LXR modulation blocks prostaglandin E2 production and matrix degradation in cartilage and alleviates pain in a rat osteoarthritis model

Abstract

Osteoarthritis (OA), the most common arthritic condition in humans, is characterized by the progressive degeneration of articular cartilage accompanied by chronic joint pain. Inflammatory mediators, such as cytokines and prostaglandin E(2) (PGE(2)) that are elevated in OA joints, play important roles in the progression of cartilage degradation and pain-associated nociceptor sensitivity. We have found that the nuclear receptor family transcription factors Liver X Receptors (LXRalpha and -beta) are expressed in cartilage, with LXRbeta being the predominant isoform. Here we show that genetic disruption of Lxrbeta gene expression in mice results in significantly increased proteoglycan (aggrecan) degradation and PGE(2) production in articular cartilage treated with IL-1beta, indicating a protective role of LXRbeta in cartilage. Using human cartilage explants, we found that activation of LXRs by the synthetic ligand GW3965 significantly reduced cytokine-induced degradation and loss of aggrecan from the tissue. Furthermore, LXR activation dramatically inhibited cytokine-induced PGE(2) production by human osteoarthritic cartilage as well as by a synovial sarcoma cell line. These effects were achieved at least partly by repression of the expression of ADAMTS4, a physiological cartilage aggrecanase, and of cyclooxygenase-2 and microsomal prostaglandin E synthase-1, key enzymes in the PGE(2) synthesis pathway. Consistent with our in vitro observations, oral administration of GW3965 potently alleviated joint pain in a rat meniscal tear model of osteoarthritis.

Fig. 1.

(A) Increased aggrecan release upon IL-1β stimulation of mouse-hip cartilage from LXRβ null mice. Hip cartilage from 3-week-old wild-type (wt) C57BL/6 and Lxrβ^−/− mice were collected and cultured in serum-free medium ± IL-1β (1.0 ng/mL) for 3 days. Treatment of mouse-hip cartilage with IL-1β alone for 3 days was sufficient to induce a significant increase of glycosaminoglycan (GAG) release to the culture medium. Amounts of GAG were measured using DMMB assay and expressed as the percentage of GAG in the media compared to the total GAG (the GAG released to the medium plus the GAG in the explant). No significant difference was observed in basal GAG release between the wt (n = 9) and Lxrβ^−/− (n = 9) groups. Cytokine-induced GAG release was significantly greater from cartilage from Lxrβ^−/− mice (n = 11) compared to wt mice (n = 10). Data points represent the mean values (±SEM) of the percentage of GAG release over 3 days of culturing. *P < 0.05, by one-way ANOVA (Bonferroni). (B) Increased PGE₂ production by mouse-hip cartilage from LXRβ null mice as compared with cartilage explants from wt mice upon IL-1β treatment. Comparable basal PGE₂ production was detected in cultures of cartilage explants from Lxrβ^−/− (n = 12) and wt (n = 9) mice. Cytokine-induced PGE₂ production from cartilage from Lxrβ^−/− mice (n = 11) was significantly greater than from wt mice (n = 11). Data points represent the mean values (±SEM) of PGE₂ levels in each group. *P < 0.05 by Mann–Whitney U test.

Fig. 2.

Protective effects of LXR agonist GW3965 against cytokine-induced loss of proteoglycan in human cartilage. (A) Cartilage explants from human OA joints were treated with IL-1β/OSM with/without GW3965. The explants were treated for 10 days, and culture media at days 2, 4, 6, 8, and 10 were collected to monitor GAG release at all time points. During the 10 days of culturing, the preweighed explants were exposed to IL-1β (1 ng/mL) plus OSM (5 ng/mL) with or without GW3965 at indicated concentrations (2, 1, and 0.5 μM). The total amount of aggrecan released to the culture medium (GAG release) and the total aggrecan content retained in the explants were measured. The percentage of cumulative GAG released into the media in each well was calculated. Values are means ± SEM (triplicate wells with each well containing ∼20 pieces of explants). *P < 0.05 by one-way ANOVA (Bonferroni) analysis. Data are representative of five separate cartilage samples. (B) LXR agonist GW3965 reduces the release of the aggrecanase-generated aggrecan AGEG fragment from cytokine-treated cartilage explants. Representative Western blot analysis of the culture medium collected on day 8 (left) and day 10 (right) from one donor is depicted. Equal volumes of culture medium samples were first concentrated and lyophilized and then analyzed by Western blot assay for AGEG-reactive fragments as described in Materials and Methods.

Fig. 3.

Inhibition of basal-level and cytokine-induced PGE₂ synthesis in human OA cartilage explants by LXR agonist GW3965. Human OA cartilage explants were treated with cytokines (IL-1β 1 ng/mL + OSM 5 ng/mL) with or without cotreatment with the specific LXR agonist GW3965 at indicated concentrations for 48 h. The accumulation of PGE₂ in each explant culture medium was measured. Bars indicate the mean ± SEM of PGE₂ production without cytokine stimulation (hatched bars in A and B) or following stimulation with IL-1β/OSM (IL-1β, 1 ng/mL; OSM, 5 ng/mL, open bars in B). ^# P < 0.05 versus cytokine-stimulated explants without GW3965 treatment; *P < 0.05 versus basal (DMSO). Data for one representative donor are shown from a total of eight human donors who provided consistently similar results.

Fig. 4.

Effects of LXR activation on the expression of ABCG1 ADAMTS4 and mPGES-1 (PTGES) genes in human cartilage. Real-time quantitative RT–PCR expression analysis of RNA samples from five donors. Human cartilage explants were pretreated with 2 μM GW3965 for 6 h and then stimulated with IL-1β/OSM (1 and 5 ng/mL, respectively) for an additional 18 h. The expression levels for each gene under various treatments are expressed as abundance relative to β-glucuronidase (GUSB) levels in the DMSO control samples. Relative expression levels of each gene with treatments in all five donors are shown as scatter plot. *P < 0.05 by paired Student’s t test.

Fig. 5.

LXR ligand GW3965 dose dependently inhibits IL-1β–induced PGE₂ production (A) and COX-2 expression (B) in SW982 cells. (A) A dose curve generated to show potent inhibition of PGE₂ production by GW3965 treament. (B) Relative expression levels of COX-2 evaluated by real-time PCR, using GAPDH as endogenous control. *P < 0.05 versus IL-1β–treated group.

Fig. 6.

Oral administration of the LXR ligand GW3965 effectively alleviates joint pain in a rat meniscal tear model of osteoarthritis. Meniscal tear surgery was performed on two groups (of male Lewis rats). With feed, dosing (20 mg/kg/day GW3965) was started right before the surgery and lasted for 13 weeks. HPWD was measure at 8 and 10 weeks. Shown are the results at the 10-week time point. Statistically significant differences were determined using one-way ANOVA (Bonferroni). (*P < 0.001 vs. vehicle). n = 20 (vehicle and GW3965 treatment) rats per group.