Karouzakis Emmanuel, Gay Renate E, Gay Steffen, Neidhart Michel
University Hospital, Zurich, Switzerland.
Arthritis Rheum. 2012 Jun;64(6):1809-17. doi: 10.1002/art.34340. Epub 2011 Dec 14.
Global DNA hypomethylation in rheumatoid arthritis synovial fibroblasts (RASFs) contributes to their intrinsic activation. The aim of this study was to investigate whether increased polyamine metabolism is associated with a decreased level of S-adenosyl methionine (SAM), causing global DNA hypomethylation.
Synovial fibroblasts were isolated from synovial tissue obtained from 12 patients with RA and from 6 patients with osteoarthritis (OA). The cells were stained for S-adenosyl methionine decarboxylase (AMD), spermidine/spermine N1-acetyltransferase (SSAT1), polyamine-modulated factor 1-binding protein 1 (PMFBP1), solute carrier family 3 member 2 (SLC3A2), DNA methyltransferase 1 (DNMT-1), α9 integrin, and β1 integrin and analyzed by flow cytometry. Nuclear 5-methylcytosine (5-MeC) was measured by flow cytometry, the expression of diacetylspermine (DASp) in cell culture supernatants and cell extracts was determined by enzyme-linked immunosorbent assay, and SAM expression in cell extracts was measured by fluorometry.
The expression of SSAT1, AMD, and PMFBP1 was significantly increased in RASFs compared with OASFs. The expression of DASp in cell culture supernatants and the expression of SLC3A2 were significantly elevated in RASFs. The levels of SAM in cell culture extracts, as well as the levels of DNMT-1 protein and 5-MeC, were significantly reduced in RASFs. Parameters of polyamine metabolism were negatively correlated with the expression of SAM, DNMT-1, and 5-MeC.
These data clearly show that intrinsic elevations of PMFBP1 and SSAT1 enhance the catabolism and recycling of polyamines in RASFs and suggest that high consumption of SAM via this pathway is an important factor contributing to global DNA hypomethylation in these cells.
类风湿关节炎滑膜成纤维细胞（RASFs）中的全基因组DNA低甲基化有助于其内在激活。本研究旨在调查多胺代谢增加是否与S-腺苷甲硫氨酸（SAM）水平降低有关，从而导致全基因组DNA低甲基化。
从12例类风湿关节炎患者和6例骨关节炎（OA）患者的滑膜组织中分离出滑膜成纤维细胞。对细胞进行S-腺苷甲硫氨酸脱羧酶（AMD）、亚精胺/精胺N1-乙酰转移酶（SSAT1）、多胺调节因子1结合蛋白1（PMFBP1）、溶质载体家族3成员2（SLC3A2）、DNA甲基转移酶1（DNMT-1）、α9整合素和β1整合素染色，并通过流式细胞术进行分析。通过流式细胞术测量细胞核5-甲基胞嘧啶（5-MeC），通过酶联免疫吸附测定法测定细胞培养上清液和细胞提取物中二乙酰亚精胺（DASp）的表达，并通过荧光法测量细胞提取物中SAM的表达。
与OA滑膜成纤维细胞（OASFs）相比，RASFs中SSAT1、AMD和PMFBP1的表达显著增加。RASFs中细胞培养上清液中DASp的表达和SLC3A2的表达显著升高。RASFs中细胞培养提取物中SAM的水平以及DNMT-1蛋白和5-MeC的水平显著降低。多胺代谢参数与SAM、DNMT-1和5-MeC的表达呈负相关。
这些数据清楚地表明，PMFBP1和SSAT1的内在升高增强了RASFs中多胺的分解代谢和再循环，并表明通过该途径对SAM的高消耗是导致这些细胞中全基因组DNA低甲基化的一个重要因素。
