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J. Biol. Chem., Vol. 280, Issue 27, 25830-25839, July 8, 2005

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Search of Factors That Intermediate Cytokine-induced Group IIA Phospholipase A₂ Expression through the Cytosolic Phospholipase A₂- and 12/15-Lipoxygenase-dependent Pathway^*

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

Received for publication, January 5, 2005 , and in revised form, April 8, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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. IL-1/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 sPLA₂-IIA, 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Phospholipase A₂s (PLA₂s)¹ are a family of enzymes that hydrolyze the sn-2 position of the ester bond in glycerophospholipids, 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 sPLA₂-IIA 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 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 cPLA₂-12/15-LOX 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

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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)-monomethyl-L-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 µg/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-LOX-transfected 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 µg 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 µl 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-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 acid-binding 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 µg) 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, 5x Denhardt's solution (Nakalai Tesque), 10% (w/v) dextran sulfate (Sigma), and 100 µg/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 µl 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 µl of Opti-MEM. 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 µg/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 µg/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 µM A23187 [GenBank] for 10 min and IL-1/TNF for 24 h, respectively, and the amounts of PGE₂ released into the supernatant were measured.

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 TBS-Tween). 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 µg/ml leupeptin, 2 µg/ml pepstatin, and 2 µg/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 x 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 µg/ml leupeptin, 2 µg/ml pepstatin, and 2 µg/ml aprotinin by vigorous mixing for 5 min at 4 °C. After centrifugation at 10,000 x 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 µg/ml leupeptin, 2 µg/ml pepstatin, 2 µg/ml aprotinin, 10% (v/v) glycerol, and 1 µg/ml poly(dI-dC)·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

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 cPLA₂-12/15-LOX pathway.

View larger version (62K): [in this window] [in a new window]   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.

View this table: [in this window] [in a new window]   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, 2.0 or 0.4) are included. A representative result of three independent experiments is shown.

Cytokine Induction of sPLA₂-IIA Is Intermediated by MIP-2—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 dramatically 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 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).

View larger version (48K): [in this window] [in a new window]   FIG. 2. Altered expression of several genes following 12/15-LOX 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.

View larger version (49K): [in this window] [in a new window]   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.

View larger version (44K): [in this window] [in a new window]   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 µg/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 µg/ml MIP-2 (lanes 1–5) or IL-1/TNF (lanes 6–9) in the presence or absence of 10 µM AACOCF3 (lanes 3 and 8), 20 µM NDGA (lanes 4 and 9), or 1 ng/ml CHX (lane 5). The expression of sPLA2-IIA (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.

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 [GenBank] , which elicits immediate PGE₂ biosynthesis via the cPLA₂ and COX-1 pathway (20), the expression of cPLA₂-(1–522) led to 80% reduction of A23187 [GenBank] -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 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.

View larger version (44K): [in this window] [in a new window]   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 [GenBank] 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.

View larger version (24K): [in this window] [in a new window]   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.

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 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-1-binding 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 IIA-AP-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.

View larger version (49K): [in this window] [in a new window]   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.

View larger version (54K): [in this window] [in a new window]   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.

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 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).

View larger version (66K): [in this window] [in a new window]   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.

View larger version (32K): [in this window] [in a new window]   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

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Regulation of the Expression of Various Genes by 12/15-LOX—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 12/15-LOX 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 12/15-LOX-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.

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-LOX-dependent 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-2-dependent 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 [GenBank] -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 sPLA₂-IIA 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-dependently 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 12/15-LOX overexpression. These results suggest that certain 12/15-LOX 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