Tài liệu Báo cáo khoa học: Roles of AP-2 transcription factors in the regulation of cartilage and skeletal development doc

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. 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. References 1 Mitchell PJ, Wang C & Tjian R (1987) Positive and negative regulation of transcription in vitro: enhancer- binding protein AP-2 is inhibited by SV40 T antigen. Cell 50, 847–861. 2 Williams T, Admon A, Luscher B & Tjian R (1988) Cloning and expression of AP-2, a cell-type-specific transcription factor that activates inducible enhancer elements. Genes Dev 2, 1557–1569. 3 Moser M, Imhof A, Pscherer A, Bauer R, Amselgruber W, Sinowatz F, Hofstadter F, Schule R & Buettner R (1995) Cloning and characterization of a second AP-2 transcription factor: AP-2 beta. Development 121, 2779– 2788. 4 Chazaud C, Oulad-Abdelghani M, Bouillet P, Decimo D, Chambon P & Dolle P (1996) AP-2.2, a novel gene related to AP-2, is expressed in the forebrain, limbs and face during mouse embryogenesis. Mech Dev 54, 83–94. 5 Oulad-Abdelghani M, Bouillet P, Chazaud C, Dolle P & Chambon P (1996) AP-2.2: a novel AP-2-related transcription factor induced by retinoic acid during dif- ferentiation of P19 embryonal carcinoma cells. Exp Cell Res 225, 338–347. 6 Zhao F, Satoda M, Licht JD, Hayashizaki Y & Gelb BD (2001) Cloning and characterization of a novel mouse AP-2 transcription factor, AP-2delta, with unique DNA binding and transactivation properties. J Biol Chem 276, 40755–40760. 7 Wang HV, Vaupel K, Buettner R, Bosserhoff AK & Moser M (2004) Identification and embryonic expres- sion of a new AP-2 transcription factor, AP-2 epsilon. Dev Dyn 231, 128–135. 8 Garcia MA, Campillos M, Ogueta S, Valdivieso F & Vazquez J (2000) Identification of amino acid residues of transcription factor AP-2 involved in DNA binding. J Mol Biol 301, 807–816. 9 Wankhade S, Yu Y, Weinberg J, Tainsky MA & Kan- nan P (2000) Characterization of the activation domains of AP-2 family transcription factors. J Biol Chem 275, 29701–29708. 10 Williams T & Tjian R (1991) Analysis of the DNA- binding and activation properties of the human tran- scription factor AP-2. Genes Dev 5, 670–682. 11 Eckert D, Buhl S, Weber S, Jager R & Schorle H (2005) The AP-2 family of transcription factors. Genome Biol 6, 246, doi:10.1186/gb-2005-6-13-246. 12 Moser M, Ruschoff J & Buettner R (1997) Comparative analysis of AP-2 alpha and AP-2 beta gene expression during murine embryogenesis. Dev Dyn 208 , 115–124. 13 Zhao F, Lufkin T & Gelb BD (2003) Expression of Tfap2d, the gene encoding the transcription factor Ap-2 delta, during mouse embryogenesis. Gene Expr Patterns 3, 213–217. 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 899 14 Feng W & Williams T (2003) Cloning and characteriza- tion of the mouse AP-2 epsilon gene: a novel family member expressed in the developing olfactory bulb. Mol Cell Neurosci 24, 460–475. 15 Wenke AK, Rothhammer T, Moser M & Bosserhoff AK (2006) Regulation of integrin alpha10 expression in chondrocytes by the transcription factors AP-2epsilon and Ets-1. Biochem Biophys Res Commun 345, 495–501. 16 Winger Q, Huang J, Auman HJ, Lewandoski M & Williams T (2006) Analysis of transcription factor AP-2 expression and function during mouse preimplantation development. Biol Reprod 75, 324–333. 17 Zhang J, Hagopian-Donaldson S, Serbedzija G, Elsemore J, Plehn-Dujowich D, McMahon AP, Flavell RA & Williams T (1996) Neural tube, skeletal and body wall defects in mice lacking transcription factor AP-2. Nature 381, 238–241. 18 Schorle H, Meier P, Buchert M, Jaenisch R & Mitchell PJ (1996) Transcription factor AP-2 essential for cranial closure and craniofacial development. Nature 381, 235\– 238. 19 Nottoli T, Hagopian-Donaldson S, Zhang J, Perkins A & Williams T (1998) AP-2-null cells disrupt morphogen- esis of the eye, face, and limbs in chimeric mice. Proc Natl Acad Sci USA 95, 13714–13719. 20 Moser M, Pscherer A, Roth C, Becker J, Mucher G, Zerres K, Dixkens C, Weis J, Guay-Woodford L, Buettner R et al. (1997) Enhanced apoptotic cell death of renal epithelial cells in mice lacking transcription factor AP-2beta. Genes Dev 11, 1938–1948. 21 Moser M, Dahmen S, Kluge R, Grone H, Dahmen J, Kunz D, Schorle H & Buettner R (2003) Terminal renal failure in mice lacking transcription factor AP-2 beta. Lab Invest 83, 571–578. 22 Werling U & Schorle H (2002) Transcription factor gene AP-2 gamma is essential for early murine development. Mol Cell Biol 22, 3149–3156. 23 Auman HJ, Nottoli T, Lakiza O, Winger Q, Donaldson S & Williams T (2002) Transcription factor AP-2gamma is essential in the extra-embryonic lineages for early postim- plantation development. Development 129, 2733–2747. 24 Luscher B, Mitchell PJ, Williams T & Tjian R (1989) Regulation of transcription factor AP-2 by the morpho- gen retinoic acid and by second messengers. Genes Dev 3, 1507–1517. 25 Grether-Beck S, Olaizola-Horn S, Schmitt H, Grewe M, Jahnke A, Johnson JP, Briviba K, Sies H & Krutmann J (1996) Activation of transcription factor AP-2 mediates UVA radiation- and singlet oxygen-induced expression of the human intercellular adhesion molecule 1 gene. Proc Natl Acad Sci USA 93, 14586–14591. 26 Huang Y & Domann FE (1998) Redox modulation of AP-2 DNA binding activity in vitro. Biochem Biophys Res Commun 249, 307–312. 27 Underhill TM, Sampaio AV & Weston AD (2001) Retinoid signalling and skeletal development. Novartis Found Symp 232, 171–185. 28 Imagawa M, Chiu R & Karin M (1987) Transcription factor AP-2 mediates induction by two different signal- transduction pathways: protein kinase C and cAMP. Cell 51, 251–260. 29 Garcia MA, Campillos M, Marina A, Valdivieso F & Vazquez J (1999) Transcription factor AP-2 activity is modulated by protein kinase A-mediated phosphoryla- tion. FEBS Lett 444, 27–31. 30 Bamforth SD, Braganca J, Eloranta JJ, Murdoch JN, Marques FI, Kranc KR, Farza H, Henderson DJ, Hurst HC & Bhattacharya S (2001) Cardiac malforma- tions, adrenal agenesis, neural crest defects and exen- cephaly in mice lacking Cited2, a new Tfap2 co-activator. Nat Genet 29, 469–474. 31 Braganca J, Eloranta JJ, Bamforth SD, Ibbitt JC, Hurst HC & Bhattacharya S (2003) Physical and functional interactions among AP-2 transcription factors, p300 ⁄ CREB-binding protein, and CITED2. J Biol Chem 278 , 16021–16029. 32 Roth C, Schuierer M, Gunther K & Buettner R (2000) Genomic structure and DNA binding properties of the human zinc finger transcriptional repressor AP-2rep (KLF12). Genomics 63, 384–390. 33 Schuierer M, Hilger-Eversheim K, Dobner T, Bosserhoff AK, Moser M, Turner J, Crossley M & Buettner R (2001) Induction of AP-2alpha expression by adenoviral infection involves inactivation of the AP-2rep transcriptional corepressor CtBP1. J Biol Chem 276, 27944–27949. 34 Ding X, Luo C, Zhou J, Zhong Y, Hu X, Zhou F, Ren K, Gan L, He A, Zhu J et al. (2009) The interaction of KCTD1 with transcription factor AP-2alpha inhibits its transactivation. J Cell Biochem 106, 285–295. 35 Kannan P, Yu Y, Wankhade S & Tainsky MA (1999) PolyADP-ribose polymerase is a coactivator for AP-2-mediated transcriptional activation. Nucleic Acids Res 27 , 866–874. 36 Li M, Naidu P, Yu Y, Berger NA & Kannan P (2004) Dual regulation of AP-2alpha transcriptional activation by poly(ADP-ribose) polymerase-1. Biochem J 382, 323–329. 37 Yokota H, Goldring MB & Sun HB (2003) CITED2- mediated regulation of MMP-1 and MMP-13 in human chondrocytes under flow shear. J Biol Chem 278, 47275–47280. 38 Agrawal A, Gajghate S, Smith H, Anderson DG, Albert TJ, Shapiro IM & Risbud MV (2008) Cited2 modulates hypoxia-inducible factor-dependent expres- sion of vascular endothelial growth factor in nucleus pulposus cells of the rat intervertebral disc. Arthritis Rheum 58 , 3798–3808. AP-2 proteins in cartilage differentiation A K. Wenke and A K. Bosserhoff 900 FEBS Journal 277 (2010) 894–902 ª 2009 The Authors Journal compilation ª 2009 FEBS 39 Zakany R, Bakondi E, Juhasz T, Matta C, Szijgyarto Z, Erdelyi K, Szabo E, Modis L, Virag L & Gergely P (2007) Oxidative stress-induced poly(ADP-ribosyl)ation in chick limb bud-derived chondrocytes. Int J Mol Med 19, 597–605. 40 Gonzalez-Rey E, Martinez-Romero R, O’Valle F, Aguilar-Quesada R, Conde C, Delgado M & Oliver FJ (2007) Therapeutic effect of a poly(ADP-ribose) poly- merase-1 inhibitor on experimental arthritis by downre- gulating inflammation and Th1 response. PLoS ONE 2, e1071, doi:10.1371/journal.pone.0001071. 41 Karjalainen JM, Kellokoski JK, Mannermaa AJ, Kujala HE, Moisio KI, Mitchell PJ, Eskelinen MJ, Alhava EM & Kosma VM (2000) Failure in post-tran- scriptional processing is a possible inactivation mechanism of AP-2alpha in cutaneous melanoma. Br J Cancer 82, 2015–2021. 42 Batsche E, Muchardt C, Behrens J, Hurst HC & Cremisi C (1998) RB and c-Myc activate expression of the E-cadherin gene in epithelial cells through interac- tion with transcription factor AP-2. Mol Cell Biol 18, 3647–3658. 43 Decary S, Decesse JT, Ogryzko V, Reed JC, Naguibne- va I, Harel-Bellan A & Cremisi CE (2002) The retino- blastoma protein binds the promoter of the survival gene bcl-2 and regulates its transcription in epithelial cells through transcription factor AP-2. Mol Cell Biol 22, 7877–7888. 44 McPherson LA, Loktev AV & Weigel RJ (2002) Tumor suppressor activity of AP2alpha mediated through a direct interaction with p53. J Biol Chem 277, 45028–45033. 45 Hilger-Eversheim K, Moser M, Schorle H & Buettner R (2000) Regulatory roles of AP-2 transcription factors in vertebrate development, apoptosis and cell-cycle con- trol. Gene 260, 1–12. 46 Pfisterer P, Ehlermann J, Hegen M & Schorle H (2002) A subtractive gene expression screen suggests a role of transcription factor AP-2 alpha in control of proliferation and differentiation. J Biol Chem 277, 6637–6644. 47 Pellikainen JM & Kosma VM (2007) Activator protein- 2 in carcinogenesis with a special reference to breast cancer – a mini review. Int J Cancer 120, 2061–2067. 48 Karsenty G (2008) Transcriptional control of skeleto- genesis. Annu Rev Genomics Hum Genet 9, 183–196. 49 Thorogood PV & Hinchliffe JR (1975) An analysis of the condensation process during chondrogenesis in the embryonic chick hind limb. J Embryol Exp Morphol 33, 581–606. 50 Kosher RA, Kulyk WM & Gay SW (1986) Collagen gene expression during limb cartilage differentiation. J Cell Biol 102, 1151–1156. 51 Foster JW, Dominguez-Steglich MA, Guioli S, Kwok C, Weller PA, Stevanovic M, Weissenbach J, Mansour S, Young ID, Goodfellow PN et al. (1994) Campomelic dysplasia and autosomal sex reversal caused by muta- tions in an SRY-related gene. Nature 372, 525–530. 52 Wagner T, Wirth J, Meyer J, Zabel B, Held M, Zimmer J, Pasantes J, Bricarelli FD, Keutel J, Hustert E et al. (1994) Autosomal sex reversal and campomelic dyspla- sia are caused by mutations in and around the SRY-related gene SOX9. Cell 79, 1111–1120. 53 Bi W, Deng JM, Zhang Z, Behringer RR & de Cromb- rugghe B (1999) Sox9 is required for cartilage forma- tion. Nat Genet 22, 85–89. 54 Bell DM, Leung KK, Wheatley SC, Ng LJ, Zhou S, Ling KW, Sham MH, Koopman P, Tam PP & Cheah KS (1997) SOX9 directly regulates the type-II collagen gene. Nat Genet 16, 174–178. 55 Xie WF, Zhang X, Sakano S, Lefebvre V & Sandell LJ (1999) Trans-activation of the mouse cartilage-derived retinoic acid-sensitive protein gene by Sox9. J Bone Miner Res 14, 757–763. 56 Smits P, Li P, Mandel J, Zhang Z, Deng JM, Behringer RR, de Crombrugghe B & Lefebvre V (2001) The tran- scription factors L-Sox5 and Sox6 are essential for cartilage formation. Dev Cell 1, 277–290. 57 Takeda S, Bonnamy JP, Owen MJ, Ducy P & Karsenty G (2001) Continuous expression of Cbfa1 in nonhyper- trophic chondrocytes uncovers its ability to induce hypertrophic chondrocyte differentiation and partially rescues Cbfa1-deficient mice. Genes Dev 15, 467–481. 58 Yoshida CA, Yamamoto H, Fujita T, Furuichi T, Ito K, Inoue K, Yamana K, Zanma A, Takada K, Ito Y et al. (2004) Runx2 and Runx3 are essential for chondrocyte maturation, and Runx2 regulates limb growth through induction of Indian hedgehog. Genes Dev 18, 952–963. 59 Ducy P (2000) Cbfa1: a molecular switch in osteoblast biology. Dev Dyn 219, 461–471. 60 Ducy P, Zhang R, Geoffroy V, Ridall AL & Karsenty G (1997) Osf2 ⁄ Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89, 747–754. 61 Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao YH, Ina- da M et al. (1997) Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to matura- tional arrest of osteoblasts. Cell 89, 755–764. 62 Otto F, Thornell AP, Crompton T, Denzel A, Gilmour KC, Rosewell IR, Stamp GW, Beddington RS, Mund- los S, Olsen BR et al. (1997) Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 89, 765–771. 63 Quack I, Vonderstrass B, Stock M, Aylsworth AS, Becker A, Brueton L, Lee PJ, Majewski F, Mulliken JB, Suri M et al. (1999) Mutation analysis of core binding factor A1 in patients with cleidocranial dysplasia. Am J Hum Genet 65, 1268–1278. 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 901 64 Huang Z, Xu H & Sandell L (2004) Negative regulation of chondrocyte differentiation by transcrip- tion factor AP-2alpha. J Bone Miner Res 19, 245– 255. 65 Davies SR, Sakano S, Zhu Y & Sandell LJ (2002) Distribution of the transcription factors Sox9, AP-2, and [delta]EF1 in adult murine articular and meniscal cartilage and growth plate. J Histochem Cytochem 50, 1059–1065. 66 Xie WF, Kondo S & Sandell LJ (1998) Regulation of the mouse cartilage-derived retinoic acid-sensitive pro- tein gene by the transcription factor AP-2. J Biol Chem 273, 5026–5032. 67 Tuli R, Seghatoleslami MR, Tuli S, Howard MS, Danielson KG & Tuan RS (2002) p38 MAP kinase regulation of AP-2 binding in TGF-beta1-stimulated chondrogenesis of human trabecular bone-derived cells. Ann NY Acad Sci 961 , 172–177. 68 Rock JR, Lopez MC, Baker HV & Harfe BD (2007) Identification of genes expressed in the mouse limb using a novel ZPA microarray approach. Gene Expr Patterns 8, 19–26. 69 Wenke AK, Grassel S, Moser M & Bosserhoff AK (2009) The cartilage-specific transcription factor Sox9 regulates AP-2epsilon expression in chondrocytes. FEBS J 276, 2494–2504. 70 Zhang J & Williams T (2003) Identification and regula- tion of tissue-specific cis-acting elements associated with the human AP-2alpha gene. Dev Dyn 228, 194–207. 71 Donner AL & Williams T (2006) Frontal nasal promi- nence expression driven by Tcfap2a relies on a con- served binding site for STAT proteins. Dev Dyn 235, 1358–1370. 72 Feng W, Huang J, Zhang J & Williams T (2008) Identi- fication and analysis of a conserved Tcfap2a intronic enhancer element required for expression in facial and limb bud mesenchyme. Mol Cell Biol 28, 315–325. 73 Shen H, Wilke T, Ashique AM, Narvey M, Zerucha T, Savino E, Williams T & Richman JM (1997) Chicken transcription factor AP-2: cloning, expression and its role in outgrowth of facial prominences and limb buds. Dev Biol 188, 248–266. 74 Barrallo-Gimeno A, Holzschuh J, Driever W & Knapik EW (2004) Neural crest survival and differentiation in zebrafish depends on mont blanc ⁄ tfap2a gene function. Development 131 , 1463–1477. 75 O’Brien EK, d’Alencon C, Bonde G, Li W, Schoene- beck J, Allende ML, Gelb BD, Yelon D, Eisen JS & Cornell RA (2004) Transcription factor Ap-2alpha is necessary for development of embryonic melanophores, autonomic neurons and pharyngeal skeleton in zebra- fish. Dev Biol 265, 246–261. 76 Knight RD, Javidan Y, Nelson S, Zhang T & Schilling T (2004) Skeletal and pigment cell defects in the lockjaw mutant reveal multiple roles for zebrafish tfap2a in neu- ral crest development. Dev Dyn 229 , 87–98. 77 Knight RD, Javidan Y, Zhang T, Nelson S & Schilling TF (2005) AP2-dependent signals from the ectoderm regulate craniofacial development in the zebrafish embryo. Development 132, 3127–3138. 78 Luo T, Xu Y, Hoffman TL, Zhang T, Schilling T & Sargent TD (2007) Inca: a novel p21-activated kinase- associated protein required for cranial neural crest development. Development 134, 1279–1289. 79 Milunsky JM, Maher TA, Zhao G, Roberts AE, Stalker HJ, Zori RT, Burch MN, Clemens M, Mulliken JB, Smith R et al. (2008) TFAP2A mutations result in branchio-oculo-facial syndrome. Am J Hum Genet 82 , 1171–1177. 80 Rahimov F, Marazita ML, Visel A, Cooper ME, Hitchler MJ, Rubini M, Domann FE, Govil M, Chris- tensen K, Bille C et al. (2008) Disruption of an AP- 2alpha binding site in an IRF6 enhancer is associated with cleft lip. Nat Genet 40, 1341–1347. AP-2 proteins in cartilage differentiation A K. Wenke and A K. 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,