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    • The activity of MMPs is regulated at the level of

    2023-12-28

    The activity of MMPs is regulated at the level of gene transcription or by the enzymatic activation of latent enzymes. Inflammatory cytokines such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α and IL-6 are elevated in synovial fluid, synovium and cartilage in patients with OA, and these cytokines are related to the production of MMPs in articular cartilage under pathologic conditions [1]. Transcription factors such as activator protein-1 (AP-1), PEA3, RUNX2 and nuclear factor (NF)-κB regulate the expression of MMPs [10]. The AP-1 binding site is an enhancer 17 alpha hydroxylase located near the TATA box in the promoter regions of most MMP family member genes [11,12]. Because mutations of the AP-1 site completely inhibit the induction of MMP expression, it is likely that the expression of this family of enzymes is AP-1 dependent [13,14]. AP-1 contains members of the Fos and Jun families, which form either c-Fos/c-Jun heterodimers or c-Jun/c-Jun homodimers. c-Fos/AP-1 directly controls expression by binding to the AP-1 site on the promoters of inflammatory cytokine and MMP genes [11,14,15]. Previous studies designed and synthesized a selective c-Fos/AP-1 inhibitor using 3D pharmacophore modeling based on the crystal structure of the AP-1-DNA complex to inhibit the c-Fos/c-Jun heterodimer [[16], [17], [18]]. One of these studies showed that the small molecule c-Fos/AP-1 inhibitor, T-5224, inhibits most members of the MMP family including MMP-3, 9, and 13, as well as IL-1β, and resolves type II collagen-induced arthritis [16]. We have been investigating the effects of T-5224 in musculoskeletal diseases [16,19]. We found that T-5224 prevents intervertebral disc degeneration in a mouse explant culture and a rat needle-puncture model [19]. Here, we further focused on the effects of c-Fos/AP-1 in articular cartilage, through upstream regulation of cartilage degrading proteinases and inflammatory cytokines. We hypothesized that selective inhibition of c-Fos/AP-1 may be an effective therapeutic target for inhibiting the progression of OA. We therefore analyzed the effects of T-5224 in human articular chondrocytes and investigated whether orally administered T-5224 prevents cartilage destruction in a mouse model of OA induced by destabilization of the medial meniscus (DMM).
    Methods
    Results To clarify the effect of T-5224 in human articular chondrocytes in vitro, we investigated the mRNA expression of MMPs, tissue inhibitors of metalloproteinases (TIMPs) and inflammatory cytokines by real-time RT-PCR. T-5224 inhibited the mRNA expression of MMPs including MMP-1, 3, and 13, and inflammatory cytokines such as IL-1β, TNF-α and IL-6 in IL-1β-stimulated human chondrocytes in a dose-dependent manner (Fig. 1a and b). TIMPs are endogenous inhibitors of MMPs [23], but T-5224 only increased TIMP-3 mRNA expression at a dose of 40 μmol/l (Fig. 1c). We created an OA model by DMM in mice, and orally administered T-5224 once a day (Fig. 2a) to assess its effect on the OA pathology. The DMM surgical instability model displayed mild mechanical stress and gradually developed OA changes. The OA changes therefore did not become apparent until 8 weeks after surgery. Safranin-O staining in the vehicle-treated group showed cartilage degradation from the superficial zone to the mid zone at 12 weeks. Cartilage loss partly extending to the tidemark became apparent at 16 weeks. In contrast, treatment with the c-Fos/AP-1 inhibitor substantially inhibited the progression of cartilage degeneration (Fig. 2b). Development of OA was quantified according to the recommended semi-quantitative OARSI grading system in the mouse. The OA histological grade was significantly higher in the vehicle-treated group than in the T-5224-treated group at 12 and 16 weeks after surgery. Furthermore, there was no significant difference between the T-5224-treated group and no-surgery group at 8, 12 and 16 weeks (Fig. 2c). These results indicate that the progression of cartilage destruction was almost completely abolished following treatment with T-5224.