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Microarray of Dexamethasone-treated primary chondrocytes identifies downstream targets of glucocorticoid signalling

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 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 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. Conclusions: Together, these results provide insight into the mechanisms and major molecular classes functioning downstream of DEX in primary chondrocytes monolayers. 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 basis for subsequent functional studies. Keywords: Time course and pharmacological treatment

ORGANISM(S): Mus musculus

PROVIDER: GSE7683 | GEO | 2007/07/04

SECONDARY ACCESSION(S): PRJNA99433

REPOSITORIES: GEO

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Project description: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 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 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. Conclusions: Together, these results provide insight into the mechanisms and major molecular classes functioning downstream of DEX in primary chondrocytes monolayers. 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 basis for subsequent functional studies. Experiment Overall Design: Primary chondrocytes isolated from the tibiae, humeri and femurs of 15.5 day old mice are treated with 10EXP-7M DEX or an equal volume of the DMSO vehicle for 24 hrs. Total RNA is isolated after 6 hrs and 24 hrs of treatment. Samples from three independent experiments independent time courses were hybridized to Affymetrix MOE 430 2.0 mouse chips. Experiment Overall Design: Number of time points: 2 (6hr and 24 hr) Experiment Overall Design: Number of treatments: 2 (DEX and the vehicle control) Experiment Overall Design: Number of Samples: 3 replicates per time point per treatment Experiment Overall Design: Affymetrix chip: MOE 430 2.0 Experiment Overall Design: Cell type: Primary chondrocyte Experiment Overall Design: Tissue or origin: Tibiae, humerus, femur Experiment Overall Design: Species E15.5 mice Experiment Overall Design: Samples: Total RNA

Project description:Objective: Joint formation begins with the establishment of an interzone within the cartilaginous anlagen of the future skeleton and both Gdf5 and Erg are proposed as regulators of chondrocyte differentiation during and post interzone formation. The aim of this study was to examine the relationship between Gdf5 and Erg expression and downstream effects on chondrocyte gene expression. Design: Erg expression was identified in mouse knee joints at E13.5. Expression and microarray analyses were performed using micromass cultures of murine C3H10T1/2 mesenchymal cells undergoing induced chondrogenesis in the presence of absence of Gdf5 and Erg. Results: At E13.5, Erg expression was found to surround epiphyseal chondrocytes and span the interzone up to the intermediate zone. Erg splice forms were expressed in micromass cultures, and their expression profile was altered by the addition of recombinant Gdf5 depending on the stage of differentiation. Overexpression of Erg-010 resulted in a downregulation of Col2a1 and Col10a1. Microarray analysis following Erg-010 overexpression identified two potential downstream targets, Ube2b and Osr2, which were also differentially regulated by Gdf5. Conclusion: Erg regulation by Gdf5 in mesenchymal cells in vitro is dependent on the stage of chondrogenesis, and its expression in vivo demarcates chondrocytes that are not destined to be consumed by endochondral ossification. Functionally, Erg expression causes downregulation of Col2a1 and Col10a1 expression and this effect is potentially mediated by Osr2, which is a known regulator of chondrocyte differentiation. The identification of Ube2b as a putative downstream target of Erg-010 suggests that it may contribute to the regulation of the ubiquitination pathway and thereby BMP2 signaling, which is essential for normal knee joint development. RNA from overexpression experiments was extracted as described above. Total RNA quality was confirmed using a Bioanalyzer 2100 (Agilent). Labelled targets were generated from total RNA and hybridized to GeneChip Mouse Genome 430 2.0 arrays (Affymatrix) (Genomics Centre, University of Manchester). The raw fluorescence intensity values were normalized using a Robust Multi-array Average approach using dChip (V2005)

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Microarray of Dexamethasone-treated primary chondrocytes identifies downstream targets of glucocorticoid signalling

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