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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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440
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
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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 3×10−8M 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
6×103cells/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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442
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
D. P. Thomas and others
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.
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443c-Fos inhibits chondrocyte differentiation
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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444
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
D. P. Thomas and others
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.
-
445c-Fos inhibits chondrocyte differentiation
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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446
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
D. P. Thomas and others
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.
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447c-Fos inhibits chondrocyte differentiation
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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448
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
D. P. Thomas and others
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449c-Fos inhibits chondrocyte differentiation
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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