Cyclin Dependent Kinase 6 in Cardiac Development

这项建议的长期目标是促进我们对胚胎发生过程中心脏细胞周期退出的调控级联的理解。这种撤回大概限制了心脏对病理性和老年性肌细胞损失的反应能力。该提议的工作假设是，细胞周期蛋白依赖性激酶cdk 6通过心肌细胞发育期间的心肌细胞特异性亚细胞区室化，是连接心肌细胞细胞周期退出与终末分化的关键节点。使用细胞分级分离，间接多标记免疫荧光和FACs分析，我们已经描述和量化的时间和空间的G1，S和G2/M调节蛋白在心脏发生的核表达模式。与已知存在的所有其他细胞周期调节蛋白相比，cdk 6和细胞周期蛋白A蛋白水平的核减少与心肌细胞细胞周期退出同步发生。进一步的研究表明：1）早期核/胞质cdk 6表达发生在持续心肌增殖期间; 2）进行性核下调有利于增加cdk 6胞质区室化; 3）在体外，cdk 6的胞质流出可受TGF β影响; 4）cdk 6与发育中的胞质肌原纤维的体内共定位; 5）cdk 6与肌球蛋白重链的特异性结合。考虑到在cdk 4敲除小鼠和其他细胞周期蛋白敲除小鼠中观察到的显著和独特的器官器官特异性缺陷，我们的初步数据与cdk 6是心肌细胞增殖和分化的组织特异性调节的假设一致。本提案将研究cdk 6在哺乳动物发育过程中的作用，特别强调其在肌细胞细胞周期退出中的作用。我们最近创建的新试剂，包括cdk 6- GFP融合质粒，稳定过表达cdk 6的CDC 12细胞和转基因cdk 6小鼠创始人，应该特别有助于验证我们的假设。我们的目标的成功完成，应进一步了解参与细胞周期调控的一般机制，并通过细胞周期调节的潜力，以重新激活心肌细胞增殖。

The long-term goal of this proposal is to advance our understanding of the regulatory cascade involved in cardiac cell cycle withdrawal during embryogenesis. This withdrawal presumably limits the heart's ability to respond to pathologic and senile myocyte loss. The working hypothesis of this proposal is that the cyclin-dependent kinase cdk6, via myocyte- specific subcellular compartmentalization during cardiomyocyte development, is a critical nodal point linking cardiomyocyte cell cycle withdrawal to terminal differentiation. Using cell fractionation, indirect multi-labeling immunofluorescence and FACs analysis, we have described and quantified the temporal and spatial nuclear expression patterns of G1, S, and G2/M regulatory proteins during cardiogenesis. In contrast to all other cell cycle regulatory proteins known to exist, the nuclear reduction in cdk6 and cyclin A protein levels occurs synchronously with cardiomyocyte cell cycle withdrawal. Further study has revealed: 1) Early nuclear/cytoplasmic cdk6 expression occurs during periods of persistent myocardial proliferation; 2) Progressive nuclear down-regulation in favor of increased cdk6 cytoplasmic compartmentalization; 3) Cytosolic egress of cdk6 can be effected in vitro by TGFbeta; 4) In vivo co- localization of cdk6 with developing cytoplasmic myofibrils ; and 5) Specific binding of cdk6 to myosin heavy chain with the maturing sarcomere. Given the striking and distinct organ organ-specific deficits in seen in cdk4 knockout mice and knockouts of other cyclins, our preliminary data are consistent with the hypothesis that cdk6 is a tissue- specific regulatory of cardiomyocyte proliferation and differentiation. This proposal will examine the role of cdk6 during mammalian development with special emphasis on its role in myocyte cell cycle withdrawal. Novel reagents we have recently created, including cdk6- GFP fusion plasmids, CDC12 cells stably over-expressing cdk6 and transgenic cdk6 mouse founders, should be especially useful in testing our hypothesis. Successful completion of our aims should further our understanding of the general mechanisms involved in cell cycle regulation and the potential to reactivate cardiomyocyte proliferation via cell cycle modulation.