Liver X receptor regulates rheumatoid arthritis fibroblast-like synoviocyte invasiveness, matrix metalloproteinase 2 activation, interleukin-6 and CXCL10

Abstract

Fibroblast-like synoviocyte (FLS) invasiveness correlates with articular damage in rheumatoid arthritis (RA), yet little is known about its regulation. In this study we aimed to determine the role of the nuclear receptor liver X receptor (LXR) in FLS invasion. FLS were isolated from synovial tissues obtained from RA patients and from DA rats with pristane-induced arthritis. Invasion was tested on Matrigel-coated chambers in the presence of the LXR agonist T0901317, or control vehicle. FLS were cultured in the presence or absence of T0901317, and supernatants were used to quantify matrix metalloproteinase 1 (MMP-1), MMP-2, MMP-3, interleukin-6 (IL-6), tumor necrosis factor-α and C-X-C motif chemokine ligand 10 (CXCL10). Nuclear factor-κB (NF-κB) (p65) and Akt activation, actin cytoskeleton, cell morphology and lamellipodia formation were also determined. The LXR agonist T0901317 significantly reduced DA FLS invasion by 99% (P ≤ 0.001), and RA FLS invasion by 96% (P ≤ 0.001), compared with control. T0901317-induced suppression of invasion was associated with reduced production of activated MMP-2, IL-6 and CXCL10 by RA FLS, and with reduction of actin filament reorganization and reduced polarized formation of lamellipodia. T0901317 also prevented both IL-1β-induced and IL-6-induced FLS invasion. NF-κB (p65) and Akt activation were not significantly affected by T0901317. This is the first description of a role for LXR in the regulation of FLS invasion and in processes and pathways implicated both in invasion as well as in inflammatory responses. These findings provide a new rationale for considering LXR agonists as therapeutic agents aimed at reducing both inflammation and FLS-mediated invasion and destruction in RA.

Figure 1

LXR and the LXR-inducible gene SCD1 are expressed in FLS. (A) LXRα and LXRβ expression in RA fibroblast–like synoviocytes is reduced by IL-1β (10 ng/mL) treatment. Pre-treatment with the LXR agonist T0901317 (10 μmol/L) prevented the IL-1β–induced reduction of LXRα (four FLS cell lines from different RA patients). Levels of LXRβ were not significantly affected by IL-1β treatment. (B) Levels of the LXR-inducible gene SCD1 were low at baseline and were not affected by IL-1β treatment. Pretreatment with the LXR agonist T0901317 significantly increased the expression of SCD1 by 26-fold, demonstrating that LXR is responsive to the synthetic agonist (^#P = 0.0014, t test; n = 7). (C) Western blot showing that both LXRα and LXRβ proteins are expressed in FLS, but levels are not changed by IL-1β treatment in the presence or absence of T0901317 during 24-h and 72-h periods. Vinculin was used as the loading control for protein normalization (representative of experiments done with five different DA FLS cell lines). No stim, no stimulation.

Figure 2

The LXR agonist significantly reduced FLS invasion. (A) The synthetic LXR agonist T0901317 significantly reduced the number of invading DA FLS in a dose-dependent manner by 64% (1 μmol/L) and 99% (10 μmol/L) (P < 0.001, Mann-Whitney test; mean ± standard deviation [SD]; n = 8). (B) Numbers of invading FLS from patients with RA were also significantly reduced by 99% with T0901317 at 10 μmol/L, compared with vehicle-treated (DMSO) controls (P ≤ 0.001, Mann-Whitney test; mean ± SD; n = 7). (C) MTT assay showing that T0901317 10 μmol/L was not toxic and did not cause increased cell death over a 24 h period, the same period of time used for the invasion assays.

Figure 3

Treatment with the LXR agonist reduces RA FLS production of active MMP-2. Supernatants from six different RA FLS cell lines (four are shown) cultured on Matrigel were used in a zymogram to determine the amount of MMP-2 in the presence or absence of the LXR agonist T0901317 10 μmol/L (D, DMSO; T, T0901317).

Figure 4

Morphologic changes induced by T0901317 in FLS. (A) T0901317 prevents morphologic changes required for FLS invasion. Insets A–C: DA FLS treated with control vehicle (DMSO) have a linearized-fusiform shape, with thick and longitudinal actin fibers, polarized formation of lamellipodia, and phospho-FAK colocalization with lamellipodia. Insets D–F: T0901317 DA FLS have a round, nonfusiform shape, with thinner actin fibers, and with either no lamellipodia formation, or a nonpolarized formation of lamellipodialike structures. These lamellipodialike structures do not colocalize with phospho-FAK. (Red, phospho-FAK; green, phalloidin–FITC–actin staining; images are representative of four different FLS cell lines). (B) Immunofluorescence morphologic scoring of FLS. DA FLS cultured on glass slides in the presence or absence of T0901317 10 μmol/L were scored for actin cytoskeleton characteristics, cell shape, lamellipodia location and phospho-FAK localization (see Methods for details). T0901317 significantly reduced all parameters compared with control (*P < 0.05; ^#P = 0.00005; ^¶P = 0.0036, t test; mean ± SD; n = 4).

Figure 5

The LXR agonist T0901317 reduces the expression of proinflammatory mediators and MMP-3 in DA and RA FLS. (A) Treatment of DA FLS with IL-1β 10 ng/mL caused a significant increase in the expression of IL-1β mRNA by 5-fold, which was completely prevented by T0901317. (B) IL-1β induced a 161-fold increase in mRNA levels of IL-6, and T0901317 reduced that to 43-fold, or a 73% reduction compared with IL-1β alone. (C) IL-1β induced a 25-fold increase in the expression of MMP-3, and T0901317 reduced that to 16-fold, compared with control, or a 36% reduction. (D, E) Levels of IL-6 in supernatants of RA FLS cultured overnight on Matrigel were significantly reduced by 74% with T0901317 treatment. (F, G) Levels of CXCL10 in supernatants of RA FLS cultured overnight on Matrigel were significantly reduced by 60% with T0901317 treatment. *P ≤ 0.0005; ^#P = 0.004; ^¶P = 0.01 (t test). No stim, no stimulation.

Figure 6

The LXR agonist T0901317 blocks IL-1β– and IL-6–induced FLS invasion. Treatment of DA FLS with IL-1β (10 ng/mL) or IL-6 (10 ng/mL) significantly increased cell invasion by 154% and 179%, respectively, compared with controls (white bar). Treatment with T0901317 (1 μmol/L) prevented the cytokine-induced increased invasion, suggesting that LXR interferes with IL-1β and IL-6 activity. T0901317 treatment reduced FLS invasion to levels below the control group, suggesting that factors other than IL-1β, IL-6 and their receptors are involved in the LXR-mediated suppression of invasion (^#P = 0.022; *P = 0.017; mean ± standard error of the mean; t test; n = 5 different FLS cell lines).