Gene expression changes in damaged osteoarthritic cartilage identify a signature of non-chondrogenic and mechanical responses

Abstract

Objectives: Joint degeneration in osteoarthritis (OA) is characterised by damage and loss of articular cartilage. The pattern of loss is consistent with damage occurring only where the mechanical loading is high. We have investigated using RNA-sequencing (RNA-seq) and systems analyses the changes that occur in damaged OA cartilage by comparing it with intact cartilage from the same joint.

Methods: Cartilage was obtained from eight OA patients undergoing total knee replacement. RNA was extracted from cartilage on the damaged distal medial condyle (DMC) and the intact posterior lateral condyle (PLC). RNA-seq was performed to identify differentially expressed genes (DEGs) and systems analyses applied to identify dysregulated pathways.

Results: In the damaged OA cartilage, there was decreased expression of chondrogenic genes SOX9, SOX6, COL11A2, COL9A1/2/3, ACAN and HAPLN1; increases in non-chondrogenic genes COL1A1, COMP and FN1; an altered pattern of secreted proteinase expression; but no expression of major inflammatory cytokines. Systems analyses by PhenomeExpress revealed significant sub-networks of DEGs including mitotic cell cycle, Wnt signalling, apoptosis and matrix organisation that were influenced by a core of altered transcription factors (TFs), FOSL1, AHR, E2F1 and FOXM1.

Conclusions: Gene expression changes in damaged cartilage suggested a signature non-chondrogenic response of altered matrix protein and secreted proteinase expression. There was evidence of a damage response in this late OA cartilage, which surprisingly showed features detected experimentally in the early response of cartilage to mechanical overload. PhenomeExpress analysis identified a hub of DEGs linked by a core of four differentially regulated TFs.

Keywords: Cartilage; Osteoarthritis; PhenomeExpress; RNA-seq; Systems biology.

Fig. 1

Histological grading and GAG content of OA articular cartilage. Safranin O staining of the PLC (A) and DMC (B) of representative OA patient samples. Modified Mankin score of the articular cartilage (C) and GAG content of the cartilage tissue (D) obtained from the PLC and the DMC and used for RNA-seq analysis and an independent patient cohort for validation (n = 16). Images acquired using a [20×/0.80 Plan Apo] objective using the 3D Histech Pannoramic 250 Flash II slide scanner. Magnification× 5. ****P < 0.0001, **P < 0.01.

Fig. 2

Comparison of intact vs damaged OA cartilage transcriptome studies. Overlap of DEGs identified by the RNA-seq data and the two existing microarray datasets Snelling et al. and Ramos et al. (A). The log2 fold change of the 22 DEGs in the three compared datasets (B).

Fig. 3

PhenomeExpress analysis. Network analysis incorporating cross-species gene-phenotype associations, identified 23 differentially expressed networks based on direct protein–protein interactions in the damaged cartilage. Sub-networks linked to OA included; mitotic cell cycle (P = 0.0001) (A), regulation of transcription (P = 0.021) (B), Wnt signalling and calcium modulating pathway (P = 0.0102) (C), apoptotic processes (P = 0.0056) (D), negative regulation of blood coagulation (P = 0.0001) (E) and ECM organisation (P = 0.0001) (F). The fold change of the proteins is shown by the node colour and OA associated phenotype annotated proteins used to generate the sub-networks are shown with a black border.

Fig. 4

Multi-layered architecture of damaged OA cartilage signalling. Network showing regulatory interactions between the differentially expressed, predicted upstream TFs and the PhenomeExpress identified pathways in damaged OA cartilage. Transcriptional links between the four upstream TFs are shown with grey arrows. Regulatory links between the TFs and significantly upregulated target genes present in PhenomeExpress pathways are shown red, green, pink and blue for FOSL1, AHR, E2F1 and FOXM1 respectively. The PhenomeExpress pathways are named by the top enriched GO biological process term. Only PhenomeExpress pathways with at least two target genes present are shown.