Background Activated fibroblast-like synoviocytes (FLS) are key effector cells in the joint in rheumatoid arthritis (RA). Local FLS proliferation is responsible for synovial hyperplasia, a key feature of the RA synovium correlating with disease activity. PDGF and IL-1b are known FLS mitogens. LBH is a transcription regulator and tumor suppressor, recently identified as a RA risk gene. We have demonstrated that LBH regulates FLS proliferation and that LBH expression is regulated by growth factors and by epigenetic mechanisms. Methotrexate (MTX) is still the first-line treatment of RA but the target cells and mechanism of action of the low dose used in rheumatic diseases is largely unknown. Increased expression of cell cycle checkpoint genes and modified DNA methylation in immune cells have recently been described.
Objectives The aim of this study was to investigate the effects of MTX on PDGF and IL1b-induced FLS proliferation in vitro and in particular on the expression of LBH, cell cycle genes (CDKN1A/p21 and CCND1/cyclinD1) and on genes regulating DNA methylation (DNMTs) in order to further understand the pharmacodynamics of this drug in RA and to identify novel markers for drug response.
Methods Primary FLS from RA patients and from patients with osteoarthritis (OA) were plated on day 0 in DMEM complete, pre-treated 24 hours with MTX or control medium day 1, and stimulated with 20ng/ml PDGF+2 ng/ml IL-1b with or without 1 uM MTX in DMEM with 1% FBS for 24–48 hours starting day 2. Cells were then harvested for qPCR for gene expression and flowcytometry for cell cycle analysis.
Results Stimulating RA-FLS cultures (n=3) with PDGF+IL-1b for 24 hours, pushed 24,5±3,5% cells into G2/M phase compared to 3,4±0,8% in unstimulated controls. Interestingly, treating PDGF+IL1b stimulated FLS with MTX, significantly inhibited cell cycle progression (4,6±1,9% in G2/M phase, p=0,02). PDGF+IL-1b stimulation of FLS for 24 hours reduced LBH mRNA expression. However, in the presence of 1uM MTX the LBH mRNA expression was significantly higher in RA-FLS (3,2±0,5 fold, p=0.002, n=5) and in OA-FLS (2,2±0,5 fold, p=0,02, n=5) after PDGF+IL-1b stimulation compared to untreated controls. In addition, MTX treatment strikingly increased the CDKN1A expression 14,3±4,4 fold (p=0,006) of treated vs untreated stimulated FLS. Furthermore, we found that 1 uM MTX restored and increased a lowered DNMT1 mRNA expression to 144±12% after PDGF+IL1b stimulation. There were no significant effects of MTX on CCND1 or DNMT3a expression at investigated time points.
Conclusions Therapeutic doses of MTX reduce mitogen induced FLS proliferation and significantly revert mitogen-induced reduction of LBH and p21 expression in RA FLS. MTX restores expression of DNMT1 suggesting that MTX might regulate gene expression and proliferation by affecting the epigenome.
Ekwall, A.K., et al., The Rheumatoid Arthritis Risk Gene LBH Regulates Growth in Fibroblast-like Synoviocytes. Arthritis Rheumatol, 2015. 67(5): p. 1193–202.
Spurlock, C.F., 3rd, et al., Methotrexate increases expression of cell cycle checkpoint genes via JNK activation. Arthritis Rheum, 2012. 64(6): p. 1780–9.
Cribbs, A.P., et al., Methotrexate Restores Regulatory T Cell Function Through Demethylation of the FoxP3 Upstream Enhancer in Patients With Rheumatoid Arthritis. Arthritis Rheumatol, 2015. 67(5): p. 1182–92.
Disclosure of Interest None declared
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