Potent Inhibition of the Master Chondrogenic FactorSox9 Gene by Interleukin-1 and Tumor Necrosis Factor-α
2000; Elsevier BV; Volume: 275; Issue: 5 Linguagem: Inglês
10.1074/jbc.275.5.3687
ISSN1083-351X
AutoresShunichi Murakami, Véronique Lefebvre, Benoît De Crombrugghe,
Tópico(s)Cell Adhesion Molecules Research
ResumoThe inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) strongly inhibit the expression of genes for cartilage extracellular matrix proteins. We have recently obtained genetic evidence indicating that the high mobility group domain containing transcription factor Sox9 is required for cartilage formation and for expression of chondrocyte-specific genes including the gene for type II collagen (Col2a1). We show here that IL-1 and TNF-α cause a marked and rapid decrease in the levels ofSox9 mRNA and/or protein in chondrocytes. A role for the transcription factor NFκB in Sox9 down-regulation was suggested by the ability of pyrrolidine dithiocarbamate, an inhibitor of the NFκB pathway, to block the effects of IL-1 and TNF-α. This role was further supported by the ability of a dominant-negative mutant of IκBα to block the IL-1 and TNF-α inhibition of Sox9-dependent Col2a1 enhancer elements. Furthermore, forced expression of the NFκB subunits p65 or p50 also inhibited Sox9-dependent Col2a1 enhancer. Because Sox9 is essential for chondrogenesis, the marked down-regulation of the Sox9 gene by IL-1 and TNF-α in chondrocytes is sufficient to account for the inhibition of the chondrocyte phenotype by these cytokines. The down-regulation ofSox9 may have a crucial role in inhibiting expression of the cartilage phenotype in inflammatory joint diseases. The inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) strongly inhibit the expression of genes for cartilage extracellular matrix proteins. We have recently obtained genetic evidence indicating that the high mobility group domain containing transcription factor Sox9 is required for cartilage formation and for expression of chondrocyte-specific genes including the gene for type II collagen (Col2a1). We show here that IL-1 and TNF-α cause a marked and rapid decrease in the levels ofSox9 mRNA and/or protein in chondrocytes. A role for the transcription factor NFκB in Sox9 down-regulation was suggested by the ability of pyrrolidine dithiocarbamate, an inhibitor of the NFκB pathway, to block the effects of IL-1 and TNF-α. This role was further supported by the ability of a dominant-negative mutant of IκBα to block the IL-1 and TNF-α inhibition of Sox9-dependent Col2a1 enhancer elements. Furthermore, forced expression of the NFκB subunits p65 or p50 also inhibited Sox9-dependent Col2a1 enhancer. Because Sox9 is essential for chondrogenesis, the marked down-regulation of the Sox9 gene by IL-1 and TNF-α in chondrocytes is sufficient to account for the inhibition of the chondrocyte phenotype by these cytokines. The down-regulation ofSox9 may have a crucial role in inhibiting expression of the cartilage phenotype in inflammatory joint diseases. interleukin tumor necrosis factor-α base pair(s) pyrrolidine dithiocarbamate Cartilage is a highly specialized connective tissue with distinct biochemical and biomechanical properties. Its extracellular matrix is composed of a series of proteins such as collagen types II, IX, and XI; link protein; and aggrecan. The coordinated regulation of the genes for these proteins is likely to be essential for normal skeletal development and maintenance of cartilage in postnatal life, as mutations in these molecules lead to chondrodysplasias and degenerative joint diseases (1.Spranger J. Winterpacht A. Zabel B. Eur. J. Pediatr. 1994; 153: 56-65PubMed Google Scholar, 2.Vikkula M. Mariman E.C. Lui V.C. Zhidkova N.I. Tiller G.E. Goldring M.B. van Beersum S.E. de Waal Malefijt M.C. van den Hoogen F.H. Ropers H.H. Mayne R. Cheah K.S.E. Olsen B.R. Warman M.L. Brunner H.G. Cell. 1995; 80: 431-437Abstract Full Text PDF PubMed Scopus (305) Google Scholar, 3.Muragaki Y. Mariman E.C. van Beersum S.E. Perälä M. van Mourik J.B. Warman M.L. Olsen B.R. Hamel B.C. Nat. Genet. 1996; 12: 103-105Crossref PubMed Scopus (173) Google Scholar, 4.Richards A.J. Yates J.R. Williams R. Payne S.J. Pope F.M. Scott J.D. Snead M.P. Hum. Mol. Genet. 1996; 5: 1339-1343Crossref PubMed Scopus (224) Google Scholar, 5.Kuivaniemi H. Tromp G. Prockop D.J. Hum. Mutat. 1997; 9: 300-315Crossref PubMed Scopus (280) Google Scholar).Sox9 is a transcription factor with a high mobility group DNA-binding domain that is expressed in all prechondrocytic and chondrocytic cells during embryonic development in a pattern that closely parallels that of the gene for type II collagen (Col2a1) (6.Zhao Q. Eberspaecher H. Lefebvre V. de Crombrugghe B. Dev. Dyn. 1997; 209: 377-386Crossref PubMed Scopus (434) Google Scholar, 7.Ng L.J. Wheatley S. Muscat G.E. Conway-Campbell J. Bowles J. Wright E. Bell D.M. Tam P.P. Cheah K.S. Koopman P. Dev. Biol. 1997; 183: 108-121Crossref PubMed Scopus (556) Google Scholar). In humans, heterozygous mutations in and around the SOX9 gene cause campomelic dysplasia, a disease that is characterized by anomalies in a number of skeletal structures and is also often associated with XY sex reversal (8.Wagner T. Wirth J. Meyer J. Zabel B. Held M. Zimmer J. Pasantes J. Bricarelli F.D. Keutel J. Hustert E. Wolf U. Tommerupd N. Schempp W. Scherer G. Cell. 1994; 79: 1111-1120Abstract Full Text PDF PubMed Scopus (1267) Google Scholar, 9.Foster J.W. Dominguez-Steglich M.A. Guioli S. Kowk G. Weller P.A. Stevanovic M. Weissenbach J. Mansour S. Young I.D. Goodfellow P.N. Brook J.D. Schafer A.J. Nature. 1994; 372: 525-530Crossref PubMed Scopus (1303) Google Scholar, 10.Cameron F.J. Hageman R.M. Cooke-Yarborough C. Kwok C. Goodwin L.L. Sillence D.O. Sinclair A.H. Hum. Mol. Genet. 1996; 5: 1625-1630Crossref PubMed Scopus (72) Google Scholar, 11.Südbeck P. Schmitz M.L. Baeuerle P.A. Scherer G. Nat. Genet. 1996; 13: 230-232Crossref PubMed Scopus (184) Google Scholar). The disease is thought to be due to SOX9 haploinsufficiency, i.e. 50% of SOX9 being insufficient to fulfill the physiological function of SOX9. Recent work from our laboratory based on mouse embryo chimeras derived from Sox9homozygous mutant embryonic stem cells obtained by gene targeting has demonstrated that Sox9 is a master regulatory factor for chondrocyte differentiation. Indeed, in these mouse embryo chimeras,Sox9 −/− mutant cells were blocked in their differentiation to become chondrocytes and persisted as mesenchymal cells; these cells were unable to express the genes for chondrocyte-specific markers such as collagen types II, IX, and XI and aggrecan (Col2a1, Col11a2, Col9a2, andaggrecan). In addition, no cartilages were formed in teratomas derived from Sox9 homozygous mutant embryonic stem cells, although the other types of tissues normally present in these tumors were formed (12.Bi W. Deng J.M. Zhang Z. Behringer R.R. de Crombrugghe B. Nat. Genet. 1999; 22: 85-89Crossref PubMed Scopus (1378) Google Scholar). SOX9 binds to and activates chondrocyte-specific enhancer elements in the Col2a1 andCol11a2 genes and ectopic expression of SOX9 in transgenic mice activates the endogenous Col2a1 gene, providing evidence that these genes are direct targets for Sox9 (13.Lefebvre V. Huang W. Harley V.R. Goodfellow P.N. de Crombrugghe B. Mol. Cell. Biol. 1997; 17: 2336-2346Crossref PubMed Google Scholar, 14.Bridgewater L.C. Lefebvre V. de Crombrugghe B. J. Biol. Chem. 1998; 273: 14998-15006Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 15.Bell D.M. Leung K.K. Wheatley S.C. Ng L.J. Zhou S. Ling K.W. Sham M.H. Koopman P. Tam P.P. Cheah K.S. Nat. Genet. 1997; 16: 174-178Crossref PubMed Scopus (760) Google Scholar).Previous studies have shown that interleukin-1 (IL-1)1 and tumor necrosis factor-α (TNF-α) are potent inhibitors of the chondrocyte phenotype. Indeed, the expression of cartilage-specific genes such as those for collagen types II, IX, and XI and aggrecan is inhibited by both IL-1 and TNF-α (16.Goldring M.B. Birkhead J. Sandell L.J. Kimura T. Krane S.M. J. Clin. Invest. 1988; 82: 2026-2037Crossref PubMed Scopus (344) Google Scholar, 17.Lefebvre V. Peeters-Joris C. Vaes G. Biochim. Biophys. Acta. 1990; 1052: 366-378Crossref PubMed Scopus (225) Google Scholar, 18.Lum Z.P. Hakala B.E. Mort J.S. Recklies A.D. J. Cell. Physiol. 1996; 166: 351-359Crossref PubMed Scopus (61) Google Scholar, 19.Bolton M.C. Dudhia J. Bayliss M.T. Biochem. J. 1996; 319: 489-498Crossref PubMed Scopus (36) Google Scholar). These inhibitory effects have been implicated in the breakdown of cartilage in arthritis, since IL-1 and TNF-α are produced by synovial cells in arthritic lesions and are present at elevated levels in synovial fluid in osteoarthritis and rheumatoid arthritis (20.Fontana A. Hengartner H. Weber E. Fehr K. Grob P.J. Cohen G. Rheumatol. Int. 1982; 2: 49-53Crossref PubMed Scopus (253) Google Scholar, 21.Wood D.D. Ihrie E.J. Dinarello C.A. Cohen P.L. Arthritis Rheum. 1983; 26: 975-983Crossref PubMed Scopus (391) Google Scholar, 22.Miossec P. Dinarello C.A. Ziff M. Arthritis Rheum. 1986; 29: 461-470Crossref PubMed Scopus (86) Google Scholar, 23.Husby G. Williams Jr., R.C. J. Autoimmun. 1988; 1: 363-371Crossref PubMed Scopus (103) Google Scholar, 24.Yocum D.E. Esparza L. Dubry S. Benjamin J.B. Volz R. Scuderi P. Cell Immunol. 1989; 122: 131-145Crossref PubMed Scopus (93) Google Scholar). In addition, IL-1 and TNF-α stimulate the synthesis of protein-degrading enzymes such as collagenases and stromelysins in cartilage (25.Okada Y. Shinmei M. Tanaka O. Naka K. Kimura A. Nakanishi I. Bayliss M.T. Iwata K. Nagase H. Lab. Invest. 1992; 66: 680-690PubMed Google Scholar, 26.Chubinskaya S Huch K. Mikecz K. Cs-Szabo G. Hasty K.A. Kuettner K.E. Cole A.A. Lab. Invest. 1996; 74: 232-240PubMed Google Scholar, 27.Mitchell P.G. Magna H.A. Reeves L.M. Lopresti-Morrow L.L. Yocum S.A. Rosner P.J. Geoghegan K.F. Hambor J.E. J. Clin. Invest. 1996; 97: 761-768Crossref PubMed Scopus (820) Google Scholar, 28.Reboul P. Pelletier J.P. Tardif G. Cloutier J.M. Martel-Pelletier J. J. Clin. Invest. 1996; 97: 2011-2019Crossref PubMed Scopus (421) Google Scholar, 29.Ohta S. Imai K. Yamashita K. Matsumoto T. Azumano I. Okada Y. Lab. Invest. 1998; 78: 79-87PubMed Google Scholar). Binding of IL-1 and TNF-α to their receptors activates several signaling pathways, including the NFκB and AP-1 pathways. While IL-1 and TNF-α signals that lead to AP-1 activation have been implicated in the up-regulation of metalloproteinase genes (30.Brenner D.A. O'Hara M. Angel P. Chojkier M. Karin M. Nature. 1989; 337: 661-663Crossref PubMed Scopus (609) Google Scholar, 31.Conca W. Kaplan P.B. Krane S.M. J. Clin. Invest. 1989; 83: 1753-1757Crossref PubMed Scopus (85) Google Scholar, 32.Sirum-Connolly K. Brinckerhoff C.E. Nucleic Acids Res. 1991; 19: 335-341Crossref PubMed Scopus (72) Google Scholar), little is known about the mechanisms whereby these cytokines inhibit expression of the chondrocyte phenotype.We show here that IL-1 and TNF-α markedly down-regulate the expression of Sox9 in chondrocytes. The activity of aCol2a1 chondrocyte-specific enhancer, which is dependent on an intact SOX9-binding site, was also strongly inhibited by these cytokines. We present evidence that these effects are both mediated by the NFκB pathway. We hypothesize that in inflammatory joint diseases, such as rheumatoid arthritis, a major mechanism by which IL-1 and TNF-α inhibit the chondrocyte phenotype is by down-regulation ofSox9, a master cartilage regulatory gene.MATERIALS AND METHODSRecombinant human IL-1β was obtained from the National Cancer Institute. Recombinant human TNF-α was purchased from PeproTech Inc. (Rocky Hill, NJ). 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole and pyrrolidine dithiocarbamate (PDTC) were purchased from Sigma.Cell CultureThe mouse chondrocytic cell line MC615 was kindly provided by Drs. Frédéric Mallein-Gerin and Bjorn R. Olsen (33.Mallein-Gerin F. Olsen B.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3289-3293Crossref PubMed Scopus (66) Google Scholar). Mouse costal chondrocytes were obtained from 1–5-day-old mice and cultured as described previously (34.Lefebvre V. Garofalo S. Zhou G. Metsäranta M. Vuorio E. de Crombrugghe B. Matrix Biol. 1994; 14: 329-335Crossref PubMed Scopus (135) Google Scholar).RNA Preparation and Northern AnalysisTotal cellular RNA was extracted from cultures with the modified guanidium thiocyanate-phenol-chloroform method described by Chomczynski and Sacchi (35.Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62983) Google Scholar) using the Trizol reagent (Life Technologies, Inc.). Aliquots of 10 or 20 μg of RNA/lane were fractionated by electrophoresis on 1% agarose gels containing 0.22 mformaldehyde, transferred onto nylon filters (Zeta-Probe GT, Bio-Rad) by capillary blotting, and cross-linked to the filters by exposure to ultraviolet light. The filters were prehybridized for 20 min in hybridization solution containing 0.25 m sodium phosphate buffer, pH 7.2, and 7% SDS. The Sox9 and Col2a1probes were as described previously (13.Lefebvre V. Huang W. Harley V.R. Goodfellow P.N. de Crombrugghe B. Mol. Cell. Biol. 1997; 17: 2336-2346Crossref PubMed Google Scholar, 36.Metsäranta M. Toman D. de Crombrugghe B. Vuorio E. Biochim. Biophys. Acta. 1991; 1089: 241-243Crossref PubMed Scopus (142) Google Scholar). The 18 S rRNA probe was from Ambion (Austin, TX). These probes were radiolabeled by random primer method using Klenow fragment (Roche Molecular Biochemicals) and [α-32P]dCTP (NEN Life Science Products) as described by Feinberg and Vogelstein (37.Feinberg A.P. Vogelstein B. Anal. Biochem. 1983; 132: 6-13Crossref PubMed Scopus (16567) Google Scholar) to a specific activity greater than 108 cpm/μg DNA. Hybridization was performed in hybridization solution supplemented with 1 × 106cpm/ml of denatured 32P-labeled probe for 16 h at 65 °C. Filters were washed in 2× SSC (1× SSC: 150 mmNaCl, 15 mm sodium citrate), 0.1% SDS at 65 °C for 20 min, followed by 20 min of washing in 0.2× SSC, 0.1% SDS at 65 °C. The filters were subjected to autoradiography using x-ray films (Fuji Photo Film Co., Minamiashigara, Japan) and intensifying screens (DuPont) at −80 °C. Hybridization signals were quantified by scanning densitometry (Intelligent Quantifier, Bio Image Systems Corp., Ann Arbor, MI). Col2a1 and Sox9 mRNA levels were corrected for RNA loading by normalization with 18 S rRNA levels.Western Blot AnalysisMouse primary chondrocytes were cultured as indicated in figure legends. Total cell lysates were prepared in a buffer containing 20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 2 mm EDTA, 1% Triton X-100, 5 μg/ml aprotinin, 5 μg/ml leupeptin, 5 μg/ml pepstatin, and 1 mm phenylmethylsulfonyl fluoride. Protein concentration was determined by the Bradford method (38.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213377) Google Scholar), and 40 μg of each sample was separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (39.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206024) Google Scholar). The proteins were electrophoretically transferred to nitrocellulose filters (Protran, Schleicher & Schuell) as described previously (40.Burnette W.N. Anal. Biochem. 1981; 112: 195-203Crossref PubMed Scopus (5867) Google Scholar). The filters were blocked overnight in 5% nonfat dry milk in Tris-buffered saline, pH 7.5, containing 0.1% Tween 20 and then incubated with antibodies against p50 (H-119, Santa Cruz Biotechnology), p65 (Rockland Inc., Gilbertsville, PA), IκBα (H-4, Santa Cruz Biotechnology), or SOX9. The SOX9 antibody was as described previously (13.Lefebvre V. Huang W. Harley V.R. Goodfellow P.N. de Crombrugghe B. Mol. Cell. Biol. 1997; 17: 2336-2346Crossref PubMed Google Scholar). Filters were then incubated with horseradish peroxidase-conjugated donkey anti-rabbit IgG or sheep anti-mouse IgG (Amersham Pharmacia Biotech) for 1 h, and the signal was detected by autoradiography using ECL (Amersham Pharmacia Biotech).Transient TransfectionCol2a1-luciferase constructions harboring 12 tandem copies of the 18-bp and 4 tandem copies of the 48-bp enhancer elements were described previously (41.Lefebvre 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). Two tandem copies of the 231-bp Col2a1 intron 1 fragment (+2113 to +2343) (42.Mukhopadhyay K. Lefebvre V. Zhou G. Garofalo S. Kimura J.H. de Crombrugghe B. J. Biol. Chem. 1995; 270: 27711-27719Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar) were cloned in the p309Col2a1-βgeo vector as described previously for other enhancer elements (43.Zhou G. Garofalo S. Mukhopadhyay K. Lefebvre V. Smith C.N. Eberspaecher H. de Crombrugghe B. J. Cell Sci. 1995; 108: 3677-3684Crossref PubMed Google Scholar). pNFκB-Luc, a reporter construct harboring 5 copies of a NFκB binding site immediately upstream of a minimal promoter, was purchased from Stratagene. RSV-p50 and RSV-p65 plasmids (44.Duckett C.S. Perkins N.D. Kowalik T.F. Schmid R.M. Huang E.S. Baldwin Jr., A.S. Nabel G.J. Mol. Cell. Biol. 1993; 13: 1315-1322Crossref PubMed Google Scholar) encoding the p50 and p65 NFκB subunits were generous gifts from Dr. Gary J. Nabel (University of Michigan Medical Center, Ann Arbor, MI). pCMX-IκBαM encoding the dominant-negative mutant of IκBα (45.Van Antwerp D.J. Martin S.J. Kafri T. Green D.R. Verma I.M. Science. 1996; 274: 787-789Crossref PubMed Scopus (2440) Google Scholar, 46.Wang C.Y. Mayo M.W. Baldwin Jr., A.S. Science. 1996; 274: 784-787Crossref PubMed Scopus (2499) Google Scholar) under the control of the cytomegalovirus promoter was kindly provided by Dr. Inder M. Verma (Salk Institute for Biological Studies, La Jolla, CA). Primary chondrocytes were transiently transfected using the FuGene6 transfection reagent (Roche Molecular Biochemicals) according to the manufacturer's instructions. Briefly, 1.7 μl of FuGene6 was mixed with a total of 600–750 ng of plasmid DNA in 50 μl of standard medium. The mixture was preincubated for 15 min and added to preestablished monolayers of 3 × 105cells/4-cm2 well. Cells were harvested after 44–48 h of transfection. Reporter plasmids and pSVβgal, pSV2βgal, and pGL3-Promoter (Promega Corp., Madison, WI), used as an internal control for transfection efficiency, were cotransfected in a 3:1 ratio. Luciferase and β-galactosidase activities were assayed as described previously (41.Lefebvre 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). Promoter activities were corrected for transfection efficiency.DISCUSSIONIn our study, both IL-1 and TNF-α markedly decreased expression of Sox9 in chondrocytic cells. This inhibition was very likely transcriptional, since IL-1 had no effect on the half-life ofSox9 mRNA when mRNA synthesis initiation was blocked. Since the levels of Sox9 mRNA returned to normal levels after IL-1 withdrawal (data not shown), it is likely that the IL-1 inhibition of Sox9 expression was not due to nonspecific effects. The cytokine-mediated decrease inCol2a1 mRNA levels occurred more slowly, probably because the half-life of Col2a1 mRNA is longer than that of the Sox9 transcript. Since our recent genetic results have demonstrated that homozygous inactivation of the Sox9gene prevents chondrocyte differentiation and Col2a1expression (12.Bi W. Deng J.M. Zhang Z. Behringer R.R. de Crombrugghe B. Nat. Genet. 1999; 22: 85-89Crossref PubMed Scopus (1378) Google Scholar), the marked IL-1 and TNF-α down-regulation ofSox9 should be sufficient to account for the inhibition ofCol2a1 expression by these cytokines.Other previous studies from our laboratory showed that SOX9 binds to a sequence essential for chondrocyte-specific enhancer activity within a 48-bp Col2a1 enhancer and a 18-bp subsegment of that enhancer (13.Lefebvre V. Huang W. Harley V.R. Goodfellow P.N. de Crombrugghe B. Mol. Cell. Biol. 1997; 17: 2336-2346Crossref PubMed Google Scholar). SOX9 also activated these enhancers in co-transfection experiments of nonchondrocytic cells, whereas a mutation that prevented SOX9 binding to these enhancers abolished their activity in chondrocytes. We, therefore, used the activity of these enhancers as a functional measurement of Sox9. Inhibition of these Col2a1enhancer elements by IL-1 and TNF-α in chondrocytic cells was very likely due to the marked decrease in Sox9 levels produced by these cytokines. In support of this hypothesis, we found that transfection of a SOX9-expressing plasmid in chondrocytes overcame the inhibitory effect of IL-1 on the activity of the 48- and 18-bp enhancers.Several lines of evidence strongly suggested that NFκB mediates the IL-1/TNF-α down-regulation of Sox9. First, addition of PDTC, an inhibitor of IκB degradation, blocked IL-1 and TNF-α inhibition of Sox9 expression. This inhibitor also blocked the effects of IL-1 and TNF-α on the Sox9-dependent 48-bpCol2a1 enhancer (data not shown). Second, expression of a dominant-negative IκBα mutant abolished the IL-1 and TNF-α inhibition of both the 48- and 18-bp SOX9-dependent cartilage-specific Col2a1 enhancers. Finally, forced expression of the p50 and p65 NFκB subunits mimicked the IL-1 inhibition of the enhancer. Interestingly, the expression of the dominant-negative IκBα mutant increased the activity of the chondrocyte-specific enhancer in the absence of IL-1. One possible explanation for this increase might be that the dominant-negative IκBα mutant inhibited endogenous NFκB activity.The precise mechanism whereby NFκB inhibits Sox9expression remains to be elucidated. One possible mechanism is that NFκB binds to regulatory sequences of the Sox9 gene. This possibility is suggested by the role of Drosophila homolog of NFκB, Dorsal, in repressing zen, dpp, andtolloid by binding to κB-like motifs in the regulatory sequences in these genes (58.Ip Y.T. Kraut R. Levine M. Rushlow C.A. Cell. 1991; 64: 439-446Abstract Full Text PDF PubMed Scopus (152) Google Scholar, 59.Kirov N. Childs S. O'Connor M. Rushlow C. Mol. Cell. Biol. 1994; 14: 713-722Crossref PubMed Google Scholar, 60.Huang J.D. Dubnicoff T. Liaw G.J. Bai Y. Valentine S.A. Shirokawa J.M. Lengyel J.A. Courey A.J. Genes Dev. 1995; 9: 3177-3189Crossref PubMed Scopus (77) Google Scholar). Moreover, in the developing chick limb bud, c-Rel, another subunit of the NFκB family, inhibits expression of the gene for BMP-4, a vertebrate homolog of dpp, indicating the conservation of this pathway in vertebrates (61.Bushdid P.B. Brantley D.M. Yull F.E. Blaeuer G.L. Hoffman L.H. Niswander L. Kerr L.D. Nature. 1998; 392: 615-618Crossref PubMed Scopus (149) Google Scholar). Alternatively, activation of NFκB could lead to the expression of a repressor for the Sox9 gene or the activation of such a repressor. The identification and detailed analysis of Sox9regulatory elements should help clarify the mechanism ofSox9 inhibition by IL-1 and TNF-α.IL-1 and TNF-α inhibit expression of a number of other genes encoding chondrocyte-specific matrix molecules including collagen types IX and XI and aggrecan (16.Goldring M.B. Birkhead J. Sandell L.J. Kimura T. Krane S.M. J. Clin. Invest. 1988; 82: 2026-2037Crossref PubMed Scopus (344) Google Scholar, 17.Lefebvre V. Peeters-Joris C. Vaes G. Biochim. Biophys. Acta. 1990; 1052: 366-378Crossref PubMed Scopus (225) Google Scholar, 18.Lum Z.P. Hakala B.E. Mort J.S. Recklies A.D. J. Cell. Physiol. 1996; 166: 351-359Crossref PubMed Scopus (61) Google Scholar, 19.Bolton M.C. Dudhia J. Bayliss M.T. Biochem. J. 1996; 319: 489-498Crossref PubMed Scopus (36) Google Scholar). The pronounced inhibition of Sox9by these cytokines, together with our recent observation that in mouse chimeric embryos Sox9 null cells are unable to express these markers, strongly suggest that down-regulation of Sox9 is sufficient to account for the inhibition of expression of these genes. Given that IL-1 and TNF-α are present at elevated levels in arthritic joints (20.Fontana A. Hengartner H. Weber E. Fehr K. Grob P.J. Cohen G. Rheumatol. Int. 1982; 2: 49-53Crossref PubMed Scopus (253) Google Scholar, 21.Wood D.D. Ihrie E.J. Dinarello C.A. Cohen P.L. Arthritis Rheum. 1983; 26: 975-983Crossref PubMed Scopus (391) Google Scholar, 22.Miossec P. Dinarello C.A. Ziff M. Arthritis Rheum. 1986; 29: 461-470Crossref PubMed Scopus (86) Google Scholar, 24.Yocum D.E. Esparza L. Dubry S. Benjamin J.B. Volz R. Scuderi P. Cell Immunol. 1989; 122: 131-145Crossref PubMed Scopus (93) Google Scholar), the inhibition of expression of the master regulatory Sox9 gene could account for the poor healing capacity of cartilage in arthritis.IL-1 and TNF-α also activate other cellular pathways distinct from the NFκB pathway that can result in changes in expression or activity of transcription factors (30.Brenner D.A. O'Hara M. Angel P. Chojkier M. Karin M. Nature. 1989; 337: 661-663Crossref PubMed Scopus (609) Google Scholar, 31.Conca W. Kaplan P.B. Krane S.M. J. Clin. Invest. 1989; 83: 1753-1757Crossref PubMed Scopus (85) Google Scholar, 32.Sirum-Connolly K. Brinckerhoff C.E. Nucleic Acids Res. 1991; 19: 335-341Crossref PubMed Scopus (72) Google Scholar). This notion is illustrated by the following results. The activity of a construct containing a 309-bpCol2a1 promoter and two tandem copies of a 231-bpCol2a1 enhancer that includes the 48-bp enhancer is chondrocyte-specific and dependent on SOX9; indeed, a 10-bp deletion in this 231-bp element that removed the SOX9 binding site resulted in loss of activity of this enhancer (13.Lefebvre V. Huang W. Harley V.R. Goodfellow P.N. de Crombrugghe B. Mol. Cell. Biol. 1997; 17: 2336-2346Crossref PubMed Google Scholar). Although IL-1 strongly inhibited the activity of this enhancer construct, this down-regulation was only partially overcome by cotransfection with a SOX9-expressing vector. In addition, coexpression of a dominant-negative IκBα mutant did not block the down-regulation of this promoter-enhancer construct (data not shown). Thus, in addition to their inhibition of Sox9, both IL-1 and TNF-α may affect the expression or activity of other transcription factors controlling the activity of the construct containing this 231-bp enhancer by mechanisms that are independent of the NFκB pathway. However, because Sox9 is completely required for expression of Col2a1 and other chondrocyte marker genes, the almost complete down-regulation of its expression by cytokines is sufficient to account for the IL-1 and TNF-α inhibition of the chondrocyte phenotype.Although IL-1 and TNF-α are not thought to have major roles in skeletal development, since mice deficient in IL-1β, TNF-α, or their receptors did not show any skeletal phenotype (62.Rothe J. Lesslauer W. Lötscher H. Lang Y. Koebel P. Köntgen F. Althage A. Zinkernagel R. Steinmetz M. Bluethmann H. Nature. 1993; 364: 798-802Crossref PubMed Scopus (1148) Google Scholar, 63.Shornick L.P. de Togni P. Mariathasan S. Goellner J. Strauss- Schoenberger J. Karr R.W. 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Further studies on the role of the NFκB pathway in regulating Sox9 may provide insights into the mechanisms of cartilage degeneration in arthritis. Cartilage is a highly specialized connective tissue with distinct biochemical and biomechanical properties. Its extracellular matrix is composed of a series of proteins such as collagen types II, IX, and XI; link protein; and aggrecan. The coordinated regulation of the genes for these proteins is likely to be essential for normal skeletal development and maintenance of cartilage in postnatal life, as mutations in these molecules lead to chondrodysplasias and degenerative joint diseases (1.Spranger J. Winterpacht A. Zabel B. Eur. J. Pediatr. 1994; 153: 56-65PubMed Google Scholar, 2.Vikkula M. Mariman E.C. Lui V.C. Zhidkova N.I. Tiller G.E. Goldring M.B. van Beersum S.E. de Waal Malefijt M.C. van den Hoogen F.H. Ropers H.H. Mayne R. Cheah K.S.E. Olsen B.R. Warman M.L. Brunner H.G. 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