REVIEW ARTICLE
Roles of AP-2 transcription factors in the regulation of
cartilage and skeletal development
Ann-Kathrin Wenke and Anja K. Bosserhoff
Institute of Pathology, University of Regensburg, Germany
The AP-2 family
AP-2a was first identified by its ability to bind to enhan-
cer regions of SV40 and human metallothionein IIA [1].
The AP-2 family of transcription factors is composed of
five members: AP-2a, AP-2b, AP-2c, AP-2d, and AP-2e
[2–7], described for humans and mice. Orthologs of
some AP-2s have also been found in frogs and fish, and
homologs occur in invertebrates. All AP-2s have a
highly conserved basic helix–span–helix DNA-binding
and dimerization domain at their C-terminus, and a less
conserved proline-rich and glutamine-rich transactiva-
tion domain at their N-terminus [8–10]. Most isoforms
also have a PY-motif (XPPXY) in the N-terminal trans-
activation domain that is important for their role as
transcriptional activators [9]. The AP-2 factors form
homodimers and heterodimers for their transcriptional
activity. A multiple alignment of all five human AP-2s,
illustrating their domain structure, is shown in Fig. 1.
A detailed and extensive overview of the AP-2 family
is given in the review of Eckert et al., [11] which also
contains a schematic illustration of the AP-2 structure.
Expression patterns of AP-2 molecules
and functional implications
The expression and function of AP-2 isoforms have
been systematically analyzed during murine embryo-
genesis and in studies of the corresponding knockout

regulators during chondrogenesis has been characterized. This review gives
an overview of AP-2s, and discusses the recent findings on the AP-2 family,
in particular AP-2a, AP-2b, and AP-2e, as regulators of cartilage and skeletal
development.
Abbreviations
NCC, neural crest cell; RA, retinoic acid; ZPA, zone of polarizing activity.
894 FEBS Journal 277 (2010) 894–902 ª 2009 The Authors Journal compilation ª 2009 FEBS
and the extraembryonic trophectoderm [4,12,13]. In
contrast to the other AP-2s, AP-2d is specifically
expressed in the central nervous system, retina, and
developing heart [6]. AP-2e expression has been
detected in the developing olfactory bulb, neural tis-
sue, especially the midbrain and hindbrain [7,14], and
hypertrophic chondrocytes during chondrogenesis [15].
Winger et al. [16] analyzed the expression of all five
mouse AP-2 family members in the unfertilized
oocyte and from zygote formation to the blastocyst
Transactivation domain
with PY-motif
Dimerization domain
DNA-binding domain
Alpha MLWKLTDNIKYEDC-EDRHDGTSNGTARLPQLGTVGQSPYTSAPPLSHT
Beta MHSPPRDQAAIMLWKLVENVKYEDIYEDRHDGVPSHSSRLSQLGSVSQGPYSSAPPLSHT
Gamma MLWKITDNVKYEEDCEDRHDGSSNGNPRVPHLSSAGQHLYSPAPPLSHT
Epsilon MLVHTYSAME RPDGLG-AAAGGARLSSLPQAAYGPAPPLCHT
Delta MSTTFPGLVHDAEIRHDGSNSYRLMQLGCLESVANSTVAYSSSSPLTYS
* :. :. . : * .:.** ::
Alpha PNA DFQPP-YFPPPY QPI-YPQSQDP YSHVN-DPYS LNPLHAQPQP Q
Beta PSS DFQPP-YFPPPY QPLPYHQSQDP YSHVN-DPYS LNPLHQ-PQ Q
Gamma GVA EYQPPPYFPPPY QQLAYSQSADP YSHLG-EAYAAAINPLHQPAPTGSQ

Epsilon FPAKAAAEYLCRQHAD-PGELHSRKSMLLAAKQICKEFADLMAQDRSPLGNSRPALILEP
Delta FPAKAVGEHLARQHME-QKEQTARKKMILATKQICKEFQDLLSQDRSPLGSSRPTPILDL
**:* *.* * * : :**.*:**::*:**** :*::***:* *.** :*:
Alpha GIQSCLTHFNLISHGFGSPAVCAAVTALQNYLTEALKAMDKMYLS NNP-NSHTDN
Beta GIQSCLTHFSLITHGFGAPAICAALTALQNYLTEALKGMDKMFLN NTTTNRHTSG
Gamma NIQNCLSHFSLITHGFGSQAICAAVSALQNYIKEALIVIDKSYMN PGD-QSPADS
Epsilon GVQSCLTHFSLITHGFGGPAICAALTAFQNYLLESLKGLDKMFLS SVG-SGHGET
Delta DIQRHLTHFSLITHGFGTPAICAALSTFQTVLSEMLNYLEKHTTHKNGGAADSGQGHANS
.:* *:**.**:**** *:***::::*. : * * ::* . .
Alpha N AKSSDKEEKHRK
Beta EGP-GSKTGDKEEKHRK
Gamma N KTLEKMEKHRK
Epsilon K ASEKDAKHRK
Delta EKAPLRKTSEAAVKEGKTEKTD
: : : *. *
Fig. 1. Multiple alignment of AP-2a, AP-2b, AP-2c, AP-2d, and AP-2e. The proline-rich and glutamine-rich N-terminus, which is important for
transactivation, is shown in yellow, and contains the PY-motif (green). The helix–span–helix domain at the C-terminus shown in blue medi-
ates dimerization and, together with the basic domain, (red) DNA-binding. ‘*’, amino acids that are identical in all sequences in the align-
ment; ‘:’, conserved substitutions have been observed; ‘.’, semiconserved substitutions.
A K. Wenke and A K. Bosserhoff AP-2 proteins in cartilage differentiation
FEBS Journal 277 (2010) 894–902 ª 2009 The Authors Journal compilation ª 2009 FEBS 895
stage of development. They found that AP-2a,
AP-2b, AP-2c and AP-2e are differentially expressed
during the preimplantation period, and, with the
exception of AP-2a, also in unfertilized oocytes.
Furthermore, they determined that functional redun-
dancy occurs between these proteins during at least
the preimplantation period [16].
However, gene knockout experiments indicate that
the AP-2s perform individual and nonredundant

during embryonic development. To date, knockout
studies concerning AP-2d or AP-2 e have not been
published.
Regulation of AP-2 and AP-2 target
genes
The expression of the AP-2a transcription factor is
induced by different signal-transducing agents, such as
retinoic acid (RA), cAMP, phorbol ester, UV light, and
singlet oxygen [2,24–26]. RA plays an important role in
the process of chondrocyte differentiation [27]. AP-2
mediates transcriptional activation in response to two
different signal transduction pathways, the phorbol
ester-activated protein kinase C pathway, or the cAMP-
dependent protein kinase A pathway [28]. Here, cAMP
may modulate AP-2 activity by protein kinase A-induced
phosphorylation of the transcription factor [29].
So far, interactions with AP-2 have been described
for many proteins. For example, CBP ⁄ p300-interacting
transactivator with ED-rich tail 2 interacts with and co-
activates all isoforms of AP-2, and the interaction with
AP-2a is suggested to be necessary for normal neural
tube and cardiac development [30,31]. The Kru
¨
ppel-
related zinc finger protein AP-2rep (Klf12) has been
characterized as a repressor of AP-2a. Repression of
AP-2a transcription by AP-2rep is dependent on an
N-terminal PVDLS motif that interacts specifically with
the corepressor CtBP1 [32,33]. Recently, it was shown
that the broad-complex, tramtrack and bric-a-brac

of this GC-rich sequence within multiple gene promot-
ers [45]. AP-2s play a dual role as transcriptional acti-
vators and repressors. By regulating target genes with
AP-2-binding sites within their promoter sequences,
the AP-2 transcription factors play important roles in
cellular processes, such as morphogenesis, in particular
proliferation, differentiation, cell cycle regulation, and
apoptosis [11,45,46]. Through suppression of genes
inducing terminal differentiation, apoptosis, and
AP-2 proteins in cartilage differentiation A K. Wenke and A K. Bosserhoff
896 FEBS Journal 277 (2010) 894–902 ª 2009 The Authors Journal compilation ª 2009 FEBS
growth retardation, AP-2s play vital roles in cell prolif-
eration. Besides the functions of AP-2s in physiological
processes, they have also crucial roles in pathological
processes such as tumorigenesis and genetic diseases
[47].
Most analyses of the regulation of AP-2 and the
interactions of the transcription factor with binding
partners, as well as of the regulation of target gene
expression, have been performed for AP-2a.Upto
now, there have been no similar studies for the other
AP-2 isoforms.
Chondrogenesis and skeletal
development
Most elements of the vertebrate skeleton are built
through enchondral ossification. This is a complex pro-
cess beginning with the migration of undifferentiated
mesenchymal cells to regions determined to differenti-
ate into bone, followed by aggregation and the forma-
tion of mesenchymal condensation [48,49]. These

chondrocytic markers type II collagen, type IX colla-
gen, type X collagen, and aggrecan. Besides type II
collagen and aggrecan, Sox9 also regulates the expres-
sion of the cartilage-derived retinoic acid-sensitive pro-
tein [54,55]. Sox5 and Sox6, members of the Sox
family, are also important for chondrocyte differentia-
tion, as embryos lacking Sox5 and Sox6 die at embry-
onic day 16.5 and display a failure of chondrocyte
progenitor cells to differentiate into hypertrophic chon-
drocytes [56].
Two members of the Runx family of transcription
factors, Runx2 and Runx3, are positive regulators of
chondrocyte hypertrophy. Runx2 is transiently
expressed in prehypertrophic chondrocytes, and
enforced expression of Runx2 in these cells in trans-
genic mice leads to ectopic chondrocyte hypertrophy
[57]. Mice lacking both Runx2 and Runx3 do not have
hypertrophic chondrocytes or type X collagen-express-
ing cells, showing that both Runx2 and Runx3 are
important regulators for hypertrophic development of
chondrocytes [58]. Alongside the important function
for chondrogenesis, Runx2 is also a key regulator for
osteoblast differentiation. In particular, Runx2 is
expressed in cells prefiguring the vertebrate skeleton as
early as embryonic day 10.5 [59]. Runx2 regulates
many genes that determine the osteoblast phenotype,
as the forced expression of Runx2 in nonosteoblast
cells is sufficient to induce the osteoblast-specific gene
osteocalcin [60]. The inactivation of both Runx2 alleles
in mice results in a lack of osteoblasts throughout the

AP-2a [64,67].
Reports on AP-2a knockout mice clearly indicate
the importance of this transcription factor in regulat-
ing bone and cartilage development during embryogen-
esis, because of the severe skeletal defects in growth
and the development of face and limbs [17–19]. Don-
ner et al. tried to link the expression of AP-2a in these
tissues to upstream signaling pathways. They assessed
the organization of a cis-regulatory region within the
fifth intron specific for directing AP-2a expression to
the developing frontal nasal process and limb bud mes-
enchyme, which they had previously identified in trans-
genic mice [70,71]. The results demonstrate that a
STAT binding site is required for robust AP-2a expres-
sion in the face and limbs. In a follow-up study, they
found that this conserved cis-acting sequence serves to
maintain a level of AP-2a expression that limits the
size of the hand plate and the associated number of
digit primordia [72].
AP-2 function was also analyzed in other species.
A similar role for AP-2a as a regulator for face and
limb bud development was described in chickens. AP-2
expression is completely downregulated after treatment
of the chick face with RA, and this is accompanied by
an increase in apoptosis [73]. The authors of this study
ascribe the regulation of outgrowth of limb buds and
patterning of the digits to the chicken AP-2.
The role of AP-2a was further studied in zebrafish.
It was confirmed that AP-2a is an essential regulator
of the development of neural crest derivates, including

Proliferative zone Resting zone
Sox9
AP-2
ε
Undifferentiated
mesenchymal cells
Differentiated
chondrocytes
Hypertrophic
chondrocytes
Condensed
mesenchymal cells
Sox9
Sox9
Sox5
Sox6
AP-2
α
Runx2
Runx3
Runx2
Runx2
Fig. 2. Functional role of AP-2a and AP-2e
in chondrogenesis. Overview of the differen-
tiation stages during chondrogenesis and
the involvement of transcription factors
(henatoxylin and eosin-stained section of an
embryonic cartilaginous limb).
AP-2 proteins in cartilage differentiation A K. Wenke and A K. Bosserhoff
898 FEBS Journal 277 (2010) 894–902 ª 2009 The Authors Journal compilation ª 2009 FEBS

thritic chondrocytes, but the exact function of AP-2e
in osteoarthritic development of cartilage is still
unknown [69].
Conclusions
AP-2 proteins, especially AP-2a and AP-2e, are impor-
tant for chondrogenic and skeletal development. Many
studies on AP-2a have been performed, analyzing the
role of this transcription factor as a main regulator
of facial and limb development in embryogenesis.
Further analyses are required to clarify the regulatory
mechanisms during early chondrocytic differentiation,
because it is still unknown how AP-2a itself is upregu-
lated in chondroprogenitor cells. The molecular rele-
vance of AP-2e in hypertrophic cartilage and in the
development of osteoarthritis also still has to be ana-
lyzed in detail. It is necessary to obtain more insights
into the transcriptional regulation of AP-2s, to under-
stand the complex story of AP-2s during embryonic
development.
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