Department of Cardiovascular Medicine, the First Hospital of China Medical University, Shenyang 110001, Liaoning, China.
Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110122, Liaoning, China.
Int J Mol Sci. 2016 Feb 23;17(3):267. doi: 10.3390/ijms17030267.
T-LAK-cell-originated protein kinase (TOPK) is a PDZ-binding kinase (PBK) that was recently identified as a novel member of the mitogen-activated protein kinase (MAPK) family. It has been shown to play an important role in many cellular functions. However, its role in cardiac function remains unclear. Thus, we have herein explored the biological function of TOPK in myocardial ischemia/reperfusion (I/R) and oxidative stress injury in H9C2 cardiomyocytes. I/R and ischemic preconditioning (IPC) were induced in rats by 3-hour reperfusion after 30-min occlusion of the left anterior descending coronary artery and by 3 cycles of 5-min I/R. Hydrogen peroxide (H₂O₂) was used to induce oxidative stress in H9C2 cardiomyocytes. TOPK expression was analyzed by western blotting, RT-PCR, immunohistochemical staining, and immunofluorescence imaging studies. The effects of TOPK gene overexpression and its inhibition via its inhibitor HI-TOPK-032 on cell viability and Bcl-2, Bax, ERK1/2, and p-ERK1/2 protein expression were analyzed by MTS assay and western blotting, respectively. The results showed that IPC alleviated myocardial I/R injury and induced TOPK activation. Furthermore, H₂O₂ induced TOPK phosphorylation in a time-dependent manner. Interestingly, TOPK inhibition aggravated the H₂O₂-induced oxidative stress injury in myocardiocytes, whereas overexpression relieved it. In addition, the ERK pathway was positively regulated by TOPK signaling. In conclusion, our results indicate that TOPK might mediate a novel survival signal in myocardial I/R, and that its effect on anti-oxidative stress involves the ERK signaling pathway.
T-LAK 细胞来源的蛋白激酶（TOPK）是一种 PDZ 结合激酶（PBK），最近被鉴定为丝裂原激活蛋白激酶（MAPK）家族的一个新成员。它在许多细胞功能中发挥着重要作用。然而，它在心脏功能中的作用尚不清楚。因此，我们在此探讨了 TOPK 在心肌缺血/再灌注（I/R）和 H9C2 心肌细胞氧化应激损伤中的生物学功能。通过 30 分钟左前降支闭塞后 3 小时再灌注和 5 分钟 I/R 的 3 个循环，在大鼠中诱导 I/R 和缺血预处理（IPC）。使用过氧化氢（H₂O₂）诱导 H9C2 心肌细胞氧化应激。通过 Western blot、RT-PCR、免疫组织化学染色和免疫荧光成像研究分析 TOPK 表达。通过 MTS 测定和 Western blot 分别分析 TOPK 基因过表达及其抑制剂 HI-TOPK-032 对细胞活力和 Bcl-2、Bax、ERK1/2 和 p-ERK1/2 蛋白表达的影响。结果表明，IPC 减轻了心肌 I/R 损伤并诱导了 TOPK 激活。此外，H₂O₂ 以时间依赖性方式诱导 TOPK 磷酸化。有趣的是，TOPK 抑制加重了 H₂O₂ 诱导的心肌细胞氧化应激损伤，而过表达则减轻了这种损伤。此外，TOPK 信号正向调节 ERK 通路。总之，我们的结果表明，TOPK 可能介导心肌 I/R 中的一种新的存活信号，其抗氧化应激作用涉及 ERK 信号通路。
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