Esculetin inhibits cartilage resorption induced by interleukin 1alpha in combination with oncostatin M

Abstract

Objective: To determine if a new inhibitor, esculetin (EST), can block resorption of cartilage.

Methods: Interleukin 1alpha (IL1alpha, 0.04-5 ng/ml) and oncostatin M (OSM, 0.4-50 ng/ml) were used to stimulate the release of proteoglycan and collagen from bovine nasal cartilage and human articular cartilage in explant culture. Proteoglycan and collagen loss were assessed by dimethylmethylene blue and hydroxyproline assays, respectively. Collagenase levels were measured by assay of bioactivity and by enzyme linked immunosorbent assay (ELISA). The effects of EST on the expression of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the transformed human chondrocyte cell line T/C28a4 were assessed by northern blot analysis. TIMP-1 protein levels were assayed by ELISA. The effect of EST on the MMP-1 promoter was assessed using a promoter-luciferase construct in transient transfection studies.

Results: EST inhibited proteoglycan and collagen resorption in a dose dependent manner with significant decreases seen at 66 microM and 100 microM EST, respectively. Collagenolytic activity was significantly decreased in bovine nasal cartilage cultures. In human articular cartilage, EST also inhibited IL1alpha + OSM stimulated resorption and decreased MMP-1 levels. TIMP-1 levels were not altered compared with controls. In T/C28a4 chondrocytes the IL1alpha + OSM induced expression of MMP-1, MMP-3, and MMP-13 mRNA was reduced to control levels by 250 microM EST. TIMP-1 mRNA levels were unaffected by EST treatment. All cytokine stimulation of an MMP-1 luciferase-promoter construct was lost in the presence of the inhibitor.

Conclusion: EST inhibits degradation of bovine nasal cartilage and human articular cartilage stimulated to resorb with IL1alpha + OSM.

Figure 1

Esculetin (6,7-dihydroxycoumarin).

Figure 2

Esculetin (EST) inhibits collagen loss from bovine nasal cartilage stimulated to resorb with interleukin 1α (IL1α) and oncostatin M (OSM). Bovine nasal cartilage discs were cultured for 14 days with IL1α (1 ng/ml) in combination with OSM (10 ng/ml) ± EST (10-330 µmol/l). Media were harvested and replenished at day 7. Collagen release is expressed as a percentage of the total present in the explant. The cumulative collagen loss over 14 days of culture is presented. Results are expressed as mean (SD) (n=4). Statistical analyses compared collagen loss seen with IL1α + OSM alone to that seen with IL1α + OSM + EST. ***p<0.001.

Figure 3

Esculetin (EST) inhibits proteoglycan loss from bovine nasal cartilage. Bovine nasal cartilage discs were incubated for 14 days with interleukin 1α (IL1α) and oncostatin M (OSM) at the concentrations indicated ± EST (100 µmol/l). Media were harvested and replenished at day 7; media and cartilage were harvested at day 14. Proteoglycan loss is expressed as a percentage of the total present in each explant; the data illustrated are for proteoglycan loss by day 7 of culture. Results are mean (SD) (n=4). Statistical analyses compared proteoglycan loss seen with IL1α + OSM treatment to that seen with IL1α + OSM + EST for each set of cytokine concentrations as indicated by the bars. ***p<0.001, *p<0.05.

Figure 4

Esculetin (EST) inhibits interleukin 1α (IL1α) + oncostatin M (OSM) induced collagenolytic activity in bovine nasal cartilage. Bovine nasal cartilage discs were incubated for 14 days with IL1α (1 ng/ml) in combination with OSM (10 ng/ml) ± EST (10-330 µmol/l), with media changes at day 7. Media were harvested at day 14 and assessed by using the diffuse collagen fibril assay for active collagenolytic activity (A) and total collagenolytic activity by the inclusion of p-aminophenylmercuric acetate (0.7 mmol/l) in the assay (B). Statistical analyses compared the activity of IL1α + OSM treatment with that of IL1α + OSM + EST treatment (n=4). *p<0.05.

Figure 5

Esculetin (EST) inhibits collagen loss and collagenolytic activity up to 72 hours after cytokine stimulation of bovine nasal cartilage. Bovine nasal cartilage discs were cultured for 14 days with interleukin 1α (IL1α; 1 ng/ml) and oncostatin M (OSM; 10 ng/ml). EST (100 µmol/l) was added simultaneously with the cytokines or up to 120 hours thereafter. Media were harvested and replenished at day 7; media and cartilage were harvested at day 14. (A) Cumulative collagen loss over 14 days of culture. Collagenolytic activity was measured in day 7 media in the presence (B) or absence (C) of the metalloproteinase activator, p-aminophenylmercuric acetate (0.7 mmol/l). Statistical analyses of collagen loss and collagenolytic activity levels compared IL1α + OSM + EST (0 hours) with IL1α + OSM + EST added at time points thereafter (n=4). ***p<0.001, *p<0.05.

Figure 6

Esculetin (EST) inhibits interleukin 1α (IL1α) + oncostatin M (OSM) stimulated resorption of human articular cartilage. Pieces of human articular cartilage (2 mm³) were cultured with IL1α (5 ng/ml) in combination with OSM (50 ng/ml) ± EST (50-500 µmol/l). Media were harvested and replenished at days 7 and 14; media and cartilage were harvested at day 21. Proteoglycan and collagen release are presented as a percentage of the total. (A) Cumulative proteoglycan loss over 21 days of culture. (B) Cumulative MMP-1 levels over 21 days of culture. (C) Cumulative collagen loss over 21 days of culture. Statistical analyses compared the data for IL1α + OSM with that for the IL1α + OSM + EST treatments (n=4). ***p<0.001, **p<0.01.

Figure 7

Esculetin (EST) reduces the interleukin 1α (IL1α) + oncostatin M (OSM) induced expression of MMP-1, MMP-3, and MMP-13 mRNA in T/C28a4 chondrocytes. T/C28a4 chondrocytes were stimulated for 24 hours with IL1α (1 ng/ml) in combination with OSM (10 ng/ml) ± EST (100-500 µmol/l). Total RNA was harvested, northern blotted, and the membranes probed for MMP-1, MMP-3, MMP-13, and TIMP-1 mRNA. Glyceraldehyde-3-phosphate dehydrogenase was used to correct for RNA loading. The data are representative of three separate experiments.

Figure 8

Esculetin (EST) inhibits matrix metalloproteinase-1 (MMP-1) promoter activity. T/C28a4 cells were transiently transfected with an MMP-1/luciferase promoter construct. Two days after transfection, cells were incubated with interleukin 1α (IL1α; 1 ng/ml) and/or OSM (10 ng/ml) ± EST (100 µmol/l) for 24 hours. Cells were then harvested and luciferase activities measured, and normalised to the amount of DNA plasmid in each well. The results represent the mean (SD) (n=3) of a representative experiment performed three times.