Cyclin-Dependent Kinase 9 Forms a Complex With GATA4 and Is Involved in the Differentiation of Mouse ES Cells Into Cardiomyocytes
Cyclin-Dependent Kinase 9 Forms a Complex With GATA4 and Is Involved in the Differentiation of Mouse ES Cells Into Cardiomyocytes
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DOI：
10.1002/jcp.22336
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发表时间：
2011-01-01
影响因子：
5.6
通讯作者：
Hasegawa, Koji
中科院分区：
文献类型：
--
作者：
Kaichi, Shinji;Takaya, Tomohide;Hasegawa, Koji
用HDAC抑制剂曲马斯他丁A（TSA）处理ES细胞，诱导GATA 4以及组蛋白的乙酰化，并促进其分化为心肌细胞。最近，我们证明了细胞周期蛋白依赖性激酶9（Cdk 9），正延伸因子-b的核心组成部分，是一种新的GATA 4结合伴侣。本研究检测了Cdk 9是否在小鼠ES细胞中与GATA 4形成复合物并参与其向心肌细胞的分化。在无饲养层明胶包被的平板上诱导小鼠ES细胞和Nkx 2.5/GFP ES细胞分化，其中绿色荧光蛋白（GFP）在心脏特异性Nkx 2.5启动子的控制下表达。免疫沉淀/蛋白质印迹法在小鼠ES细胞核提取物证明，Cdk 9以及细胞周期蛋白T1与GATA 4在心肌分化过程中相互作用。TSA处理增加Nkx2.5/GFP阳性细胞和Nkx2.5和心房钠尿因子的内源性mRNA水平。为了确定Cdk 9在心肌细胞分化中的作用，我们研究了失去激酶活性的显性负性形式Cdk 9（DN-Cdk 9）和Cdk 9激酶抑制剂5，6-二氯-1-β-呋喃核糖基-苯并咪唑（DRB）对TSA诱导的心肌细胞分化的影响。DN-Cdk 9的引入抑制了TSA诱导的Nkx2.5/GFP ES细胞中GFP表达的增加。将DRB施用到ES细胞中显著抑制TSA诱导的ES细胞中内源性Nkx2.5 mRNA水平的增加以及Nkx2.5/GFP ES细胞中GFP表达。这些发现表明Cdk 9通过与GATA 4相互作用参与小鼠ES细胞向心肌细胞的分化。J.细胞。226：248-254，2010。(C)2010 Wiley-Liss，Inc.
The treatment of ES cells with trichostatin A (TSA), an HDAC inhibitor, induces the acetylation of GATA4 as well as histones, and facilitates their differentiation into cardiomyocytes. Recently, we demonstrated that cyclin-dependent kinase 9 (Cdk9), a core component of positive elongation factor-b, is a novel GATA4-binding partner. The present study examined whether Cdk9 forms a complex with GATA4 in mouse ES cells and is involved in their differentiation into cardiomyocytes. Mouse ES cells and Nkx2.5/GFP ES cells, in which green fluorescent protein (GFP) is expressed under the control of the cardiac-specific Nkx2.5 promoter, were induced to differentiate on feeder-free gelatin-coated plates. Immunoprecipitation/Western blotting in nuclear extracts from mouse ES cells demonstrated that Cdk9 as well as cyclin T1 interact with GATA4 during myocardial differentiation. TSA treatment increased Nkx2.5/GFP-positive cells and endogenous mRNA levels of Nkx2.5 and atrial natriuretic factor. To determine the role of Cdk9 in myocardial cell differentiation, we examined the effects of a dominant-negative form of Cdk9 (DN-Cdk9), which loses its kinase activity, and a Cdk9 kinase inhibitor, 5,6-dichloro-1-beta-ribofuranosyl-benzimidazole (DRB) on TSA-induced myocardial cell differentiation. The introduction of the DN-Cdk9 inhibited TSA-induced increase in GFP expression in Nkx2.5/GFP ES cells. The administration of DRB into ES cells significantly inhibited TSA-induced increase of endogenous Nkx2.5 mRNA levels in ES cells as well as GFP expression in Nkx2.5/GFP ES cells. These findings demonstrate that Cdk9 is involved in the differentiation of mouse ES cells into cardiomyocytes by interacting with GATA4. J. Cell. Physiol. 226: 248-254, 2010. (C) 2010 Wiley-Liss, Inc.