Chien Peter Tzu-Yu, Hsieh Hsi-Lung, Chi Pei-Ling, Yang Chuen-Mao
Graduate Institute of Biomedical Science, Chang Gung University, Tao-Yuan, Taiwan; Department of Physiology and Pharmacology and Health Ageing Research Center, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan.
Br J Pharmacol. 2014 Oct;171(19):4504-19. doi: 10.1111/bph.12794. Epub 2014 Sep 5.
Different protease-activated receptors (PARs) activated by thrombin are involved in cardiovascular disease, via up-regulation of inflammatory proteins including COX-2. However, the mechanisms underlying thrombin-regulated COX-2 expression in human cardiomyocytes remain unclear.
Human cardiomyocytes were used in the study. Thrombin-induced COX-2 protein and mRNA expression, and signalling pathways were determined by Western blot, real-time PCR and COX-2 promoter luciferase reporter assays, and pharmacological inhibitors or siRNAs. PGE2 generation and cell proliferation were also determined.
Thrombin-induced COX-2 protein and mRNA expression, promoter activity and PGE2 release was attenuated by the PAR1 antagonist (SCH79797) or the inhibitors of proteinase activity (PPACK), MEK1/2 (U0126), p38 MAPK (SB202190) or JNK1/2 (SP600125), and transfection with small interfering RNA (siRNA) of PAR1, p38, p42 or JNK2. These results suggested that PAR1-dependent MAPKs participate in thrombin-induced COX-2 expression in human cardiomyocytes. Moreover, thrombin stimulated phosphorylation of MAPKs, which was attenuated by PPACK and SCH79797. Furthermore, thrombin-induced COX-2 expression was blocked by the inhibitors of AP-1 (tanshinone IIA) and NF-κB (helenalin). Moreover, thrombin-stimulated phosphorylation of c-Jun/AP-1 and p65/NF-κB was attenuated by tanshinone IIA and helenalin, respectively, suggesting that thrombin induces COX-2 expression via PAR1/MAPKs/AP-1 or the NF-κB pathway. Functionally, thrombin increased human cardiomyocyte proliferation through the COX-2/PGE2 system linking to EP2 receptors, as determined by proliferating cell nuclear antigen and cyclin D1 expression.
These findings demonstrate that MAPKs-mediated activation of AP-1/NF-κB pathways is, at least in part, required for COX-2/PGE2 /EP2 -triggered cell proliferation in human cardiomyocytes.
凝血酶激活的不同蛋白酶激活受体（PARs）通过上调包括COX-2在内的炎症蛋白参与心血管疾病。然而，凝血酶调节人心肌细胞中COX-2表达的机制仍不清楚。
本研究使用人心肌细胞。通过蛋白质印迹法、实时定量PCR以及COX-2启动子荧光素酶报告基因检测，结合药理学抑制剂或小干扰RNA（siRNA），测定凝血酶诱导的COX-2蛋白和mRNA表达以及信号通路。同时也测定了前列腺素E2（PGE2）的生成和细胞增殖情况。
PAR1拮抗剂（SCH79797）、蛋白酶活性抑制剂（PPACK）、MEK1/2抑制剂（U0126）、p38丝裂原活化蛋白激酶（MAPK）抑制剂（SB202190）或JNK1/2抑制剂（SP600125），以及PAR1、p38、p42或JNK2的小干扰RNA转染，均可减弱凝血酶诱导的COX-2蛋白和mRNA表达、启动子活性及PGE2释放。这些结果表明，PAR1依赖性的MAPKs参与了凝血酶诱导的人心肌细胞中COX-2的表达。此外，凝血酶刺激了MAPKs的磷酸化，而PPACK和SCH79797可减弱这种磷酸化。此外，AP-1抑制剂（丹参酮IIA）和核因子κB（NF-κB）抑制剂（海伦内酯）可阻断凝血酶诱导的COX-2表达。而且，丹参酮IIA和海伦内酯分别减弱了凝血酶刺激的c-Jun/AP-1和p65/NF-κB的磷酸化，这表明凝血酶通过PAR1/MAPKs/AP-1或NF-κB途径诱导COX-2表达。在功能上，通过增殖细胞核抗原和细胞周期蛋白D1表达测定发现，凝血酶通过与EP2受体相连的COX-2/PGE2系统增加人心肌细胞增殖。
这些发现表明，MAPKs介导的AP-1/NF-κB途径的激活至少在一定程度上是COX-2/PGE /EP2触发的人心肌细胞增殖所必需的。 22
