Kim Youn-Jung, Song Mee, Ryu Jae-Chun
Cellular and Molecular Toxicology Laboratory, Korea Institute of Science and Technology, P.O. Box 131, Cheongryang, Seoul, Republic of Korea.
Toxicology. 2009 Feb 27;256(3):183-90. doi: 10.1016/j.tox.2008.11.016. Epub 2008 Nov 28.
Methotrexate (MTX) has been widely used for the treatment of inflammatory diseases and rheumatoid arthritis (RA), as well as a variety of tumors. However, MTX-induced toxicity is a serious and unpredictable side effect of this therapy and an important clinical problem. We used microarray analysis to examine MTX-induced gene expression in a human lung epithelial cell line (BEAS-2B) and identified 10 differentially expressed genes related to the p38 mitogen-activated protein kinase (MAPK) pathway, including IL-1beta, MKK6, and MAPKAPK2. Differential gene expression was confirmed via real-time RT-PCR. To determine the functional significance of MTX-induced p38 MAPK activation, we used a p38 MAPK inhibitor (SB203580) to block the p38 MAPK cascade. We also used protein array technology to investigate the modulated expression of pro- and anti-inflammatory cytokines in BEAS-2B cells. MTX activated IL-1beta expression and induced the phosphorylation of various proteins in the p38 MAPK cascade, including TAK1, MKK3/MKK6, p38 MAPK, MAPKAPK2, and HSP27. Finally, HSP27 activation may increase IL-8 secretion, resulting in a pulmonary inflammatory response such as pneumonitis. Although IL-1beta and IL-8 expression increased, the expression of IL-4, IL-6, IL-12, TNF-alpha, MIP-1alpha, and MIP-1beta decreased in a dose-dependent manner. These results suggest that the modulation of cytokine expression may play an important role in MTX-induced pulmonary toxicity.
甲氨蝶呤（MTX）已被广泛用于治疗炎症性疾病和类风湿性关节炎（RA）以及多种肿瘤。然而，MTX诱导的毒性是这种治疗方法严重且不可预测的副作用，也是一个重要的临床问题。我们使用微阵列分析来检测MTX在人肺上皮细胞系（BEAS-2B）中诱导的基因表达，并鉴定了10个与p38丝裂原活化蛋白激酶（MAPK）途径相关的差异表达基因，包括IL-1β、MKK6和MAPKAPK2。通过实时逆转录聚合酶链反应（RT-PCR）证实了差异基因表达。为了确定MTX诱导的p38 MAPK激活的功能意义，我们使用p38 MAPK抑制剂（SB203580）来阻断p38 MAPK级联反应。我们还使用蛋白质阵列技术研究BEAS-2B细胞中促炎和抗炎细胞因子的调节表达。MTX激活了IL-1β的表达，并诱导了p38 MAPK级联反应中各种蛋白质的磷酸化，包括TAK1、MKK3/MKK6、p38 MAPK、MAPKAPK2和HSP27。最后，HSP27的激活可能会增加IL-8的分泌，导致如肺炎等肺部炎症反应。虽然IL-1β和IL-8的表达增加，但IL-4、IL-6、IL-12、肿瘤坏死因子-α（TNF-α）、巨噬细胞炎性蛋白-1α（MIP-1α）和巨噬细胞炎性蛋白-1β（MIP-1β）的表达呈剂量依赖性下降。这些结果表明，细胞因子表达的调节可能在MTX诱导的肺部毒性中起重要作用。
