Hakuno Daihiko, Takahashi Tomosaburo, Lammerding Jan, Lee Richard T
Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02139, USA.
J Biol Chem. 2005 Nov 25;280(47):39534-44. doi: 10.1074/jbc.M505575200. Epub 2005 Sep 11.
The signaling steps that induce cardiac differentiation in embryonic stem (ES) cells are incompletely understood. We examined the effect of adhesion signaling including Src and focal adhesion kinase (FAK) on cardiogenesis in mouse ES cells using alpha-myosin heavy chain promoter-driven enhanced green fluorescent protein or luciferase as reporters. Cardiac transcription factors including Nkx2.5 and Tbx5 mRNA were first expressed at day 4 in hanging drop embryoid bodies, and adhesion of embryoid bodies to surfaces at or before that day strongly inhibited differentiation of ES cells to cardiomyocytes. Since adhesion signaling could suppress cardiogenesis through Src kinases, embryoid bodies were exposed to the small molecule PP2, known as a Src family kinase inhibitor. PP2 during embryoid body adhesion dramatically increased cardiomyocyte differentiation and decreased mRNA expression of neuronal cellular adhesion molecule and alpha-fetoprotein, neuroectodermal, and endodermal markers, respectively. Surprisingly, although there was an interaction between Src and FAK in cardiogenesis, the procardiogenic effect of PP2 appeared incompletely explained by Src kinase inhibition, since another Src family kinase inhibitor, SU6656, failed to induce cardiogenesis. Instead, PP2 specifically inhibited adhesion-induced FAK phosphorylation. In ES cells stably expressing FAK-related nonkinase, which functions as a dominant negative FAK, cell migration from embryoid bodies was inhibited, whereas alpha-myosin heavy chain expression and myosin-stained cardiomyocytes were increased, suggesting that reducing cell motility may contribute to cardiogenesis. These data indicate that FAK is a key regulator of cardiogenesis in mouse ES cells and that FAK signaling within embryoid bodies can direct stem cell lineage commitment.
诱导胚胎干细胞（ES细胞）发生心脏分化的信号传导步骤尚未完全明确。我们使用α-肌球蛋白重链启动子驱动的增强型绿色荧光蛋白或荧光素酶作为报告基因，研究了包括Src和粘着斑激酶（FAK）在内的粘着信号对小鼠ES细胞心脏发生的影响。包括Nkx2.5和Tbx5 mRNA在内的心脏转录因子在悬滴胚胎体培养第4天首次表达，而在该日或该日之前胚胎体与表面的粘着强烈抑制了ES细胞向心肌细胞的分化。由于粘着信号可通过Src激酶抑制心脏发生，因此将胚胎体暴露于已知为Src家族激酶抑制剂的小分子PP2中。胚胎体粘着期间的PP2显著增加了心肌细胞的分化，并分别降低了神经细胞粘着分子和甲胎蛋白、神经外胚层和内胚层标志物的mRNA表达。令人惊讶的是，尽管Src和FAK在心脏发生过程中存在相互作用，但PP2的促心脏发生作用似乎不能完全用Src激酶抑制来解释，因为另一种Src家族激酶抑制剂SU6656未能诱导心脏发生。相反，PP2特异性抑制粘着诱导的FAK磷酸化。在稳定表达作为显性负性FAK发挥作用的FAK相关非激酶的ES细胞中，胚胎体的细胞迁移受到抑制，而α-肌球蛋白重链表达和肌球蛋白染色的心肌细胞增加，这表明降低细胞运动性可能有助于心脏发生。这些数据表明，FAK是小鼠ES细胞心脏发生的关键调节因子，并且胚胎体内的FAK信号传导可指导干细胞谱系定向分化。
