The regulation of human MMP-13 by licofelone, an inhibitor of cyclo-oxygenases and 5-lipoxygenase, in human osteoarthritic chondrocytes is mediated by the inhibition of the p38 MAP kinase signalling pathway (original) (raw)

Abstract

Background: MMP-13 is one of the most important metalloproteases (MMP) involved in osteoarthritis. Licofelone, a novel dual inhibitor of cyclo-oxygenases (COX) and 5-lipoxygenase (5-LOX), can modulate MMP-13 production in human osteoarthritis chondrocytes.

Objective: To evaluate the impact of licofelone on MMP-13 expression/production, promoter, and major MAP kinase signalling pathways and transcription factors.

Methods: Human osteoarthritis chondrocytes were stimulated by interleukin 1ß (IL1ß) and treated with or without: licofelone (0.3, 1, or 3 µg/ml); NS-398 (10 µM; a specific COX-2 inhibitor); or BayX-1005 (10 µM; a specific 5-LOX inhibitor). MMP-13 synthesis was determined by specific enzyme linked immunosorbent assay, and expression by real time polymerase chain reaction. The effect of licofelone on the MMP-13 promoter was studied through transient transfection; dexamethasone (10–7 M) was used as comparison. The effect on IL1ß induced MMP-13 signalling pathways was determined using specific ELISA for phosphorylated MAP kinases and transcription factors.

Results: Licofelone dose dependently inhibited the IL1ß stimulated production and expression of MMP-13. NS-398 and BayX-1005 had very little effect. Licofelone also inhibited MMP-13 transcription on each of the promoter constructs used. The licofelone inhibition was comparable to that obtained with dexamethasone. Licofelone had no effect on phosphorylated p44/42 or JNK1/2; however, it decreased phosphorylated c-jun and inhibited phosphorylated p38, CREB, and AP-1 activity.

Conclusions: Licofelone inhibited MMP-13 production under proinflammatory conditions on human osteoarthritis chondrocytes, through inhibition of the p38/AP-1 pathway and the transcription factor CREB. This may explain some of the mechanisms whereby licofelone exerts its positive effect on osteoarthritic changes.

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Figure 1.

Figure 1

Interleukin (IL) 1ß dose and time response study of matrix metalloprotease 13 (MMP-13) production (A) and expression (B) in the presence or absence of licofelone. Human osteoarthritis chondrocytes were incubated for (A) 24 or 72 hours, or (B) 24 hours with or without 1–100 pg/ml IL1ß or 3 µg/ml licofelone. MMP-13 production was determined in the conditioned medium using a specific enzyme linked immunosorbent assay (ELISA) (A), and MMP-13 expression was determined by real time polymerase chain reaction (B). (C) Licofelone dose and time response study of MMP-13 production. Human osteoarthritis chondrocytes were incubated for 24 to 96 hours in the absence or presence of 100 pg/ml IL1ß and 0.3, 1.0, or 3.0 µg/ml licofelone or NS-398 at 10 µM and Bay-X-1005 at 10 µM. MMP-13 production was determined in the conditioned medium using a specific ELISA. Data are expressed as means of (A) four, (B) six to seven, and (C) four independent experiments. Error bars = SEM; p values indicate significant differences from the respective controls.

Figure 2.

Figure 2

Functional analysis of the matrix metalloprotease 13 (MMP-13) promoter in human osteoarthritis chondrocytes. Three different constructs of the MMP-13 promoter fused to a luciferase reporter are shown in schematic representation (left). Transfection was carried out in the presence of 100 ng/ml PMA with or without licofelone at 3 µg/ml or dexamethasone at 10–7 M. The cells were also co-transfected with pCMV-ß-galactosidase. Data are expressed as the mean percentage of six to seven independent experiments. Error bars = SEM; p values indicate significant difference from the control.

Figure 3.

Figure 3

Effect of licofelone on cell signalling pathways. (A) Phosphorylated forms of p44/42, JNK1/2, and p38. (B) Phosphorylated forms of CREB and c-jun. (C) AP-1 data from the electrophoretic mobility shift assay (EMSA). Specificity of the EMSA binding was assayed through competition of the oligonucleotides with 200-fold of excess unlabelled AP-1 oligonucleotides (competitor) in the presence of the nuclear extract of interleukin (IL) 1ß (100 pg/ml) stimulated chondrocytes. The top panel is a representative EMSA and the lower panel is a histogram of the independent experiments. Human osteoarthritis chondrocytes were incubated for (A) and (B) 15 to 120 minutes or (C) 24 hours with or without 100 pg/ml IL1ß in the presence or absence of 3 µg/ml licofelone. Data are calculated as time-fold expression of the unstimulated control, which was given the arbitrary unit of 1. Data are expressed as the mean of four independent experiments for (A) and (B), and three for (C). Error bars = SEM; p values indicate significant differences. Significant differences between unstimulated and IL1ß stimulated cells were: *p<0.04 for the phosphorylated p44/42 at 45 to 60 minutes; **p<0.03 for phosphorylated CREB at 15 to 60 minutes; and ***p<0.01 for phosphorylated p38 at 15 to 60 minutes. Statistical differences between IL1ß induced phosphorylated p38 and CREB were as indicated in the figure.

Selected References

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