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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:2559.)
© 2000 American Heart Association, Inc.

Vascular Biology

Protein Kinase A–Dependent Stimulation of Rat Type II Secreted Phospholipase A₂ Gene Transcription Involves C/EBP-ß and - in Vascular Smooth Muscle Cells

Cyril Couturier; Valérie Antonio; Arthur Brouillet; Gilbert Béréziat; Michel Raymondjean; Marise Andréani

From Unité Propre de Recherche de l’Université Pierre et Marie Curie, associée au CNRS (ESA7079).

Correspondence to Marise Andréani, Université Pierre et Marie Curie, ESA 7079, Université Pierre et Marie Curie, Case 256, 7 quai Saint Bernard 75005 Paris, France. E-mail andreani{at}ccr.jussieu.fr

	Abstract

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Abstract—Type II secreted phospholipase A₂ (sPLA₂) releases precursors of important inflammatory lipid mediators from phospholipids. Some observations have indicated that the sPLA₂, which has been implicated in chronic inflammatory conditions such as arthritis, contributes to atherosclerosis in the arterial wall. sPLA₂ was not detected in control vascular smooth muscle cells (VSMC). Treatment of VSMC with agents that increase intracellular cAMP (eg, forskolin, dibutyryl [db]-cAMP) resulted in a time- and concentration-dependent increase in sPLA₂ gene expression. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) showed a marked dose-dependent inhibition of forskolin-induced mRNA by protein kinase A inhibitor. Electrophoretic mobility shift analysis of nuclear proteins from forskolin-treated and db–cAMP-treated VSMC with C/EBP consensus oligonucleotides and C/EBP oligonucleotides from the rat promoter revealed greater binding than in control VSMC. Incubation of VSMC with H89, a specific protein kinase inhibitor, also blocked the binding of nuclear C/EBP to the C/EBP site of the rat promoter induced by db-cAMP and forskolin. Binding was unchanged with the use of CRE consensus oligonucleotides. Antibodies revealed the specific formation of C/EBP/DNA complexes, the majority of which were supershifted by C/EBP-ß and - antibodies. Functional activation of C/EBP was confirmed by a luciferase reporter gene assay. A construct comprising 4 tandem repeat copies of the C/EBP element from the rat sPLA₂ promoter linked to luciferase was transcriptionally activated in VSMC by cotransfection with expression vector for the protein kinase A catalytic subunit. It was also significantly activated in transfected VSMC treated by forskolin or db-cAMP. H89 inhibited this activations. We therefore conclude that the increases in sPLA₂ mRNA and enzyme activity produced by cAMP-elevating agents is controlled by a mechanism involving nuclear C/EBP-ß and - acting through a protein kinase A signaling pathway.

Key Words: gene regulation • protein kinase A • secreted type II phospholipase A₂ • smooth muscle cells • C/EBP

	Introduction

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Secreted type II phospholipase A₂ (type II sPLA₂) has been implicated in the pathophysiology of atherosclerosis. It has been found in human atherosclerotic plaques.¹ Transgenic mice overproducing type II sPLA₂ develop atherosclerotic lesions when they are maintained on a low fat diet, and their lesions increase dramatically when they are fed a high fat, high cholesterol diet.²

The precise physiological substrate of type II sPLA₂ is still unclear. Indirect evidence indicates that type II sPLA₂ can hydrolyze mammalian cell phospholipids in vivo to generate lipid mediators.³ Phospholipase A₂–derived mediators are thought to be involved in smooth muscle cell proliferation. Lysophosphatidylcholine,⁴ cyclooxygenase and lipoxygenase products,⁵ and arachidonic acid itself⁶ may all mediate the proliferative effect of IL-1ß.

Vascular smooth muscle cells (VSMC) undergo phenotypic changes during the onset of atherosclerosis, switching from a contractile phenotype to a proliferating, secretory phenotype.⁷ The proliferation of these cells is implicated in the pathogenesis of atherosclerosis and the restenosis that may follow revascularization.⁷ Early atherosclerotic lesions have many characteristics in common with inflammatory reactions,⁸ and VSMC contribute to the local inflammatory processes involved in the development of lesions and the formation of new intima.⁹ IL-1ß, which is thought to play an important role in this process, also triggers the synthesis and secretion of type II sPLA₂ by VSMC.¹⁰

In recent years, it has become evident that agents that increase intracellular cAMP, in addition to inflammatory cytokines such as IL-1ß, influence the activity of the type II sPLA₂ gene in rat aortic smooth muscle cells.¹⁰

cAMP regulates the expression of specific genes in most cells by mediating the protein kinase A–dependent phosphorylation of the cAMP-responsive element binding transcription factor (CREB) at serine 133.¹¹ However, the human type II sPLA₂ promoter that we found to respond to forskolin and cAMP has no CREB binding site, and the gene is thought to be induced primarily by CCAAT box enhancer binding proteins (C/EBP).¹² The ß- and -isoforms of C/EBP are mainly produced in cells involved in inflammatory reactions and participate in the acute phase response (for review, see Reference ¹³ ).

We have therefore examined the process by which cAMP induces type II sPLA₂ gene expression in rat VSMC. We found that forskolin and cAMP transactivate the gene through the activation of C/EBP-ß and - and protein kinase A but not through the CREB pathway.

	Methods

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Reagents
Type I collagen from calf skin, glutamine, penicillin, streptomycin, fatty acid–free bovine serum albumin, and Naja naja type II secreted PLA₂ were purchased from Sigma Chemical Co. FCS was from Boehringer Mannheim. Antibodies to smooth muscle cell -actin from hybridoma cells, clone 1A4, were from DAKO SA. Murine mammary lentivirus reverse transcriptase and random primers and lipofectamine were from Life Technologies, and oligonucleotides were from Oligo Express. Hybon N+ nylon membranes, ECL direct nucleic acid labeling system, and ECL reagents kit for HRP were from Amersham. The fluorescent substrate 1-hexadecanoyl-2-(1-pyrenyldecanoyl)-sn-glycero-3-phosphoglycerol was from Interchim. IL-1ß was purchased from Immugenex Corp. The PGL3 basic vector, pRL-SV40 vector, and dual-luciferase reporter assay kit were purchased from Promega Inc. Antibodies to C/EBP-, -ß, an - were purchased from Santa Cruz Biotechnology, Inc.

Isolation and Culture of Rat Aortic Smooth Muscle Cells
VSMC were isolated by enzymatic digestion of thoracic aortic media from male Wistar rats (weight, 300 g; Elevage Janvier) by the method of Michel (Battle et al¹⁴ ). The cells were seeded on dishes coated with type I collagen from calf skin and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS, 4 mmol/L glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. The purity of the smooth muscle cell preparation was evaluated by staining the cells with monoclonal antibodies to smooth muscle cell -actin. More than 96% of cells were immunoreactive. Smooth muscle cells were subcultured every 7 days, and experiments were performed on cells at 3 to 6 passages after primary culture.

Confluent cells were made quiescent by incubating them for 24 hours in serum-free medium and then in the same medium containing 0.2% fatty acid–free bovine serum albumin and appropriate agents (see figure legends). The medium was then removed and assayed for phospholipase A₂ activity; the cells were treated for reverse transcription–polymerase chain reaction (RT-PCR) analysis, protein determinations, or nuclear extract preparations.

Reverse Transcription–Polymerase Chain Reaction
Total RNA was extracted according to Chomczynski and Sacchi¹⁵ and 1.5 µg was used as template for RT. First-strand cDNA was synthesized by means of murine mammary lentivirus RT and random primers. sPLA₂ cDNA was amplified together with GAPDH cDNA as an internal control for semiquantitative PCR, and the linear amplification was determined for each experiment (currently 20 to 24 cycles). The primers were designed as described previously,¹⁶ with sequences for rat sPLA2 cDNA corresponding to nucleotides 155 to 438 from the start codon and sequences for rat GAPDH cDNA corresponding to nucleotides 305 to 599 from the start codon. The primers used for type II sPLA₂ were GTG GCA GAG GAT CCC CCA AGG (CS 10, forward) and GCA ACT GGG CGT GTT CCC TCT GCA (CS 11, reverse). Those used for GAPDH were CCA TGG AGA AGG CTG GGG (GS, forward) and CAA AGT TGT CAT GGA TGA CC (GAS, reverse).

The following standard conditions were used for PCR in a volume of 25 µL: 2.5 µL of cDNA template generated from RT reactions, 1.25 U of Taq DNA polymerase, 20 to 24 amplification cycles with 160 nmol/L CS 10 and CS 11 primers, 120 nmol/L GS and GAS primers. PCR amplifications were performed in a thermocycler (Hybaid Omnigene): denaturation (3 minutes at 95°C) followed by cycles of PCR (denaturation for 1 minute at 95°C, annealing 1 minute at 64°C, extension 1 minute at 72°C), and a final extension for 4 minutes at 72°C.

The PCR products (5 µL each sample) were electrophoresed in 2% agarose gel, blotted, and transferred to a Hybon N+ nylon membrane. The identity of amplified cDNA products was confirmed by hybridization with 5'-CAA CCG TCT GGA GAA ACG TGG ATG TGG CAC-3' (nucleotides 216 to 245 from ATG) for rat type II sPLA₂ cDNA and 5'-GTG AAC CAC GAG AAA TAT GAC AAC TCC CTC-3' (nucleotides 397 to 426 from ATG) for GAPDH cDNA. The oligonucleotide probes were labeled with the ECL direct nucleic acid labeling system. The hybridized membranes were washed, revealed by the ECL reagents kit for HRP, and autoradiographed.

Phospholipase A₂ Assay
Phospholipase A₂ activity was measured with a selective fluorometric assay. The activity secreted into 400-µL samples of medium was assayed with 4 nmol fluorescent substrate 1-hexadecanoyl-2-(1-pyrenyldecanoyl)-sn-glycero-3-phosphoglycerol. Total hydrolysis of the substrate by 0.1 U phospholipase A₂ from Naja naja was used as a reference to calculate the phospholipase A₂ activity of the samples. Spontaneous hydrolysis of the substrate was assayed with fresh culture medium and subtracted from test values.

Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared from smooth muscle cells by the method of Dignam et al,¹⁷ with minor modifications. Cells were washed in 5 mL of ice-cold PBS, harvested, and centrifuged at 1000g for 5 minutes. The cell pellet was suspended in 500 µL buffer A (5 mmol/L HEPES, pH 7.9, 1.5 mmol/L MgCl₂, 10 mmol/L KCl, 0.5% NP40, 50 mmol/L NaF, 0.5 mmol/L DTT, 0.1 mmol/L PMSF, 5 µg/mL leupeptin). Cell lysates were placed on ice for 10 minutes and centrifuged at 3000g for 10 minutes. The nuclear pellet was then suspended in 100 µL buffer B (20 mmol/L HEPES, pH 7.9, 25% glycerol, 0.5 mol/L NaCl, 1.5 mmol/L MgCl₂, 0.5 mmol/L EDTA, 50 mmol/L NaF, 0.5 mmol/L DTT, 0.5 mmol/L PMSF, 5 µg/mL leupeptin) for 30 minutes at 4°C. Nuclear debris was removed by centrifugation at 15 000g for 30 minutes. The supernatant (nuclear extract) was distributed in 15-µL aliquots, which were stored at -80°C until analysis by electrophoretic mobility shift assay (EMSA). Protein concentration was determined as previously described.¹⁶

The C/EBP double-stranded oligonucleotides corresponded to the sequence 5'-GGG ATG AAC TTT CGA AAT CAG CT-3' (C/EBP site 1), corresponding to the region [-227/-240] of the rat type II sPLA₂ promoter or the sequence 5'-GGG ATG GGC TTT TGG AAA GTT CTC-3' (C/EBP site 2), corresponding to the region [-282/-295] of the rat type II sPLA₂ promoter.¹⁸ They were annealed, and 100 ng of double strand of oligonucleotides was end-labeled with the T4 polynucleotide kinase with 50 µCi [³²P]dATP. Unincorporated nucleotides were removed with Sephadex G50. Binding reactions were carried out in 20 µL of binding reaction mixture (10 mmol/L HEPES, pH 7.9, 50 mmol/L NaCl, 1 mmol/L DTT, 10% glycerol, 0.2% NP40, 0.5 mmol/L EDTA) containing 7 µg nuclear proteins and 1 ng of the C/EBP site 1 probe or the C/EBP site 2 probe (50 000 cpm). Samples were incubated at room temperature for 25 minutes and fractionated on 6% denaturing polyacrylamide gels in 0.25xTBE (45 mmol/L Tris-borate, 1 mmol/L EDTA) by preelectrophoresis for 30 minutes at 180 V followed by 180 V for 3 hours. Gels were placed on 3 MM paper (Whatman Ltd), dried in a gel dryer under vacuum at 80°C, and then exposed to Amersham x-ray film. The specificity of the DNA protein complexes was determined in competition assays with a 100-fold molar excess of an unlabeled double-stranded oligonucleotide corresponding to each of the C/EBP site 1 and site 2 probes and to a probe corresponding to the human C/EBP binding site¹² (specific inhibitors) or a 100-fold molar excess of a double-stranded oligonucleotide corresponding to an AP1 binding site consensus sequence: 5'-GGG AGC CGC AAG TGA GTC AGC GCG GGG CTG GTG CA-3', or to an NF-B binding site consensus sequence (non specific competitors). The CRE doubled-stranded oligonucleotides corresponded to a CREBP binding site consensus sequence¹¹ : 5' GGG GAT CCG GCG CCT CCT TGG CTG ACG GAG AGA GAG A. An aliquot (1 µL) of specific antibody to C/EBP-, -ß, or - was added to the binding mixture for C/EBP in some experiments, and the mix was incubated for 15 to 25 minutes before adding radiolabeled probe. EMSA was performed as described above.

Transfection and Luciferase Assays
The [-54/+11] region of the L-type pyruvate kinase promoter was inserted into the pGL3 basic vector at the Mlu I and Xho I sites. The resulting vector was checked by DNA sequencing and used as a control in transfection assays. A double-stranded oligonucleotide corresponding to C/EBP binding site 1 containing an Mlu I site at its end was synthesized and used in ligase reactions with the [-54/+11] L-type pyruvate kinase promoter luciferase vector digested by Mul I. The plasmid was circularized with T4 ligase, and the clones generated were checked by DNA sequencing to select a [(C/EBP binding site)x4] pyruvate kinase promoter luciferase vector.

Cultured rat smooth muscle cells were seeded in 6-well dishes 24 hours before transfection at a concentration giving 70% confluence. Smooth muscle cells were transfected by incubation with 4 µL lipofectamine plus, 800 ng of a plasmid construct containing the [(C/EBP binding site 1)x4] pyruvate kinase promoter region fused to luciferase reporter gene, and 2 ng of pRSV-Renilla per well (as a control of transfection efficiency) for 3 hours, as recommended by the manufacturer (Life Technologies, Inc). The transfected cells were washed twice with PBS, cultured for 24 hours in serum-free medium, and then incubated for 6 hours in the same medium containing appropriate agents, as indicated in the figure legends.

The firefly and Renilla luciferase activities were determined by a dual-luciferase reporter assay kit, with signal detection for 12 seconds by a luminometer (Berthold, Inc).

	Results

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Dose-Dependent and Time-Course Effects of Dibutyryl-cAMP and Forskolin on Type II sPLA₂ Gene Expression in VSMC
The type II sPLA₂ gene is not expressed under resting conditions in rat aortic smooth muscle cells, as in other cells in primary culture. Dibutyryl-cAMP (db-cAMP) and forskolin induce gene expression in these cells.¹⁰ We first determined the kinetic parameters of type II sPLA₂ induction to identify the signal transduction events involved in this effect. Type II sPLA₂ gene expression gradually increased from zero with time, as indicated by the concentration of type II sPLA₂ mRNA and sPLA₂ activity in the supernatant (Figure 1A). Addition of 10 µmol/L forskolin or 0.5 mmol/L db-cAMP resulted in the appearance of type II sPLA₂ mRNA after 4 hours, and a maximum mRNA concentration was reached at 12 hours (Figure 1A). This effect was not inhibited by cycloheximide, the inhibitor of protein synthesis (not shown). In contrast, the enzymatic activity was barely detected in the supernatant during the first 6 hours and then increased linearly until 24 hours (Figure 1A). This suggests that the secretion of type II sPLA₂ is delayed after mRNA and protein synthesis in smooth muscle cells. Type II sPLA₂ mRNA was detected 24 hours after adding as little as 1 µmol/L forskolin or 0.25 mmol/L db-cAMP (Figure 1B), but the enzyme activity was detectable after incubation with 0.1 µmol/L forskolin or 0.1 mmol/L db-cAMP at this time (not shown).

View larger version (42K): [in this window] [in a new window]   Figure 1. Time course and dose response of type II sPLA2 [infi] gene induction by forskolin and db-cAMP in VSMC. Serum-starved cells were incubated in DMEM containing 10 µmol/L forskolin (FK, ) or 0.5 mmol/L db-cAMP () or vehicle alone (•) for various times. A, Phospholipase A2 activity in extracellular medium was determined by spectrofluorometric assay. Maximal activity was obtained after 24 hours, 3.0±0.5 mmol/min per milligram of protein for forskolin and 3.2±0.6 mmol/min per milligram of protein for db-cAMP and is expressed as 100%. Results are mean±SD of 2 independent experiments. Inserted figure: Cells were washed at end of each incubation time and total RNA was extracted for analysis by RT-PCR. Representative autoradiogram of 3 independent experiments is shown. B, Serum-starved cells were incubated for 24 hours in DMEM containing increasing concentrations of forskolin (0.1 to 100 µmol/L) or db-cAMP (0.1 to 1 mmol/L) RT-PCR of mRNA.

Characterization of Induction of Type II sPLA₂ Gene by Forskolin
The reversibility of type II sPLA₂ induction was tested by incubating VSMC with 10 µmol/L forskolin for 6 hours. The forskolin was then washed out and the cells were reincubated for various times. There was a rapid decrease in type II sPLA₂ mRNA concentration, so that more than two thirds of the mRNA was lost after 16 hours. Very little mRNA was detected at 24 hours, and none was found after 48 hours of reincubation, (Figure 2A).

View larger version (31K): [in this window] [in a new window]   Figure 2. Induction of type II sPLA2 gene by forskolin. A, Reversible effect of forskolin on type II sPLA2 gene. Cells were incubated with 10 µmol/L forskolin for 6 hours. Forskolin was removed and cells were incubated in DMEM for various times. mRNA was assessed by RT-PCR. C indicates control cells; T, treated cells. B, Effect of protein kinase A inhibitor on type II sPLA2 gene induction by forskolin in VSMC. Cells were incubated for 30 minutes with increasing amounts of protein kinase A inhibitor H89 and then for 6 hours with 10 µmol/L forskolin in medium. mRNA was assessed by RT-PCR as indicated in Figure 1. One representative autoradiogram from 2 independent experiments is shown. Numbers given in frame represent percentage inhibition by each dose calculated from sPLA2 mRNA/GAPDH mRNA ratios obtained by densitometer scanning.

Forskolin generally activates genes by stimulating adenylate cyclase, which generates cAMP and stimulates protein kinase A. We therefore investigated the role of protein kinase A in type II sPLA₂ gene induction by forskolin by incubating cells for 30 minutes with increasing amounts of H89, a specific protein kinase A inhibitor, and then with 10 µmol/L forskolin for 6 hours. Analysis of type II sPLA₂ mRNA showed a dose-dependent inhibition of the forskolin effect (Figure 2B). Total inhibition was obtained with 40 µmol/L H89.

Increased Binding of C/EBP to Specific Binding Sites in Type II sPLA₂ Promoter by Forskolin and db-cAMP Acting Through Protein Kinase
The common pathway leading to gene expression through protein kinase A is the phosphorylation of the transcription factor CREB, which becomes bound to the cAMP responsive element (CRE).¹¹ The rat type II sPLA₂ promoter does not have a typical CRE or a related sequence. We used electromobility gel shift and competition assays with nuclear extracts of stimulated and unstimulated cells. There was no increase in CREB binding to an oligonucleotide having a CRE-related sequence (Figure 3B, lanes 11 to 14) in forskolin-treated VSMC. As alternative pathways might involve C/EBP factors, we investigated their role in forskolin and db-cAMP type II sPLA₂ gene induction. Sequence analyses of the rat type II sPLA₂ promoter revealed 2 putative C/EBP binding sites: C/EBP binding site 1 (C/EBP 1) [-227/-240] and C/EBP binding site 2 (C/EBP 2) [-282/-295] (Figure 3A). Oligonucleotides bearing C/EBP 1 and C/EBP 2 binding sites and the binding site of the human type II sPLA₂ promoter (C/EBP C)¹² were synthesized. Nuclear extracts from smooth muscle cells treated for 6 hours by 10 µmol/L forskolin or 0.5 mmol/L db-cAMP had more nuclear proteins that bound to an oligonucleotide bearing C/EBP 1 than did control nuclear extracts (Figure 3B, lanes 1 to 8). This binding was abolished by excess cold oligonucleotides bearing either C/EBP 1 or C/EBP C. By contrast, an oligonucleotide bearing C/EBP 2 did not form complexes with either control or stimulated nuclear extracts and the corresponding cold oligonucleotide only weakly displaced the complexes with C/EBP 1 (Figure 3B, lane 6). This indicates that C/EBP 2 binds poorly C/EBP factors. Cold oligonucleotides bearing either an NF-B consensus site or an AP1 binding site did not displace the complexes with the C/EBP 1 probe. Nuclear extracts were prepared from cells incubated for 30 minutes with or without 20 µmol/L H89 before adding forskolin and db-cAMP to confirm the role of protein kinase A in the stimulation of C/EBP. The protein kinase A inhibitor H89 completely blocked the increase in nuclear factor binding induced by both agents (Figure 4).

View larger version (48K): [in this window] [in a new window]   Figure 3. Effect of forskolin and db-cAMP on DNA-binding activity of C/EBP in VSMC. A, Sequence alignment of C/EBP consensus site, C/EBP site from domain C of type II sPLA2 by human promoter, C/EBP binding site 1 [-227/-240], and C/EBP binding site 2 [-282/-295] from type II sPLA2 rat promoter. B, Nuclear extracts were prepared from untreated cells and from cells incubated for 6 hours with 10 µmol/L forskolin or 0.5 mmol/L db-cAMP. C/EBP binding was detected by electromobility shift assays with radiolabeled oligonucleotides bearing C/EBP binding site 1 (lanes 1 to 8), C/EBP binding site 2 (lanes 9, 10), or CRE binding site (lanes 11 to 14). Labeled probes were incubated with nuclear extracts from untreated cells (lane 1) or cells incubated with forskolin (lanes 2, 10, 13, and 14) or db-cAMP (lanes 3 to 8). Large excesses (x100) of cold oligonucleotides bearing C/EBP binding site 1 (lane 4), human type II sPLA2 C/EBP binding site 2 (lane 5), C/EBP binding site 2 (lane 6), AP1 binding site (lane 7), or NF-B binding site (lane 8) or CRE binding site (lanes 12 and 14) were used to check specificity of binding.

View larger version (67K): [in this window] [in a new window]   Figure 4. Effect of protein kinase A inhibitor H89 on increased C/EBP binding to DNA in VSMC induced by forskolin and db-cAMP. Nuclear extracts were prepared from serum-starved cells incubated for 30 minutes with or without 20 µmol/L H29 and then for 6 hours with 10 µmol/L forskolin, 0.5 mmol/L db-cAMP, or vehicle alone. Electromobility shift assays were performed with labeled probe bearing C/EBP binding site 1. Nuclear extracts were prepared from control cells (lanes 1 and 2) or from cells treated with forskolin (lanes 3 and 4) or db-cAMP (lanes 5 and 6) after incubation with H89 (lanes 2, 4, and 6) or vehicle alone (lanes 1, 3, and 5).

Increased Function of a Promoter Bearing the C/EBP Site 1 in Smooth Muscle Cells After Stimulation of the Protein Kinase A Pathway
We performed experiments with a reporter gene containing several repeats of C/EBP 1 to determine whether the increased binding of C/EBP to C/EBP 1 was responsible for the increased function of the rat type II sPLA2 promoter in response to forskolin or db-cAMP. We constructed a plasmid containing 4 repeats of C/EBP 1 fused to a minimal promoter of the [-54/+11] L-type pyruvate kinase in front of the luciferase gene. Smooth muscle cells transiently transfected with this reporter gene were incubated with 10 µmol/L forskolin or 0.5 mmol/L db-cAMP, or cotransfected with an expression vector for the catalytic subunit of protein kinase A with or without increasing amounts of H89. We found a 4-fold stimulation of luciferase synthesis by forskolin or db-cAMP and a 13-fold stimulation in the cells cotransfected with the protein kinase A expression vector (Figure 5). These stimulatory effects were also dose-dependently inhibited by the protein kinase A inhibitor H89.

View larger version (33K): [in this window] [in a new window]   Figure 5. C/EBP binding site 1 of type II sPLA2 promoter in VSMC. Vascular smooth muscle cells were transiently cotransfected with plasmid construct containing 4 repeats of C/EBP binding site 1 in front of the [-54/+11] L-type pyruvate kinase promoter region fused to firefly luciferase reporter gene and pRLSV40 Renilla luciferase vector as control of transfection efficiency with lipofectamine technique. Third plasmid corresponding to expression vector of catalytic protein kinase A subunit was transfected in some experiments. One day later, cells were incubated for 30 minutes with increasing amounts of the protein kinase A inhibitor H89 or with vehicle alone and then with 10 µmol/L forskolin (FK), 0.5 mmol/L db-cAMP, or vehicle alone for 6 hours. Luciferase activities were then measured. Results are expressed in arbitrary units (X, control). Results are representative of 3 independent experiments performed in duplicate.

Involvement of C/EBP-ß and C/EBP- Isoforms in Forskolin and db-cAMP–Stimulated Type II sPLA2 Gene Expression
There are several isoforms of C/EBP that are involved in the control of the human type II sPLA2 gene in HepG₂ cells.¹² We evaluated the involvement of these isoforms in the response of the type II sPLA2 gene to forskolin and db-cAMP in rat smooth muscle cells by using supershift assays with specific antibodies to -, ß-, and -C/EBP isoforms (Figure 6). Only the antibodies specific for the ß- and -isoforms supershifted proteins from the complexes obtained with nuclear extracts and the oligonucleotide bearing C/EBP 1.

View larger version (60K): [in this window] [in a new window]   Figure 6. Characteristics of the C/EBP isoforms involved in forskolin (FK) and db-cAMP induction of type II sPLA2 gene. Smooth muscle cells were incubated in serum-free DMEM for 6 hours without (lanes 1 and 7) or with 10 µmol/L forskolin (lanes 2 to 6) or 0.5 mmol/L db-cAMP (lanes 8 to 12). Nuclear extracts were prepared and electromobility shift assays were performed to detect C/EBP binding. Nuclear extracts were incubated for 15 minutes with specific antibodies against C/EBP- (lanes 3 and 9), C/EBP-ß (lanes 4 and 10), or C/EBP- (lanes 5 and 11) before adding labeled oligonucleotide bearing C/EBP binding site 1. Specificity was checked by competition with 100-fold excess of cold oligonucleotide (lanes 6 and 12).

	Discussion

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Type II sPLA₂ is the major phospholipase A₂ secreted by many cell types in response to inflammatory agents, and it is thought to contribute to inflammation by the delayed production of various lipid mediators.¹⁹ Immunoreactive type II sPLA2 is found in -actin–positive VSMC in both normal and atherosclerotic human arteries.²⁰ It has been suggested that type II sPLA₂ is important in early atherosclerosis because it is present in the preatherosclerotic arterial wall, where it may cause changes in LDL²¹ and foam cell formation.²² Our results confirm that the type II sPLA₂ gene, which is silent in rat aortic smooth muscle cells under resting conditions, is markedly stimulated by forskolin and db-cAMP in a time-dependent and dose-dependent manner (Figure 1). This contrasts with the effect of forskolin on guinea pig alveolar macrophages, in which this drug inhibits LPS-induced type II sPLA₂ gene expression.²³ The forskolin effect does not need priming by an inflammatory cytokine, in agreement with previous studies on rat smooth muscle cells.¹⁰ Smooth muscle cells have various adenylate cyclase–coupled receptors whose stimulation might increase cAMP in response to ß₂-receptor agonists,²⁴ prostaglandins,²⁵ vasopressin,²⁶ angiotensin II,²⁷ and aldosterone.²⁸ In addition to stimulating the type II sPLA₂ gene, cAMP-elevating agents stimulate the production of vascular endothelial growth factor.²⁹ Our results also indicate that the induction of type II sPLA₂ needs the sustained increase in cAMP, because removal of forskolin quickly reverses the forskolin-induced increase in type II sPLA₂ mRNA. Approximately two thirds of mRNA was lost within 16 hours of removing the drug (Figure 2). The plasma concentrations of several cAMP-elevating hormones (angiotensin, vasopressin, PGE₂, aldosterone) are increased in pathological states such as hypertension and renal diseases, which are known to be risk factors for atherosclerosis. Therefore, the sustained production of cAMP-elevating agents in the neighborhood of VSMC can cooperate with inflammatory cytokines to stimulate type II sPLA₂ synthesis. The overproduction of this enzyme and the subsequent sustained release of inflammatory lipid mediators during the onset of atherosclerosis may trigger positive regulatory feedback loops that accelerate the atherosclerotic process.

cAMP acts by activating protein kinase A in most cells. This is also the case in smooth muscle cells, because the specific protein kinase A inhibitor H89 completely blocks the effects of forskolin and db-cAMP on endogenous type II sPLA₂ mRNA (Figure 2). Protein kinase A is generally believed to control gene expression through the translocation of its catalytic subunit into the nucleus, where it phosphorylates the transcription factor CREB at serine 133, inducing increased binding to the CRE. However, the promoter of type II sPLA₂ does not have any obvious CRE-related sequences,³⁰ and we found no increase in binding to a typical CRE oligonucleotide by nuclear extract prepared from smooth muscle cells treated with forskolin or db-cAMP (Figure 3). By contrast, this nuclear extract had an increased ability to form complexes with an oligonucleotide bearing the C/EBP binding site 1 of the rat type II sPLA₂ promoter (Figure 3). These complexes were competitively destroyed by cold oligonucleotides bearing the C/EBP binding site of human type II sPLA₂ promoter (Figure 3) and were supershifted by specific antibodies to C/EBP-ß or C/EBP- but not by those to C/EBP- (Figure 6). Finally, a reporter gene bearing 4 copies of the C/EBP binding site 1 is strongly stimulated by both forskolin and db-cAMP and by cotransfection of the catalytic subunit protein kinase A (Figure 5). These events were inhibited by the protein kinase A inhibitor H89. Thus, we have demonstrated that the effects of forskolin and cAMP on the type II sPLA₂ gene in rat aortic smooth muscle cells are mediated by C/EBP-ß, C/EBP-, or both.

Several mechanisms are involved in the transactivation of genes by cAMP acting through C/EBP factors. In some cases, cAMP increases the concentration of C/EBP-ß and C/EBP- by a CREB-dependent transactivation of their respective genes.^{31 32} The quantity of C/EBP-ß is above normal in synoviocytes and macrophages from patients with rheumatoid arthritis and/or osteoarthritis.³³ This does not appear to be the mechanism at work in our case because (1) we found no increase in CRE binding activity in the nuclear extracts from forskolin or db-cAMP–treated smooth muscle cells, and (2) the protein synthesis inhibitor cycloheximide did not block the increase in type II sPLA₂ mRNA induced by forskolin or db-cAMP. There is cooperation between C/EBP-ß and various CREB family members in the stimulation of the CHOP (Gadd153, C/EBP) gene in PC12 cells by arsenite.³⁴ Even though forskolin and cAMP do not activate CREB in smooth muscle cells, it is possible that the basal CREB protein content of the cells is enough to interact with C/EBP-ß or C/EBP- and transactivate type II sPLA₂ promoter.

The organization of the coactivator complex in the promoter region that interacts with the basic transcription machinery is an emerging concept in the regulation of gene expression in eukaryotic cells.³⁵ This complex recruits coactivator proteins, such as CREB-binding protein (CBP/P300), to increase the transcription rate.³⁶ C/EBP family members interact with CBP/P300.^{37 38} The coactivator complex might also be implicated in interactions with other transactivating factors. For example, NF-B and PPAR- cooperate to drive type II sPLA₂ gene expression induced by IL-1ß in rat smooth muscle cells.¹⁶ We now need to check whether NF-B, PPAR- and C/EBPs also cooperate with C/EBP-ß or - to mediate the IL1ß/cAMP synergistic effect that occurs in this cell type.¹⁰

Our experiments have shown C/EBP-ß and C/EBP- in the complexes formed by nuclear extracts from forskolin-stimulated or db-cAMP–stimulated smooth muscle cells. The increased production of the PDGF- receptor in genetically hypertensive rats has been linked to the overproduction of C/EBPs in aortic smooth muscle cells, but overproduction of C/EBP-ß has an inhibitory effect.³⁹ We have also identified the two C/EBP isoforms as being responsible for stimulating type II sPLA₂ gene activity in rabbit chondrocytes (manuscript in preparation). We now then plan to determine whether these two isoforms can replace each other for type II sPLA₂ gene stimulation in smooth muscle cells or whether C/EBP- is the only isoform involved in the cAMP effect.

	Acknowledgments

 
Financial support for this research was provided by Association de la Recherche contre le Cancer and Fondation de la recherche médicale. We are grateful to Sylvie Demaretz and Brigitte Janvier for technical assistance. Owen Parkes edited the English text.

Received November 30, 1999; accepted July 10, 2000.

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