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GRNs driving cartilage and bone formation interact via averaging and synergism during cartilage maturation

ABSTRACT: Transcriptional control of any biological process occurs through the action of one to many gene regulatory networks (GRNs), but studies analyzing GRN interaction are lacking. To address this, we tested for the first time in vivo the hypothesis that two independent GRNs, which are active during formation of two discrete skeletal cell types (immature chondrocytes and osteoblasts), interact during differentiation of a third skeletal cell type (mature chondrocytes). These three cell types were isolated from the mouse embryo specifically using laser capture microdissection, and then RNA was extracted. Multiple analyses of corresponding RNA-seq data supported the hypothesis. Gene co-expression network analyses of differentially expressed genes suggested that one GRN containing the chondrogenic transcription factor Sox9 characterizes immature chondrocytes, one GRN containing the osteogenic transcription factor Runx2 characterizes osteoblasts, and both GRNs operate in mature chondrocytes. Indeed, mature chondrocytes differentially expressed fewer genes than the other cell types, consistent with the idea that overlapping actions of the immature chondrocyte and osteoblast GRNs regulate mature chondrocytes. Clustering analyses provided molecular insights into potential GRN interactions. Several genes in mature chondrocytes had expression levels that represented an averaging between levels in the immature chondrocyte and osteoblast. Interestingly, expression levels of one gene cluster, containing the hallmark mature chondrocyte genes Collagen type 10a1 and Indian hedgehog, suggested a synergistic interaction between immature chondrocyte and osteoblast GRNs. In addition to identifying novel genes expressed in mature chondrocytes, these results outline a novel in vivo experimental system through which to understand GRN organization and interaction.

ORGANISM(S): Mus musculus

PROVIDER: GSE110051 | GEO | 2018/03/01

REPOSITORIES: GEO

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Project description:Rheumatoid arthritis (RA) leads to progressive destruction of articular structures. Despite recent progress in controlling inflammation and pain, little cartilage repair has yet been observed. This in vitro study aims to determine the role of chondrocytes in RA-related cartilage destruction and antirheumatic drug-related regenerative processes. Human chondrocytes were three-dimensionally cultured in alginate beads. To determine the RA-induced gene expression pattern, human chondrocytes were stimulated with supernatant of RA synovial fibroblasts (RASF) and normal donor synovial fibroblasts (NDSF), respectively. To examine antirheumatic drug response signatures, human chondrocytes were stimulated with supernatant of RASF that have been treated with disease-modifying antirheumatic drugs (DMARD; azathioprine, sodium aurothiomalate, chloroquine phosphate, methotrexate), non-steroidal anti-inflammatory drugs (NSAID; piroxicam, diclofenac) or steroidal anti-inflammatory drugs (SAID; methylprednisolone, prednisolone). Genome-wide expression profiling with oligonucleotide microarrays was used to determine differentially expressed genes. Real-time RT-PCR and ELISA were performed for validation of microarray data. Following antirheumatic treatment, microarray analysis disclosed a reverted expression of 94 RA-induced chondrocyte genes involved in inflammation/NF-ÎºB signalling, cytokine/chemokine activity, immune response, proliferation/differentiation and matrix remodelling. Hierarchical clustering analysis showed that treatment of RASF with the DMARD azathioprine, gold sodium thiomalate and methotrexate resulted in chondrocyte gene expression signatures that were closely related to the âhealthyâ pattern. Treatment with the SAID methylprednisolone and prednisolone strongly reverted the RA-related chondrocyte gene expression, in particular the expression of genes involved in inflammation/NF-ÎºB and cytokine/chemokine activity. The NSAID piroxicam and diclofenac and the DMARD chloroquine phosphate had only moderate to marginal effects. Pathway analysis determined major mechanisms of drug action, for example pathways of cytokine-cytokine receptor interaction, TGF-Î²/TLR/Jak-STAT signalling and ECM-receptor interaction were targeted. This in vitro study provides a comprehensive molecular insight into the antirheumatic drug response signatures in human chondrocytes, thereby revealing potential molecular targets, pathways and mechanisms of drug action involved in chondrocyte regeneration. Thus, the present study may contribute to the development of novel therapeutic chondro-protective compounds and strategies. Experiment Overall Design: Drug-related suppression of gene expression in activated chondrocytes was determined by genome-wide microarray analysis. Chondrocytes were stimulated with supernatant of RASF and NDSF. Effect of treatment with DMARDs, NSAIDs and glucocorticoids was tested by treating RASF prior to collection of supernatant. Two RNA pools were analyzed for each group (RASF-stimulated NDSF stimulated and RASF-treated), each pool consisting of equal amounts of RNA from three different donors.

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GRNs driving cartilage and bone formation interact via averaging and synergism during cartilage maturation

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