Biomedical Research Institute and Department of Biochemistry & Molecular Biology, College of Medicine, Hanyang University, Seoul 133-791, Republic of Korea.
Biomedical Research Institute and Department of Biochemistry & Molecular Biology, College of Medicine, Hanyang University, Seoul 133-791, Republic of Korea.
Cytokine. 2014 Mar;66(1):69-77. doi: 10.1016/j.cyto.2013.12.018. Epub 2014 Jan 25.
The purpose of this study was to identify the role of phospholipase D1 (PLD1) in lipopolysaccharide (LPS)-induced tumor necrosis factor-α (TNF-α) expression and production. LPS-induced TNF-α expression and production were TLR4 (Toll-like receptor 4)/Myd88 dependent in Raw 264.7 cells. LPS enhanced PLD activation, which was attenuated by TLR4 inhibitor (Polymixin B) or knockdown of Myd88 with siRNA treatment. To investigate the role of PLD in LPS-induced TNF-α expression and production, we transfected PLD1 and PLD2 siRNAs to Raw 264.7 cells, respectively. Interestingly, only knockdown of PLD1 decreased TNF-α expression but not PLD2. Next, we investigated the S6K1-JNK-c-Jun signaling pathway in LPS-induced TNF-α expression mechanism. Knockdown of PLD1 also decreased phosphorylation of S6K1, JNK and c-Jun induced by LPS. Furthermore, we found that activated c-Jun63/73 bound to TNF-α promoter and turned on TNF-α expression. Taken together, our results demonstrate that PLD1 is activated by LPS/TLR4/Myd88 pathway and regulates TNF-α expression and production through S6K1/JNK/c-Jun in Raw 264.7 cells.
本研究旨在确定磷脂酶 D1（PLD1）在脂多糖（LPS）诱导的肿瘤坏死因子-α（TNF-α）表达和产生中的作用。LPS 诱导的 TNF-α表达和产生在 Raw 264.7 细胞中依赖于 TLR4（Toll 样受体 4）/Myd88。LPS 增强了 PLD 的激活，而 TLR4 抑制剂（多粘菌素 B）或 siRNA 处理下调 Myd88 可减弱其激活。为了研究 PLD 在 LPS 诱导的 TNF-α表达和产生中的作用，我们分别用 PLD1 和 PLD2 siRNA 转染 Raw 264.7 细胞。有趣的是，只有 PLD1 的下调降低了 TNF-α的表达，但不影响 PLD2。接下来，我们研究了 LPS 诱导的 TNF-α表达机制中的 S6K1-JNK-c-Jun 信号通路。PLD1 的下调也降低了 LPS 诱导的 S6K1、JNK 和 c-Jun 的磷酸化。此外，我们发现激活的 c-Jun63/73 与 TNF-α启动子结合并开启 TNF-α表达。总之，我们的结果表明，PLD1 被 LPS/TLR4/Myd88 途径激活，并通过 S6K1/JNK/c-Jun 在 Raw 264.7 细胞中调节 TNF-α的表达和产生。
