Division of Translational Research, Kyoto Medical Center, National Hospital Organization, Fushimi-ku, Kyoto, Japan.
J Cell Physiol. 2011 Jan;226(1):248-54. doi: 10.1002/jcp.22336.
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-β-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.
用组蛋白去乙酰化酶抑制剂曲古抑菌素 A（TSA）处理 ES 细胞可诱导 GATA4 乙酰化以及组蛋白乙酰化，并促进其向心肌细胞分化。最近，我们证明细胞周期蛋白依赖性激酶 9（Cdk9）是正延伸因子-b 的核心成分，是 GATA4 的一个新的结合伙伴。本研究旨在探讨 Cdk9 是否在小鼠 ES 细胞中与 GATA4 形成复合物，并参与其向心肌细胞的分化。将小鼠 ES 细胞和 Nkx2.5/GFP ES 细胞（其在心脏特异性 Nkx2.5 启动子的控制下表达绿色荧光蛋白（GFP））在无饲养细胞的铺有明胶的平板上诱导分化。从小鼠 ES 细胞的核提取物中进行免疫沉淀/Western 印迹分析表明，在心肌分化过程中，Cdk9 以及 cyclin T1 与 GATA4 相互作用。TSA 处理增加了 Nkx2.5/GFP 阳性细胞以及内源性 Nkx2.5 和心钠素因子的 mRNA 水平。为了确定 Cdk9 在心肌细胞分化中的作用，我们研究了一种失去激酶活性的 Cdk9 显性失活形式（DN-Cdk9）和 Cdk9 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。引入 DN-Cdk9 抑制了 TSA 诱导的 Nkx2.5/GFP ES 细胞中 GFP 表达的增加。将 DRB 给药到 ES 细胞中，可显著抑制 TSA 诱导的 ES 细胞中内源性 Nkx2.5 mRNA 水平以及 Nkx2.5/GFP ES 细胞中 GFP 表达的增加。这些发现表明，Cdk9 通过与 GATA4 相互作用参与了小鼠 ES 细胞向心肌细胞的分化。
