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The transcription factor ATF3 is upregulated during chondrocyte differentiation and represses cyclin… James, Claudine G; Woods, Anita; Underhill, T M; Beier, Frank Sep 19, 2006

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ralssBioMed CentBMC Molecular BiologyOpen AcceResearch articleThe transcription factor ATF3 is upregulated during chondrocyte differentiation and represses cyclin D1 and A gene transcriptionClaudine G James†1,2, Anita Woods†1,2, T Michael Underhill1,3 and Frank Beier*1,2Address: 1CIHR Group in Skeletal Development and Remodeling, University of Western Ontario, London, ON, Canada, 2Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada and 3Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, CanadaEmail: Claudine G James - claudine.james@schulich.uwo.ca; Anita Woods - pitawoods@hotmail.com; T Michael Underhill - tunderhi@interchange.ubc.ca; Frank Beier* - fbeier@uwo.ca* Corresponding author    †Equal contributorsAbstractBackground: Coordinated chondrocyte proliferation and differentiation are required for normalendochondral bone growth. Transcription factors binding to the cyclicAMP response element(CRE) are known to regulate these processes. One member of this family, Activating TanscriptionFactor 3 (ATF3), is expressed during skeletogenesis and acts as a transcriptional repressor, but thefunction of this protein in chondrogenesis is unknown.Results: Here we demonstrate that Atf3 mRNA levels increase during mouse chondrocytedifferentiation in vitro and in vivo. In addition, Atf3 mRNA levels are increased in response tocytochalasin D treatment, an inducer of chondrocyte maturation. This is accompanied by increasedAtf3 promoter activity in cytochalasin D-treated chondrocytes. We had shown earlier thattranscription of the cell cycle genes cyclin D1 and cyclin A in chondrocytes is dependent on CREs.Here we demonstrate that overexpression of ATF3 in primary mouse chondrocytes results inreduced transcription of both genes, as well as decreased activity of a CRE reporter plasmid.Repression of cyclin A transcription by ATF3 required the CRE in the cyclin A promoter. In parallel,ATF3 overexpression reduces the activity of a SOX9-dependent promoter and increases theactivity of a RUNX2-dependent promoter.Conclusion: Our data suggest that transcriptional induction of the Atf3 gene in maturingchondrocytes results in down-regulation of cyclin D1 and cyclin A expression as well as activationof RUNX2-dependent transcription. Therefore, ATF3 induction appears to facilitate cell cycle exitand terminal differentiation of chondrocytes.BackgroundGrowth and development of endochondral bones is con-trolled through the highly coordinated proliferation andparacrine and autocrine hormones and growth factorsthat, to a large part, act on chondrocyte cell surface recep-tors. The intracellular signaling pathways mediating thesePublished: 19 September 2006BMC Molecular Biology 2006, 7:30 doi:10.1186/1471-2199-7-30Received: 05 July 2006Accepted: 19 September 2006This article is available from: http://www.biomedcentral.com/1471-2199/7/30© 2006 James et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 11(page number not for citation purposes)differentiation of growth plate chondrocytes [1-3]. Theseprocesses are regulated by a large number of endocrine,effects are not completely understood; however, over thelast 10 years many of the key transcriptional regulators ofBMC Molecular Biology 2006, 7:30 http://www.biomedcentral.com/1471-2199/7/30chondrocyte differentiation have been identified. TheSox9 gene is required for the differentiation of mesenchy-mal precursor cells to chondrocytes and, together with therelated L-Sox5 and Sox6 proteins, controls chondrocyte-specific gene expression [4,5]. Sox9 also inhibits terminaldifferentiation of chondrocytes to the hypertrophic phe-notype [6]. In contrast, the Runx2 gene (also known asCbfa1) is essential for differentiation of osteoblasts, butalso promotes hypertrophic chondrocyte differentiation[5,7].In addition to these key regulators of chondrocyte matu-ration, numerous other transcription factors have beenimplicated in this process. One example is the activatingtranscription factor/cyclicAMP response element-bindingprotein (ATF/CREB) family that is defined by the ability ofits members to bind to the cyclicAMP response element(CRE) in target promoters. Mice with inactivation of theAtf2 gene display chondrodysplasia and reducedchondrocyte proliferation [8], a similar phenotype totransgenic mice overexpressing a dominant-negative formof CREB in cartilage [9]. We and others have shown thatATF2 and CREB regulate the transcription of the cell cyclegenes cyclin D1 and cyclin A in chondrocytes throughCRE-dependent mechanisms [10-15]. However, the ATF/CREB family contains additional members [16,17], someof which (such as ATF3) act as transcriptional repressors.These repressors could down-regulate CRE-dependenttranscription and thus cause delay of cell cycle progres-sion and/or promote cell cycle exit during terminal differ-entiation.Atf3 expression has been shown to be induced by a largevariety of cellular stresses, including radiation, DNA-dam-aging agents, adenoviral infection and others [18-22].Recent evidence also suggests induction of ATF3 by anumber of physiological stimuli such as growth hormone[23], transforming growth factor β[24] and ligands forseveral G-protein-coupled receptors [25]. In someinstances (e.g. in certain heterodimers with other proteinsor certain isoforms of ATF3), ATF3 has been shown to acti-vate transcription [26,27]. However, in most cases ATF3has been found to repress transcription of target genes[17]. Similarly, the effects of ATF3 on cellular physiologyappear to be very context- and cell type-dependent; forexample, ATF3 has been shown to both promote [27-29]and inhibit [21,30,31] cellular proliferation and cell cycleprogression, and to have both pro- and anti-apoptoticeffects [31,32].We recently performed microarray analyses of an in vitrosystem of mouse chondrocyte differentiation in three-dimensional micromass cultures [33]. We now extendedthese studies and demonstrated that numerous ATF/CREBfamily members are expressed in chondrocytes. Mostnotably, ATF3 expression is markedly upregulated duringchondrocyte differentiation. Validation of ATF3 expres-sion and functional studies strongly suggest an importantrole for ATF3 in cell cycle exit and terminal differentiation.ResultsAtf3 mRNA levels increase during chondrocyte differentiationBased on the prominent role of ATF2 (gene name Atf2)and CREB (Creb1) in cartilage development, we analyzedthe expression of ATF-CREB family members in our tran-scriptional profiles of differentiating micromass cultures[33]. Numerous family members were expressed duringmicromass differentiation (Table 1). Most genes, includ-ing Atf2 and Creb1, did not display marked changes inTable 1: Microarray Expression Profiles of ATF/CREB Family MembersNormalized Signal Intensity ValuesGenbank Gene Symbol Day 3 Day 6 Day 9 Day 12 Day 15NM_007497 Atf1 0.62 0.87 1.13 1.27 1.158BM119623 Atf2 1.10 0.95 0.98 1.00 0.91BC019946 Atf3 0.84 0.42 0.96 3.40 3.92AV314773 Atf4 0.99 0.87 1.03 1.42 1.03AF375476 Atf5 0.40 0.74 1.16 2.54 1.735AV270913 Atf6 0.74 0.73 1.01 1.75 1.818BC026483 Atf7 1.09 1.07 0.95 1.08 0.875NM_016767 Batf 0.90 0.97 1.01 1.04 1.058NM_009952 Creb1 1.17 0.88 0.76 0.77 0.77BG070002 Creb3 0.97 1.07 1.00 0.99 0.94BC013534 Crebl1 1.58 1.21 1.01 0.91 0.860BC016447 Creb3l1 0.30 0.71 1.09 1.34 1.037BC010786 Creb3l3 0.94 1.03 1.15 0.99 1.002BC022605 Creb3l4 0.92 0.93 1.08 0.86 0.862Page 2 of 11(page number not for citation purposes)AI467599 Crem 1.21 1.09 0.90 0.79 0.890BMC Molecular Biology 2006, 7:30 http://www.biomedcentral.com/1471-2199/7/30expression during micromass differentiation, and nogenes were down-regulated two-fold or more. However,Atf3, Atf5, Atf6 and Creb3l1 genes displayed upregulationtowards the end of the time course. The strongest effectswere observed for Atf3 transcript levels that increased 4.7-fold from day 3 to 15 of micromass culture (Fig. 1A). Atf5,Atf6 and Creb3l1 mRNA levels increased 4.3-, 2.5- and 3.5-fold over the same period, respectively. Because it showedthe largest increase, we focused subsequent studies on theAtf3 gene. We performed real-time PCR analyses of inde-pendent micromass cultures from mouse embryonic limbbud cells and demonstrated a strong increase of Atf3mRNA expression during micromass differentiation, thusvalidating the microarray data (Fig. 1B).To examine the expression of Atf3 during chondrocyte dif-ferentiation in vivo, we dissected tibiae from embryonicday 15.5 CD1 mice into the resting/proliferating, pre-hypertrophic and hypertrophic areas (at this stage, CD1tibiae are not vascularized yet; the center of the bone isthus still comprised of hypertrophic cartilage). RNA wasdirectly isolated from dissected tibiae, without subcultur-ing of cells. Real-time PCR analyses demonstrated expres-sion of collagen II (Col2a1) in the resting/proliferatingarea that declines significantly in the prehypertrophic andhypertrophic areas (Fig. 2A). In contrast, collagen X(Col10a1) mRNA was virtually undetectable in the rest-ing/proliferating area and increased strongly in the otherzones (Fig. 2B). These expression profiles of known mark-ers of early and late chondrocyte differentiation con-firmed efficient separation of the different growth patezones by microdissection. Atf3 mRNA levels increasedmore than five-fold from the resting/proliferating to thehypertrophic zone (Fig. 2C). Atf3 expression thus dis-played a similar pattern as Col10a1, although the induc-tion was less pronounced for Atf3.Atf3 is upregulated by cytochalasin D, an inhibitor of actin polymerizationWe had shown earlier that inhibition of actin polymeriza-tion by cytochalasin D promotes chondrogenic differenti-ation [34,35]. Thus, we examined whether cytochalasin Daffects Atf3 expression. Indeed, incubation of primarychondrocytes with cytochalasin D caused a more than 30-fold induction of Atf3 mRNA levels (Fig. 3A). To examinewhether this upregulation is due to transcriptional effects,primary chondrocytes were transfected with an Atf3 pro-moter – firefly luciferase construct. Cytochalasin D treat-ment resulted in five-fold upregulation of Atf3 promoteractivity (Fig. 3B). Thus, cytochalasin D induces Atf3through transcriptional mechanisms, at least in part.ATF3 represses SOX9-dependent and increases RUNX2-dependent transcriptionWe next asked whether increased expression of Atf3 affectschondrocyte-specific gene expression. Primary chondro-cytes were cotransfected with reporter vectors for SOX9and RUNX2 activity and an ATF3 expression vector. Over-expression of ATF3 resulted in an approximately 40 %reduction in SOX9-dependent transcription (Fig. 4A) andan 80 % increase in RUNX2-dependent transcription (Fig.4B), suggesting that upregulation of ATF3 can promotethe transition from proliferating to hypertrophic chondro-cytes.Atf3 expression is upregulated during chondrocyte differenti-a ion in vitroFigure 1Atf3 expression is upregulated during chondrocyte differentiation in vitro. Atf3 mRNA expression during chondrogenic differentiation of mouse embryonic limb bud cells in micromass culture was examined by microarray (A) and real-time PCR (B) analyses (different cell isolations were used for the experiments in A and B). Microarray data sets represent averages from three independent experiments. Real-time data were normalized to Gapdh levels and present average and SEM from four independent experiments. Both approaches show similar expression patterns with a strong     'D\V$7)P51$/HYHOV$    'D\V$7)P51$/HYHOV%0LFURDUUD\T3&5Page 3 of 11(page number not for citation purposes)increase in Atf3 expression during micromass differentiation.BMC Molecular Biology 2006, 7:30 http://www.biomedcentral.com/1471-2199/7/30Page 4 of 11(page number not for citation purposes)Atf3 expression is upregulated during chondrocyte differentiation in vivoFigure 2Atf3 expression is upregulated during chondrocyte differentiation in vivo. Tibia isolated from embryonic day 15.5. mice were microdissected into the resting/proliferating, prehypertrophic and hypertrophic areas, and RNA was isolated directly out of cartilage. Expression of type II collagen II (Col2a1), type X collagen (Col10a1) and Atf3 was examined by real-time PCR analyses and normalized to Gapdh expression. All data represent averages and SEM from four independent experiments (*: p < 0.05). Col2a1 was strongly expressed in the resting/proliferating area and declined subsequently (A). Col10a1 was virtu-ally undetectable in the resting/proliferating area and strongly induced in the prehypertrophic and hypertrophic areas (B). Atf3 was already expressed in the resting/proliferating area, but expression was significantly increased in the more mature zones of the growth plate.5HODWLYH*HQH([SUHVVLRQ=RQHV5HVWLQJ 3UHK\SHUWURSKLF +\SHUWURSKLF5HVWLQJ 3UHK\SHUWURSKLF +\SHUWURSKLF5HODWLYH*HQH([SUHVVLRQ$&ROODJHQ;$7)%&&ROODJHQ,,D5HVWLQJ 3UHK\SHUWURSKLF +\SHUWURSKLF5HODWLYH*HQH([SUHVVLRQBMC Molecular Biology 2006, 7:30 http://www.biomedcentral.com/1471-2199/7/30ATF3 represses transcription of the cyclin D1 and cyclinA genesATF3 acts as a transcriptional repressor through the CRE.We had demonstrated a requirement for the CRE for max-imal activity of the cyclin D1 and cyclin A promoters inchondrocytes [11,12,14,15]. We thus examined the effectsof ATF3 overexpression on CRE-dependent transcriptionactivity of the CRE-dependent reporter by approximately50 % (Fig. 5A) and cyclin D1 promoter activity by 38 %(Fig. 5B). ATF3 overexpression most strongly repressedcyclin A promoter activity which decreased to less than10% of control values (Fig. 5C). To determine whether theeffects of ATF3 on cyclin A transcription were mediated bythe CRE, we performed parallel experiments using a cyclinA promoter construct with a mutated CRE (Fig. 5C). ATF3overexpression did not affect activity of this promoterfragment, suggesting that ATF3 represses cyclin A tran-scription directly through the CRE.DiscussionThis study is the first to demonstrate an important role ofATF3 suppresses Sox9-dependent transcription and stimu-lates Runx2-dependent transcriptionFigure 4ATF3 suppresses Sox9-dependent transcription and stimulates Runx2-dependent transcription. Primary chondrocytes were cotransfected with SOX9 (A) or RUNX2 (B) reporter plasmids, pcDNA3 (control) or an ATF3 expression vector, and pRLCMV. 24 hours after transfection, cells were harvested, firefly luciferase activity was measured and normalized to Renilla luciferase activity. Data represent averages and SEM from three independent experiments, per-formed in quadruplicate each (*: p < 0.05). ATF3 overexpres-sion suppressed SOX9-dependent transcription and stimulated RUNX2-dependent transcription.9HFWRU $7)5HODWLYH/LJKW8QLWV$ 6R[5HSRUWHU% 5XQ[5HSRUWHU9HFWRU $7)5HODWLYH/LJKW8QLWV Inhibition of actin polymerization induces Atf3 expression through trans r ptional mechanismsFigure 3Inhibition of actin polymerization induces Atf3 expression through transcriptional mechanisms. A) Primary chondrocytes were cultured for 24 hours with DMSO (control) or 1 μM cytochalasin D before harvest and RNA isolation. Real-time PCR demonstrated marked induc-tion of Atf3 mRNA levels by cytochalasin D. Data represent averages and SEM from three independent experiments, nor-malized to Gapdh (*: p < 0.05). B) Primary chondrocytes were transfected with an Atf3 promoter plasmid and pRL-CMV, followed by incubation for 24 hours with DMSO (con-trol) or 1 μM cytochalasin D. Cells were then harvested, firefly luciferase activity was measured and normalized to Renilla luciferase activity. Data represent averages and SEM from three independent experiments, performed in quadru-plicate each (*: p < 0.05). Cytochalasin D induced Atf3 pro-moter activity.$7)3URPRWHU%9HKLFOH &\WRFKDODVLQ'5HODWLYH/LJKW8QLWV7UHDWPHQWV$ $7)P51$7UHDWPHQWV5HODWLYH*HQH([SUHVVLRQ9HKLFOH &\WRFKDODVLQ'Page 5 of 11(page number not for citation purposes)and the activity of these two cyclin promoters. Transienttransfection of an ATF3 expression plasmid decreased theATF3 in the control of endochondral bone growth andskeletal development. Our data demonstrate upregulationBMC Molecular Biology 2006, 7:30 http://www.biomedcentral.com/1471-2199/7/30Page 6 of 11(page number not for citation purposes)ATF3 suppresses CRE-dependent transcriptionFigure 5ATF3 suppresses CRE-dependent transcription. Primary chondrocytes were cotransfected with a CRE reporter (A), a cyclin D1 promoter (-1745CD1LUC, B) or cyclin A promoter plasmids (p707cycAluc and p707cycAlucMut; C), pcDNA3 (con-trol) or an ATF3 expression vector, and pRLCMV. 24 hours after transfection, cells were harvested, firefly luciferase activity was measured and normalized to Renilla luciferase activity. Data represent averages and SEM from three independent experi-ments, performed in quadruplicate each (*: p < 0.05). ATF3 overexpression suppressed the CRE reporter, cyclin D1 and cyclin A wild type promoters significantly, whereas the mutant cyclin A construct showed no response to ATF3 overexpression.9HFWRU $7)5HODWLYH/LJKW8QLWV&&5(GHSHQGHQW5HSRUWHU&\FOLQ'3URPRWHU$9HFWRU $7)5HODWLYH/LJKW8QLWV%&\FOLQ$3URPRWHU5HODWLYH/LJKW8QLWV&\FOLQ$$7)&\FOLQ$PXW9HFWRU&\FOLQ$PXW$7)&\FOLQ$9HFWRUBMC Molecular Biology 2006, 7:30 http://www.biomedcentral.com/1471-2199/7/30of Atf3 during chondrogenic differentiation in vivo and invitro. Atf3 expression is known to be induced by a numberof different cellular stressors, including hypoxia [22].Hypoxia has been demonstrated in the cartilage growthplate in vivo [36], raising the possibility that low oxygentension is one of the physiological inducers of ATF3 indeveloping cartilage. Interestingly, two of the other ATF/CREB family members found to be upregulated in ourstudies, ATF6 and CREB3L1 (also known as OASIS), havealso been shown to be induced by cellular stress (in par-ticular endoplasmatic reticulum stress) [37-41]. Thesedata suggest that upregulation of stress-responsive tran-scription factors is a common theme during late stagechondrogenic differentiation in micromass culture. SinceAtf3 transcript levels display similar upregulation duringchondrocyte differentiation in vivo, this pattern does notappear to be an artifact of the micromass culture system,but to reflect the physiological processes of cartilagedevelopment.ATF2 and CREB, the prototype members of the ATF-CREBfamily, have been shown to play crucial roles in chondro-cyte cell cycle gene expression and proliferation [42,43].While their activities are essential to ensure adequate ratesof proliferation, physiological bone development alsorequires regulated cell cycle exit and onset of postmitoticchondrocyte differentiation. These requirements necessi-tate tight control of ATF2 and CREB activity that can beachieved through several mechanisms. For example, bothtranscription factors are regulated by posttranslationalmodifications such as phosphorylation. Changes in phos-phorylation status could therefore repress their activitywhen chondrocytes exit the proliferative phase. For exam-ple, we and others have shown that extracellular signalssuch as Transforming Growth Factor β, Parathyroid Hor-mone-related Peptide and prostaglandins induce phos-phorylation of ATF2 and CREB in chondrocytes,respectively [10,13,44]. Our data presented here also sug-gest that Atf2 and Creb1 mRNA levels do not change dur-ing chondrocyte differentiation, suggesting that they areregulated at the posttranscriptional level (e.g. throughphosphorylation as discussed above and/or by regulationof protein stability). Another possibility to limit ATF2/CREB activity is the expression of transcriptional repres-sors that compete with them for binding elements on tar-get genes. One such repressor is ATF3 [26].Our data show that Atf3 mRNA expression is markedlyupregulated during chondrocyte differentiation in a three-dimensional micromass culture system in vitro and inmouse embryonic tibiae in vivo. In addition, Atf3 isstrongly induced in response to cytochalasin D, an actin-modifying drug that induces early [34,35,45,46] and lateinduction of Atf3 expression by cytochalasin D occurs, atleast in part, at the transcriptional level; however, induc-tion of mRNA levels is markedly higher than stimulationof promoter activity. This could be due to posttranscrip-tional mechanisms such as increased mRNA stability ashas been described for Atf3 before [47]. Alternatively,transcriptional activation of the endogenous Atf3 genethrough promoter/enhancer elements not present in ourreporter plasmid is possible.The expression pattern of Atf3 is therefore consistent witha function during cell cycle exit and terminal differentia-tion. Furthermore, our data suggest that ATF3 repressesCRE-dependent transcription, for example the activity ofthe cyclin D1 and cyclin A promoters. We have shown ear-lier that the activities of these promoters correlates withthe corresponding protein levels in chondrocytes [11-13].Induction of ATF3 should therefore result in reduced lev-els of these cyclins and other ATF2/CREB targets. The cyc-lin A promoter showed a much stronger response to ATF3overexpression than the cyclin D1 promoter and the CREreporter. The most plausible explanation for this differ-ence is that the cyclin A promoter, in addition to directregulation through the CRE, is also controlled byupstream cell cycle proteins such as cyclin D1 and E2F[48,49]. Reduced cyclin D1 levels and E2F activity inresponse to ATF3 overexpression could therefore contrib-ute to the observed strong downregulation of cyclin Atranscription by ATF3.Mutation of the CRE in the cyclin A promoter completelyabolished the response to ATF3 overexpression, providingstrong evidence that the effects of ATF3 on cyclin A tran-scription are mediated by direct binding to the CRE. Inter-estingly, overexpression of ATF3 repressed cyclin Apromoter more than mutation of CRE. This suggests thatbinding of ATF3 to the CRE also suppresses the activity ofother elements in the promoter, for example the E2Fresponse element discussed above.In growth plate physiology, Sox9 suppresses and Runx2promotes chondrocyte hypertrophy. In this study, weobserved repression of Sox9 activity and enhancement ofRunx2 activity in response to ATF3 overexpression, inagreement with the postmitotic role of ATF3 suggested byus. However, the mechanism(s) involved remain to beelucidated. One possibility is that ATF3, indirectly ordirectly, regulates expression of Sox9 and/or Runx2, orrequired cofactors. A second possibility is that ATF3 phys-ically interacts with each or both of these factors, therebymodulating their activity. Both Sox9 and Runx2 areknown to undergo protein-protein interactions withmany other transcription factors. For example, Sox9 hasPage 7 of 11(page number not for citation purposes)(Woods and Beier, in prep.) chondrocyte differentiation.Our transient transfection assays demonstrate that thebeen shown to interact with c-Maf [50], steroidogenic fac-tor 1 [51] and β-catenin [52]. Runx2 has been shown toBMC Molecular Biology 2006, 7:30 http://www.biomedcentral.com/1471-2199/7/30interact with, among others, Nrf2 [53] and Smad [54] pro-teins. Most notably, Runx2 has been demonstrated tobind to ATF4, another member of the ATF/CREB family,although this interaction appears to be indirect [55]. Athird possibility is that the effects of ATF3 on Sox9 andRunx2 activity are secondary to ATF3's effect on cell cycleprogression. It has been shown that Runx2 activity fluctu-ates throughout the cell cycle and is controlled by the cellcycle machinery [56-59]. For example, cyclin D1 togetherwith cyclin-dependent kinase 4 has been shown to pro-mote ubiquitination and degradation of Runx2 [60].Repression of cyclin D1 expression by ATF3 would thusresult in stabilization of Runx2 and increased differentia-tion. Our model for the effects of ATF3 on chondrocytecell cycle gene expression and Runx2 activity is illustratedin Fig. 6. To our knowledge, no cell cycle control mecha-nisms for Sox9 activity have been reported, but are con-ceivable.ConclusionIn summary, this study identifies ATF3 as a novel player inthe complex networks controlling growth plate chondro-cyte proliferation and differentiation. Induction of Atf3expression by differentiation stimuli is likely to contributeto the coordination of cell cycle exit and hypertrophic dif-ferentiation during endochondral bone growth. Analysesof the in vivo roles of ATF3 in endochondral ossificationand identification of the physiological inducers and targetgenes of ATF3 during skeletogenesis will be importantsteps towards a better understanding of cartilage develop-ment.MethodsMaterialsTimed-pregnant CD1 mice (embryonic day 11.5 [E11.5]or E15.5) were obtained from Charles River. Real-timePCR reagents were obtained from Applied Biosystems.Cell culture reagents and the pcDNA3 plasmid were fromInvitrogen, and cytochalasin D was purchased from Calbi-ochem. Fugene 6 was obtained from Roche, the dual luci-ferase assay and the plasmid pRLCMV were fromPromega. The CRE reporter plasmid was from Stratagene.The SOX9 reporter plasmid has been described [61]; theRUNX2 reporter plasmid was constructed in a similarfashion (8 copies of a Runx2 binding site cloned upstreamof a minimal osteocalcin promoter in pGl3basic[Promega]). The Atf3 promoter plasmid [23], the ATF3expression plasmid [62], the cyclin D1 promoter plasmid-1745CD1Luc [63] and the cyclin A promoter plasmidspcycAluc707 and pcycAluc707mut [64] plasmid weregenerously provided by Drs. J. Schwartz (University ofMichigan), D. Steiner (University of Chicago), R. Pestell(Georgetown University) and K. Oda (Science Universityof Tokyo), respectively.RNA isolation, microarray analyses and real-time PCRSample generation, RNA isolation, microarrays and bioin-formatics analyses for these data have been describedrecently [33]. For microdissections, embryonic day 15.5tibiae were dissected into the resting/proliferating, pre-hypertrophic and hypertrophic areas under a stereomicro-scope, and RNA was isolated following the RNeasy ® LipidTissue Extraction protocol from Qiagen (Mississauga).RNA integrity was verified using the Agilent 2100 Bioana-lyzer. Real-time PCR was performed as described, usingAssays-on-Demand (Applied Biosystems) and Gapdh forstandardization [33,34,65]. All reactions were run inquadruplicate from at least three independent experi-ments.Micromass cultures and primary chondrocyte culturesMicromass cultures from E11.5 mouse limb buds and iso-lation of primary murine chondrocytes from E15.5 longbones were performed as recently described [33,66].Micromass cultures were differentiated for 15 days, andsamples were harvested every three days for RNA isola-Model for ATF3 action in chondrocyte differentiationFigure 6Model for ATF3 action in chondrocyte differentia-tion. We suggest that induction of ATF3 by differentiation stimuli antagonizes the CRE-dependent expression of cyclin D1 and cyclin A, which are induced by mitogenic stimuli through ATF2 and CREB. Reduced cyclin-dependent kinase activity and hypophosphorylation of pocket proteins in response to ATF3 upregulation then results both in cell cycle exit and in increased activity of Runx2, promoting chondro-mitogenic stimulidifferentiation signalsATF2/CREBATF3cyclin D1, Acell cycle progressionRunx2Page 8 of 11(page number not for citation purposes)tion. Primary chondrocytes were plated in monolayer cul-ture in medium containing 10 % fetal bovine serum andcyte differentiation.BMC Molecular Biology 2006, 7:30 http://www.biomedcentral.com/1471-2199/7/30incubated for 24 hours with DMSO (control) or 1 μMcytochalasin D before harvest for RNA as described [35].Transient transfections and luciferase assaysPrimary chondrocytes in monolayer culture were tran-siently transfected using Fugene 6 as described [14,35,67].For Atf3 promoter analyses, cells were transfected with theAtf3 promoter plasmid and pRLCMV for standardization.After transfection, cells were incubated with DMSO or 1μM cytochalasin D for 24 hours before harvesting for luci-ferase assays. For cotransfections, cells were transfectedwith the respective promoter plasmid (cyclin D1, cyclin Aor the CRE, SOX9 or RUNX2 reporters), pRLSV40 andempty expression vector (pcDNA3) or the ATF3 expres-sion vector. Cells were harvested 24 hours after transfec-tion. Firefly luciferase activity was determined andnormalized to Renilla luciferase activity. All data shownpresent averages and SEM from three independent experi-ments done in quadruplicate each.Statistical analysesStatistical significance of real-time PCR and luciferaseresults was determined by two-way ANOVA with Bonfer-roni post-test using GraphPad Prism version 3.00 for Win-dows, GraphPad Software, San Diego California USA.AbbreviationsATF, activating transcription factor; CRE, cyclic AMPresponse element, CREB, CRE-binding protein; PCR,polymerase chain reactionAuthors' contributionsCGJ and AW performed experiments and contributed tothe writing of the manuscript. TMU provided input intothe design of the study and the manuscript. FB conceivedof the study, performed selected experiments and contrib-uted to the writing of the manuscript. All authors read andapproved the final manuscript.AcknowledgementsWe are grateful to Drs. Schwartz, Steiner, Pestell and Oda for the gift of plasmids. And to H. Agoston and Dr. L.-A. Stanton for RNA samples. C.G.J. and A.W. are recipients of Doctoral Awards from the Canadian Institutes of Health Research (CIHR). A.W. held a graduate student award from the Canadian Arthritis Network, and C.G.J held Ontario Graduate Scholarships for Science and Technology. T.M.U is the recipient of a New Investigator Award from CIHR, and F.B. holds a New Investigator Award from The Arthritis Society and a Canada Research Chair. Work in the laboratory of F.B. is supported by operating grants from CIHR, The Arthritis Society, the Canadian Arthritis Network and the Natural Sciences and Engineering Research Council.References1. van der Eerden BCJ, Karperien M, Wit JM: Systemic and LocalRegulation of the Growth Plate.  Endocr Rev 2003,3. Ballock RT, O'Keefe RJ: Physiology and pathophysiology of thegrowth plate.  Birth Defects Res C Embryo Today 2003,69(2):123-143.4. 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