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J Biol Chem, Vol. 275, Issue 5, 3687-3692, February 4, 2000


Potent Inhibition of the Master Chondrogenic Factor Sox9 Gene by Interleukin-1 and Tumor Necrosis Factor-alpha *

Shunichi Murakami, Véronique LefebvreDagger , and Benoit de Crombrugghe§

From the Department of Molecular Genetics, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha ) 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-alpha cause a marked and rapid decrease in the levels of Sox9 mRNA and/or protein in chondrocytes. A role for the transcription factor NFkappa B in Sox9 down-regulation was suggested by the ability of pyrrolidine dithiocarbamate, an inhibitor of the NFkappa B pathway, to block the effects of IL-1 and TNF-alpha . This role was further supported by the ability of a dominant-negative mutant of Ikappa Balpha to block the IL-1 and TNF-alpha inhibition of Sox9-dependent Col2a1 enhancer elements. Furthermore, forced expression of the NFkappa B 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-alpha in chondrocytes is sufficient to account for the inhibition of the chondrocyte phenotype by these cytokines. The down-regulation of Sox9 may have a crucial role in inhibiting expression of the cartilage phenotype in inflammatory joint diseases.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (1-5).

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, 7). 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 (8-11). 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 Sox9 homozygous 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, and aggrecan). 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). SOX9 binds to and activates chondrocyte-specific enhancer elements in the Col2a1 and Col11a2 genes and ectopic expression of SOX9 in transgenic mice activates the endogenous Col2a1 gene, providing evidence that these genes are direct targets for Sox9 (13-15).

Previous studies have shown that interleukin-1 (IL-1)1 and tumor necrosis factor-alpha (TNF-alpha ) 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-alpha (16-19). These inhibitory effects have been implicated in the breakdown of cartilage in arthritis, since IL-1 and TNF-alpha are produced by synovial cells in arthritic lesions and are present at elevated levels in synovial fluid in osteoarthritis and rheumatoid arthritis (20-24). In addition, IL-1 and TNF-alpha stimulate the synthesis of protein-degrading enzymes such as collagenases and stromelysins in cartilage (25-29). Binding of IL-1 and TNF-alpha to their receptors activates several signaling pathways, including the NFkappa B and AP-1 pathways. While IL-1 and TNF-alpha signals that lead to AP-1 activation have been implicated in the up-regulation of metalloproteinase genes (30-32), little is known about the mechanisms whereby these cytokines inhibit expression of the chondrocyte phenotype.

We show here that IL-1 and TNF-alpha markedly down-regulate the expression of Sox9 in chondrocytes. The activity of a Col2a1 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 NFkappa B pathway. We hypothesize that in inflammatory joint diseases, such as rheumatoid arthritis, a major mechanism by which IL-1 and TNF-alpha inhibit the chondrocyte phenotype is by down-regulation of Sox9, a master cartilage regulatory gene.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Recombinant human IL-1beta was obtained from the National Cancer Institute. Recombinant human TNF-alpha was purchased from PeproTech Inc. (Rocky Hill, NJ). 5,6-dichloro-1-beta -D-ribofuranosylbenzimidazole and pyrrolidine dithiocarbamate (PDTC) were purchased from Sigma.

Cell Culture-- The mouse chondrocytic cell line MC615 was kindly provided by Drs. Frédéric Mallein-Gerin and Bjorn R. Olsen (33). Mouse costal chondrocytes were obtained from 1-5-day-old mice and cultured as described previously (34).

RNA Preparation and Northern Analysis-- Total cellular RNA was extracted from cultures with the modified guanidium thiocyanate-phenol-chloroform method described by Chomczynski and Sacchi (35) 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 M formaldehyde, 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 Col2a1 probes were as described previously (13, 36). 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 [alpha -32P]dCTP (NEN Life Science Products) as described by Feinberg and Vogelstein (37) to a specific activity greater than 108 cpm/µg DNA. Hybridization was performed in hybridization solution supplemented with 1 × 106 cpm/ml of denatured 32P-labeled probe for 16 h at 65 °C. Filters were washed in 2× SSC (1× SSC: 150 mM NaCl, 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 Analysis-- Mouse 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), and 40 µg of each sample was separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (39). The proteins were electrophoretically transferred to nitrocellulose filters (Protran, Schleicher & Schuell) as described previously (40). 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), Ikappa Balpha (H-4, Santa Cruz Biotechnology), or SOX9. The SOX9 antibody was as described previously (13). 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 Transfection-- Col2a1-luciferase constructions harboring 12 tandem copies of the 18-bp and 4 tandem copies of the 48-bp enhancer elements were described previously (41). Two tandem copies of the 231-bp Col2a1 intron 1 fragment (+2113 to +2343) (42) were cloned in the p309Col2a1-beta geo vector as described previously for other enhancer elements (43). pNFkappa B-Luc, a reporter construct harboring 5 copies of a NFkappa B binding site immediately upstream of a minimal promoter, was purchased from Stratagene. RSV-p50 and RSV-p65 plasmids (44) encoding the p50 and p65 NFkappa B subunits were generous gifts from Dr. Gary J. Nabel (University of Michigan Medical Center, Ann Arbor, MI). pCMX-Ikappa Balpha M encoding the dominant-negative mutant of Ikappa Balpha (45, 46) 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 × 105 cells/4-cm2 well. Cells were harvested after 44-48 h of transfection. Reporter plasmids and pSVbeta gal, pSV2beta gal, and pGL3-Promoter (Promega Corp., Madison, WI), used as an internal control for transfection efficiency, were cotransfected in a 3:1 ratio. Luciferase and beta -galactosidase activities were assayed as described previously (41). Promoter activities were corrected for transfection efficiency.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Inhibition of Sox9 Expression-- We first examined the effects of IL-1beta on Sox9 gene expression in mouse primary chondrocytes. IL-1beta treatment resulted in a dose-dependent decrease in the levels of Sox9, which paralleled decrease in Col2a1 mRNA (Fig. 1, A and B). A similar parallel decrease in Sox9 and Col2a1 mRNA was also observed in a chondrocytic cell line MC615 (data not shown). In both types of cells, the levels of Sox9 mRNA dropped to about 10% or less of control levels at concentrations of IL-1beta between 3 and 10 ng/ml. The time course of this effect is shown in Fig. 1 (C and D). IL-1 decreased the levels of Sox9 mRNA in a time-dependent manner in both primary chondrocytes and MC615 cells (data not shown). The maximal decrease of Sox9 mRNA levels was observed as early as 3-6 h after addition of IL-1 in both cell types, whereas the decrease in Col2a1 mRNA levels occurred later.


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  		Fig. 1.   IL-1beta decreases Sox9 and Col2a1 mRNA levels in chondrocytes in dose-dependent and time-dependent manners. A and B, mouse primary chondrocytes were treated with the indicated doses of IL-1beta for 24 h. C and D, mouse primary chondrocytes were cultured for the indicated periods of time in the absence (-) or presence (+) of IL-1beta . Total RNA was subjected to Northern blot analysis with Col2a1 and Sox9 probes as well as an 18 S rRNA probe as loading control. Quantification of Col2a1 and Sox9 signals by scanning densitometry is shown in B and D. The figure presents data from one of two experiments that produced similar results.

The IL-1-induced inhibition of Sox9 expression was further confirmed at the protein level in primary chondrocytes (Fig. 2A). The decrease in the level of Sox9 protein was detectable as early as 3 h after addition of IL-1 and became more pronounced thereafter. Thus, IL-1 rapidly decreased the levels of Sox9 mRNA and protein. In parallel experiments, addition of TNF-alpha to primary chondrocytes in culture also produced a marked decrease in the level of Sox9 protein (Fig. 2B).


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  		Fig. 2.   IL-1 and TNF-alpha decrease Sox9 protein levels in chondrocytes. A, primary chondrocytes were cultured at confluence in the absence (-) or presence (+) of either 10 ng/ml IL-1beta (A) or 20 ng/ml TNF-alpha (B). Total cell lysates were examined by Western blot analysis at the indicated times after addition of cytokines. The molecular mass in kilodaltons of protein standards and the position of Sox9 are indicated. The figure presents data from one of two experiments with similar results.

To analyze the mode of action of IL-1 on Sox9 and Col2a1 mRNA levels, the effect of IL-1 on mRNA stability was investigated using the transcriptional inhibitor 5,6-dichloro-1-beta -D-ribofuranosylbenzimidazole in MC615 cells. In these experiments, the inhibitor was added 15 min before either IL-1 or the solvent alone and the levels of Sox9 mRNA were examined at various times thereafter (Fig. 3). The decay of Sox9 mRNA was unchanged by addition of IL-1, indicating that IL-1 did not affect the stability of this mRNA. The estimated half-life of Sox9 mRNA was about 40 min, suggesting a rapid turnover. This experiment strongly suggests that the down-regulation by IL-1 of the RNA levels for the master transcription factor Sox9 was transcriptional. The longer half-life of Col2a1 mRNA, about 9 h, also unaffected by IL-1 could account for the slower decrease of Col2a1 mRNA levels after addition of IL-1; this result is also consistent with previous reports showing that the effects of IL-1 on Col2a1 expression are mediated by a transcriptional mechanism (47, 48).


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  		Fig. 3.   Effect of IL-1 on the stability of Col2a1 and Sox9 mRNAs. 5,6-Dichloro-1-beta -D-ribofuranosylbenzimidazole (25 µg/ml) was added to the culture medium of confluent MC615 cells 15 min prior to the addition of vehicle (-) or 2 ng/ml IL-1 (+). A, total RNA was isolated at indicated periods of time and subjected to Northern blot analysis with Col2a1, Sox9, and 18 S rRNA probes. B and C, quantification of Col2a1 and Sox9 mRNA levels, respectively, by scanning densitometry. The figure presents data from one of two experiments with similar results.

Inhibition of the Activity of Sox9-dependent Enhancers-- In previous experiments, we delineated a 48-bp element present in intron 1 of the mouse Col2a1 gene that displayed strong chondrocyte-specific enhancer activity both in transient expression experiments and in transgenic mice (41). We also showed that SOX9 binds to a segment of this enhancer that is essential for activity and strongly activates this enhancer in nonchondrocytic cells (13). A mutant 48-bp enhancer that abolishes Sox9 binding was completely inactive. Thus, we use the activity of this 48-bp enhancer as a functional assay for Sox9. As shown previously, a construct containing four copies of this 48-bp element placed 5' to a short 89-bp Col2a1 promoter (Fig. 4A) was very active in primary chondrocytes and other chondrocytic cells, whereas the 89-bp promoter had very little activity by itself and no cell specificity. Addition of IL-1 to either primary chondrocytes or MC615 cells caused a dose-dependent inhibition of this enhancer (Fig. 4B, and data not shown). A construct containing 12 copies of a 18-bp subsegment of the 48-bp element that bound SOX9 (Fig. 4A) was also very active in chondrocytes, and its activity was shown to be strongly stimulated by SOX9 in nonchondrocytic cells (13, 41). The activity of this enhancer was also markedly inhibited by IL-1 in primary chondrocytes (Fig. 4C). Hence, the inhibition of the activity of these enhancer constructions is likely due to the severe decrease in Sox9 levels produced by IL-1.


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  		Fig. 4.   Inhibition by IL-1 of the activity of Col2a1 enhancer constructs in chondrocytes. A, schematic of Col2a1 constructions. The 48- and 18-bp chondrocyte-specific enhancer elements are aligned, and their positions relative to the Col2a1 transcription start site and the Sox9 binding site (black box) are indicated. B, dose-dependent inhibition of the 48-bp Col2a1 enhancer element by IL-1. Primary chondrocytes were transiently transfected with pSV2beta gal and either p89 or 4x48-p89. IL-1beta at indicated concentrations was added to the culture medium 2 h after the start of transfection. C, inhibition of the 18-bp Col2a1 enhancer element by IL-1. Primary chondrocytes were transfected as described in B, but with 12x18-p89. IL-1beta was added at 3 ng/ml. D, inhibition of the effects of IL-1 on the 18- and 48-bp enhancer elements by coexpression of SOX9. One nanogram of SOX9 plasmid or empty vector was cotransfected in primary chondrocytes with pSVbeta gal and either 12x18-p89 or 4x48-p89. The cells were incubated in the presence or absence of 3 ng/ml IL-1beta . In all panels, promoter activities are average values ± standard deviations for three independently transfected cultures from one representative experiment.

In order to further test this hypothesis, a SOX9-expressing plasmid was cotransfected with the Col2a1 enhancer constructs in primary chondrocytes. The results in Fig. 4D show that coexpression of SOX9 increased the activities of the 48- and 18-bp enhancers and blocked the IL-1 inhibition of these enhancers. These results support the notion that the inhibition of these enhancers by IL-1 was caused by the decrease in the endogenous levels of Sox9.

Involvement of the NFkappa B Pathway-- NFkappa B is a ubiquitous transcription factor that can be activated by a variety of stimuli including IL-1 and TNF-alpha (49-51). It is a homo- or heterodimer of DNA binding subunits, the most prototypical of which are p50 and p65. The activity of NFkappa B is regulated by the Ikappa B proteins. In unstimulated cells, NFkappa B dimers are retained in an inactive form in the cytoplasm through association with one of the Ikappa B inhibitory proteins. Upon stimulation, Ikappa B molecules are rapidly phosphorylated and degraded, allowing the NFkappa B dimers to translocate to the nucleus and regulate transcription through binding to DNA at kappa B sites (52-55). We verified by Western blotting that in primary chondrocytes p65 is present and that Ikappa Balpha , a major member of the Ikappa B family, is indeed degraded within 30 min after addition of IL-1beta (data not shown). We were unable to detect p50 in these cells.

To examine the possible involvement of the NFkappa B pathway in the IL-1-mediated down-regulation of Sox9 and the inhibition of the cartilage-specific Col2a1 enhancer, several types of experiments were performed. In a first experiment, we added PDTC, an inhibitor of Ikappa B degradation (52, 53, 56, 57), to primary chondrocytes. We verified by an electrophoretic mobility shift assay experiment that addition of this inhibitor blocked the increase in the NFkappa B DNA binding activity produced by IL-1 (data not shown). We also verified that PDTC blocked the nuclear translocation of p65 after IL-1 stimulation in primary chondrocytes as visualized by immunofluorescence using an antibody against p65 (data not shown). Under the same conditions, PDTC blocked the decrease in Sox9 protein level produced by IL-1 and TNF-alpha (Fig. 5). This result strongly suggests that down-regulation of Sox9 by IL-1 and TNF-alpha was mediated by NFkappa B. In a second experiment, we used a dominant-negative mutant of Ikappa Balpha . This mutant contained serine to alanine substitutions at amino acids 32 and 36 that prevent signal-induced phosphorylation and subsequent degradation (45, 46). Expression of this mutant Ikappa Balpha prevents NFkappa B translocation into the nucleus. Indeed, co-expression of this dominant-negative mutant of Ikappa Balpha reduced the basal activity of pNFkappa B-Luc, a reporter construct harboring 5 copies of a consensus NFkappa B binding site, and completely inhibited the increase in the activity by IL-1 and TNF-alpha in primary chondrocytes (data not shown). Under the same conditions, expression of the dominant-negative Ikappa Balpha mutant increased the activity of the 18- and 48-bp Col2a1 enhancers in primary chondrocytes about 2-fold, but completely blocked the IL-1 inhibition of these enhancers (Fig. 6A). In additional experiments, we tested the effects of TNF-alpha on the activity of the 18-bp Col2a1 enhancer. TNF-alpha also caused inhibition of this enhancer, and this effect was also abolished by co-expression of the dominant-negative Ikappa Balpha mutant (Fig. 6B). The ability of two different inhibitors of the NFkappa B pathway to block either the down-regulation of Sox9 by IL-1 and TNF-alpha or the decrease in activity of Sox9-dependent Col2a1 enhancers supports the hypothesis that the down-regulation of Sox9 is mediated by the NFkappa B pathway.


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  		Fig. 5.   Inhibition of IL-1 and TNF-alpha effects on Sox9 expression by PDTC. Confluent primary chondrocytes were incubated with or without 200 µM PDTC for 1 h before addition of 10 ng/ml IL-1beta (I), 20 ng/ml TNF-alpha (T), or control vehicle (C). Total cell lysates were prepared 9 h later and analyzed in Western blot using Sox9 antibody.


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  		Fig. 6.   Inhibition of the 18- and 48-bp enhancer elements by IL-1 and TNF-alpha is released by expression of dominant-negative mutant of Ikappa Balpha . Primary chondrocytes were cotransfected with 30 ng of pCMX-Ikappa Balpha M or empty vector, pSVbeta gal and either 12x18-p89 or 4x48-p89. Cells were incubated in the presence (+) or absence (-) of IL-1beta at 3 ng/ml (A) or TNF-alpha at 20 ng/ml (B). Promoter activities are average values ± standard deviations for three independently transfected cultures from one representative experiment.

To further examine the role of the NFkappa B pathway in the IL-1 inhibition of the cartilage-specific enhancer, we co-transfected expression plasmids encoding the p50 and p65 subunits of NFkappa B. The 48-bp enhancer was strongly inhibited by co-transfection of either the p65 or p50 subunit, providing additional evidence for the hypothesis that NFkappa B inhibits Sox9 expression (Fig. 7).


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  		Fig. 7.   Expression of p50 and p65 NFkappa B subunits inhibited the activity of the 48-bp enhancer element in primary chondrocytes. Seventy-five nanograms of empty vector, RSV-p50, and/or RSV-p65 was cotransfected with pSVbeta gal and either p89 or 4x48-p89. Promoter activities are average values ± standard deviations for three independently transfected cultures from one representative experiment.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In our study, both IL-1 and TNF-alpha markedly decreased expression of Sox9 in chondrocytic cells. This inhibition was very likely transcriptional, since IL-1 had no effect on the half-life of Sox9 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 in Col2a1 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 Sox9 gene prevents chondrocyte differentiation and Col2a1 expression (12), the marked IL-1 and TNF-alpha down-regulation of Sox9 should be sufficient to account for the inhibition of Col2a1 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). 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 Col2a1 enhancer elements by IL-1 and TNF-alpha 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 NFkappa B mediates the IL-1/TNF-alpha down-regulation of Sox9. First, addition of PDTC, an inhibitor of Ikappa B degradation, blocked IL-1 and TNF-alpha inhibition of Sox9 expression. This inhibitor also blocked the effects of IL-1 and TNF-alpha on the Sox9-dependent 48-bp Col2a1 enhancer (data not shown). Second, expression of a dominant-negative Ikappa Balpha mutant abolished the IL-1 and TNF-alpha inhibition of both the 48- and 18-bp SOX9-dependent cartilage-specific Col2a1 enhancers. Finally, forced expression of the p50 and p65 NFkappa B subunits mimicked the IL-1 inhibition of the enhancer. Interestingly, the expression of the dominant-negative Ikappa Balpha 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 Ikappa Balpha mutant inhibited endogenous NFkappa B activity.

The precise mechanism whereby NFkappa B inhibits Sox9 expression remains to be elucidated. One possible mechanism is that NFkappa B binds to regulatory sequences of the Sox9 gene. This possibility is suggested by the role of Drosophila homolog of NFkappa B, Dorsal, in repressing zen, dpp, and tolloid by binding to kappa B-like motifs in the regulatory sequences in these genes (58-60). Moreover, in the developing chick limb bud, c-Rel, another subunit of the NFkappa B family, inhibits expression of the gene for BMP-4, a vertebrate homolog of dpp, indicating the conservation of this pathway in vertebrates (61). Alternatively, activation of NFkappa B 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 Sox9 regulatory elements should help clarify the mechanism of Sox9 inhibition by IL-1 and TNF-alpha .

IL-1 and TNF-alpha inhibit expression of a number of other genes encoding chondrocyte-specific matrix molecules including collagen types IX and XI and aggrecan (16-19). The pronounced inhibition of Sox9 by 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-alpha are present at elevated levels in arthritic joints (20-22, 24), 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-alpha also activate other cellular pathways distinct from the NFkappa B pathway that can result in changes in expression or activity of transcription factors (30-32). This notion is illustrated by the following results. The activity of a construct containing a 309-bp Col2a1 promoter and two tandem copies of a 231-bp Col2a1 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). 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 Ikappa Balpha 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-alpha 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 NFkappa B 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-alpha inhibition of the chondrocyte phenotype.

Although IL-1 and TNF-alpha are not thought to have major roles in skeletal development, since mice deficient in IL-1beta , TNF-alpha , or their receptors did not show any skeletal phenotype (62-66), the NFkappa B pathway may play an important role in the physiological regulation of Sox9. Recent reports demonstrated a role for c-rel in chick limb bud development as regulator of the expression of BMP-4, Sonic Hedgehog, and twist (61, 67). Since expression of c-rel appears to be inhibited prior to cartilage formation, it is possible that the NFkappa B pathway is involved in the control of the Sox9 gene during embryonic development.

In conclusion, our results demonstrate that IL-1 and TNF-alpha markedly down-regulate the gene for Sox9, a factor that is required for chondrocyte differentiation, and also inhibit the activity of Sox9-dependent Col2a1 chondrocyte-specific enhancer elements in chondrocytes. Furthermore, our results strongly suggest that inhibition of Sox9 is mediated by the NFkappa B pathway. Down-regulation of Sox9 should be a major mechanism by which expression of Col2a1 and other chondrocyte-specific genes is inhibited by IL-1 and TNF-alpha . Further studies on the role of the NFkappa B pathway in regulating Sox9 may provide insights into the mechanisms of cartilage degeneration in arthritis.

    ACKNOWLEDGEMENTS

We thank Frédéric Mallein-Gerin and Bjorn R. Olsen for MC615 cells, Gary J. Nabel for p50 and p65 expression plasmids, and Inder M. Verma for the dominant-negative Ikappa Balpha plasmid. We are grateful to Sandra McKinney for technical assistance. We also thank Janie Finch and Pat Arubaleze for editorial assistance.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants RO1 AR42909 and PO1 AR42919-02 (to B. d. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of an Arthritis Investigator Award from the Arthritis Foundation.

§ To whom correspondence should be addressed: Dept. of Molecular Genetics, M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-792-2590; Fax: 713-794-4295; E-mail: benoit_decrombrugghe@molgen.mdacc.tmc.edu.

    ABBREVIATIONS

The abbreviations used are: IL, interleukin; TNF-alpha , tumor necrosis factor-alpha ; bp, base pair(s); PDTC, pyrrolidine dithiocarbamate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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