CCAAT/Enhancer-binding Protein β and NF-κB Mediate High Level Expression of Chemokine Genes CCL3 and CCL4 by Human Chondrocytes in Response to IL-1β*

A large set of chemokines is highly up-regulated in human chondrocytes in response to IL-1β (Sandell, L. J., Xing, X., Franz, C., Davies, S., Chang, L. W., and Patra, D. (2008) Osteoarthr. Cartil. 16, 1560–1571). To investigate the mechanism of transcriptional regulation, deletion constructs of selected chemokine gene promoters, the human CCL3 (MIP-1α) and CCL4 (MIP-1β), were transfected into human chondrocytes with or without IL-1β. The results show that an IL-1β-responsive element is located between bp −300 and −140 of the CCL3 promoter and between bp −222 and −100 of the CCL4 promoter. Because both of these elements contain CCAAT/enhancer-binding protein β (C/EBPβ) motifs, the function of C/EBPβ was examined. IL-1β stimulated the expression of C/EBPβ, and the direct binding of C/EBPβ to the C/EBPβ motif was confirmed by EMSA and ChIP analyses. The −300 bp CCL3 promoter and −222 bp CCL4 promoter were strongly up-regulated by co-transfection with the C/EBPβ expression vector. Mutation of the C/EBPβ motif and reduction of C/EBPβ expression by siRNA decreased the up-regulation. Additionally, another cytokine-related transcription factor, NF-κB, was also shown to be involved in the up-regulation of chemokines in response to IL-1β, and the binding site was identified. The regulation of C/EBPβ and NF-κB was confirmed by the inhibition by C/EBPβ and NF-κB and by transfection with C/EBPβ and NF-κB expression vectors in the presence or absence of IL-1β. Taken together, our results suggest that C/EBPβ and NF-κB are both involved in the IL-1β-responsive up-regulation of chemokine genes in human chondrocytes. Time course experiments indicated that C/EBPβ gradually and steadily induces chemokine up-regulation, whereas NF-κB activity was highest at the early stage of chemokine up-regulation.

mokines are known to be important in inflammatory diseases, including rheumatoid arthritis, osteoarthritis (OA), 2 inflammatory bowel disease, multiple sclerosis and transplant rejection (1,2). Chemokines are a specific class of cytokines that classically mediate chemoattraction (chemotaxis) between cells. However, the production and function of chemokines in organs and tissues have recently been recognized, and knock-out of the chemokine receptors CXCR4 and CXCR2 suggests a role in development and cell senescence (3,4), and the chemokine CCL3 is a potent osteoclastogenic factor (5). There exist over 50 chemokine ligands and 20 G protein-coupled receptors (1). Chemokines have similar protein structures, being 8 -10 kDa, with two major subclasses having conserved cysteine residues either adjacent (CC) or separated by one amino acid (CXC) (2).
IL-1␤ is an important cytokine in rheumatoid and osteoarthritic joint diseases. Generally, IL-1␤ is viewed as a catabolic factor for cartilage, inducing enzymes that degrade the extracellular matrix and reducing synthesis of the primary cartilage components type II collagen (COL2A1) and aggrecan (6 -8), although it can also induce BMP-2 (bone morphogenetic protein 2) potentially to initiate a repair response (9). In joint diseases, IL-1␤ is synthesized by synovial cells (10) and cartilage chondrocytes (6,11); therefore, its effect on chondrocytes is highly relevant to the fate of cartilage.
We have shown by microarray analysis, that a large set of chemokine genes is up-regulated by the proinflammatory cytokine IL-1␤ in adult normal cartilage and from patients with OA (6). It can be expected that this increase in a wide range of chemokines will have a significant impact on the cells of cartilage and other related joint tissues and should be considered in the pathophysiology of OA. Recently, we demonstrated that the adipokine, resistin, present in injured joints (12), also increased chemokine genes at both the transcriptional and post-transcriptional levels (13) in temporal patterns similar to the IL-1␤ patterns (6). The post-transcriptional regulation of chemokines was further investigated by us in chondrocytes with resistin (13) and by others in fibroblasts (14).
Plasmid Constructs-A series of CCL3 and CCL4 promoter 5Ј-deletion constructs were made by PCR and subcloned into pGL3-basic vector using the pGL2-CCL3(Ϫ1972/ ϩ75) and pGL3-CCL4(Ϫ1281/ϩ12) as described previously (26,27). The CCL3 and CCL4 promoter constructs, C/EBP␤ and IKK2 (IB kinase 2) expression vector, and pNF-B lucif-erase reporter were provided by the following. The human pGL2-CCL3(Ϫ1972/ϩ75) was from Dr. G. David Roodman (University of Pittsburgh) (26); human pGL3-CCL4(Ϫ1281/ ϩ12) was obtained from Dr. Sheau-Farn Yeh (National Yang-Ming University, Taipei) (27); human IKK2 in the pCDNA3 vector and pNF-B luciferase reporter were from Dr. Yousef Abu-Amer (Washington University) (28); full-length human C/EBP in the pCDNA3 vector was from Dr. Erika Crouch (Washington University) (29). The empty expression vectors were made by excision of cDNAs from the corresponding C/EBP expression vectors (22). To facilitate subcloning of the amplified fragments, the antisense primer contained a HindIII restriction site adaptor and the sense primer contained an XhoI or SmaI site. The PCR fragments and the luciferase expression vector pGL3-basic vector were digested with XhoI or SmaI and HindIII before ligation. All constructs were confirmed by DNA sequence analysis using a GL2 primer and RV3 primer.
Cell Culture-Human primary chondrocytes were obtained from articular cartilage obtained at the time of total joint replacement or from above the knee amputation, with approval of the Washington University Human Studies Review Board and permission of the patient (Institutional Review Board 05-0279). Chondrocytes were isolated following previously published procedures (6) and plated at a density of 2.5 ϫ 10 5 cells/ cm 2 in DMEM/F-12 medium plus 10% fetal bovine serum (FBS), 50 g/ml ascorbate, and antibiotics (50 units/ml penicillin and 50 g/ml streptomycin). Cells were allowed to rest for 24 h, and IL-1␤ was added at the concentrations and times indicated. IL-1␤ was reconstituted in sterile phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin. The T/C-28a2 human chondrocyte cell line was also used (a gift from Dr. Mary B. Goldring, Cornell University) and cultured like human articular chondrocytes. For RNA or nuclear protein extraction, the T/C-28 human chondrocyte cell line was plated in 3 ϫ 10 4 cells/cm 2 densities and cultured overnight. Human IL-1␤ was then added to the medium at the concentrations indicated.
Transient Transfection and Luciferase Assay-DNA transfections of T/C-28a2 cells were performed using FuGENE 6 TM transfection reagent or X-tremeGENE siRNA transfection reagent. 2 ϫ 10 5 of T/C-28a2 cells were cultured in a 6-well plate overnight. The transfection mixture containing 3-9 l of transfection reagent (6:1 ratio of transfection reagent (l) to DNA (g)), 500 ng of various promoter constructs, and 100 or 200 ng of pCMV-␤-gal was then added, and the cells were cultured for 8 or 24 h with or without IL-1␤ as indicated. For the co-transfection assay, 500 ng of C/EBP␤ expression vectors or empty vector and a 162 pM concentration of siRNAs were added to the 100-l transfection mixture as indicated. The sequences of siRNAs used were described previously (21). Due to low translation efficiency of C/EBP␤, we used higher amounts of plasmid (500 ng) for transfection, as shown previously (22,24). FBS was added to transfection medium 4 h later to a final concentration of 10%. After 24 h of incubation, cells were replaced with fresh complete medium and incubated for an additional 8 or 24 h with or without IL-1␤. The cells were then harvested with Reporter Lysis Buffer TM , and the lysate was analyzed for luciferase activity using Promega Luciferase Assay Reagent TM . The ␤-galactosidase activities were also measured to normalize variations in transfection efficiency. Each transfection experiment was performed in duplicate or triplicate and repeated at least three times.
Preparation of Nuclear Extracts-Nuclear extracts from T/C-28a2 cells were isolated using Nu-Clear Extraction Kit TM according to the manufacturer's instructions. Protein concentration of nuclear extract was determined using the Bio-Rad protein assay kit.
Electrophoretic Mobility Shift Assay (EMSA)-Fragment A (between Ϫ300 and Ϫ141 bp relative to the human CCL3 translation start site) and Fragment B (between Ϫ222 and Ϫ101 bp relative to the human CCL4 translation start site) were amplified by PCR. All oligonucleotides were synthesized by Invitrogen, and complementary oligonucleotide was annealed to make double-stranded oligonucleotide (30). The Fragment A, Fragment B, and various double-stranded oligonucleotides were end-labeled using T4 polynucleotide kinase and [␥-32 P]dATP. Bandshifts were performed by incubating 5 g of nuclear extracts in the mobility shift buffer (10 mM Hepes-KOH, pH 7.9, 100 mM NaCl, 1 mM EDTA, 10% glycerol, and 1 mM DTT) and 2 g of poly(dI-dC) with the DNA probe on ice for 30 min. For the competition studies, the cold DNA fragments were added at a 100-fold molar excess compared with the probe and incubated for 30 min on ice before adding the DNA probe. For the antibody interference experiments, the nuclear extracts and 2 l of antibody were preincubated in the buffer for 1 h at 4°C. DNA protein complexes were resolved on a 5% polyacrylamide gel at 100 V for about 90 min. The gels were dried and autoradiographed.
Western Blotting-Fifteen g of nuclear extracts were denatured in SDS sample buffer containing 0.1 M dithiothreitol at 100°C for 3 min and separated on a 12% Bio-Rad Ready Gel in Tris/glycine/SDS buffer. The gels were then transferred to Immobilon-P transfer membrane in Tris/glycine buffer, pH 8.3, containing 20% methanol. The membranes were saturated in 5% nonfat dry milk in PBS at 4°C overnight and reacted with anti-C/EBP␤ antibody diluted to 1:1000 or actin antibody diluted to 1:500 in PBS containing 0.05% Tween 20. The bound antibodies were recognized by IgG antibodies coupled to horseradish peroxidase, and the secondary antibodies were detected by autoradiograph using SuperSignal chemiluminescent substrate from Thermo Scientific. For Western blotting using the Odyssey imaging system, 10 g of nuclear extracts were used, and the gels were transferred and scanned according to the protocol recommended by the manufacturer (LI-COR).
Chromatin Immunoprecipitation (ChIP) Assay-Chromatin immunoprecipitations (IPs) were carried out using the EZ ChIP TM assay kit following the protocol recommended by the manufacturer. One 15-cm dish of T/C-28 cells (4 ϫ 10 6 ) in 15-cm culture dishes was used for one IP reaction. Cross-linking of DNA-proteins was induced by the addition of formaldehyde (1% final concentration) directly to the culture medium for 10 min at 37°C. Cells were lysed, and DNA in the supernatant was sheared by sonication. A 10-l aliquot of the sonicated chromatin sample ("input") was removed for PCR analysis, and the remainder was used for IPs. To each IP was added either 5 g of C/EBP␤ antibody or normal rabbit IgG, and reactions were incubated overnight with constant mixing at 4°C. Protein G beads were collected by centrifugation, and protein-DNA complexes were eluted from the beads followed by a cross-link reversal step by the addition of 5 M NaCl (8 l) to the eluted sample (200 l) overnight at 65°C. DNA from each IP reaction (2 l) was used for PCR with primer pairs to amplify the 140-bp region of interest in CCL3 (Ϫ300/Ϫ140) and the 110 bp in CCL4 (Ϫ222/Ϫ100): CCL3, 5Ј-TCATGCACAGACCA-GTTCTTATGA-3Ј (sense primer) and 5Ј-CTCTAACTCTCAGCTC-TC-AACTCA-3Ј (antisense primer); CCL4, 5Ј-CTGTACCACTTCCCT-TTTCTTCTC-3Ј (sense primer) and 5Ј-CTGAAGCTAGCTGAGT-GAGGAGTT-3Ј (antisense primer). Input or immunoprecipitated DNA was amplified by PCR (94°C, 20 s; 59°C, 30 s; 72°C, 2 min) for 32 cycles (CCL3) or 34 cycles (CCL4). Immunofluorescence-5 ϫ 10 4 human articular chondrocytes were cultured in each well of 8-well chamber slides (from Lab Tek). Cells were allowed to rest for 24 h, and IL-1␤ was then added at the times indicated. Cells were incubated in 4% paraformaldehyde in PBS for 10 min, 0.2% Triton X-100 in PBS for 5 min, and 10% normal goat serum in PBS for 2 h   OCTOBER 22, 2010 • VOLUME 285 • NUMBER 43 at room temperature. Cells were reacted with rabbit anti-C/EBP␤ and mouse anti-c-Rel antibodies diluted to 1:400 in 2% normal goat serum in PBS for overnight at 4°C. The secondary antibodies, Alexa fluor 488 dye-labeled goat antimouse IgG diluted to 1:250 and Alexa fluor 594 dye-labeled goat anti-rabbit IgG diluted to 1:400 in 2% normal goat serum in PBS, were then added to the cells for 1 h at room temperature. Immunoreactivity was detected by fluorescence microscopy.

IL-1␤ Stimulates the Expression of Chemokine Genes CCL3 and CCL4 in Normal Human Articular Chondrocytes and the
Chondrocyte-derived Cell Line, T/C-28-We have reported that a large set of chemokines that are up-regulated by the IL-1␤ in adult normal cartilage and from patients with OA (6). Here, we showed that two representative genes, CCL3 and CCL4, were increased in human articular chondrocytes and in the cell line T/C-28 with the same kinetics in the 24-h time period, although the expression level of CCL3 and CCL4 in human primary chondrocytes was higher than in the T/C-28 chondrocyte cell line (Fig. 1, A-C). CCL3 demonstrated a continuous increase to 24 h to an approximately 80-fold increase in primary cells. CCL4 was up-regulated by 4 h but then decreased during the remaining time period to about a 45-fold increase at 24 h. A dose-response test of IL-1␤ showed that 1 ng/ml is adequate for significant up-regulation (Fig. 1C). The primary chondrocytes and T/C-28 chondrocyte cell line showed a similar pattern of induction (Fig. 1, A and B). FIGURE 4. C/EBP␤ binds the sequence between ؊251 and ؊238 bp of the CCL3 promoter and between ؊168 and ؊155 bp of the CCL4 promoter. A, a diagram showing DNA fragments used for EMSA. For CCL3, Fragment A from Ϫ300 to Ϫ141 bp and the relative locations of oligonucleotides 1, 2, and 3 are shown. For CCL4, Fragment B from Ϫ222 to Ϫ101 bp and the relative locations of oligonucleotides 4, 5, and 6 are shown. Oligonucleotide 2 from Ϫ255 to Ϫ232 bp and oligonucleotide 5 from Ϫ175 to Ϫ151 bp contain the C/EBP␤ motif (underlined). Mu, mutant oligonucleotides 2 and 5 containing two base pair mutations (AA to CC) is underlined within the C/EBP␤ motif. B, EMSA for T/C-28a2 nuclear extracts using Fragment A or B as a probe and various cold competitors at a 100-fold molar excess. Oligonucleotides 2 and 5 competed with the binding of nuclear proteins to the probe. C, EMSA and supershift analysis for T/C-28a2 nuclear extracts using oligonucleotides 2 and 5 as the probe. Mutant oligonucleotides 2 and 5 did not compete with the binding of nuclear proteins to the probe. Antibodies against C/EBP␤ or SREBP1 were preincubated with the nuclear extracts. The lines show the shift band of the complex of C/EBP␤ antibody (Ab) and specific retarded bands (C, control; I, IL-1␤ treatment).

An IL-1␤-responsive Element Is Located between Ϫ300 and
Ϫ140 bp of the CCL3 Promoter and between Ϫ222 and Ϫ100 bp of the CCL4 Promoter-To identify the IL-1␤-responsive element within CCL3 and CCL4 promoters, five 5Ј-deletion constructs were transiently transfected into T/C-28 cells and incubated in the absence or presence of IL-1␤ (Fig. 2, A and B). Although there are many exceptions, most transcriptional regulation occurs within the first 1000 nucleotides of the promoter (33). For the CCL3 promoter constructs, the Ϫ1395, Ϫ522, Ϫ420, and Ϫ300 bp CCL3 promoter constructs were up-regulated by the IL-1␤ treatment; however, the response was lost in the Ϫ140 bp CCL3 constructs ( Fig. 2A). For CCL4 promoter constructs, the expression of the Ϫ1281, Ϫ521, Ϫ372, and Ϫ222 bp CCL4 promoter constructs were up-regulated by the IL-1␤ treatment, whereas the response was lost in the Ϫ100 bp CCL4 construct (Fig. 2B). These data suggest that in these constructs, the IL-1␤-responsive element is located between Ϫ300 and Ϫ140 bp of the CCL3 promoter and between Ϫ222 and Ϫ100 bp of the CCL4 promoter.
IL-1␤ Stimulates the Expression of C/EBP␤ in Human Articular Chondrocytes and T/C-28 Cells-Our computational data base of regulatory motifs revealed that there were potential binding sites for C/EBP␤ between Ϫ300 and Ϫ140 bp of the CCL3 promoter (2 sites) and between Ϫ222 and Ϫ100 bp of the CCL4 promoter (1 site); therefore, we examined whether IL-1␤ stimulates the expression of C/EBP␤ in primary chondrocytes and T/C-28 cells. The expression level of C/EBP␤ mRNA was stimulated by IL-1␤ over 24 h (Fig. 3, A-C). Type II collagen mRNA decreased to about 60% within 24 h in human chondrocytes as expected (Fig. 3A) as we have shown previously for rat chondrosarcoma cells (22) and human articular chondrocytes (6). Western blots for C/EBP␤ revealed that in the absence of IL-1␤ treatment, C/EBP␤ protein was present in the nuclei. When cells were stimulated with IL-1␤, C/EBP␤ protein expression was increased about 3-fold (Fig. 3D). Multiple C/EBP␤ isoforms with stimulatory or inhibitory activity can be trans-lated from a single mRNA by use of alternative translation initiation sites within the same open reading frame (34,35). The IL-1␤ treatment stimulated the expression of two of C/EBP␤ forms, 38-and 36-kDa liver-enriched transcriptional activator proteins (LAP), and 16-kDa liver-enriched inhibitory protein (LIP) (23,36); however, the up-regulation of protein expression of C/EBP␤ LAP was highest.
C/EBP␤ Binds to the IL-1␤responsive Element of the CCL3 and CCL4 Promoters-To determine whether C/EBP␤ functions within the IL-1␤-responsive element, EMSA was carried out using nuclear extract from the T/C-28 cells. Fragment A, containing the sequence between Ϫ300 and Ϫ141 bp of the CCL3, and Fragment B, containing the sequence between Ϫ222 and Ϫ101 bp of the CCL4, were used as probes, and various cold oligonucleotides were used as competitors (Fig. 4A). Competitors 2 and 5, which have a potential binding site for C/EBP␤ at Ϫ251/Ϫ238 bp of CCL3 (site S3 in Fig.  9) and Ϫ168/Ϫ155 bp of CCL4 (site S14 in Fig. 9), competed for the binding of nuclear proteins and the probe (Fig. 4B). As expected, the mutant oligonucleotides 2 and 5, in which the C/EBP␤-binding sites were altered, did not compete for the binding. Supershift analysis confirmed that C/EBP␤ bound to the sequence of oligonucleotide 2, in the CCL3 gene and oligonucleotide 5 in the CCL4 gene (Fig. 4C). Site 4 in CCL3 did not bind to the Fragment A (supplemental Fig. 1).
To further confirm that C/EBP␤ binds to the specific CCL3 and CCL4 promoter regions, we performed ChIP assays using anti-C/EBP␤ antibody. Endogenous binding of C/EBP␤ was stimulated by treatment with IL-1␤ up to 24 h (Fig. 5A). Treatment with IL-1␤ enhanced the binding of C/EBP␤ to the CCL3 promoter region from Ϫ300 to Ϫ140 bp and the CCL4 promoter region from Ϫ222 to Ϫ100 bp (Fig. 5, A and B), quantified by real-time PCR (Fig. 5, C and D). As a control, the ChIP analysis with and without IL-1␤ at 1 h is shown in Fig. 5B. IL-1␤ significantly increased binding of C/EBP␤ to its cognate sites. IL-1␤ stimulation gradually increased the binding activity of C/EBP␤ over time (Fig. 5E).
C/EBP␤ Functions as an Activator for the CCL3 and CCL4 Promoter Activities in Response to IL-1␤ Treatment-To confirm the function of C/EBP␤-responsive sites on the CCL3 and CCL4 genes, the C/EBP␤-binding sites were mutated in the Ϫ300 bp CCL3 construct and Ϫ222 bp CCL4 construct using mutant oligonucleotides 2 and 5, respectively. The constructs were transfected into T/C-28 cells and incubated in the absence or presence of IL-1␤ for an additional 8 or 24 h. The promoter activity of the wild type Ϫ300 bp CCL3 construct was about 9-fold stronger than that of mutant Ϫ300 bp CCL3 construct, and the Ϫ222 bp CCL4 construct was about 6.5-fold stronger than the mutant Ϫ222 bp construct after transient transfection  into T/C-28 cells, confirming that C/EBP␤ is acting as an activator (Fig. 6). The mutation greatly decreased the response to IL-1␤ in both gene constructs (Fig. 6).
In order to demonstrate the requirement for C/EBP␤, we examined the induction of CCL3 and CCL4 after reducing the C/EBP␤ expression by siRNA. Knockdown of C/EBP␤ expression was confirmed by Western blot analysis (Fig. 7A). The knockdown of C/EBP␤ suppressed the IL-1␤-induced CCL3 and CCL4 promoter activities (Fig. 7B) as well as CCL3 and CCL4 mRNA expression (Fig. 7C). Taken together, these results indicate that the C/EBP␤ sites at Ϫ251/Ϫ238 bp of CCL3 and Ϫ168/Ϫ155 bp of CCL4 promoters are required for the functional response to IL-1␤ and suggest that C/EBP␤ is a key factor in this response.

NF-B Is Involved in the Up-regulation of Chemokine Genes by Human Chondrocytes in
Response to IL-1␤-Because the transcription complex NF-B has been shown to be involved in the regulation of multiple cytokine and chemokine genes, we sought to determine the relative roles of C/EBP␤ and NF-B. The expression of NF-B in normal human articular chondrocytes was examined. The levels of NF-B1 (p50), NF-B2 (p52), RelA (p65), c-Rel, RelB, and IB␣ mRNAs were investigated in IL-1␤-treated chondrocytes at various times. NF-B isoforms were rapidly increased, but they decreased after 4 h (Fig. 8A). When compared with the concentration of C/EBP␤ in cells, we show that the NF-B isoforms are lower. In human chondrocytes, ⌬C of C/EBP␤ averages 3.98 and thus is lower than ⌬C of NF-B1 (6.09), NF-B2 (6.81), RelA (5.11), c-Rel (9.16), and RelB (5.92). However, these studies also provided insight into the likely NF-B subunits used in chondrocytes in response to IL-1␤. p50 and c-Rel showed the highest relative increase.
Due to the complicated regulatory mechanism of NF-B, we also looked directly at NF-B function by using pNF-B luciferase reporter. The activity of pNF-B luciferase reporter was up-regulated at 1 h, peaked at 8 h, and was reduced by 24 h (Fig. 8B).
To further evaluate the potential role of NF-B in primary human articular chondrocyte IL-1␤-induced chemokine activation, IKK-NBD, a specific NF-B inhibitor, was added to the culture before the addition of IL-1␤. Following 4 h of IL-1␤ treatment, the mRNA from these cells showed a modest but dose-dependent suppression of chemokine activity in the medium of cultures treated with IKK-NBD compared with non-inhibited cultures, whereas control IKK-NBD had no significant effect (Fig. 8C). The modest suppression can be attributed to the use short time periods and the use of primary chondrocytes in the present study as opposed to previous experiments, where only cell lines were used (13). Therefore, these results indicated that NF-B is involved in the expression of chemokines, particularly at 4 -8 h of exposure.

C/EBP␤ and NF-B Enhance the Expression of CCL3 and CCL4 in Human Chondrocytes in Response to IL-1␤-
The previous results indicated that both C/EBP␤ and NF-B contributed to the transcriptional up-regulation of CCL3 and CCL4. In order to determine the relative roles of C/EBP␤ and NF-B in transcription of CCL3 and CCL4, we determined the location of binding sites on the genes and performed transfection studies with CCL3 (Ϫ1395) constructs (26), CCL4 (Ϫ1281) constructs (27), C/EBP␤ expression vector (29), and IKK2 expression vector (28). The CCL3 (Ϫ1395) and CCL4 (Ϫ1281) constructs contain several high probability candidate C/EBP␤ and NF-B binding sites as assessed by our computational data base (6,37,38) (Fig. 9A), although only a limited number of sites are present in the IL-1␤ response domain. Based on the reported NF-B binding studies in chemokines (20), the IL-1␤-responsive elements had c-Rel binding sites at Ϫ210 to Ϫ206 bp of the CCL3 promoter (site S5) and Ϫ174 to Ϫ169 bp of the CCL4 promoter (site S13).
To investigate the C/EBP␤ and NF-B function on CCL3 and CCL4 in detail, C/EBP␤ and IKK2 (IKK␤) expression vectors were co-transfected with the Ϫ1395 bp CCL3 and Ϫ1281 bp CCL4 promoter constructs. IKK-2 is a protein subunit of IB kinase complex (39,40). Activated IKK-2 phosphorylates IB␣, which binds NF-B and inhibits its function (40,41). Phosphorylated IB␣ undergoes proteasomal degradation, thus permitting nuclear translocation of NF-B subunits, where they activate various genes involved in inflammation and other immune responses (40,42).
The promoter activities of the Ϫ1395 bp CCL3 and Ϫ1281 bp CCL4 constructs were up-regulated in a dose-dependent manner with or without IL-1␤ stimulation after being transfected with C/EBP␤ and IKK2, suggesting that C/EBP␤ and IKK2 function as activators (Fig. 9B). Experiments were terminated at the peak of mRNA expression (i.e. 24 h for CCL3 and 8 h for CCL4). Consequently, CCL3 appears more responsive to C/EBP␤, whereas CCL4 is responsive to C/EBP␤ and NF-B.
The involvement of NF-B in the IL-1␤-responsive elements was further confirmed by inhibiting its activity. The promoter activities were significantly decreased in the CCL3 (Ϫ300) and CCL4 (Ϫ222) by the IKK-NBD following 4 h of IL-1␤ treat-  ment, whereas the control IKK-NBD had no significant effect, and there was no effect on the shorter promoter constructs (Fig. 10, A and B).
The effects of inhibition of C/EBP␤ and NF-B were tested by inhibition of each transcription factor separately and together. SB303580, an inhibitor of C/EBP␤ via inhibition of p38 MAPK (21,43), and IKK-NBD reduced the expression of CCL3 and CCL4 with IL-1␤ stimulation (Fig. 11, A and B). IKK-NBD and SB303580 added together further decreased mRNA expression of CCL3 and CCL4 (Fig. 11A). Last, to confirm that both transcription factors are functional in transcription, promoter activity was determined after inhibition. The result showed that the promoter activities of CCL3 and CCL4 were significantly decreased by the inhibition of both factors (Fig. 11B).
Immunohistochemistry of C/EBP␤ and NF-B-Our biochemical studies provided evidence that the regulation of CCL3 and CCL4 is coordinated in time with the NF-B response early and transient and that the C/EBP␤ response is early and sustained. To support this conclusion, we traced the location of these transcription factors over time by immunohistochemistry (Fig. 12). Fig. 12 shows double immunohistochemistry with antibodies to C/EBP␤ and c-Rel after stimulation with IL-1␤. At zero time, c-Rel was localized in the cytoplasm, and then it was localized in the nucleus at 4 h and was greatly reduced by 24 h. C/EBP␤ was present at the beginning and was increased at 4 and 24 h.
Our studies strongly suggest that both C/EBP␤ and NF-B are important for expression and response to IL-1␤ in human chondrocytes (Fig. 13). The transcription factors, C/ EBP␤ and NF-B, are both involved in the up-regulation of chemokine genes in human chondrocytes in a time-ordered manner; NF-B provides an initial up-regulation, and C/EBP␤ sustains increased gene expression. Because C/EBP␤ is present in untreated chondrocytes and can stimulate chemokine gene expression in the absence of IL-1␤ (13), we predict that C/EBP␤ participates in basal regulation of these chemokine genes.

DISCUSSION
In this study, we demonstrate a critical role for C/EBP␤ in the increased expression of chemokine genes in response to IL-1␤ in human chondrocytes. We studied in detail the regulation of two representative chemokines that are known to be increased in osteoarthritis and many other inflammatory conditions, CCL3 and CCL4. We also show the cooperative regulation of these genes by both C/EBP␤ and NF-B that is both time-and function-dependent. We conclude from these data that C/EBP␤ is involved in constitutive regulation of chemokine genes and is also responsive to IL-1␤. From this work on chemokine gene stimulation and our previous work on extracellular matrix gene suppression by  C/EBP␤, we hypothesize that C/EBP␤ is one of the most important regulators of gene activity in chondrocytes in response to IL-1␤. In fact, C/EBP␤ may be one of the pivotal regulators of chondrocyte function in that we have shown that C/EBP␤ is responsible for the suppression of cartilage matrix genes in noncartilaginous tissues (25), and Kawaguchi and co-workers (44) have shown that C/EBP␤ is critical for chondrocyte hypertrophy, where it increases type X collagen and MMP-13 (matrix metalloproteinase 13), suppresses type II collagen synthesis, and activates p57 kip2 . This group also showed that removal of C/EBP␤ protects against osteoarthritis.
In addition to C/EBP␤, we demonstrate a complementary role for the classic proinflammatory mediator, NF-B. Like C/EBP␤, NF-B activity is increased by IL-1␤; however, in con-trast to C/EBP␤, the increase in NF-B activity is transient. Fig. 13 demonstrates the timing and functional activity of each transcription factor. Considering the time course of expression of CCL3 and the gradual increase of C/EBP␤ binding to DNA, we suggest that C/EBP␤ is involved in the up-regulation of chemokine genes over time, especially for the regulation of chemokine genes like CCL3, which are induced more slowly and where the mRNA was gradually and steadily increased, not reaching peak expression even in the 24-h observation period. In contrast, NF-B appears to be involved in the upregulation of chemokines in the early stage, with involvement gradually decreasing in the late stage. In CCL4, which was up-regulated by 4 h but then quickly decreased during the remaining time period, NF-B is probably more important than C/EBP␤. In addition, the level of gene expression is regulated at both transcriptional and post-transcriptional levels in eukaryotic cells, including fibroblasts and chondrocytes (14,45,46). As Baltimore and colleagues have reported (14), the expression of genes like CCL4 is also highly regulated by mRNA stability.
We showed that the IL-1␤-responsive elements of CCL3 and CCL4 promoters have C/EBP␤ binding sites, and co-transfection with C/EBP␤ could enhance the promoter activity. Within the IL-1␤-responsive elements of these genes were also NF-B-binding sites. Of note is that the increase in gene expression due to C/EBP␤ was greater than that induced by the same concentration of IKK2. Because the NF-B inhibitor, IKK-NBD, inhibited only about 30% of the CCL3 and CCL4 mRNA expression, C/EBP␤ could be the more important regulator of the expression of chemokine genes in this cell type.
Co-regulation by NF-B and C/EBP␤ was recently shown in several genes in different cells (4,21,47,48). Teti and colleagues (21) reported that Pseudomonas aeruginosa induced IL-8 (CXCL8) promoter expression and protein production in conjunctival epithelial cells by activating RelA and C/EBP␤ and by promoting the cooperative binding of these transcription factors to the IL-8 promoter that in turn activates transcription. They also showed that C/EBP␤ regulated the IL-8 basal transcriptional activity but not NF-B, which is consistent with our  data on the IL-1␤ effect in chondrocytes. Gil and colleagues (4) reported that IL-8, CXCL1, and CXCL5, which are CXCR2binding chemokines, are up-regulated during replicative and oncogene-induced senescence. They demonstrated a function of NF-B and C/EBP␤ in controlling the secretory program associated with cell senescence, although they did not investigate the mechanism.
Studies have shown that the RelA (p65) subunit of NF-B is involved in regulation of some lipopolysaccharide (LPS)-stimulated chemokines in human monocytic cells (17,18). However, we show in chondrocytes that, of the NF-B subunits, c-Rel mRNA was increased more than p65 with IL-1␤ stimulation, and the computational analysis in chemokine genes CCL3 and CCL4 showed that c-Rel binding sites were predicted in the IL-1␤-responsive elements. Ohashi and colleagues (49) recently reported that c-Rel was an important transcription factor in the regulation of the induction of proinflammatory cytokines; therefore, c-Rel would be an interesting target for further investigation.
Additional transcription factors have been shown to regulate CCL3 and CCL4. In multiple myeloma, AML-1A (acute myeloid leukemia 1A) and AML-1B (also known as Runx1 (Runtrelated transcription factor 1)) are principle regulators of CCL3 (26), although C/EBP␤ was later found to be important (50). In our experiments, Runx1 transfection did not increase gene expression. 3 C/EBP␤ is thought to be responsible for regulating growth, proliferation, and antiapoptotic responses by regulating the expression of other key transcription factors. In an early publication, Grove and Plumb (20) demonstrated that C/EBP, NF-B, and cEts family members were important in the LPSinduced stimulation of CCL3.
Levels of the proinflammatory cytokine IL-1␤ are increased in synovial fluid in joint diseases, such as OA and rheumatoid arthritis, and can induce cartilage damage and bone resorption. Chemokines function in the recruitment of neutrophils, monocytes, immature dendritic cells, B cells, and activated T cells (51). However, chemokines also have functions at the individual tissue level as it has been reported that the CXC family of chemokines are important in the regulation of angiogenesis, and CCL2, CCL3, and CCR2 stimulate osteoclastogenesis (52)(53)(54). In summary, the data presented here show that chondrocytes react in a cell-specific manner to IL-1␤, utilizing C/EBP␤ and NF-B in a combinatorial regulation of chemokine gene expression. The activity of C/EBP␤ is augmented by a transient increase in activity of NF-B, and both transcription factors act independently on the chemokine genes, CCL3 and CCL4. These studies provide the foundation for the control of chemokine gene expression in chronic joint disease.