Dexamethasone Attenuates the Expression of MMP-13 in Chondrocytes through MKP-1

Abstract

Mitogen-activated protein kinase phosphatase-1 (MKP-1) is upregulated in inflammation and reduces the activity of proinflammatory mitogen-activated protein kinases (MAP kinases) by dephosphorylation. MAP kinases are intracellular signaling pathways that mediate the cellular effects of proinflammatory cytokines. In the present study, we investigated the effects of the glucocorticoid dexamethasone on the expression of catabolic enzymes in chondrocytes and tested the hypothesis that these effects are mediated through MKP-1. Dexamethasone was found to significantly attenuate the expression of matrix metalloproteinase (MMP)-13 in human OA chondrocytes as well as in chondrocytes from MKP-1 WT mice, but not in chondrocytes from MKP-1 KO mice. Dexamethasone also increased the expression of MKP-1 in murine and human OA chondrocytes. Furthermore, p38 MAP kinase inhibitors significantly attenuated MMP-13 expression in human OA chondrocytes, while JNK MAP kinase inhibitors had no effect. The results indicate that the effect of dexamethasone on MMP-13 expression in chondrocytes was mediated by an MKP-1 and p38 MAP kinase-dependent manner. These findings, together with previous results, support the concept of MKP-1 as a protective factor in articular chondrocytes in inflammatory conditions and as a potential drug target to treat OA.

Keywords: MKP-1; MMP-13; chondrocyte; dexamethasone; inflammation; osteoarthritis.

Figure 1

Dexamethasone attenuated the expression of MMP-13 mRNA (A) and protein (B) in primary chondrocytes from MKP-1 WT mice, but not in chondrocytes from MKP-1 KO mice. Chondrocytes were treated with IL-1β (100 pg/mL) alone or together with dexamethasone (1 μM) for 24 h. The mRNA levels of MMP-13 were measured with quantitative RT-PCR and normalized against GAPDH mRNA levels. The MMP-13 protein levels were determined with ELISA. MMP-13 expression levels in IL-1β-treated samples of each genotype were set at 100%, and all other values are expressed in relation to those values. The results are expressed as mean + SEM, n = 8. Two-way ANOVA followed by Tukey’s post-test was performed; **** p < 0.0001, ns: not significant.

Figure 2

Dexamethasone increased the expression of MKP-1 in IL-1β-stimulated primary murine (A) and human OA (B–D) chondrocytes. (A–C) Primary murine and human OA chondrocytes were treated with IL-1β (100 pg/mL) alone or together with dexamethasone (1 μM) for 90 min. In (A,B), the mRNA levels of MKP-1 were measured with qRT-PCR and normalized against GAPDH mRNA levels. In (C), protein levels of MKP-1 and actin were determined by Western blotting. MKP-1 protein expression levels were normalized against actin levels. In (A–C), normalized MKP-1 expression levels in IL-1β-stimulated samples were set as 100%, and all other values are expressed in relation to those values. The results are expressed as mean + SEM, n = 4–6. Analysis of variance followed by Bonferroni post-test was performed; * p < 0.05, ** p < 0.01, *** p < 0.001. In (D), one representative Western blot result is shown.

Figure 3

Dexamethasone (A,B) and p38 MAP kinase inhibitors (C,D) reduced the expression of MMP-13 mRNA and protein in human OA chondrocytes. In A and B, the chondrocytes were treated with IL-1β (100 pg/mL) alone and together with dexamethasone (1 μM) for 24 h. In (C,D), the chondrocytes were treated with IL-1β (100 pg/mL) alone and together with the selective p38 MAP kinase inhibitors SB203580 (1 µM) or BIRB796 (100 nM), or with the selective JNK inhibitors SP600125 (10 µM) or JNK inhibitor VIII (1 µM) for 24 h. The mRNA levels of MMP-13 were measured with quantitative RT-PCR and normalized against GAPDH mRNA levels. MMP-13 protein levels were determined with ELISA. MMP-13 expression levels in IL-1β-treated samples were set at 100%, and all other values are presented in relation to those values. The results are given as mean + SEM, n = 8 (A,B) or n = 5 (C,D). Samples were obtained from eight (A,B) or five (C,D) patients with OA, and the experiments were carried out in duplicate. Repeated measures ANOVA followed by Bonferroni post-test was performed; **** p < 0.0001, ns = not significant.

Figure 4

Schematic presentation of the mechanism of action of dexamethasone in chondrocytes according to the present study. Dexamethasone enhances the expression of MKP-1, leading to dephosphorylation (i.e., inactivation) of p38 MAP kinase. Decreased activity of p38 MAP kinase attenuates MMP-13 expression, resulting in decreased cartilage matrix degradation.