The cartilage-specific transcription factor Sox9 regulates AP-2e expression in chondrocytes Ann-Kathrin Wenke1, Susanne Grassel2, Markus Moser3 and Anja K Bosserhoff1 ¨ Institute of Pathology, University Regensburg, Germany Department of Orthopedics, University Regensburg, Germany Max-Planck-Institute of Biochemistry, Martinsried, Germany Keywords AP-2e; cartilage; differentiation; osteoarthritis; transcriptional regulation Correspondence A.-K Bosserhoff, Institute of Pathology, University of Regensburg, Franz-JosefStrauss-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 a10 expression in cartilage In this study, we analyzed 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 cartilage, consistent with immunohistochemical stainings of mouse embryo sections Furthermore, osteoarthritic chondrocytes, resembling a hypertrophic phenotype, have high AP-2e levels The AP-2e promoter harbors binding sites for the transcription factors AP-2a and Sox9 Both transcription 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 confirmed 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 The family of activating enhancer-binding protein (AP)-2 transcription factors regulate their target genes through binding to the palindromic recognition sequence 5¢-GCCN3GGC-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 physiological processes during development, cell cycle regulation, 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-terminus [9–11] So far, the most recently identified AP-2 transcription 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 regulates the expression of integrin a10, the predominant collagen-binding integrin during cartilage development [13] The axial skeleton is formed by a process named endochondral bone formation This complex process 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 A.-K Bosserhoff et al starts with the migration of undifferentiated mesenchymal 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) containing 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 cartilagespecific 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 differentiation, chondrocytes become hypertrophic and express increased levels of type X collagen and reduced levels of type II collagen [19–21] Finally, osteoblasts infiltrate 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 problem in osteoarthritis (OA), a degenerative joint disorder, leading to destruction of the cartilage The onset of this disease might be triggered by multiple factors such as mechanical overload, defects in the composition of the ECM, or altered expression of transcription factors controlling the production of matrix molecules [27] Here, we analyzed AP-2e expression and its regulation during cartilage differentiation and in osteoarthritic chondrocytes Our data provide evidence that AP-2e is directly regulated by the transcription factor Sox9 and has a role in cartilage differentiation Results Sox9 regulates AP-2e in hypertrophic chondrocytes human mesenchymal stem cells (HMSCs), and differentiated them either to chondrocytes or to osteoblasts [29] Figure 1A shows that AP-2e is highly expressed in human chondrocytes and in chondrogenically differentiated HMSCs, as compared with untreated or osteoblastically differentiated HMSCs To further analyze at which stages during chondrocyte differentiation the expression of AP-2e increases, we used an in vitro model system for HMSC differentiation into chondrocytes 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 differentiation (Fig 1B) These stages correspond to the hypertrophic phase of chondrogenesis, which is characterized by increased expression of the hypertrophic marker gene type X collagen The expression of AP-2a, which is known to be expressed during cartilage 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 differentiation, at around day 17 (Fig 1D) To confirm AP-2e expression in hypertrophic regions of the developing cartilage, immunohistochemical 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 sections from AP-2e knockout mice (M Moser, unpublished 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 is expressed in hypertrophic cartilage Previous studies demonstrated that the transcription factor AP-2e is expressed in chondrocytes and regulates gene expression of integrin a10, which plays an important role in cartilage development [13,28] To determine the functional role of AP-2e in human chondrocytes, we used dedifferentiated chondrocytes and AP-2e expression in osteoarthritic chondrocytes Osteoarthritic cartilage often resembles a hypertrophic phenotype [32,33] To address whether AP-2e expression is altered in osteoarthritic chondrocytes in comparison with differentiated chondrocytes, we quantified their mRNA expression, and detected significantly FEBS Journal 276 (2009) 2494–2504 ª 2009 The Authors Journal compilation ª 2009 FEBS 2495 Sox9 regulates AP-2e in hypertrophic chondrocytes A.-K Bosserhoff et al Fig Expression of AP-2e in human chondrocytes 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 chondrogenic 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 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 a10, an AP-2e target gene, was also measured, and was found to be strongly upregulated in osteoarthritic chondrocytes 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 osteoarthritic cartilage with the AP-2e antiserum Figure 3E shows AP-2e-positive cells within the osteoarthritic cartilage tissue sections We also analyzed the expression of Sox9 in these tissue sections, and found Sox9 expression in the osteoarthritic cartilage (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 chondrocytes, we studied the AP-2e promoter One thousand base pairs upstream of the translation start site of the AP-2e gene were analyzed in detail to identify 2496 binding sites for known transcription factors that might regulate AP-2e expression Two potential binding sites for the transcription factor Sox9 at positions )973 ⁄ )970 and )448 ⁄ )445 and three putative AP-2abinding 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 expression 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 transfected 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 promoterless 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 FEBS Journal 276 (2009) 2494–2504 ª 2009 The Authors Journal compilation ª 2009 FEBS A.-K Bosserhoff et al Sox9 regulates AP-2e in hypertrophic chondrocytes the 604 bp wild-type construct The remaining activation 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 Fig 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 hypertrophic cartilage (B) Tissue slides of an AP-2e knockout (ko) mouse were stained as a control, and were clearly negative (C) Immunohistochemical staining of Sox9 in day 14.5 mouse embryos showed Sox9 expression in the early stages of hypertrophic chondrocytes wt, wild-type 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 compared with cells transfected with a control plasmid Additional transfection of AP-2a or Sox9 expression plasmids showed an increase of AP-2e promoter activity in SW1353 cells (Fig 4C) The cotransfection of AP-2a and Sox9 further increased the promoter activity 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 In the following studies, we focused on Sox9 as a regulator of AP-2e expression, because AP-2a is expressed at a constant level during chondrocyte differentiation, 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 further analyzed using small interfering RNA (siRNA) against Sox9 SW1353 cells were transfected with control 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 fragments 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 Sox9binding sites within the AP-2e promoter was confirmed 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 oligonucleotides containing the Sox9-binding sites resulted in a strong DNA–protein interaction (Fig 5D, lanes and 7) The specificity of these complexes was shown in competition studies using unlabeled oligonucleotides in a 400-molar excess (Fig 5D, lanes and 8) Incubation with a 400-molar excess of unlabeled oligonucleotides harboring a mutated Sox9-binding site did not lead to competition of the complexes (Fig 5D, lanes and 9) FEBS Journal 276 (2009) 2494–2504 ª 2009 The Authors Journal compilation ª 2009 FEBS 2497 Sox9 regulates AP-2e in hypertrophic chondrocytes A.-K Bosserhoff et al Fig Expression of AP-2e, AP-2a, integrin a10 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), integrin a10 (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 Discussion Recently, we showed that the transcription factor AP-2e is a positive regulator of integrin a10 expression in chondrocytes [13] In this study, we wanted to determine 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 differentiation To further investigate the time point of AP-2e induction during chondrocyte differentiation, chondrogenic 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 differentiation, chondrocytes undergo a process of terminal differentiation, by which they become hypertrophic and express hypertrophic marker genes such as type X collagen [34,35] Immunohistochemical staining of embryonic tissues at day 14.5 and day 17.5 confirmed clear 2498 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, induction of AP-2e expression is seen upon a further increase in Sox9 expression at later stages of chondrocyte differentiation Therefore, we suggest that Sox9 is FEBS Journal 276 (2009) 2494–2504 ª 2009 The Authors Journal compilation ª 2009 FEBS A.-K Bosserhoff et al Sox9 regulates AP-2e in hypertrophic chondrocytes Fig 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 constructs 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 Data are given as mean ± SEM; *P < 0.05 an important regulator of AP-2e expression in hypertrophic chondrocytes and in osteoarthritis The transcription factor Sox9 is known to be a regulator of chondrogenesis It is expressed in all chondrogenic progenitor cells and chondrocytes Sox9 is essential for the early steps of chondrogenesis in mesenchymal 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 chondrocytes [25,26], but in these studies no subdivision was made into early and late hypertrophy Our expression analyses using quantitative real-time PCR and immunohistochemical staining of Sox9 demonstrated that Sox9 is expressed in early chondrogenic development 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 FEBS Journal 276 (2009) 2494–2504 ª 2009 The Authors Journal compilation ª 2009 FEBS 2499 Sox9 regulates AP-2e in hypertrophic chondrocytes A.-K Bosserhoff et al Fig 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 and 8) and mutated oligonucleotides (lanes and 9) were used Lanes and show the labeled oligonucleotides incubated without protein 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 hypertrophic phase of chondrocyte differentiation A hypertrophic 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 chondrocytes For the first time, we found that the transcription factor Sox9 is a positive regulator of AP-2e expression at the beginning of the hypertrophic development of cartilage and of osteoarthritic chondrocytes 2500 The dramatic increase in AP-2e expression and that of its target gene integrin a10 in OA suggests an important 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 a10, 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 supplemented with penicillin (400 mL)1), streptomycin (50 lgỈmL)1), l-glutamine (300 lgỈmL)1), and 10% fetal bovine serum (Sigma, Deisenhofen, Germany), and split at a : ratio every days Primary chondrocytes were obtained from Cambrex (Iowa, IA, USA), and cultured as FEBS Journal 276 (2009) 2494–2504 ª 2009 The Authors Journal compilation ª 2009 FEBS A.-K Bosserhoff et al suggested by the manufacturers The proliferating cells are dedifferentiated in culture To differentiate these cells, they were stimulated with transforming growth factor-b1 (10 ngỈmL)1) for week HMSCs from CellSystems (St Katharinen, Germany) were cultivated in MSCGM medium (CellSystems) under a humidified atmosphere of 5% CO2 at 37 °C [41] To stimulate 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 Commission of the University of Regensburg Full-thickness cartilage slices were aseptically dissected from healthy aspects of femoral condyles of patients aged 50–76 years with osteoarthritis who had undergone total knee arthroplasty OA chondrocytes were prepared from osteoarthritic cartilage slices obtained as described above, and are therefore defined as osteoarthritic chondrocytes; no biochemical marker was used for characterization, except for the diagnosis from the orthopedic surgeon according to accepted orthopedic 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 transcriptase reaction performed in a 20 lL reaction volume containing lg of total cellular RNA, lL of 5· firststrand buffer (Invitrogen, Groningen, the Netherlands), lL of 0.1 m dithiothreitol, lL of dN6-primer (10 mm), lL of dNTPs (10 mm), and diethylpyrocarbonate ⁄ water The reaction mixture was incubated for 10 at 70 °C, 200 U of Superscript II reverse transcriptase (Invitrogen) were added, and RNAs were transcribed for h at 37 °C The reverse transcriptase was inactivated at 70 °C for 10 min, and the RNA was degraded by digestion with lL of RNaseA (10 mgỈmL)1) at 37 °C for 30 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 a10 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 Sox9rev, 5¢-AGGTGAAGGTGGAGTAGAGGC-3¢ (annealing temperature 58 °C); integrin alpha10-for, 5¢-CATGAGGTT Sox9 regulates AP-2e in hypertrophic chondrocytes 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 immunohistochemistry 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 °C, with the secondary antibody (biotin-labeled anti-rabbit; DAKO, Hamburg, Germany) for 30 at room temperature, and then with streptavidin-POD (DAKO) for 30 Antibody 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 transcription factor-binding sites, we screened approximately 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 isoforms 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 fragment, the reverse primer contained a HindIII 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 mutagenesis 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 mutSox9447_rev, 5Â-GCGTGAATTCAGCCCACAGCAATCAGAG CCGCCTTCTGG-3Â FEBS Journal 276 (2009) 24942504 ê 2009 The Authors Journal compilation ª 2009 FEBS 2501 Sox9 regulates AP-2e in hypertrophic chondrocytes A.-K Bosserhoff et al 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 transfection solutions, according to the manufacturer’s instructions 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 independent 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 Transfection Reagent (Qiagen), according to the manufacturer’s protocol Cells were transfected in six-well culture plates, and RNA was isolated 24 h after transfection ChIP assay The ChIP assay was performed following the manufacturer’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 analysis by PCR PCR was performed on four DNA templates: 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 (H2O) Four sets 2502 of specific primer pairs were used: the glyceraldehyde-3phosphate 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-2e promoter 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 [32P]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 MgCl2, mm EDTA, 25 mm dithiothreitol, 250 mm KCl, 25 mm Hepes ⁄ KOH, pH 7.9) with the DNA probe for 10 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 at room temperature before addition of the DNA probe DNprotein 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) FEBS Journal 276 (2009) 2494–2504 ª 2009 The Authors Journal compilation ª 2009 FEBS A.-K Bosserhoff et al Acknowledgement This work was partly supported by a DFG grant assigned to S Grassel (GR 1301 ⁄ 7-1) ¨ References Hilger-Eversheim K, Moser M, Schorle H & Buettner R (2000) Regulatory roles of AP-2 transcription factors in vertebrate development, apoptosis and cell-cycle control Gene 260, 1–12 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 Bosher JM, Williams T & Hurst HC (1995) The developmentally regulated transcription factor AP-2 is 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Biol 19, 194–204 46 Ho SN, Hunt HD, Horton RM, Pullen JK & Pease LR (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction Gene 77, 51–59 FEBS Journal 276 (2009) 2494–2504 ª 2009 The Authors Journal compilation ª 2009 FEBS ... 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 Sox9binding... AP-2e promoter (Sox9_ 1 and Sox9_ 2) Incubation of in vitro-synthesized Sox9 with the labeled oligonucleotides containing the Sox9- binding sites resulted in a strong DNA–protein interaction (Fig... 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