4-Methylumbelliferone suppresses catabolic activation in anterior cruciate ligament-derived cells via a mechanism independent of hyaluronan inhibition

Abstract

Background: The anterior cruciate ligament (ACL) has a key role as a dynamic stabilizer of the knee joints, and ACL dysfunction caused by traumatic or degenerative rupture accelerates osteoarthritis progression. Thus, it is important to prevent the degenerative rupture of the ACL. 4-Methylumbelliferone (4-MU), a pre-approved drug, exerts anti-inflammatory effects in osteoarthritis chondrocytes. It was originally used as an inhibitor of hyaluronan synthesis in chondrocytes.

Methods: In this study, we investigated whether 4-MU affects the expression of catabolic factors, such as matrix metalloproteinase (MMP)-1, MMP-3, and interleukin (IL)-6, in ACL-derived cells and ACL explant cultures using immunohistochemistry, real-time RT-qPCR, and capillary western immunoassay. Furthermore, the hyaluronan concentration was evaluated using a colorimetric assay. Statistical analyses were conducted using analysis of variance for multi-group comparisons, followed by Tukey or Tukey-Kramer post hoc test.

Results: Our results revealed, for the first time, that 4-MU suppressed the IL-β-induced upregulation of pro-catabolic factors, such as MMP-1, MMP-3, and IL-6, in ACL-derived cells. This suppressive effect was also observed in the cultured ligament tissues in ex vivo experiments. 4-MU also reversed an enhanced dependence on glycolysis in IL-1β-activated ACL-derived cells. Furthermore, we found that the suppressive effects of 4-MU were exerted directly and not through the inhibition of hyaluronan synthesis.

Conclusions: We conclude that 4-MU could be an effective and useful treatment for knee osteoarthritis, owing to its anti-inflammatory effect on, not only chondrocytes but also on ligament cells.

Keywords: 4-Methylumbelliferone; Anterior cruciate ligament; Hyaluronan; Interleukin-6; Matrix metalloproteinase; Osteoarthritis.

Fig. 1

Effect of 4-methylumbelliferone (4-MU) on the anterior cruciate ligament (ACL)-derived cells stimulated with interleukin (IL)-1β. ACL-derived cells were treated with 0 to 10 ng/mL of IL-1β for 12 h. (A) To examine cytotoxicity of 4-MU, ACL-derived cells were treated with different concentrations of 4-MU for 12 h and cytotoxicity LDH Assay was performed (n = 3). (B) qRT-PCR was performed to determine the mRNA expression of matrix metalloproteinase 1 (MMP-1), MMP-3, and IL-6 (n = 3). ACL-derived cells were treated with or without 0.1 ng/mL IL-1β in the presence or absence of different concentrations of 4-MU for 12 h. (C) qRT-PCR (n = 6) and (D) capillary western immunoassay (n = 3) were performed to determine the MMP-1, MMP-3, and IL-6 mRNA and MMP-1, MMP-3, and IL-6 protein expression, respectively. (E) Statistical quantification of each protein expression. The values are presented as mean ± SD. *p < 0.05; **p < 0.01. n.s., not significant

Fig. 2

Matrix metalloproteinase (MMP)-1, MMP-3, and interleukin (IL)-6 immunohistochemistry in the anterior cruciate ligament (ACL) explants. ACL explants were cultured in a medium with or without 2.0 ng/mL IL-1β in the presence or absence of 1.0 mM 4-MU for 7 days. Here, we show the representative images of MMP-1, MMP-3, and IL-6 staining of the ACL explants. Scale bars represent 20 μm. Staining for MMP-1, MMP-3, and IL-6 indicated the highest protein expression in the 2.0 ng/mL IL-1β-treated explants (white arrows). The expression in explants that were co-treated with 2.0 ng/mL IL-1β and 1.0 mM 4-MU was substantially reduced (black arrows). (B) Statistical quantification for staining positivity of each antibody (n = 3). The values are presented as mean ± SD. *p < 0.05; **p < 0.01

Fig. 3

The effect of exogenous hyaluronan (HA) on inhibition of interleukin (IL)-1β by 4-methylumbelliferone (4-MU). (A) The HA concentrations in media (ng/mL) were determined using the HA ELISA (n = 6). Exogenous HA (1.0 mg/mL) was added to the cultured anterior cruciate ligament (ACL)-derived cells during incubation without or with 0.1 ng/mL IL-1β in the presence or absence of 1.0 mM 4-MU. After a 12 h incubation, the cells were lysed and analyzed by (B) real-time qRT-PCR (n = 6) and (C) capillary western immunoassay for determining the MMP-1, MMP-3, and IL-6 mRNA and matrix metalloproteinase (MMP)-1, MMP-3, and IL-6 protein expression, respectively. (D) Statistical quantification of each protein expression (n = 3). The values are presented as mean ± SD. *p < 0.05; **p < 0.01. n.s., not significant

Fig. 4

4-Methylumbelliferone (4-MU) reverses enhanced glycolysis dependence in the interleukin (IL)-1β-activated anterior cruciate ligament (ACL)-derived cells. ACL-derived cells were incubated for 12 h with or without IL-1β (0.1 ng/mL) in the presence or absence of 4-MU (1.0 mM). (A) The data from the ATP rate assay, which evaluates the contribution rates of the glycolysis (red bars; n = 3) and mitochondrial respiration (gray bars; n = 3) toward ATP production, are shown as stacked bars. (B) The concentration of l-lactate in the conditioned medium from the culture of the ACL-derived cells treated with IL-1β and 4-MU is indicated. To examine the 4-MU effect on early signaling events during interleukin (IL)-1β treatment of anterior cruciate ligament (ACL)-derived cells. (C) Phosphorylation of nuclear factor-kappa B (NF-κB) and AKT at 0-60 min in IL-1β treated ACL-derived cells. (D) Phosphorylation of NF-κB at 15 min and AKT at 5min in IL-1β treated ACL-derived cells in the absence or presence of 1 mM 4-MU. (E) For quantification, phospho NF-κB and AKT was normalized to total-NF-κB or AKT, respectively and presented as values relative to control (n = 3). The values are presented as mean ± SD. *p < 0.05; **p < 0.01