Expression profiling of Dexamethasone-treated primary chondrocytes identifies targets of glucocorticoid signalling in endochondral bone development

  • Claudine G James1,

    Affiliated with

    • Veronica Ulici1,

      Affiliated with

      • Jan Tuckermann2,

        Affiliated with

        • T Michael Underhill3 and

          Affiliated with

          • Frank Beier1Email author

            Affiliated with

            BMC Genomics20078:205

            DOI: 10.1186/1471-2164-8-205

            Received: 26 February 2007

            Accepted: 01 July 2007

            Published: 01 July 2007

            Abstract

            Background

            Glucocorticoids (GCs) are widely used anti-inflammatory drugs. While useful in clinical practice, patients taking GCs often suffer from skeletal side effects including growth retardation in children and adolescents, and decreased bone quality in adults. On a physiological level, 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 limb bud mesenchyme micromass cultures. Some targets of GC regulation in chondrogenesis are known, but the global effects of pharmacological GC doses on chondrocyte gene expression have not been comprehensively evaluated.

            Results

            This study systematically identifies a spectrum of GC target genes in embryonic growth plate 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 with extracellular matrix genes. In contrast, a subset of growth factors and cytokines were negatively correlated with DEX treatment. Comparing DEX-induced gene expression data to developmental changes in gene expression in micromass cultures revealed an additional layer of complexity in which DEX maintains the expression of certain chondrocyte marker genes while inhibiting factors that promote vascularization and ultimately ossification of the cartilaginous template.

            Conclusion

            Together, these results provide insight into the mechanisms and major molecular classes functioning downstream of DEX in primary chondrocytes. In addition, comparison of our data with microarray studies of DEX treatment in other cell types demonstrated that the majority of DEX effects are tissue-specific. This study provides novel insights into the effects of pharmacological GC on chondrocyte gene transcription and establishes the foundation for subsequent functional studies.

            Background

            Cartilage provides a scaffold for the deposition of osteoblast precursors and ultimately the development of long bones. This process, termed endochondral ossification, describes a coordinated developmental series that involves commitment of mesenchymal precursor cells to the chondrogenic lineage and subsequent alternating phases of proliferation and differentiation, which culminate in the replacement of the cartilage by bone tissue [14]. In the first phase of this process, multipotent mesenchymal progenitors condense and initiate expression of the pro-chondrogenic Sox family members 9, 5 and 6 [5, 6]. A subset of cells at the center of these aggregates differentiates into chondrocytes. Newly formed chondrocytes secrete an extracellular matrix rich in type II collagen (Col2a1), proliferate and ultimately terminally differentiate into hypertrophic chondrocytes [7]. Chondrocyte hypertrophy precedes the end of the chondrocyte life cycle by apoptosis and is accompanied by vascularization of the hypertrophic template and mineralization of the cartilaginous extracellular matrix [812]. Concomitantly, osteoclasts degrade the calcified cartilage extracellular matrix, making way for the invasion and deposition of an osteoprogenitor population that form the primary ossification center [13].

            These events take place in a region called the growth plate that illustrates the organization of different phases of cartilage development into distinct zones. The resting zone delineates newly differentiated chondrocytes with low mitotic activity and the cellular reserve for subsequent stages of chondrocyte differentiation. Proliferative zone chondrocytes exhibit higher mitotic activity resulting in distinct columns containing cells reminiscent of stacked coins. The hypertrophic zone demarcates terminally differentiated chondrocytes which are identified by high cytoplasm to nuclear ratio and the expression of type X collagen (Col10a1) [1416]. Terminally differentiated chondrocytes are fated for programmed cell death after which primary ossification occurs by way of vascularization of the remaining cartilaginous matrix and the deposition of osteoprogenitor cells [1719].

            Glucocorticoids (GC) are among various endocrine molecules including growth hormone (GH) and thyroid hormone (TH) known to regulate linear growth [2023]. Regulation of linear growth follows the paradigm in which steroid hormones affect target tissue through both local and systemic mechanisms [2427]. Indirect effects occur through modulation of other endocrine systems such as the GH/IGF-I axis. Generally, GC decrease IGF-I, GH receptor and IGF receptor 1 expression and also abrogate the release of GH from the pituitary [20, 28, 29]. Direct regulation of growth occurs through GC receptor (GR)-mediated gene transcription in chondrocytes [24, 30, 31].

            GC functions are primarily mediated by the glucocorticoid receptor (GR) that is encoded by the Nr3c1 gene. The GR is ubiquitously expressed in mammalian tissues, including the growth plate, and is essential for life [3136]. Many studies have examined GC regulation of the skeleton and have led to various theories on potential modes of GC function in cartilage [3740]. The specific function of the receptor in terms of its transcriptional regulation in cartilage, however, remains enigmatic.

            While endogenous GCs have been shown to promote the differentiation of both chondrocytes and osteoblasts, exogenous GCs in pharmacological doses which are also widely used in clinical practice to treat inflammatory disorders [4146]. Their have different effects. Indeed, their utility in treating various diseases is, however, limited by numerous side effects such as growth failure and decreased bone quality [47]. GC-target genes including C-type natriuretic peptide and VEGF have been identified in chondrocytes [28, 48, 49]; however, the cartilage-specific transcriptional consequences of high-GC-doses in the growth plate have not been studied comprehensively.

            Work in our laboratory identified GR amongst factors that were up-regulated during chondrocyte maturation [50] Thus, to comprehensively understand the transcriptional effects of pharmacological GC doses in growth plate, we completed a genomic screen of gene expression changes in chondrocytes derived from E15.5 day old mouse embryos. Primary monolayer chondrocytes were treated with a synthetic GC, dexamethasone (DEX), and RNA was isolated for microarray analysis. We complemented traditional microarray analysis methods with the gene set enrichment algorithm to correlate the behaviour of specific molecular classes with DEX treatment [51, 52].

            Results and Discussion

            Microarray screen of dexamethasone-treated primary chondrocyte monolayers

            We identified the GR as a candidate for the regulation of chondrocyte hypertrophy in a previous expression profiling screen using primary micromass cultures [50]. The Nr3c1 probe set which encodes the GR was up-regulated 4-fold from day 3 to day 15 of micromass culture (Figure 1A, top panel). Confirmation of the GR expression profile with qRT-PCR showed an approximately 8-fold increase over the same time course (Figure 1A, bottom panel). Studies in our laboratory and others have implicated GCs in chondrocyte differentiation and growth plate function [25, 26, 47, 48, 53, 54]. In addition, our cell counting experiments revealed that DEX consistently decreases cell numbers after 24 hrs (Figure 1B), in agreement with other studies that show increased apoptosis [38, 55] and reduced proliferation [56] in response to GCs. We therefore aimed at extending this analysis to examine pharmacological effects of GCs on growth plate chondrocytes by systematically identifying downstream effector genes of DEX. Primary chondrocytes derived from the long bones of 15.5 day old embryonic mice were treated with DEX or the vehicle control, and total RNA was isolated after 6 and 24 hrs of culture, respectively.
            http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-8-205/MediaObjects/12864_2007_Article_918_Fig1_HTML.jpg
            Figure 1

            Gene 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).

            Gene expression was evaluated using Affymetrix MOE 430 2.0 mouse genome chips using three independent cell isolations. We first analyzed gene expression using conventional analysis functions in GeneSpring GX*. After pre-processing the data set using the GC-RMA algorithm and eliminating probe sets showing expression levels close to background, 22 091 probe sets remained, reducing the data set by 48% (Table 1). Significance testing with one-Way ANOVA analysis identified probe sets differentially expressed between DEX and vehicle-treated cultures over the entire time course (Figure 1C, left panel). The resulting list contained 1158 probe sets, which is 2% of the data set's original size. Approximately 70% of significantly changed probe sets exhibited upregulation in response to DEX treatment. This data set was further subdivided by using 1.5-, 5- and 10- fold change filters which generated lists of 162, 21 and 7 probe sets for the 6 hr time point and 399, 53 and 19 probe sets for the 24 hr time point, respectively (Table 1). Examination of the overall differences between the mean normalized signal intensities associated with each condition showed minimal changes in gene expression (Figure 1C, right panel), indicating that GC treatment affects the expression of only a small subset of all expressed genes in this system. A distribution of fold differences between 6 and 24 hrs showed that the majority of gene expression changes did not exceed 2-fold (Figure 1D). In each case, both time points exhibited the same overall trends in gene expression, but, as expected, the 24 hr time point consistently showed a higher proportion of probe sets altered by DEX treatment.
            Table 1

            Microarray analysis of DEX-treated primary chondrocyte monolayers.

            Specifications

            Probe sets at 6 hrs

            Probe sets at 24 hrs

            Total number of probe sets

            45101

            45101

            Significantly expressed

            22091

            22091

            Differentially expressed

            1158

            1158

            1.5-fold changed

            162

            399

            5-fold changed

            21

            53

            10-fold changed

            7

            33

            1.5-fold up-regulated

            141

            342

            5-fold up-regulated

            20

            50

            10-fold up-regulated

            7

            19

            1.5-fold down-regulated

            21

            57

            5-fold down-regulated

            1

            3

            10-fold down-regulated

            0

            0

            Probe set validation

            To confirm the accuracy of the microarrays in identifying biologically significant differences, we selected a variety of expressed transcripts for qRT-PCR analysis (Figure 2A). Transcripts that either belonged to a functional class implicated in cartilage development or exhibited marked changes with DEX treatment were chosen. Markers exhibiting marginal changes in gene expression were also selected for control purposes. Specifically, we evaluated the expression patterns of Indian hedgehog (Ihh), Tissue inhibitor of matrix metalloproteinase 4 (Timp4), Cyclin-dependent kinase inhibitor 1C (Cdkn1c), which contains a GC response element in its promoter [57], Integrin beta like 1 protein (Itgbl1), GC receptor (Nr3c1), Integrin beta 1 (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 hrs while Ihh, Timp4, Cdkn1c and Itgbl1 all increased after the 6 hr time point. Nr3c1, which encodes the GR, was not affected by DEX-treatment at both 6 and 24 hrs, but does contain a putative GRE [58]. Transcripts such as Itgb1 that exhibited less than 1.5-fold change in our arrays were also confirmed with qRT-PCR, providing further evidence that the microarray data represented authentic gene expression data. Interestingly, the fold change difference varied according to the experimental method. In cases such as Timp4 and to a lesser extent Cdkn1c, qtPCR data showed higher fold change increases with the DEX treatment than in microarrays. In contrast, the expression pattern for Klf15 exhibits a higher fold-change difference in the microarrays compared to the control. While data normalization using the RMA algorithm provides excellent estimates of reliable signal intensities, other methods such as the M.A.S. 5.0 algorithm are known to outperform RMA in its ability to accurately estimate fold change differences in transcript levels [59].
            http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-8-205/MediaObjects/12864_2007_Article_918_Fig2_HTML.jpg
            Figure 2

            Identification 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 inhibitor 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 expression 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.

            GSEA to identify the effects of dexamethasone on gene expression in chondrocytes

            Traditional microarray analysis methods are useful for the identification of probe sets exhibiting transcriptional responses to DEX-treatment, but are limited in certain capacities. Alternate statistical methods such as ANOVA testing produced transcript lists that, while effectively reducing the dimensionality or sample size of the data set, increased the rate of false negative data thus hampering our ability to generate meaningful hypotheses from the data (Figure 1). Also, the overall effect of DEX treatment on gene expression was modest, which may have reduced the significance of biologically relevant genes because their signal intensities were close to background levels. Accordingly, we did not have a clear concept of the central pathways and biological categories affected by DEX treatment. Similarly, Gene Ontology annotations were not sufficiently robust to detect differences in the representation of specific molecular categories (data not shown). We therefore implemented GSEA [52], an algorithm that is designed to effectively evaluate the effect of a specific experimental condition on known biological pathways and functional categories. These analyses show whether a given treatment (e.g. DEX stimulation) results in enrichment of genes sets involved in the regulation of a specific phenotype (see materials and methods for details).

            We created a gene set consisting of 77 gene lists representing different tissue types, functional categories and pathways derived from other microarray studies in the literature (Table 2). We drew conclusions from the top gene sets that had a false discovery rate (FDR) less than 25% and a p-value less than 0.001, both of which are acceptable cut-offs for the identification of biologically relevant probe sets. This cut-off, although relatively high, was optimized to reduce the occurrence of false negative data in data sets interrogating a small number of gene sets. Additionally, the FDR compensates for the inherent lack of coherence microarray data sets exhibit between gene expression and specific experimental conditions [52]. Enriched gene sets were identified in both DEX and vehicle data (Table 3). Specifically, the highest statistical confidence and correlation with the DEX phenotype was assigned to metabolism and extracellular matrix, which contained 196 and 228 genes, respectively (Figure 3, left panels, Table 4 and 5). In each case, the expression of genes positively correlated with the DEX phenotype at the 24 hr time point exceeded the number of genes at the 6 hr time point (Figure 3, right panels). Metabolic genes included aldehyde and alcohol dehydrogenases (Table 4), among others, and were identified in accordance with previously documented roles for GC in various metabolic processes and tissues [60, 61]. Closer examination of the genes contributing to the enrichment scores for the ECM gene set revealed that Dentin matrix protein 1 (Dmp1) was the top ranking gene (Table 5). DMP1 belongs to the SIBLING family of matrix molecules and has been linked to chondrocyte differentiation. Dmp1 knockout mice display disordered postnatal chondrogenesis, among other skeletal abnormalities [62]. Interestingly, integrin binding sialoprotein (Ibsp) [6366]), another SIBLING family member, and osteocalcin (Bglap2) both contain putative GRE sequences, but did not contribute to the enrichment score for this category [63, 66]. They did, however, belong to the core group of genes that were enriched when a micromass culture gene set was used to interrogate the DEX data (Figure 4).
            Table 2

            Gene sets used in GSEA.

            Category name

            Number of genes

            Category name

            Number of genes

            Adipose

            70

            Nucleus_3

            510

            Apoptosis

            39

            Fkbp

            33

            Bone

            116

            3vs15_1.5x_1

            497

            Cartilage

            28

            3vs15_1.5x_2

            497

            Catalytic

            245

            3vs15_1.5x_3

            497

            Chaperone

            81

            3vs15_1.5x_4

            497

            Chemokine

            31

            3vs15_1.5x_5

            76

            Chromatin/Hdacs

            24

            Igf

            48

            Cyclin

            225

            Cart_2

            299

            Cytokine

            127

            Cart_3

            352

            1_Dnabind

            500

            Liver_1

            260

            2_Dnabind

            448

            Liver_2

            260

            Ecm

            228

            Blood

            111

            Electron_Transp

            40

            Protease_1

            269

            Gf Receptor

            327

            Protease_2

            269

            Gluconeogen

            31

            Phosphatase

            473

            Growth Factor

            106

            Dusp

            20

            Gtpase Activator

            46

            Kinase_1

            499

            Gtpase Activ

            73

            Kinase_2

            499

            Heparin Bind

            37

            Kinase_3

            227

            Hormone

            75

            Integrin_Rel

            173

            Muscle

            198

            Brain_Rel

            379

            Neg_Apoptosis

            50

            Hepatocyte

            19

            Oncogene

            154

            Obl_Oclast

            16

            Pos_Apoptosis

            79

            Interleukinrelated

            175

            Related_Apoptosis

            311

            Rgs_Related

            44

            Structure

            151

            Caspase_Related

            47

            Sugar_Bind

            104

            Creb_Atf3

            32

            Tf_Activ

            56

            Nuclear Receptor

            138

            Tf_Repress

            55

            Nuc_Hormone_Receptor

            55

            Tgfb

            45

            Mapkrelated

            267

            Tnf_Receptor

            69

            Membrane

            260

            Tumor Suppressor

            48

            Metabolism

            196

            Wnt

            53

            Nucleus_1

            494

            Actin_Cytoskel

            38

            Nucleus_2

            494

            Angiogen

            57

            Pzhorton.Farnum

            413

            Bmprelated

            62

            Hzhorton.Farnum

            407

            Cytoplasm

            411

              

            Erk_Related

            40

              

            Fgf_Related

            64

              
            Table 3

            GSEA of DEX-treated primary chondrocytes.

            Gene set name

            Size

            ES

            NES

            NOM p-val

            FDR q-val

            Metabolism

            196

            0.471

            1.935

            <0.001

            0.016

            Extracellualr Matrix

            228

            0.451

            1.878

            <0.001

            0.016

            Fkbp

            33

            0.559

            1.696

            0.011

            0.054

            Integrin_Related

            173

            0.407

            1.643

            <0.001

            0.001

            Angiogenesis

            57

            0.479

            1.610

            0.012

            0.065

            Kinase_1

            499

            0.343

            1.549

            <0.001

            0.092

            Tumor Suppressor

            48

            0.457

            1.492

            0.037

            0.126

            Catalytic

            245

            0.337

            1.420

            0.008

            0.172

            Hepatocyte

            19

            0.529

            1.406

            0.104

            0.161

            D3 Vs D15_2

            497

            0.304

            1.368

            0.004

            0.194

            Igf

            48

            0.412

            1.348

            0.093

            0.208

            Cyclin

            224

            0.322

            1.344

            0.028

            0.199

            Actin_Cytoskel

            38

            0.426

            1.325

            0.124

            0.213

            Structure

            151

            0.332

            1.312

            0.053

            0.219

            Cytoplasm

            411

            0.292

            1.300

            0.023

            0.224

            Adipose

            70

            0.368

            1.285

            0.116

            0.232

            Gtpase Activity

            73

            0.363

            1.280

            0.113

            0.230

            Cartilage

            28

            0.432

            1.262

            0.169

            0.246

            Chemokine

            31

            -0.779

            -2.40

            <0.001

            0

            Cytokine

            127

            -0.579

            -2.31

            <0.001

            0

            Growth Factor

            106

            -0.517

            -2.01

            <0.001

            7.698E-04

            Interleukinrelated

            175

            -0.469

            -1.98

            <0.001

            9.475E-04

            Bone

            16

            -0.577

            -1.51

            0.051

            8.945E-02

            Creb_Atf3

            30

            -0.469

            -1.43

            0.065

            1.300E-01

            Dusp

            20

            -0.508

            -1.40

            0.102

            1.418E-01

            Blood

            111

            -0.351

            -1.37

            0.037

            1.425E-01

            3vs15_1.5x_3

            496

            -0.288

            -1.35

            0.002

            1.518E-01

            Protease_2

            268

            -0.306

            -1.35

            0.015

            1.411E-01

            Nuc_Hormone_Receptor

            55

            -0.381

            -1.32

            0.086

            1.570E-01

            Tf_Repress

            55

            -0.380

            -1.32

            0.091

            1.498E-01

            3vs15_1.5x_4

            497

            -0.272

            -1.28

            0.011

            1.817E-01

            Erk_Related

            40

            -0.385

            -1.25

            0.157

            2.169E-01

            ES, enrichment score

            NES, normalized enrichment score

            FDR q-val, false discovery rate and multiple testing corrections (q-value)

            NOM p-val; the uncorrected p-value

            http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-8-205/MediaObjects/12864_2007_Article_918_Fig3_HTML.jpg
            Figure 3

            Enrichment 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.

            Table 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

            0.426

            Eya2

            40

            0.355

            0.099

            Ltbp2

            1721

            0.056

            0.428

            Vcl

            106

            0.228

            0.125

            Hsd17b1

            1783

            0.055

            0.432

            Adhfe1

            116

            0.222

            0.154

            P4ha2

            1783

            0.055

            0.432

            Ids

            123

            0.212

            0.181

            Mut

            1850

            0.053

            0.443

            Cbr3

            133

            0.204

            0.207

            Pde3a

            2195

            0.048

            0.432

            Aldh6a1

            202

            0.165

            0.225

            Sulf2

            2200

            0.048

            0.438

            Bcat2

            224

            0.157

            0.245

            Prep

            2316

            0.046

            0.438

            Pmm1

            278

            0.145

            0.261

            Plod3

            2387

            0.045

            0.441

            Pcx

            553

            0.105

            0.261

            1110013G13RIK

            2510

            0.043

            0.440

            Fthfd

            554

            0.105

            0.275

            Pld1

            2669

            0.041

            0.437

            Atp1a1

            560

            0.104

            0.288

            Au041707

            2721

            0.040

            0.440

            Gstm1

            619

            0.099

            0.298

            Decr1

            2837

            0.039

            0.439

            Gstm2

            742

            0.088

            0.303

            Gstm5

            2872

            0.038

            0.443

            1700061G19RIK

            787

            0.086

            0.312

            Bckdha

            2932

            0.038

            0.445

            Slc38a4

            833

            0.084

            0.321

            Atp11a

            2951

            0.038

            0.449

            Pyp

            847

            0.083

            0.331

            Gstp1

            2967

            0.037

            0.453

            Aacs

            901

            0.080

            0.339

            Dhrs7

            3014

            0.037

            0.455

            Plod1

            934

            0.079

            0.348

            Cbr2

            3147

            0.035

            0.453

            Acas2

            983

            0.077

            0.355

            Echdc3

            3152

            0.035

            0.458

            Auh

            1068

            0.074

            0.361

            Acy3

            3254

            0.035

            0.457

            Gcat

            1109

            0.072

            0.368

            Dhrs1

            3483

            0.032

            0.450

            Dhrs8

            1184

            0.070

            0.373

            Itgb1

            3527

            0.032

            0.452

            Egln3

            1232

            0.068

            0.380

            4933406E20RIK

            3553

            0.031

            0.454

            Mthfs

            1268

            0.067

            0.387

            Plod2

            3574

            0.031

            0.458

            Mvk

            1298

            0.066

            0.394

            Pmm2

            3582

            0.031

             

            Aup1

            1325

            0.065

            0.401

            Ugp2

            3583

            0.031

             

            Spr

            1456

            0.062

            0.403

            Gnpat

            3633

            0.031

             

            Sc5dl

            1462

            0.062

            0.411

            1110003P22RIK

            3636

            0.031

             

            1300018J18RIK

            1516

            0.061

            0.416

            Dbt

            3710

            0.030

             

            Agpat3

            1524

            0.061

            0.423

                

            Rank = position of genes in the context of the ranked list of array genes

            RMS = the ranked metric score

            RES = the running enrichment score

            Table 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

            0.420

            Omd

            27

            0.409

            0.068

            Lama3

            886

            0.081

            0.427

            Itga5

            38

            0.358

            0.095

            Nyx

            992

            0.077

            0.427

            Adamts1

            57

            0.305

            0.118

            Lamb2

            1082

            0.073

            0.429

            Timp4

            61

            0.296

            0.141

            Bsg

            1100

            0.072

            0.433

            Col4a1

            86

            0.268

            0.161

            Fbn2

            1242

            0.068

            0.432

            Col4a2

            98

            0.247

            0.180

            Ntn4

            1245

            0.068

            0.437

            Adam12

            112

            0.225

            0.197

            5730577E14RIK

            1381

            0.064

            0.435

            Prelp

            139

            0.200

            0.211

            Col6a2

            1405

            0.064

            0.439

            Postn

            142

            0.195

            0.227

            Ntn3

            1415

            0.063

            0.443

            Chad

            176

            0.174

            0.239

            Tgfb2

            1531

            0.060

            0.442

            Mgp

            195

            0.168

            0.251

            Mia1

            1575

            0.059

            0.445

            Col1a1

            232

            0.154

            0.261

            Mmp14

            1803

            0.054

            0.438

            Mfap5

            233

            0.153

            0.273

            Col15a1

            1845

            0.053

            0.440

            Col10a1

            266

            0.146

            0.283

            Ctgf

            1882

            0.052

            0.442

            Smoc2

            279

            0.145

            0.294

            Col6a1

            1942

            0.052

            0.443

            Aspn

            294

            0.141

            0.304

            Gpld1

            1946

            0.051

            0.447

            Col4a5

            367

            0.128

            0.310

            Emid2

            2043

            0.050

            0.446

            Adamts15

            385

            0.126

            0.319

            Col7a1

            2047

            0.050

            0.450

            Tgfb1

            394

            0.125

            0.329

            Adam10

            2107

            0.049

            0.451

            Sparcl1

            440

            0.119

            0.336

            Col9a2

            605

            0.100

            0.370

            Adam17

            483

            0.112

            0.343

            Matn3

            610

            0.099

            0.377

            Lama5

            508

            0.110

            0.350

            Col11a2

            636

            0.097

            0.384

            Lamc1

            517

            0.109

            0.358

            Hapln1

            650

            0.096

            0.391

            Spock2

            581

            0.102

            0.363

            Lama2

            685

            0.092

            0.396

            Lama1

            688

            0.092

            0.403

            Gpc3

            796

            0.086

            0.412

            Ltbp4

            704

            0.091

            0.410

            Lama4

            827

            0.084

            0.417

            *RMS = the ranked metric score

            **RES = the running enrichment score

            http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-8-205/MediaObjects/12864_2007_Article_918_Fig4_HTML.jpg
            Figure 4

            Comparison of DEX-treated primary chondrocytes to a time course of chondrocyte differentiation in micromass 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 chondrocytes (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-regulation in the day 3 samples of the micromass (MM) culture data set.

            Osteomodulin, an additional matrix molecule shown to be structurally similar to IBSP [67], ranked second in the list of enriched ECM genes. Additional ECM molecules expressed in terminally differentiated chondrocytes such as collagen 10 (Col10a1) and osteonectin (Spock1) were identified, suggesting that this molecular classification is important for transmitting GC signaling in the growth plate.

            Interestingly, the normalized enrichment scores for factors down-regulated by DEX treatment were higher than those positively correlated with DEX, but contained fewer probe sets contributing to the scores. Gene sets composed of 127 and 106 genes associated with cytokine and growth factor activity, respectively, were negatively correlated with DEX treatment (Figure 4, Table 6, 7). In other studies, cytokines such as Il-8 and GROα were found to promote the hypertrophy of osteoarthritic cartilage, and excess interleukins 1β(IL-1β), interleukin 6 (IL-6) and Tumor Necrosis Factor alpha (TNF-α) cause growth failure in children [6870]. Our studies identified three members of the GP-130 family of cytokines, namely interleukins -11,-6 (Il11, Il6) and leukemia inhibitory factor (Lif), as part of the core enrichment group for cytokines (Table 6). Transgenic mice overexpressing Il-6 exhibit growth retardation, and LIF is thought to regulate the rate at which terminally differentiated cartilage is calcified and vascularized [71, 72].
            Table 6

            Cytokine transcripts enriched in vehicle-treated chondrocytes. I.

            HUGO gene symbol

            Rank

            RMS

            RES

            Cklfsf2b

            16971

            -0.0424

            -0.574

            Il7

            16981

            -0.0425

            -0.569

            Il1f9

            17007

            -0.0427

            -0.566

            Grn

            17130

            -0.0439

            -0.567

            Il1f6

            17153

            -0.0442

            -0.563

            Ifna2

            17418

            -0.0468

            -0.571

            Tslp

            17503

            -0.0477

            -0.570

            Il17

            17568

            -0.0483

            -0.568

            A730028g07rik

            17606

            -0.0486

            -0.564

            Cxcl11

            17634

            -0.0490

            -0.560

            Ctf1

            17857

            -0.0519

            -0.565

            Lta

            17864

            -0.0519

            -0.559

            Il1a

            18018

            -0.0539

            -0.561

            Ccl20

            18038

            -0.0542

            -0.556

            Ccl17

            18334

            -0.0584

            -0.564

            Ccl12

            18384

            -0.0592

            -0.560

            Cklf

            18618

            -0.0639

            -0.564

            Ifna11

            18855

            -0.0688

            -0.568

            Cklfsf6

            18874

            -0.0693

            -0.561

            Il15

            18955

            -0.0719

            -0.557

            Ltb

            19146

            -0.0779

            -0.558

            Ccl3

            19220

            -0.0814

            -0.552

            Tnfsf9

            19228

            -0.0816

            -0.543

            Cx3cl1

            19523

            -0.0975

            -0.547

            Gdf15

            19660

            -0.1100

            -0.541

            Bmp5

            19775

            -0.1238

            -0.533

            Cxcl14

            19798

            -0.1289

            -0.519

            Cxcl1

            19928

            -0.1698

            -0.507

            Cxcl10

            19951

            -0.1807

            -0.487

            Ccl7

            19956

            -0.1849

            -0.466

            Gdf5

            19973

            -0.2066

            -0.444

            Cxcl12

            19978

            -0.2104

            -0.420

            Areg

            19983

            -0.2189

            -0.395

            Cxcl2

            19996

            -0.2421

            -0.369

            Ppbp

            20014

            -0.2944

            -0.336

            Lif

            20024

            -0.3296

            -0.299

            Ccl2

            20030

            -0.3589

            -0.258

            Il11

            20035

            -0.4036

            -0.213

            Cxcl5

            20039

            -0.5406

            -0.152

            Tnfsf11

            20041

            -0.5835

            -0.085

            Il6

            20043

            -0.7529

             

            Rank = position of genes in the context of the ranked list of array genes

            RMS = the ranked metric score

            RES = the running enrichment score

            Table 7

            Growth factor transcripts vehicle in DEX-treated chondrocytes.

            HUGO gene symbol

            Rank

            RMS

            RES

            Fgf21

            18968

            -0.073

            -0.508

            Nrg3

            19132

            -0.077

            -0.506

            Fgf5

            19190

            -0.080

            -0.499

            Ereg

            19507

            -0.096

            -0.502

            Fgf7

            19581

            -0.102

            -0.493

            Gdf15

            19660

            -0.110

            -0.483

            Igf1

            19679

            -0.111

            -0.469

            Bmp5

            19775

            -0.124

            -0.458

            Nov

            19848

            -0.144

            -0.443

            Vegf

            19877

            -0.150

            -0.425

            Ptn

            19885

            -0.153

            -0.406

            Cxcl1

            19928

            -0.170

            -0.386

            Bdnf

            19939

            -0.176

            -0.364

            Inhba

            19971

            -0.204

            -0.340

            Gdf5

            19973

            -0.207

            -0.313

            Cxcl12

            19978

            -0.210

            -0.287

            Areg

            19983

            -0.219

            -0.259

            Hbegf

            20006

            -0.264

            -0.226

            Ngfb

            20013

            -0.287

            -0.189

            Lif

            20024

            -0.330

            -0.148

            Il11

            20035

            -0.404

            -0.096

            Il6

            20043

            -0.753

            0.000

            Rank = position of genes in the context of the ranked list of array genes

            RMS = the ranked metric score

            RES = the running enrichment score

            This group also contained the gene encoding Tumor necrosis factor (ligand) superfamily, member 11 (Tnfsf11, RANKL), which has been localized to mature chondrocytes and is thought to promote degradation of the calcified cartilage ECM and ultimately endochondral ossification through activation of osteoclasts [7375]. It is important to note that several independent gene sets connected to inflammation such as cytokines, chemokines and interleukins exhibit some overlap and showed similar enrichment patterns, which provides additional confirmation that DEX is indeed downregulating inflammatory molecules in chondrocytes. GC have been previously reported to down-regulate the expression of VEGF, one of the central growth factors involved in vascularization of calcified 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 processes during endochondral ossification, our data suggest that DEX prevents the replacement of hypertrophic cartilage by bone. GC have been shown to delay chondrocyte maturation while retaining their capacity to re-engage in their developmental program [21]. This could account for upregulation of genes typically associated with the chondrocyte phenotype, such as ECM genes and the coordinated downregulation of factors that promote the transition from cartilage into bone.

            Identification of cartilage-specific dexamethasone-effects

            Identification of cartilage-specific gene sets affected by DEX treatment provided further insight into the complex nature of GC functions in cartilage. We knew from other studies that DEX effects on chondrogenic differentiation are dependent on cell source, experimental system and DEX concentration [40, 42, 7678]. We aimed to systematically characterize the effects of DEX on growth plate chondrocytes. To ensure that our DEX data set was expressing bona fide cartilage markers, we compared the DEX data to our previously generated micromass culture data set [50]. We compared all expressed probe sets in the DEX array to probe sets exhibiting a minimum 1.5-fold change in expression between days 3 and 15 of micromass cultures that encompass the various stages of the chondrocyte life cycle. Day 3 of micromass culture likely coincides with the onset of the cartilage developmental program and early chondrogenesis. After 15 days of culture, the cell population is comprised primarily of terminally differentiated chondrocytes and thus corresponds mostly to the hypertrophic zone of the growth plate [50, 79], although small numbers of other cells are present at all stages. Out of the 2119 probe sets displaying at least 1.5-fold changes in expression in the micromass culture data set (a probe set list generated from the pair-wise comparison of day 3 versus day 15 of micromass culture), 1730 were also expressed in the DEX array. This shows that our primary chondrocyte monolayers do exhibit prototypical chondrocyte gene expression patterns in both the presence and absence of DEX treatment.

            To complete more robust classification of the data in which we could correlate chondrocyte gene expression to the DEX phenotype, we created a gene set from this list of 2119 probe sets (Table 8, 9). The micromass derived gene list was enriched in this study; however, the list was found to correlate both positively and negatively with different aspects of the DEX phenotype. We therefore proceeded to evaluate both the micromass (MM) data set and the DEX data set using GSEA analysis and the previously created gene sets. If both the micromass time course and the DEX data sets show the same enrichment pattern, we would have evidence to suggest that pharmacological DEX doses promote chondrocyte differentiation. Normalized enrichment scores for gene sets common to both culture methods were therefore compared to identify differences and similarities between DEX-treated chondrocytes and the chondrocyte phenotype (Figure 4B).
            Table 8

            Micromass culture-derived gene sets are enriched in DEX-treated primary chondrocytes (d3 vs d15_2). I.

            HUGO gene symbol

            Rank

            RMS

            RES

            Itgbl1

            32

            0.391

            0.015

            Adrb2

            54

            0.308

            0.026

            Bst1

            80

            0.271

            0.036

            Gpx3

            83

            0.269

            0.047

            Myocd

            90

            0.259

            0.058

            Grk5

            105

            0.229

            0.066

            Ids

            123

            0.212

            0.074

            Ms4a6b

            140

            0.200

            0.082

            1810057c19rik

            146

            0.193

            0.090

            Igfbp2

            149

            0.190

            0.097

            Zfp36

            218

            0.159

            0.100

            Serpina3n

            222

            0.158

            0.107

            P2ry6

            225

            0.157

            0.113

            Adm

            228

            0.156

            0.120

            Crym

            277

            0.145

            0.123

            Ppap2a

            303

            0.139

            0.128

            Pycard

            307

            0.138

            0.133

            Kcns1

            320

            0.134

            0.138

            Cd80

            321

            0.134

            0.144

            Trim24

            330

            0.133

            0.149

            C1qtnf6

            339

            0.131

            0.154

            A330049m08rik

            377

            0.127

            0.157

            Adamts15

            385

            0.126

            0.162

            Elovl4

            398

            0.124

            0.167

            C1qa

            402

            0.124

            0.172

            Sox9

            434

            0.119

            0.175

            Htra3

            455

            0.116

            0.179

            Adam17

            483

            0.112

            0.182

            Mgll

            493

            0.112

            0.186

            Ibsp

            507

            0.110

            0.190

            C1qb

            511

            0.109

            0.194

            Bambi

            516

            0.109

            0.199

            Anxa4

            551

            0.105

            0.201

            Cd109

            555

            0.105

            0.206

            Nrk

            559

            0.104

            0.210

            Gstm1

            619

            0.099

            0.211

            Asb4

            634

            0.097

            0.214

            Pygl

            654

            0.095

            0.217

            Rasl11b

            655

            0.095

            0.221

            Cdc42ep4

            674

            0.093

            0.224

            Slc9a3r2

            683

            0.092

            0.227

            Lama1

            688

            0.092

            0.231

            Bb146404

            707

            0.091

            0.234

            Ai194308

            724

            0.090

            0.237

            Smn1

            752

            0.088

            0.239

            Alcam

            772

            0.087

            0.242

            Cst3

            790

            0.086

            0.244

            Pyp

            847

            0.083

            0.245

            2700017m01rik

            870

            0.082

            0.247

            Fgfr3

            884

            0.081

            0.250

            Mrpl34

            912

            0.080

            0.252

            C9orf46

            972

            0.077

            0.252

            Maf

            981

            0.077

            0.255

            8430420c20rik

            1028

            0.075

            0.255

            Gfm2

            1030

            0.075

            0.259

            Anxa6

            1041

            0.075

            0.261

            Isg20

            1064

            0.074

            0.263

            Auh

            1068

            0.074

            0.266

            Bsg

            1100

            0.072

            0.267

            Peg3

            1179

            0.070

            0.266

            Adam23

            1208

            0.069

            0.268

            Ezh1

            1213

            0.069

            0.270

            2810022l02rik

            1214

            0.069

            0.273

            0610011i04rik

            1248

            0.068

            0.274

            Pbx2

            1257

            0.067

            0.277

            Jup

            1291

            0.066

            0.278

            Zcwcc2

            1301

            0.066

            0.280

            Whsc2

            1317

            0.066

            0.282

            2410004l22rik

            1344

            0.065

            0.283

            Lmnb2

            1388

            0.064

            0.284

            Fndc1

            1435

            0.063

            0.284

            Rarres2

            1460

            0.062

            0.285

            Tap2

            1512

            0.061

            0.285

            Ctbs

            1559

            0.060

            0.285

            Jdp2

            1574

            0.059

            0.287

            Hck

            1712

            0.056

            0.282

            5031400m07rik

            1792

            0.054

            0.281

            Pkn1

            1839

            0.053

            0.280

            Dag1

            1929

            0.052

            0.278

            Fth1

            1976

            0.051

            0.278

            1110001e17rik

            1979

            0.051

            0.280

            Rbp4

            1984

            0.051

            0.282

            Pdcd6ip

            2044

            0.050

            0.281

            Siat7d

            2050

            0.050

            0.283

            Kcnd2

            2074

            0.050

            0.284

            2310004k06rik

            2076

            0.050

            0.286

            D19ertd678e

            2106

            0.049

            0.286

            Npdc1

            2114

            0.049

            0.288

            Fts

            2116

            0.049

            0.290

            Prickle1

            2123

            0.049

            0.291

            1110037f02rik

            2171

            0.048

            0.291

            Cdc42se1

            2246

            0.047

            0.289

            Chpt1

            2261

            0.047

            0.290

            Wwp2

            2341

            0.045

            0.288

            Dact1

            2363

            0.045

            0.289

            Rragd

            2380

            0.045

            0.290

            Irf5

            2406

            0.044

            0.291

            Nrbf2

            2414

            0.044

            0.292

            Cox4i2

            2436

            0.044

            0.293

            Bmp7

            2456

            0.044

            0.294

            1810008a18rik

            2517

            0.043

            0.292

            Asph

            2533

            0.043

            0.293

            Stat2

            2550

            0.042

            0.294

            Hoxa11

            2560

            0.042

            0.296

            Bax

            2599

            0.042

            0.295

            Sspn

            2611

            0.042

            0.297

            Ifngr2

            2612

            0.042

            0.298

            Glrx1

            2672

            0.041

            0.297

            Gba

            2739

            0.040

            0.295

            Fzd2

            2759

            0.040

            0.296

            Crtap

            2772

            0.040

            0.297

            Slc1a5

            2786

            0.040

            0.298

            Slco3a1

            2831

            0.039

            0.297

            3110040n11rik

            2833

            0.039

            0.299

            Tep1

            2845

            0.039

            0.300

            Fastk

            2860

            0.039

            0.301

            Tmed3

            2869

            0.038

            0.302

            Ephb4

            2876

            0.038

            0.303

            Asah2

            2908

            0.038

            0.303

            Pold4

            2989

            0.037

            0.301

            1110001a07rik

            2995

            0.037

            0.302

            Pcp4

            3010

            0.037

            0.303

            Mab21l2

            3025

            0.037

            0.304

            Rank = position of genes in the context of the ranked list of array genes

            RMS = the ranked metric score

            RES = the running enrichment score

            Table 9

            Micromass culture-derived transcripts enriched in vehicle-treated primary chondrocytes (d3 vs d15_3/4). I.

            HUGO gene symbol

            Rank

            RMS

            RES

            Rabggtb

            16734

            -0.040

            -0.271

            Ube2e2

            16759

            -0.041

            -0.270

            Cd68

            16769

            -0.041

            -0.269

            H2-T23

            16830

            -0.041

            -0.270

            Derl1

            16834

            -0.041

            -0.268

            Smarcc1

            16853

            -0.041

            -0.267

            Srxn1

            16856

            -0.041

            -0.266

            Klf10

            16868

            -0.042

            -0.264

            Zfhx1b

            16879

            -0.042

            -0.263

            H2afy3

            16929

            -0.042

            -0.264

            Wisp2

            16973

            -0.042

            -0.264

            Tbl1xr1

            16976

            -0.042

            -0.262

            Ppp1r3c

            16979

            -0.042

            -0.260

            D11lgp2e

            17036

            -0.043

            -0.261

            Smpdl3b

            17079

            -0.043

            -0.262

            Dock2

            17125

            -0.044

            -0.262

            Purb

            17127

            -0.044

            -0.260

            Grn

            17130

            -0.044

            -0.258

            1110035l05rik

            17139

            -0.044

            -0.257

            Kiaa1008

            17185

            -0.045

            -0.257

            E430025l02rik

            17195

            -0.045

            -0.256

            Timm8a

            17293

            -0.046

            -0.259

            C130006e23

            17307

            -0.046

            -0.257

            Rbm10

            17319

            -0.046

            -0.256

            A230103n10rik

            17347

            -0.046

            -0.255

            Cd151

            17401

            -0.047

            -0.256

            Srf

            17409

            -0.047

            -0.254

            Cacna1s

            17507

            -0.048

            -0.257

            Ythdf1

            17529

            -0.048

            -0.256

            Ppp2r1b

            17539

            -0.048

            -0.254

            Tead2

            17545

            -0.048

            -0.252

            Igsf7

            17590

            -0.049

            -0.252

            Per3

            17604

            -0.049

            -0.251

            G1p2

            17739

            -0.050

            -0.256

            Slco2a1

            17786

            -0.051

            -0.256

            Coq7

            17918

            -0.053

            -0.260

            Rarb

            17940

            -0.053

            -0.259

            Lcp1

            17954

            -0.053

            -0.257

            Dnaja1

            17987

            -0.053

            -0.256

            Thoc3

            17993

            -0.054

            -0.254

            Cd44

            18041

            -0.054

            -0.254

            Slc41a1

            18171

            -0.056

            -0.258

            Kif11

            18232

            -0.057

            -0.259

            Hspa5bp1

            18235

            -0.057

            -0.257

            Ncf4

            18290

            -0.058

            -0.257

            Bub1b

            18292

            -0.058

            -0.254

            Cap2

            18295

            -0.058

            -0.252

            Aig1

            18340

            -0.059

            -0.251

            Rfc3

            18361

            -0.059

            -0.250

            Stmn1

            18396

            -0.060

            -0.249

            9130213b05rik

            18408

            -0.060

            -0.247

            Tyms-Ps

            18432

            -0.060

            -0.245

            Timp3

            18513

            -0.062

            -0.247

            Tiparp

            18564

            -0.063

            -0.247

            Thbs4

            18627

            -0.064

            -0.247

            Wasf1

            18652

            -0.064

            -0.245

            Nupr1

            18686

            -0.065

            -0.244

            Ezh2

            18706

            -0.066

            -0.242

            Fbxl14

            18709

            -0.066

            -0.239

            Prim1

            18780

            -0.067

            -0.240

            Insig2

            18805

            -0.068

            -0.238

            B3gnt5

            18858

            -0.069

            -0.238

            Fam60a

            18963

            -0.072

            -0.240

            H2-M3

            18972

            -0.073

            -0.237

            Gja7

            18974

            -0.073

            -0.234

            Bex2

            18987

            -0.073

            -0.231

            Tk1

            19043

            -0.074

            -0.231

            1200015n20rik

            19109

            -0.076

            -0.231

            Clecsf5

            19114

            -0.077

            -0.228

            Ms4a7

            19141

            -0.078

            -0.226

            Cdca5

            19163

            -0.079

            -0.223

            C730042f17rik

            19180

            -0.079

            -0.220

            Trim25

            19194

            -0.080

            -0.218

            Efnb2

            19207

            -0.081

            -0.215

            Apex1

            19236

            -0.082

            -0.212

            Ddah2

            19243

            -0.082

            -0.209

            Bub1

            19262

            -0.083

            -0.206

            Nup43

            19263

            -0.083

            -0.203

            Rdh10

            19270

            -0.083

            -0.199

            2610201a13rik

            19330

            -0.086

            -0.199

            Rp2h

            19406

            -0.089

            -0.198

            Tnni1

            19407

            -0.089

            -0.195

            Myog

            19423

            -0.091

            -0.191

            Osmr

            19486

            -0.095

            -0.190

            Mmp9

            19524

            -0.097

            -0.188

            Tnnt1

            19525

            -0.098

            -0.184

            Fhod3

            19528

            -0.098

            -0.179

            D930038m13rik

            19537

            -0.099

            -0.175

            Nes

            19567

            -0.101

            -0.172

            Sbk1

            19571

            -0.102

            -0.168

            Dusp9

            19594

            -0.103

            -0.165

            Akr1b8

            19622

            -0.106

            -0.161

            Pdgfrb

            19663

            -0.110

            -0.158

            Tfrc

            19667

            -0.111

            -0.154

            Moxd1

            19670

            -0.111

            -0.149

            1810008k03rik

            19681

            -0.112

            -0.145

            Cpeb1

            19710

            -0.115

            -0.141

            6720475j19rik

            19716

            -0.116

            -0.136

            Ripk4

            19718

            -0.116

            -0.131

            Itga6

            19756

            -0.121

            -0.127

            Bmp5

            19775

            -0.124

            -0.123

            Lhx9

            19776

            -0.124

            -0.117

            Pkp2

            19797

            -0.129

            -0.113

            Chrna1

            19808

            -0.131

            -0.108

            Bhlhb2

            19837

            -0.142

            -0.103

            Gp49a

            19847

            -0.144

            -0.097

            Clecsf10

            19893

            -0.155

            -0.092

            Gch1

            19902

            -0.159

            -0.086

            D0h4s114

            19908

            -0.161

            -0.079

            Cxcl1

            19928

            -0.170

            -0.072

            Ch25h

            19946

            -0.178

            -0.065

            Mkrn3

            19988

            -0.228

            -0.057

            Ptprc

            20016

            -0.297

            -0.046

            Car6

            20017

            -0.298

            -0.032

            Nr1d2

            20031

            -0.368

            -0.017

            Evi2a

            20033

            -0.393

            0.001

            Plxnc1

            18075

            -0.055

            -0.286

            Cilp2

            18106

            -0.055

            -0.285

            Brca1

            18148

            -0.056

            -0.285

            Litaf

            18149

            -0.056

            -0.283

            Bc027246

            18154

            -0.056

            -0.281

            6820424l24rik

            18268

            -0.057

            -0.285

            Hrb

            18272

            -0.057

            -0.283

            Nnat

            18303

            -0.058

            -0.282

            P2ry12

            18329

            -0.058

            -0.282

            Cdca4

            18343

            -0.059

            -0.280

            6030404e16rik

            18367

            -0.059

            -0.279

            Tfec

            18429

            -0.060

            -0.280

            Nfe2l2

            18440

            -0.060

            -0.278

            Gtf2h2

            18467

            -0.061

            -0.277

            4930469p12rik

            18504

            -0.062

            -0.277

            Cul4b

            18535

            -0.062

            -0.276

            H2afy2

            18547

            -0.063

            -0.274

            1190002n15rik

            18582

            -0.063

            -0.274

            B430218l07rik

            18591

            -0.063

            -0.272

            Rgs18

            18607

            -0.064

            -0.270

            Frk

            18631

            -0.064

            -0.269

            Slc6a9

            18633

            -0.064

            -0.267

            Tgfbr2

            18687

            -0.065

            -0.267

            Tia1

            18802

            -0.068

            -0.270

            Lgr5

            18844

            -0.068

            -0.270

            Sgpp1

            18909

            -0.071

            -0.271

            Matn2

            18924

            -0.071

            -0.269

            Sox11

            18931

            -0.071

            -0.266

            Hus1

            18980

            -0.073

            -0.266

            D930015e06rik

            19028

            -0.074

            -0.266

            Apob48r

            19032

            -0.074

            -0.263

            Av344025

            19045

            -0.074

            -0.261

            Eno2

            19047

            -0.074

            -0.258

            2610024e20rik

            19053

            -0.075

            -0.256

            Chd1l

            19093

            -0.076

            -0.255

            Emr1

            19145

            -0.078

            -0.255

            Rgs4

            19200

            -0.081

            -0.254

            D030028o16rik

            19211

            -0.081

            -0.252

            Kif2c

            19216

            -0.081

            -0.249

            Ccl3

            19220

            -0.081

            -0.246

            Trim30

            19232

            -0.082

            -0.244

            Qrsl1

            19242

            -0.082

            -0.241

            Nr3c1

            19281

            -0.083

            -0.240

            Trip13

            19282

            -0.084

            -0.237

            Dna2l

            19317

            -0.085

            -0.236

            Tcf8

            19335

            -0.086

            -0.233

            Clecsf8

            19341

            -0.086

            -0.230

            Lyzs

            19422

            -0.090

            -0.231

            Palmd

            19475

            -0.095

            -0.230

            Tjp2

            19487

            -0.095

            -0.227

            D430019h16rik

            19493

            -0.096

            -0.224

            Sesn3

            19501

            -0.096

            -0.221

            Ereg

            19507

            -0.096

            -0.218

            Cx3cl1

            19523

            -0.097

            -0.215

            Fzd6

            19529

            -0.098

            -0.211

            Sod3

            19564

            -0.101

            -0.209

            Tnnt2

            19580

            -0.102

            -0.206

            Satb1

            19599

            -0.104

            -0.203

            Cd14

            19606

            -0.104

            -0.200

            Gbp2

            19607

            -0.104

            -0.196

            Tgfbi

            19609

            -0.105

            -0.192

            Chek1

            19652

            -0.109

            -0.190

            Tm4sf1

            19653

            -0.109

            -0.186

            Igf1

            19679

            -0.111

            -0.183

            Enpp1

            19695

            -0.113

            -0.180

            Slc15a3

            19704

            -0.114

            -0.176

            Pdpn

            19725

            -0.117

            -0.173

            Dkk1

            19747

            -0.119

            -0.169

            Slk

            19759

            -0.121

            -0.166

            Ankrd1

            19794

            -0.128

            -0.163

            Trp53bp1

            19801

            -0.129

            -0.158

            C79407

            19804

            -0.130

            -0.153

            2210010l05rik

            19809

            -0.131

            -0.149

            Eps8

            19815

            -0.133

            -0.144

            Dkk2

            19862

            -0.147

            -0.141

            Arhgap18

            19863

            -0.147

            -0.136

            Twist2

            19878

            -0.151

            -0.131

            Pcdha8

            19915

            -0.164

            -0.126

            Il4r

            19926

            -0.169

            -0.121

            Mdm1

            19931

            -0.172

            -0.115

            Phlda1

            19957

            -0.188

            -0.109

            Bhlhb5

            19960

            -0.192

            -0.102

            C130076o07rik

            19964

            -0.196

            -0.095

            5830411e10rik

            19974

            -0.207

            -0.088

            Ptpre

            19989

            -0.228

            -0.080

            Trib3

            19990

            -0.235

            -0.071

            9230117n10rik

            19994

            -0.241

            -0.062

            Pcdhb7

            19998

            -0.249

            -0.053

            Mmp3

            20001

            -0.252

            -0.044

            Cd34

            20009

            -0.274

            -0.034

            Thbd

            20022

            -0.310

            -0.023

            A830016g23rik

            20023

            -0.323

            -0.011

            Ahr

            20028

            -0.336

            0.001

            Rank = position of genes in the context of the ranked list of array genes

            RMS = the ranked metric score

            RES = the running enrichment score

            Four different patterns were observed when comparing DEX treatment and micromass differentiation data sets for gene enrichments scores (Figure 4B). First, similar gene sets were indeed enriched in both day 15 micromass and DEX-treated monolayer cultures, and core genes contributing to the normalized enrichment scores were similarly overlapping between the two data sets in results with low FDR. For example, ECM genes were enriched with both DEX treatment and the day 15 micromass phenotype. Other gene sets following this enrichment pattern included genes involved in integrin function, angiogenesis, catalytic activity, IGF related, adipocyte and cartilage, all of which have a precedent for being involved in chondrocyte maturation [28, 49, 80, 81]. The enrichment of angiogenic transcripts with DEX treatment was unexpected since DEX was shown to have anti-angiogenic roles in cartilage; however, upon closer examination of the genes contributing to the enrichment score, Vegf, which is thought to be a central angiogenic factor in endochondral ossification [82], was excluded from the core enrichment genes and had the lowest correlation with the DEX phenotype in that gene set. In contrast, Vegf was enriched in the growth factor data set which positively correlated with the vehicle control and not DEX treatment (Table 7).

            Gene sets associated with the actin cytoskeleton, tumour suppressors, structure, cytoplasmic genes, hepatocyte markers and dual specificity phosphatases (DUSPs) were enriched in the DEX data set and the phenotype positively correlated with day 3 of micromass culture. The identification of DUSPs was particularly interesting since DEX has been shown to induce genes encoding for these proteins [77, 83, 84]. DUSPS counteract the activation of MAP kinase pathways, known regulators of chondrocyte differentiation [85], and are thought to mediate DEX's anti-inflammatory functions and to influence hepatic gluconeogenesis [83, 86, 87].

            Additional comparisons identified genes that show enrichment in day 15 micromass cultures and downregulation with DEX treatment. These include the previously identified chemokines, cytokines and interleukins. A final trend in similarly enriched gene sets identified lists that were negatively correlated both with the DEX phenotype and day 15 of micromass cultures. Only transcriptional repressors and molecules involved in the extracellular signal-regulated kinase (ERK) pathway were identified. This pattern is consistent with DEX's anti-proliferative functions, as another study showed that DEX decreases ERK phosphorylation and thus cell cycle progression in a pre-osteoblast cell line [77]. Altogether this analysis shows that DEX regulation of growth plate chondrocyte differentiation is multifaceted. The patterns identified here are in agreement with a dual role of DEX in maintenance of the cartilage phenotype and delay in the cartilage-to-bone transition, as we suggested above.

            We also wanted to determine whether DEX target genes identified in the current study were similar to DEX-responsive genes identified in alternate studies, in different cell types [88]. Out of a total of twelve microarray studies evaluating the transcriptional effects of DEX treatment on a specific tissue or cell type, only ten genes were common to at least three of the chosen DEX studies. Specifically, bone morphogenetic protein 2 (Bmp2), delta sleep inducing factor 1 (Dsip1), beta-2 microglobulin (B2m), neuroepithelial cell transforming gene 1 (Net1), TNFAIP3 interacting protein 1 (Tnip1), bone marrow stromal cell antigen 2 (Bst2), B-cell leukemia/lymphoma 6 (Bcl6), nuclear factor of kappa light chain gene enhancer in B-cells inhibitor, alpha (Nfkbia), FK506 binding protein 5 (Fkbp5) and B-cell translocation gene 1, anti-proliferative (Btg1) were identified. It therefore appears that while DEX affects similar functional categories across various species, tissue types and experimental conditions, the individual genes that respond to DEX treatment are variable. These results also reinforce the paradigm that GC regulation is inextricably linked to its physiological context [8899].

            Analyses of GC response elements in dexamethasone target genes in chondrocytes

            Classical genomic GC action is thought to be mediated by a cytoplasmic GR that modifies transcription upon binding its cognate ligand and translocating to the nucleus. In the nucleus, the GR binds a GRE sequence. GR can both activate and repress transcription, depending on the GRE variant present in the regulatory regions of GC target genes [100]. Binding to composite GREs involves homodimerization of the GR to bind a non-palindromic consensus sequence comprised of two GR binding sites and is generally associated with transcriptional activation. In some instances, however, GR can function to block access or activity of transcription factors within promoter regions of certain genes, thus impeding transcription [101]. GR are also able to bind a modified GRE consisting of composite GRE half-sites, termed negative GREs, since they have documented roles in transcriptional repression. Variations on the genomic functions of GC include transcriptional regulation at the level of protein-protein interactions 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 with other co-activators and repressors to influence transcription indirectly [102, 103].

            The 100 most highly expressed probe sets with greatest enrichment in the DEX or vehicle-treated chondrocytes are shown in Figure 5. Probe sets identified in this analysis included both known cartilage markers and established DEX target genes such as Vegf, Ibsp, Bglap2 and Fkbp5 [49, 6366, 104, 105]. We examined the proximal promoter regions of three separate gene lists, the top 100 DEX-responsive transcripts generated by GSEA analysis (Figure 5), the 22 091 probe sets deemed expressed in primary chondrocyte cultures and the 1158 transcripts deemed differentially expressed between DEX and vehicle treated cultures by one-Way ANOVA. Specifically, we searched the 9990 base pairs upstream regulatory regions in this list for the composite GRE consensus sequence. We identified putative 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-regulated probe sets such as prostaglandin-endoperoxide synthase 2 (Ptgs2), phosphodiesterase 4A (Pde4), Vegf, Period homolog 1 (Per1) and Krüppel like factor 15 (Klf15) do not appear to contain a GRE in the first 10 kilobases and may by regulated by DEX via a GRE-independent mechanism, through a GRE that deviates from the consensus GRE sequence or through GREs at other locations in the gene.
            http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-8-205/MediaObjects/12864_2007_Article_918_Fig5_HTML.jpg
            Figure 5

            Heat 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 replicates 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).

            Examination of all lists generated similar results in that approximately 16–20% of all probes contained the consensus GRE. Consequently, we cannot exclude the presence of less conventional GRE loci in the transcripts, or the presence of GREs that deviate from the consensus sequence or are located outside the queried sequence. Since many of the genes affected at the 6 hr time point encode transcription factors, it is likely that a large proportion of the genes that only change after 24 hrs are regulated indirectly by DEX, through altered expression of these transcription factors and other regulatory proteins (e.g. phosphatases and cytokines, as discussed above).

            Functional analysis is required to unequivocally evaluate the contribution of GRE-dependent mechanisms to GC regulation in chondrocytes. In addition to the genomic functions of GC, non-genomic modes of GC regulation have been documented. Non-genomic mechanisms are thought to occur through specific and non-specific mechanisms. Specific non-genomic GC regulation occurs through the classical GR and its cytoplasmic heteroprotein complex or non-classical GRs such as membrane GR [106109]. Conversely, non-specific non-genomic mechanisms rely on the physiochemical properties of GC and the phospholipid bilayer (Buttgereit and Scheffold, 2002). Further, studies in which candidate molecules are selected and characterized in depth are imperative to discern the specific regulatory mechanisms occurring in chondrocytes.

            Conclusion

            This study elucidates the downstream transcriptional impact of pharmacological GC exposure on developing chondrocytes. We have identified a small subset of transcripts containing putative GREs in cartilage, but it appears that GRE-independent or indirect mechanisms of GC regulation also contribute to GC regulation in primary chondrocyte monolayer cultures. In addition, traditional microarray analysis methods and gene class testing point to a dual role for pharmacological GC doses in chondrocytes. DEX acts in a gene class-specific manner in cartilage in which it promotes the expression of ECM and metabolic transcripts necessary for maintaining the chondrocyte phenotype while simultaneously downregulating cytokines and growth factors which stimulate the cartilage to bone transition. Understanding the implications of gene expression changes and integrating them into the network of molecules controlling cartilage development continues to be challenging, but robust analytical methods will prove to be useful in constructing the networks of gene interactions and understanding the complex nature of GC signaling in the skeleton. The ultimate objective of this study will be to translate these findings into more efficacious therapeutic GCs.

            Methods

            Animals and Materials

            Timed-pregnant CD1 mice were purchased from Charles River Laboratories at embryonic day E15.5 mice (E15.5). Dexamethasone was obtained from Calbiochem and reconstituted in Dimethyl sulfoxide (DMSO, vehicle) according to the manufacturer's instructions. Cell culture materials and general chemicals were obtained from Invitrogen, Sigma or VWR unless otherwise stated.

            Primary cell culture and dexamethasone-treatment

            Tibiae, femurs and humeri were isolated from E15.5 mouse 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 penicillin/streptomycin and incubated at 37°C in a humidified 5% CO2 incubator overnight. The following morning media was removed and the bones placed in 4 ml of 0.25% trypsin-EDTA (Invitrogen) for 15 min at 37°C. Trypsin was subsequently replaced with 1 mg/ml collagenase P (Roche) in DMEM/10% fetal bovine serum (Invitrogen), and cells were incubated at 37°C with rotation at 100 rpm for 90 min. Following digestion, the cell suspension was centrifuged for 5 min at 1000 rpm, and the collagenase containing supernatant was decanted. Chondrocytes were resuspended in media containing 2:3 DMEM:F12, 10% fetal bovine serum, 0.5 mM L-glutamine, and penicillin/streptomycin (25 units/ml). Cells were seeded in 6-well NUNC plates at a density of 2.5 × 104 cells per ml and incubated overnight. Primary monolayer chondrocytes were treated with 10-7 M dexamethasone (DEX) or the DMSO control (vehicle) diluted in fresh media supplemented with 0.25 mM ascorbic acid (Sigma) and 1 mM β-glycerophosphate (Sigma) and incubated for up to 24 hrs. Micromass cultures were completed as previously described [50].

            Cell counting studies

            Chondrocytes were isolated and seeded in 24-well NUNC plates (Nunc Inc.) at a density of 16 000 cells/cm2. Cells were cultured, treated and enzymatically digested as described with some modifications. Collagenase digestion occurred for 5 minutes followed by mechanical digestion to liberate cells from the ECM. Cells were counted with a hemocytometer in triplicate with a minimum of 3 individual wells per treatment and three independent cell isolations.

            RNA isolations and quantitative real-time PCR

            All RNA protocols were completed as previously outlined [50]. Total RNA was isolated at 6 hrs and 24 hrs after treatment using the RNeasy mini extraction kit (Qiagen) according to the manufacturer's instructions. RNA quantity and integrity was assessed using the Bioanalyzer 2000 system (Agilent). Quantitative real-time polymerase chain reaction (qRT-PCR) amplification was completed using the ABI Prism 7900 Sequence Detection System (Applied Biosystems). Triplicate reactions were executed for each sample of each of three independent trials. The TaqMan one-step master mix kit (Applied Biosystems) with gene-specific target primers and probes were used for amplification. The collagen X (Col10a1) probe and primer set (forward primer 5'-ACGCCTACGATGTACACGTATGA-3', reverse primer 5'-ACTCCCTGAAGCCTGATCCA-3', 6-FAM-5'-AGTACAGCAAAGGCTAC-MGBNFQ) was designed with PrimerDesigner 2.0 software (Applied Biosystems) [79]. TaqMan GAPDH control reagents for house-keeping gene glyceraldehyde-3-phosphate dehydrogenase (Gapdh, forward primer 5'-GAAGGTGAAGGTCGGAGTC; reverse primer 5'-GAAGATGGTGATGGGATTTC; probe JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA) was used as an internal amplification 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 in accordance with the manufacturers directions. Amplified transcripts were quantified using the standard curve method, and the relative transcript abundance was determined by calculating the quotient of the gene of interest and equivalent Gapdh values.

            Microarray analysis

            Total RNA was extracted from control and DEX-treated cultures at 6 hr and 24 hr following treatment, in three independent experiments. RNA integrity and quantity was assessed using the Agilent 2000 Bioanalyzer system, and RNA samples were subsequently hybridized to the MOE 430 2.0 mouse chip from Affymetrix© containing 45 101 probe sets as described [50]. Bioanalysis, microarray hybridization, scanning and preliminary MAS 5.0 normalizations were completed at the London Regional Genomics Facility. Data were deposited in the GEO database (NCBI; accession number GSE7683).

            Data normalization

            Microarray data were pre-processed using the GC-RMA algorithm in Genespring GX*. Expression values were further filtered by retaining only those probe sets with expression values of at least 50 in at least 25% of all conditions, thus generating a list of 22 091 probe sets. To assess differential gene expression between treatments at both the 6 and 24 hr time points, a Welch ANOVA test with a p-value cut-off of 0.01 and a 5% false discovery rate (FDR) reduced the data to 1158 probe sets. Subsequent 1.5-, 5- and 10-fold change filters produced lists of 162, 21 and 7 probe sets for the 6 hr time point and 399, 53 and 19 probe sets for the 24 hr time point, respectively.

            The same data set was normalized in parallel using Robust Multichip Analysis using RMAEXPRESS software v.0.4.1 developed by B. Bolstad, University of California, Berkeley [110]. Background adjustment and quantile normalization parameters were selected for data processing. Logarithmically transformed expression values were used to implement Gene Set Enrichment Analysis (GSEA).

            Gene set enrichment analysis (GSEA)

            The GSEA algorithm was implemented with GSEA v2.0 software [51, 52]. Ranked expression lists were derived from RMAEXPRESS and GeneSpring GX® 7.3.1. Briefly, the GSEA algorithm ranks all array genes according to their expression under each experimental condition. The resulting ranked metric score (RMS) is therefore a function of the correlation between a gene's signal intensity, the experimental conditions in question and all other genes in the data set. An enrichment score (ES) is then calculated for an a priori gene list or gene set that is associated with a particular molecular classification. In our analysis, gene sets were created from different functional groupings, molecular classifications, tissues, and other microarray screens. A Ranked enrichment score (RES) which determines the extent to which a given gene from a gene set is represented at the extremes of the ranked gene list is then calculated. Specifically, this value is obtained by walking along the ranked list using a cumulative sum statistic which increases when a member of a particular gene set is found in the ranked gene list and is coordinately penalized when it does not appear in the gene set. A null distribution of ES is subsequently generated by permutation filtering to evaluate the statistical significance of the observed RES values. Permutation filtering randomly assigns 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 in gene set size. The false discovery rate (FDR) is then calculated relative to the NES values to determine the false-positive rate. Significant FDR and p-values were less than 25% and 0.001, respectively in accordance with GSEA recommendations.

            Gene set creation

            Gene sets were generated using the probe set search tool and the molecular function class of Gene Ontology annotations in GeneSpring GX. Additional gene sets were created using lists from pairwise comparisons between day 3 and 15 of a previously generated micromass data set (James et al., 2005), and publications that identified DEX target genes in other cartilage array screens, other tissue types and experimental systems. A total of 2119 probe sets showing a minimum 1.5-fold change in gene expression were used in the analysis. Probe set redundancy was eliminated in all gene sets using the CollapseDataset function in the GSEA program. All probe set identifiers were assimilated to the Human Genome Organization (HUGO) annotations. Probe sets lacking corresponding HUGO annotations were excluded from the analysis. Default parameters were used to execute the analysis and median values taken to represent the range of duplicated probe sets for a given gene. A total of 77 user-defined gene sets were generated from GeneSpring derived Gene Ontology annotations for various molecular classifications and probe sets of differentially expressed genes between days 3 and 15 of micromass culture (James et al., 2005).

            Glucocorticoid response element (GRE) analysis

            Putative GRE were identified with the GenespringGX mouse genome9999 application which allows sequences up to 9999 bp upstream of the transcriptional start sites of all annotated MOE4302.0 transcripts to be interrogated for transcription factor binding sites. The GR consensus sequence GGTACAnnntgttCT [111] was queried from 10 bp to 10 000 bp upstream of the transcriptional start sites of available probe sets. The GRE consensus sequence was screened against 10 748 probe sets derived from the list of 22 091 reliably expressed probe sets exhibiting homology to upstream regulatory regions annotated in the program. Only exact matches were retained for subsequent analyses out a total of 1,073,741,824 tests.

            Abbreviations

            DEX: 

            Dexamethasone

            GSEA: 

            gene set enrichment analysis

            RES: 

            ranked enrichment score

            RMS: 

            ranked metric score

            ES: 

            enrichment scores

            NES: 

            normalized enrichment score

            SOM: 

            self-organizing maps

            FDR: 

            false discovery rate

            GR: 

            glucocorticoid receptor

            Declarations

            Acknowledgements

            CGJ is supported by a doctoral award from the Canadian Institutes of Health Research (CIHR) and previously by an Ontario Graduate Scholarship in Science and Technology. V.U. is the recipient of a graduate scholarship 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.

            Authors’ Affiliations

            (1)
            CIHR Group in Skeletal Development and Remodelling, Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario
            (2)
            Group of Tissue Specific Hormone Action, Leibniz Institute for Age Research -Fritz Lipmann Institute
            (3)
            Department of Cellular & Physiological Sciences, University of British Columbia

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            © James et al. 2007

            This article is published under license to 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.

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