Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of mouse 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. 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

ORGANISM(S): Mus musculus

SUBMITTER: Claudine James

PROVIDER: E-GEOD-7683 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Publications

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

James Claudine G CG Ulici Veronica V Tuckermann Jan J Underhill T Michael TM Beier Frank F

BMC genomics 20070701

<h4>Background</h4>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 ...[more]

PMID: 17603917

Similar Datasets

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. Keywords: Time course and pharmacological treatment

Project description:A variety of cell cultures models and in vivo approaches have been used to study gene expression during chondrocyte differentiation. The extent to which the in vitro models reflect bona fide gene regulation in the growth plate has not been quantified. In addition, studies that evaluate global gene expression changes among different growth plate zones are limited. To address these issues, we completed a microarray screen of three growth plate zones derived from manually segmented embryonic mouse tibiae. Classification of genes differentially expressed between each respective growth plate zone, functional categorization as well as characterization of gene expression patterns, cytogenetic loci, signaling pathways and functional motifs confirmed documented data and pointed to novel aspects of chondrocyte differentiation. Parallel comparisons of the microdissected tibiae data set to our previously completed micromass culture screen further corroborated the suitability of micromass cultures for modeling gene expression in chondrocyte development. The micromass culture system demonstrated striking similarities to the in vivo microdissected tibiae screen; however, the micromass system was unable to accurately distinguish gene expression differences in the hypertrophic and mineralized zones of the growth plate. These studies will allow us to better understand zone-specific gene expression patterns in the growth plate. Ultimately, this work will help define both the genomic context in which genes are expressed in the long bones and the extent to which the micromass culture system is able to recapitulate chondrocyte development in endochondral ossification. Experiment Overall Design: Tibiae from 15.5 day old mouse embryos were isolated and partitioned into three distinct zones. Total RNA was isolated from each segment and the individual segments pooled within a litter. Experiment Overall Design: Samples were hybridized to Affymetrix MOE 430 2.0 mouse chips for analysis. Four independent trials were executed for each zone. Experiment Overall Design: Number of time points: 1 Experiment Overall Design: Number of treatments: 0 Experiment Overall Design: Number of Samples: 4 replicates per zone Experiment Overall Design: Affymetrix chip: MOE 430 2.0 Experiment Overall Design: Tissue or origin: Tibiae Experiment Overall Design: Species E15.5 mice Experiment Overall Design: Samples: Total RNA

Project description:While the hypothalamo-pituitary-adrenal axis (HPA) activates a general stress response by increasing glucocorticoid (Gc) synthesis, biological stress resulting from infections triggers the inflammatory response through production of cytokines. The pituitary gland integrates some of these signals by responding to the pro-inflammatory cytokines IL6 and LIF and to a negative Gc feedback loop. The present work used whole-genome approaches to define the LIF/STAT3 regulatory network and to delineate cross-talk between this pathway and Gc action. Genome-wide ChIP-chip identified 3 449 STAT3 binding sites, whereas 2 396 genes regulated by LIF and/or Gc were found by expression profiling. Surprisingly, LIF on its own changed expression of only 85 genes but the joint action of LIF and Gc potentiated the expression of more than a thousand genes. Accordingly, activation of both LIF and Gc pathways also potentiated STAT3 and GR recruitment to many STAT3 targets. Our analyses revealed an unexpected gene cluster that requires both stimuli for delayed activation: 83% of the genes in this cluster are involved in different cell defense mechanisms. Thus, stressors that trigger both general stress and inflammatory responses lead to activation of a stereotypic innate cellular defense response. Experiment Overall Design: 1) Pituitary corticotrophs cell line (AtT-20) response to glucocorticoids and LIF: Experiment Overall Design: AtT-20 cells mRNA extracted after treatment with PBS as control, LIF for 18h, Dex+LIF for 3h and Dex+LIF for 18h. Each treatment were made in duplicate. [GSM471246-GSM471253] Experiment Overall Design: 2) Pituitary corticotrophs cell line (AtT-20) response to LIF after 3h treatment: Experiment Overall Design: AtT-20 cells mRNA extracted after treatment with PBS as control or LIF for 3h. Each treatment were made in duplicate. [GSM471254-GSM471257] Experiment Overall Design: 3) Pituitary corticotrophs cell line (AtT-20) response to Dex after 3h treatment: Experiment Overall Design: AtT-20 cells mRNA extracted after treatment with PBS as control or Dex for 3h. Each treatment were made in duplicate. [GSM471258-GSM471261] Experiment Overall Design: 4) Pituitary corticotrophs cell line (AtT-20) response to Dex after 18h treatment: Experiment Overall Design: AtT-20 cells mRNA extracted after treatment with PBS as control or Dex for 18h. Each treatment were made in duplicate. [GSM471262-GSM471269]

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)

Dataset Information

Transcription profiling of mouse dexamethasone-treated primary chondrocytes identifies downstream targets of glucocorticoid signalling

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure