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*
2010; Elsevier BV; Volume: 285; Issue: 43 Linguagem: Inglês
10.1074/jbc.m110.130377
ISSN1083-351X
AutoresZhiqi Zhang, Jennifer L. Bryan, Elizabeth DeLassus, Li‐Wei Chang, Wei‐Ming Liao, Linda J. Sandell,
Tópico(s)interferon and immune responses
ResumoA 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. 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. IntroductionChemokines are associated with several diseases, including cardiovascular diseases, neuroinflammation, cancer, and HIV-associated diseases (1Gerard C. Rollins B.J. Nat. Immunol. 2001; 2: 108-115Crossref PubMed Scopus (1199) Google Scholar). As mediators of cell recruitment, chemokines are known to be important in inflammatory diseases, including rheumatoid arthritis, osteoarthritis (OA), 2The abbreviations used are: OAosteoarthritisC/EBPβCCAAT/enhancer-binding proteinIKK-NBDcell-permeable NEMO binding domainLAPliver-enriched transcriptional activator proteinsLIPliver-enriched inhibitory proteinIPimmunoprecipitation. inflammatory bowel disease, multiple sclerosis and transplant rejection (1Gerard C. Rollins B.J. Nat. Immunol. 2001; 2: 108-115Crossref PubMed Scopus (1199) Google Scholar, 2Haringman J.J. Ludikhuize J. Tak P.P. Ann. Rheum. Dis. 2004; 63: 1186-1194Crossref PubMed Scopus (108) Google Scholar). 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 (3Ma Q. Jones D. Borghesani P.R. Segal R.A. Nagasawa T. Kishimoto T. Bronson R.T. Springer T.A. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 9448-9453Crossref PubMed Scopus (1410) Google Scholar, 4Acosta J.C. O'Loghlen A. Banito A. Guijarro M.V. Augert A. Raguz S. Fumagalli M. Da Costa M. Brown C. Popov N. Takatsu Y. Melamed J. d'Adda di Fagagna F. Bernard D. Hernando E. Gil J. Cell. 2008; 133: 1006-1018Abstract Full Text Full Text PDF PubMed Scopus (1153) Google Scholar), and the chemokine CCL3 is a potent osteoclastogenic factor (5Han J.H. Choi S.J. Kurihara N. Koide M. Oba Y. Roodman G.D. Blood. 2001; 97: 3349-3353Crossref PubMed Scopus (275) Google Scholar). There exist over 50 chemokine ligands and 20 G protein-coupled receptors (1Gerard C. Rollins B.J. Nat. Immunol. 2001; 2: 108-115Crossref PubMed Scopus (1199) Google Scholar). 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) (2Haringman J.J. Ludikhuize J. Tak P.P. Ann. Rheum. Dis. 2004; 63: 1186-1194Crossref PubMed Scopus (108) Google Scholar).IL-1β is an important cytokine in rheumatoid and osteoarthritic joint diseases. Generally, IL-1β 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 (6Sandell L.J. Xing X. Franz C. Davies S. Chang L.W. Patra D. Osteoarthr. Cartil. 2008; 16: 1560-1571Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 7Yamasaki K. Nakasa T. Miyaki S. Ishikawa M. Deie M. Adachi N. Yasunaga Y. Asahara H. Ochi M. Arthritis Rheum. 2009; 60: 1035-1041Crossref PubMed Scopus (271) Google Scholar, 8Miyaki S. Nakasa T. Otsuki S. Grogan S.P. Higashiyama R. Inoue A. Kato Y. Sato T. Lotz M.K. Asahara H. Arthritis Rheum. 2009; 60: 2723-2730Crossref PubMed Scopus (346) Google Scholar), although it can also induce BMP-2 (bone morphogenetic protein 2) potentially to initiate a repair response (9Fukui N. Zhu Y. Maloney W.J. Clohisy J. Sandell L.J. J. Bone Joint Surg. Am. 2003; 85: 59-66Crossref PubMed Scopus (189) Google Scholar). In joint diseases, IL-1β is synthesized by synovial cells (10Martel-Pelletier J. Alaaeddine N. Pelletier J.P. Front. Biosci. 1999; 4: D694-D703Crossref PubMed Google Scholar) and cartilage chondrocytes (6Sandell L.J. Xing X. Franz C. Davies S. Chang L.W. Patra D. Osteoarthr. Cartil. 2008; 16: 1560-1571Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 11Tiku K. Thakker-Varia S. Ramachandrula A. Tiku M.L. Cell. Immunol. 1992; 140: 1-20Crossref PubMed Scopus (38) Google Scholar); 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-1β in adult normal cartilage and from patients with OA (6Sandell L.J. Xing X. Franz C. Davies S. Chang L.W. Patra D. Osteoarthr. Cartil. 2008; 16: 1560-1571Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). 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 (12Lee J.H. Ort T. Ma K. Picha K. Carton J. Marsters P.A. Lohmander L.S. Baribaud F. Song X.Y. Blake S. Osteoarthr. Cartil. 2009; 17: 613-620Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), also increased chemokine genes at both the transcriptional and post-transcriptional levels (13Zhang Z. Xing X. Hensley G. Chang L.W. Liao W. Abu-Amer Y. Sandell L.J. Arthritis Rheum. 2010; 162: 1193-2003Google Scholar) in temporal patterns similar to the IL-1β patterns (6Sandell L.J. Xing X. Franz C. Davies S. Chang L.W. Patra D. Osteoarthr. Cartil. 2008; 16: 1560-1571Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). The post-transcriptional regulation of chemokines was further investigated by us in chondrocytes with resistin (13Zhang Z. Xing X. Hensley G. Chang L.W. Liao W. Abu-Amer Y. Sandell L.J. Arthritis Rheum. 2010; 162: 1193-2003Google Scholar) and by others in fibroblasts (14Hao S. Baltimore D. Nat. Immunol. 2009; 10: 281-288Crossref PubMed Scopus (374) Google Scholar).In the present study, we focused on the transcriptional regulation of chemokine genes. As we reported (6Sandell L.J. Xing X. Franz C. Davies S. Chang L.W. Patra D. Osteoarthr. Cartil. 2008; 16: 1560-1571Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), IL-1β increased expression of chemokines CCL3, CCL4, CCL20, CCL3L1, CXCL1, CXCL2, CXCL3, CXCL6, and CXCL8 (IL-8) from 25- to 75-fold in human articular chondrocytes. A computational analysis of these co-regulated genes identified NF-κB, C/EBPβ, and MEF-3 (myocyte enhancer binding factor 3), as candidate transcriptional regulators. NF-κB has been shown to regulate a specific subset of chemokines (15Pulai J.I. Chen H. Im H.J. Kumar S. Hanning C. Hegde P.S. Loeser R.F. J. Immunol. 2005; 174: 5781-5788Crossref PubMed Scopus (161) Google Scholar, 16Rezzonico R. Imbert V. Chicheportiche R. Dayer J.M. Blood. 2001; 97: 2932-2940Crossref PubMed Scopus (75) Google Scholar, 17Lakshmanan U. Porter A.G. J. Immunol. 2007; 179: 8480-8490Crossref PubMed Scopus (47) Google Scholar, 18Lim C.A. Yao F. Wong J.J. George J. Xu H. Chiu K.P. Sung W.K. Lipovich L. Vega V.B. Chen J. Shahab A. Zhao X.D. Hibberd M. Wei C.L. Lim B. Ng H.H. Ruan Y. Chin K.C. Mol. Cell. 2007; 27: 622-635Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar); however, Amos et al. (19Amos N. Lauder S. Evans A. Feldmann M. Bondeson J. Rheumatology. 2006; 45: 1201-1209Crossref PubMed Scopus (70) Google Scholar) recently demonstrated that inhibition of NF-κB activity did not inhibit all inflammatory mediators; therefore, there are probably other transcriptional mechanisms involved. C/EBP has been shown to regulate a set of chemokines (4Acosta J.C. O'Loghlen A. Banito A. Guijarro M.V. Augert A. Raguz S. Fumagalli M. Da Costa M. Brown C. Popov N. Takatsu Y. Melamed J. d'Adda di Fagagna F. Bernard D. Hernando E. Gil J. Cell. 2008; 133: 1006-1018Abstract Full Text Full Text PDF PubMed Scopus (1153) Google Scholar, 20Grove M. Plumb M. Mol. Cell. Biol. 1993; 13: 5276-5289Crossref PubMed Google Scholar, 21Venza I. Cucinotta M. Visalli M. De Grazia G. Oliva S. Teti D. J. Biol. Chem. 2009; 284: 4191-4199Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). We have previously shown that C/EBPβ is associated with IL-1β-induced and tumor necrosis factor-α (TNF-α)-induced down-regulation of matrix genes in chondrocytes and the repression of cartilage gene expression in non-cartilaginous tissues (22Okazaki K. Li J. Yu H. Fukui N. Sandell L.J. J. Biol. Chem. 2002; 277: 31526-31533Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 23Imamura T. Imamura C. Iwamoto Y. Sandell L.J. J. Biol. Chem. 2005; 280: 16625-16634Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 24Imamura T. Imamura C. McAlinden A. Davies S.R. Iwamoto Y. Sandell L.J. Arthritis Rheum. 2008; 58: 1366-1376Crossref PubMed Scopus (11) Google Scholar, 25Okazaki K. Yu H. Davies S.R. Imamura T. Sandell L.J. J. Cell. Biochem. 2006; 97: 857-868Crossref PubMed Scopus (14) Google Scholar). Here, we investigated the roles of C/EBPβ and NF-κB in the up-regulation of two chemokine genes, CCL3 (MIP-1α; macrophage inflammatory protein 1α) and CCL4 (MIP-1β), in human chondrocytes in response to IL-1β.DISCUSSIONIn this study, we demonstrate a critical role for C/EBPβ in the increased expression of chemokine genes in response to IL-1β 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/EBPβ and NF-κB that is both time- and function-dependent. We conclude from these data that C/EBPβ is involved in constitutive regulation of chemokine genes and is also responsive to IL-1β. From this work on chemokine gene stimulation and our previous work on extracellular matrix gene suppression by C/EBPβ, we hypothesize that C/EBPβ is one of the most important regulators of gene activity in chondrocytes in response to IL-1β. In fact, C/EBPβ may be one of the pivotal regulators of chondrocyte function in that we have shown that C/EBPβ is responsible for the suppression of cartilage matrix genes in non-cartilaginous tissues (25Okazaki K. Yu H. Davies S.R. Imamura T. Sandell L.J. J. Cell. Biochem. 2006; 97: 857-868Crossref PubMed Scopus (14) Google Scholar), and Kawaguchi and co-workers (44Hirata M. Kugimiya F. Fukai A. Ohba S. Kawamura N. Ogasawara T. Kawasaki Y. Saito T. Yano F. Ikeda T. Nakamura K. Chung U.I. Kawaguchi H. PLoS One. 2009; 4: e4543Crossref PubMed Scopus (75) Google Scholar) have shown that C/EBPβ is critical for chondrocyte hypertrophy, where it increases type X collagen and MMP-13 (matrix metalloproteinase 13), suppresses type II collagen synthesis, and activates p57kip2. This group also showed that removal of C/EBPβ protects against osteoarthritis.In addition to C/EBPβ, we demonstrate a complementary role for the classic proinflammatory mediator, NF-κB. Like C/EBPβ, NF-κB activity is increased by IL-1β; however, in contrast to C/EBPβ, the increase in NF-κB 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/EBPβ binding to DNA, we suggest that C/EBPβ 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-κB appears to be involved in the up-regulation 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-κB is probably more important than C/EBPβ. In addition, the level of gene expression is regulated at both transcriptional and post-transcriptional levels in eukaryotic cells, including fibroblasts and chondrocytes (14Hao S. Baltimore D. Nat. Immunol. 2009; 10: 281-288Crossref PubMed Scopus (374) Google Scholar, 45Fukui N. Ikeda Y. Ohnuki T. Hikita A. Tanaka S. Yamane S. Suzuki R. Sandell L.J. Ochi T. J. Biol. Chem. 2006; 281: 27229-27241Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 46Tebo J.M. Datta S. Kishore R. Kolosov M. Major J.A. Ohmori Y. Hamilton T.A. J. Biol. Chem. 2000; 275: 12987-12993Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). As Baltimore and colleagues have reported (14Hao S. Baltimore D. Nat. Immunol. 2009; 10: 281-288Crossref PubMed Scopus (374) Google Scholar), the expression of genes like CCL4 is also highly regulated by mRNA stability.We showed that the IL-1β-responsive elements of CCL3 and CCL4 promoters have C/EBPβ binding sites, and co-transfection with C/EBPβ could enhance the promoter activity. Within the IL-1β-responsive elements of these genes were also NF-κB-binding sites. Of note is that the increase in gene expression due to C/EBPβ was greater than that induced by the same concentration of IKK2. Because the NF-κB inhibitor, IKK-NBD, inhibited only about 30% of the CCL3 and CCL4 mRNA expression, C/EBPβ could be the more important regulator of the expression of chemokine genes in this cell type.Co-regulation by NF-κB and C/EBPβ was recently shown in several genes in different cells (4Acosta J.C. O'Loghlen A. Banito A. Guijarro M.V. Augert A. Raguz S. Fumagalli M. Da Costa M. Brown C. Popov N. Takatsu Y. Melamed J. d'Adda di Fagagna F. Bernard D. Hernando E. Gil J. Cell. 2008; 133: 1006-1018Abstract Full Text Full Text PDF PubMed Scopus (1153) Google Scholar, 21Venza I. Cucinotta M. Visalli M. De Grazia G. Oliva S. Teti D. J. Biol. Chem. 2009; 284: 4191-4199Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 47El-Asmar B. Giner X.C. Tremblay J.J. J. Mol. Endocrinol. 2009; 42: 131-138Crossref PubMed Scopus (21) Google Scholar, 48Sow F.B. Alvarez G.R. Gross R.P. Satoskar A.R. Schlesinger L.S. Zwilling B.S. Lafuse W.P. J. Leukocyte Biol. 2009; 86: 1247-1258Crossref PubMed Scopus (66) Google Scholar). Teti and colleagues (21Venza I. Cucinotta M. Visalli M. De Grazia G. Oliva S. Teti D. J. Biol. Chem. 2009; 284: 4191-4199Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) reported that Pseudomonas aeruginosa induced IL-8 (CXCL8) promoter expression and protein production in conjunctival epithelial cells by activating RelA and C/EBPβ 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/EBPβ regulated the IL-8 basal transcriptional activity but not NF-κB, which is consistent with our data on the IL-1β effect in chondrocytes. Gil and colleagues (4Acosta J.C. O'Loghlen A. Banito A. Guijarro M.V. Augert A. Raguz S. Fumagalli M. Da Costa M. Brown C. Popov N. Takatsu Y. Melamed J. d'Adda di Fagagna F. Bernard D. Hernando E. Gil J. Cell. 2008; 133: 1006-1018Abstract Full Text Full Text PDF PubMed Scopus (1153) Google Scholar) reported that IL-8, CXCL1, and CXCL5, which are CXCR2-binding chemokines, are up-regulated during replicative and oncogene-induced senescence. They demonstrated a function of NF-κB and C/EBPβ 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-κB is involved in regulation of some lipopolysaccharide (LPS)-stimulated chemokines in human monocytic cells (17Lakshmanan U. Porter A.G. J. Immunol. 2007; 179: 8480-8490Crossref PubMed Scopus (47) Google Scholar, 18Lim C.A. Yao F. Wong J.J. George J. Xu H. Chiu K.P. Sung W.K. Lipovich L. Vega V.B. Chen J. Shahab A. Zhao X.D. Hibberd M. Wei C.L. Lim B. Ng H.H. Ruan Y. Chin K.C. Mol. Cell. 2007; 27: 622-635Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). However, we show in chondrocytes that, of the NF-κB subunits, c-Rel mRNA was increased more than p65 with IL-1β stimulation, and the computational analysis in chemokine genes CCL3 and CCL4 showed that c-Rel binding sites were predicted in the IL-1β-responsive elements. Ohashi and colleagues (49Lu Y.C. Kim I. Lye E. Shen F. Suzuki N. Suzuki S. Gerondakis S. Akira S. Gaffen S.L. Yeh W.C. Ohashi P.S. J. Immunol. 2009; 182: 7212-7221Crossref PubMed Scopus (86) Google Scholar) 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 (Runt-related transcription factor 1)) are principle regulators of CCL3 (26Choi S.J. Oba T. Callander N.S. Jelinek D.F. Roodman G.D. Blood. 2003; 101: 3778-3783Crossref PubMed Scopus (38) Google Scholar), although C/EBPβ was later found to be important (50Pal R. Janz M. Galson D.L. Gries M. Li S. Jöhrens K. Anagnostopoulos I. Dörken B. Mapara M.Y. Borghesi L. Kardava L. Roodman G.D. Milcarek C. Lentzsch S. Blood. 2009; 114: 3890-3898Crossref PubMed Scopus (63) Google Scholar). In our experiments, Runx1 transfection did not increase gene expression. 3Z. Zhang and L. J. Sandell, unpublished results. C/EBPβ 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 (20Grove M. Plumb M. Mol. Cell. Biol. 1993; 13: 5276-5289Crossref PubMed Google Scholar) demonstrated that C/EBP, NF-κB, and cEts family members were important in the LPS-induced stimulation of CCL3.Levels of the proinflammatory cytokine IL-1β 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 (51Borzì R.M. Mazzetti I. Cattini L. Uguccioni M. Baggiolini M. Facchini A. Arthritis Rheum. 2000; 43: 1734-1741Crossref PubMed Scopus (127) Google Scholar). 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 (52Binder N.B. Niederreiter B. Hoffmann O. Stange R. Pap T. Stulnig T.M. Mack M. Erben R.G. Smolen J.S. Redlich K. Nat. Med. 2009; 15: 417-424Crossref PubMed Scopus (142) Google Scholar, 53Miyamoto K. Ninomiya K. Sonoda K.H. Miyauchi Y. Hoshi H. Iwasaki R. Miyamoto H. Yoshida S. Sato Y. Morioka H. Chiba K. Egashira K. Suda T. Toyama Y. Miyamoto T. Biochem. Biophys. Res. Commun. 2009; 383: 373-377Crossref PubMed Scopus (71) Google Scholar, 54Rudolph E.H. Woods J.M. Curr. Pharm. Des. 2005; 11: 613-631Crossref PubMed Scopus (27) Google Scholar). In summary, the data presented here show that chondrocytes react in a cell-specific manner to IL-1β, utilizing C/EBPβ and NF-κB in a combinatorial regulation of chemokine gene expression. The activity of C/EBPβ is augmented by a transient increase in activity of NF-κB, 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. IntroductionChemokines are associated with several diseases, including cardiovascular diseases, neuroinflammation, cancer, and HIV-associated diseases (1Gerard C. Rollins B.J. Nat. Immunol. 2001; 2: 108-115Crossref PubMed Scopus (1199) Google Scholar). As mediators of cell recruitment, chemokines are known to be important in inflammatory diseases, including rheumatoid arthritis, osteoarthritis (OA), 2The abbreviations used are: OAosteoarthritisC/EBPβCCAAT/enhancer-binding proteinIKK-NBDcell-permeable NEMO binding domainLAPliver-enriched transcriptional activator proteinsLIPliver-enriched inhibitory proteinIPimmunoprecipitation. inflammatory bowel disease, multiple sclerosis and transplant rejection (1Gerard C. Rollins B.J. Nat. Immunol. 2001; 2: 108-115Crossref PubMed Scopus (1199) Google Scholar, 2Haringman J.J. Ludikhuize J. Tak P.P. Ann. Rheum. Dis. 2004; 63: 1186-1194Crossref PubMed Scopus (108) Google Scholar). 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 (3Ma Q. Jones D. Borghesani P.R. Segal R.A. Nagasawa T. Kishimoto T. Bronson R.T. Springer T.A. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 9448-9453Crossref PubMed Scopus (1410) Google Scholar, 4Acosta J.C. O'Loghlen A. Banito A. Guijarro M.V. Augert A. Raguz S. Fumagalli M. Da Costa M. Brown C. Popov N. Takatsu Y. Melamed J. d'Adda di Fagagna F. Bernard D. Hernando E. Gil J. Cell. 2008; 133: 1006-1018Abstract Full Text Full Text PDF PubMed Scopus (1153) Google Scholar), and the chemokine CCL3 is a potent osteoclastogenic factor (5Han J.H. Choi S.J. Kurihara N. Koide M. Oba Y. Roodman G.D. Blood. 2001; 97: 3349-3353Crossref PubMed Scopus (275) Google Scholar). There exist over 50 chemokine ligands and 20 G protein-coupled receptors (1Gerard C. Rollins B.J. Nat. Immunol. 2001; 2: 108-115Crossref PubMed Scopus (1199) Google Scholar). 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) (2Haringman J.J. Ludikhuize J. Tak P.P. Ann. Rheum. Dis. 2004; 63: 1186-1194Crossref PubMed Scopus (108) Google Scholar).IL-1β is an important cytokine in rheumatoid and osteoarthritic joint diseases. Generally, IL-1β 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 (6Sandell L.J. Xing X. Franz C. Davies S. Chang L.W. Patra D. Osteoarthr. Cartil. 2008; 16: 1560-1571Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 7Yamasaki K. Nakasa T. Miyaki S. Ishikawa M. Deie M. Adachi N. Yasunaga Y. Asahara H. Ochi M. Arthritis Rheum. 2009; 60: 1035-1041Crossref PubMed Scopus (271) Google Scholar, 8Miyaki S. Nakasa T. Otsuki S. Grogan S.P. Higashiyama R. Inoue A. Kato Y. Sato T. Lotz M.K. Asahara H. Arthritis Rheum. 2009; 60: 2723-2730Crossref PubMed Scopus (346) Google Scholar), although it can also induce BMP-2 (bone morphogenetic protein 2) potentially to initiate a repair response (9Fukui N. Zhu Y. Maloney W.J. Clohisy J. Sandell L.J. J. Bone Joint Surg. Am. 2003; 85: 59-66Crossref PubMed Scopus (189) Google Scholar). In joint diseases, IL-1β is synthesized by synovial cells (10Martel-Pelletier J. Alaaeddine N. Pelletier J.P. Front. Biosci. 1999; 4: D694-D703Crossref PubMed Google Scholar) and cartilage chondrocytes (6Sandell L.J. Xing X. Franz C. Davies S. Chang L.W. Patra D. Osteoarthr. Cartil. 2008; 16: 1560-1571Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 11Tiku K. Thakker-Varia S. Ramachandrula A. Tiku M.L. Cell. Immunol. 1992; 140: 1-20Crossref PubMed Scopus (38) Google Scholar); 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-1β in adult normal cartilage and from patients with OA (6Sandell L.J. Xing X. Franz C. Davies S. Chang L.W. Patra D. Osteoarthr. Cartil. 2008; 16: 1560-1571Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). 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 (12Lee J.H. Ort T. Ma K. Picha K. Carton J. Marsters P.A. Lohmander L.S. Baribaud F. Song X.Y. Blake S. Osteoarthr. 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Here, we investigated the roles of C/EBPβ and NF-κB in the up-regulation of two chemokine genes, CCL3 (MIP-1α; macrophage inflammatory protein 1α) and CCL4 (MIP-1β), in human chondrocytes in response to IL-1β.
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