Sequential alterations in catabolic and anabolic gene expression parallel pathological changes during progression of monoiodoacetate-induced arthritis

Abstract

Chronic inflammation is one of the major causes of cartilage destruction in osteoarthritis. Here, we systematically analyzed the changes in gene expression associated with the progression of cartilage destruction in monoiodoacetate-induced arthritis (MIA) of the rat knee. Sprague Dawley female rats were given intra-articular injection of monoiodoacetate in the knee. The progression of MIA was monitored macroscopically, microscopically and by micro-computed tomography. Grade 1 damage was observed by day 5 post-monoiodoacetate injection, progressively increasing to Grade 2 by day 9, and to Grade 3-3.5 by day 21. Affymetrix GeneChip was utilized to analyze the transcriptome-wide changes in gene expression, and the expression of salient genes was confirmed by real-time-PCR. Functional networks generated by Ingenuity Pathways Analysis (IPA) from the microarray data correlated the macroscopic/histologic findings with molecular interactions of genes/gene products. Temporal changes in gene expression during the progression of MIA were categorized into five major gene clusters. IPA revealed that Grade 1 damage was associated with upregulation of acute/innate inflammatory responsive genes (Cluster I) and suppression of genes associated with musculoskeletal development and function (Cluster IV). Grade 2 damage was associated with upregulation of chronic inflammatory and immune trafficking genes (Cluster II) and downregulation of genes associated with musculoskeletal disorders (Cluster IV). The Grade 3 to 3.5 cartilage damage was associated with chronic inflammatory and immune adaptation genes (Cluster III). These findings suggest that temporal regulation of discrete gene clusters involving inflammatory mediators, receptors, and proteases may control the progression of cartilage destruction. In this process, IL-1β, TNF-α, IL-15, IL-12, chemokines, and NF-κB act as central nodes of the inflammatory networks, regulating catabolic processes. Simultaneously, upregulation of asporin, and downregulation of TGF-β complex, SOX-9, IGF and CTGF may be central to suppress matrix synthesis and chondrocytic anabolic activities, collectively contributing to the progression of cartilage destruction in MIA.

Figure 1. Progression of MIA at the distal femoral ends by macroscopic, microscopic, and µCT analyses.

Right knees of rats were given an intra-articular injection of MIA on day 0, and distal ends of right femurs examined on post-injection days 5 (Grade 1 damage, MIA5), 9 (Grade 2 damage, MIA9) and 21 (Grade 3–3.5 damage, MIA21) and compared to saline-injected sham control (Cont). Macroscopic view of condyles, patellar grooves of cartilage, histology, and subchondral bone imaging by µCT of: (a, b) Cont femur showing smooth surface, (c) normal histology and no bone lesions on the femoral condyles and patellar grove and (d) lack of lesions in the subchondral bone (Movie S1); (e, f) MIA5 cartilage showing superficial abrasions on the condyles (black arrows) and patellar groove (white arrows), (g) superficial fibrillation (black arrow), chondrocyte clustering and disorientation (blue arrow), and (h) no bone lesions in µCT images (Movie S2); (i, j) MIA9 cartilage exhibiting lesions at the apexes of condyles (black arrow) and ridges of the patellar groove (white arrow), (k) thinning of cartilage, matrix and cell loss above the tidal layer with large disarrayed chondrocytes (black arrow), and some multinucleated chondrocytes (blue arrow), subchondral bone marrow/fibrous tissue extension in the cartilage typical of Grade 2 damage (white arrow), and (l) scattered subchondral bone lesions on the femoral condyles and patellar groove in µCT images (Movie S3); (m, n) MIA21 cartilage exhibiting increased lesions and damage on the condyles (black arrows) and patellar groove and ridges (white arrow), (o) delamination of surface, full depth cartilage lesions and denuded cartilage layer at some places (black arrow), and (p) increased subchondral bone lesions on the femoral condyles and patellar groove in µCT images (Movie S4). Each figure shows representative right femur from separate rats from each group (n = 10). Arrows indicate cartilage damages. The distal ends of femurs showing 360° µCT projection can be found in Movie files S1 to S4.

Figure 2. Transcriptome-wide microarray analysis of cartilage from Cont, MIA5, MIA9, or MIA21 afflicted joints.

(A) PCA analysis showing reproducible overall gene expression in the articular cartilage from the right knee joint of 3 separate rats from Cont, MIA5, MIA9, or MIA21. (B) Overall gene expression profiles of articular cartilage from 3 separate rats in each experimental group as compared to Cont. Hierarchical clustering representing the transcripts that were significantly (p<0.05) and differentially up- or downregulated at one or more time points by more than two-fold change. Note the maximal changes in overall gene expression occurred in MIA5, followed by MIA 21 and MIA9 as compared to gene expression in cont cartilage.

Figure 3. Partition clustering of significantly regulated genes.

Partition clustering analysis of the genes that showed two fold or greater changes in their expression at one or more time points (p<0.05). The graphs represent 10 most regulated genes in each cluster. Identification of five gene clusters that exhibited maximal upregulation on day 5 (Grade 1 damage) followed by their downregulation (Cluster I); upregulation on day 9 (Grade 2 damage) followed by their downregulation (Cluster II); upregulation in a sustained manner showing maximal expression on day 21 (Grade 3–3.5 damage, Cluster III); downregulation of genes on day 5 followed by their upregulation (Cluster IV); and downregulation of genes on day 9 followed by their upregulation (Cluster V). Detailed description of these genes is given in Tables 1, 2, 3, 4, 5, and 6, and in Tables S1, S2, S3, S4, and S5.

Figure 4. Confirmation of salient gene expression by rt-PCR.

Quantitative rt-PCR analyses of specific transcripts of genes from articular cartilage obtained from Cont, MIA5, MIA9, or MIA21. Two genes from Cluster I (A; Ctss and Il1b), Custer II (B; Mmp12 and Ccr1), Cluster III (C; Alox5 and Vcam1), Cluster IV (D; Cilp and Sox9), and Cluster V (E; Col9a1 and Col2a1) were analyzed to verify the data obtained by microarray analysis (n = 5, * p<0.05 as compared to Cont, ** p<0.01 as compared to Cont, # p<0.05 as compared to MIA5, ## p<0.01 as compared to MIA5, † p<0.05 as compared to MIA9, †† p<0.01 as compared to MIA9, ‡ p<0.05 as compared to MIA21, ‡‡ p<0.01 as compared to MIA21).

Figure 5. Distribution of genes in each cluster according to their functions.

Relative distribution of genes in each cluster subdivided according to their functions. Cell division, genes involved in cell division, proliferation, apoptosis; Growth factors, genes for growth factors and their receptors; GF reg, growth factor regulatory molecules and transcription factors; Inflammation, cytokines, chemokines and their receptors; Inflam reg, inflammatory mediators, signaling molecules, transcription factors, and regulators; ECM, extracellular matrix proteins; ECM reg, Proteases, regulators of ECM synthesis and breakdown; Others genes involved in cell metabolism, transporters and ion channels and genes of unknown function (Tables S1, S2, S3, S4, and S5).

Figure 6. Molecular networks generated from the genes in each cluster by Ingenuity Pathways Analysis.

The molecular networks generated from genes in: (A) Cartilage with Grade 1 damage (Cluster I) – Immunological disease network, showing upregulation of genes associated with acute/innate immune response; (B) Cartilage with Grade 1 damage (Clusters IV) - Skeletal & muscular development and function network, showing downregulation of transcription factors and growth factors associated with matrix synthesis. The symbols under individual mediators are defined in Figure 8B. Red, green, and white colors represent upregulation, downregulation and no regulation as compared to cont cartilage, respectively. The shading of each color represents fold change in gene expression; dark, higher changes and light lower changes.

Figure 7. Molecular networks generated from the genes in each cluster by Ingenuity Pathways Analysis.

The molecular networks generated from genes in: (A) Cartilage with Grade 2 damage (Cluster II) – Inflammatory response/Immune cell trafficking network, showing upregulation of genes associated with chronic inflammation and immune cell trafficking; (B) Cartilage with Grade 2 damage (Clusters V) – Skeletal and muscular disease network showing suppression of genes for growth factors and major matrix proteins. The symbols under individual mediators are defined in Figure 8B. Red, green, and white colors represent upregulation, downregulation and no regulation as compared to cont cartilage, respectively. The shading of each color represents fold change in gene expression; dark, higher changes and light lower changes.

Figure 8. Molecular networks generated from the genes in each cluster by Ingenuity Pathways Analysis.

The molecular networks generated from genes in: (A) Cartilage with Grade 3–3.5 damage (Cluster III) - Inflammatory disease network showing upregulation of many genes involved in immune suppression and adaptation. Each cluster is based on the genes that were significantly up or downregulated (p<0.05, over ±2-fold change) in articular cartilage from Cont, MIA5, MIA9, and MIA21 specimens. The symbols under individual mediators are defined in (B). Red, green, and white colors represent upregulation, downregulation and no regulation as compared to cont cartilage, respectively. The shading of each color represents fold change in gene expression; dark, higher changes and light lower changes.

Figure 9. Schematic presentation of collective catabolic and anabolic gene regulation during the progression of MIA.

Grade 1 damage in the cartilage was associated with induction of genes required for acute inflammation and innate immunity, broad specificity proteases, and cell cycle/division and suppression of genes for proteoglycan synthesis. Grade 2 damage in the cartilage was associated with induction of gene for NF-κB signaling cascade, inflammatory mediators/cytokines, metallopeptidases, and immune trafficking, and suppression of growth factors and collagens. Grade 3–3.5 damage in the cartilage exhibited upregulation of anti-inflammatory genes, and simultaneous reduction in the suppression of matrix-associated proteins and growth factors as compared to cartilage with Grade 1 or Grade 2 damage. Collective and sequential up and down regulation of these gens may be important in the cartilage damage during the progression of MIA.