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Identification of priming factors that determine cardiac lineage of cardiac stem cells

确定心脏干细胞心脏谱系的启动因子的鉴定

基本信息

批准号：
20249046
负责人：
MATSUBARA Hiroaki
金额：
$ 31.12万
依托单位：
Kyoto Prefectural University of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2008
资助国家：
日本
起止时间：
2008 至 2010
项目状态：
已结题

项目摘要

Increasing evidence indicates that bone morphogenetic proteins (BMPs) are crucial for cardiac induction, specification, and development. Although signaling of BMPs is tightly regulated through soluble BMP-binding proteins, how they regulate BMP signaling during cardiac differentiation remains unknown. To identify molecules responsible for BMP signaling during early cardiomyocyte differentiation of P19 cells, cDNA subtraction was performed. We found a bimodal expression of the BMP-binding protein Crossveinless-2 (Cv2) during cardiomyocyte differentiation ; Cv2 is temporally expressed earlier than cardiac transcription factors such as Nkx2.5 and Tbx5 and acts as a suppressor for BMP signaling in P19 cells. We established a P19 clonal cell line harboring a cardiac alpha-myosin heavy chain promoter-driven enhanced green fluorescent protein gene to monitor cardiac differentiation by flow cytometry. Treatment with BMP2 during the first 2 days of differentiation suppressed cardiomyocyte differentiation through activation of down-stream targets Smadl/5/8 protein and Idl gene, whereas treatment with Cv2 conversely inhibited Smadl/5/8 activation and Idl expression, leading to increased generation of cardiac cells. RNA interference-mediated knockdown (KD) of endogenous Cv2 showed increased Smadl/5/8 activation and impaired cardiomyocyte differentiation. Expression of cardiac mesoderm markers was reduced, whereas expression of Idl and endoderm markers such as Sox7, Hnf4, and E-cadherin was induced in Cv2-kinase dead cells. These phenotypes were rescued by the addition of Cv2 protein to the culture media during the first 2 days of differentiation or co-culture with parental cells. These data suggest that Cv2 may specify cardiac mesodermal lineage through inhibition of BMP signaling at early stage of cardiogenesis.

越来越多的证据表明，骨形态发生蛋白（BMPs）对心脏的诱导、规范和发育至关重要。尽管BMP的信号通过可溶性BMP结合蛋白受到严格调节，但它们在心脏分化过程中如何调节BMP信号仍然未知。为了鉴定在P19细胞早期心肌细胞分化过程中负责BMP信号传导的分子，我们进行了cDNA减法。我们发现在心肌细胞分化过程中bmp结合蛋白Crossveinless-2 （Cv2）的双峰表达；Cv2的暂时表达早于心脏转录因子如Nkx2.5和Tbx5，并在P19细胞中作为BMP信号的抑制因子。我们建立了一株含有心肌α -肌球蛋白重链启动子驱动的增强型绿色荧光蛋白基因的P19克隆细胞系，通过流式细胞术监测心脏分化。在分化的前2天，BMP2通过激活下游靶点Smadl/5/8蛋白和Idl基因来抑制心肌细胞分化，而Cv2则反过来抑制Smadl/5/8的激活和Idl的表达，导致心肌细胞的生成增加。RNA干扰介导的内源性Cv2敲低（KD）显示Smadl/5/8激活增加和心肌细胞分化受损。心肌中胚层标志物的表达减少，而Idl和内胚层标志物如Sox7、Hnf4和E-cadherin在cv2激酶死亡细胞中被诱导表达。在分化的前2天，将Cv2蛋白添加到培养基中或与亲本细胞共培养，可以挽救这些表型。这些数据表明，Cv2可能通过在心脏发生早期抑制BMP信号来指定心脏中胚层谱系。