Involvement of protein kinase C and RhoA in protease-activated receptor 1-mediated F-actin reorganization and cell growth in rat cardiomyocytes.
Involvement of protein kinase C and RhoA in protease-activated receptor 1-mediated F-actin reorganization and cell growth in rat cardiomyocytes.
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发表时间：
2011-02
影响因子：
3.5
通讯作者：
H. Otani;K. Yoshioka;H. Nishikawa;C. Inagaki;Tomoyuki Nakamura
中科院分区：
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作者：
H. Otani;K. Yoshioka;H. Nishikawa;C. Inagaki;Tomoyuki Nakamura
蛋白酶激活受体1（PAR 1）可被丝氨酸蛋白酶如凝血酶激活，已被证明有助于心肌损伤后心脏重塑和肥大的发展。在这里，我们研究的机制，PAR 1导致肥大心肌细胞生长培养的新生大鼠心室肌细胞。用凝血酶（1 U/ml）或合成激动剂肽（TFLLR-NH（2），50 µM）刺激PAR 1 48 h，诱导细胞大小和与BNP（脑钠肽）产生相关的肌原纤维形成增加。通过异硫氰酸荧光素（FITC）标记的鬼笔环肽染色评估的肌动蛋白重组出现在PAR 1刺激后1小时，蛋白激酶C（PKC）抑制剂白屈菜红碱、Rho（辛伐他汀）抑制剂和Rho相关激酶（ROCK）（Y-27632）可降低该反应，但百日咳毒素（PTX）不降低该反应。Western blot分析显示，在凝血酶或TFLLR-NH（2）刺激2 - 5 min后，PKCα或PKCε从胞浆向胞膜转移。此外，PAR 1刺激3 - 5 min可增加活性RhoA的水平。此外，PKC和ROCK和Rho的抑制剂消除了PAR 1介导的细胞大小的增加。通过特异性小干扰RNA去除PKCα或PKCε也抑制肌动蛋白重组和细胞生长。这些结果表明，PAR 1刺激心肌细胞诱导细胞肥大，肌动蛋白细胞骨架重组通过激活PKCα和PKCε亚型和RhoA通过PTX不敏感的G蛋白。
Protease-activated receptor 1 (PAR1) that can be activated by serine proteinases such as thrombin has been demonstrated to contribute to the development of cardiac remodeling and hypertrophy after myocardial injury. Here, we investigated the mechanisms by which PAR1 leads to hypertrophic cardiomyocyte growth using cultured rat neonatal ventricular myocytes. PAR1 stimulation with thrombin (1 U/ml) or a synthetic agonist peptide (TFLLR-NH(2), 50 µM) for 48 h induced an increase in cell size and myofibril formation associated with BNP (brain natriuretic peptide) production. This actin reorganization assessed by fluorescein isothiocyanate (FITC)-conjugated phalloidin staining appeared at 1 h after PAR1 stimulation, and this response was reduced by a protein kinase C (PKC) inhibitor, chelerythrine, inhibitors of Rho (simvastatin) and Rho-associated kinase (ROCK) (Y-27632), but not by pertussis toxin (PTX). By Western blot analysis, translocation of PKCα or PKCε from the cytosol to membrane fractions was observed in cells stimulated with thrombin or TFLLR-NH(2) for 2 - 5 min. In addition, PAR1 stimulation for 3 - 5 min increased the level of active RhoA. Furthermore, inhibitors of PKC and ROCK and Rho abrogated PAR1-mediated increase in cell size. Depletion of PKCα or PKCε by specific small interfering RNA also suppressed both actin reorganization and cell growth. These results suggest that PAR1 stimulation of cardiomyocytes induces cell hypertrophy with actin cytoskeletal reorganization through activation of PKCα and PKCε isoforms and RhoA via PTX-insensitive G proteins.