Metalloproteinase and inhibitor expression profiling of resorbing cartilage reveals pro-collagenase activation as a critical step for collagenolysis

Abstract

Excess proteolysis of the extracellular matrix (ECM) of articular cartilage is a key characteristic of arthritis. The main enzymes involved belong to the metalloproteinase family, specifically the matrix metalloproteinases (MMPs) and a group of proteinases with a disintegrin and metalloproteinase domain with thrombospondin motifs (ADAMTS). Chondrocytes are the only cell type embedded in the cartilage ECM, and cell-matrix interactions can influence gene expression and cell behaviour. Thus, although the use of monolayer cultures can be informative, it is essential to study chondrocytes encapsulated within their native environment, cartilage, to fully assess cellular responses. The aim of this study was to profile the temporal gene expression of metalloproteinases and their endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), reversion-inducing cysteine-rich protein with Kazal motifs (RECK), and alpha2-macroglobulin (alpha2M), in actively resorbing cartilage. The addition of the pro-inflammatory cytokine combination of interleukin-1 (IL-1) + oncostatin M (OSM) to bovine nasal cartilage induces the synthesis and subsequent activation of pro-metalloproteinases, leading to cartilage resorption. We show that IL-1+OSM upregulated the expression of MMP-1, -2, -3, -9, 12, -13, -14, TIMP-1, and ADAMTS-4, -5, and -9. Differences in basal expression and the magnitude of induction were observed, whilst there was no significant modulation of TIMP-2, -3, RECK, or ADAMTS-15 gene expression. IL-1+OSM downregulated MMP-16,TIMP-4, and alpha2M expression. All IL-1+OSM-induced metalloproteinases showed marked upregulation early in the culture period, whilst inhibitor expression was reduced throughout the stimulation period such that metalloproteinase production would be in excess of inhibitors. Moreover, although pro-collagenases were upregulated and synthesized early (by day 5), collagenolysis became apparent later with the presence of active collagenases (day 10) when inhibitor levels were low. These findings indicate that the activation cascades for pro-collagenases are delayed relative to collagenase expression, further confirm the coordinated regulation of metalloproteinases in actively resorbing cartilage, and support the use of bovine nasal cartilage as a model system to study the mechanisms that promote cartilage degradation.

Figure 1

Profiling aggrecanase gene expression relative to aggrecanolysis in resorbing cartilage. Bovine nasal cartilage chips were cultured in medium ± IL-1+OSM (1 and 10 ng/ml, respectively) for 14 days. At day 7, medium was removed and the cartilage replenished with identical reagents. Cartilage and medium were harvested at days 0, 1, 3, 5, 7, 8, 10, 12, and 14. Each time point and condition were performed in triplicate. As a measure of proteoglycan, the levels of GAG released into the media from unstimulated (control) and IL-1+OSM-stimulated cartilage were assayed; cumulative GAG release is shown (n = 3). RNA was extracted from the cartilage, and ADAMTS-1, -4, -5, -9, and -15 gene expression was determined by real-time polymerase chain reaction (n = 3) as described in Materials and methods. The data are presented relative to 18S. Values are the mean ± standard error of the mean. ◇ = control; ▲ = IL-1+OSM. ADAMTS, a disintegrin and metalloproteinase domain with thrombospondin motifs; GAG, glycosaminoglycan; IL-1, interleukin-1; OSM, oncostatin M.

Figure 2

Profiling collagenase gene expression, collagenase activity, and collagenolysis in resorbing cartilage. Bovine nasal cartilage chips were cultured in medium ± interleukin-1 (IL-1) + oncostatin M (OSM) for 14 days exactly as described in the legend to Figure 1. As a measure of collagen, the levels of hydroxyproline (OHPro) released into the media from unstimulated (control) and IL-1+OSM-stimulated cartilage were assayed (n = 3); cumulative OHPro release is shown. Active collagenase activity in the media was assayed using the ³H-acetylated collagen diffuse fibril assay. Aminophenylmercuric acetate (0.67 mM) was used to activate pro-collagenases in order to measure the total collagenase activity (pro + active). RNA was extracted from cartilage, and matrix metalloproteinase (MMP) -1, -13, and -14 gene expression was determined by real-time polymerase chain reaction (n = 3) as described in Materials and methods. The data are presented relative to 18S. Values are the mean ± standard error of the mean. ◇ = control; ▲ = IL-1+OSM.

Figure 3

Profiling gelatinase gene expression and gelatinolytic activity in resorbing cartilage. Bovine nasal cartilage chips were cultured in medium ± interleukin-1 (IL-1) + oncostatin M (OSM) for 14 days exactly as described in the legend to Figure 1. RNA was extracted from cartilage, and matrix metalloproteinase (MMP)-2 and -9 gene expression determined by real-time polymerase chain reaction (n = 3) as described in Materials and methods. The data are presented relative to 18S. As a measure of gelatinase activity, the culture media were analysed by gelatin zymography. Values are the mean ± standard error of the mean. ◇ = control; ▲ = IL-1+OSM.

Figure 4

Profiling other matrix metalloproteinases (MMPs) in resorbing cartilage. Bovine nasal cartilage chips were cultured in medium ± interleukin-1 (IL-1) + oncostatin M (OSM) for 14 days exactly as described in the legend to Figure 1. RNA was extracted from cartilage, and MMP-3, -12, and -16 gene expression determined by real-time polymerase chain reaction (n = 3) as described in Materials and methods. The data are presented relative to 18S. Values are the mean ± standard error of the mean. ◇ = control; ▲ = IL-1+OSM.

Figure 5

Profiling metalloproteinase inhibitor gene expression and inhibitory activity in resorbing cartilage. Bovine nasal cartilage chips were cultured in medium ± IL-1+OSM for 14 days exactly as described in the legend to Figure 1. RNA was extracted from cartilage, and TIMP-1, -2, -3, and -4, RECK, and α₂M gene expression determined by real-time polymerase chain reaction (n = 3) as described in Materials and methods. The data are presented relative to 18S. Inhibitory activity was assayed in the culture media by the addition of samples to a known amount of active matrix metalloproteinase-1 (MMP-1) in the diffuse fibril assay (n = 3). Values are the mean ± standard error of the mean. ◇ = control; ▲ = IL-1+OSM. *p < 0.05 using the Student's t test. α₂M, alpha 2 macroglobulin; IL-1, interleukin-1; OSM, oncostatin M; RECK, reversion-inducing cysteine-rich protein with Kazal motifs; TIMP, tissue inhibitor of metalloproteinase.

Figure 6

Relative differential expression of MMPs, ADAMTS, and metalloproteinase inhibitors in resorbing cartilage. Bovine nasal cartilage chips were cultured in medium ± IL-1+OSM for 14 days exactly as described in the legend to Figure 1. RNA was extracted from the cartilage, and metalloproteinase and inhibitor gene expression were determined by real-time polymerase chain reaction as described in Materials and methods. The mean 2^-ΔCT of each gene (where ΔC_T is calculated as [C_T gene - C_T 18S]) was used as a measure of relative gene expression to allow simultaneous comparisons. The heat map was generated using GeneSpring GX 7.3 (Agilent Technologies, Palo Alto, CA, USA) with the expression range set at 0.025 (high), 5 × 10^-6 (normal), and 1 × 10^-10 (low) arbitrary units. ADAMTS, a disintegrin and metalloproteinase domain with thrombospondin motifs; C_T, cycle threshold; IL-1, interleukin-1; MMP, matrix metalloproteinase; OSM, oncostatin M.