Tài liệu Báo cáo khoa học: The cartilage-specific transcription factor Sox9 regulates AP-2e expression in chondrocytes pptx

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

at their C-terminus, and a less conserved proline and
glutamine-rich transactivation domain at their N-ter-
minus [9–11].
So far, the most recently identified AP-2 transcrip-
tion factor, AP-2e, has been only poorly characterized
[4,12]. Expression of AP-2e was first described in the
olfactory system [4], in skin, and in in vitro-cultured
keratinocytes [12]. Previously, we demonstrated that
AP-2e is also expressed in chondrocytes, where it regu-
lates the expression of integrin a
10
, the predominant
collagen-binding integrin during cartilage development
[13].
The axial skeleton is formed by a process named
endochondral bone formation. This complex process
Keywords
AP-2e; cartilage; differentiation;
osteoarthritis; transcriptional regulation
Correspondence
A K. Bosserhoff, Institute of Pathology,
University of Regensburg, Franz-Josef-
Strauss-Allee 11, D-93053 Regensburg,
Germany
Fax: +49 941 944 6602
Tel: +49 941 944 6705
E-mail: anja.bosserhoff@klinik.
uni-regensburg.de
(Received 14 January 2009, revised 13
February 2009, accepted 18 February 2009)

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

start to produce an extracellular matrix (ECM) con-
taining type I collagen, hyaluronic acid, tenascin, and
fibronectin [16–18]. Subsequent differentiation of the
mesenchymal cells to chondrocytes causes a change in
ECM composition. Chondrocytes express cartilage-
specific type II, type IX and type XI collagen,
proteoglycans, aggrecan, and cartilage-derived retinoic
acid-sensitive protein (CD-RAP), whereas expression
of type I collagen stops. After further steps of differen-
tiation, chondrocytes become hypertrophic and express
increased levels of type X collagen and reduced levels
of type II collagen [19–21]. Finally, osteoblasts infil-
trate into the cartilage and start to displace it with
mineralized bone.
Sox9 represents an essential transcription factor of
the chondrogenic lineage, and regulates the expression
of chondrocyte-specific genes such as those encoding
type II collagen and CD-RAP [22,23]. Sox9 belongs to
the HMG-box superfamily of transcription factors,
which bind in the minor groove of the DNA to the
consensus sequence (A⁄ T)(A ⁄ T)CAA(A ⁄ T)G [24].
Sox9 expression has been detected in all chondrogenic
progenitor cells and chondrocytes [25,26].
The cartilage, which mainly consists of chondrocytes
and ECM, serves as a protective layer for the joints.
Degradation of the articular cartilage is a major prob-
lem in osteoarthritis (OA), a degenerative joint disor-
der, leading to destruction of the cartilage. The onset
of this disease might be triggered by multiple factors
such as mechanical overload, defects in the composi-

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

analyzed to demonstrate differentiation stages [29].
Using this model system, the expression of AP-2e
mRNA was followed by quantitative real-time PCR
over 40 days. Interestingly, the expression of AP-2e
increased relatively late during chondrogenic differenti-
ation (Fig. 1B). These stages correspond to the
hypertrophic phase of chondrogenesis, which is charac-
terized by increased expression of the hypertrophic
marker gene type X collagen. The expression of
AP-2a, which is known to be expressed during carti-
lage development [30], was analyzed as a control
(Fig. 1C). AP-2a expression increased early during
chondrocyte differentiation and then remained at a
moderate level. The expression of the transcription
factor Sox9, a key regulator of chondrogenesis, was
also analyzed as a marker. Sox9 was expressed early
during chondrogenic differentiation, but its expression
increased up to two-fold at later stages of differentia-
tion, at around day 17 (Fig. 1D).
To confirm AP-2e expression in hypertrophic
regions of the developing cartilage, immunohistochem-
ical stainings of tissue sections from 14.5-day-old and
17.5-day-old mouse embryos were performed using a
specific polyclonal antiserum against AP-2e [31]
(Fig. 2A). AP-2e was detected in hypertrophic areas of
the cartilage. To verify the specificity, we stained sec-
tions from AP-2e knockout mice (M. Moser, unpub-
lished data), and did not find any signal (Fig. 2B). In
parallel, we also analyzed Sox9 expression in these
tissue sections. Immunohistochemical staining with a

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,

and found Sox9 expression in the osteoarthritic carti-
lage (Fig. 3F).
AP-2a and Sox9 activate the AP-2e promoter
To obtain insights into the regulatory mechanisms
leading to the upregulation of AP-2e in the late stages
of cartilage differentiation and in osteoarthritic chon-
drocytes, we studied the AP-2e promoter. One thou-
sand base pairs upstream of the translation start site
of the AP-2e gene were analyzed in detail to identify
binding sites for known transcription factors that
might regulate AP-2e expression. Two potential bind-
ing sites for the transcription factor Sox9 at positions
)973 ⁄ )970 and )448 ⁄ )445 and three putative AP-2a-
binding sites at positions )322 ⁄ )312, )170 ⁄ )162
and )86 ⁄ )78 relative to the translation start site were
identified (Fig. 4A).
To test whether Sox9 or AP-2a regulates the expres-
sion of AP-2e, the chondrosarcoma cell line SW1353
was transfected with expression constructs for each
AP-2a and Sox9 or with both of them. As a control,
cells were transfected with expression constructs for
Sox5. The expression of endogenous AP-2e mRNA
was measured 24 h after transfection by quantitative
real-time PCR. Figure 4B shows that AP-2a or Sox9
transfection alone resulted in only low induction of
AP-2e expression, but when AP-2a or Sox9 were trans-
fected together, they strongly increased the expression
of AP-2e, up to 32-fold (Fig. 4B). These data were
confirmed by luciferase promoter assays. First, a
302 bp construct of the AP-2e promoter sequence

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.

Additional transfection of AP-2a or Sox9 expression
plasmids showed an increase of AP-2e promoter activ-
ity in SW1353 cells (Fig. 4C). The cotransfection of
AP-2a and Sox9 further increased the promoter activ-
ity of AP-2e. Transfection with an AP-2e expression
plasmid did not influence promoter activity, implying
that AP-2e does not regulate its own expression (data
not shown). A promoter construct with a mutation
within the Sox9-binding site (prom604mut) showed
decreased promoter activity as compared with the
wild-type 604 bp promoter construct. Transfection
with an expression construct for Sox9 resulted in a
minor increase in promoter activity as compared with
the 604 bp wild-type construct. The remaining activa-
tion could be due to additional Sox9-binding sites
within the AP-2e promoter that are less conserved.
Sox9 is an activator of AP-2e expression
In the following studies, we focused on Sox9 as a regu-
lator of AP-2e expression, because AP-2a is expressed
at a constant level during chondrocyte differen-
tiation, and Sox9 is upregulated in the later stages
of differentiation. Additionally, in OA, we found
AP-2e and Sox9 to be upregulated but not AP-2a
(Fig. 3).
The influence of Sox9 on AP-2e expression was fur-
ther analyzed using small interfering RNA (siRNA)
against Sox9. SW1353 cells were transfected with con-
trol siRNA, siRNAs against Sox9 (siSox9_2 and
siSox9_5), or siRNAs against Sox5 (siSox5_1 and
siSox5_4) as a second control. First, Sox9 expression

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-

bation with a 400-molar excess of unlabeled oligonu-
cleotides harboring a mutated Sox9-binding site did
not lead to competition of the complexes (Fig. 5D,
lanes 4 and 9).
Fig. 2. Expression of AP-2e and Sox9 in tissue slides of mouse
embryos. (A) Immunohistochemical staining of AP-2e day 14.5 and
day 17.5 mouse embryos revealed strong signals in areas of hyper-
trophic cartilage. (B) Tissue slides of an AP-2e knockout (ko) mouse
were stained as a control, and were clearly negative. (C) Immuno-
histochemical staining of Sox9 in day 14.5 mouse embryos showed
Sox9 expression in the early stages of hypertrophic chondrocytes.
wt, wild-type.
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 2497
Discussion
Recently, we showed that the transcription factor
AP-2e is a positive regulator of integrin a
10
expression
in chondrocytes [13]. In this study, we wanted to deter-
mine the role of AP-2e expression during cartilage
development.
Our results demonstrate that the transcription factor
AP-2e is expressed in human chondrocytes and in
HMSCs stimulated to undergo chondrogenic differenti-
ation. To further investigate the time point of AP-2e
induction during chondrocyte differentiation, chondro-
genic differentiation of HMSCs was analyzed over a
time course of 40 days. Expression data showed
increased expression of AP-2e in the late stages of

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

and Sox9 in differentiated
chondrocytes as compared with that in
osteoarthritic chondrocytes. (A–D) Using
quantitative real-time PCR analyses, the
expression of AP-2e (A), AP-2a (B), inte-
grin a
10
(C) and Sox9 (D) was measured in
differentiated chondrocytes in comparison
with osteoarthritic chondrocytes (n = 5).
(E, F) Immunohistochemical staining of
AP-2e (E) and Sox9 (F) in tissue slides of
osteoarthritic cartilage revealed strong
signals. Black arrows indicate positively
stained cells. Data are given as
mean ± SEM; *P < 0.05.
Sox9 regulates AP-2e in hypertrophic chondrocytes A K. Bosserhoff et al.
2498 FEBS Journal 276 (2009) 2494–2504 ª 2009 The Authors Journal compilation ª 2009 FEBS
an important regulator of AP-2e expression in hyper-
trophic chondrocytes and in osteoarthritis.
The transcription factor Sox9 is known to be a regu-
lator of chondrogenesis. It is expressed in all chondro-
genic progenitor cells and chondrocytes. Sox9 is
essential for the early steps of chondrogenesis in mes-
enchymal condensation [38,39]. In the later stages,Sox9
regulates the differentiation markers type II collagen
[22] and CD-RAP [23]. Several groups have described
a reduction of Sox9 expression in hypertrophic chon-
drocytes [25,26], but in these studies no subdivision
was made into early and late hypertrophy. Our expres-

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

factor in inducing AP-2e expression in the early hyper-
trophic phase of chondrocyte differentiation. A hyper-
trophic phenotype is also characteristic for
osteoarthritic cartilage [32,33]. Expression analyses of
osteoarthritic chondrocytes showed a strong increase
in AP-2e expression in these cells. Sox9 expression is
also highly increased in osteoarthritic chondrocytes as
compared with differentiated chondrocytes, whereas
that of AP-2a is not.
In summary, we demonstrated increased expression
of AP-2e in hypertrophic and osteoarthritic chondro-
cytes. For the first time, we found that the transcrip-
tion factor Sox9 is a positive regulator of AP-2e
expression at the beginning of the hypertrophic devel-
opment of cartilage and of osteoarthritic chondrocytes.
The dramatic increase in AP-2e expression and that of
its target gene integrin a
10
in OA suggests an impor-
tant functional role of AP-2e in the development of
hypertrophic chondrocytes. To determine the role of
AP-2e as a modulator of hypertrophy in cartilage,
additional target genes of AP-2e, besides integrin a
10
,
have to be determined.
Experimental procedures
Cell culture
The chondrosarcoma cell line SW1353 was obtained from
the American Type Culture Collection (ATCC, #HTB-94).

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

)1
) for 1 week.
HMSCs from CellSystems (St Katharinen, Germany)
were cultivated in MSCGM medium (CellSystems) under a
humidified atmosphere of 5% CO
2
at 37 °C [41]. To stimu-
late HMSCs to undergo either chondrogenic or osteoblastic
differentiation, cells were seeded in a 15 mL Falcon tube
and treated as previously described [29].
The cartilage samples used for immunohistology are of
human origin. Cartilage was obtained from patients giving
informed consent following the standards of the Ethics Com-
mission of the University of Regensburg. Full-thickness car-
tilage slices were aseptically dissected from healthy aspects of
femoral condyles of patients aged 50–76 years with osteo-
arthritis who had undergone total knee arthroplasty.
OA chondrocytes were prepared from osteoarthritic carti-
lage slices obtained as described above, and are therefore
defined as osteoarthritic chondrocytes; no biochemical mar-
ker was used for characterization, except for the diagnosis
from the orthopedic surgeon according to accepted ortho-
pedic standards, which resulted in total joint replacement
surgery.
RNA isolation, reverse transcription, and
quantitative real-time PCR
Total cellular RNA was isolated from cultured cells or
from tissues using the RNeasy kit (Qiagen, Hilden,
Germany), and cDNAs were generated by a reverse trans-
criptase reaction performed in a 20 lL reaction volume

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

CAAAGGTCACAGTCAAGG-3¢ (annealing temperature
64 °C). The expression ratios of the analyzed genes
were calculated using an internal control standard curve of
b-actin levels.
Immunohistochemical staining
Paraffin sections of osteoarthritic cartilage and whole
mouse day 14.5 and day 17.5 embryos were screened for
AP-2e and Sox9 protein expression by immunohistochem-
istry. The tissues were fixed, and subsequently incubated
with specific primary AP-2e antiserum [31] (1 : 200) or
primary Sox9 antibody (Chemicon International Inc.,
Temecula, CA, USA) (1 : 100) overnight at 4 °C, with the
secondary antibody (biotin-labeled anti-rabbit; DAKO,
Hamburg, Germany) for 30 min at room temperature, and
then with streptavidin-POD (DAKO) for 30 min. Anti-
body binding was visualized using AEC solution (DAKO).
Finally, the tissues were counterstained with hemalaun
solution (DAKO).
Plasmid constructs
Expression constructs for Sox9 and Sox5 were kind gifts
from V. Lefebvre (Department of Cell Biology, Cleveland
Clinic, Cleveland, OH, USA) [44]. An AP-2a expression
plasmid was generated according to Moser et al. [45].
For analyses of the AP-2e promoter for putative tran-
scription factor-binding sites, we screened approximately
1 kb of DNA of the upstream regulatory region, using the
matinspector (Genomatix Software GmbH, Munich,
Germany). We determined the start site of transcription
by extrapolation from cDNA clones and available expressed
sequence tags, by analogy with the other four AP-2 iso-

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

instructions. The cells were harvested 24 h later, and RNA
was isolated.
For measurement of luciferase promoter activity, each
cationic lipid ⁄ plasmid DNA suspension was prepared by
mixing 0.2 or 0.5 lg of the luciferase reporter plasmid and
0.1 lg of the internal control plasmid pRL-TK with trans-
fection solutions, according to the manufacturer’s instruc-
tions. The cells were harvested 24 h later, and the lysate
was analyzed for luciferase activity with a luminometer,
using Promega dual-luciferase assay reagent (Promega
Corporation, Madison, WI, USA). At least three indepen-
dent transfection experiments were performed for each
construct.
siRNA transfection
The siRNAs against Sox9 (siSox9_2, siSox9_5) and the
control siRNAs (siSox5_1, siSox5_4 and control siRNA)
were synthesized by Qiagen. Cells of the chondrosarcoma
cell line SW1353 were grown to 70–80% confluence in
culture dishes, and harvested in the proliferative growth
phase. Cells were transfected with the HiPerFect Trans-
fection Reagent (Qiagen), according to the manufac-
turer’s protocol. Cells were transfected in six-well
culture plates, and RNA was isolated 24 h after trans-
fection.
ChIP assay
The ChIP assay was performed following the manu-
facturer’s instructions (ChIP-IT Express; Active Motif,
Carlsbad, CA, USA). SW1353 cells grown to 70–80%
confluence on three 15 cm plates were used for chromatin
isolation. Samples were immunoprecipitated with a

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).

EMSA
The EMSA was based on the binding of Sox9 protein to a
32
P-labeled oligonucleotide containing a Sox9-binding site.
Two double-stranded oligomeric binding sites for Sox9,
specific for the AP-2e promoter (Sox9_1, 5¢-GCGG
CTCTGATCAATGTGGGCTGAATTC-3¢; and Sox9_2,
5¢-CATGCCCACACTCAATCAGCCCAGGACCC-3¢)were
generated. The fragments correspond to the AP-2 e pro-
moter regions from )458 to )432 (Sox9_1) and from )985
to )957 (Sox9_2) upstream of the ATG. The fragments
were end-labeled with T4 polynucleotide kinase (Roche)
and [
32
P]ATP[cP] (Amersham, GE Healthcare, Munich,
Germany). Band shifts were performed by incubating
in vitro-synthesized Sox9 in the 5· mobility shift buffer
[1 lg of poly(dI-dC)(dI-dC), 40% glycerol, 25 mm MgCl
2
,
1mm EDTA, 25 mm dithiothreitol, 250 mm KCl, 25 mm
Hepes ⁄ KOH, pH 7.9) with the DNA probe for 10 min
before separation on a 6% nondenaturing polyacrylamide
gel. For the competition studies, the cold oligonucleotides
were added at a 400-fold molar excess and incubated for
10 min at room temperature before addition of the
DNA probe. DNAÆprotein complexes were resolved on a
nondenaturing polyacrylamide gel at 250 V, 50 mA and
100 W for 1.5 h. In vitro-synthesized protein was used to
demonstrate the specificity of Sox9.

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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