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 mice. AP-2a, AP-2b and AP-2c show partially overlap- ping expression patterns in neural crest cells (NCCs), the peripheral nervous system, the facial mesenchyme, the limbs, various epithelia of the developing embryo, Keywords AP-2; cartilage; chondrogenesis; limb; 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-regens burg.de (Received 12 October 2009, revised 13 November 2009, accepted 20 November 2009) doi:10.1111/j.1742-4658.2009.07509.x During embryogenesis, most of the mammalian skeletal system is preformed as cartilaginous structures that ossify later. The different stages of cartilage and skeletal development are well described, and several molecular factors are known to influence the events of this enchondral ossification, especially transcription factors. Members of the AP-2 family of transcription factors play important roles in several cellular processes, such as apoptosis, migra- tion and differentiation. Studies with knockout mice demonstrate that a main function of AP-2s is the suppression of terminal differentiation during embryonic development. Additionally, the specific role of these molecules as 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 PAATAAAEFQPP-YFPPPYPQPPLPYGQAPDAAAAFPHLAGDPYGG-LAPLAQPQPP Delta TTG TEFASP-YFSTNHQYTPL-HHQSFHYEFQHSHPAVTPDAYSLNSLHHSQQYYQQ . :: .* ** : : : *: . * . . : .* Alpha HPGWPGQRQ SQESGLLHTHRGLPHQLSG-LDP RRDY RRHEDLLHGP-HA Beta HPWGQRQRQEVGSEAGSLLPQPRAALPQLSG-LDP RRDYHSVRRPDVLLHSAHHG Gamma QQAWPGRQSQEGAGLPSHHGRPAGLLPHLSG-LEAGAVSARRDAY RRSDLLLPHAHAL Epsilon QAAWAAPRAAARAHEE PPGLLAPPARALG-LDP RRDYA TAVPRLLHGLADG Delta IHHGEPTDFINLHNARALKSSCLDEQRRELGCLDAYR RHDLS LMSHGSQYGMHPD : * *:. *:* Alpha LSSGLGD-LSIHSLPH AIEEVPHVEDP GINIPDQT-VIKKGPVSLSKSNSNAVSA Beta LDAGMGDSLSLHGLGHP-GMEDVQSVEDANNSGMNLLDQS-VIKKVPVPP KSVTS Gamma DAAGLAENLGLHDMPH QMDEVQNVDDQ HLLLHDQT-VIRKGPISMT KNPLN Epsilon AHGLADAPLGLPGLAAAPGLEDLQAMDEP GMSLLDQS-VIKKVPIPSK ASSLSA Delta QRLLPGPSLGLAAAGA DDLQGSVEAQ-CGLVLNGQGGVIRRG *.: ::: : : : .* **:: Alpha IPINKDNLFGGV-VNPNEVFCSVPGRLSLLSSTSKYKVTVAEVQRRLSPPECLNASLLGG Beta LMMNKDGFLGGMSVNTGEVFCSVPGRLSLLSSTSKYKVTVGEVQRRLSPPECLNASLLGG Gamma LPCQKE LVGAVMNPTEVFCSVPGRLSLLSSTSKYKVTVAEVQRRLSPPECLNASLLGG Epsilon LSLAKDS-LVGGITNPGEVFCSVPGRLSLLSSTSKYKVTVGEVQRRLSPPECLNASLLGG Delta GTCVVNPTDLFCSVPGRLSLLSSTSKYKVTIAEVKRRLSPPECLNASLLGG *. ::********************:.**:**************** Alpha VLRRAKSKNGGRSLREKLDKIGLNLPAGRRKAANVTLLTSLVEGEAVHLARDFGYVCETE Beta VLRRAKSKNGGRSLRERLEKIGLNLPAGRRKAANVTLLTSLVEGEAVHLARDFGYICETE Gamma VLRRAKSKNGGRSLREKLDKIGLNLPAGRRKAAHVTLLTSLVEGEAVHLARDFAYVCEAE Epsilon VLRRAKSKNGGRCLRERLEKIGLNLPAGRRKAANVTLLTSLVEGEAVHLARDFGYVCETE Delta ILRRAKSKNGGRCLREKLDRLGLNLPAGRRKAANVTLLTSLVEGEALHLARDFGYTCETE :***********.***:*:::************:************:******.* **:* Alpha FPAKAVAEFLNRQHSD-PNEQVTRKNMLLATKQICKEFTDLLAQDRSPLGNSRPNPILEP Beta FPAKAVSEYLNRQHTD-PSDLHSRKNMLLATKQLCKEFTDLLAQDRTPIGNSRPSPILEP Gamma FPSKPVAEYLTRPHLGGRNEMAARKNMLLAAQQLCKEFTELLSQDRTPHGTSRLAPVLET 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 functions during mouse development. Analyses of AP-2a-null mice have demonstrated that AP-2a is a fundamental regulator of mammalian craniofacial development. AP-2a knockout mice die perinatally with craniofacial defects, thoracoabdominoschisis, and severe skeletal defects in the head and trunk region [17,18]. Studies of earlier embryonic stages of these mice indicate a failure of cranial neural tube closure and defects in cranial ganglia development. Another role of AP-2a previously masked in the knockout mice became apparent in chimeric mice composed of both wild-type and AP-2a-null cells [19]. These chimeras reveal the major influence of AP-2a on eye forma- tion and limb pattern formation typified by limb duplications. In contrast to these defects, the lack of AP-2b leads to enhanced apoptotic cell death of renal epi- thelial cells. AP-2b knockout mice die shortly after birth because of polycystic kidney disease and termi- nal renal failure [20,21]. The targeted deletion of AP-2c also has severe consequences. The loss of AP-2c is already lethal in early embryogenic develop- ment directly after implantation during gastrulation, because AP-2c controls proliferation and differentia- tion of extraembryonic trophectodermal cells [22,23]. So far, nothing is known about chondrogenic defects mediated by knocking out AP-2b or AP-2c. However, all these types of grave damage after deletion of AP-2 transcription factors demonstrate the importance of the AP-2s for several functions 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 domain containing protein KCTD1 directly binds to AP-2a and acts as a negative regulator for AP-2a trans- activation [34]. It was also demonstrated in other studies that the nuclear protein poly(ADP-ribose) polymerase-1 interacts with the C-terminus of AP-2a and enhances its transcriptional activity in normal circumstances, whereas its enzymatic activity is used as a temporary shut-off mechanism during unfavorable conditions [35,36]. Little is known about the interaction of AP-2 and its binding partners in cartilage. However, at least CBP ⁄ p300-interacting transactivator with ED-rich tail 2 and protein poly(ADP-ribose) polymerase-1 are expressed in this tissue [37–40]. It would be interesting to further analyze their interactions with AP-2 and the functional role of these in chondrocytes. Furthermore, it is speculated that in melanoma, where AP-2a acts as a tumor suppressor, the loss of AP-2a is caused by a failure in post-transcriptional processing of the protein [41]. Additionally, it is evi- dent that AP-2 transcription factors can indirectly modulate genes by functional interactions with other transcription factors, e.g. c-myc, rBP, and p53 [42–44]. The formation of AP-2 homodimers and heterodimers could also be important for their regulatory activity, but no studies have been published so far. For the regulation of target gene expression, the AP-2 transcription factors bind to the palindromic recognition sequence 5¢-GCCN 3 GGC-3¢ or variations 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 resting and proliferating chondrocytes produce an extracellular matrix mainly consisting of aggrecan and type II collagen. As skeletogenesis proceeds, proliferat- ing chondrocytes exit the cell cycle, become hypertro- phic, express type X collagen, and reduce the expression of type II collagen [50]. Hypertrophic chon- drocytes undergo terminal differentiation before they finally become apoptotic. Through the invasion of blood vessels from the perichondrium, the cartilage becomes vascularized. Additionally, osteoblasts invade the cartilage and start to replace it with mineralized bone [48]. Many molecules and signaling cascades are neces- sary to regulate these molecular processes of chondro- genic and skeletal development, including transcription factors. Essential transcription factors in chondrocyte differentiation are Sox9 and Runx2. Sox9 plays a key role in chondrogenesis, as an inactivating mutation in the gene encoding Sox9 leads to severe cartilage abnor- malities called campomelic dysplasia [51,52]. The effect of a complete loss of Sox9 during chondrogenesis was analyzed using a model of mice chimeras injected with homozygous embryonic Sox9 ) ⁄ ) stem cells into wild- type blastocysts, because Sox9 knockout mice are not viable [53]. The Sox9 ) ⁄ ) cells were excluded from mes- enchymal condensation and had no expression of the 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 skeleton [61,62]. It has also been shown that deletions resulting in the heterozygous loss of runx2 cause cleid- ocranial dysplasia [63]. Role of AP-2 a,AP-2b and AP-2e in chondrogenesis and skeletal development In addition to Sox and Runx transcription factors, members of the AP-2 family also have important func- tions in chondrogenesis and development of the verte- brate skeleton during embryogenesis. Especially for AP-2a, but also for AP-2b and AP-2e, a role as a reg- ulator of cartilage differentiation has been shown [64–69]. The functional and important roles of AP-2 transcription factors during chondrogenesis are illus- trated in Fig. 2. AP-2a is expressed in the growth plate and in articu- lar cartilage, and has been described as a negative regulator of chondrocyte differentiation [64]. The expression of cartilage-derived retinoic acid-sensitive protein and type II collagen is negatively correlated with AP-2a expression, and AP-2a thus acts as a sup- pressor of these two cartilage matrix genes during car- tilage differentiation [64–66] (Fig. 2). High expression levels of AP-2a in chondroprogenitor cells maintain these cells in an early differentiation phenotype and inhibit the transition to differentiated chondrocytes. The induction of Sox5 and Sox6 as well as that of chondrocytic matrix genes such as type II collagen, 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 897 aggrecan and type X collagen are also delayed by 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 embryonic cartilage and neurons, as well as pigmented cells [74–76]. Knight et al. [77] demonstrated essential functions for zebrafish AP-2a (tfap2a) and also AP-2b (tfap2b) in the development of the facial ectoderm, and for signals from this epithelium that induce skeletogen- esis in NCCs. Zebrafish embryos lacking both tfap2a and tfap2b have defects in epidermal cell survival and deficient NCC-derived cartilage. The authors propose that AP-2s have two distinct functions in cranial NCCs: they play an early cell-autonomous role in cell specification and survival, and a later nonautonomous role as regulators of ectodermal signals that induce skeletogenesis [77]. Luo et al. [78] characterized Inca (induced in the neural crest by AP-2) as a target gene upregulated by AP-2a in Xenopus embryos. Knockdown experiments for Inca in frog and fish revealed essential functions in a subset of NCCs that form craniofacial cartilage. Cells deficient for Inca show normal migration but fail to condense into skeletal primordia. This is an interest- ing aspect, as, for murine embryonic development, AP-2a is described as a suppressor of cartilage differ- entiation, maintaining cells in an early differentiated phenotype. For AP-2b, expression in murine limbs has also been demonstrated. AP-2b is expressed in the zone of polar- izing activity (ZPA), the signaling center of the devel- oping vertebrate limb [68]. A microarray approach comparing gene expression in the ZPA with that in the Hypertrophic zone 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 rest of the limb showed that AP-2b expression is increased in the ZPA. The fifth member of the AP-2 family, AP-2e,is expressed in human articular cartilage, where it has been shown to be a regulator of integrin a10 expres- sion [15]. Recently, it was reported that the transcrip- tion factor Sox9 induces AP-2e expression in the hypertrophic stage of chondrocytic differentia- tion through direct binding to the AP-2e promoter [69] (Fig. 2). Additionally, osteoarthritis chondrocytes show increased expression of AP-2e as compared with differentiated chondrocytes [69]. Further studies are required to identify AP-2e target genes other than integrin a10, to clarify the role of AP-2e in chon- drocyte differentiation and in the development of osteoarthritis. Role of AP-2 in chondrocytic diseases A role for AP-2s as regulators has been shown for sev- eral chondrogenic diseases. For example, mutations in tfap2a are known to cause branchio-oculo-facial syn- drome [79]. The characteristic craniofacial features of this disease are dolichocephaly, malformed pinnae, thick nasal tip, and cleft lip. Moreover, it has been reported that branchio-oculo-facial syndrome has over- lapping features, such as orofacial clefting and occa- sional lip pits, with Van der Woude syndrome, in which disruption of an AP-2a-binding site within an interferon regulatory factor 6 enhancer is strongly associated with cleft lip [80]. Recently, it has been demonstrated that AP-2e is overexpressed in osteoar- 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. 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Bosserhoff 902 FEBS Journal 277 (2010) 894–902 ª 2009 The Authors Journal compilation ª 2009 FEBS . 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,. 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,