Department of Physiology and Pharmacology and Health Aging Research Center, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan.
Biochem Pharmacol. 2013 Apr 15;85(8):1115-23. doi: 10.1016/j.bcp.2013.01.013. Epub 2013 Jan 24.
TNF-α plays a critical mediator in the pathogenesis of chronic heart failure contributing to cardiac remodeling and peripheral vascular disturbances. The implication of TNF-α in inflammatory responses has been shown to be mediated through up-regulation of inflammatory genes, including matrix metalloproteinase-9 (MMP-9). However, the detailed mechanisms of TNF-α-induced MMP-9 expression are largely unclear in the heart cells. Here, we demonstrated that in rat embryonic-heart derived H9c2 cells, TNF-α could induce MMP-9 mRNA expression associated with an increase in the secretion of MMP-9, determined by real-time PCR, zymography, and promoter activity assays. TNF-α-mediated responses were attenuated by pretreatment with the inhibitor of c-Src (PP1), EGFR (AG1478), PDGFR (AG1296), PI3K (LY294002), Akt (SH-5), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) and transfection with siRNA of c-Src, EGFR, PDGFR, p110, Akt, or c-Jun. TNF-α stimulated c-Src, PDGFR, and EGFR phosphorylation, which were reduced by PP1. In addition, TNF-α-stimulated Akt phosphorylation was inhibited by PP1, AG1478, AG1296, or LY294002. We further demonstrated that TNF-α markedly stimulated p38 MAPK, p42/p44 MAPK, and JNK1/2 phosphorylation via a c-Src/EGFR, PDGFR/PI3K/Akt pathway. Finally, we showed that, in H9c2 cells, TNF-α-stimulated AP-1 promoter activity, c-Jun mRNA expression, and c-Jun phosphorylation were attenuated by PP1, AG1478, AG1296, LY294002, SB202190, SP600125, or U0126. These results suggested that TNF-α-induced MMP-9 expression is mediated through a c-Src/EGFR, PDGFR/PI3K/Akt/MAPKs/AP-1 cascade in H9c2 cells. Consequently, MMP-9 induction may contribute to cell migration and cardiovascular inflammation.
TNF-α 在慢性心力衰竭的发病机制中起着关键的介质作用，导致心肌重构和外周血管紊乱。TNF-α 在炎症反应中的作用是通过上调炎症基因来介导的，包括基质金属蛋白酶-9（MMP-9）。然而，TNF-α 诱导 MMP-9 表达的详细机制在心脏细胞中还不清楚。在这里，我们证明在大鼠胚胎心脏衍生的 H9c2 细胞中，TNF-α 可以诱导 MMP-9 mRNA 表达，同时通过实时 PCR、酶谱分析和启动子活性测定确定 MMP-9 的分泌增加。用 c-Src（PP1）、EGFR（AG1478）、PDGFR（AG1296）、PI3K（LY294002）、Akt（SH-5）、MEK1/2（U0126）、p38 MAPK（SB202190）、JNK1/2（SP600125）或 AP-1（丹参酮 IIA）抑制剂预处理或用 c-Src、EGFR、PDGFR、p110、Akt 或 c-Jun 的 siRNA 转染，可减弱 TNF-α 介导的反应。TNF-α 刺激 c-Src、PDGFR 和 EGFR 磷酸化，而 PP1 可减少这种磷酸化。此外，PP1、AG1478、AG1296 或 LY294002 抑制了 TNF-α 刺激的 Akt 磷酸化。我们进一步证明，TNF-α 通过 c-Src/EGFR、PDGFR/PI3K/Akt 途径显著刺激 p38 MAPK、p42/p44 MAPK 和 JNK1/2 的磷酸化。最后，我们表明在 H9c2 细胞中，TNF-α 刺激的 AP-1 启动子活性、c-Jun mRNA 表达和 c-Jun 磷酸化被 PP1、AG1478、AG1296、LY294002、SB202190、SP600125 或 U0126 减弱。这些结果表明，TNF-α 诱导的 MMP-9 表达是通过 H9c2 细胞中的 c-Src/EGFR、PDGFR/PI3K/Akt/MAPKs/AP-1 级联反应介导的。因此，MMP-9 的诱导可能有助于细胞迁移和心血管炎症。
