The cartilage-specific transcription factor Sox9 regulates
AP-2
e
expression in chondrocytes
Ann-Kathrin Wenke
1
, Susanne Gra
¨
ssel
2
, Markus Moser
3
and Anja K. Bosserhoff
1
1 Institute of Pathology, University Regensburg, Germany
2 Department of Orthopedics, University Regensburg, Germany
3 Max-Planck-Institute of Biochemistry, Martinsried, Germany
The family of activating enhancer-binding protein
(AP)-2 transcription factors regulate their target genes
through binding to the palindromic recognition
sequence 5¢-GCCN
3
GGC-3¢ or variations of this
GC-rich sequence within multiple gene promoters [1].
Both in vitro and in vivo data from AP-2 knockout
mice have shown their importance in numerous physi-
ological processes during development, cell cycle regu-
lation, and cell survival [1,2]. The AP-2 family consists
of five members: AP-2a, AP-2b, AP-2c, AP-2d and
AP-2e [3–8]. They all share a conserved basic-helix–
span–helix DNA-binding and dimerization domain

doi:10.1111/j.1742-4658.2009.06973.x
Activating enhancer-binding protein (AP)-2e was previously described as a
new regulator of integrin a
10
expression in cartilage. In this study, we ana-
lyzed the expression of AP-2e in differentiated chondrocytes and in human
mesenchymal stem cells (HMSCs), which have been differentiated into
chondrocytes in vitro. AP-2e is predominantly expressed during the late
stages of chondrocyte differentiation, mainly in early hypertrophic carti-
lage, consistent with immunohistochemical stainings of mouse embryo
sections. Furthermore, osteoarthritic chondrocytes, resembling a hyper-
trophic phenotype, have high AP-2e levels. The AP-2e promoter harbors
binding sites for the transcription factors AP-2a and Sox9. Both transcrip-
tion factors strongly activate AP-2e expression in a cooperative manner in
the chondrosarcoma cell line SW1353. The inhibition of Sox9 expression
by small interfering RNA resulted in decreased AP-2e expression. In
addition, direct interaction of Sox9 with the AP-2e promoter could be con-
firmed by chromatin immunoprecipitation and electromobility shift assays.
This is the first study to prove the direct regulation of AP-2e by the
transcription factor Sox9, and to indicate that AP-2e potentially has an
important role as a modulator of hypertrophic cartilage.
Abbreviations
AP, activating enhancer-binding protein; CD-RAP, cartilage-derived retinoic acid-sensitive protein; ChIP, chromatin immunoprecipitation; ECM,
extracellular matrix; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HMSC, human mesenchymal stem cell; OA, osteoarthritis; SEM,
standard error of the mean; siRNA, small interfering RNA.
2494 FEBS Journal 276 (2009) 2494–2504 ª 2009 The Authors Journal compilation ª 2009 FEBS
starts with the migration of undifferentiated mesenchy-
mal cells to regions that are destined to differentiate
into bones. These progenitor cells condense and stick
together without increased proliferation [14,15]. They

tion of the ECM, or altered expression of transcription
factors controlling the production of matrix molecules
[27].
Here, we analyzed AP-2e expression and its regula-
tion during cartilage differentiation and in osteo-
arthritic chondrocytes. Our data provide evidence that
AP-2e is directly regulated by the transcription factor
Sox9 and has a role in cartilage differentiation.
Results
AP-2e is expressed in hypertrophic cartilage
Previous studies demonstrated that the transcription
factor AP-2e is expressed in chondrocytes and regu-
lates gene expression of integrin a
10
, which plays an
important role in cartilage development [13,28]. To
determine the functional role of AP-2e in human chon-
drocytes, we used dedifferentiated chondrocytes and
human mesenchymal stem cells (HMSCs), and differ-
entiated them either to chondrocytes or to osteoblasts
[29]. Figure 1A shows that AP-2e is highly expressed
in human chondrocytes and in chondrogenically differ-
entiated HMSCs, as compared with untreated or
osteoblastically differentiated HMSCs. To further ana-
lyze at which stages during chondrocyte differentiation
the expression of AP-2e increases, we used an in vitro
model system for HMSC differentiation into chondro-
cytes established in our laboratory. Marker genes for
different stages of chondrogenesis, such as collagen
type II, collagen type X, CD-RAP, and aggrecan, were

specific Sox9 antibody demonstrated an increase of
Sox9 in the early hypertrophic stages of cartilage
development (Fig. 2C).
AP-2e expression in osteoarthritic chondrocytes
Osteoarthritic cartilage often resembles a hypertrophic
phenotype [32,33]. To address whether AP-2e expres-
sion is altered in osteoarthritic chondrocytes in com-
parison with differentiated chondrocytes, we quantified
their mRNA expression, and detected significantly
A K. Bosserhoff et al. Sox9 regulates AP-2e in hypertrophic chondrocytes
FEBS Journal 276 (2009) 2494–2504 ª 2009 The Authors Journal compilation ª 2009 FEBS 2495
higher expression in osteoarthritic chondrocytes than
in differentiated chondrocytes (Fig. 3A). Interestingly,
AP-2a expression was not increased in osteoarthritic
cartilage (Fig. 3B). The expression of integrin a
10
,an
AP-2e target gene, was also measured, and was found
to be strongly upregulated in osteoarthritic chondro-
cytes as compared with differentiated chondrocytes
(Fig. 3C). Furthermore, expression of Sox9 was
strongly increased in osteoarthritic chondrocytes as
compared with the control (Fig. 3D).
Next, we wanted to confirm AP-2e expression in
cartilage from osteoarthritic patients. To this end, we
performed immunohistochemical stainings of osteo-
arthritic cartilage with the AP-2e antiserum.
Figure 3E shows AP-2e-positive cells within the
osteoarthritic cartilage tissue sections. We also ana-
lyzed the expression of Sox9 in these tissue sections,

without a binding site for Sox9 (prom302) was cloned
into a reporter gene plasmid containing a promoter-
less luciferase gene. SW1353 cells were transiently
transfected with the AP-2e
promoter construct, and
luciferase activity was measured. The 302 bp promoter
construct showed no increased promoter activity as
compared with the control (Fig. 4C). In comparison
Fig. 1. Expression of AP-2e in human chon-
drocytes and human mesenchymal stem
cells stimulated to undergo chondrogenic
differentiation. (A) Quantitative real-time
PCR to measure the expression of AP-2e
mRNA in human chondrocytes as compared
with that in dedifferentiated chondrocytes,
and in HMSCs stimulated to undergo chon-
drogenic or osteoblastic differentiation in
comparison with untreated cells. (B–D)
HMSCs were stimulated to undergo
chondrogenic differentiation, and RNA was
analyzed over 40 days. The expression of
AP-2e (B), AP-2a (C) and Sox9 (D) mRNA
was analyzed using quantitative real-time
PCR.
Sox9 regulates AP-2e in hypertrophic chondrocytes A K. Bosserhoff et al.
2496 FEBS Journal 276 (2009) 2494–2504 ª 2009 The Authors Journal compilation ª 2009 FEBS
with this, an AP-2e promoter construct of 604 bp
(prom604) containing binding sites for AP-2a and
Sox9 was clearly active in the cell line SW1353 as com-
pared with cells transfected with a control plasmid.

was measured after siRNA transfection (Fig. 5A). A
clear reduction of Sox9 expression could be shown
after transfection with both Sox9 siRNAs, but not
after transfection with control siRNA or siRNAs
against Sox5. The reduction of Sox9 expression using
siRNA strategies also caused a significant reduction of
AP-2e expression (Fig. 5B), suggesting that Sox9 is a
positive regulator of AP-2e expression in chondrocytes.
To demonstrate the direct interaction of Sox9 with
the AP-2e promoter, chromatin immunoprecipitation
(ChIP) assays were performed using SW1353 cells and
a specific Sox9 antibody. DNA samples were analyzed
by PCR using specific primer pairs generating frag-
ments spanning the first (Sox9_1) or the second
(Sox9_2) Sox9-binding site of the AP-2e promoter.
Sox9 binding to both binding sites (Sox9_1 and
Sox9_2) within the AP-2e promoter was observed
in vivo (Fig. 5C).
Finally, the direct binding of Sox9 to the two Sox9-
binding sites within the AP-2e promoter was con-
firmed by electrophoretic mobility shift assays
(EMSAs). Here, radioactively labeled oligonucleotides
were used that harbored the Sox9-binding sites of the
AP-2e promoter (Sox9_1 and Sox9_2). Incubation of
in vitro-synthesized Sox9 with the labeled oligonucleo-
tides containing the Sox9-binding sites resulted in a
strong DNA–protein interaction (Fig. 5D, lanes 2 and
7). The specificity of these complexes was shown in
competition studies using unlabeled oligonucleotides
in a 400-molar excess (Fig. 5D, lanes 3 and 8). Incu-

chondrocyte development. At these stages of differenti-
ation, chondrocytes undergo a process of terminal dif-
ferentiation, by which they become hypertrophic and
express hypertrophic marker genes such as type X col-
lagen [34,35]. Immunohistochemical staining of embry-
onic tissues at day 14.5 and day 17.5 confirmed clear
AP-2e expression in the hypertrophic cartilage. Thus,
AP-2e expression seems to correlate with hypertrophic
cartilage differentiation.
To determine how the increased expression of AP-2e
in hypertrophic chondrocytes is regulated, the sequence
of the AP-2e promoter was analyzed, and binding sites
for the transcription factors AP-2a and Sox9 were
identified. Both transcription factors are known to
play an important role in chondrocyte differentiation.
AP-2a is essential for skeletal development, and is
expressed in limb buds during early embryogenesis, in
the growth plate, and in chondrocytes of the joints
[36]. The AP-2a knockout mouse died at birth, with
severe malformations of the craniofacial skeleton and
defects in the development of the extremities [30,37].
We showed that moderate AP-2a levels might be
important for AP-2e expression, as both Sox9 and
AP-2a are needed to induce expression. Thus, induc-
tion of AP-2e expression is seen upon a further
increase in Sox9 expression at later stages of chondro-
cyte differentiation. Therefore, we suggest that Sox9 is
Fig. 3. Expression of AP-2e, AP-2a,
integrin a
10

sion analyses using quantitative real-time PCR and
immunohistochemical staining of Sox9 demonstrated
that Sox9 is expressed in early chondrogenic develop-
ment and that expression is increased again at the
beginning of the hypertrophic phase of differentiation,
which is in accordance with other data [39,40]. In
detail, the study of Tchetina et al. also proved that, in
growth plates, Sox9 expression increased in the early
hypertrophic zones of cartilage together with that of
the hypertrophic marker gene type X collagen, and did
not decrease until the late hypertrophic phase. Thus,
these experiments support our findings that Sox9 can
positively regulate the expression of AP-2e in early
hypertrophic chondrocytes.
Using ChIP experiments and EMSAs, we confirmed
the direct binding of the transcription factor Sox9 to
Fig. 4. Promoter sequence of AP-2e, and regulation of AP-2e by AP-2a and Sox9. (A) Schematic illustration of the AP-2e promoter region.
Binding sites for the transcription factors AP-2a and Sox9 are indicated. (B) SW1353 cells were transiently transfected with expression con-
structs for AP-2a, Sox5 and Sox9, or with AP-2a and Sox9. The expression of AP-2e was measured using quantitative real-time PCR. (C)
Three hundred and two base pairs, 604 and 604 bp containing a mutated Sox9-binding site of the AP-2e promoter region were subcloned
into pGL3-basic, and promoter activity was analyzed in SW1353 cells. Additionally, expression constructs for AP-2a, Sox9 or both together
were transiently transfected into SW1353 cells, together with the AP-2e promoter constructs, and promoter activity was measured.
pGL3-basic is set as 1. Data are given as mean ± SEM; *P < 0.05.
A K. Bosserhoff et al. Sox9 regulates AP-2e in hypertrophic chondrocytes
FEBS Journal 276 (2009) 2494–2504 ª 2009 The Authors Journal compilation ª 2009 FEBS 2499
the AP-2e promoter. Further studies showed that Sox9
activates the promoter of AP-2e in cooperation with
AP-2a, resulting in an increase in AP-2e expression.
Because AP-2a is expressed during chondrogenesis at a
constant level, we suppose that Sox9 is the crucial

Cells were maintained in high-glucose DMEM supple-
mented with penicillin (400 UÆmL
)1
), streptomycin
(50 lgÆmL
)1
), l-glutamine (300 lgÆmL
)1
), and 10% fetal
bovine serum (Sigma, Deisenhofen, Germany), and split at
a 1 : 5 ratio every 3 days. Primary chondrocytes were
obtained from Cambrex (Iowa, IA, USA), and cultured as
Fig. 5. Expression of AP-2e in SW1353 cells after silencing of Sox9 by siRNA transfection. Expression levels of Sox9 (A) and AP-2e (B) were
analyzed by quantitative real-time PCR after transfection of SW1353 cells with siRNAs (siSox9_2, siSox9_5), and compared with those in
cells transfected with control siRNAs (control) or siRNAs against Sox5 (siSox5_1, siSox5_4). Data are given as mean ± SEM; *P < 0.05, ns,
not significant. Sox9 binds to the AP-2e promoter in vivo. (C) A ChIP assay demonstrates the direct binding of Sox9 to the two Sox9-binding
sites within the AP-2e promoter. DNA samples of the ChIP reaction (Pol II, IgG, and Sox9) and the input DNA were used in PCR reactions
with different primer pairs (GAPDH, negative control primers, Sox9_1 and Sox9_2). All PCR fragments could be detected in the input DNA
sample. A clear product of Sox9_1 and Sox9_2 was detected in the Sox9 ChIP DNA sample. (D) EMSA to confirm the binding of Sox9 to
the AP-2e promoter. The contents of the reaction mixtures are marked above the image of the gel shift. The Sox9 binding was shown using
oligonucleotides spanning the two Sox9 regions Sox9_1 (lane 2) and Sox9_2 (lane 7) of the AP-2e promoter and in vitro-synthesized Sox9
protein. For competition experiments, unlabeled wild-type oligonucleotides (lanes 3 and 8) and mutated oligonucleotides (lanes 4 and 9) were
used. Lanes 1 and 5 show the labeled oligonucleotides incubated without protein.
Sox9 regulates AP-2e in hypertrophic chondrocytes A K. Bosserhoff et al.
2500 FEBS Journal 276 (2009) 2494–2504 ª 2009 The Authors Journal compilation ª 2009 FEBS
suggested by the manufacturers. The proliferating cells are
dedifferentiated in culture. To differentiate these cells, they
were stimulated with transforming growth factor-b
1
(10 ngÆmL

containing 2 lg of total cellular RNA, 4 lLof5· first-
strand buffer (Invitrogen, Groningen, the Netherlands),
2 lL of 0.1 m dithiothreitol, 1 lLofdN
6
-primer (10 mm),
1 lL of dNTPs (10 mm), and diethylpyrocarbonate ⁄ water.
The reaction mixture was incubated for 10 min at 70 °C,
200 U of Superscript II reverse transcriptase (Invitrogen)
were added, and RNAs were transcribed for 1 h at 37 °C.
The reverse transcriptase was inactivated at 70 °C for
10 min, and the RNA was degraded by digestion with 1 lL
of RNaseA (10 mgÆmL
)1
)at37°C for 30 min.
To precisely quantify the expression of cDNAs, the
real-time PCR LightCycler system (Roche, Mannheim,
Germany) was used as described previously [42,43]. The
quantitative real-time PCR analysis of AP-2e, AP-2a, Sox9
and integrin a
10
expression was performed using specific
primers: AP-2e-for, 5¢-GAAATAGGGACTTAGCTCTTG
G-3¢, and AP-2e-rev, 5¢-CCAAGCCAGATCCCCAACT
CTG-3¢ (annealing temperature 59 ° C); AP-2a-for, 5¢-GAT
CCTCGCAGGGACTACA-3¢, and AP-2a-rev, 5¢-GTTGG
ACTTGGACAGGGAC-3¢ (annealing temperature 60 °C);
Sox9-for, 5¢-CGAACGCACATCAAGACGA-3¢, and Sox9-
rev, 5 ¢-AGGTGAAGGTGGAGTAGAGGC-3¢ (annealing
temperature 58 °C); integrin alpha10-for, 5¢-CATGAGGTT
CACCGCATCACT-3¢, and integrin alpha10-rev, 5¢-AAGG

forms. For generation of the AP-2e promoter constructs,
the human genomic region was amplified by PCR with a
3¢-reverse primer (rev_promAP-2e,5¢-GACAAGCTTGT
AGGTGTGCACCAGCAT-3¢) in conjunction with two
different 5¢-forward primers (for_promAP-2e_604, 5¢-GAC
GCTAGCGAGGCCAGCGAAGAATAG-3¢; for_prom
AP-2e_302, 5¢-GACGCT AGCTGGAGTGCATGGAG
CAGGC-3¢). To facilitate subcloning of the amplified frag-
ment, the reverse primer contained a Hin dIII restriction site
adaptor, and the forward primers contained an NheI site.
The PCR fragments and the luciferase expression vector
pGL3-basic were digested separately with HindIII and NheI
before ligation. For generation of the promoter construct
containing a mutated Sox9-binding site, site-directed muta-
genesis with overlap extension was performed [46]. For
insertion of the mutated binding site, the following primers
were used: mutSox9-447_for, 5¢-CCAGAAGGCGGCTCT
GATTGCTGTGGGCTGAATTCACGC-3¢; and mutSox9-
447_rev, 5¢-GCGTGAATTCAGCCCACAGCAATCAGAG
CCGCCTTCTGG-3¢.
A K. Bosserhoff et al. Sox9 regulates AP-2e in hypertrophic chondrocytes
FEBS Journal 276 (2009) 2494–2504 ª 2009 The Authors Journal compilation ª 2009 FEBS 2501
Transient transfection and luciferase assay
DNA transfection of the SW1353 cells was performed
using Lipofectamine plus (Invitrogen, Carlsbad, CA,
USA). Briefly, the procedure was as follows. Cells were
cultured in six-well plates. For transient transfection with
expression plasmids, each cationic lipid ⁄ plasmid DNA
suspension was prepared with 0.5 lg of plasmid in the
transfection solutions, according to the manufacturer’s

specific Sox9 antibody (2 lg of anti-Sox9; Chemicon
International). An RNA polymerase II antibody was used
as a positive control, and an IgG antibody as a negative
control, following the protocol provided with the control
kit (ChIP-IT control Kit-human; Active Motif). DNA
samples from the ChIP experiments were used for analy-
sis by PCR. PCR was performed on four DNA tem-
plates: the input DNA (1 : 5), DNA isolated through
RNA polymerase II ChIP (Pol II), DNA isolated through
the negative control IgG ChIP (IgG), and DNA isolated
through the Sox9 ChIP (Sox9). A control reaction with
no DNA template was also performed (H
2
O). Four sets
of specific primer pairs were used: the glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) and the negative
control primer pairs provided by the kit, and primer
pairs spanning the two Sox9-binding sites of the AP-2e
promoter: Sox9_1prom_for, 5¢-GAGGCCAGCGAAGA
ATAGTG-3¢, and Sox9_1prom_rev, 5¢-GTTCTCTC
CCTTTTCCCCAGC-3¢ (234 bp fragment); Sox9_2prom_
for, 5¢-CAGTCACTCAACAGTCTCTGG-3¢, and Sox9_2
prom_rev, 5¢-CACTTCGCTCTCAGGCTTC-3¢ (213 bp
fragment). PCR fragments were analyzed on a 1.5%
agarose gel.
Synthesis of Sox9 protein in vitro
Sox9 protein was synthesized by in vitro transcription–
translation with the Sox9 expression vector and the TNT
Quick Coupled Transcription ⁄ Translation System (Promega
Corporation, Madison, USA).

Statistical analysis
Results are expressed as mean ± standard deviation
(range) or percentage. Comparison between groups was
made using Student’s paired t-test. A P-value < 0.05 was
considered to be statistically significant. All calculations
were performed using graphpad prism software (GraphPad
Software Inc., San Diego, CA, USA).
Sox9 regulates AP-2e in hypertrophic chondrocytes A K. Bosserhoff et al.
2502 FEBS Journal 276 (2009) 2494–2504 ª 2009 The Authors Journal compilation ª 2009 FEBS
Acknowledgement
This work was partly supported by a DFG grant
assigned to S. Gra
¨
ssel (GR 1301 ⁄ 7-1).
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