Regulated Expression and Role of c-Myb in the Cardiovascular-Directed Differentiation of Mouse Embryonic Stem Cells
Regulated Expression and Role of c-Myb in the Cardiovascular-Directed Differentiation of Mouse Embryonic Stem Cells
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DOI：
10.1161/circresaha.111.259499
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发表时间：
2012-01-20
影响因子：
20.1
通讯作者：
Husain, Mansoor
中科院分区：
文献类型：
--
作者：
Ishida, Masayoshi;El-Mounayri, Omar;Husain, Mansoor
目的：明确c-Myb在拟胚体分化为心肌细胞中的作用。方法与结果：在野生型拟胚体中，分化后0-2天高表达c-Myb的胚胎干细胞在2.5-3天经历泛素介导的蛋白酶体降解，在4-6天恢复表达，但不改变c-Myb基因表达水平。用流式细胞仪分离激活素A和骨形态发生蛋白4诱导的心血管祖细胞，检测血管内皮生长因子受体2和血小板衍生生长因子受体α的表达。到第3.75天，具有造血功能的VEGFR2+细胞较少，而心肌细胞定向的VEGFR2+/PDGFRα+细胞在基因敲除和WT EB的丰度上没有差异。重要的是，c-Myb在VEGFR2+和VEGFR2+/PDGFRα+细胞中的表达水平分别最高和最低。蛋白酶体抑制剂MG132和能够诱导c-myb表达或下调c-myb的慢病毒被用来调节WT和敲除EB中的c-Myb。这些实验表明，c-Myb通过PDGFRα促进VEGFR2的表达，染色质免疫沉淀和启动子-报告分析定义了VEGFR2启动子中特定的c-Myb反应结合部位。其次，与VEGFR2+/PDGFRα+细胞相比，FACS分选的VEGFR2+细胞在RA后7-14天分别表达最高和最低水平的SMC和成纤维细胞特异性标记。相反，未加RA培养的VEGFR2+/PDGFRα+细胞在培养7-14天时会自发搏动，就像心肌细胞一样，并表达心肌肌钙蛋白。值得注意的是，RA需要更充分地分化SMC和VEGFR2+细胞，并完全阻止心肌细胞从VEGFR2+/PDGFRα+细胞分化。结论：c-Myb在ESC心血管定向分化过程中受到蛋白酶体降解的严格调控，扩增早期能够形成RA反应的SMC的VEGFR2+前体细胞。(中国保监会决议2012；110：253-264。)
Rationale: c-myb null (knockout) embryonic stem cells (ESC) can differentiate into cardiomyocytes but not contractile smooth muscle cells (SMC) in embryoid bodies (EB).Objective: To define the role of c-Myb in SMC differentiation from ESC.Methods and Results: In wild-type (WT) EB, high c-Myb levels on days 0-2 of differentiation undergo ubiquitin-mediated proteosomal degradation on days 2.5-3, resurging on days 4-6, without changing c-myb mRNA levels. Activin-A and bone morphogenetic protein 4-induced cardiovascular progenitors were isolated by FACS for expression of vascular endothelial growth factor receptor (VEGFR) 2 and platelet-derived growth factor receptor (PDGFR)alpha. By day 3.75, hematopoesis-capable VEGFR2+ cells were fewer, whereas cardiomyocyte-directed VEGFR2+/PDGFR alpha+ cells did not differ in abundance in knockout versus WT EB. Importantly, highest and lowest levels of c-Myb were observed in VEGFR2+ and VEGFR2+/PDGFR alpha+ cells, respectively. Proteosome inhibitor MG132 and lentiviruses enabling inducible expression or knockdown of c-myb were used to regulate c-Myb in WT and knockout EB. These experiments showed that c-Myb promotes expression of VEGFR2 over PDGFR alpha, with chromatin immunopreciptation and promoter-reporter assays defining specific c-Myb-responsive binding sites in the VEGFR2 promoter. Next, FACS-sorted VEGFR2+ cells expressed highest and lowest levels of SMC- and fibroblast-specific markers, respectively, at days 7-14 after retinoic acid (RA) as compared with VEGFR2+/PDGFR alpha+ cells. By contrast, VEGFR2+/PDGFR alpha+ cells cultured without RA beat spontaneously, like cardiomyocytes between days 7 and 14, and expressed cardiac troponin. Notably, RA was required to more fully differentiate SMC from VEGFR2+ cells and completely blocked differentiation of cardiomyocytes from VEGFR2+/PDGFR alpha+ cells.Conclusions: c-Myb is tightly regulated by proteosomal degradation during cardiovascular-directed differentiation of ESC, expanding early-stage VEGFR2+ progenitors capable of RA-responsive SMC formation. (Circ Res. 2012;110:253-264.)