Ding Ling, Liang Xing-Guang, Lou Yi-Jia
Institute of Pharmacology and Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.
Acta Pharmacol Sin. 2007 May;28(5):634-42. doi: 10.1111/j.1745-7254.2007.00558.x.
To investigate the possible roles of peroxisome proliferator-activated receptor alpha(PPAR alpha) and the signal pathway regulating the transcription of PPAR alpha in the cardiomyocyte differentiation course of murine embryonic stem (ES) cells in vitro.
The expression of PPAR alpha during cardiomyocyte differentiation was analyzed using both Western blotting and immunofluorescence. Cardiac specific genes and sarcomeric proteins were evaluated when embryoid bodies were challenged with PPAR alpha specific inhibitor GW6471 at different time courses. The phosphorylation of p38 mitogen-activated protein kinase (MAPK) was studied in the differentiation process, and its specific inhibitor SB203580 was employed to study the function of p38 MAPK on cardiac differentiation and the expression of PPAR alpha.
The expression of PPAR alpha was observed to be at a low level in undifferentiated ES cells and markedly induced with the appearance of beating clusters. The inhibition of PPAR alpha by its specific inhibitor GW6471 (1X10(-5) mol/L) significantly prevented cardiomyocyte differentiation and resulted in the reduced expression of cardiac sarcomeric proteins (ie alpha-actinin, troponin-T) and specific genes (ie alpha-MHC, MLC2v) in a time-dependent manner. In the differentiation course, p-p38 MAPK was maintained at a high level from d 3 followed by a decrease from d 10. The inhibition of the p38 MAPK pathway by SB203580 between d 3 and d 7 efficiently prevented cardiomyocyte differentiation and resulted in the capture of the upregulation of PPAR alpha.
Taken together, these results showed a close association between PPAR alpha and cardiomyocyte differentiation in vitro, and p38 MAPK was partly responsible for the regulation of PPAR alpha.
探讨过氧化物酶体增殖物激活受体α（PPARα）及其转录调控信号通路在小鼠胚胎干细胞（ES细胞）体外心肌细胞分化过程中的可能作用。
采用蛋白质免疫印迹法和免疫荧光法分析心肌细胞分化过程中PPARα的表达。在不同时间点用PPARα特异性抑制剂GW6471处理拟胚体，评估心脏特异性基因和肌节蛋白。研究分化过程中p38丝裂原活化蛋白激酶（MAPK）的磷酸化情况，并使用其特异性抑制剂SB203580研究p38 MAPK对心脏分化及PPARα表达的作用。
未分化的ES细胞中PPARα表达水平较低，随着搏动细胞团的出现而显著诱导表达。其特异性抑制剂GW6471（1×10⁻⁵ mol/L）抑制PPARα可显著阻止心肌细胞分化，并导致心脏肌节蛋白（即α-辅肌动蛋白、肌钙蛋白-T）和特异性基因（即α-肌球蛋白重链、肌球蛋白轻链2v）的表达呈时间依赖性降低。在分化过程中，p-p38 MAPK从第3天开始维持在高水平，随后从第10天开始下降。在第3天至第7天之间用SB203580抑制p38 MAPK信号通路可有效阻止心肌细胞分化，并导致PPARα上调受阻。
综上所述，这些结果表明PPARα与体外心肌细胞分化密切相关，p38 MAPK部分参与PPARα的调控。
