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J. Biol. Chem., Vol. 275, Issue 30, 22686-22694, July 28, 2000

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Induction of Secreted Type IIA Phospholipase A₂ Gene Transcription by Interleukin-1

ROLE OF C/EBP FACTORS^*

Charbel Massaad, Michel Paradon, Claire Jacques, Colette Salvat, Gilbert Bereziat, Francis Berenbaum, and Jean-Luc Olivier

From UPRES-A CNRS 7079, UFR Saint Antoine, UPRES-A CNRS 7079, Université Pierre et Marie Curie, 7 quai Saint Bernard 75252 Paris Cedex 05, France

Received for publication, February 15, 2000, and in revised form, April 26, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Secreted type IIA phospholipase A₂, which is involved in arachidonic acid release, is abundantly produced by chondrocytes and secreted in the synovial fluids of patients affected by rheumatoid arthritis. Transfection experiments showed that interleukin-1 stimulates the phospholipase A₂ [1614; +20] promoter activity by 6-7-fold and that the [210; 176] fragment is critical for this stimulation. CAAT enhancer-binding protein (C/EBP)  and C/EBP transcription factors bind to this element as shown by bandshift experiments. Interleukin-1 increased the levels of C/EBP mRNA as soon as 2 h and up to 24 h whithout affecting those of C/EBP. Higher amounts of C/EBP proteins correlate with the stimulation of C/EBP mRNA. Mutations or 5' deletions in the upstream [247; 210] region reduced by 2-fold the basal and interleukin-1-stimulated transcription activities. Two types of factors bind to overlapping sequences on this fragment: NF1-like proteins and the glucocorticoid receptor. The glucocorticoid receptor is responsible for a moderate stimulation of the promoter activity by dexamethasone and may interact with C/EBP factors to achieve a full transcription activity in basal conditions and in the presence of interleukin-1. A [114; 85] proximal regulatory element forms three complexes in bandshift experiments, the slowest mobility one involving the Sp1 zinc finger factor. Mutation of this sequence reduced to 2-fold the stimulation of the promoter activity by interleukin-1 or the C/EBP factors. Induction of the transcription of secreted type IIA phospholipase A₂ gene by interleukin-1 in chondrocytes absolutely requires C/EBP and C/EBP factors but does not involve NF-B.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The synovial fluid of patients suffering from rheumatoid arthritis or osteoarthritis contains large quantities of prostaglandin E2. Such quantities inhibit collagen synthesis and therefore contribute to joint destruction. These inflammatory lipid mediators are produced by a cascade of enzymes among which phospholipases A₂ (PLA₂)¹ play a key role by releasing arachidonic acid from membrane phospholipids. Two calcium-dependent PLA₂ are involved in the release of arachidonic acid. Cytosolic PLA₂, a ubiquitous 85-kDa enzyme, is activated by MAP kinases and translocated from the cytosol to membrane (1, 2). Type IIA secreted PLA₂ (sPLA₂-IIA) was originally purified from the synovial fluid of patients with rheumatoid arthritis, which contains high quantities of this enzyme (3). sPLA₂-IIA belongs to a large group of 13-15-kDa secreted enzymes present in mammalian fluids and in the venoms of snakes and insects. According to a recent classification (4), the pancreatic version of the enzyme has been included in a type I group whereas the synovial PLA₂ is referred as type IIA sPLA₂. Three other recently cloned mammalian PLA₂ were classified in IIC, V, and X groups, but their involvement in the arachidonic acid release remains unknown.

Purified or recombinant sPLA₂-IIA triggers joint inflammation when it is injected intra-articularly in rabbits (5). The number of rheumatoid arthritis-affected joints and the presence of destructive erosion have been correlated with the amount of sPLA₂-type IIA in the serum of patients (6). Mice with both the TNF and sPLA₂-IIA transgenes exhibit more joint destruction than do those with TNF alone (7). Interleukin-1 (IL-1) is the most abundant cytokine in inflammatory synovial fluids. It stimulates the expression of numerous genes in articular cells (8) and increases the level of sPLA₂-IIA mRNA in chondrocytes. We have previously demonstrated that prostaglandin E2 production by rabbit articular chondrocytes is related to plasma membrane-associated sPLA₂-IIA activity and that the transcription rate of sPLA₂-IIA gene is stimulated by IL-1 (9-11).

Three main classes of transcriptional factors have been shown to mediate the effect of IL-1 on gene transcription: (i) NF-B is a dimer of p50 and p65 subunits and belongs to the Rel family. (ii) A member of the STAT family of transcription factors, which are activated through phosphorylation by Jak kinases and translocated to the cell nucleus within a few minutes, was characterized as an IL-1-stimulated factor but can also be activated by IL-6 and lipopolysaccharide (12). (iii) AP-1 can also be involved in the IL-1 pathway because IL-1 induces the transcription of the c-Jun and c-Fos genes in some cell models (13).

C/EBP transcription factors are involved in the regulation of gene transcription by IL-6. The C/EBP family includes three main members: C/EBP, C/EBP, and C/EPB. This last member is transcriptionally induced by IL-6 (14, 15), whereas C/EBP is mainly regulated at the post-transcriptional level by this cytokine in hepatoma cell lines (16, 17), although a transcriptional regulation of C/EBP by the inflammatory cytokines has also been described (for review see Ref. 18). In contrast to IL-6, the relationship between IL-1 and C/EBP factors has been poorly studied, and some positive regulations in interaction with NF-B have been reported (19, 20). However, induction of C/EBP binding to DNA by pro-inflammatory cytokines correlates with the accumulation of prostaglandin E2, and both effects are reversed by anti-inflammatory cytokines (21). C/EBP factors act with NF-B to induce the transcription of many acute phase response genes in response to pro-inflammatory cytokines, and this effect is based on direct protein-protein interactions (22, 23). Similar interactions have also been reported between C/EBP proteins and the glucocorticoid receptor (GR), which may explain the co-induction of the transcription of acute phase response genes by glucocorticoids and cytokines (24). The GR is a member of the steroid/nuclear receptor superfamily and binds to the glucocorticoid-responsive element (GRE) on gene promoters (25).

We have previously shown that the activity of the sPLA₂-IIA promoter is controlled by three regulatory elements in human hepatoma HepG2 cells (26) (see Fig. 1A). The [210; 176] element C is critical for the stimulation of the promoter by IL-6 and binds C/EBP family members, whereas an adjacent [247; 210] element D is recognized by several factors, some of which belonging to the NF1 family (27). The [114; 85] element B is responsible for a high basal activity when the region upstream of the C/EBP-binding site is deleted (28). We have demonstrated that C/EBP factors can mediate the stimulation of transcription by IL-6 in HepG2 cells by suppressing the basal inhibition of transcription, a process that may involve single strand binding activities (27).

In this study, we have identified the sequences and transcription factors involved in the stimulation of the sPLA₂-IIA promoter by IL-1 in chondrocytes. We have found that C/EBP plays a critical role in this stimulation and is transcriptionally induced by IL-1 in chondrocytes. We have also shown that the glucocorticoid receptor and the NF1/CTF family members bind to overlapping sites in the previously identified regulatory D element. Moreover Sp1 and other unknown factors bind to the regulatory elements B. These last factors and the glucocorticoid receptor potentiate the transactivation of the sPLA₂-IIA promoter by C/EBP and C/EBP to achieve full stimulation by IL-1.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Restriction enzymes, T4 kinase, ligase, and Taq polymerase were purchased from New England Biolabs. Oligonucleotides were provided by Oligoexpress (Paris, France). Materials for cell culture and protein molecular weight markers were provided from Sigma (Dulbecco's supplemented modified Eagle medium, Ham's F-12 medium, fetal calf serum, HEPES, trypsin), Life Technologies, Inc. (Gey medium) and Costar (town, state) (flasks and Petri dishes). The -galactosidase expression vector CMV--gal was obtained from CLONTECH, and poly(dI-dC) and deoxynucleotides were from Amersham Pharmacia Biotech. Radioactive products were supplied by ICN or Amersham Pharmacia Biotech. Collagenase, hyaluronidase, and trypsin for the preparation of monolayers of rabbit chondrocytes were supplied by Roche Molecular Biochemicals. IL-1 was provided by Immungenex (Los Angeles, CA).

Plasmid Constructions and Chondrocyte Cultures and Transfections-- The various CAT constructs containing wild type and mutant fragments of the sPLA₂-IIA promoter have been described elsewhere (26, 27). PHD expression vectors containing C/EBP and C/EBP were a gift from Dr. Ciliberto (Rome, Italy).

Three-week-old female Fauve de Bourgogne rabbits were killed and the shoulders, knees, and femoral heads were dissected out under sterile conditions as described by Jacques et al. (9). The articular cartilage was removed, cut into small pieces, and digested at 37 °C with 0.05% hyaluronidase in Gey medium for 15 min and then with 0.25% trypsin for 30 min and finally with 0.2% collagenase for 90 min. The chondrocytes were then washed with Ham's F-12 medium for 60 min. The suspension of chondrocytes was seeded into 60 mm dishes (1.5 × 10⁵ cells per dish) in Ham's F-12 medium supplemented with 10% fetal calf serum. The cells were maintained at 37 °C in 5% CO₂, and the culture medium was changed every 2-3 sday. The cells reached preconfluency within 6-7 days.

Chondrocytes were transfected using the calcium phosphate DNA co-precipitation method. Cells were changed by Dulbecco's modified Eagle's medium before transfection. Cells were incubated with the transfection mixture containing 12 µg of pUC-SH-CAT constructs and 2.5 µg of plasmids bearing the -galactosidase gene for 4 h and then shocked with HBS buffer (21 mM HEPES, pH 7.1, 16 mM dextrose, 0.8 mM NA₂HPO4, 5 mM KCl, and 137 mM NaCl) containing 15% glycerol for 90 s. The cells were incubated 20 h in Ham's F-12 supplemented with 0.2% bovine serum albumin and grown for an additional 24 h in the presence or absence of IL-1 (10 ng/ml). The harvested cells were lysed by incubation with 50 µl of 100 mM Tris, pH 7.8, 0.7% Nonidet P-40 for 15 min at 4 °C. CAT activities were measured by the two-liquid phases method as described by Fan et al. (27). -Galactosidase activities were measured to normalize variations in transfection efficiency. Transfection experiments were performed in duplicate and repeated four times with two different preparations of plasmids.

Preparation of Nuclear Extracts and Cell Lysates-- Chondrocytes nuclear extracts were prepared as described previously (27). Briefly, confluent cells from 3 P100 dishes were grown in Ham's F-12 without fetal calf serum and then incubated in the presence or absence of IL-1 (10 ng/ml) for 24 h in Ham's F-12 containing 10% fetal calf serum. They were then washed and scraped off into phosphate-buffered saline. The cells were centrifuged at 1500 × g for 5 min, and the pellet was suspended in 500 µl of buffer A (5 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.5% Nonidet P-40, 0.5 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 50 mM NaF). The cells were incubated at 4 °C for 15 min, centrifuged at 6000 × g for 10 min, and the pellet was suspended in 100 µl of buffer C (20 mM HEPES, pH 7.9, 25% glycerol, 0.5 M NaCl, 1.5 mM MgCl₂, 0.5 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 50 mM NaF). The nuclei were lysed by pipetting up and down four times and incubating for 30 min at 4 °C. The lysates were centrifuged at 100,000 × g for 30 min at 4 °C in a TLC centrifuge (Beckman). The supernatants were collected, and the protein concentrations were measured according Olivier et al. (26). The nuclear protein batches were stored at 80 °C. Lysates of COS-1 cells were prepared 40 h after transfection of the cells with the C/EBP expression vectors according to Olivier et al. (26).

Bandshift Assays-- We used the double-stranded CWT and DWT as wild type probes, corresponding to the [210; 176] sequence of the element C and to the [247; 210] sequence of the element D. These double-stranded oligonucleotides (100 ng) were labeled using T4 kinase and 50 µCi of [-³²P]ATP. Free nucleotides were separated from the labeled probe on a Sephadex G50 column. The specific activity of the probe was estimated by spotting 1 µl of the labeling mixture (before the G50 column) on to a TLC plate, separating the labeled probe and free nucleotide by chromatography, and counting them. The specific activities were 1-2 10⁸ cpm/µg. Chondrocyte nuclear extracts (6-9 µg) were incubated at 4 °C for 15 min in 20 ml of solution containing 25 mM HEPES, pH 7.6, 8% Ficoll, 40 mM KCl, 5 mM MgCl₂, 1 mM dithiothreitol, and 3 µg of double-stranded poly(dI-dC). When double-stranded competitor oligonucleotides were used, they were added in a volume of 1 µl, and the reaction mixture was incubated with the nuclear extracts at 4 °C for 15 min before adding the probe. As competitors in bandshift assays, we used the Sp1 oligonucleotide 5'-GCAGTGTTTCTCCGCCCCGATACGCGTAT-3' (29), the consensus glucocorticoid responsive element 5'-AGCTGCTCAGCTGGTACACTCCGTCCTCTACT (30), the HNF3 oligonucleotide 5'-GTTGACTAAGTCAATAATCAGA-3' corresponding to the HNF3-binding element of the transthyretin promoter (31), the NF1 oligonucleotide 5'-ACAATTTTTTGGCAAGAATATTAT-3' and the C/EBP oligonucleotide 5'-TGGTATGATTTTGTAATGGGGTAGGA-3', which correspond to the elements E and D, respectively, of the murine albumin promoter (32). The sequences of the other oligonucleotides used as competitors are indicated on the corresponding figures. Double-stranded oligonucleotide probes (60,000 cpm) were then added, and the incubation was continued for 30 min at 4 °C. Free DNA and DNA-protein complexes were resolved by electrophoresis in 5% or 7% polyacrylamide gels in 6.7 mM Tris-HCl, 3.3 mM sodium acetate, 1 mM EDTA, pH 7.9. The gels were dried and used to expose X-OMAT® films (Eastman Kodak Co.). In supershift experiments, chondrocyte nuclear extracts were preincubated for 15 min at 4 °C with 1 µl of antibodies raised against Sp1, C/EBP, C/EBP, or C/EBP (Santa Cruz Biotechnology Inc., Santa Cruz, CA).

Northern Blotting-- Total RNA was isolated from cultured chondrocytes using guanidinium isothiocyanate. The total RNA content was measured by spectrophotometry, and its integrity was assessed by agarose gel electrophoresis; 15 µg of total RNA/lane were separated on 1% agarose/2.2 M formaldehyde gels and transferred to nylon filters (Hybond N; Amersham Pharmacia Biotech). The membranes were prehybridized for 15 min, and then hybridized at 65 °C for 2 h with the various probes in the rapid hyb-buffer medium (Amersham Pharmacia Biotech). The specific C/EBP and C/EBP probes were obtained by digestion of the corresponding expression vectors with PstI and XhoI (New England Biolabs, Boston, MA) respectively. The digestion products were separated on 1% agarose gels, the 500- and 850-bp-length bands corresponding to fragments of the C/EBP and C/EBP cDNA, respectively, were sliced and extracted using the Gene-Clean kit (Bio101, La Jolla, CA). The C/EBP probes were labeled using the random-primed labeling system (Amersham Pharmacia Biotech) and [-³²P]dCTP (3000 Ci/mmol). An oligonucleotide hybridizing to the 28 S RNA was labeled with [-³²P]ATP by T4 kinase and used as probe to take into account the variations in loaded and transferred RNA. The hybridized filters were washed twice in 2× SSC (150 mM NaCl, 17 mM trisodium citrate) 0.1% SDS at room temperature for 15 min and then twice in 0.1× SSC, 0.1% SDS for 15 min at room temperature and at 50 °C. Autoradiography was performed for 10 days for C/EBP probes and 24 h for the 28 S RNA probe. The blots were successively hybridized with the C/EBP, C/EBP, and 28 S RNA probes. The filters were washed in 0.1× SSC, 0.1% SDS at 85 °C for 10 min before rehybridization.

Western Blotting-- Aliquots (50 µg) of nuclear proteins were separated on a 12% SDS-polyacrylamide gel electrophoresis in 0.38 M Tris-HCl, 0.1% SDS, pH 8.8, and electroblotted on to Protran BA83 nitrocellulose membranes (Schleicher & Schull). The membranes were saturated in 10 mM Tris, pH 7.5, 100 mM NaCl, 0.1% Tween 20, 5% nonfat milk at 4 °C overnight, hybridized with anti-C/EBP or anti-C/EBP antibodies (Santa Cruz) for 1 h at 4 °C, washed in the saturation buffer, and then developed using the ECL system (Amersham Pharmacia Biotech). The abundance of C/EBP protein in nuclear extracts from untreated and IL-1-treated chondrocytes was calculated by quantitative scanning of autoradiograms using a CCD video camera and the Densylab system (Quantum Bioprobe, Montreuil, France).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Regulatory Element C of the sPLA₂-IIA Promoter Is Critical for the Stimulation of Its Activity by IL-1 and Binds C/EBP and C/EBP-- Rabbit primary culture chondrocytes were transfected with CAT constructs containing various 5' deleted fragments of the sPLA₂-IIA promoter. A 24-h treatment of the cells by IL-1 increased by 6.6 ± 2 the activity of the [1614; +20] fragment of the sPLA₂-IIA promoter (Fig. 1B). Deletion of the [247; 225] fragment decreased by 2-fold the basal and IL-1-stimulated transcription activities. The relative stimulation of the transcription activities by IL-1 was not significantly modified by the 5' deletions from the 225 to the 203 positions because the basal and stimulated activities were similarly decreased. Deletion downstream from the position 195 completely suppressed the stimulation of the transcription activity by IL-1. Deletion of the 159/138 fragment produced an additional 2-fold reduction of the transcription activity, and the promoter activity was suppressed by a further deletion to the 87 position (Fig. 1B).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Regulatory elements of the sPLA2-IIA promoter (A) and induction of its activity and that of 5' deleted mutants by IL-1 in rabbit primary culture chondrocytes (B). The regulatory elements were previously identified by footprint, bandshift, and transfection assays (26-28). The P60 dishes of 60% confluent chondrocytes were transfected by the calcium phosphate co-precipitation method using 12 µg of the various CAT constructs and 2 µg of the CMV--gal expression vector. 24 h after transfection, the cells were incubated with or without IL-1 (10 ng/ml) for a further 24 h and then harvested for the measurement of CAT and -galactosidase activity. The activities of untreated (white bars) or IL-1-stimulated (solid bars) chondrocytes were calculated relative to that induced by the [326; +20] fragment in untreated cells. Results are expressed as the means ± S.E. of four independent transfections performed in duplicate.

Electrophoretic mobility shift assays showed that chondrocyte nuclear extracts formed two complexes, C1 and C2, with the CWT probe, which corresponds to the [210; 176] (Fig. 2B, lanes 1 and 7). The slowest electrophoretic mobility complex C1 could be clearly observed only when the chondrocytes were treated with IL-1 prior the extraction of the nuclear proteins (Fig. 2B, compare lanes 1 and 7). The C2 complex was formed when the extracts of both the untreated and IL-1-stimulated chondrocytes were used, but its intensity was higher when the cells were treated by IL-1 (Fig. 2B, compare lanes 1 and 7). Because this region was previously shown to correspond to a C/EBP-binding site in hepatocytes (26), we used as competitors the C/EBP-binding oligonucleotide corresponding to the D element of the murine albumin promoter (32) and the Cmut oligonucleotide in which the 199/197 5'-TTT-3' triplet was mutated into an 5'-GCC-3' sequence (Fig. 2A). All the complexes were suppressed when the chondrocyte nuclear extracts were preincubated with the C/EBP-binding oligonucleotide prior to the addition of the CWT probe (Fig. 2B, lanes 2 and 8). By contrast, none of the complexes were competed out by the Cmut oligonucleotide (Fig. 2B, lanes 3 and 9). An antibody raised against C/EBP did not alter any of the complexes formed between the CWT probe and the chondrocyte nuclear extracts (Fig. 2B, lanes 4 and 10). Because all the C/EBP family members share the same binding site, it is likely that rabbit articular chondrocytes lack C/EBP expression. Antibodies to C/EBP and C/EBP supershifted the C1 complex (Fig. 2B, lanes 5, 6, 11, and 12). The upper part of the C2 complex was displaced by the antibody to C/EBP complex (Fig. 2B, lanes 5 and 11), whereas its lower part was shifted by the antibody to C/EBP (Fig. 2B, lanes 6 and 12). Because C/EBP and C/EBP bind to DNA by forming heterodimers as well as homodimers, these data indicate that the C1 complex is formed by C/EBP-C/EBP heterodimers, whereas the C2 complex is heterogeneous and corresponds to co-migrating C/EBP-C/EBP and C/EBP-C/EBP homodimers.

View larger version (60K): [in this window] [in a new window]   Fig. 2.   Binding of the [210; 176] regulatory element C to C/EBP and C/EBP. A, sequences of the oligonucleotide CWT and its mutant Cmut. Dashes indicate the nucleotides unchanged in the Cmut mutant. The mutations of the 199/197 positions are indicated. B, the 5' end 32P-labeled probe CWT was incubated with 6 µg of nuclear extracts of untreated (lanes 1-6) or IL-1-stimulated (lanes 7-12) chondrocytes. Treatment with 10 ng/ml IL-1 was performed for 24 h prior to the extraction of nuclear proteins. Incubation of the CWT probe was carried out for 30 min at 4 °C in a mixture containing 6 µg of rabbit chondrocyte nuclear extracts and 3 µg of poly(dI-dC) in 16 mM HEPES, pH 7.6, 5% Ficoll, 40 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, and 0.1 mM EDTA. 250-fold excesses of C/EBP and Cmut oligonucleotides over the CWT concentration were incubated with the nuclear extracts for 15 min before the addition of the probe (lanes 2, 3, 8, and 9). Antibodies specifically raised against C/EBP (lanes 4 and 10) C/EBP (lanes 5 and 11) or C/EBP (lanes 6 and 12) were incubated with chondrocyte nuclear extracts for 15 min at 4 °C prior to the addition of poly(dI-dC). NS indicates a nonspecific band, and the asterisk represents a variable complex. C1 and C2 complexes are indicated by the arrows. Electrophoresis was run on a 5% 30:1 bisacrylamide/acrylamide gel.

The mutations of the whole [204; 181] sequence or the 199/197 triplet abolished the stimulation of the promoter activity by IL-1 (Fig. 3). The 6-fold stimulation of the wild type [326; +20] sPLA₂-IIA promoter activity by IL-1 was mimicked by co-transfecting the C/EBP and C/EBP expression vectors with the [326; +20]-pUC-SH-CAT construct in the absence of cytokine treatment (Fig. 3). By contrast co-transfection had no effect with the mutated CAT constructs for the element C. Treatment of the cells with IL-1, after co-transfection of the C/EBP expression vectors with the wild type [326; +20]-pUC-SH-CAT construct, did not increase the stimulation of the [326; +20] promoter activity by the overexpressed C/EBP and C/EBP factors (Fig. 3), suggesting a pivotal role for the C/EBP factors in the induction of the sPLA₂-IIA promoter activity by IL-1.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   The C/EBP-binding site on element C is critical for the stimulation of sPLA2-IIA promoter activity by IL-1. Rabbit primary culture chondrocytes were transfected (as indicated under "Experimental Procedures" and in Fig. 1) with the mutant [204; 181)- and [199; 197]-sPLA2-pUC-SH-CAT and wild type (WT) [326,+20]-sPLA2-pUC-SH-CAT constructs. Mutations performed in the CAT constructs and the wild type element C are shown at the top of the figure. The CAT constructs were co-transfected with 1 µg of PHD-based expression vectors of C/EBP and C/EBP or the wild type PHD plasmid in control experiments. Cells were cultivated in the absence or presence of IL-1 (10 ng/ml) as indicated in Fig. 1. CAT activities were calculated relatively to those induced by the wild type [326,+20]-sPLA2-pUC-SH-CAT construct in the absence of treatment with IL-1 and co-transfected C/EBP expression vector. Results are expressed as the means ± S.E. of four independent experiments performed in duplicate.

IL-1 Stimulates the Expression of the C/EBP Gene-- Messenger RNA levels of C/EBP and C/EBP were measured in chondrocytes at various times after treatment by IL-1. The C/EBP mRNAs were barely detectable by Northern blot in absence of IL-1. The C/EBP mRNA levels were increased by IL-1 after a 2-h treatment, reached maximal values between 14-16 h of treatment, and were maintained over a 24-h period. By contrast IL-1 did not affect the levels of C/EBP mRNAs (Fig. 4). Western blot experiments confirmed that IL-1 increased by 3-4-fold the concentration of C/EBP proteins (Fig. 5).

View larger version (39K): [in this window] [in a new window]   Fig. 4.   IL-1 induces expression of the C/EBP gene but does not affect that of C/EBP. Confluent rabbit primary culture chondrocytes in P100 dishes were incubated with 10 ng/ml IL-1 for 2 to 24 h. In control experiments (time 0), the cells were cultivated for an additional 24 h without the cytokine. After extraction, 10 µg of total RNA were used for Northern blot experiments, as indicated under "Experimental Procedures." Hybridization to the 28 S ribosomal fraction was used to normalize any variations in mRNA quantity and integrity.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   IL-1 increases the quantities of C/EBP protein in primary culture chondrocyte. A, confluent rabbit primary culture chondrocytes in P100 dishes were incubated with 10 ng/ml IL-1 or the carrier (phosphate saline buffer) for 24 h. 50 µg of nuclear proteins were separated by electrophoresis and transferred on to a nitrocellulose membrane as indicated under "Experimental Procedures." The membrane was incubated with antibodies raised against C/EBP. A lysate (5 µg proteins) of COS-I cells expressing C/EBP was used as a control of migration and hybridization. NS indicates a nonspecific band cross-hybridizing with the antibodies against C/EBP. The intensity of this band was measured and taken as reference. MW, molecular mass. B, densitometric analysis of the intensities of the nonspecific band (NS) and the band corresponding to C/EBP in the absence or in presence of IL-1.

The [247; 210] Regulatory Element D Binds CTF/NF1 Family Members and the Glucocorticoid Receptor, Which Potentiates the Transactivation of the sPLA₂-IIA Promoter by the C/EBP and C/EBP Factors-- The [247; 210] element D displayed a consensus CTF/NF1 hemi-site 5'-TGGCA-3' located between the positions 224 and 220 and overlapping an upstream [229; 224] 5'-TGTTTT-3' sequence, which is homologous to the consensus 5' half-site for the glucocorticoid receptor 5'-TGTTCT-3' (33) (Fig. 6A). The DWT probe (Fig. 6A) formed, with nuclear extracts from untreated chondrocytes, two specific complexes, D1 and D2 (Fig. 6B, lanes 1 and 2). The formation of complex D1 was suppressed by the addition of a 250-fold excess of a consensus GRE (Fig. 6B, lane 4), which suggests that this complex correspond to the binding of the glucocorticoid receptor. Complex D2 was competed out by the same excess of a NF1-binding oligonucleotide, which indicates the involvement of CTF/NF1 family members in the formation of this complex (Fig. 6B, lane 3).

View larger version (56K): [in this window] [in a new window]   Fig. 6.   The [247;  210] element D binds CTF/NF1 family members and glucocorticoid receptors on overlapping sequences. A, sequences of the oligonucleotides DWT, Dm1, Dm2, Dm3 and Dm4. The GRE and NF1 hemi-sites are indicated at the top of the figure. Dashes indicate the nucleotides unchanged in the Dm1, Dm2, Dm3, and Dm4 mutants. The mutations in these oligonucleotides are listed. B, the 5' end 32P-labeled probes (60,000 cpm) DWT was incubated for 30 min at 4 °C in a mixture containing 9 µg of nuclear extracts from untreated chondrocytes and 3 µg of poly(dI-dC). The unlabeled oligonucleotides Dwt (lane 2), NF1 (lane 3), GRE (lane 4), DM1 (lane 5), Dm2 (lane 6), DM3 (lane 7), and Dm4 (lane 8) were used as competitors at 250-fold excesses over the probe concentration. The D1 and D2 complexes are indicated by arrows. They were separated from the free probe on a 5% 30/1 acrylamide/bisacrylamide gel. C, the GRE (lanes 1 and 2), Dm1 (lane 3), Dm3 (lane 4), and Dm4 (lane 5) probes were incubated with 9 µg of nuclear extracts from untreated (lanes 1 and 3-5) or IL-1-treated (lane 2) chondrocytes and the migration was performed as in B. The complexes formed with glucocorticoid receptor are indicated.

To further delineate the binding sites of the NF1 proteins and the glucocorticoid receptor, four oligonucleotides corresponding to a mutation of the element D (Fig. 6A) were used in bandshift experiments. The Dm1 oligonucleotide, in which the [224; 220] CTF/NF1 hemi-site was mutated (Fig. 6A), competed out the D1 complex but did not affect the formation of the D2 one (Fig. 6B, lane 5), confirming that this latter involves CTF/NF1 family members. The Dm2 oligonucleotide which contains two mutations, respectively, on the 228 G nucleotide and on the 238/236 sequence (Fig. 6A), competed out the D1 complex but not the D2 one (Fig. 6B, lane 6). The involvement of the [229; 224] 5'-TGTTTT-3' sequence in the formation of the D1 complex was confirmed by the lack of competition of this complex by the Dm3 oligonucleotide (Fig. 6B, lane 7) in which the 229/228 TG nucleotides were mutated into CA (Fig. 6A). In addition the 250-fold excess of Dm3 oligonucleotide did not fully displaced the D2 complex, indicating that the sequence upstream from the [224; 220] CTF/NF1 hemi-site plays a role in the binding of the CTF/NF1 family members to the element D (Fig. 6B, lane 7). Finally the mutation of the whole [231; 224] 5'-TGTGTTTT-3' sequence abolished the ability of the Dm4 oligonucleotide to compete with the DWT probe for the formation of both the D1 and D2 complexes (Fig. 6B, lane 8). The Dm1 oligonucleotide was also used as probe and formed the D1 complex but not the D2 one (Fig. 6C, lane 3). The GRE probe formed two complexes with the nuclear extracts from untreated and IL-1-stimulated chondrocytes. The upper complex, which was the most intense, co-migrated with the D1 complex formed with the DM1 probe (Fig. 6C, compare lanes 1 and 2 with lane 3). This result supports the involvement of the glucocorticoid receptor in the formation of the D1 complex and also indicates that this receptor is present in nuclei in absence of dexamethasone treatment. The Dm3 oligonucleotide used as probe did not form either the D1 or the D2 complex, confirming the involvement of the sequence upstream the [224; 220] CTF/NF1 hemi-site in the binding of the CTF/NF1 family members to the D element (Fig. 6C, lane 4). Treatment of chondrocytes with IL-1 for 24 h did not affect the formation or the intensity of the complex formed with the GRE probe (Fig. 6C, lane 2).

A 24-h treatment of transfected chondrocytes by dexamethasone (10⁷ M) induced a moderate stimulation of the [326; +20] sPLA₂-IIA promoter activity by 160 ± 12%, and this stimulation was suppressed by co-incubation with the synthetic glucocorticoid analog and inhibitor RU486 (10⁵ M) (Fig. 7A). Co-incubation with dexamethasone (10⁷ M) had no effect on the stimulation of the [326; +20] sPLA₂-IIA promoter activity by IL-1 (Fig. 7A). Similarly, RU486 did not modify the induction of transcription activity by IL-1 (Fig. 7A). Dexamethasone elicited a 5-fold induction of a promoter containing a GRE sequence upstream of the thymidine kinase promoter, and this induction was inhibited by the anti-glucocorticoid RU486. The dexamethasone-stimulated transcription was equivalent in the case of the GRE-TK and [326/+20] CAT construct, whereas the basal activities of these two plasmids were different.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   The [326; +20] sPLA2-IIA promoter is inducible by dexamethasone in chondrocytes, and this stimulation as well as that by IL-1 depend on the 5'-TGTTTT-3' GRE. A, rabbit primary culture chondrocytes were transfected with either the wild type [326,+20]-pUC-SH-CAT or GRE-TK (30) constructs, and the cells were treated by dexamethasone (Dex, 107 M) or IL-1 (10 ng/ml) for 24 h in the absence or presence of RU486 (105 M). CAT activities are expressed relative to that measured in untreated chondrocytes. Results are expressed as the means ± S.E. of three independent experiments performed in duplicate. B, rabbit primary culture chondrocytes were transfected with the wild type [326,+20]-pUC-SH-CAT construct or CAT constructs containing various 5' deleted fragments of the promoter or the substitution mutants of the [240; 217] and [231; 224] sequences. These mutations were similar to those contained in the Dm1 and Dm4 oligonucleotides (Fig. 6A). After transfection the cells were incubated in the absence or presence of dexamethasone (107 M) or IL-1 (10 ng/ml) for 24 h. CAT activities are expressed relative to that of the wild type [326; +20]-pUC-SH-CAT construct in the absence of treatment. Results are expressed as the means ± S.E. of three independent experiments performed in duplicate.

Stimulation of the sPLA₂-IIA transcription by dexamethasone in transient transfection experiments of chondrocytes was suppressed by the deletion of the region upstream the 225 position (Fig. 7B), which altered the GRE (Fig. 6A). This last 5' deletion also decreased the basal transcription activity as previously shown in Fig. 1B. The substitution of the [231; 224] 5'-TGTGTTTT-3' fragment overlapping the NF1 and GR hemi-sites by the nonspecific sequence 5'-GGTACCCG-3' drastically reduced the basal transcription activity of the resulting [231; 224]-pUC-SH-CAT construct to 18 ± 9% of that of the wild type promoter and suppressed its stimulation by IL-1 and dexamethasone (Fig. 7B). The mutation of the 5'-TGGCA-3' CTF/NF1 hemi-site in the [223; 218]-pUC-SH-CAT construct did not affect either the basal transcription activity or its stimulation by IL-1 but abolished that by dexamethasone (Fig. 7B). Taken together, these results indicate that the stimulation of the sPLA₂-IIA transcription by IL-1 and dexamethasone are not mediated in the same way, although both involve the GR hemi-site on the element D. The GRE located on the element D is required to achieve full basal and IL-1-stimulated transcription activities.

The Zinc Finger Protein Sp1 Is Involved in the Chondrocyte Nuclear Proteins Bound to the [114; 87] Regulatory Element B of the sPLA₂-IIA Promoter-- We have demonstrated that the [107; 99] 5' GACCACGCC-3' sequence is critical for the sPLA₂-IIA promoter activity in HepG2 cells (28). The BWT probe, which corresponds to the [114; 87] sequence of the sPLA₂-IIA promoter (Fig. 8A), formed three complexes with the chondrocyte nuclear proteins (Fig. 8B, lane 1). A 500-fold excess of the unlabeled BMut oligonucleotide, in which the [107; 99] sequence was mutated (Fig. 8A), did not displace any of the complexes B1, B1', or B2 (Fig. 8B, lane 3). The complex B1 was supershifted by a specific antibody to Sp1 (Fig. 8B, lane 5), indicating the involvement of Sp1 in the formation of this complex. The pretreatment of the chondrocytes by IL-1 did not change the mobility or the intensity of any of the three complexes formed with the BWT probe (Fig. 8B, lane 6).

View larger version (71K): [in this window] [in a new window]   Fig. 8.   Sp1 is involved in the chondrocyte transcription factors bound to the [114; 85] element B. A, sequences of the oligonucleotide BWT and its mutant Bmut. Dashes indicate the nucleotides unchanged in the Cmut mutant. On the contrary, the nucleotides replacing the wild type sequence between the 104 and 97 positions are indicated. B, the BWT probe was incubated with 6 µg of rabbit primary culture chondrocyte nuclear extracts. In lane 5, the cells were incubated with 10 ng/ml IL-1 for 24 h prior to the extraction of the nuclear proteins. Unlabeled oligonucleotides, BWT (lane 2), Bmut (lane 3), or Sp1 (lane 4), were added to the nuclear proteins at ×500 probe concentration and incubated for 15 min before addition of the BWT probe. In lane 5, a specific antibody to Sp1 was incubated for 15 min at 4 °C with rabbit chondrocyte nuclear extract before the addition of poly(dI-dC). In lane 6 nuclear extracts of IL-1-treated chondrocytes were used. The electrophoresis was performed in a 5% polyacrylamide gel. The arrows indicate the free probe and the three specific complexes (B1, B1', and B2) formed between the BWT oligonucleotide and rabbit chondrocyte nuclear extracts.

Transfection experiments showed that the mutation of the native [107; 99] sequence in the [107; 99]-pUC-SH-CAT plasmid reduced the stimulation of the [326; +20] promoter activity by IL-1 2-fold (Fig. 9). This mutation had a moderate effect on the basal activity of the sPLA₂-IIA promoter activity, i.e. a decrease by 30% was observed (Fig. 9). When the expression vectors of C/EBP was co-transfected with the constructs containing the wild type and the mutant promoters, respectively, the stimulation of the transcription activity dropped from 7-8-fold to 2.5-3.5-fold. By contrast, overexpressed C/EBP stimulated the activity of the wild type promoter 7-8-fold and that of the mutant promoter 5-fold (Fig. 9). These results show that the factors bound to the regulatory element B are involved in the stimulation of the sPLA₂-IIA promoter activity by IL-1 and reinforce the transactivation by the C/EBP factors.

View larger version (37K): [in this window] [in a new window]   Fig. 9.   Transcription factors bound to element B contribute to the stimulation of sPLA2-IIA promoter activity by IL-1 and C/EBP factors. Rabbit primary culture chondrocytes were transfected with wild type (WT) [326,+20]- and mutant [104,-97]-pUC-SH-CAT constructs. CAT constructs were co-transfected with 1 µg of PHD plasmid or expression vectors of C/EBP and C/EBP. After transfection the cells were incubated in the absence or in presence of 10 ng/ml IL-1, as indicated under "Experimental Procedures." CAT activities are expressed relative to that of the wild type [326; +20]-pUC-SH-CAT construct in the absence of treatment by IL-1. Results are expressed as the means ± S.E. of three independent experiments performed in duplicate.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We present evidence that C/EBP factors are central to the control of sPLA₂-IIA gene transcription by IL-1. The major complex C2, formed by C/EBP and C/EBP homodimers, was observed with extracts from both untreated and IL-1-treated chondrocytes, whereas a second complex C1 was formed with extracts from IL-1-treated cells. The amounts of C/EBP mRNA and protein in rabbit chondrocytes are increased by IL-1, which does not, in contrast, modify the amounts of C/EBP mRNA. The newly synthesized C/EBP protein can form higher amounts of homodimers and heterodimers with C/EBP, and this could explain the fact that the intensities of both the C1 and C2 complexes were reinforced when the nuclear proteins were extracted from IL-1-treated chondrocytes. The stimulation of sPLA₂-IIA transcription by IL-1 is therefore mediated through the increase of the C/EBP gene expression itself. The increase of C/EBP mRNA levels after 2 h is consistent with the delayed increase of the sPLA2 mRNA levels, which we previously measured in rabbit primary culture chondrocytes (9).

Although the element C is the key element for the regulation of sPLA₂-IIA promoter activity by IL-1 in chondrocytes, intact regulatory elements B and D are required to achieve full basal and stimulated transcription activity. We found that the mutation of the [107; 99] sequence abolished the binding of nuclear proteins to the element B and strongly reduced the stimulation of the activity of sPLA₂-IIA promoter by IL-1. However, overexpression of C/EBP compensates for the effect of the mutation of the [107; 99] sequence. This suggests that C/EBP and C/EBP may interact differently with the general transcription machinery in terms of their affinity for the various co-activators such as CBP/P300 (34) or Nopp140 (35). We have shown that the 5'-TGTTTT-3' sequence of element D is critical for the formation of a slow electrophoretic mobility complex D1 and for full sPLA₂-IIA basal and dexamethasone- and IL-1-stimulated transcription activity. The D1 complex is competed out by a consensus GRE and co-migrates with the main complex formed between the GRE probe and the chondrocyte nuclear extracts. The glucocorticoid receptor typically binds to two hexameric inverted repeats separated by 3 bp. The [229; 224] 5'-TGTTTT-3' sequence of element D is highly homologous to the consensus 3' hemi-site 5'-TGTTCT-3' for GREs. The G and T nucleotides on positions 2 and 3, which interact with Arg-447 and Val-443 residues of GR, are conserved, and we have observed in a previous study that the 228 G nucleotide is a major site of interference with a liver protein forming an intense complex with a mobility similar to that of the D1 complex (26). However, the C nucleotide on position 5, which interacts with the Lys-442 residue of GR in consensus GRE (36) was replaced by a T. The element D lacks a typical 5' half-site, but the [241; 236] 5'-CTGCCT-3' sequence, which is 6 bp upstream of the 5'-TGTTTT-3' site, is weakly homologous to the consensus sequence (36). Furthermore, variations of orientation and spacing between the two hemi-sites up to 8-9 bp have been described (37, 38), and many GRE lack 5' hemi-sites, such as those of mouse IL-2 receptor , human elastin, and rat chromogranin promoters (39-41).

We have shown that both the GRE and DWT probes form complexes with nuclear extracts from untreated chondrocytes. This indicates that the glucocorticoid receptor was present in chondrocyte nuclei in the absence of treatment by dexamethasone or IL-1. Several studies have reported the localization of unliganded glucocorticoid receptors inside the nuclei of several cell types (42-45). More recently, two types of glucocorticoid receptor have been described in humans, hGR and hGR, which differ at their C-terminal end beyond amino acid 727; hGR, which has 50 additional residues; and hGR, which contains 15 nonhomologous residues that are generated through alternative splicing of the last exon (46). hGR is sequestered in the cytosol by heat shock protein hsp90 in basal conditions, binds glucocorticoids or RU486, and is translocated into the nuclei; hGR in the nuclei does not bind dexamethasone or RU486 but can bind to GRE (46, 47). This factor has been described as a putative repressor of hGR (47-49), but this hypothesis is a subject of debate (50). Furthermore RU486 does not impede the binding of GR to DNA (51, 52) but inhibits the transactivation ability of GR in the presence of dexamethasone. The glucocorticoid receptor(s) expressed in chondrocytes have been poorly studied, and their regulation remains to be examined in further detail in this cell model. Dexamethasone decreases the level and activity of sPLA₂-IIA mRNA in vascular smooth muscle cells (53), but conflicting data have been reported in mesangial cells (54, 55). Furthermore these data have been obtained in rats, and the rat promoter does not contain any sequence homologous to a GRE in the region corresponding to the human element D. The only study in humans was performed in the hepatoma HepG2 cell line by Haselmann and Goppelt-Struebe (56), who described a partial decrease of oncostatin M-stimulated expression in the presence of dexamethasone. Modulation of sPLA₂-IIA gene by glucocorticoids are therefore cell- and species-specific and may act at different levels in opposite ways. Regarding the regulation of the sPLA₂-IIA promoter, we assume that unliganded GRs bind to the 5'-TGTTTT-3' sequence and increase the basal transcription activity without affecting the relative stimulation by IL-1, as shown by deletions through the element D. The suppression of the IL-1-induced stimulation of the transcription activity, which was observed by using the [326; +20] promoter mutated on the [231; 224] 5'-TGTTTT-3' sequence, might be due to the activity of inhibitory factors such as those described in HepG2 cells (27). The binding of GR to the element D also explains the stimulation of the promoter activity by dexamethasone, which is abolished by mutation of the 5'-TGTTTT-3' sequence; the low level of homology with the consensus GRE and the absence of repeated GRE may explain the low level of stimulation.

Mutation of the [224; 220] 5'-TGGCA-3' CTF/NF1 hemi-site suppressed the stimulation of sPLA₂-IIA transcription activity by dexamethasone without affecting its basal and IL-1-stimulated levels. The CTF/NF1 family members are encoded by four genes, NFI-A, NFI-B, NFI-C, and NFI-X, and their diversity is increased by alternative splicing or cleavage of larger polypeptides (57-60). The products of the four genes have different transactivation abilities and generate heterodimers with intermediate activation potentials (61). Stimulations of the human papillomavirus type 16 and aspartate aminotransferase promoters by glucocorticoid require the presence of intact NF1-binding sites (62, 63). The glucocorticoid receptor favors binding of CTF/NF1 factors to the murine mammary tumor virus promoter in vivo by stimulating the nucleosome disrupting activity of the SWI/SNF complex (64). In the context of the sPLA₂-IIA promoter, its main action would be to allow the C/EBP factors to achieve full transcription activities. Such effects are conditioned by interactions between the GR bound to the element D and the C/EBP factors bound to the element C. It is interesting to note that the GR recruits C/EBP to the rat 1 acid glycoprotein promoter (65) and that the downstream GRE is separated from the upstream C/EBP-binding sites by 23 bp in this promoter (24), the same distance as that between the GR and C/EBP-binding sites in the sPLA₂-IIA promoter. Boruk et al. (66) demonstrated that the GR interacts with C/EBP through their AF2 domain and an intermediary factor. Chang et al. (67) have suggested that this intermediary factor is, in the context of the 1 acid glycoprotein promoter, the co-activator TIF1, which belongs to the RING protein family. The identification of the factor(s) ensuring the interaction between the GR and the C/EBP proteins bound to sPLA₂-IIA promoter in chondrocytes requires further studies. The hypothesis of the putative regulatory mechanisms of the various factors that bind to human sPLA₂-IIA promoter are summarized in Fig. 10.

View larger version (23K): [in this window] [in a new window]   Fig. 10.   Hypothetical model of the interactions between transcription factors bound to the regulatory elements of the human sPLA2-IIA promoter, and general transcription factors bound to the TATA box.

Increased synthesis of prostaglandin E2 by chondrocytes and synoviocytes in response to IL-1 is a key event of the inflammatory process in joints. Prostaglandin E2 is produced through the functional interaction between cyclooxygenase-2 and sPLA₂ in cell types that express both of these genes (68, 69). We have shown in this study that C/EBP and C/EBP play a critical role in the regulation of the secreted type IIA sPLA₂ promoter activity by IL-1. We and others have demonstrated that these factors are also essential for the regulation of cyclooxygenase-2 transcription. C/EBP and C/EBP may therefore be considered as putative targets of therapeutic strategies to inhibit icosanoid synthesis in chondrocytes.

	FOOTNOTES

^* This work was supported by the French Association pour la Recherche sur la Polyarthrite Rhumatoïde and the Société Française de Rhumatologie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: UPRES-A CNRS 7079, Université Pierre et Marie Curie, 7 quai Saint Bernard 75252 Paris Cedex 05, France. Tel.: 33-1-44273256; Fax: 33-1-44275140; E-mail: olivier@ccr.jussieu.fr.

Published, JBC Papers in Press, May 1, 2000, DOI 10.1074/jbc.M001250200

	ABBREVIATIONS

The abbreviations used are: PLA₂, phospholipase A₂; sPLA₂-IIA, type IIA secreted PLA₂; IL, interleukin; CMV, cytomegalovirus; C/EBP, CAAT enhancer-binding protein; GR, glucocorticoid receptor; GRE, glucocorticoid-responsive element; CAT, chloramphenicol acetyltransferase; bp, base pair(s).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES