Inhibition of chondrocyte differentiation in vitro by constitutive

INTRODUCTION

The control of proliferation and differentiation of chondrogeniccells is central to the coordinated development of the vertebrateskeleton. Vertebrate long bones develop by the process ofendochondral ossification, which is initiated in the embryowith the condensation of undifferentiated mesenchymal cellsand progresses with their commitment and differentiation intochondrogenic cells. By late embryonic development theepiphyseal growth plate has developed with distinguishable,well-organized and spatially distinct zones of resting,proliferating and post-proliferative hypertrophic chondrocytes(for review see Erlebacher et al., 1995). Chondrocyteproliferation and differentiation continue at the growth platethrough juvenile growth and are partly responsible forregulating the rate of expansion of the long bones. Theconsequences of failure of the regulation of chondrocytegrowth and differentiation can be seen in human

chondrodysplasias. Interestingly, specific genetic defectsassociated with several chondrodysplasias have been mappedto genes involved in endocrine/paracrine signalling. Forexample, the fibroblast growth factor (FGF) receptor 3(FGFR3) is disrupted in achondroplasia (Shiang et al., 1994),the transforming growth factor β (TGF-β) superfamily memberCDMP-1 in Hunter-Thomson-type chondrodysplasia (Thomaset al., 1996) and the common parathyroid hormone (PTH) andPTH-related peptide (PTHrP) receptor, PTH1R, in Jansen-typeand Blomstrand-type chondrodysplasias (Schipani et al., 1995;Karaplis et al., 1998; Jobert et al., 1998). Moreover, analysisof chondrodysplastic mouse models allows more detaileddefinition of the roles of these pathways. Thus, geneticdisruption of the PTH1R gene (Karaplis et al., 1994; Amizukaet al., 1994), overexpression of PTHrP or PTH1R (Weir et al.,1996; Schipani et al., 1997), disruption of FGFR3 (Deng et al.,1996), or mutations of members of the TGF-β superfamily(Kingsley et al., 1992; Storm et al., 1994) all result in altered

439Journal of Cell Science 113, 439-450 (2000)Printed in Great Britain © The Company of Biologists Limited 2000JCS0911

We have investigated the role of c-Fos in chondrocytedifferentiation in vitro using both constitutive andinducible overexpression approaches in ATDC5chondrogenic cells, which undergo a well-defined sequenceof differentiation from chondroprogenitors to fullydifferentiated hypertrophic chondrocytes. Initially, weconstitutively overexpressed exogenous c-fos in ATDC5cells. Several stable clones expressing high levels ofexogenous c-fos were isolated and those also expressing thecartilage marker type II collagen showed a markeddecrease in cartilage nodule formation. To investigatefurther whether c-Fos directly regulates cartilagedifferentiation independently of potential clonal variation,we generated additional clones in which exogenous c-fosexpression was tightly controlled by a tetracycline-regulatable promoter. Two clones, DT7.1 and DT12.4 werecapable of nodule formation in the absence of c-fos.However, upon induction of exogenous c-fos, differentiationwas markedly reduced in DT7.1 cells and was virtuallyabolished in clone DT12.4. Pulse experiments indicatedthat induction of c-fos only at early stages ofproliferation/differentiation inhibited nodule formation,

and limiting dilution studies suggested that overexpressionof c-fos decreased the frequency of chondroprogenitorcells within the clonal population. Interestingly, rates ofproliferation and apoptosis were unaffected by c-fosoverexpression under standard conditions, suggesting thatthese processes do not contribute to the observed inhibitionof differentiation. Finally, gene expression analysesdemonstrated that the expression of the cartilage markerstype II collagen and PTH/PTHrP receptor were down-regulated in the presence of exogenous c-Fos and correlatedwell with the differentiation status. Moreover, induction ofc-fos resulted in the concomitant increase in the expressionof fra-1 and c-jun, further highlighting the importance ofAP-1 transcription factors in chondrocyte differentiation.These data demonstrate that c-fos overexpression directlyinhibits chondrocyte differentiation in vitro, and thereforethese cell lines provide very useful tools for identifyingnovel c-Fos-responsive genes that regulate thedifferentiation and activity of chondrocytes.

Key words: c-Fos, Chondrocyte, In vitro, Tetracycline,Differentiation

SUMMARY

Inhibition of chondrocyte differentiation in vitro by constitutive and inducible

overexpression of the c-fos proto-oncogene

David P. Thomas, Andrew Sunters, Aleksandra Gentry and Agamemnon E. Grigoriadis*

Departments of Orthodontics and Pediatric Dentistry & Craniofacial Development, King’s College London, Guy’s Hospital, LondonBridge, London SE1 9RT, UK*Author for correspondence (e-mail: [email protected])

Accepted 18 November 1999; published on WWW 19 January 2000

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chondrocyte proliferation and/or differentiation. However, lessis known about the molecular events downstream ofendocrine/paracrine signalling and thus it is of interest thatgenetic disruption of several nuclear proteins, such as (i)transcription factors like ATF-2 (Reimold et al., 1996), theproto-oncogene Ets-2 (Sumarsono et al., 1996), and the runt-domain protein cbfa-1 (Otto et al., 1997; Komori et al., 1997;Inada et al., 1999), (ii) cell cycle control proteins such as theretinoblastoma (Rb)-related proteins p107 and p130 (Cobriniket al., 1996) and (iii) the cyclin dependent kinase inhibitor(CKI) p57 (Zhang et al., 1997), also result in skeletalabnormalities in mice, with specific effects on chondrogeniccells.

One additional nuclear transcription factor that has a provenrole in chondrocyte differentiation is c-Fos. The proto-oncogene c-fos was first identified as the cellular homologueof the v-fos oncogene from the FBJ- and FBR-murine sarcomaviruses. The c-Fos gene product is a member of the AP-1family of transcription factors, which includes the other Fos-related (FosB, Fra-1, Fra-2) and Jun-related (c-Jun, JunB,JunD) proteins (for review see Angel and Karin, 1991). Thefunctional activity of c-Fos, and of the other Fos familymembers is dependent on the formation of heterodimers withproteins of the Jun family. Fos proteins are unable to formhomodimers, but Jun proteins can additionally dimerise witheach other and with the related ATF-2 transcription factor.Dimerised complexes can then modulate transcription bybinding to AP-1 consensus binding sites in the promoterregions of target genes (Angel and Karin, 1991). c-fos andother AP-1 family members have been shown to display animmediate early gene pattern of expression being rapidly andtransiently induced by external mitogenic signals such asserum stimulation, suggesting a role for c-Fos in signaltransduction of mitogenic stimuli (see also Morgan and Curran,1991). The downstream effects of c-Fos induction arenumerous and in addition to roles in proliferation,transformation and apoptosis, c-Fos has been implicated in thedifferentiation of several cell types: Clear c-Fos-dependentdifferentiation effects have been demonstrated by theoverexpression or inactivation of c-fos in teratocarcinoma stemcells (Müller and Wagner, 1984), adipocytes (Abbott andHolt, 1997) and cells of the osteoclast/macrophage lineage(Grigoriadis et al., 1994).

Although c-Fos expression can be induced in mosttissues, gain-of-function and loss-of-function studies havedemonstrated a specific role in bone/cartilage biology(Grigoriadis et al., 1995). The earliest embryonic expression ofc-Fos is restricted specifically to the growth regions of fetalbones (Dony and Gruss, 1987; DeTogni et al., 1988) and post-natal expression has been detected in osteoblasts and growthplate chondrocytes (Grigoriadis et al., 1993; Lee et al., 1994;Sunters et al., 1998). In transgenic mice expressing exogenousc-fos from a ubiquitous promoter the primary phenotype is thetransformation of osteoblasts leading to bone tumour formation(Grigoriadis et al., 1993), whilst inactivation of the c-fos genecauses severe osteopetrosis with a complete block in osteoclastdifferentiation (Johnson et al., 1992; Wang et al., 1992;Grigoriadis et al., 1994). A specific role for c-Fos inchondrocytes in vivo is highlighted by several further lines ofevidence. Infection of embryonic chick limb buds with c-fosexpressing retroviruses results in truncation of the long bones

due to severe retardation of the differentiation of proliferatingchondrocytes into mature hypertrophic chondrocytes(Watanabe et al., 1997). Furthermore, c-fos-overexpressingembryonic stem (ES) cell chimeric mice demonstrate thetransformation of chondrocytes and the development ofchondrosarcomas, and transformed, type II collagen (coll II)expressing cell lines derived from these tumours failed toprogress to hypertrophy (Wang et al., 1991, 1993). Finally, c-fos knockout mice display shortened limbs and disruptedgrowth plate architecture, with a significantly depleted zone ofproliferating cells and an expanded zone of hypertrophic cells(Wang et al., 1992).

These in vivo models indicate a clear role for c-Fos inchondrogenesis in the context of an intact animal, withoverexpression of c-fos inhibiting differentiation andendochondral progression and the absence of c-fos apparentlyaccelerating this process. However, further cellular andmolecular dissection of such a role by in vitro analysis hasproved more difficult due to the relative difficulty inestablishing stable, continuously growing, non-transformedchondrogenic cell lines which differentiate with reproduciblekinetics. Some useful cell lines have indeed been isolatedwhich display varying degrees of chondrogenic potential (e.g.Grigoriadis et al., 1989, 1996; Atsumi et al., 1990; Bernierand Goltzman, 1993; Lefebvre et al., 1995). However, nosystematic approach to analysing the effect of c-fosoverexpression on the differentiation of in vitro chondrocytecultures has yet been undertaken. We have used an establishedmodel of chondrocyte differentiation in which the ATDC5embryonal carcinoma derived cell line can be induced toundergo a reproducible, time-dependent in vitro progressionfrom early precursors to hypertrophic chondrocytes (Atsumi etal., 1990; Shukunami et al., 1997). Using this model we haveshown that stable overexpression of c-fos, either constitutivelyor by using an inducible promoter clearly demonstrates aninhibition of chondrogenic progression and that phenotypicand molecular characterisation demonstrates that c-Fos directlyregulates chondrocyte differentiation.

MATERIALS AND METHODS

Construction of pJMF2-c-fosThe expression vector pJMF2 was obtained as a gift from Dr J.Feingold (UCHC, Farmington, CT), generated as a one-componentconstruct based on the Tet-off system of Gossen and Bujard (1992).Briefly, this vector had been constructed by the removal of thepromoter region of the expression vector pREP9 (Invitrogen,Groningen, Netherlands) and its replacement with a tetracycline-repressed transactivator (tTA) expression cassette (PCMV tTA), aminimal tTA responsive promoter (tetO), and a multiple cloning siteupstream of the SV40 poly A site of pREP9 (Lang and Feingold,1996). The c-fos cDNA was excised by BamHI as a 1.35 kb fragmentfrom pX-c-fos (Superti-Furga et al., 1991) and ligated into the BamHIsite upstream of the tetO promoter region of pJMF2. Clones wereisolated and the orientation of the c-fos cDNA insert was determinedusing the BglII and NcoI sites within the c-fos cDNA sequence. Thesize of the c-fos cDNA-SV40 poly A transcript is estimated to be ~1.8kb (Fig. 1). The construction of the vector p76/21 containing MT-c-fosLTR has been previously described and gives rise to 2 exogenoustranscripts of 3.0 kb and 2.0 kb (Rüther et al., 1985; Wang et al.,1991).

D. P. Thomas and others

441c-Fos inhibits chondrocyte differentiation

Cell culture and cloningATDC5 cells were obtained from the RIKEN cell bank (Japan) andmaintained as described by Atsumi et al. (1990). Cells were culturedin a standard medium of DMEM/Hams’ F12 (1:1) (Gibco BRL,Paisley, UK) supplemented with 5% FCS (M. D. Meldrum, Hants,UK), 10 µg/ml bovine insulin, 10 µg/ml human transferrin and 3×10−8

M sodium selenite (ITS) (Sigma, Poole, UK) and antibiotics (50 U/mlpenicillin and 50 µg/ml streptomycin; Gibco BRL). Stabletransfections were performed using either SuperFect (Qiagen,Crawley, UK; DT8 and DT7.1) or Effectene (Qiagen; DT12.4)according to the manufacturer’s instructions. After transfection, cellswere plated at varying densities and transfected clones selected instandard media supplemented with 0.5 mg/ml G418 (Gibco BRL) andisolated by ring-cloning. Additionally, in transfections with pJMF2-c-fos the media were supplemented with 1 µg/ml tetracycline (Tc)(Sigma) throughout the selection, expansion and maintenance ofclones, in order to repress exogenous c-fos expression. Exogenous c-fos expression levels of all clones were determined by northern blotanalyses following Tc withdrawal. In all, 6 stable clones transfectedwith p76/21 were analysed (DT8 series) and 22 pJMF-c-fos-transfected clones, of which 2 were strongly positive (DT7.1 andDT12.4), and one was weakly positive (DT12.5, data not shown).

Differentiation, proliferation and apoptosisFor all cell biological analyses cells were plated at a density of 6×103

cells/cm2 in 6-well plates (Nunc, Roskilde, Denmark) unlessotherwise stated and the standard media were replaced every two daysfor 21 or 30 days. For differentiation assays cultures were fixed in 4%paraformaldehyde in PBS and stained with 0.25% Alcian blue at pH0.75. For quantification, stain was solubilised in 4 M guanidine HCl(pH 1.5) and the optical density measured at 595 nm. For expressionanalyses cells were plated in 140 cm culture dishes (Nunc), and wereharvested by trypsinisation at least 24 hours after feeding. For longerterm cultures after 21 days, the media were changed to αMEM (GibcoBRL) with the same additives to induce hypertrophic differentiation(Shukunami et al., 1997).

Analyses of pJMF2-c-fos clones were performed in the presence orabsence of 1 µg/ml Tc (± Tc) as stated. For pulse experiments,induction of c-fos was performed by washing cell cultures 3-4 timesin PBS to remove Tc, prior to feeding with Tc-deficient media.Cultures were ‘pulsed’ for 4-day time periods as inductionexperiments suggested that maximal c-fos expression is not achieveduntil 48-72 hours after the removal of Tc (data not shown). Limitingdilution analysis was performed by plating DT12.4 cells in 96-wellplates with densities per well as indicated. Cells were cultured for 21days ± Tc, fixed and stained with Alcian blue as stated. The fractionof wells without cartilage was plotted against cell density. From theequation F0=e−x, where x is the number of chondroprogenitors perwell, the probability of no nodule at the 1/e level (1/e=0.37, asindicated in Fig. 6C) determines the frequency of chondroprogenitorsin each population (see also Grigoriadis et al., 1996).

For proliferation and apoptosis assays cells were plated at standarddensity and cultured for 24 hours in full medium (5% FCS), thenwashed in PBS and fed with low serum media ± Tc as indicated. For

proliferation assays cells were trypsinised every 48 hours andcounted using a haemocytometer. For apoptosis assays, after a further24 hours, non-adherent and adherent cells (after trypsinisation) werecytospun onto TESPA-coated slides, fixed in 4% paraformaldehydein PBS and stained with haematoxylin and eosin. The cells wereviewed microscopically and the proportion of morphologicallyapoptotic cells (condensed or fragmented nuclei) was calculated bycounting at least 300 cells from random fields, from each of triplicatecell cultures. The frequency of apoptosis was also measured bystaining cytospin preparations with Acridine Orange and by TUNELassays using standard protocols. Similar results were obtained by allmethods (data not shown). All apoptosis experiments were carriedout in the absence of ITS except in 5% FCS, in order to mimicdifferentiation assay conditions. However, c-fos expression caused nodifference in apoptosis rates at 5% FCS in the absence of ITS (datanot shown).

Northern blot analysisCell cultures for expression analyses were harvested by trypsinisationeither at confluence or at the day indicated and cell pellets were storedat −80°C prior to processing. Poly(A)+ RNA isolation, northern blotanalysis and hybridisation in Church buffer were performed aspreviously described (Wang et al., 1991). Probes were labelled with[32P]dCTP (NEN, Boston, MA) to a specific activity of 4×108 cpm/µg DNA using Ready-To-Go oligo-labelling beads (AmershamPharmacia, St Albans, UK). The following probes were used: v-fos/fox (0.8 kb), which hybridises to c-fos and additionally, to c-fox, anabundant RNA found in mouse tissues but unrelated to c-fos (see alsoWang et al., 1991), which was used here as a loading control; murineprobes for fosB (0.27 kb), fra-1 (0.23 kb), fra-2 (0.24 kb), c-jun (0.45kb), junB (0.48 kb), and junD (0.3 kb) were all obtained as gifts fromDr R. Bravo (Bristol-Myers Squibb, Princeton, NJ); murine coll II (0.4kb), coll X (1.2 kb) and aggrecan (0.47 kb) were gifts from DrE.Vuorio (University of Turku, Finland); murine PTH1R (2 kb), fromDr B. Lanske (MPI, Martinsried, Germany); murine Sox-9 (0.52 kb),from Dr P. T. Sharpe (Guy’s Hospital, London, UK); and humanGAPDH (1 kb), from Dr J. Beresford (University of Bath, UK).

Protein analyses Total cellular proteins were extracted for use in western blotting andelectromobility shift analysis (EMSA) studies. Briefly, cells werewashed twice in ice-cold PBS then lysed in Tween lysis buffer (50mM HEPES, 1 mM EDTA, 2.5 mM EGTA, 150 mM NaCl, 1 mMDTT, 0.1% Tween-20, 1 mM NaF, 0.1 mM NaVO4, 100 µg/ml PMSF,1 µg/ml aprotinin, and 1 µg/ml leupeptin, pH 8.0). For western blotanalysis 50 µg of protein/lane was resolved on a 7.5% SDS-PAGE gel(National Diagnostics, Atlanta, GA) and proteins were transferredonto Immobilon P PVDF membranes (Millipore, Watford, UK),which were incubated in block buffer (5% low fat milk powder, 2%bovine serum albumin (BSA) in Tris-buffered saline (TBS)). Blockedfilters were incubated with a 1:1000 dilution of a rabbit polyclonalanti c-Fos antibody (sc-52 Santa Cruz, Santa Cruz, CA) in blockbuffer for 1 hour, and subsequently with a 1:1000 dilution of apolyclonal goat anti-rabbit antibody conjugated to horseradish

Fig. 1. The pJMF2c-fos construct. The murine c-fos cDNA(1.35 kb) was excised from the plasmid pX-c-fos (Superti-Furga et al., 1991) and ligated into the unique BamHIcloning site of the plasmid pJMF2 (Lang and Feingold,1996). Regulation of expression is provided byconstitutive expression of the tetracycline-repressedtransactivator from a CMV promoter (PCMV tTA). In theabsence of Tc the tTA protein binds to the tetracyclineoperator sequence fused to a minimal human CMVpromoter (tetO), directing expression of the cloned target gene, in this case c-fos. Transcription terminates at the SV40 poly A site (SV40 pA)of the expression vector pREP9 (see Materials and Methods).

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peroxidase (Dako, Denmark) in block buffer. c-Fos was visualisedusing enhanced chemiluminescence (ECL) (Amersham Pharmacia).

For electromobility shift assay (EMSA), 5 µg extracted protein wasincubated with 1-2×105 cpm (~35 fmol) of [32P]ATP end-labelled AP-1 specific oligo (5′-GCGTTGATGAGTCAGCCGGAA-3′ – Promega,Southampton, UK), in band shift buffer (50 mM NaCl, 5 mM DTT,10 mM Tris-HCl (pH 7.5), 4% glycerol, 0.5 mM EDTA, 1 mM MgCl2and 10 µg/ml poly(dI-dC)) for 1 hour at 25°C. For supershift analyses,complexes were subsequently incubated with 10 µg of c-Fos specificantibody (sc-52X- Santa Cruz), pan-Fos specific antibody (sc-523X –Santa Cruz), or non-c-Fos reactive rabbit immunoglobulins (Dako) for1 hour at 4°C. Complexes were resolved by electrophoresis on 4%non-denaturing polyacrylamide gels in 0.5× TBE. Gels were dried andexposed for autoradiography.

RESULTS

Gene expression during ATDC5 chondrogenesisWe have analysed the expression of cartilage marker genes, andof c-fos and c-jun family members, throughout the in vitrodifferentiation of ATDC5 cells. Expression was analysed at thefollowing stages of cartilage differentiation: pre-confluentcultures (day 3), confluent cultures (day 5), the appearance ofvisible multi-layered nodules (day 11), matrix accumulation(day 15) and the onset of chondrocyte hypertrophy (day 21).Additionally, some cultures were continued until day 30 todemonstrate the further accumulation of hypertrophic cells

(Shukunami et al., 1997). Type II collagen (coll II) is thepredominant collagenous protein of cartilage matrix and itsexpression is highly specific for chondrogenic cells. Coll IIexpression was detectable in early stage cultures but levelsshowed a significant increase from day 11, reached a maximumat day 21, and decreased slightly with hypertrophicdifferentiation (day 30; Fig. 2). Aggrecan, which is the majornon-collagenous protein in cartilage matrix, showed an almostidentical temporal expression profile to that of coll II (Fig. 2).Expression of type X collagen (coll X), a marker forhypertrophic chondrocytes, was undetectable in early stageATDC5 cultures, but was observed at low levels from day 11,and increased to a maximum in day 30 cultures (Fig. 2). TheHMG-domain gene Sox-9, a known marker of earlychondrogenic commitment (Wright et al., 1995), was expressedat detectable levels in proliferating ATDC5 cells (day 3) and atsimilar levels throughout the culture period (Fig. 2). We alsoobserved a continuous increase over time in levels of mRNAexpression for PTH1R (Fig. 2) which has previously beenshown to be a marker for chondrogenic progression of ATDC5cells (Shukunami et al., 1996). Additionally, the visibledevelopment of cuboidal cells into multi-layered nodules, andthe deposition of cartilage matrix, as assessed by Alcian bluestaining (data not shown), accelerated significantly from day 11in parallel with the expression of coll II and aggrecan. Thus, asassessed by both gene expression and morphological criteria,the ATDC5 cells used in this study recapitulate the well-established sequence of cartilage differentiation fromchondroprogenitor cells to hypertrophic chondrocytes.

Within the context of this in vitro differentiation, levels ofexpression of mRNA for the c-fos and c-jun family memberswere assessed. c-fos expression was detectable at all timepoints albeit at low levels with a slight increase at later timepoints (Fig. 3A), and this was also seen at the protein level (Fig.3C). Similarly, levels of fosB mRNA appeared generally absentthroughout differentiation but became detectable at later stages(Fig. 3A). In contrast, fra-1 expression was detectablethroughout differentiation at significant levels and fra-2showed differentiation-dependent variation in expression, withlevels elevated prior to confluence (day 3) and during matrixdeposition (day 15; Fig. 3A). Members of the c-jun family ofgenes were readily expressed at significant levels throughoutdifferentiation (Fig. 3B). Levels of junB mRNA were initiallyelevated (day 3), but in post-confluent cultures both c-jun andjunB showed similar profiles with levels increasing until day15, then decreasing during hypertrophic differentiation (Fig.3B). In fact, c-jun mRNA levels were further decreased at day30 (data not shown). Expression of junD was initially low (day3) but maintained steady-state levels throughout the remainderof the time course. To investigate the functionality of thedifferent Fos and Jun proteins expressed throughout ATDC5cell differentiation AP-1 binding complexes were assessed byelectro-mobility shift assay (EMSA). AP-1 complexes weredetected at all time points (Fig. 3D), and the specificity ofbinding was determined by oligo competition studies (data notshown). Supershift analyses demonstrated that no c-Fos-specific supershift was detected at any of the time points, butwas seen in a stable clone constitutively overexpressing c-Fos(clone 8.6; see also Fig. 4A). In contrast an antibody thatrecognises all Fos family members (pan-Fos) retarded all AP-1 complexes, whereas non-specific rabbit immunoglobulins


Fig. 2. Expression of cartilage markers throughout ATDC5differentiation. Northern blot analysis was performed on 5 µg ofpoly(A)+ RNA, extracted from ATDC5 cells at 3, 5, 11, 15, 21 and30 days of differentiation (see Materials and Methods). Filters wereserially hybridised with cDNA probes for coll II, aggrecan, coll X,Sox9, and PTH1R (see Materials and Methods) with transcript sizesindicated on the left. Additionally, filters were probed with v-fos/foxwhich hybridises to the abundant fox transcripts, and which are usedas a loading control.


failed to elicit any supershift (Fig. 3D). This demonstrates thatall active AP-1 complexes act as Fos-family:Jun-familyheterodimers and Jun:Jun dimers are either low or absent.Since c-Fos and FosB expression were apparently low, the AP-1 complexes of ATDC5 cells most likely consist of Fra proteinsdimerised with specific Jun protein partners.

Constitutive overexpression of c-fos in ATDC5 cellsPrevious evidence from in vivo studies has suggested thatelevated c-fos expression may have an effect on the

differentiation of chondrocytes (Wang et al., 1991; Watanabeet al., 1997). We initially investigated this in vitro bytransfection of ATDC5 cells with the construct MT-c-fosLTR(Rüther et al., 1985), which has previously been shown toexpress high levels of exogenous c-fos in chondrocytes in vivo(Wang et al., 1991, 1993). Six stable clones were selected inG418 and analysed for gene expression and differentiationpotential. Three of the clones (DT8.2, DT8.5, DT8.6)expressed the transgene at high levels, whilst three (DT8.1,DT8.4, DT8.8) expressed no detectable exogenous c-fostranscripts (Fig. 4). All of the c-fos expressing clones failed todemonstrate significant levels of cartilage nodule formationimplying that c-fos expression is inhibitory to chondrogenicdifferentiation (Fig. 4). One c-fos-negative clone (DT8.4) diddifferentiate to levels comparable to wild-type ATDC5 cells,although others (DT8.1, DT8.8) did not (Fig. 4). This mayreflect clonal variation and we investigated the possiblemolecular basis for this by analysing markers of chondrocyte

Fig. 3. Expression analyses of c-fos and c-jun family membersthroughout ATDC5 differentiation. (A) mRNA expression of c-fosfamily members. Northern blot analysis of poly(A)+ RNA extractedfrom ATDC5 cells at the time points indicated. Filters were seriallyprobed with v-fos/fox, fosB, fra-1 and fra-2. (B) mRNA expressionof c-jun family genes. Northern blot of RNA samples as in A seriallyhybridised with cDNA probes for c-jun, junB, junD and GAPDH as aloading control. All mRNA transcript sizes are indicated to the left ofthe figures. (C) Western blot analysis of 50 µg total protein extractedfrom ATDC5 cells at the time points indicated. In addition, 50 µg ofprotein from clone DT8.6, which constitutively expresses c-Fos (seeFig. 4), was loaded as a positive control (8.6). The indicated c-Fosspecific bands are 55-62 kDa in size. (D) Supershift analysis of 5 µgof total protein extracted from differentiating cultures of ATDC5cells on the days indicated (d). DNA binding complexes wereincubated with an antibody specific for c-Fos (F), an antibodyrecognising all Fos members (p) or non-specific rabbitimmunoglobulins (n). The first lane contains no protein extract. Thepositions of free probe (P) and AP-1 specific shifts are indicated (A),as are supershift bands (S). Only the overexpressing clone DT8.6(8.6) (see Fig. 4) demonstrated a c-Fos-specific supershift. Note thatthis consists of 2 bands suggestive of multiple complexes.

Fig. 4. Expression analysis and nodule formation of stable clonestransfected with MT-c-fosLTR. Poly (A)+ RNA was isolated from 6clones (DT8.1, DT8.2, DT8.4, DT8.5, DT8.6 and DT8.8) after 5days in culture, subjected to northern blot analysis and hybridisedwith cDNA probes for v-fos/fox, Sox9 and coll II. Additionallypoly(A)+ RNA from wild-type ATDC5 cells at day 15 (AT(d15)) wasloaded as a positive control for endogenous c-fos and for coll IIexpression (see Figs 2 and 3A). The positions of endogenous (en.c-fos) and exogenous (ex. c-fos) c-fos transcripts are indicated.Although high expression of coll II can only be seen for samplesDT8.4 and DT8.6, all clones displayed detectable coll II expressionafter a longer autoradiographic exposure. All mRNA transcript sizesare indicated to the left of the figure. The bottom panel indicatessemi-quantitative analysis of levels of nodule formation in DT8clones by Alcian blue staining after 21 days in culture. ‘+++’represents levels equivalent to wild-type, whilst ‘–’ representscomplete absence of Alcian blue staining.

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differentiation: Sox-9, was expressed in all clones at similarlevels, but coll II was variable with highest expression in clonesDT8.4 and DT8.6 (Fig. 4). Interestingly, these clones were theonly two that demonstrated significant nodule formation andthey also displayed a clear negative correlation between levelsof exogenous c-fos and the extent of differentiation.

Inducible expression of c-fos in ATDC5chondrocytesTo overcome the problem of clonal variation we sought totransfect ATDC5 cells with a regulatable expression construct,where levels of c-fos could be varied within single clones andtherefore the effect of high and low expression can be assessedon a stable background of known differentiation potential. Tothis end, we have cloned the murine c-fos cDNA into pJMF2(Lang and Feingold, 1996), a single vector system based on theTet-off system as originally derived by Gossen and Bujard(1992), where expression of a cloned gene is repressed in thepresence of tetracycline (Tc), but can be induced to high levelsupon withdrawal of Tc from the culture medium of stablytransfected cells. We transfected this construct (pJMF2-c-fos(Fig. 1)) into ATDC5 cells and selected clones in G418 and inthe continuous presence of Tc. Two clones (DT7.1 andDT12.4) derived from independent transfections showed highlevels of exogenous c-Fos induction upon withdrawal of Tc:western blot analyses demonstrated strong induction of c-Fosprotein in clone DT7.1 (~10-fold) and clone DT12.4 (~100-fold) (Fig. 5A) and similar levels of induction were observedat the RNA level (see Fig. 8-top).

In order to assess the effects of induced c-fos expression onchondrocyte differentiation we performed the differentiationassay on clones DT7.1 and DT12.4 in the presence and absenceof Tc. Both clones formed significant numbers of Alcian blue-positive nodules with levels of exogenous c-fos repressed (1µg/ml Tc). However, in both clones there was a significantdecrease in the levels of differentiation upon induction ofexogenous c-fos (withdrawal of Tc), with an estimated 30-50%decrease in the number of nodules in clone DT7.1, and analmost complete abolition of nodule formation in clone DT12.4(Fig. 5B). As with the stably transfected clones, there appearedto be variation in the basal levels of nodule formation (+Tc),most probably due to clonal variation, however, the inhibitionof nodule formation in the absence of Tc in these clones wasindependent of such variation and thus dependent on the levelsof exogenous c-fos expression. These results clearlydemonstrate that chondrocyte differentiation is inhibited byexogenous c-fos expression and this effect is independent ofclonal variability.

Cellular effects of exogenous c-fos expressionWe have used the tightly regulatable expression of exogenousc-fos in DT12.4 cells to define more completely the role of c-fos in ATDC5 cell differentiation in vitro. Initially we analysedthe effect of c-fos overexpression at different stages of thedifferentiation process. Thus, DT12.4 cells were cultured eitherin the continuous presence of Tc (i.e. low c-fos), or in itsabsence (i.e. high c-fos) for 4 day periods as indicated. Elevatedexogenous c-fos levels during the pre-confluence stage (day 0-4) had a significant inhibitory effect with an ~50% decrease innodule formation, whereas, induction of high c-fos levels afterconfluence (after day 4) failed to cause an effect (Fig. 6A,B).

This suggests that the inhibitory effects of c-fos are manifestedprimarily at the early stages of differentiation, possibly byaffecting specific chondroprogenitor cell populations. Toaddress this possibility, we have used limiting dilution analysisto define the effects of c-fos on the number ofchondroprogenitors (Fig. 6C). The results showed that in theabsence of c-fos (+Tc) approximately 1 in every 22 cells platedis able to form cartilage, however, in the presence of elevatedc-fos (−Tc) only ~1 in 65 cells forms cartilage, anapproximately 3-fold decrease in the proportion ofchondroprogenitors (Fig. 6C). These data thereforedemonstrate that the inhibition of differentiation in vitro byexogenous c-fos is a direct effect on the frequency ofchondroprogenitor cells within the cell population.

In addition to direct roles for c-fos in chondrocytedifferentiation, the possibility remains that exogenous c-fosexpression additionally perturbs other cellular processes, suchas proliferation and apoptosis, and these may contribute, atleast in part, to the observed inhibition of differentiation.Indeed, roles for c-fos in proliferation and apoptosis have beencited in a number of systems (see Angel and Karin, 1991;Smeyne et al., 1993; Pandey and Wang, 1995). We haveinitially investigated a role for c-fos in proliferation of DT12.4cells by growth curve analysis at different serum


Fig. 5. Expression of c-fos in clones transfected with the pJMF2-c-fos construct inhibits chondrocyte differentiation. (A) Western blotanalysis of total protein extracted from clones DT7.1 and DT12.4after 5 days of culture in the presence (+Tc) or absence (−Tc) of Tc,probed with a c-Fos specific antibody. Note the strong induction ofmultiple c-Fos-reactive bands at ~55-62 kDa in size in the absence ofTc presumably representing different phosphorylation states.(B) Cultures of clones DT7.1 and DT12.4 were stained with Alcianblue after differentiation for 21 days in either the continuouspresence (+Tc) or absence (−Tc) of 1 µg/ml Tc. c-fos inductionclearly results in an inhibition of chondrogenesis in both clones.Additionally, non-transfected ATDC5 cultures were processed inparallel to demonstrate that Tc at 1 µg/ml has no effect onchondrogenesis.


concentrations. Interestingly, in full serum conditions (5%FCS), there is no difference in cell number in the presence orabsence of c-fos expression (Fig. 7A). However, at lowerserum concentrations (0.5% FCS and 0.1% FCS), c-fosexpression does appear to be mitogenic (Fig. 7A). Likewise,the effect of elevated c-fos expression on apoptosis of pre-confluent DT12.4 cells was quantified at different serumconcentrations. Again, in standard culture conditions (5%FCS), apoptosis rates were low and unaffected by theexpression of c-fos. However, in low serum conditions, ratesof apoptosis are decreased approximately twofold in thepresence of exogenous c-fos expression (Fig. 7B). As the rates

of both proliferation and apoptosis were unaffected by c-fosexpression in standard culture conditions (5% FCS) then itseems unlikely that these processes are contributing towardsthe observed inhibition of differentiation. Nevertheless, takentogether, the proliferation and apoptosis data do imply thatectopic c-fos expression can lead to decreased serumdependence in DT12.4 cells.

Finally, the high levels of c-fos expression induced in cloneDT12.4 resulted in a clear change, from a polygonal to aspindle-shaped morphology exhibiting elongated processespossibly indicating transformation (data not shown). Thismorphological change was reversible upon readdition of Tcand appears consistent with the previously reported effects ofc-Fos on fibroblast morphology (Miao and Curran, 1994) and,more importantly, with the demonstration that chondrocytes

Fig. 6. The role of c-fos expression in chondrocyte differentiation.(A) DT12.4 cultures were maintained in the continuous presence ofTc, with withdrawal of Tc for 4 day periods as indicated (d=day)resulting in a pulse of elevated c-fos expression only for theseperiods. All cultures were then stained for Alcian blue. (B) Afterstaining the amount of stain in each plate was solubilised and theoptical density measured at 595 nm (OD 595). The data from eachtime point represent the mean ± s.e.m. of triplicate wells. Controlcultures in the continuous presence of Tc (+Tc) are included.(C) Limiting dilution analysis of chondroprogenitor frequency inDT12.4 cells (see Materials and Methods). From the graph thechondroprogenitor frequency in the presence of Tc (+Tc) isestimated at 1 in 22 cells, and in the absence of Tc (−Tc) at 1 in 65.

Fig. 7. The effects of exogenous c-fos on proliferation and apoptosis.(A) Growth curve analysis of DT12.4 cells. Cells were plated atstandard density with Tc and serum (FCS) conditions as indicated tothe right of the figure. Cell numbers were counted every 2 days for16 days. (B) Apoptosis rates of DT12.4 cells. Cells were cultured for24 hours in the serum concentrations indicated, in either the presence(+Tc) of absence (−Tc) of Tc. The percentage of apoptotic cells wascalculated after staining of cells with heamatoxylin and eosin, andare expressed as mean ± s.e.m. of triplicate counts.

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were target cells for c-Fos-induced transformation in ES cellchimeric mice (Wang et al., 1991, 1993).

Modulation of gene expression by exogenous c-fosThe pattern of gene expression of DT7.1 and DT12.4 cell linesin the presence and absence of induced exogenous c-fosexpression was assessed by northern blot analysis at both early(day 5) and late (day 21) time points of differentiation.Expression of the early chondrocyte marker Sox-9 wasunaffected by either the levels of exogenous c-fos expressionor the stage of differentiation in either cell line (Fig. 8).Interestingly, in DT12.4 cells at day 5, there was a cleardecrease in the levels of coll II expression with high c-fos(−Tc; Fig. 8), whilst levels of PTH1R appeared unaffected atthis stage. At day 21 the expression of coll II and PTH1R weremarkedly decreased in both cell lines in the presence of

exogenous c-fos (−Tc; Fig. 8) and correlated well with thedifferentiation status (Fig. 5B).

With respect to AP-1-related genes mRNA levels of both fra-1 and c-jun were upregulated in the presence of high levels ofexogenous c-fos (−Tc) in both DT7.1 and DT12.4 cell lines andat both early and late stages of chondrocyte differentiation (Fig.8). The elevation of c-jun appeared to be most significant at day5 in DT12.4 cells, whereas c-fos-dependent increases in fra-1expression were comparable between both cell lines at both earlyand late time points.

DISCUSSION

In this paper we have demonstrated that overexpression of c-Fosdirectly inhibits differentiation in ATDC5 chondrocytes. Initiallythis was achieved by assessing differentiation in ATDC5 clonesstably overexpressing c-Fos from a constitutive promoter andsubsequently, by expression from an inducible promoter we haveshown that elevated c-Fos levels inhibit differentiation withinindividual clones independently of clonal variation inchondrogenic potential. No previous study has investigated theeffects of exogenous c-Fos expression on chondrocytedifferentiation in vitro, although negative effects on matrixdeposition have previously been observed in HCS chondrocytes(Tsuji et al., 1996). This in vitro inhibition is in accordance withthe previous in vivo evidence for a role of c-Fos. Thus, gain-of-function experiments have shown that the progression ofproliferating chondrocytes to hypertrophy was severely retardedwhen chick limb buds were infected with c-fos expressingretroviruses (Watanabe et al., 1997), whilst chondrocytesisolated from c-fos-induced chondrosarcomas of ES cellchimeric mice likewise were unable to progress to hypertrophy(Wang et al., 1991, 1993). In addition, loss-of-function studiesin c-fos knockout mice demonstrated a diminished zone ofproliferating chondrocytes at the epiphyseal growth plate (Wanget al., 1992), which may be due to altered chondrocyteprogression in the absence of c-Fos.

Initially, we correlated the phenotypic differentiation of wild-type ATDC5 cells with the pattern of expression of a numberof chondrogenic markers. Both Sox-9 and coll II, at low levels,were expressed in pre-confluent cultures, indicative of analready committed chondroprogenitor population. Cartilagedifferentiation was evident from day 11 as indicated by thedramatic rise in coll II expression, and this was coincident withthe deposition of Alcian blue-staining matrix and with theexpression of aggrecan and PTH1R. In addition, wedemonstrated hypertrophic differentiation of longer termcultures (day 30), by elevated expression of coll X and decreasedlevels of coll II and aggrecan.

AP-1 gene expression in differentiating ATDC5 cellsThe analysis of the expression levels of c-fos and c-jun familygenes throughout ATDC5 chondrogenesis revealed someinteresting expression patterns. The most prominent fos familymembers expressed during differentiation were the fra genes,whilst c-fos and fosB were expressed at low levels whichincreased only slightly with differentiation. Expression of the junfamily members was approximately constitutive throughout,with c-jun and junB elevated during matrix deposition, butdecreased during hypertrophic differentiation. Interestingly, a


Fig. 8. Gene expression analysis of clone DT7.1 and DT12.4 withrepressed and induced c-fos expression at early and late stages ofdifferentiation. Poly (A)+ RNA was extracted from DT7.1 andDT12.4 cells grown either in the presence (+Tc) or absence (−Tc) ofTc at either early (d5) or late (d21) stages of differentiation. Filterswere serially probed with v-fos/fox, Sox-9, PTH1R, coll II, fra-1 andc-jun. Transcript sizes are indicated on the left of the figure. For allprobes, exposure times were identical between both clones at bothtime points. However, in DT12.4 cells the film for coll II expressionwas overexposed compared to DT7.1 cells, to allow a bettercomparison of levels ±Tc and to demonstrate inhibited coll IIexpression at d5; basal coll II expression in DT7.1 cells issignificantly higher than in DT12.4 cells.


decrease in c-jun expression has been observed duringhypertrophic differentiation of embryonic chick chondrocytes(Kameda et al., 1997). These expression data suggest that themajority of AP-1-specifc DNA binding complexes in wild-typeATDC5 cells consist of Fra:Jun heterodimers, and this issupported by supershift analyses. However, it is likely that AP-1 complex composition will change during the differentiationprocess, which could result in differential regulation of targetgenes. This possibility is supported by evidence from ananalogous osteoblast differentiation model, where lowendogenous c-fos expression, and stage-specific changes in AP-1 complex composition have been demonstrated (McCabe et al.,1995, 1996). Whether comparable changes in complexcomposition occur during ATDC5 chondrogenesis remains to bedetermined by further supershift experiments using antibodiesspecific for each AP-1 family member.

The low levels of c-fos expression observed do not necessarilyimply that c-fos has no function in chondrogenesis. The earliestembryonic expression of c-Fos is seen in chondrogenic cells ofthe developing limbs (Dony and Gruss, 1987; DeTogni et al.,1988), and postnatal expression has been detected inproliferating and articular chondrocytes (Grigoriadis et al., 1993;Sunters et al., 1998) as well as in hypertrophic chondrocytes byreporter gene expression in c-fos-lacZ transgenic mice (Smeyneet al., 1993). More importantly, it seems that high level c-fosexpression can be induced in chondrocytes when required, forexample, in response to specific extracellular stimuli, such asPTH (Lee et al., 1994; see also below). Basal levels ofendogenous c-fos may not be important in its role indifferentiation, as elevated levels of c-fos appear to inhibitchondrogenesis even when higher basal levels are detectable,both in vivo (Wang et al., 1991, 1993; Watanabe et al., 1997)and in vitro in C5.18 chondrocytes (D. P. Thomas and A. E.Grigoriadis, unpublished results). In fact, lower steady statelevels of c-Fos may be preferable if c-fos expression is indeedinhibitory to chondrocyte differentiation. Moreover, previousevidence in vivo demonstrated that elevated c-Fos expression ona background of high endogenous expression may initiate acascade of oncogenic transformation (Wang et al., 1991, 1993).Thus, it is probable that there exists a threshold of c-Fosexpression, above which chondrocytes are susceptible totransformation, but below which c-Fos functions to regulatechondrocyte differentiation. As c-Fos can only act as onecomponent of AP-1 complexes, the mechanisms whereby c-Foscauses inhibition of differentiation are likely to depend on theavailability of dimerisation partners (e.g. Jun proteins) and theextent to which c-Fos can compete with other Fos-relatedproteins for dimerisation, resulting in the formation of newcomplexes, and presumably differential regulation of specifictarget genes.

Overexpression of exogenous c-fos inhibitschondrocyte differentiation Constitutive overexpression experiments using a c-fos constructwhich caused chondrosarcomas in ES cell chimeric mice (Wanget al., 1991, 1993) yielded three ATDC5 clones which expressedhigh levels of exogenous c-fos but failed to differentiate. Theseclones thus provide an initial indication that c-fos overexpressionis inhibitory to chondrocyte differentiation. However, otherclones did not express exogenous c-fos, and despite beingcommitted to the chondrocyte lineage, as judged by Sox-9 and

coll II expression, they also failed to differentiate. The reasonsfor this clonal variation are presumably due to subpopulationheterogeneity which is not uncommon when clones are derivedfrom essentially non-homogeneous progenitor populations, forexample, C3H10T1/2 (Taylor and Jones, 1979), RCJ 3.1(Grigoriadis et al., 1988), and RCJ 3.1C5 clones (Grigoriadis etal., 1996).

We subsequently derived ATDC5 clones where high levelexpression of exogenous c-fos could be induced by withdrawalof Tc. Such a regulatable system permits differentiation assaysand expression analyses to be carried out at both high and lowlevels of c-fos in the same clone allowing unambiguousassessment of the potential role of c-Fos in differentiation andcartilage-specific gene expression. Additionally, by adding orremoving Tc at different time points it is possible to assess theeffect of c-fos expression at various stages of differentiation.

Clones DT7.1 and DT12.4 demonstrated tight regulation ofexogenous c-fos expression in the presence and absence ofTc and exogenous c-fos induction significantly inhibitedchondrogenesis in both of these clones with a 30-50% decreasein nodule formation for clone DT7.1 and an almost completeabolition of differentiation in clone DT12.4. This analysis furtherconfirms the observations of the constitutively c-fosoverexpressing clones. In clone DT12.4, there was a significantdecrease following c-fos induction in the expression levels ofcoll II at day 5, whilst both clones displayed downregulation ofcoll II and PTH1R at late stages of differentiation. As both ofthese genes are markers of chondrocyte differentiation, then theirdown-regulation at late stages may be secondary to the inhibitionof differentiation. However, down-regulation of coll II at earlystages of DT12.4 differentiation, may indicate more directregulation by c-fos that may, at least in part, contribute towardsthe inhibited differentiation. Whether c-fos can directly modulatecoll II expression remains to be determined (see also below). Inboth of the regulatable clones, fra-1 and c-jun expression wereupregulated together with c-fos at both at early and late stagesof differentiation. This suggests a potentially more complexpattern of AP-1 transcription factor activity after elevation of c-fos that may have implications for the control of downstreamtargets. Fra-1 is considered to be a c-Fos target gene as it hasbeen associated with high c-Fos levels in fibroblasts andosteoblasts (Braselmann et al., 1992; Grigoriadis et al., 1993;Schreiber et al., 1997). Moreover, ectopic fra-1 expression invitro can stimulate osteoclastogenesis (Owens et al., 1999) andfra-1 transgenic mice have specific osteoblastic defects and canrescue the block in osteoclast differentiation in c-fos knockoutmice (Jochum et al., 1999; Matsuo et al., 1999). Based on ourinducible overexpression system it appears that fra-1 also lies inthe pathway induced by c-Fos in chondrocytes. In contrast, thecorrelation between c-jun and c-fos expression in different celltypes is not so well established. Exogenous c-fos expression inosteoblasts in transgenic mice, does not result in enhanced c-junexpression (Grigoriadis et al., 1993), however, in ES cellchimeric mice there is a clear correlation between c-fos and c-jun expression: All chimeric tissues expressing exogenous c-fos,including chondrosarcomas and chondrogenic cell lines derivedfrom these tumours, demonstrate high c-jun levelsconcomitantly with exogenous c-fos (Wang et al., 1991, 1993).In addition, like c-fos, c-jun expression in chick chondrocytesdelays their differentiation (Kameda et al., 1997). Although fra-1 and c-jun appear to be regulated by c-Fos in chondrocytes,

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whether they have specific roles in chondrocyte biology remainsto be determined, for example, by specific gain-of-function andloss-of-function analyses.

In order to define the specific time points duringdifferentiation in which the inhibitory action of c-Fos ismanifested, DT12.4 cells were ‘pulsed’ with elevated c-fos levelsfor 4 day periods. Interestingly, only when exogenous c-fos wasexpressed during the subconfluent phase (days 0-4) was adecrease in nodule formation observed, whilst elevation of c-fosat later time points had no significant effect. The reduction in thenumber of differentiated nodules and the putative window of c-Fos action suggests that c-Fos acts directly on chondroprogenitorcells to inhibit their progression. This was confirmed by limitingdilution analysis of DT12.4 cells, which demonstrated an ~3-fold decrease in the number of cells competent to differentiateinto cartilage. Although, c-Fos clearly affects the frequency ofchondroprogenitor cells within ATDC5 cell populations, highlevels are nevertheless not completely incompatible withchondrocyte differentiation, as some nodule formation persistsin these clones. These data imply that there may exist asubpopulation of precursors that are not affected by elevated c-Fos. Alternatively, together with the pulse experimentsdemonstrating an early effect, our observations may point to thepresence of a restriction point, prior to which c-Fos expressioncan inhibit the differentiation of chondroprogenitors, but oncepassed c-Fos has no apparent effect. Whilst the effects of c-Foson early chondroprogenitors are clear, at this point we can notexclude the possibility that, under different experimentalconditions, other effects may be uncovered, for example,alterations in proteoglycan synthesis or changes in rates ofmatrix deposition.

Effects of c-fos on chondrocyte proliferation andapoptosisHaving defined a role for c-fos expression in the differentiationof chondrocytes in vitro, we have also sought to analyse whetheror not c-fos affects other cellular processes such as proliferationand apoptosis as have been demonstrated in various othersystems (Angel and Karin, 1991; Smeyne et al., 1993; Pandeyand Wang, 1995). One important reason for doing so is thatdecreases in chondroprogenitor proliferation and/or increases inapoptosis may to some degree contribute towards the observeddecrease in nodule formation. In analysing the rates ofproliferation and apoptosis of DT12.4 cells in the presence andabsence of c-fos expression, we demonstrated that under theconditions whereby c-Fos inhibited differentiation (5% FCS)neither the growth rate nor the apoptotic index were significantlyaffected. However, modulation of growth rates and apoptosiswere nevertheless observed under conditions of reduced serumconcentrations, with rates of proliferation increased and rates ofapoptosis decreased in the presence of exogenous c-fos.Therefore, under these conditions, c-Fos both increases themitogenicity of these chondrocytes, and protects them fromapoptosis, indicative of decreased serum dependence. We havealso observed similar effects in osteoblastic MC3T3-E1 cellsoverexpressing c-fos (A. Sunters, D. P. Thomas and A. E.Grigoriadis, unpublished), and the roles of c-fos expression onthe molecular mechanisms of proliferation and apoptosis inchondrocytes in vitro are currently under investigation. Thus,although c-Fos has the potential to regulate the rates ofcell growth and programmed cell death of ATDC5

chondroprogenitors, these processes apparently do notcontribute to the inhibitory effect of c-Fos on chondrocytedifferentiation.

The role of c-Fos expression in chondrocytesThe in vitro evidence presented here clearly defines a role for c-Fos in inhibiting the differentiation of ATDC5 chondrocytes, buthow does this fit into previously described models ofchondrocyte differentiation? One strong candidate for aphysiologically relevant stimulus of c-Fos expression inchondrocytes would be PTHrP. In this regard, it is extremelyinteresting that the endochondral growth plates of PTHrPknockout mice (Karaplis et al., 1994) and c-fos knockout mice(Wang et al., 1992) look very similar, suggesting that the normalrole of both of these molecules is to inhibit the differentiation ofgrowth plate chondrocytes. Signalling via the PTH1R has beenshown to upregulate c-Fos expression in osteoblasts in vitro(Pearman et al., 1996; McCauley et al., 1997) and in growth platechondrocytes in vivo (Lee et al., 1994), and using transgenic andknockout mice the PTH1R signalling cascade has been shownto regulate normal chondrocyte differentiation in vivo (Weir etal., 1996; Vortkamp et al., 1996: Lanske et al., 1996; Schipaniet al., 1997). Interestingly, in ATDC5 cells, our preliminaryresults indicate that PTH efficiently stimulates c-fos expression,and inhibits cartilage differentiation with similar kinetics to theeffects demonstrated by pulsed elevation of c-fos in our Tc-regulatable clones (D. P. Thomas and A. E. Grigoriadis, data notshown). Thus, it is likely that c-fos represents a specificphysiological target for PTHrP signalling and may mediate atleast some of the phenotypic effects of PTHrP action inchondrocytes. In addition, it is possible that c-fos expression isalso regulated by other signalling pathways important inchondrogenic cells, such as those induced by bonemorphogenetic proteins (BMPs) and BMP receptors (Zou et al.,1997), hedgehog proteins (Vortkamp et al., 1996; Iwasaki et al.,1997), and FGF receptors (Peters et al., 1992; Naski et al., 1998).

The identification of c-fos responsive genes in chondrocyteswill be important in understanding the molecular roles of c-fosin mediating the observed phenotype. In particular, the earlyeffects of c-Fos provide a useful time window for analysis ofgenes that are potentially direct transcriptional targets of c-Fos,and the tight regulation of c-fos expression in our clones providesan excellent system for the screening of such targets. Candidatec-Fos-regulated genes may be proposed on the grounds eitherthat they have demonstrated important roles in chondrocytebiology, for example from gene deletion studies, or that theyhave been shown previously to be regulated by c-Fos. Suchgenes fall into several categories: Firstly, cellular transcriptionfactors such as additional members of the Sox family (Sox-5 andSox-6; Lefebvre et al., 1998), as well as ATF-2 (Reimold et al.,1996), Ets2 (Sumarsono et al., 1996), and cbfa-1 (Inada et al.,1999) affect cartilage differentiation and some of these havebeen shown to interact with AP-1 complexes and modulate geneexpression (De Cesare et al., 1995; Basuyaux et al., 1997;Selvamurugan et al., 1998; Porte et al., 1999). Secondly,profound effects on chondrogenesis have been reported inresponse to autocrine or paracrine signalling mediated by BMPs(see Hogan, 1996), hedgehog proteins (Vortkamp et al, 1996)and FGFs (Naski et al., 1998; for review see Tickle and Eichele,1994), as well as by inhibitors, e.g. Noggin (Brunet et al., 1998;Ito et al., 1999). Thirdly, several genes associated with cell cycle



control have demonstrated specific roles in chondrocytedifferentiation, such as the Rb-related genes p107 and p130(Cobrinik et al., 1996), and the CKI p57 (Zhang et al., 1997),whilst cyclin D1 has been shown to be regulated by c-Fos andFos-related genes in fibroblasts and chondrocytes in vitro(Brown et al., 1998; Beier et al., 1999), and in osteoblasts in vivo(Sunters et al., 1998). Finally, regulation of the apoptosis genes,Bcl-2 and Bax by PTH has been demonstrated in growth platechondrocytes in vivo (Amling et al., 1997), and thereforerepresent potential c-Fos targets. Besides elucidating whetherknown genes are affected by altered c-Fos levels, this inducibleexpression model allows for the identification of novel targetsby cDNA subtractive hybridisation techniques, specificallyduring early stages of differentiation and these studies arecurrently underway.

We thank Dr Peter Angel (DKFZ, Heidelberg, Germany) and DrBernd Baumann (University of Würzburg, Germany) for helpful advicewith EMSA analyses and Dr Chris Healy for discussions and criticalreview of the manuscript. This work was generously supported by theArthritis Research Campaign (G0519).

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Inhibition of chondrocyte differentiation in vitro by constitutive