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Expression profiling of Dexamethasone-treated primary chondrocytes identifies targets of glucocorticoid… James, Claudine G; Ulici, Veronica; Tuckermann, Jan; Underhill, T M; Beier, Frank Jul 1, 2007

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ralssBioMed CentBMC GenomicsOpen AcceResearch articleExpression profiling of Dexamethasone-treated primary chondrocytes identifies targets of glucocorticoid signalling in endochondral bone developmentClaudine G James1, Veronica Ulici1, Jan Tuckermann2, T Michael Underhill3 and Frank Beier*1Address: 1CIHR Group in Skeletal Development and Remodelling, Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Canada, 2Group of Tissue Specific Hormone Action, Leibniz Institute for Age Research -Fritz Lipmann Institute, Beutenbergstraße 11, D-07745 Jena, Germany and 3Department of Cellular & Physiological Sciences, University of British Columbia, Vancouver, British Columbia, CanadaEmail: Claudine G James - cjames9@uwo.ca; Veronica Ulici - vulici@uwo.ca; Jan Tuckermann - jan@fli-leibniz.de; T Michael Underhill - tunderhi@interchange.ubc.ca; Frank Beier* - fbeier@uwo.ca* Corresponding author    AbstractBackground: Glucocorticoids (GCs) are widely used anti-inflammatory drugs. While useful inclinical practice, patients taking GCs often suffer from skeletal side effects including growthretardation in children and adolescents, and decreased bone quality in adults. On a physiologicallevel, GCs have been implicated in the regulation of chondrogenesis and osteoblast differentiation,as well as maintaining homeostasis in cartilage and bone. We identified the glucocorticoid receptor(GR) as a potential regulator of chondrocyte hypertrophy in a microarray screen of primary limbbud mesenchyme micromass cultures. Some targets of GC regulation in chondrogenesis areknown, but the global effects of pharmacological GC doses on chondrocyte gene expression havenot been comprehensively evaluated.Results: This study systematically identifies a spectrum of GC target genes in embryonic growthplate chondrocytes treated with a synthetic GR agonist, dexamethasone (DEX), at 6 and 24 hrs.Conventional analysis of this data set and gene set enrichment analysis (GSEA) was performed.Transcripts associated with metabolism were enriched in the DEX condition along withextracellular matrix genes. In contrast, a subset of growth factors and cytokines were negativelycorrelated with DEX treatment. Comparing DEX-induced gene expression data to developmentalchanges in gene expression in micromass cultures revealed an additional layer of complexity inwhich DEX maintains the expression of certain chondrocyte marker genes while inhibiting factorsthat promote vascularization and ultimately ossification of the cartilaginous template.Conclusion: Together, these results provide insight into the mechanisms and major molecularclasses functioning downstream of DEX in primary chondrocytes. In addition, comparison of ourdata with microarray studies of DEX treatment in other cell types demonstrated that the majorityof DEX effects are tissue-specific. This study provides novel insights into the effects ofPublished: 1 July 2007BMC Genomics 2007, 8:205 doi:10.1186/1471-2164-8-205Received: 26 February 2007Accepted: 1 July 2007This article is available from: http://www.biomedcentral.com/1471-2164/8/205© 2007 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 27(page number not for citation purposes)pharmacological GC on chondrocyte gene transcription and establishes the foundation forsubsequent functional studies.BMC Genomics 2007, 8:205 http://www.biomedcentral.com/1471-2164/8/205BackgroundCartilage provides a scaffold for the deposition of osteob-last precursors and ultimately the development of longbones. This process, termed endochondral ossification,describes a coordinated developmental series thatinvolves commitment of mesenchymal precursor cells tothe chondrogenic lineage and subsequent alternatingphases of proliferation and differentiation, which culmi-nate in the replacement of the cartilage by bone tissue [1-4]. In the first phase of this process, multipotent mesen-chymal progenitors condense and initiate expression ofthe pro-chondrogenic Sox family members 9, 5 and 6[5,6]. A subset of cells at the center of these aggregates dif-ferentiates into chondrocytes. Newly formed chondro-cytes secrete an extracellular matrix rich in type II collagen(Col2a1), proliferate and ultimately terminally differenti-ate into hypertrophic chondrocytes [7]. Chondrocytehypertrophy precedes the end of the chondrocyte life cycleby apoptosis and is accompanied by vascularization of thehypertrophic template and mineralization of the cartilag-inous extracellular matrix [8-12]. Concomitantly, osteo-clasts degrade the calcified cartilage extracellular matrix,making way for the invasion and deposition of an osteo-progenitor population that form the primary ossificationcenter [13].These events take place in a region called the growth platethat illustrates the organization of different phases of car-tilage development into distinct zones. The resting zonedelineates newly differentiated chondrocytes with lowmitotic activity and the cellular reserve for subsequentstages of chondrocyte differentiation. Proliferative zonechondrocytes exhibit higher mitotic activity resulting indistinct columns containing cells reminiscent of stackedcoins. The hypertrophic zone demarcates terminally dif-ferentiated chondrocytes which are identified by highcytoplasm to nuclear ratio and the expression of type Xcollagen (Col10a1) [14-16]. Terminally differentiatedchondrocytes are fated for programmed cell death afterwhich primary ossification occurs by way of vasculariza-tion of the remaining cartilaginous matrix and the depo-sition of osteoprogenitor cells [17-19].Glucocorticoids (GC) are among various endocrine mole-cules including growth hormone (GH) and thyroid hor-mone (TH) known to regulate linear growth [20-23].Regulation of linear growth follows the paradigm inwhich steroid hormones affect target tissue through bothlocal and systemic mechanisms [24-27]. Indirect effectsoccur through modulation of other endocrine systemssuch as the GH/IGF-I axis. Generally, GC decrease IGF-I,GH receptor and IGF receptor 1 expression and also abro-gate the release of GH from the pituitary [20,28,29].(GR)-mediated gene transcription in chondrocytes[24,30,31].GC functions are primarily mediated by the glucocorti-coid receptor (GR) that is encoded by the Nr3c1 gene. TheGR is ubiquitously expressed in mammalian tissues,including the growth plate, and is essential for life [31-36]. Many studies have examined GC regulation of theskeleton and have led to various theories on potentialmodes of GC function in cartilage [37-40]. The specificfunction of the receptor in terms of its transcriptional reg-ulation in cartilage, however, remains enigmatic.While endogenous GCs have been shown to promote thedifferentiation of both chondrocytes and osteoblasts,exogenous GCs in pharmacological doses which are alsowidely used in clinical practice to treat inflammatory dis-orders [41-46]. Their have different effects. Indeed, theirutility in treating various diseases is, however, limited bynumerous side effects such as growth failure anddecreased bone quality [47]. GC-target genes including C-type natriuretic peptide and VEGF have been identified inchondrocytes [28,48,49]; however, the cartilage-specifictranscriptional consequences of high-GC-doses in thegrowth plate have not been studied comprehensively.Work in our laboratory identified GR amongst factors thatwere up-regulated during chondrocyte maturation [50]Thus, to comprehensively understand the transcriptionaleffects of pharmacological GC doses in growth plate, wecompleted a genomic screen of gene expression changesin chondrocytes derived from E15.5 day old mouseembryos. Primary monolayer chondrocytes were treatedwith a synthetic GC, dexamethasone (DEX), and RNA wasisolated for microarray analysis. We complemented tradi-tional microarray analysis methods with the gene setenrichment algorithm to correlate the behaviour of spe-cific molecular classes with DEX treatment [51,52].Results and DiscussionMicroarray screen of dexamethasone-treated primary chondrocyte monolayersWe identified the GR as a candidate for the regulation ofchondrocyte hypertrophy in a previous expression profil-ing screen using primary micromass cultures [50]. TheNr3c1 probe set which encodes the GR was up-regulated4-fold from day 3 to day 15 of micromass culture (Figure1A, top panel). Confirmation of the GR expression profilewith qRT-PCR showed an approximately 8-fold increaseover the same time course (Figure 1A, bottom panel).Studies in our laboratory and others have implicated GCsin chondrocyte differentiation and growth plate function[25,26,47,48,53,54]. In addition, our cell counting exper-Page 2 of 27(page number not for citation purposes)Direct regulation of growth occurs through GC receptor iments revealed that DEX consistently decreases cell num-bers after 24 hrs (Figure 1B), in agreement with otherBMC Genomics 2007, 8:205 http://www.biomedcentral.com/1471-2164/8/205studies that show increased apoptosis [38,55] andreduced proliferation [56] in response to GCs. We there-fore aimed at extending this analysis to examine pharma-cological effects of GCs on growth plate chondrocytes bysystematically identifying downstream effector genes ofDEX. Primary chondrocytes derived from the long bonesof 15.5 day old embryonic mice were treated with DEX orthe vehicle control, and total RNA was isolated after 6 and24 hrs of culture, respectively.Gene expression was evaluated using Affymetrix MOE 4302.0 mouse genome chips using three independent cell iso-lations. We first analyzed gene expression using conven-tional analysis functions in GeneSpring GX*. After pre-processing the data set using the GC-RMA algorithm andeliminating probe sets showing expression levels close tobackground, 22 091 probe sets remained, reducing thedata set by 48% (Table 1). Significance testing with one-Way ANOVA analysis identified probe sets differentiallyexpressed between DEX and vehicle-treated cultures overthe entire time course (Figure 1C, left panel). The resultinglist contained 1158 probe sets, which is 2% of the dataset's original size. Approximately 70% of significantlychanged probe sets exhibited upregulation in response toDEX treatment. This data set was further subdivided byusing 1.5-, 5- and 10- fold change filters which generatedlists of 162, 21 and 7 probe sets for the 6 hr time point and399, 53 and 19 probe sets for the 24 hr time point, respec-tively (Table 1). Examination of the overall differencesbetween the mean normalized signal intensities associ-ated with each condition showed minimal changes ingene expression (Figure 1C, right panel), indicating thatGC treatment affects the expression of only a small subsetof all expressed genes in this system. A distribution of folddifferences between 6 and 24 hrs showed that the majorityof gene expression changes did not exceed 2-fold (Figure1D). In each case, both time points exhibited the sameoverall trends in gene expression, but, as expected, the 24hr time point consistently showed a higher proportion ofprobe sets altered by DEX treatment.Probe set validationTo confirm the accuracy of the microarrays in identifyingbiologically significant differences, we selected a variety ofexpressed transcripts for qRT-PCR analysis (Figure 2A).Transcripts that either belonged to a functional classimplicated in cartilage development or exhibited markedchanges with DEX treatment were chosen. Markers exhib-iting marginal changes in gene expression were alsoselected for control purposes. Specifically, we evaluatedthe expression patterns of Indian hedgehog (Ihh), Tissueinhibitor of matrix metalloproteinase 4 (Timp4), Cyclin-dependent kinase inhibitor 1C (Cdkn1c), which contains1 (Itgb1) and Kruppel-like factor 15 (Klf15) over 0, 6, 12,and 24 hrs of culture with or without DEX treatment.Transcripts for Klf15 were up-regulated from 0 to 6 hrswhile Ihh, Timp4, Cdkn1c and Itgbl1 all increased after the6 hr time point. Nr3c1, which encodes the GR, was notaffected by DEX-treatment at both 6 and 24 hrs, but doescontain a putative GRE [58]. Transcripts such as Itgb1 thatexhibited less than 1.5-fold change in our arrays were alsoconfirmed with qRT-PCR, providing further evidence thatthe microarray data represented authentic gene expressiondata. Interestingly, the fold change difference variedaccording to the experimental method. In cases such asTimp4 and to a lesser extent Cdkn1c, qtPCR data showedhigher fold change increases with the DEX treatment thanin microarrays. In contrast, the expression pattern forKlf15 exhibits a higher fold-change difference in themicroarrays compared to the control. While data normal-ization using the RMA algorithm provides excellent esti-mates of reliable signal intensities, other methods such asthe M.A.S. 5.0 algorithm are known to outperform RMAin its ability to accurately estimate fold change differencesin transcript levels [59].GSEA to identify the effects of dexamethasone on gene expression in chondrocytesTraditional microarray analysis methods are useful for theidentification of probe sets exhibiting transcriptionalresponses to DEX-treatment, but are limited in certaincapacities. Alternate statistical methods such as ANOVAtesting produced transcript lists that, while effectivelyreducing the dimensionality or sample size of the data set,increased the rate of false negative data thus hamperingour ability to generate meaningful hypotheses from thedata (Figure 1). Also, the overall effect of DEX treatmenton gene expression was modest, which may have reducedthe significance of biologically relevant genes becausetheir signal intensities were close to background levels.Accordingly, we did not have a clear concept of the centralpathways and biological categories affected by DEX treat-ment. Similarly, Gene Ontology annotations were not suf-ficiently robust to detect differences in the representationof specific molecular categories (data not shown). Wetherefore implemented GSEA [52], an algorithm that isdesigned to effectively evaluate the effect of a specificexperimental condition on known biological pathwaysand functional categories. These analyses show whether agiven treatment (e.g. DEX stimulation) results in enrich-ment of genes sets involved in the regulation of a specificphenotype (see materials and methods for details).We created a gene set consisting of 77 gene lists represent-ing different tissue types, functional categories and path-ways derived from other microarray studies in thePage 3 of 27(page number not for citation purposes)a GC response element in its promoter [57], Integrin betalike 1 protein (Itgbl1), GC receptor (Nr3c1), Integrin betaliterature (Table 2). We drew conclusions from the topgene sets that had a false discovery rate (FDR) less thanBMC Genomics 2007, 8:205 http://www.biomedcentral.com/1471-2164/8/205Page 4 of 27(page number not for citation purposes)Gene expression changes in DEX-treated primary chondrocytesFigur  1Gene expression changes in DEX-treated primary chondrocytes. Microarray and quantitative RT-PCR expression profiles of the Glucocorticoid receptor (Nr3c1) in primary mesenchymal micromass cultures (A). Primary chondrocytes are plated in high density monolayers and treated with DEX or vehicle for 24 hrs and counted with a hemocytometer (B). Ordered list of global microarray data set derived from the hybridization of RNA isolated from primary chondrocytes treated with 10-7 M DEX and the vehicle (v) control (C, left panel). One-Way ANOVA testing for significantly expressed probe sets between DEX-treated samples and the vehicle control resulted in a list of 1158 transcripts. Mean normalized signal intensities for all 1158 probe sets are shown (C, right panel). Fold change filtering of these transcripts reveal that the majority of probe sets vary in the range of 1 to 2-fold (D).$ %&         ++)ROGFKDQJHVWH6HERU3IRUHEPX1 YHKLFOH'(;KRXUV\WLVQHWQ,ODQJL6QDH0'(;KYK'(;KYK1RUPDOL]HG6LJQDO,QWHQVLW\'D\QRLVVHUS[HQLHJQDKFGOR)T573&5$UUD\    1UFYHKLFOH '(;7UHDWPHQW&HOOQXPEHU;'BMC Genomics 2007, 8:205 http://www.biomedcentral.com/1471-2164/8/20525% and a p-value less than 0.001, both of which areacceptable cut-offs for the identification of biologicallyrelevant probe sets. This cut-off, although relatively high,was optimized to reduce the occurrence of false negativedata in data sets interrogating a small number of gene sets.Additionally, the FDR compensates for the inherent lackof coherence microarray data sets exhibit between geneexpression and specific experimental conditions [52].Enriched gene sets were identified in both DEX and vehi-cle data (Table 3). Specifically, the highest statistical con-fidence and correlation with the DEX phenotype wasassigned to metabolism and extracellular matrix, whichcontained 196 and 228 genes, respectively (Figure 3, leftpanels, Table 4 and 5). In each case, the expression ofgenes positively correlated with the DEX phenotype at the24 hr time point exceeded the number of genes at the 6 hrtime point (Figure 3, right panels). Metabolic genesincluded aldehyde and alcohol dehydrogenases (Table 4),among others, and were identified in accordance with pre-viously documented roles for GC in various metabolicprocesses and tissues [60,61]. Closer examination of thegenes contributing to the enrichment scores for the ECMgene set revealed that Dentin matrix protein 1 (Dmp1) wasthe top ranking gene (Table 5). DMP1 belongs to the SIB-LING family of matrix molecules and has been linked tochondrocyte differentiation. Dmp1 knockout mice displaydisordered postnatal chondrogenesis, among other skele-tal abnormalities [62]. Interestingly, integrin binding sia-loprotein (Ibsp) [63-66]), another SIBLING familymember, and osteocalcin (Bglap2) both contain putativeGRE sequences, but did not contribute to the enrichmentscore for this category [63,66]. They did, however, belongto the core group of genes that were enriched when amicromass culture gene set was used to interrogate theDEX data (Figure 4).Osteomodulin, an additional matrix molecule shown toexpressed in terminally differentiated chondrocytes suchas collagen 10 (Col10a1) and osteonectin (Spock1) wereidentified, suggesting that this molecular classification isimportant for transmitting GC signaling in the growthplate.Interestingly, the normalized enrichment scores for fac-tors down-regulated by DEX treatment were higher thanthose positively correlated with DEX, but contained fewerprobe sets contributing to the scores. Gene sets composedof 127 and 106 genes associated with cytokine and growthfactor activity, respectively, were negatively correlatedwith DEX treatment (Figure 4, Table 6, 7). In other stud-ies, cytokines such as Il-8 and GROα were found to pro-mote the hypertrophy of osteoarthritic cartilage, andexcess interleukins 1β(IL-1β), interleukin 6 (IL-6) andTumor Necrosis Factor alpha (TNF-α) cause growth fail-ure in children [68-70]. Our studies identified three mem-bers of the GP-130 family of cytokines, namelyinterleukins -11,-6 (Il11, Il6) and leukemia inhibitory fac-tor (Lif), as part of the core enrichment group forcytokines (Table 6). Transgenic mice overexpressing Il-6exhibit growth retardation, and LIF is thought to regulatethe rate at which terminally differentiated cartilage is cal-cified and vascularized [71,72].This group also contained the gene encoding Tumornecrosis factor (ligand) superfamily, member 11 (Tnfsf11,RANKL), which has been localized to mature chondro-cytes and is thought to promote degradation of the calci-fied cartilage ECM and ultimately endochondralossification through activation of osteoclasts [73-75]. It isimportant to note that several independent gene sets con-nected to inflammation such as cytokines, chemokinesand interleukins exhibit some overlap and showed similarenrichment patterns, which provides additional confir-mation that DEX is indeed downregulating inflammatoryTable 1: Microarray analysis of DEX-treated primary chondrocyte monolayers.Specifications Probe sets at 6 hrs Probe sets at 24 hrsTotal number of probe sets 45101 45101Significantly expressed 22091 22091Differentially expressed 1158 11581.5-fold changed 162 3995-fold changed 21 5310-fold changed 7 331.5-fold up-regulated 141 3425-fold up-regulated 20 5010-fold up-regulated 7 191.5-fold down-regulated 21 575-fold down-regulated 1 310-fold down-regulated 0 0Page 5 of 27(page number not for citation purposes)be structurally similar to IBSP [67], ranked second in thelist of enriched ECM genes. Additional ECM moleculesmolecules in chondrocytes. GC have been previouslyreported to down-regulate the expression of VEGF, one ofBMC Genomics 2007, 8:205 http://www.biomedcentral.com/1471-2164/8/205Page 6 of 27(page number not for citation purposes)Identification of significantly expressed probe sets and subsequent validation with real-time RT-PCRFigure 2Identification of significantly expressed probe sets and subsequent validation with real-time RT-PCR. Expression profiles for selected transcripts in vehicle- or DEX-treated chondrocytes are confirmed with real-time RT-PCR at 0, 6, 12 and 24 hr time points. Indian hedgehog (Ihh), tissue inhibitor of matrix metalloproteinase 4 (Timp4), cyclin-dependent kinase inhibi-tor 1C (Cdkn1c, p57), integrin beta like 1 protein (Itgbl1), glucocorticoid receptor (Nr3c1), integrin beta 1 (Itgb1) and kruppel-like factor 15 (Klf15) microarray data are shown on the left at the 6 and 24 hr time points and corresponding real-time expres-sion values are shown on the right. P-values less than 0.01 are deemed significant. Specifically, Ihh, Timp4, Itgbl1 and Klf15 exhibit significant differences between the 6 and 24 hr time point and between treatments. Dotted lines indicate the control and solid lines denote DEX treatment.5HDOWLPH3&5H[SUHVVLRQSURILOH,KK7LPS&GNQF,WJEO1UF,WJE.OI   KRXUV0LFURDUUD\H[SUHVVLRQSURILOH7LPS,KK1UF,WJE.OI&GNQF ,WJEO KRXUV,KKQRLVVHUS[(QLHJQDK&GOR)QRLVVHUS[(QLHJQDK&GOR)BMC Genomics 2007, 8:205 http://www.biomedcentral.com/1471-2164/8/205the central growth factors involved in vascularization ofcalcified cartilage matrix [49], in agreement with our data(Table 7). Since some of these factors, such as RANKL,VEGF and LIF, promote normal tissue remodeling proc-esses during endochondral ossification, our data suggestthat DEX prevents the replacement of hypertrophic carti-lage by bone. GC have been shown to delay chondrocytematuration while retaining their capacity to re-engage intheir developmental program [21]. This could account forupregulation of genes typically associated with thechondrocyte phenotype, such as ECM genes and the coor-Identification of cartilage-specific dexamethasone-effectsIdentification of cartilage-specific gene sets affected byDEX treatment provided further insight into the complexnature of GC functions in cartilage. We knew from otherstudies that DEX effects on chondrogenic differentiationare dependent on cell source, experimental system andDEX concentration [40,42,76-78]. We aimed to systemat-ically characterize the effects of DEX on growth platechondrocytes. To ensure that our DEX data set wasexpressing bona fide cartilage markers, we compared theDEX data to our previously generated micromass cultureTable 2: Gene sets used in GSEA.Category name Number of genes Category name Number of genesAdipose 70 Nucleus_3 510Apoptosis 39 Fkbp 33Bone 116 3vs15_1.5x_1 497Cartilage 28 3vs15_1.5x_2 497Catalytic 245 3vs15_1.5x_3 497Chaperone 81 3vs15_1.5x_4 497Chemokine 31 3vs15_1.5x_5 76Chromatin/Hdacs 24 Igf 48Cyclin 225 Cart_2 299Cytokine 127 Cart_3 3521_Dnabind 500 Liver_1 2602_Dnabind 448 Liver_2 260Ecm 228 Blood 111Electron_Transp 40 Protease_1 269Gf Receptor 327 Protease_2 269Gluconeogen 31 Phosphatase 473Growth Factor 106 Dusp 20Gtpase Activator 46 Kinase_1 499Gtpase Activ 73 Kinase_2 499Heparin Bind 37 Kinase_3 227Hormone 75 Integrin_Rel 173Muscle 198 Brain_Rel 379Neg_Apoptosis 50 Hepatocyte 19Oncogene 154 Obl_Oclast 16Pos_Apoptosis 79 Interleukinrelated 175Related_Apoptosis 311 Rgs_Related 44Structure 151 Caspase_Related 47Sugar_Bind 104 Creb_Atf3 32Tf_Activ 56 Nuclear Receptor 138Tf_Repress 55 Nuc_Hormone_Receptor 55Tgfb 45 Mapkrelated 267Tnf_Receptor 69 Membrane 260Tumor Suppressor 48 Metabolism 196Wnt 53 Nucleus_1 494Actin_Cytoskel 38 Nucleus_2 494Angiogen 57 Pzhorton.Farnum 413Bmprelated 62 Hzhorton.Farnum 407Cytoplasm 411Erk_Related 40Fgf_Related 64Page 7 of 27(page number not for citation purposes)dinated downregulation of factors that promote the tran-sition from cartilage into bone.data set [50]. We compared all expressed probe sets in theDEX array to probe sets exhibiting a minimum 1.5-foldBMC Genomics 2007, 8:205 http://www.biomedcentral.com/1471-2164/8/205change in expression between days 3 and 15 of micromasscultures that encompass the various stages of the chondro-cyte life cycle. Day 3 of micromass culture likely coincideswith the onset of the cartilage developmental programand early chondrogenesis. After 15 days of culture, the cellpopulation is comprised primarily of terminally differen-tiated chondrocytes and thus corresponds mostly to thehypertrophic zone of the growth plate [50,79], althoughsmall numbers of other cells are present at all stages. Outof the 2119 probe sets displaying at least 1.5-fold changesin expression in the micromass culture data set (a probeset list generated from the pair-wise comparison of day 3versus day 15 of micromass culture), 1730 were alsoexpressed in the DEX array. This shows that our primarychondrocyte monolayers do exhibit prototypicalchondrocyte gene expression patterns in both the pres-To complete more robust classification of the data inwhich we could correlate chondrocyte gene expression tothe DEX phenotype, we created a gene set from this list of2119 probe sets (Table 8, 9). The micromass derived genelist was enriched in this study; however, the list was foundto correlate both positively and negatively with differentaspects of the DEX phenotype. We therefore proceeded toevaluate both the micromass (MM) data set and the DEXdata set using GSEA analysis and the previously createdgene sets. If both the micromass time course and the DEXdata sets show the same enrichment pattern, we wouldhave evidence to suggest that pharmacological DEX dosespromote chondrocyte differentiation. Normalized enrich-ment scores for gene sets common to both culture meth-ods were therefore compared to identify differences andsimilarities between DEX-treated chondrocytes and theTable 3: GSEA of DEX-treated primary chondrocytes.Gene set name Size ES NES NOM p-val FDR q-valMetabolism 196 0.471 1.935 <0.001 0.016Extracellualr Matrix 228 0.451 1.878 <0.001 0.016Fkbp 33 0.559 1.696 0.011 0.054Integrin_Related 173 0.407 1.643 <0.001 0.001Angiogenesis 57 0.479 1.610 0.012 0.065Kinase_1 499 0.343 1.549 <0.001 0.092Tumor Suppressor 48 0.457 1.492 0.037 0.126Catalytic 245 0.337 1.420 0.008 0.172Hepatocyte 19 0.529 1.406 0.104 0.161D3 Vs D15_2 497 0.304 1.368 0.004 0.194Igf 48 0.412 1.348 0.093 0.208Cyclin 224 0.322 1.344 0.028 0.199Actin_Cytoskel 38 0.426 1.325 0.124 0.213Structure 151 0.332 1.312 0.053 0.219Cytoplasm 411 0.292 1.300 0.023 0.224Adipose 70 0.368 1.285 0.116 0.232Gtpase Activity 73 0.363 1.280 0.113 0.230Cartilage 28 0.432 1.262 0.169 0.246Chemokine 31 -0.779 -2.40 <0.001 0Cytokine 127 -0.579 -2.31 <0.001 0Growth Factor 106 -0.517 -2.01 <0.001 7.698E-04Interleukinrelated 175 -0.469 -1.98 <0.001 9.475E-04Bone 16 -0.577 -1.51 0.051 8.945E-02Creb_Atf3 30 -0.469 -1.43 0.065 1.300E-01Dusp 20 -0.508 -1.40 0.102 1.418E-01Blood 111 -0.351 -1.37 0.037 1.425E-013vs15_1.5x_3 496 -0.288 -1.35 0.002 1.518E-01Protease_2 268 -0.306 -1.35 0.015 1.411E-01Nuc_Hormone_Receptor55 -0.381 -1.32 0.086 1.570E-01Tf_Repress 55 -0.380 -1.32 0.091 1.498E-013vs15_1.5x_4 497 -0.272 -1.28 0.011 1.817E-01Erk_Related 40 -0.385 -1.25 0.157 2.169E-01ES, enrichment scoreNES, normalized enrichment scoreFDR q-val, false discovery rate and multiple testing corrections (q-value)NOM p-val; the uncorrected p-valuePage 8 of 27(page number not for citation purposes)ence and absence of DEX treatment. chondrocyte phenotype (Figure 4B).BMC Genomics 2007, 8:205 http://www.biomedcentral.com/1471-2164/8/205Four different patterns were observed when comparingDEX treatment and micromass differentiation data sets forgene enrichments scores (Figure 4B). First, similar genesets were indeed enriched in both day 15 micromass andDEX-treated monolayer cultures, and core genes contrib-uting to the normalized enrichment scores were similarlyoverlapping between the two data sets in results with lowFDR. For example, ECM genes were enriched with bothDEX treatment and the day 15 micromass phenotype.Other gene sets following this enrichment patternincluded genes involved in integrin function, angiogen-esis, catalytic activity, IGF related, adipocyte and cartilage,all of which have a precedent for being involved inchondrocyte maturation [28,49,80,81]. The enrichmentof angiogenic transcripts with DEX treatment was unex-genes contributing to the enrichment score, Vegf, which isthought to be a central angiogenic factor in endochondralossification [82], was excluded from the core enrichmentgenes and had the lowest correlation with the DEX pheno-type in that gene set. In contrast, Vegf was enriched in thegrowth factor data set which positively correlated with thevehicle control and not DEX treatment (Table 7).Gene sets associated with the actin cytoskeleton, tumoursuppressors, structure, cytoplasmic genes, hepatocytemarkers and dual specificity phosphatases (DUSPs) wereenriched in the DEX data set and the phenotype positivelycorrelated with day 3 of micromass culture. The identifica-tion of DUSPs was particularly interesting since DEX hasbeen shown to induce genes encoding for these proteinsTable 4: Metabolic transcripts enriched in DEX-treated chondrocytes. I.HUGO symbol Rank RMS* RES** HUGO symbol Rank RMS* RES**Aldh1a1 26 0.417 0.053 Slc27a4 1616 0.058 0.426Eya2 40 0.355 0.099 Ltbp2 1721 0.056 0.428Vcl 106 0.228 0.125 Hsd17b1 1783 0.055 0.432Adhfe1 116 0.222 0.154 P4ha2 1783 0.055 0.432Ids 123 0.212 0.181 Mut 1850 0.053 0.443Cbr3 133 0.204 0.207 Pde3a 2195 0.048 0.432Aldh6a1 202 0.165 0.225 Sulf2 2200 0.048 0.438Bcat2 224 0.157 0.245 Prep 2316 0.046 0.438Pmm1 278 0.145 0.261 Plod3 2387 0.045 0.441Pcx 553 0.105 0.261 1110013G13RIK2510 0.043 0.440Fthfd 554 0.105 0.275 Pld1 2669 0.041 0.437Atp1a1 560 0.104 0.288 Au041707 2721 0.040 0.440Gstm1 619 0.099 0.298 Decr1 2837 0.039 0.439Gstm2 742 0.088 0.303 Gstm5 2872 0.038 0.4431700061G19RIK787 0.086 0.312 Bckdha 2932 0.038 0.445Slc38a4 833 0.084 0.321 Atp11a 2951 0.038 0.449Pyp 847 0.083 0.331 Gstp1 2967 0.037 0.453Aacs 901 0.080 0.339 Dhrs7 3014 0.037 0.455Plod1 934 0.079 0.348 Cbr2 3147 0.035 0.453Acas2 983 0.077 0.355 Echdc3 3152 0.035 0.458Auh 1068 0.074 0.361 Acy3 3254 0.035 0.457Gcat 1109 0.072 0.368 Dhrs1 3483 0.032 0.450Dhrs8 1184 0.070 0.373 Itgb1 3527 0.032 0.452Egln3 1232 0.068 0.380 4933406E20RIK 3553 0.031 0.454Mthfs 1268 0.067 0.387 Plod2 3574 0.031 0.458Mvk 1298 0.066 0.394 Pmm2 3582 0.031Aup1 1325 0.065 0.401 Ugp2 3583 0.031Spr 1456 0.062 0.403 Gnpat 3633 0.031Sc5dl 1462 0.062 0.411 1110003P22RIK 3636 0.0311300018J18RIK 1516 0.061 0.416 Dbt 3710 0.030Agpat3 1524 0.061 0.423Rank = position of genes in the context of the ranked list of array genesRMS = the ranked metric scoreRES = the running enrichment scorePage 9 of 27(page number not for citation purposes)pected since DEX was shown to have anti-angiogenic rolesin cartilage; however, upon closer examination of the[77,83,84]. DUSPS counteract the activation of MAPkinase pathways, known regulators of chondrocyte differ-BMC Genomics 2007, 8:205 http://www.biomedcentral.com/1471-2164/8/205entiation [85], and are thought to mediate DEX's anti-inflammatory functions and to influence hepatic glucone-ogenesis [83,86,87].Additional comparisons identified genes that showenrichment in day 15 micromass cultures and downregu-lation with DEX treatment. These include the previouslyidentified chemokines, cytokines and interleukins. A finaltrend in similarly enriched gene sets identified lists thatwere negatively correlated both with the DEX phenotypeand day 15 of micromass cultures. Only transcriptionalrepressors and molecules involved in the extracellular sig-nal-regulated kinase (ERK) pathway were identified. Thispattern is consistent with DEX's anti-proliferative func-tions, as another study showed that DEX decreases ERKphosphorylation and thus cell cycle progression in a pre-osteoblast cell line [77]. Altogether this analysis showsthat DEX regulation of growth plate chondrocyte differen-tiation is multifaceted. The patterns identified here are inagreement with a dual role of DEX in maintenance of thecartilage phenotype and delay in the cartilage-to-bonetransition, as we suggested above.responsive genes identified in alternate studies, in differ-ent cell types [88]. Out of a total of twelve microarraystudies evaluating the transcriptional effects of DEX treat-ment on a specific tissue or cell type, only ten genes werecommon to at least three of the chosen DEX studies. Spe-cifically, bone morphogenetic protein 2 (Bmp2), deltasleep inducing factor 1 (Dsip1), beta-2 microglobulin(B2m), neuroepithelial cell transforming gene 1 (Net1),TNFAIP3 interacting protein 1 (Tnip1), bone marrow stro-mal cell antigen 2 (Bst2), B-cell leukemia/lymphoma 6(Bcl6), nuclear factor of kappa light chain gene enhancerin B-cells inhibitor, alpha (Nfkbia), FK506 binding pro-tein 5 (Fkbp5) and B-cell translocation gene 1, anti-prolif-erative (Btg1) were identified. It therefore appears thatwhile DEX affects similar functional categories across var-ious species, tissue types and experimental conditions, theindividual genes that respond to DEX treatment are varia-ble. These results also reinforce the paradigm that GC reg-ulation is inextricably linked to its physiological context[88-99].Analyses of GC response elements in dexamethasone target genes in chondrocytesTable 5: ECM-related transcripts enriched in DEX-treated chondrocytes.HUGO symbol Rank RMS RES HUGO symbol Rank RMS* RES**Dmp1 18 0.470 0.036 Matn4 882 0.081 0.420Omd 27 0.409 0.068 Lama3 886 0.081 0.427Itga5 38 0.358 0.095 Nyx 992 0.077 0.427Adamts1 57 0.305 0.118 Lamb2 1082 0.073 0.429Timp4 61 0.296 0.141 Bsg 1100 0.072 0.433Col4a1 86 0.268 0.161 Fbn2 1242 0.068 0.432Col4a2 98 0.247 0.180 Ntn4 1245 0.068 0.437Adam12 112 0.225 0.197 5730577E14RIK 1381 0.064 0.435Prelp 139 0.200 0.211 Col6a2 1405 0.064 0.439Postn 142 0.195 0.227 Ntn3 1415 0.063 0.443Chad 176 0.174 0.239 Tgfb2 1531 0.060 0.442Mgp 195 0.168 0.251 Mia1 1575 0.059 0.445Col1a1 232 0.154 0.261 Mmp14 1803 0.054 0.438Mfap5 233 0.153 0.273 Col15a1 1845 0.053 0.440Col10a1 266 0.146 0.283 Ctgf 1882 0.052 0.442Smoc2 279 0.145 0.294 Col6a1 1942 0.052 0.443Aspn 294 0.141 0.304 Gpld1 1946 0.051 0.447Col4a5 367 0.128 0.310 Emid2 2043 0.050 0.446Adamts15 385 0.126 0.319 Col7a1 2047 0.050 0.450Tgfb1 394 0.125 0.329 Adam10 2107 0.049 0.451Sparcl1 440 0.119 0.336 Col9a2 605 0.100 0.370Adam17 483 0.112 0.343 Matn3 610 0.099 0.377Lama5 508 0.110 0.350 Col11a2 636 0.097 0.384Lamc1 517 0.109 0.358 Hapln1 650 0.096 0.391Spock2 581 0.102 0.363 Lama2 685 0.092 0.396Lama1 688 0.092 0.403 Gpc3 796 0.086 0.412Ltbp4 704 0.091 0.410 Lama4 827 0.084 0.417*RMS = the ranked metric score**RES = the running enrichment scorePage 10 of 27(page number not for citation purposes)We also wanted to determine whether DEX target genesidentified in the current study were similar to DEX-Classical genomic GC action is thought to be mediated bya cytoplasmic GR that modifies transcription upon bind-BMC Genomics 2007, 8:205 http://www.biomedcentral.com/1471-2164/8/205Page 11 of 27(page number not for citation purposes)Enrichment plots for statistically significant gene sets identified by GSEAFigure 3Enrichment plots for statistically significant gene sets identified by GSEA. User-defined gene sets enriched with the DEX or vehicle conditions are depicted. Black bars illustrate the position of probe sets belonging to metabolic, extracellular matrix (A), cytokine and growth factor (B) gene sets in the context of all probes on the DEX array. The running enrichment score (RES) plotted as a function of the position within the ranked list of array probes is shown in green. The ranked list metric shown in gray illustrates the correlation between the signal to noise values of all individually ranked genes according and the class labels (experimental conditions). Metabolic and ECM genes are overrepresented in the left side of the enrichment plot indicating correlation to differential expression in DEX-treated chondrocytes. In contrast, cytokines and growth factor genes are enriched in the right side of the plots and correspond to the vehicle control. Significantly enriched data sets are defined according to GSEA default settings i.e., a p < 0.001 and a false discovery rate (FDR) < 0.25. Individual expression profiles for probe sets contributing to the normalized enrichment score are shown in the right panel. R.L.M = ranked list metric, E.S. = enrichment score.$%(QULFKPHQW3ORW5DQNLQJPHWULFVFRUHV+LWV(QULFKPHQWSURILOH([SUHVVLRQ3URILOH\WLVQHWQ,ODQJL6GH]LODPUR1'(;KYK'(;KYK0HWDEROLVP'(;KYK'(;KYK(&0'(;KYK'(;KYK&\WRNLQH'(;KYK'(;KYK*URZWK)DFWRU'(;YHKLFOH6(0/5'(;YHKLFOH6(0/5'(;YHKLFOH=(52&5266$76(0/55DQNLQ2UGHUHG'DWDVHW6(0/5'(;YHKLFOHBMC Genomics 2007, 8:205 http://www.biomedcentral.com/1471-2164/8/205Page 12 of 27(page number not for citation purposes)Comparison of DEX-treated primary chondrocytes to a time course of chondrocyte differentiation in micromass cultureFigure 4Comparison of DEX-treated primary chondrocytes to a time course of chondrocyte differentiation in micro-mass culture. The Venn diagram depicts probe sets that are common between the list of 2119 probe sets differentially expressed between days 3 and 15 of micromass culture and the list of 22 091 significantly expressed probe sets in primary chondrocyte monolayer cultures (A). The matrix of 77 user-defined gene sets are used to interrogate microarray data from days 15 and day 3 of micromass culture. Normalized enrichment scores (NES) generated from this analysis are then compared to NES scores derived from the DEX study to evaluate similarities in the regulation of different groups of genes in chondro-cytes (B). Positive enrichment scores (ES) indicate gene sets that are enriched and up-regulated in DEX-treated chondrocytes or d15 of micromass culture. Negative ES indicate gene set enrichment and down-regulation in the DEX-treatment or up-reg-ulation in the day 3 samples of the micromass (MM) culture data set. '(;00$%(&0,17(*5,15(/$7('<26+,'$$1*,2*(1(6,67802568335(6625&$7$/<7,&$&7,9,7<,*)$&7,1B&<726.(/(7216758&785(&<723/$60$',326(&$57,/$*(&+(02.,1(&<72.,1(,17(5/(8.,15(/$7('52=6$'8$/63(&,),&,7<3+263+$7$6(6%/22'75$16&5,37,21$/5(35(66256(5.3$7+:$<%21(52*$76.<+(3$72&<7(*HQH6HWVHURF6WQHPKFLUQ(GH]LODPUR1           '(;00BMC Genomics 2007, 8:205 http://www.biomedcentral.com/1471-2164/8/205ing its cognate ligand and translocating to the nucleus. Inthe nucleus, the GR binds a GRE sequence. GR can bothactivate and repress transcription, depending on the GREvariant present in the regulatory regions of GC targetgenes [100]. Binding to composite GREs involveshomodimerization of the GR to bind a non-palindromicconsensus sequence comprised of two GR binding sitesand is generally associated with transcriptional activation.In some instances, however, GR can function to blockaccess or activity of transcription factors within promoterof composite GRE half-sites, termed negative GREs, sincethey have documented roles in transcriptional repression.Variations on the genomic functions of GC include tran-scriptional regulation at the level of protein-protein inter-actions between the GR and other transcription factors,co-activators or co-repressors. In addition to the GRE-dependent roles, the GR is capable of interacting withother co-activators and repressors to influence transcrip-tion indirectly [102,103].Table 6: Cytokine transcripts enriched in vehicle-treated chondrocytes. I.HUGO gene symbol Rank RMS RESCklfsf2b 16971 -0.0424 -0.574Il7 16981 -0.0425 -0.569Il1f9 17007 -0.0427 -0.566Grn 17130 -0.0439 -0.567Il1f6 17153 -0.0442 -0.563Ifna2 17418 -0.0468 -0.571Tslp 17503 -0.0477 -0.570Il17 17568 -0.0483 -0.568A730028g07rik 17606 -0.0486 -0.564Cxcl11 17634 -0.0490 -0.560Ctf1 17857 -0.0519 -0.565Lta 17864 -0.0519 -0.559Il1a 18018 -0.0539 -0.561Ccl20 18038 -0.0542 -0.556Ccl17 18334 -0.0584 -0.564Ccl12 18384 -0.0592 -0.560Cklf 18618 -0.0639 -0.564Ifna11 18855 -0.0688 -0.568Cklfsf6 18874 -0.0693 -0.561Il15 18955 -0.0719 -0.557Ltb 19146 -0.0779 -0.558Ccl3 19220 -0.0814 -0.552Tnfsf9 19228 -0.0816 -0.543Cx3cl1 19523 -0.0975 -0.547Gdf15 19660 -0.1100 -0.541Bmp5 19775 -0.1238 -0.533Cxcl14 19798 -0.1289 -0.519Cxcl1 19928 -0.1698 -0.507Cxcl10 19951 -0.1807 -0.487Ccl7 19956 -0.1849 -0.466Gdf5 19973 -0.2066 -0.444Cxcl12 19978 -0.2104 -0.420Areg 19983 -0.2189 -0.395Cxcl2 19996 -0.2421 -0.369Ppbp 20014 -0.2944 -0.336Lif 20024 -0.3296 -0.299Ccl2 20030 -0.3589 -0.258Il11 20035 -0.4036 -0.213Cxcl5 20039 -0.5406 -0.152Tnfsf11 20041 -0.5835 -0.085Il6 20043 -0.7529Rank = position of genes in the context of the ranked list of array genesRMS = the ranked metric scoreRES = the running enrichment scorePage 13 of 27(page number not for citation purposes)regions of certain genes, thus impeding transcription[101]. GR are also able to bind a modified GRE consistingThe 100 most highly expressed probe sets with greatestenrichment in the DEX or vehicle-treated chondrocytesBMC Genomics 2007, 8:205 http://www.biomedcentral.com/1471-2164/8/205are shown in Figure 5. Probe sets identified in this analysisincluded both known cartilage markers and establishedDEX target genes such as Vegf, Ibsp, Bglap2 and Fkbp5[49,63-66,104,105]. We examined the proximal promoterregions of three separate gene lists, the top 100 DEX-responsive transcripts generated by GSEA analysis (Figure5), the 22 091 probe sets deemed expressed in primarychondrocyte cultures and the 1158 transcripts deemeddifferentially expressed between DEX and vehicle treatedcultures by one-Way ANOVA. Specifically, we searchedthe 9990 base pairs upstream regulatory regions in this listfor the composite GRE consensus sequence. We identifiedputative GRE sequences in many genes, including Fkbp5,pyruvate dehydrogenasekinase (Pdk4), RANKL (Tnfsf11),Interleukin 6 (Il6) and prostaglandin I2 synthase (Ptgis)(bold in Figure 5). However, the majority of DEX-regu-lated probe sets such as prostaglandin-endoperoxide syn-thase 2 (Ptgs2), phosphodiesterase 4A (Pde4), Vegf, Periodhomolog 1 (Per1) and Krüppel like factor 15 (Klf15) donot appear to contain a GRE in the first 10 kilobases andmay by regulated by DEX via a GRE-independent mecha-nism, through a GRE that deviates from the consensusGRE sequence or through GREs at other locations in thegene.sensus GRE. Consequently, we cannot exclude the pres-ence of less conventional GRE loci in the transcripts, orthe presence of GREs that deviate from the consensussequence or are located outside the queried sequence.Since many of the genes affected at the 6 hr time pointencode transcription factors, it is likely that a large propor-tion of the genes that only change after 24 hrs are regu-lated indirectly by DEX, through altered expression ofthese transcription factors and other regulatory proteins(e.g. phosphatases and cytokines, as discussed above).Functional analysis is required to unequivocally evaluatethe contribution of GRE-dependent mechanisms to GCregulation in chondrocytes. In addition to the genomicfunctions of GC, non-genomic modes of GC regulationhave been documented. Non-genomic mechanisms arethought to occur through specific and non-specific mech-anisms. Specific non-genomic GC regulation occursthrough the classical GR and its cytoplasmic heteropro-tein complex or non-classical GRs such as membrane GR[106-109]. Conversely, non-specific non-genomic mecha-nisms rely on the physiochemical properties of GC andthe phospholipid bilayer (Buttgereit and Scheffold,2002). Further, studies in which candidate molecules areselected and characterized in depth are imperative to dis-Table 7: Growth factor transcripts vehicle in DEX-treated chondrocytes.HUGO gene symbol Rank RMS RESFgf21 18968 -0.073 -0.508Nrg3 19132 -0.077 -0.506Fgf5 19190 -0.080 -0.499Ereg 19507 -0.096 -0.502Fgf7 19581 -0.102 -0.493Gdf15 19660 -0.110 -0.483Igf1 19679 -0.111 -0.469Bmp5 19775 -0.124 -0.458Nov 19848 -0.144 -0.443Vegf 19877 -0.150 -0.425Ptn 19885 -0.153 -0.406Cxcl1 19928 -0.170 -0.386Bdnf 19939 -0.176 -0.364Inhba 19971 -0.204 -0.340Gdf5 19973 -0.207 -0.313Cxcl12 19978 -0.210 -0.287Areg 19983 -0.219 -0.259Hbegf 20006 -0.264 -0.226Ngfb 20013 -0.287 -0.189Lif 20024 -0.330 -0.148Il11 20035 -0.404 -0.096Il6 20043 -0.753 0.000Rank = position of genes in the context of the ranked list of array genesRMS = the ranked metric scoreRES = the running enrichment scorePage 14 of 27(page number not for citation purposes)Examination of all lists generated similar results in thatapproximately 16–20% of all probes contained the con-cern the specific regulatory mechanisms occurring inchondrocytes.BMC Genomics 2007, 8:205 http://www.biomedcentral.com/1471-2164/8/205Table 8: Micromass culture-derived gene sets are enriched in DEX-treated primary chondrocytes (d3 vs d15_2). I.HUGO gene symbol Rank RMS RESItgbl1 32 0.391 0.015Adrb2 54 0.308 0.026Bst1 80 0.271 0.036Gpx3 83 0.269 0.047Myocd 90 0.259 0.058Grk5 105 0.229 0.066Ids 123 0.212 0.074Ms4a6b 140 0.200 0.0821810057c19rik 146 0.193 0.090Igfbp2 149 0.190 0.097Zfp36 218 0.159 0.100Serpina3n 222 0.158 0.107P2ry6 225 0.157 0.113Adm 228 0.156 0.120Crym 277 0.145 0.123Ppap2a 303 0.139 0.128Pycard 307 0.138 0.133Kcns1 320 0.134 0.138Cd80 321 0.134 0.144Trim24 330 0.133 0.149C1qtnf6 339 0.131 0.154A330049m08rik 377 0.127 0.157Adamts15 385 0.126 0.162Elovl4 398 0.124 0.167C1qa 402 0.124 0.172Sox9 434 0.119 0.175Htra3 455 0.116 0.179Adam17 483 0.112 0.182Mgll 493 0.112 0.186Ibsp 507 0.110 0.190C1qb 511 0.109 0.194Bambi 516 0.109 0.199Anxa4 551 0.105 0.201Cd109 555 0.105 0.206Nrk 559 0.104 0.210Gstm1 619 0.099 0.211Asb4 634 0.097 0.214Pygl 654 0.095 0.217Rasl11b 655 0.095 0.221Cdc42ep4 674 0.093 0.224Slc9a3r2 683 0.092 0.227Lama1 688 0.092 0.231Bb146404 707 0.091 0.234Ai194308 724 0.090 0.237Smn1 752 0.088 0.239Alcam 772 0.087 0.242Cst3 790 0.086 0.244Pyp 847 0.083 0.2452700017m01rik 870 0.082 0.247Fgfr3 884 0.081 0.250Mrpl34 912 0.080 0.252C9orf46 972 0.077 0.252Maf 981 0.077 0.2558430420c20rik 1028 0.075 0.255Gfm2 1030 0.075 0.259Anxa6 1041 0.075 0.261Isg20 1064 0.074 0.263Auh 1068 0.074 0.266Bsg 1100 0.072 0.267Peg3 1179 0.070 0.266Adam23 1208 0.069 0.268Ezh1 1213 0.069 0.270Page 15 of 27(page number not for citation purposes)2810022l02rik 1214 0.069 0.273BMC Genomics 2007, 8:205 http://www.biomedcentral.com/1471-2164/8/2050610011i04rik 1248 0.068 0.274Pbx2 1257 0.067 0.277Jup 1291 0.066 0.278Zcwcc2 1301 0.066 0.280Whsc2 1317 0.066 0.2822410004l22rik 1344 0.065 0.283Lmnb2 1388 0.064 0.284Fndc1 1435 0.063 0.284Rarres2 1460 0.062 0.285Tap2 1512 0.061 0.285Ctbs 1559 0.060 0.285Jdp2 1574 0.059 0.287Hck 1712 0.056 0.2825031400m07rik 1792 0.054 0.281Pkn1 1839 0.053 0.280Dag1 1929 0.052 0.278Fth1 1976 0.051 0.2781110001e17rik 1979 0.051 0.280Rbp4 1984 0.051 0.282Pdcd6ip 2044 0.050 0.281Siat7d 2050 0.050 0.283Kcnd2 2074 0.050 0.2842310004k06rik 2076 0.050 0.286D19ertd678e 2106 0.049 0.286Npdc1 2114 0.049 0.288Fts 2116 0.049 0.290Prickle1 2123 0.049 0.2911110037f02rik 2171 0.048 0.291Cdc42se1 2246 0.047 0.289Chpt1 2261 0.047 0.290Wwp2 2341 0.045 0.288Dact1 2363 0.045 0.289Rragd 2380 0.045 0.290Irf5 2406 0.044 0.291Nrbf2 2414 0.044 0.292Cox4i2 2436 0.044 0.293Bmp7 2456 0.044 0.2941810008a18rik 2517 0.043 0.292Asph 2533 0.043 0.293Stat2 2550 0.042 0.294Hoxa11 2560 0.042 0.296Bax 2599 0.042 0.295Sspn 2611 0.042 0.297Ifngr2 2612 0.042 0.298Glrx1 2672 0.041 0.297Gba 2739 0.040 0.295Fzd2 2759 0.040 0.296Crtap 2772 0.040 0.297Slc1a5 2786 0.040 0.298Slco3a1 2831 0.039 0.2973110040n11rik 2833 0.039 0.299Tep1 2845 0.039 0.300Fastk 2860 0.039 0.301Tmed3 2869 0.038 0.302Ephb4 2876 0.038 0.303Asah2 2908 0.038 0.303Pold4 2989 0.037 0.3011110001a07rik 2995 0.037 0.302Pcp4 3010 0.037 0.303Mab21l2 3025 0.037 0.304Rank = position of genes in the context of the ranked list of array genesRMS = the ranked metric scoreTable 8: Micromass culture-derived gene sets are enriched in DEX-treated primary chondrocytes (d3 vs d15_2). I. (Continued)Page 16 of 27(page number not for citation purposes)RES = the running enrichment scoreBMC Genomics 2007, 8:205 http://www.biomedcentral.com/1471-2164/8/205Table 9: Micromass culture-derived transcripts enriched in vehicle-treated primary chondrocytes (d3 vs d15_3/4). I.HUGO gene symbol Rank RMS RESRabggtb 16734 -0.040 -0.271Ube2e2 16759 -0.041 -0.270Cd68 16769 -0.041 -0.269H2-T23 16830 -0.041 -0.270Derl1 16834 -0.041 -0.268Smarcc1 16853 -0.041 -0.267Srxn1 16856 -0.041 -0.266Klf10 16868 -0.042 -0.264Zfhx1b 16879 -0.042 -0.263H2afy3 16929 -0.042 -0.264Wisp2 16973 -0.042 -0.264Tbl1xr1 16976 -0.042 -0.262Ppp1r3c 16979 -0.042 -0.260D11lgp2e 17036 -0.043 -0.261Smpdl3b 17079 -0.043 -0.262Dock2 17125 -0.044 -0.262Purb 17127 -0.044 -0.260Grn 17130 -0.044 -0.2581110035l05rik 17139 -0.044 -0.257Kiaa1008 17185 -0.045 -0.257E430025l02rik 17195 -0.045 -0.256Timm8a 17293 -0.046 -0.259C130006e23 17307 -0.046 -0.257Rbm10 17319 -0.046 -0.256A230103n10rik 17347 -0.046 -0.255Cd151 17401 -0.047 -0.256Srf 17409 -0.047 -0.254Cacna1s 17507 -0.048 -0.257Ythdf1 17529 -0.048 -0.256Ppp2r1b 17539 -0.048 -0.254Tead2 17545 -0.048 -0.252Igsf7 17590 -0.049 -0.252Per3 17604 -0.049 -0.251G1p2 17739 -0.050 -0.256Slco2a1 17786 -0.051 -0.256Coq7 17918 -0.053 -0.260Rarb 17940 -0.053 -0.259Lcp1 17954 -0.053 -0.257Dnaja1 17987 -0.053 -0.256Thoc3 17993 -0.054 -0.254Cd44 18041 -0.054 -0.254Slc41a1 18171 -0.056 -0.258Kif11 18232 -0.057 -0.259Hspa5bp1 18235 -0.057 -0.257Ncf4 18290 -0.058 -0.257Bub1b 18292 -0.058 -0.254Cap2 18295 -0.058 -0.252Aig1 18340 -0.059 -0.251Rfc3 18361 -0.059 -0.250Stmn1 18396 -0.060 -0.249Page 17 of 27(page number not for citation purposes)9130213b05rik 18408 -0.060 -0.247BMC Genomics 2007, 8:205 http://www.biomedcentral.com/1471-2164/8/205Tyms-Ps 18432 -0.060 -0.245Timp3 18513 -0.062 -0.247Tiparp 18564 -0.063 -0.247Thbs4 18627 -0.064 -0.247Wasf1 18652 -0.064 -0.245Nupr1 18686 -0.065 -0.244Ezh2 18706 -0.066 -0.242Fbxl14 18709 -0.066 -0.239Prim1 18780 -0.067 -0.240Insig2 18805 -0.068 -0.238B3gnt5 18858 -0.069 -0.238Fam60a 18963 -0.072 -0.240H2-M3 18972 -0.073 -0.237Gja7 18974 -0.073 -0.234Bex2 18987 -0.073 -0.231Tk1 19043 -0.074 -0.2311200015n20rik 19109 -0.076 -0.231Clecsf5 19114 -0.077 -0.228Ms4a7 19141 -0.078 -0.226Cdca5 19163 -0.079 -0.223C730042f17rik 19180 -0.079 -0.220Trim25 19194 -0.080 -0.218Efnb2 19207 -0.081 -0.215Apex1 19236 -0.082 -0.212Ddah2 19243 -0.082 -0.209Bub1 19262 -0.083 -0.206Nup43 19263 -0.083 -0.203Rdh10 19270 -0.083 -0.1992610201a13rik 19330 -0.086 -0.199Rp2h 19406 -0.089 -0.198Tnni1 19407 -0.089 -0.195Myog 19423 -0.091 -0.191Osmr 19486 -0.095 -0.190Mmp9 19524 -0.097 -0.188Tnnt1 19525 -0.098 -0.184Fhod3 19528 -0.098 -0.179D930038m13rik 19537 -0.099 -0.175Nes 19567 -0.101 -0.172Sbk1 19571 -0.102 -0.168Dusp9 19594 -0.103 -0.165Akr1b8 19622 -0.106 -0.161Pdgfrb 19663 -0.110 -0.158Tfrc 19667 -0.111 -0.154Moxd1 19670 -0.111 -0.1491810008k03rik 19681 -0.112 -0.145Cpeb1 19710 -0.115 -0.1416720475j19rik 19716 -0.116 -0.136Ripk4 19718 -0.116 -0.131Itga6 19756 -0.121 -0.127Bmp5 19775 -0.124 -0.123Lhx9 19776 -0.124 -0.117Pkp2 19797 -0.129 -0.113Chrna1 19808 -0.131 -0.108Table 9: Micromass culture-derived transcripts enriched in vehicle-treated primary chondrocytes (d3 vs d15_3/4). I. (Continued)Page 18 of 27(page number not for citation purposes)Bhlhb2 19837 -0.142 -0.103BMC Genomics 2007, 8:205 http://www.biomedcentral.com/1471-2164/8/205Gp49a 19847 -0.144 -0.097Clecsf10 19893 -0.155 -0.092Gch1 19902 -0.159 -0.086D0h4s114 19908 -0.161 -0.079Cxcl1 19928 -0.170 -0.072Ch25h 19946 -0.178 -0.065Mkrn3 19988 -0.228 -0.057Ptprc 20016 -0.297 -0.046Car6 20017 -0.298 -0.032Nr1d2 20031 -0.368 -0.017Evi2a 20033 -0.393 0.001Plxnc1 18075 -0.055 -0.286Cilp2 18106 -0.055 -0.285Brca1 18148 -0.056 -0.285Litaf 18149 -0.056 -0.283Bc027246 18154 -0.056 -0.2816820424l24rik 18268 -0.057 -0.285Hrb 18272 -0.057 -0.283Nnat 18303 -0.058 -0.282P2ry12 18329 -0.058 -0.282Cdca4 18343 -0.059 -0.2806030404e16rik 18367 -0.059 -0.279Tfec 18429 -0.060 -0.280Nfe2l2 18440 -0.060 -0.278Gtf2h2 18467 -0.061 -0.2774930469p12rik 18504 -0.062 -0.277Cul4b 18535 -0.062 -0.276H2afy2 18547 -0.063 -0.2741190002n15rik 18582 -0.063 -0.274B430218l07rik 18591 -0.063 -0.272Rgs18 18607 -0.064 -0.270Frk 18631 -0.064 -0.269Slc6a9 18633 -0.064 -0.267Tgfbr2 18687 -0.065 -0.267Tia1 18802 -0.068 -0.270Lgr5 18844 -0.068 -0.270Sgpp1 18909 -0.071 -0.271Matn2 18924 -0.071 -0.269Sox11 18931 -0.071 -0.266Hus1 18980 -0.073 -0.266D930015e06rik 19028 -0.074 -0.266Apob48r 19032 -0.074 -0.263Av344025 19045 -0.074 -0.261Eno2 19047 -0.074 -0.2582610024e20rik 19053 -0.075 -0.256Chd1l 19093 -0.076 -0.255Emr1 19145 -0.078 -0.255Rgs4 19200 -0.081 -0.254D030028o16rik 19211 -0.081 -0.252Kif2c 19216 -0.081 -0.249Ccl3 19220 -0.081 -0.246Trim30 19232 -0.082 -0.244Qrsl1 19242 -0.082 -0.241Table 9: Micromass culture-derived transcripts enriched in vehicle-treated primary chondrocytes (d3 vs d15_3/4). I. (Continued)Page 19 of 27(page number not for citation purposes)Nr3c1 19281 -0.083 -0.240BMC Genomics 2007, 8:205 http://www.biomedcentral.com/1471-2164/8/205Trip13 19282 -0.084 -0.237Dna2l 19317 -0.085 -0.236Tcf8 19335 -0.086 -0.233Clecsf8 19341 -0.086 -0.230Lyzs 19422 -0.090 -0.231Palmd 19475 -0.095 -0.230Tjp2 19487 -0.095 -0.227D430019h16rik 19493 -0.096 -0.224Sesn3 19501 -0.096 -0.221Ereg 19507 -0.096 -0.218Cx3cl1 19523 -0.097 -0.215Fzd6 19529 -0.098 -0.211Sod3 19564 -0.101 -0.209Tnnt2 19580 -0.102 -0.206Satb1 19599 -0.104 -0.203Cd14 19606 -0.104 -0.200Gbp2 19607 -0.104 -0.196Tgfbi 19609 -0.105 -0.192Chek1 19652 -0.109 -0.190Tm4sf1 19653 -0.109 -0.186Igf1 19679 -0.111 -0.183Enpp1 19695 -0.113 -0.180Slc15a3 19704 -0.114 -0.176Pdpn 19725 -0.117 -0.173Dkk1 19747 -0.119 -0.169Slk 19759 -0.121 -0.166Ankrd1 19794 -0.128 -0.163Trp53bp1 19801 -0.129 -0.158C79407 19804 -0.130 -0.1532210010l05rik 19809 -0.131 -0.149Eps8 19815 -0.133 -0.144Dkk2 19862 -0.147 -0.141Arhgap18 19863 -0.147 -0.136Twist2 19878 -0.151 -0.131Pcdha8 19915 -0.164 -0.126Il4r 19926 -0.169 -0.121Mdm1 19931 -0.172 -0.115Phlda1 19957 -0.188 -0.109Bhlhb5 19960 -0.192 -0.102C130076o07rik 19964 -0.196 -0.0955830411e10rik 19974 -0.207 -0.088Ptpre 19989 -0.228 -0.080Trib3 19990 -0.235 -0.0719230117n10rik 19994 -0.241 -0.062Pcdhb7 19998 -0.249 -0.053Mmp3 20001 -0.252 -0.044Cd34 20009 -0.274 -0.034Thbd 20022 -0.310 -0.023A830016g23rik 20023 -0.323 -0.011Ahr 20028 -0.336 0.001Rank = position of genes in the context of the ranked list of array genesRMS = the ranked metric scoreTable 9: Micromass culture-derived transcripts enriched in vehicle-treated primary chondrocytes (d3 vs d15_3/4). I. (Continued)Page 20 of 27(page number not for citation purposes)RES = the running enrichment scoreBMC Genomics 2007, 8:205 http://www.biomedcentral.com/1471-2164/8/205Page 21 of 27(page number not for citation purposes)Heat map of top 100 probe sets determined by GSEA analysisFigure 5Heat map of top 100 probe sets determined by GSEA analysis. GSEA-derived heat maps of the top 100 differentially expressed probe sets enriched with DEX or the vehicle control are shown (B). Expression profiles for all experimental repli-cates are shown for each time point. Genes containing a putative GRE are shown in bold, and examples of genes that do not contain GREs but have been documented as targets of DEX regulation are depicted by bold gray lettering. Signal intensities are illustrated by varying shades of red (up-regulation) and blue (down-regulation).'(;9HKLFOH,(53735(71)56)%,/,(5$+559(*)9&$0(9,$'26=)3/&+671*)%7*,)3+/'$*')$+5,/581;0,7)137;/+)3/':68(1).%,=$517$5+*$3&;&/*5,.,/5/,/562'35,.*5%15&',5,.&2&5,.)26/'%:*2(3'./,061'5*3(5+5,.$'$076).%3716%&$73%*5,.0&)/.575256&(/37*,6&%5'6,3*/8/6,$)$%367$5'3(5-5,.33$3$30$,3./)/5,.07$=%7%.&1.(*)/6267'&+3')2;2$7*00(57.&30*5,.6%3/,$)67/$5,.+63%+,)$&7'63/536.$3,0&;&/&&/*-%71)6)+$63&'+&;&/7+%'/,)37*63'(%67&3,.5KU KUKU KU\WLVQHWQ,ODQJL6GH]LODPUR1.,$$07BMC Genomics 2007, 8:205 http://www.biomedcentral.com/1471-2164/8/205ConclusionThis study elucidates the downstream transcriptionalimpact of pharmacological GC exposure on developingchondrocytes. We have identified a small subset of tran-scripts containing putative GREs in cartilage, but itappears that GRE-independent or indirect mechanisms ofGC regulation also contribute to GC regulation in primarychondrocyte monolayer cultures. In addition, traditionalmicroarray analysis methods and gene class testing pointto a dual role for pharmacological GC doses in chondro-cytes. DEX acts in a gene class-specific manner in cartilagein which it promotes the expression of ECM and meta-bolic transcripts necessary for maintaining the chondro-cyte phenotype while simultaneously downregulatingcytokines and growth factors which stimulate the cartilageto bone transition. Understanding the implications ofgene expression changes and integrating them into thenetwork of molecules controlling cartilage developmentcontinues to be challenging, but robust analytical meth-ods will prove to be useful in constructing the networks ofgene interactions and understanding the complex natureof GC signaling in the skeleton. The ultimate objective ofthis study will be to translate these findings into more effi-cacious therapeutic GCs.MethodsAnimals and MaterialsTimed-pregnant CD1 mice were purchased from CharlesRiver Laboratories at embryonic day E15.5 mice (E15.5).Dexamethasone was obtained from Calbiochem andreconstituted in Dimethyl sulfoxide (DMSO, vehicle)according to the manufacturer's instructions. Cell culturematerials and general chemicals were obtained from Inv-itrogen, Sigma or VWR unless otherwise stated.Primary cell culture and dexamethasone-treatmentTibiae, femurs and humeri were isolated from E15.5mouse embryos and placed in α-MEM media (Invitrogen)containing 0.2% Bovine Serum Albumin (BSA), 1 mM β-glycerophosphate, 0.05 mg/ml ascorbic acid and penicil-lin/streptomycin and incubated at 37°C in a humidified5% CO2 incubator overnight. The following morningmedia was removed and the bones placed in 4 ml of0.25% trypsin-EDTA (Invitrogen) for 15 min at 37°C.Trypsin was subsequently replaced with 1 mg/ml colla-genase P (Roche) in DMEM/10% fetal bovine serum (Inv-itrogen), and cells were incubated at 37°C with rotationat 100 rpm for 90 min. Following digestion, the cell sus-pension was centrifuged for 5 min at 1000 rpm, and thecollagenase containing supernatant was decanted.Chondrocytes were resuspended in media containing 2:3DMEM:F12, 10% fetal bovine serum, 0.5 mM L-glutamine, and penicillin/streptomycin (25 units/ml).monolayer chondrocytes were treated with 10-7 M dexam-ethasone (DEX) or the DMSO control (vehicle) diluted infresh media supplemented with 0.25 mM ascorbic acid(Sigma) and 1 mM β-glycerophosphate (Sigma) and incu-bated for up to 24 hrs. Micromass cultures were com-pleted as previously described [50].Cell counting studiesChondrocytes were isolated and seeded in 24-well NUNCplates (Nunc Inc.) at a density of 16 000 cells/cm2. Cellswere cultured, treated and enzymatically digested asdescribed with some modifications. Collagenase diges-tion occurred for 5 minutes followed by mechanical diges-tion to liberate cells from the ECM. Cells were countedwith a hemocytometer in triplicate with a minimum of 3individual wells per treatment and three independent cellisolations.RNA isolations and quantitative real-time PCRAll RNA protocols were completed as previously outlined[50]. Total RNA was isolated at 6 hrs and 24 hrs after treat-ment using the RNeasy mini extraction kit (Qiagen)according to the manufacturer's instructions. RNA quan-tity and integrity was assessed using the Bioanalyzer 2000system (Agilent). Quantitative real-time polymerase chainreaction (qRT-PCR) amplification was completed usingthe ABI Prism 7900 Sequence Detection System (AppliedBiosystems). Triplicate reactions were executed for eachsample of each of three independent trials. The TaqManone-step master mix kit (Applied Biosystems) with gene-specific target primers and probes were used for amplifica-tion. The collagen X (Col10a1) probe and primer set (for-ward primer 5'-ACGCCTACGATGTACACGTATGA-3',reverse primer 5'-ACTCCCTGAAGCCTGATCCA-3', 6-FAM-5'-AGTACAGCAAAGGCTAC-MGBNFQ) wasdesigned with PrimerDesigner 2.0 software (Applied Bio-systems) [79]. TaqMan GAPDH control reagents forhouse-keeping gene glyceraldehyde-3-phosphate dehy-drogenase (Gapdh, forward primer 5'-GAAGGTGAAG-GTCGGAGTC; reverse primer 5'-GAAGATGGTGATGGGATTTC; probe JOE-CAAGCTTC-CCGTTCTCAGCC-TAMRA) was used as an internalamplification control. Probes for Indian hedgehog (Ihh),Tissue inhibitor of matrix metalloproteinase 4 (Timp4),Cyclin-dependent kinase inhibitor 1C (Cdkn1c, p57),Integrin beta like 1 protein (Itgbl1), GC receptor (Nr3c1),Integrin beta 1 (Itgb1) and Kruppel-like factor 15 (Klf15)were assayed using the TaqMan® gene expression assays inaccordance with the manufacturers directions. Amplifiedtranscripts were quantified using the standard curvemethod, and the relative transcript abundance was deter-mined by calculating the quotient of the gene of interestand equivalent Gapdh values.Page 22 of 27(page number not for citation purposes)Cells were seeded in 6-well NUNC plates at a density of2.5 × 104 cells per ml and incubated overnight. PrimaryBMC Genomics 2007, 8:205 http://www.biomedcentral.com/1471-2164/8/205Microarray analysisTotal RNA was extracted from control and DEX-treatedcultures at 6 hr and 24 hr following treatment, in threeindependent experiments. RNA integrity and quantity wasassessed using the Agilent 2000 Bioanalyzer system, andRNA samples were subsequently hybridized to the MOE430 2.0 mouse chip from Affymetrix© containing 45 101probe sets as described [50]. Bioanalysis, microarrayhybridization, scanning and preliminary MAS 5.0 nor-malizations were completed at the London RegionalGenomics Facility. Data were deposited in the GEO data-base (NCBI; accession number GSE7683).Data normalizationMicroarray data were pre-processed using the GC-RMAalgorithm in Genespring GX*. Expression values were fur-ther filtered by retaining only those probe sets withexpression values of at least 50 in at least 25% of all con-ditions, thus generating a list of 22 091 probe sets. Toassess differential gene expression between treatments atboth the 6 and 24 hr time points, a Welch ANOVA testwith a p-value cut-off of 0.01 and a 5% false discovery rate(FDR) reduced the data to 1158 probe sets. Subsequent1.5-, 5- and 10-fold change filters produced lists of 162,21 and 7 probe sets for the 6 hr time point and 399, 53and 19 probe sets for the 24 hr time point, respectively.The same data set was normalized in parallel using RobustMultichip Analysis using RMAEXPRESS software v.0.4.1developed by B. Bolstad, University of California, Berke-ley [110]. Background adjustment and quantile normali-zation parameters were selected for data processing.Logarithmically transformed expression values were usedto implement Gene Set Enrichment Analysis (GSEA).Gene set enrichment analysis (GSEA)The GSEA algorithm was implemented with GSEA v2.0software [51,52]. Ranked expression lists were derivedfrom RMAEXPRESS and GeneSpring GX® 7.3.1. Briefly, theGSEA algorithm ranks all array genes according to theirexpression under each experimental condition. The result-ing ranked metric score (RMS) is therefore a function ofthe correlation between a gene's signal intensity, theexperimental conditions in question and all other genesin the data set. An enrichment score (ES) is then calculatedfor an a priori gene list or gene set that is associated witha particular molecular classification. In our analysis, genesets were created from different functional groupings,molecular classifications, tissues, and other microarrayscreens. A Ranked enrichment score (RES) which deter-mines the extent to which a given gene from a gene set isrepresented at the extremes of the ranked gene list is thencalculated. Specifically, this value is obtained by walkingfound in the ranked gene list and is coordinately penal-ized when it does not appear in the gene set. A null distri-bution of ES is subsequently generated by permutationfiltering to evaluate the statistical significance of theobserved RES values. Permutation filtering randomlyassigns the experimental conditions or class labels (i.e.,DEX versus vehicle) to the different microarray samples.After this procedure has been repeated for each gene set,the ES are normalized (NES) to account for differences ingene set size. The false discovery rate (FDR) is then calcu-lated relative to the NES values to determine the false-pos-itive rate. Significant FDR and p-values were less than 25%and 0.001, respectively in accordance with GSEA recom-mendations.Gene set creationGene sets were generated using the probe set search tooland the molecular function class of Gene Ontology anno-tations in GeneSpring GX. Additional gene sets were cre-ated using lists from pairwise comparisons between day 3and 15 of a previously generated micromass data set(James et al., 2005), and publications that identified DEXtarget genes in other cartilage array screens, other tissuetypes and experimental systems. A total of 2119 probe setsshowing a minimum 1.5-fold change in gene expressionwere used in the analysis. Probe set redundancy was elim-inated in all gene sets using the CollapseDataset functionin the GSEA program. All probe set identifiers were assim-ilated to the Human Genome Organization (HUGO)annotations. Probe sets lacking corresponding HUGOannotations were excluded from the analysis. Defaultparameters were used to execute the analysis and medianvalues taken to represent the range of duplicated probesets for a given gene. A total of 77 user-defined gene setswere generated from GeneSpring derived Gene Ontologyannotations for various molecular classifications andprobe sets of differentially expressed genes between days3 and 15 of micromass culture (James et al., 2005).Glucocorticoid response element (GRE) analysisPutative GRE were identified with the GenespringGXmouse genome9999 application which allows sequencesup to 9999 bp upstream of the transcriptional start sites ofall annotated MOE4302.0 transcripts to be interrogatedfor transcription factor binding sites. The GR consensussequence GGTACAnnntgttCT [111] was queried from 10bp to 10 000 bp upstream of the transcriptional start sitesof available probe sets. The GRE consensus sequence wasscreened against 10 748 probe sets derived from the list of22 091 reliably expressed probe sets exhibiting homologyto upstream regulatory regions annotated in the program.Only exact matches were retained for subsequent analysesout a total of 1,073,741,824 tests.Page 23 of 27(page number not for citation purposes)along the ranked list using a cumulative sum statisticwhich increases when a member of a particular gene set isBMC Genomics 2007, 8:205 http://www.biomedcentral.com/1471-2164/8/205AbbreviationsDEX: Dexamethasone; GSEA: gene set enrichment analy-sis; RES: ranked enrichment score; RMS: ranked metricscore, ES: enrichment scores; NES: normalized enrich-ment score, SOM: self-organizing maps; FDR: false discov-ery rate; GR: glucocorticoid receptorCompeting interestsThe author(s) declare that they have no competing inter-ests.Authors' contributionsCGJ completed cell culture experiments, data analysis,real-time PCR and drafted the manuscript. VU completedcell culture experiments. JT and TMU contributed to thedesign of the study and the writing of the manuscript. FBconceived of the study and contributed to the writing ofthe manuscript. All authors read and approved the finalmanuscript.AcknowledgementsCGJ is supported by a doctoral award from the Canadian Institutes of Health Research (CIHR) and previously by an Ontario Graduate Scholar-ship in Science and Technology. V.U. is the recipient of a graduate scholar-ship from the Canadian Arthritis Network. FB is the recipient of a Canada Research Chair. Operating funds for these studies were provided by the CIHR, the Canadian Arthritis Network and the Hospital for Sick Children Foundation to FB.References1. Cancedda R, Descalzi Cancedda F, Castagnola P: Chondrocyte dif-ferentiation.  Int Rev Cytol 1995, 159:265-358.2. Karsenty G, Wagner EF: Reaching a genetic and molecularunderstanding of skeletal development.  Dev Cell 2002,2(4):389-406.3. 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