Cyclin I and p53 are differentially expressed during the terminal differentiation of the postnatal mouse heart
Cyclin I and p53 are differentially expressed during the terminal differentiation of the postnatal mouse heart
复制标题
DOI：
10.1002/pmic.200600456
复制
发表时间：
2007-01-01
期刊：
影响因子：
3.4
通讯作者：
Lee, Kenneth K. H.
中科院分区：
文献类型：
--
作者：
Liu, Ye;Tang, Mei Kuen;Lee, Kenneth K. H.
在这项研究中，我们使用Ki-67和MF20单抗来确定心肌细胞在出生后小鼠心脏中增殖的广泛性。在2日龄的心脏中，心肌细胞分裂迅速。但在第13天，大多数心肌细胞已进入终末生长停滞和分化阶段。我们利用这一发现来确定与心肌细胞生长和分化相关的蛋白质。用双向电泳法建立了2日龄和13日龄心脏的蛋白质图谱，并进行了比较。在第13天发现了17个差异表达的蛋白质点，其中8个上调，其余9个下调。我们将注意力集中在MALDI-TOF MS鉴定的两种蛋白质上，即细胞周期蛋白I和p53，因为它们都被认为参与了细胞周期的调节。Western印迹分析证实，这两种蛋白在出生后13天的心脏中均呈正上调表达。为了直接确定这些蛋白是否与细胞增殖有关，我们检测了它们在体外培养的H9c2心肌细胞中的表达模式。我们发现细胞周期蛋白I在H9c2培养的生长期表达较低，在生长停滞/分化阶段表达较高。相反，P53的表达在两个阶段都没有变化。细胞周期蛋白A和生长停滞特异的1蛋白的存在证实了不同的生长阶段。我们研究了用细胞周期蛋白I-siRNA沉默细胞周期蛋白I的表达是否能促进H9c2细胞的增殖。研究表明，沉默细胞周期蛋白I可以促进H9c2细胞分裂的微小但显著的增加。从13天大的心脏中提取的心肌细胞也得到了类似的结果。这些结果表明，13天龄心脏心肌细胞周期蛋白I表达增加的原因可能与终末生长停滞有关。然而，P53表达的增加可能与心肌细胞分化有关，而不是与生长停滞有关。
In this study, we have used Ki-67 and MF20 mAb to determine how extensively cardiomyocytes proliferate in the postnatal mouse heart. It was established that the cardiomyocytes divided rapidly in 2-day-old hearts. However, at 13 days, the majority of cardiomyocytes had entered into terminal growth arrest and differentiation. We exploited this finding in order to identify proteins that were associated with cardiomyocyte growth and differentiation. The protein profiles of 2- and 13-day-old hearts were established by two-dimensional electrophoresis and compared. Seventeen protein spots were found to be differentially expressed at day 13. Eight of them were up-regulated while the remaining nine protein spots were down-regulated. We focused our attention on 2 of the proteins identified by MALDI-TOF MS, cyclin I and p53, because they are both believed to be involved in cell cycle regulation. Western blot analysis confirmed that both proteins were positively up-regulated in the 13-day-old postnatal heart. To determine directly whether these proteins were associated with cell proliferation, we examined their expression patterns in H9c2 cardiomyocytes maintained in vitro. We established that cyclin I expression was low during the growing phase of H9c2 culture and high during the growth arrest/differentiation phases. In contrast, p53 expression was unchanged during both phases. The various growth phases were confirmed by the presence of cyclin A and growth arrest-specific 1 proteins. We investigated whether silencing cyclin I expression using cyclin I-siRNA could promote an increase in H9c2 cell proliferation. It was determined that silencing cyclin I could enhance a small, but significant, increase in H9c2 cell division. Similar results were obtained for cardiomyocytes extracted from 13-day-old hearts. These results imply that the reason why cardiomyocytes in 13-day-old hearts increased cyclin I expression was probably associated with terminal growth arrest. However, the increase in p53 expression was probably associated with cardiomyocyte differentiation, rather than growth arrest.