from cPLA2␣ knock-out mice produce eicosanoids only
minimally (9), whereas there is also ⬃50% reduction of eicosanoid production in macrophages derived from sPLA2-V-deficient mice (57). In the present study, we showed that overexpression of the dominant-negative form of cPLA2␣ markedly
suppressed A23187-induced immediate PGE2 generation,
whereas its suppressive effect on IL-1␤/TNF␣-induced delayed
PGE2 generation was only partial although significant. The
latter effect is most likely to be because of suppression of
sPLA2-IIA induction. These observations thus substantiate our
current proposal that the immediate response in 3Y1 cells
depends largely on cPLA2␣, whereas the delayed response depends on the concerted action of sPLA2-IIA and cPLA2␣. However, generalization of this concept still needs caution, because
functional synergism between cPLA2␣ and sPLA2s appears to
be cell type-specific and stimulus-specific and perhaps in many,
even if not all, situations cPLA2␣ is nearly sufficient for eliciting (even delayed) eicosanoid biosynthesis, as has been shown
by various cell biological and cPLA2␣ knock-out studies (9, 53).
Thus, efforts to identify specific cell types (like 3Y1 cells) that
exhibit sPLA2-dependent eicosanoid biosynthesis, particularly
in vivo, would be important to shed light on the as yet ambiguous pathophysiological roles of sPLA2-IIA and other sPLA2
family members.
Summary—We have shown here that the induction of
sPLA2-IIA by proinflammatory cytokines is regulated by complex cross-talk between various signaling pathways, such as
up-regulation of chemokine signaling and down-regulation of
Regulation of Group IIA sPLA2 Induction by MIP-2
growth factor signaling, that lie downstream of the product(s)
of the cPLA2␣-12/15-LOX pathway. Although identification of
the particular bioactive lipid products that dictate this signal
amplification cascade will need further study, the development
and use of specific inhibitors of the cPLA2␣-12/15-LOX pathway may provide novel approaches to the treatment of inflammatory diseases by attenuating signal pathways involving
proinflammatory cytokines, chemokines, and even sPLA2s.
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