Department of Rheumatology and Inflammation Research, Institute of Medicine, The Sahlgrenska Academy, University of Gothenburg, Box 480, 405 30, Gothenburg, Sweden.
Centre for Bone and Arthritis Research, University of Gothenburg, Gothenburg, Sweden.
Arthritis Res Ther. 2018 Mar 20;20(1):49. doi: 10.1186/s13075-018-1554-7.
A key feature of joints in rheumatoid arthritis (RA) is the formation of hyperplastic destructive pannus tissue, which is orchestrated by activated fibroblast-like synoviocytes (FLS). We have demonstrated that the RA risk gene and tumor suppressor Limb bud and heart development (LBH) regulates cell cycle progression in FLS. Methotrexate (MTX) is the first-line treatment for RA, but its mechanisms of action remain incompletely understood. Here, we studied the effects of MTX on mitogen-induced FLS proliferation and expression of cell cycle regulators in vitro.
Primary FLS from patients with RA or osteoarthritis were stimulated with the mitogen platelet-derived growth factor (PDGF) and the cytokine interleukin-1β (IL-1β) in the presence or absence of MTX. Cells were then subjected to qPCR for gene expression and cell cycle analysis by flow cytometry.
Stimulation with PDGF and IL-1β increased the percentage of FLS in the G2/M phase and shifted the cell morphology to a dendritic shape. These effects were inhibited by MTX. Furthermore, PDGF + IL-1β reduced LBH mRNA expression. However, MTX treatment yielded significantly higher transcript levels of LBH, and of CDKN1A (p21) and TP53 (p53), compared to untreated samples upon mitogen stimulation. The expression of DNA methyltransferase-1 (DNMT1) was also higher in the presence of MTX and there was strong correlation between DNMT1 and LBH expression.
Therapeutic concentrations of MTX abolish the effects of PDGF and IL-1β on tumor suppressor expression and inhibit mitogen-promoted FLS proliferation. These data demonstrate novel and important effects of MTX on pathogenic effector cells in the joint, which might involve epigenetic mechanisms.
类风湿关节炎（RA）关节的一个主要特征是形成增生性破坏性血管翳组织，这是由激活的成纤维细胞样滑膜细胞（FLS）协调的。我们已经证明，RA 风险基因和肿瘤抑制因子肢芽和心脏发育（LBH）调节 FLS 的细胞周期进程。甲氨蝶呤（MTX）是 RA 的一线治疗药物，但作用机制仍不完全清楚。在这里，我们研究了 MTX 对有丝分裂原诱导的 FLS 增殖和细胞周期调节剂表达的体外作用。
来自 RA 或骨关节炎患者的原代 FLS 在有丝分裂原血小板衍生生长因子（PDGF）和细胞因子白细胞介素-1β（IL-1β）的存在或不存在的情况下被刺激。然后通过 qPCR 进行基因表达分析，通过流式细胞术进行细胞周期分析。
PDGF 和 IL-1β 的刺激增加了 FLS 在 G2/M 期的百分比，并使细胞形态向树突状转变。这些作用被 MTX 抑制。此外，PDGF + IL-1β 降低了 LBH mRNA 的表达。然而，与未经处理的样本相比，在有丝分裂原刺激下，MTX 处理产生了 LBH、CDKN1A（p21）和 TP53（p53）的转录本水平显著升高。MTX 存在时，DNA 甲基转移酶-1（DNMT1）的表达也更高，并且 DNMT1 与 LBH 表达之间存在强烈的相关性。
治疗浓度的 MTX 消除了 PDGF 和 IL-1β 对肿瘤抑制因子表达的影响，并抑制了丝裂原促进的 FLS 增殖。这些数据表明 MTX 对关节中的致病效应细胞具有新的和重要的作用，这可能涉及表观遗传机制。
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