EZH2 inhibition reduces cartilage loss and functional impairment related to osteoarthritis

Abstract

Histone methyltransferase EZH2 is upregulated during osteoarthritis (OA), which is the most widespread rheumatic disease worldwide, and a leading cause of disability. This study aimed to assess the impact of EZH2 inhibition on cartilage degradation, inflammation and functional disability. In vitro, gain and loss of EZH2 function were performed in human articular OA chondrocytes stimulated with IL-1β. In vivo, the effects of EZH2 inhibition were investigated on medial meniscectomy (MMX) OA mouse model. The tissue alterations were assayed by histology and the functional disabilities of the mice by actimetry and running wheel. In vitro, EZH2 overexpression exacerbated the action of IL-1β in chondrocytes increasing the expression of genes involved in inflammation, pain (NO, PGE2, IL6, NGF) and catabolism (MMPs), whereas EZH2 inhibition by a pharmacological inhibitor, EPZ-6438, reduced IL-1β effects. Ex vivo, EZH2 inhibition decreased IL-1β-induced degradation of cartilage. In vivo, intra-articular injections of the EZH2 inhibitor reduced cartilage degradation and improved motor functions of OA mice. This study demonstrates that the pharmacological inhibition of the histone methyl-transferase EZH2 slows the progression of osteoarthritis and improves motor functions in an experimental OA model, suggesting that EZH2 could be an effective target for the treatment of OA by reducing catabolism, inflammation and pain.

Figure 1

EZH2 increases IL-1 induced inflammation. Primary human chondrocytes were transfected with a vector of expression for EZH2 (pEZH2) or with empty vector. After 24 h, cells were treated with IL-1β (1 ng/ml) for 48 h. After treatment, RNA and proteins were extracted. (A) mRNA relative expression of EZH2 was determined by real-time RT-PCR. Data were normalized to IL-1 stimulated-chondrocytes, and are expressed as means ± SEM (n = 4). (B) EZH2 protein expression was analyzed by western blot. (C) and (D) PGE2 release in medium was determined by ELISA assay and Griess assay, respectively. Data are expressed as means ± SEM (n = 3). (E) mRNA relative expression of IL-6 was determined by RT-PCR. Data are expressed as means ± SEM (n = 4). *p value < 0.05.

Figure 2

EZH2 overexpression increases IL-1 induced expression and release of MMPs. Primary human chondrocytes were transfected and treated with IL-1β (1 ng/ml). MMP-1, 3 and 13 expression and release were determined by RT-PCR (A)–(C) and ELISA (D)–(F). Data are expressed as means ± SEM (n = 4). *p value < 0.05; **p value < 0.01.

Figure 3

EZH2 inhibition attenuates IL-1β-mediated inflammation. Primary human chondrocytes were treated with IL-1β (1 ng/ml) in the presence of EPZ-6438 (10 µM) for 48 h. PGE2 release in medium was determined by ELISA assay. mRNA relative expression of IL-6 was determined by RT-PCR. NO release was evaluated by Griess assay. Data are expressed as means ± SEM (n = 4). *p value < 0.05; **p value < 0.01.

Figure 4

EZH2 inhibition reduces IL-1β-induced cartilage degradation. (A-B) Primary human chondrocytes were treated with IL-1β (1 ng/ml) in the presence of EPZ-6438 (10 μM) or of its vehicule (DMSO, 0.1% (v/v)) for 48 h. (A) After treatment, RNA was extracted and medium collected. mRNA relative expression of MMPs was determined by real-time RT-PCR, and normalized to IL-1 stimulated-chondrocytes. Data are expressed as means ± SEM (n = 4). (B) MMP1, 3 and 13 release in medium was determined by ELISA assay. Data from ELISA are expressed as means ± SEM (n = 3). *p value < 0.05. **p value < 0.01. (C) Human articular cartilage explants were maintained in culture for 7 days in the presence or absence of EPZ-6438 and/or IL-1β. Explants were then sectioned and stained with safranin-O. Scale bare indicates 100 µm. Safranin-O intensity was quantified using Image J. *p value < 0.05.

Figure 5

EZH2 inhibition attenuates cartilage degradation in OA mice. (A) Time course of the experiments. (B) Osteoarthritis (OA) was surgically induced in mice. One, two and four weeks after OA induction, mice were injected with vehicle (0.1% (v/v) DMSO diluted in saline, 50 µl, n = 9) and EPZ-6438 (10 µM, 50 µl, n = 14). Eight weeks later, mice were euthanized and knee sections were stained with safranin O. Scale bars represent 500 µm and 100 µm. (C) Knee OARSI score. The cumulative score of tibia plateau and femoral condyle was used to score the entire articulation. Data are expressed as means ± SEM. *p value < 0.05.

Figure 6

EPZ-6438 decreases functional disability in OA mice. (A) Time course of the experiments. OA was surgically induced in mice at day 0. Then, mice were injected with vehicle (0.1% (v/v) DMSO diluted in saline, 50 µl, n = 9) or EPZ6438 (10 µM, 50 µl, n = 7) one and two weeks after OA induction. Behavioral tests were performed before OA induction (baseline), then 2–3 days after OA induction and injections. (B) and (C) Actimetry and running wheel. Data are expressed as means ± SEM. (D) OA chondrocytes were transfected with an EZH2 expression vector or incubated with EPZ-6438 in the presence of IL-1β. NGF mRNA relative expression was determined by real-time RT-PCR. Data are expressed as means ± SEM (n = 4). *p value < 0.05; **p value < 0.01.