DcR3 通过 MAPK 信号通路诱导骨关节炎软骨细胞增殖。
DcR3 induces cell proliferation through MAPK signaling in chondrocytes of osteoarthritis.
机构信息
Department of Orthopaedic Surgery, Kobe University, Graduate School of Medicine, Kobe, Japan.
出版信息
Osteoarthritis Cartilage. 2011 Jul;19(7):903-10. doi: 10.1016/j.joca.2011.03.005. Epub 2011 Mar 21.
INTRODUCTION
Decoy receptor 3 (DcR3), a soluble receptor belonging to the tumor necrosis factor (TNF) receptor superfamily, competitively binds and inhibits the TNF family including Fas-ligand (Fas-L), lymphotoxin-like inducible protein that competes with glycoprotein D for binding herpesvirus entry mediator on T-cells (LIGHT) and TNF-like ligand 1A (TL1A). In this study, we investigated the functions of DcR3 on osteoarthritis (OA) chondrocytes.
METHODS
Expressions of DcR3 in chondrocytes were measured by realtime Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). Expression of DcR3 in sera and joint fluids was measured by enzyme-linked immunosorbent assay (ELISA). Chondrocytes were incubated with DcR3-Fc chimera protein (DcR3-Fc) before induction of apoptosis by Fas-L and apoptosis was detected with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling labeling (TUNEL) staining and Western blotting of caspase 8 and poly (ADP-ribose) polymerase (PARP). Chondrocytes were incubated with DcR3-Fc and the proliferation was analyzed by 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST) assay. Phosphorylation of Extracellular Signal-Regulated Kinase (ERK), P38 mitogen-activated protein kinase (MAPK) and Jun N-terminal Kinase (JNK) in chondrocytes was measured by Western blotting after incubation with DcR3-Fc, Mitogen-activated protein kinase kinase (MEK1/2) inhibitor, or P38 MAPK inhibitor. Chondrocytes were treated with DcR3-Fc after pre-incubation with blocking antibody of Fas-L, LIGHT and TL1A, and proliferation or phosphorylation of ERK was analyzed.
RESULTS
DcR3 was expressed in OA and normal chondrocytes. DcR3-Fc protects chondrocytes from Fas-induced apoptosis. DcR3-Fc increased chondrocytes proliferation and induced the phosphorylation of ERK specifically. DcR3-induced chondrocytes proliferation was inhibited by pre-incubation of PD098059 or blocking Fas-L antibody. DcR3 increased chondrocytes proliferation in OA chondrocytes, but did not in normal.
CONCLUSION
DcR3 regulates the proliferation of OA chondrocytes via ERK signaling and Fas-induced apoptosis.
简介
凋亡受体 3（DcR3）是肿瘤坏死因子（TNF）受体超家族的一种可溶性受体，竞争性结合并抑制 TNF 家族，包括 Fas 配体（Fas-L）、淋巴毒素样诱导蛋白，该蛋白与糖蛋白 D 竞争结合 T 细胞上的疱疹病毒进入介质（LIGHT）和 TNF 样配体 1A（TL1A）。在这项研究中，我们研究了 DcR3 在骨关节炎（OA）软骨细胞中的功能。
方法
通过实时逆转录聚合酶链反应（RT-PCR）测量软骨细胞中 DcR3 的表达。通过酶联免疫吸附试验（ELISA）测量血清和关节液中 DcR3 的表达。在 Fas-L 诱导凋亡之前，用 DcR3-Fc 嵌合体蛋白（DcR3-Fc）孵育软骨细胞，并用末端脱氧核苷酸转移酶（TdT）介导的 dUTP 缺口末端标记（TUNEL）染色和 caspase 8 和多聚（ADP-核糖）聚合酶（PARP）的 Western blot 检测凋亡。用 4-[3-（4-碘苯基）-2-（4-硝基苯基）-2H-5-四唑]-1,3-苯二磺酸钠（WST）测定 DcR3-Fc 孵育后软骨细胞的增殖。用 DcR3-Fc 孵育后，通过 Western blot 测量软骨细胞中细胞外信号调节激酶（ERK）、p38 丝裂原激活蛋白激酶（MAPK）和 Jun N 末端激酶（JNK）的磷酸化，然后用丝裂原激活蛋白激酶激酶（MEK1/2）抑制剂或 p38 MAPK 抑制剂处理。用 Fas-L、LIGHT 和 TL1A 的阻断抗体预孵育 DcR3-Fc 后，处理软骨细胞，分析 ERK 的增殖或磷酸化。
结果
DcR3 在 OA 和正常软骨细胞中表达。DcR3-Fc 可保护软骨细胞免受 Fas 诱导的凋亡。DcR3-Fc 增加软骨细胞增殖并特异性诱导 ERK 磷酸化。PD098059 或 Fas-L 抗体预孵育可抑制 DcR3 诱导的软骨细胞增殖。DcR3 增加 OA 软骨细胞但不增加正常软骨细胞的增殖。
结论
DcR3 通过 ERK 信号和 Fas 诱导的凋亡调节 OA 软骨细胞的增殖。
Zotero 插件