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Definitions

• mice engineered to express only one allele of the Sox9 gene represent a near phenocopy of the human skeletal syndrome (Bi W, Huang W, Whitworth DJ, Deng JM, Zhang Z, Behnnger RR, de Crombrugghe B Haploinsufficiency of Sox9 results in defective cartilage pnmordia and premature skeletal mineralization Proc Natl Acad Sci U S A 98 6698- 6703, 2001), displaying hypoplasia of all cartilage pnmordia and bones derived therefrom Furthermore, mice derived by chime ⁇ sm of wild-type and Sox9 -/- ES cells (expressing B- galactosidase) showed that Sox9 expression is a obligatory for cells to be incorporated into mesenchymal condensations that give rise to cartilage structures (Bi W, Deng JM, Zhang Z, Behnnger RR, de Crombrugghe B Sox9 is required for cartilage formation Nat Gene
• mice having impaired or enhanced Sox9 expression showed XY or XX sex reversal, respectively (Bishop CE, Whitworth DJ, Qin Y, Agoulnik Al, Agoulnik IU, Harrison WR, Behnnger RR, Overbeek PA A transgenic msedion upstream of sox9 is associated with dominant XX sex reversal in the mouse Nat Genet 26 490-494, 2000, Vidal VP, Chaboissier MC, de Rooij DG, Schedl A Sox9 induces testis development in XX transgenic mice Nat Genet 28 216-217, 2001), confirming the role of Sox9 in sex determination in mice as well as humans
• Sox9 is a member of a family of HMG-box proteins This family of proteins is unusual in that they bind to the minor groove of DNA and usually cooperate with a protein partner(s) to regulate gene expression (reviewed in Kamachi Y, Uchikawa M, Kondoh H Pairing SOX off with partners in the regulation of embryonic development Trends Genet 16 182-187, 2000)
• ⁇ LSox5 and Sox6 appear to play a partly overlapping role in collaborating with Sox9 to regulate the expression of chondrocyte target genes including the Col2a1 and aggrecan (Smits P, Li P, Mandel J, Zhang Z, Deng JM, Behnnger RR, de Croumbrugghe B, Lefebvre V
• the transcription factors L-Sox5 and Sox6 are essential for cartilage formation Dev Cell 1 277-290, 2001, Lefebvre V, Behnnger RR, de Crombrugghe B L-Sox5, Sox6 and Sox ⁇ control essential steps of the chon
• Sox ⁇ also regulates the "minor cartilage collagen", Col11a2, by interactions with HMG-like sites in both the promoter (Bndgewater LC, Lefebvre V, de Crombrugghe B Chondrocyte-specific enhancer elements in the Col11a2 gene resemble the Col2a1 tissue- specific enhancer J Biol Chem 273 14998-15006, 1998) and within an intronic enhancer ( Liu Y, Li H, Tanaka K, Tsumaki N, Yamada Y Identification of an enhancer sequence within the first mtron required for cartilage-specific transcription of the alpha2(XI) collagen gene J Biol Chem 275 12712-12718, 2000)
• Sox ⁇ appears to integrate the many signaling pathways regulating the expression of cartilage matrix proteins This includes signals that positively regulate the cartilage phenotype, such as a FGFs, and BMP-2, as well as parathyroid hormone in prehypertrophic chondrocytes (Murakami S, Kan M, McKeehan WL, de Crombrugghe B Up-regulation of the chondrogenic Sox9 gene by fibroblast growth factors is mediated by the mitogen-activated protein kinase pathway.
• Inhibition of matrix expression by cytokines and retinoic acid also involves inhibition of Sox ⁇ (Murakami S, Lefebvre V, de Crombrugghe B: Potent inhibition of the master chondrogenic factor Sox9 gene by interleukin-1 and tumor necrosis factor-alpha. J Biol Chem 275:3687-3692, 2000); Sekiya I, Koopman P, Tsuji K, Mertin S, Harley V, Yamada Y, Shinomiya K, Niguji A, Noda M: Transcriptional suppression of Sox9 expression in chondrocytes by retinoic acid. J Cell Biochem 81:71-78, 2001).
• Sox ⁇ activity is regulated by both the level of expression and by post-translational modification ( Huang W, Zhou X, Lefebvre V, de Crombrugghe B: Phosphorylation of SOX ⁇ by Cyclic AMP-Dependent Protein Kinase A Enhances SOX9's Ability To Transactivate a Col2a1 Chondrocyte-Specific Enhancer. Mol Cell Biol 20:4149- 4158, 2000).
• Sox ⁇ has been termed the "Master Chondrogenic Transcription Factor” (Murakami S, Lefebvre V, de Crombrugghe B: Potent inhibition of the master chondrogenic factor Sox ⁇ gene by interleukin-1 and tumor necrosis factor-alpha. ( J Biol Chem 275.3687-36 ⁇ 2, 2000). Therefore, understanding Sox ⁇ function in cartilage development, tissue maintenance, and disease processes is important in considering strategies to halt cartilage destruction and induce repair.
• the Sox ⁇ transcription factor has emerged as an important determinant of chondrocyte development and the regulation of type II collagen and aggrecan gene expression.
• the Sox ⁇ transcription factor is known to play a role in the expression/activity of Col2 by chondrocytes.
• the present invention relates to a method of predicting the effect of a test compound on human chondrocyte activity
• the method comprises the steps of transfectmg SW1353 human chondrosarcoma cells with a reporter plasmid comprising Sox ⁇ DNA binding sites, contacting the transfected cells with a test compound for a period of from about 2 to about 24 hours, and determining the activity of the reporter
• the present invention also provides a chime ⁇ c cell line that is capable of mimicking the effect of various compounds on Col2 expression/activity
• the cell line comprises a SW1353 cell transfected with a plasmid containing Sox ⁇ DNA reporter binding sites It has been determined that the SW1353 cell line is capable of modeling chondrocyte behavior in response the regulation of Col2 expression by various compounds
• SW1353 cells are transfected with a reporter plasmid containing Sox ⁇ DNA binding sites
• the cells can be used as an assay to determine the effectiveness of the introduced compound in changing the level of Sox ⁇ and indirectly the level of Col2a expression/activity, and thereby acting as a tool to a lesser or greater degree predictive of the effect of the introduced compound on chondrocyte differentiation
• the SW1353 cell line recapitulates many of the effects of cytokines and growth factors on Col2 enhancer activity observed with primary mouse chondrocytes including stimulation by FGF-1 and FGF-2 and repression by IL-1/? and TNF ⁇ These effects have been correlated with changes in Sox ⁇ mRNA levels
• FGF- ⁇ stimulates Sox ⁇ expression
• Col2 enhancer activity shows that very low levels of cytokines actually stimulate enhancer activity while higher doses, sufficient to stimulate IL-8 expression, are inhibitory
• Figure 1 A shows the stimulation of basal expression of the 48bp Co/2a Luc enhancer reporter by co-expression of Sox ⁇ in transiently transfected SW1353 cells
• Figure 1 B shows western blot of SW1353 cells transfected with the Sox ⁇ expression plasmid
• Figure 2 shows the response of SW1353 cells to growth factors and cytokines with activation or repression of the 48bp Col2a1 enhancer reporter
• Figure 3 shows the opposing effects of FGFs and cytokines on the expression of Sox ⁇ in SW 1353 cells
• Figure 4 shows the dynamic range of SW1353 biological response as a function of IL- 1 ⁇ concentration.
• the present invention relates to a method of predicting the effect of a test compound on human chondrocyte differentiation
• the method comprises the steps of transfecting SW1353 human chondrosarcoma cells with a reporter plasmid comprising Sox ⁇ DNA binding sites, contacting the transfected cells with a test compound for a period of from about 2 to about 24 hours, and determining the activity of the reporter
• the present invention also provides a chimenc cell line that is capable of mimicking the effect of various compounds on Col2 expression/activity
• the cell line comprises a SW1353 cell transfected with a plasmid containing Sox ⁇ DNA reporter binding sites
• the present invention demonstrates that SW1353 cells model many of the signaling pathways identified in primary murine chondrocytes and are therefore useful in translating experimental results obtained in murine systems to human systems
• FGF-1 and FGF-2 were found to increase Sox ⁇ mRNA levels and the corresponding activity of the Sox ⁇ -dependent 48bp Col2a1 enhancer construct This has relevance to joint repair either by the normal response of chondrocytes in attempting the normal repair of damaged cartilage or when considering strategies for therapeutic intervention
• Sox ⁇ expression is elevated in the joints of mice attempting to repair damage induced by the expression of a mutant Type IIA collagen gene (Salmmen H, Vuorio E, Saamanen AM Expression of Sox ⁇ and type IIA procollagen during attempted repair of articular cartilage damage in a transgenic mouse model of osteoadhritis. Arthritis Rheum 44: ⁇ 47- ⁇ 55, 2001).
• Sox ⁇ expression corresponds with the most proliferative and metabolically active chondrocytes present in the repair tissue of this model.
• the expression of endogenous FGF-2 is also correlated with the repair of joint damage, as a neutralizing antibody to FGF-2 blocks the repair of full-thickness cartilage defects in a rabbit model, while the infusion of FGF-2 leads to improved repair (Salminen H, Vuorio E, Saamanen AM: Expression of Sox ⁇ and type IIA procollagen during attempted repair of articular cartilage damage in a transgenic mouse model of osteoarthritis. Arthritis Rheum 44:947-955, 2001).
• Sox ⁇ provides a direct link between the input of prochondrogenic growth factors and the expression of chondrocytic genes needed for repair.
• the present invention discloses that FGF- ⁇ stimulates Sox ⁇ expression and activity in a human chondrocyte-like cell line.
• FGF- ⁇ and FGF-2 were found to be the most potent of all the FGF family members in terms of stimulating the growth and matrix production of chicken chondrocyte cultures (Praul CA, Ford BC, Leach RM: Effect of fibroblast growth factors 1, 2, 4, 5, 6, 7, 8, 9, and 10 on avian chondrocyte proliferation. J Cell Biochem 84:35 ⁇ -366, 2002).
• FGF- ⁇ expression is associated with the growth of benign cartilage nodules in the joints of chondromatosis patients.
• FGF- ⁇ signals through both FGFR2c and FGFR3b (Santos-Ocampo S, Colvin JS, Chellaiah A, Ornitz DM: Expression and biological activity of mouse fibroblast growth factor- ⁇ . J Biol Chem 271:1726-1731, 1996), and mice lacking FGFR3 have severe achondroplasia (Colvin JS, Bohne BA, Harding GW, McEwen DG, Ornitz DM: Skeletal overgrowth and deafness in mice lacking fibroblast growth factor receptor 3. Nat Genet 12:390-397, 19 ⁇ 6)).
• mice lacking FGF ⁇ have normal cartilage-derived structures, but instead show XY sex reversal and lung hypoplasia ( Colvin JS, Green RP, Schmahl J, Capel B, Ornitz DM: Male-to-female sex reversal in mice lacking fibroblast growth factor ⁇ . Cell 104:875-88 ⁇ , 200; Colvin JS, White AC, Pratt SJ, Ornitz DM: Lung hypoplasia and neonatal death in Fgf ⁇ - null mice identify this gene as an essential regulator of lung mesenchyme. Development 128:20 ⁇ 5-2106, 2001).
• SW1353 cells Although we have not seen an effect of PTHrP on Sox ⁇ activity in SW1353 cells transfected with the 48bp Col2a1 enhancer, they do respond to cAMP analogs with about a 2- fold increase in enhancer expression (LB and JS, unpublished observations) SW1353 cells also appear unresponsive to TGF ⁇ , BMPs-2, -4, and -6 (data not shown), and is consistent with their phenotype being more cartilage-like and less bone-like Therefore, SW1353 cells are considered to be a useful model for studying the Sox ⁇ signaling pathways, especially those most relevant to cartilage FGF/FGF receptor signaling has a profound effect on the growth and differentiation of chondrocytes in vitro However, determining the physiologically relevant interactions is made enormously complex since there are four FGF receptors and 22 ligands in this family In murine chondrocytes and immortal chondrocyte model cell-lines (ATDC5 and C3H10T1/2), the 48bp Luc reporter is induced by pro
• This reporter plasmid includes five tandem repeats of the 48bp Col2a1 enhancer and encompasses the minimal sequence, including Sox ⁇ binding sites, sufficient to direct chondrocyte specific expression in vivo (Zhou G, Lefebvre V, Zhang Z, Eberspaecher H, de Crombrugghe B: Three high mobility group-like sequences within a 48-base pair enhancer of the Col2a1 gene are required for cartilage-specific expression in vivo. J Biol Chem 273:14 ⁇ 8 ⁇ -14 ⁇ 7, 1 ⁇ 8).
• FGF- ⁇ is potent chondrogenic factor and signals through FGFR3b and FGFR2c. Mutations that result in a constitutively active FGFR3 cause skeletal malformations including achondroplasia, hypochondroplasia and thanatorphoric dysplasia and disturb the balance between chondrocyte growth and differentiation (Weksler NB, Lunstrum GP, Reid ES, Hodon WA: Differential effects of fibroblast growth factor (FGF) 9 and FGF2 on proliferation, differentiation and terminal differentiation of chondrocytic cells in vitro. Biochem J 342 Pt 3:677-682, 1 ⁇ ).
• FGF fibroblast growth factor
• FGF- ⁇ over-expression was implicated in a rare disease, chondromatosis, characterized by cartilaginous nodule formation of the synovium. These studies suggest a central role for FGF- ⁇ in controlling the differentiation of stem cells into chondrocytes. Previous studies showed that Col2 enhancer activity correlates with Sox ⁇ expression.
• Sox ⁇ expression construct For instance, FGF-2 stimulates and cytokines repress the expression of Sox ⁇ mRNA and protein in primary mouse chondrocytes results that point to Sox ⁇ as being a key intermediate for in regulating expression of Col2 and aggrecan expression.
• Sox ⁇ expression construct
• mRNA from cultures of immortalized human chondrocyte-like cells T/C-28a4 was purified by RNeasy kit (Qiagen).
• the entire 1.5 Kb coding region (GenBank database, accession No. Z4662 ⁇ ) was obtained by reverse transcription (Clontech Advantage RT Kit) followed by PCR reaction (Roche Expand High Fidelity PCR Kit) using primers: sense 5'-
• SW1353 human chondrosarcoma cells ATCC #HTB- ⁇ 4
• DMEM 10% FBS 10 ug/ml Gentamicin passaged (2 times a week) using with 0 25% Trypsin-EDTA
• RNA was prepared from 75 cm dishes using RNeasy Kits with DNAse treatment (Qiagen) according to the manufacturer's protocol except a second application of DNase was used
• cDNA was prepared from 10-20ul of RNA (1-4 ug) using random hexamer primers and PE Biosystems Reverse Transcription reagents Taqman reactions used 8 ul cDNA and 2x PE Universal PCR Master Mix (PE Biosystems), ⁇ 00 nM PCR primers and 200nM FAM- TAMRA probe Reactions (50 ul) were cycled and quantified using the PE 5700 with standard conditions for 45 cycles
• the data was analyzed using GeneAmp5700 software Mix (PE Biosystems), adjusting the threshold to linear range for all samples
• the ⁇ Ct is calculated by subtracting norma zer (GAPDH) from corresponding gene-specific values
• the ⁇ Ct is calculated by subtracting treated ⁇ Ct from control ⁇ Ct Fold induction is determined by calculating 2 ⁇ Ct Data is
• FRET probe FAM-CCCACGAAGGGCGACGATGG-TAMRA
• Sox ⁇ expression was confirmed by preparing Western blots from cell extracts prepared from duplicate transfections (Fig 1 B) At the highest DNA concentration (0 25ug), an immunoreactive band appeared, migrating with the 66 kD marker, in good agreement with 68 kD size predicted for Sox ⁇ (lane 5) Protein of the same size was observed in transfections having 2 and 10 fold less expression plasmid (lanes 4 and 3) On longer exposures, anti-Sox ⁇ immunoreactive proteins were detected faintly with the 20 1 transfection and for those cells receiving no Sox ⁇ expression plasmid 2 SW1353 cells are responsive to pro-chondroqenic Fibroblast Growth Factors
• FGF- ⁇ was added to cultures of SW1353 cells that had been transfected with the Col2 reporter FGF-9 (200 ng/ml) stimulated enhancer expression up to 4 fold (Fig 2). This result suggests that the SW1353 cells express functional FGFR3b and/or FGFR2c receptors and further validates their utility in studying chondrocyte biology.
• Pro-inflammatory cytokines repress Col2 enhancer activity in SW1353
• the ability of IL-1 ? and TNFoc to repress Col2 enhancer activity in primary murine chondrocytes is NFkB dependent and coincides with reduced Sox9 expression.
• SW1353 cells were transfected with the 48bp Col2a1 reporter and then treated with vehicle, TNF ⁇ (10 ng/ml) or IL-1 ? (10 ng/ml) (Fig. 2A).
• TNF ⁇ 10 ng/ml
• IL-1 ? 10 ng/ml
• Fig. 2A The results demonstrate that SW1353 cells express functional TNF and IL-1 receptors by showing these agents can stimulate the expression of IL-8 (shown) and matrix metalloproteases (not shown).
• Enhancer driven luciferase expression was reduced approximately 60% by TNF ⁇ and
• Col2 enhancer activity correlates with relative Sox ⁇ mRNA expression in SW1353
• Col2 type II collagen
• Col2 An increase in type II collagen (Col2) expression has been observed in both man and dogs during the course of osteoarthritic disease (Budon-Wurster N, Hui-Chou CS, Greisen HA, Lust G: Reduced deposition of collagen in the degenerated articular cartilage of dogs with degenerative joint disease.
• Col2 expression appears to be greatest in early to moderate disease and this appears to reflect a response by the chondrocytes to attempt repair of the degenerating cartilage
• chondrocytes from OA patients with early to moderate disease also express increased levels of IL- ⁇ and IL-1 5 and these also decline with disease progression (Towle CA, Hung HH, Bonassar LJ, Treadwell BV, Mangham DC Detection of ⁇ nterleuk ⁇ n-1 in the cartilage of patients with osteoarthritis a possible autocrine/paracrme role in pathogenesis.
• IL-1 expression appears to overlap with Col2 expression
• the IL-1 levels found in osteoarthritic joints is derived from chondrocytes and these levels are distinguished from the very high levels of cytokines found in the rheumatoid joint and principally derived macrophages Unlike rheumatoid joints, IL-1 levels are lowest in OA when cartilage degradation is highest, suggesting that IL-1 does not play the same role in cartilage destruction in the two diseases

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