Sp1 and Sp3 Transcription Factors Mediate Interleukin-1β Down-regulation of Human Type II Collagen Gene Expression in Articular Chondrocytes
2003; Elsevier BV; Volume: 278; Issue: 41 Linguagem: Inglês
10.1074/jbc.m303541200
ISSN1083-351X
AutoresChristos Chadjichristos, Chafik Ghayor, Magdalini Kypriotou, Grégoire Martin, Emmanuelle Renard, Leena Ala‐Kokko, Guntram Suske, Benoît De Crombrugghe, Jean‐Pierre Pujol, Philippe Galéra,
Tópico(s)NF-κB Signaling Pathways
ResumoInterleukin-1β (IL-1β) is a pleiotropic cytokine that was shown to inhibit the biosynthesis of articular cartilage components. Here we demonstrate that IL-1β inhibits the production of newly synthesized collagens in proliferating rabbit articular chondrocytes and that this effect is accompanied by a decrease in the steady-state levels of type II collagen mRNA. IL-1β down-regulates COL2A1 gene transcription through a –41/–33 bp sequence that binds a multimeric complex including Sp1 and Sp3 transcription factors. Specificity of IL-1β effects on COL2A1 promoter activity was demonstrated in experiments in which transfection of a wild type –50/+1 sequence of COL2A1 promoter as a decoy oligonucleotide abolished the IL-1β inhibition of a –63/+47 COL2A1-mediated transcription. By contrast, transfection of the related oligonucleotide harboring a targeted mutation in the –41/–33 sequence did not modify the negative effect the cytokine. Because we demonstrated previously that Sp1 was a strong activator of COL2A1 gene expression via the –63/+1 promoter region, whereas Sp3 overexpression blocked Sp1-induced promoter activity and inhibited COL2A1 gene transcription, we conclude that IL-1β down-regulation of that gene, as we found previously for transforming growth factor-β1, is mediated by an increase in the Sp3/Sp1 ratio. Moreover, IL-1β increased steady-state levels of Sp1 and Sp3 mRNAs, whereas it enhanced Sp3 protein expression and inhibited Sp1 protein biosynthesis. Nevertheless, IL-1β decreased the binding activity of both Sp1 and Sp3 to the 63-bp short COL2A1 promoter, suggesting that the cytokine exerts a post-transcriptional regulatory mechanism on Sp1 and Sp3 gene expressions. Altogether, these data indicate that modulation of Sp3/Sp1 ratio in cartilage could be a potential target to prevent or limit the tissue degradation. Interleukin-1β (IL-1β) is a pleiotropic cytokine that was shown to inhibit the biosynthesis of articular cartilage components. Here we demonstrate that IL-1β inhibits the production of newly synthesized collagens in proliferating rabbit articular chondrocytes and that this effect is accompanied by a decrease in the steady-state levels of type II collagen mRNA. IL-1β down-regulates COL2A1 gene transcription through a –41/–33 bp sequence that binds a multimeric complex including Sp1 and Sp3 transcription factors. Specificity of IL-1β effects on COL2A1 promoter activity was demonstrated in experiments in which transfection of a wild type –50/+1 sequence of COL2A1 promoter as a decoy oligonucleotide abolished the IL-1β inhibition of a –63/+47 COL2A1-mediated transcription. By contrast, transfection of the related oligonucleotide harboring a targeted mutation in the –41/–33 sequence did not modify the negative effect the cytokine. Because we demonstrated previously that Sp1 was a strong activator of COL2A1 gene expression via the –63/+1 promoter region, whereas Sp3 overexpression blocked Sp1-induced promoter activity and inhibited COL2A1 gene transcription, we conclude that IL-1β down-regulation of that gene, as we found previously for transforming growth factor-β1, is mediated by an increase in the Sp3/Sp1 ratio. Moreover, IL-1β increased steady-state levels of Sp1 and Sp3 mRNAs, whereas it enhanced Sp3 protein expression and inhibited Sp1 protein biosynthesis. Nevertheless, IL-1β decreased the binding activity of both Sp1 and Sp3 to the 63-bp short COL2A1 promoter, suggesting that the cytokine exerts a post-transcriptional regulatory mechanism on Sp1 and Sp3 gene expressions. Altogether, these data indicate that modulation of Sp3/Sp1 ratio in cartilage could be a potential target to prevent or limit the tissue degradation. Articular cartilage is a highly specialized tissue composed of a complex extracellular matrix of proteoglycans, collagens, and noncollagenous glycoproteins. Cartilage collagens include type II as the major form and types VI, IX, and XI as minor components (1Mayne R. Irwin M.H. Kuettner K. Schleyerbach R. Hascall V.C. Articular Cartilage Biochemistry. Raven Press, New York1986: 23-35Google Scholar). Type II collagen is an homotrimer composed of α1(II) chains encoded by the COL2A1 gene. Previous studies have delineated minimal sequences in the first intron of human, mouse, and rat COL2A1 genes which are sufficient to direct chondrocyte-specific expression in cultured chondrocytes and transgenic mice (2Lefebvre V. Zhou G. Mukhopadhyay K. Smith C.N. Zhang Z. Eberspaecher H. Zhou X. Sinha S. Maity S.N. de Crombrugghe B. Mol. Cell. Biol. 1996; 16: 4512-4523Crossref PubMed Google Scholar, 3Krebsbach P.H. Nakata K. Bernier S.M. Hatano O. Miyashita T. Rhodes C.S. Yamada Y. J. Biol. 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EMBO J. 1998; 17: 5715-5718Crossref Scopus (673) Google Scholar), and also with factors having less tissue-specific expression, such as Sp1, Sp3, and C-KROX (5Ghayor C. Herrouin J.-F. Chadjichristos C. Ala-Kokko L. Takigawa M. Pujol J.-P. Galéra P. J. Biol. Chem. 2000; 275: 27421-27438Abstract Full Text Full Text PDF PubMed Google Scholar, 8Ghayor C. Chadjichristos C. Herrouin J.-F. Ala-Kokko L. Suske G. Pujol J.-P. Galéra P. J. Biol. Chem. 2001; 276: 36881-36895Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Indeed, promoter sequences are also implicated through interaction with the intronic enhancer sequence, for tissue-specific expression during in vivo and in vitro chondrogenesis (7Lefebvre V. Li P. de Crombrugghe B. EMBO J. 1998; 17: 5715-5718Crossref Scopus (673) Google Scholar, 9Savagner P. Krebsbach P.H. Miyashita T. Liebman J. Yamada Y. DNA Cell Biol. 1995; 14: 501-510Crossref PubMed Scopus (29) Google Scholar, 10Leung K.K.H. Ng L.J. Ho K.K.Y. Tam P.P.L. Cheah K.S.E. J. Cell Biol. 1998; 141: 1291-1300Crossref PubMed Scopus (45) Google Scholar). In a 266-bp promoter of the human COL2A1 gene mediating enhanced transcription activity, we identified several binding sites for Sp1, Sp3, and C-KROX (5Ghayor C. Herrouin J.-F. Chadjichristos C. Ala-Kokko L. Takigawa M. Pujol J.-P. Galéra P. J. Biol. Chem. 2000; 275: 27421-27438Abstract Full Text Full Text PDF PubMed Google Scholar, 8Ghayor C. Chadjichristos C. Herrouin J.-F. Ala-Kokko L. Suske G. Pujol J.-P. Galéra P. J. Biol. Chem. 2001; 276: 36881-36895Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 11Chadjichristos C. Ghayor C. Herrouin J.-F. Ala-Kokko L. Suske G. Pujol J.-P. Galéra P. J. Biol. Chem. 2002; 277: 43903-43917Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Sp1 was found to activate COL2A1 transcription through the promoter binding sites, whatever the differentiation state of chondrocytes, suggesting that this factor may be capable of restoring the altered chondrocyte phenotype (8Ghayor C. Chadjichristos C. Herrouin J.-F. Ala-Kokko L. Suske G. Pujol J.-P. Galéra P. J. Biol. Chem. 2001; 276: 36881-36895Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). By contrast, Sp3 was shown to prevent Sp1-transactivating effects by binding to the same cis-elements. In osteoarthritis (OA), 1The abbreviations used are: OA, osteoarthritis; AP, activator protein; CMV, cytomegalovirus; DMEM, Dulbecco's modified Eagle's medium; EMSA, electrophoretic mobility shift assay; FCS, fetal calf serum; β-gal, β-galactosidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IκB, inhibitor κB; IL, interleukin; Luc, luciferase; NF-κB, nuclear factor-κB; RAC, rabbit articular chondrocyte(s); RT, reverse transcription; TGF, transforming growth factor; wt, wild type.1The abbreviations used are: OA, osteoarthritis; AP, activator protein; CMV, cytomegalovirus; DMEM, Dulbecco's modified Eagle's medium; EMSA, electrophoretic mobility shift assay; FCS, fetal calf serum; β-gal, β-galactosidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IκB, inhibitor κB; IL, interleukin; Luc, luciferase; NF-κB, nuclear factor-κB; RAC, rabbit articular chondrocyte(s); RT, reverse transcription; TGF, transforming growth factor; wt, wild type. erosion of articular cartilage is associated with structural and functional alterations of the extracellular matrix macromolecules and phenotypic changes of the resident chondrocytes. Pathogenesis of OA involves multiple etiologies that contribute to cartilage damage, but the mechanisms of tissue destruction are not fully understood. It is known that proinflammatory cytokines induce chondrocytes to secrete metalloproteases that can cleave most of the cartilage matrix macromolecules (12Andrews H.J. Bunning R.A. Dinarello C.A. Russel R.G.G. Biochim. Biophys. Acta. 1989; 1012: 128-134Crossref PubMed Scopus (32) Google Scholar). IL-1β has been shown to play a key role in this mechanism because it is capable of increasing catabolic activity of chondrocytes and inhibiting their macromolecule synthesis (13van de Loo F.A. Joosten L.A. van Lent P.L. Arntz O.J. van den Berg W.B. Arthritis Rheum. 1995; 38: 164-172Crossref PubMed Scopus (380) Google Scholar, 14Joosten L.A. Helsen M.M. van de Loo F.A. van den Berg W.B. Arthritis Rheum. 1996; 39: 797-809Crossref PubMed Scopus (453) Google Scholar). IL-1β induces the expression of many genes whose promoters are regulated through interacting transcription factors, including NF-κB, AP1, activating transcription factor, or CCAAT/enhancer-binding protein. The cytokine signals via activation of protein kinase cascades involving the mitogen-activated protein kinase or NF-κB pathways (15Stylianou E. Saklatvala J. Biochem. Cell Biol. 1998; 30: 1075-1079Crossref Scopus (199) Google Scholar, 16Robbins J.R. Thomas B. Tan L. Choy B. Arbiser J.L. Berenbaum F. Goldring M.B. Arthritis Rheum. 2000; 43: 2189-2201Crossref PubMed Scopus (103) Google Scholar). In most of the cellular models studied to date, IL-1β was found to initiate a signaling cascade leading to the activation of NF-κB, a homo- or heterodimeric complex composed of p50, p52, p65 (RelA), c-Rel, or RelB proteins, the prototypical p50/p65 heterodimer being generally the effector of the transduction. NF-κB is normally present in the cytoplasm as an inactive element of a complex, with members of the IκB inhibitor protein family. In this complexed form, the nuclear localization sequence found on NF-κB is masked by IκB. Under IL-1β treatment two IκB kinases, IκBα and IκBβ, are activated and phosphorylate IκB at specific serine residues. In the last steps of this signal transduction, the phosphorylated and ubiquitinated IκB is degraded, and the exposed nuclear localization sequence of NF-κB interacts with the nuclear import machinery and then translocates to the nucleus where it binds to its target gene and modulates its transcription (for review, see Ref. 17Mercurio F. Manning A.M. Oncogene. 1999; 18: 6163-6171Crossref PubMed Scopus (359) Google Scholar). The importance of transcriptional regulation of cartilage markers, such as type II collagen gene, has been addressed in the studies of chondrocyte response to cytokines and growth factors, to understand the regulatory mechanisms that govern COL2A1 gene expression and further define new therapeutic approaches in OA treatment (11Chadjichristos C. Ghayor C. Herrouin J.-F. Ala-Kokko L. Suske G. Pujol J.-P. Galéra P. J. Biol. Chem. 2002; 277: 43903-43917Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 18Goldring M.B. Fukuo K. Birkhead J.R. Dudek E. Sandell L.J. J. Cell. Biochem. 1994; 54: 85-99Crossref PubMed Scopus (141) Google Scholar). Earlier studies have demonstrated that IL-1β suppresses type II collagen expression in cultured chondrocytes by reducing the transcriptional activity of the COL2A1 gene (19Pujol J.-P. Loyau G. Life Sci. 1987; 41: 1187-1198Crossref PubMed Scopus (85) Google Scholar, 20Goldring M.B. Birkead J. Kimura T. Krane S.M. J. Clin. 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Invest. 1994; 94: 2307-2316Crossref PubMed Scopus (385) Google Scholar). These data suggest that IL-1β exerts its control on type II collagen production through binding sequences of the promoter and/or first intron regions of the COL2A1 gene. Recent studies on mouse costal chondrocytes and the MC615 mouse chondrocytic cell line also revealed that IL-1β decreases the amounts of COL2A1 mRNA, SOX9 mRNA, and SOX9 protein. NF-κB is involved in SOX9 down-regulation, a mechanism that accounts for the inhibition of mouse SOX9-dependent COL2A1 enhancer elements and subsequent decrease of type II collagen biosynthesis (25Murakami S. Lefebvre V. de Crombrugghe B. J. Biol. Chem. 2000; 275: 3687-3692Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). However, the nature of transcription factors mediating IL-1β inhibition of human COL2A1 gene transcription is not completely elucidated because a recent study on that gene in a human immortalized chondrocytic cell line demonstrated that the cytokine-induced repression of transcription is mediated by two upstream Egr1 binding sites found in the promoter, but the authors cannot exclude the involvement of downstream sequences binding Sp3, which acts as a transcriptional repressor in their system (26Tan L. Peng H. Osaki M. Choy B.K. Auron P.E. Sandell L.J. Goldring M.B. J. Biol. Chem. 2003; 278: 17688-17700Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). We have shown previously that TGF-β1 decreases type II collagen production in primary RAC by a transcriptional regulatory mechanism, which implicates a –41/–33 bp promoter sequence binding Sp1 and Sp3. The repressive effect of the cytokine results from an increase in Sp3/Sp1 ratio which prevents Sp1-induced transactivating effects (11Chadjichristos C. Ghayor C. Herrouin J.-F. Ala-Kokko L. Suske G. Pujol J.-P. Galéra P. J. Biol. Chem. 2002; 277: 43903-43917Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Interestingly, we show here that IL-1β down-regulates COL2A1 gene transcription by the same promoter region and that this effect implies both Sp1 and Sp3. We propose that down-regulation of type II collagen expression under IL-1β and TGF-β1 exposure, two cytokines generally thought to have antagonistic effects on chondrocyte metabolism, involved similar transcriptional regulatory mechanisms that converge on the same complex in which the Sp3/Sp1 ratio is increased. Cell Cultures—RAC were prepared from the shoulders and the knees of 3-week-old rabbits, as described previously (5Ghayor C. Herrouin J.-F. Chadjichristos C. Ala-Kokko L. Takigawa M. Pujol J.-P. Galéra P. J. Biol. Chem. 2000; 275: 27421-27438Abstract Full Text Full Text PDF PubMed Google Scholar). Cells were seeded at 2 × 104 cells/cm2 in 6-well plates, 100-mm dishes, or 75-, 150-, and 175-cm2 flasks and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 IU/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml Fungizone in a 5% CO2 atmosphere. The medium was changed twice a week. Collagen Labeling—To assay newly synthesized collagen, RAC cultures at 80% confluence were preincubated in 10% FCS-containing DMEM in 9.6-cm2 dishes for 15 h in the presence of 50 μg/ml sodium ascorbate. Then, the medium was replaced by the same fresh medium supplemented with 100 μg/ml β-aminopropionitrile and 2 μCi/ml [3H]proline (PerkinElmer Life Sciences) containing 10 ng/ml IL-1β (a generous gift from Dr. Soichiro Sato, Shizuoka, Japan) or not. Cultures were pulsed for 24 h. Then, the culture medium was collected, and the amount of labeled collagen was assayed with pure bacterial collagenase (27Peterkofsky B. Diegelmann R. Biochemistry. 1971; 10: 988-994Crossref PubMed Scopus (1172) Google Scholar). The cell layer-associated collagens were also assayed after scraping and sonication of the cells because that fraction generally contains large amounts of type II collagen (28Galéra P. Rédini F. Vivien D. Bonaventure J. Penfornis H. Loyau G. Pujol J.-P. Exp. Cell Res. 1992; 200: 379-392Crossref PubMed Scopus (83) Google Scholar, 29Galéra P. Vivien D. Pronost S. Bonaventure J. Rédini F. Loyau G. Pujol J.-P. J. Cell. Physiol. 1992; 153: 596-606Crossref PubMed Scopus (75) Google Scholar). Levels of collagen and noncollagenous proteins were corrected to protein amounts determined by the Bradford colorimetric method. RT-PCR Analysis—For RT-PCR analysis, 2 μg of total RNA from primary RAC cultures treated or not with 10 ng/ml IL-1β at 80% of confluence were reverse transcribed into cDNA as described already (8Ghayor C. Chadjichristos C. Herrouin J.-F. Ala-Kokko L. Suske G. Pujol J.-P. Galéra P. J. Biol. Chem. 2001; 276: 36881-36895Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The amplification reaction was carried out using specific sense and antisense primers for COL2A1, Sp1, Sp3, and GAPDH genes as reported previously (11Chadjichristos C. Ghayor C. Herrouin J.-F. Ala-Kokko L. Suske G. Pujol J.-P. Galéra P. J. Biol. Chem. 2002; 277: 43903-43917Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). RT-PCR products were analyzed on a 2% agarose electrophoresis gel in the presence of ethidium bromide. After photography, the intensities of the COL2A1 and GAPDH cDNA bands were quantified by densitometric scanning, using ImageQuant software (Amersham Biosciences). The amount of COL2A1 cDNA was normalized to GAPDH cDNA levels. Transfection Experiments—For luciferase assays, primary proliferative RAC were transiently transfected at 80% of confluence by the calcium phosphate precipitation method using chimeric COL2A1-luciferase reporter vectors and pSV40β-gal plasmid, as described previously (5Ghayor C. Herrouin J.-F. Chadjichristos C. Ala-Kokko L. Takigawa M. Pujol J.-P. Galéra P. J. Biol. Chem. 2000; 275: 27421-27438Abstract Full Text Full Text PDF PubMed Google Scholar, 8Ghayor C. Chadjichristos C. Herrouin J.-F. Ala-Kokko L. Suske G. Pujol J.-P. Galéra P. J. Biol. Chem. 2001; 276: 36881-36895Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). In some experiments, reporter plasmids were also cotranfected with Sp1 and/or Sp3 expression vectors (pEVR2/Sp1 and/or pRC/CMV/Sp3, respectively) as described previously (11Chadjichristos C. Ghayor C. Herrouin J.-F. Ala-Kokko L. Suske G. Pujol J.-P. Galéra P. J. Biol. Chem. 2002; 277: 43903-43917Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The corresponding insertless expression plasmids were used as controls (pEVR2 and pRC/CMV). In these experiments, the pSV40β-gal construct has not been cotransfected because Sp1 is able to increase SV40 promoter activity because of the presence of several Sp1 DNA-binding sites in this promoter as reported already (11Chadjichristos C. Ghayor C. Herrouin J.-F. Ala-Kokko L. Suske G. Pujol J.-P. Galéra P. J. Biol. Chem. 2002; 277: 43903-43917Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In other experiments, a pNF-κB-Luc vector was used. It represents a reporter plasmid harboring five copies of a NF-κB binding site located immediately upstream from a minimal promoter purchased from Stratagene (kindly provided by Dr. B. de Crombrugghe, University of Texas M. D. Anderson Cancer Center). After overnight transfection, the culture medium was changed to a fresh one, and chondrocyte cultures were incubated for a further 24 h in DMEM + 10% FCS containing 10 ng/ml IL-1β or not. At the end of the experiment, cells were harvested, and luciferase and β-galactosidase activities were assayed as described previously (5Ghayor C. Herrouin J.-F. Chadjichristos C. Ala-Kokko L. Takigawa M. Pujol J.-P. Galéra P. J. Biol. Chem. 2000; 275: 27421-27438Abstract Full Text Full Text PDF PubMed Google Scholar). Finally, transcriptional activity was expressed as relative luciferase units after normalization to transfection efficiency and protein amounts as described previously (5Ghayor C. Herrouin J.-F. Chadjichristos C. Ala-Kokko L. Takigawa M. Pujol J.-P. Galéra P. J. Biol. Chem. 2000; 275: 27421-27438Abstract Full Text Full Text PDF PubMed Google Scholar). Decoy Oligonucleotide Assays—For decoy assays, double-stranded oligonucleotides were transfected into RAC cultures in an attempt to interfere with Sp1 and Sp3 binding to their cognate cis-acting elements within the 63-bp short promoter of the COL2A1 gene. The sequences used as a decoy element were a multicopy (two copies) of the –50/+1 wild type (wt) and mutant sequence found in the COL2A1 promoter (Table I). The decoy oligonucleotides were also added in the culture medium during the incubation period with 10 ng/ml IL-1β. 24 h later, cell lysates were prepared and assayed for luciferase and β-galactosidase activities and protein amounts.Table IOligonucleotides used in this studyKROXwt: 5′-CTC TGT ACG CGG GGG CGG TTA GAG-3′KROXmut: 5′-CTC TGT ACG CGG AAG TTG TTA GAG-3′NF-κBwt: 5′-GAT CCG GCT GGA AAA GTC CCC A-3′-49/-28wt: 5′-TCG AAA GGG GCC GGG CGC ATA CG-3′-67/-30wt: 5′-TTG GGC GAG TTC GCC AGC CTC GAA AGG GGC CGG GCG C-3′-35/+1wt: 5′-GGG CGC ATA TAA CGG GCG CCG CGG GGA GAA GAC-3′-35/+1mut: 5′-GGG CGC AGC TAA CGG GCG CCG CGG CGG GGA GAA GAC-3′-50/+1wt: 5′-CCT CGA AAG GGG CCG GGC GCA TAT AAC GGG CGC CGC GGC GGG GAG AAG ACG-3′-50/+1mc wt: 5′-GCC TCG AAA GGG GCC GGG CGC ATA TAA CGG GCG CCG CGG CGG GGA GAA GAC GCC TCG AAA GGG GCC GGG CGC ATA TAA CGG GCG CCG CGG CGG GGA GAA GAC-3′-50/+1mc mut: 5′-GCC TCG AAA GGT TCC TTG CGC ATA TAA CGG GCG CCG CGG CGG GGA GAA GAC GCC TCG AAA GGT TCC TTG CGC ATA TAA CGG GCG CCG CGG CGG GGA GAA GAC-3′-30/+1wt: 5′-TAT AAC GGG CGC CGC GGC GGG GAG AAG ACG-3′-15/+1wt: 5′-CGG CGG GGA GAA GAC G-3′ Open table in a new tab Nuclear Extracts and DNA Binding Analysis—Nuclear extracts were prepared as maxi- or minipreparations (30Dignam J.D. Lebowitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9150) Google Scholar, 31Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 19: 2499Crossref PubMed Scopus (2210) Google Scholar). EMSAs were performed with the oligonucleotides presented in Table I, as described previously (5Ghayor C. Herrouin J.-F. Chadjichristos C. Ala-Kokko L. Takigawa M. Pujol J.-P. Galéra P. J. Biol. Chem. 2000; 275: 27421-27438Abstract Full Text Full Text PDF PubMed Google Scholar, 8Ghayor C. Chadjichristos C. Herrouin J.-F. Ala-Kokko L. Suske G. Pujol J.-P. Galéra P. J. Biol. Chem. 2001; 276: 36881-36895Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 11Chadjichristos C. Ghayor C. Herrouin J.-F. Ala-Kokko L. Suske G. Pujol J.-P. Galéra P. J. Biol. Chem. 2002; 277: 43903-43917Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In the antibody interference assays, 1 μl of anti-Sp1, anti-Sp3, anti-p50, anti-p65, anti-Smad3, anti-AP2, and anti-Egr2 antibodies (Santa Cruz) was added to each reaction mixture for 15–20 min at room temperature then 15 min at 4 °C. The probe was finally added to the binding reaction, a further 15-min incubation at room temperature was performed, and the samples were finally electrophoresed on a polyacrylamide gel that was processed by autoradiography (5Ghayor C. Herrouin J.-F. Chadjichristos C. Ala-Kokko L. Takigawa M. Pujol J.-P. Galéra P. J. Biol. Chem. 2000; 275: 27421-27438Abstract Full Text Full Text PDF PubMed Google Scholar). For DNase I footprint experiments, the SmaI-HindIII fragment of the pGL2-0.110kb plasmid was end labeled to its 5′-SmaI extremity. Further processing of the probe, including gel purification and elution, were performed as described previously (5Ghayor C. Herrouin J.-F. Chadjichristos C. Ala-Kokko L. Takigawa M. Pujol J.-P. Galéra P. J. Biol. Chem. 2000; 275: 27421-27438Abstract Full Text Full Text PDF PubMed Google Scholar, 8Ghayor C. Chadjichristos C. Herrouin J.-F. Ala-Kokko L. Suske G. Pujol J.-P. Galéra P. J. Biol. Chem. 2001; 276: 36881-36895Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Western Blotting—Western blot analyses of Sp1 and Sp3 were performed on RAC nuclear extracts treated with 10 ng/ml IL-1β or not as previously described (8Ghayor C. Chadjichristos C. Herrouin J.-F. Ala-Kokko L. Suske G. Pujol J.-P. Galéra P. J. Biol. Chem. 2001; 276: 36881-36895Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). A Short COL2A1 63-Base Pair Proximal Promoter Mediates IL-1β-induced Inhibition of Type II Collagen Expression in Articular Chondrocytes—We first determined the effect of IL-1β on collagen biosynthesis in RAC primary cultures. Newly synthesized collagens were assayed after [3H]proline labeling of chondrocytes, treated or not with the cytokine (10 ng/ml). IL-1β treatment was found to decrease total collagen neosynthesis (essentially type II collagen) by 55% (Fig. 1A). The reduction was greater in the cell layer-associated fraction because this latter contains the major part of neosynthesized type II collagen. To determine whether IL-1β-decreased type II collagen synthesis was accompanied by a similar effect at the transcriptional level, steady-state levels of COL2A1 mRNA were estimated by semiquantitative RT-PCR performed on total RNA extracts of proliferating primary RAC treated or not with IL-1β. As shown in Fig. 1B, the levels of COL2A1 mRNA were reduced by ∼40% under IL-1β treatment of RAC cultures. To investigate further the molecular mechanisms whereby IL-1β down-regulates type II procollagen production and COL2A1 mRNA levels, the transcriptional activity of the human COL2A1 gene constructs containing deletions in both promoter and/or first intron regions was assayed. Transient cotransfections performed on RAC cultures showed that IL-1β inhibits the transcriptional activity of all of these constructs, except for the shortest construction, which contains only 35 bp upstream the transcription start site (Fig. 1C). These results suggest that a –63/–35 bp sequence of the proximal promoter mediates IL-1β-induced inhibition of COL2A1 gene transcription. Delineation of the DNA Binding Sites That Mediate IL-1β Inhibitory Effect on the –63/–35 Base Pair Fragment of the COL2A1 Promoter—To determine cis-acting elements that mediate the inhibitory effect of IL-1β on type II collagen expression, DNase I footprinting assays were performed on the 63-bp proximal promoter, using nuclear extracts from primary RAC treated or not with the cytokine. As shown in Fig. 2A, a region of the 63-bp fragment was protected by proteins present in nuclear extract from proliferating RAC. The protected area is located between –35 and –63 bp. Sequences within –8/–63 bp also bound Sp1 recombinant protein. A similar protection intensity was observed whatever nuclear extracts from control RAC or IL-1β-treated cells were used. To identify the transcription factors interacting with this proximal promoter, EMSA analysis was performed using the wild type oligonucleotidic sequences identified in DNase I footprint experiments on the 63-bp promoter fragment and their mutant counterparts (Table I). Three wild type double-stranded labeled oligonucleotides were generated and incubated with nuclear extracts from primary RAC. As shown in Fig. 2B, three major complexes called a, b, and c were formed upon incubation with the –50/+1wt probe when the binding reaction was performed with control nuclear extracts. Only complexes a and b are detected with the –67/–30wt probe. DNA binding activity of the transcription factors involved in a complex is decreased when nuclear extracts from RAC treated with IL-1β were used. Complex c is nonspecific as we demonstrated previously (11Chadjichristos C. Ghayor C. Herrouin J.-F. Ala-Kokko L. Suske G. Pujol J.-P. Galéra P. J. Biol. Chem. 2002; 277: 43903-43917Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) because it is not always detected, even when the same RAC nuclear extracts and the same probes are used (see Fig. 2, C and E). With the –35/+1wt probe, only complex b was formed, and IL-1β treatment of the RAC did not modulate DNA binding activity of this complex, suggesting that b does not have a functional transcriptional effect, as we have already found for the TGF-β1 effect, and that b could bound nonspecifically (11Chadjichristos C. Ghayor C. Herrouin J.-F. Ala-Kokko L. Suske G. Pujol J.-
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