Suppression of Hyaluronan Synthesis Alleviates Inflammatory Responses in Murine Arthritis and in Human Rheumatoid Synovial Fibroblasts

Abstract

Objective. To clarify the roles of hyaluronan (HA) in joint inflammation and the process of joint destruction, using 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis, in a mouse model of collagen-induced arthritis (CIA) and in a monolayer culture of fibroblastlike synoviocytes (FLS) derived from patients with rheumatoid arthritis.Methods. DAB/1J mice were immunized with type II collagen. The effects of 4-MU were evaluated by the physiologic arthritis score, paw swelling, the histologic arthritis score, and expression of matrix metalloproteinase 3 (MMP-3) and MMP-13 in chondrocytes and synovial tissue. In vitro, the effect of 4-MU on messenger RNA and protein expression of MMP-1 and MMP-3 was determined. The effects of 4-MU on HA deposition and on serum/medium concentrations of HA were analyzed using biotinylated HA binding protein staining and an HA binding assay, respectively.Results. Treatment with 4-MU in mice with CIA dramatically decreased the severity of arthritis (based on the arthritis score), paw thickness, and histopathologic changes. MMP-3 and MMP-13 expression in chondrocytes and synovial cells was significantly inhibited by 4-MU in vivo. Treatment with 4-MU also inhibited MMP-1 and MMP-3 expression in tumor necrosis factor ␣-stimulated FLS, in a dose-dependent manner. The 4-MU-induced decreases in the serum HA concentration in mice with CIA and in "medium" and "pericellular" HA concentrations in cultured FLS support the contention that the inhibitory mechanism of 4-MU is mediated by HA suppression.Conclusion. Reduced disease activity induced by 4-MU in mice with CIA revealed HA to be a crucial regulator in the course of arthritis. Therefore, 4-MU is a potential therapeutic agent in arthritis, and its inhibitory mechanism is possibly mediated by suppression of HA synthesis.

Involvement of hyaluronan synthesis in ovarian follicle growth in rats

“…4-MU is known to inhibit HA synthesis by depletion of UDP-glucuronic acid and downregulation of HAS activity (Kakizaki et al 2004, Kultti et al 2009). At doses used in the present study, 4-MU alleviates inflammatory responses caused by excess HA synthesis in the collagen-induced arthritis mouse model in vivo and in vitro (Yoshioka et al 2013). We also observed that the glutamine analog DON, which inhibits glucosamine and consequent HA synthesis (Tempel et al 2000), acted on ovaries in the same manner as in vivo 4-MU.…”

“…Even when eCG was administered in combination with 4-MU or DON, HA production, normal follicle growth, and follicular avoidance of atresia were not observed. These two chemicals have been well known to reduce HA synthesis and have been frequently used for HA reduction studies in various cell lines and organs including ovary (Clark et al 1987, Tempel et al 2000, Kakizaki et al 2004, Kultti et al 2009, Tzuman et al 2010, Yoshioka et al 2013. On the other hand, these chemicals do not specifically exert effects on HASs, and therefore the relationship between follicular HA reduction and follicular atresia is still unclear.…”

Inhibition of Hyaluronan Synthesis Protects against Central Nervous System (CNS) Autoimmunity and Increases CXCL12 Expression in the Inflamed CNS

“…Vivo-We used 4MU, a well established and specific inhibitor of hyaluronan production (3,7,(13)(14)(15)(16)(17)20), to modulate hyaluronan synthesis. First, we confirmed the inhibitory influence of 4MU on hyaluronan synthesis by in vitro experiments.…”

“…In the present study, we assessed the effect of hyaluronan in EAE by inhibiting hyaluronan synthesis with 4-methylumbelliferone (4MU) administration (3,(13)(14)(15)(16)(17). 4MU inhibits hyaluronan synthesis in a dose-dependent manner by two mechanisms: depletion of cellular UDP-glucuronic acid and down-regulation of hyaluronan synthases 2 and 3 (14).…”

4-Methylumbelliferone Diminishes Catabolically Activated Articular Chondrocytes and Cartilage Explants via a Mechanism Independent of Hyaluronan Inhibition

“…Additionally, 4-MU effects a pronounced reduction of HA synthase 2 (HAS2) transcription but by mechanisms that are currently unknown (32)(33)(34). As a potent inhibitor of HA, 4-MU has been used to examine the role of HA in epithelial-mesenchymal transitions (35), collagen-induced arthritis (36), invasion and metastasis of osteosarcoma (37) and breast cancer cells (38), inflammation and autoimmunity (31), chondrogenic differentiation (39), myofibroblast differentiation (40,41), and keratinocyte activation (42). We have shown that 4-MU blocks the assembly of pericellular coats on bovine, rat, and mouse chondrocytes and diminishes the responsiveness of chondrocytes to bone morphogenetic protein-7 similar to results obtained by hyaluronidase treatment (43).…”