Involvement of p38MAPK and reactive oxygen species in icariin-induced cardiomyocyte differentiation of murine embryonic stem cells in vitro.
We previously reported that treatment of icariin could significantly induce cardiomyocyte differentiation of murine embryonic stem (ES) cells in vitro. In the present study, the exact activity initiated by icariin was further confirmed and the underlying molecular mechanism was investigated. We found that cardiomyocyte differentiation was efficiently stimulated only if icariin was administrated between days 5 and 8 in differentiation course, which indicated with elevated percentage of embryoid bodies (EB) and with beating areas and up- regulated expression of alpha-actinin and troponin T. Exposure of icariin triggered intracellular reactive oxygen species (ROS) generation of EBs in 3 h, which was abolished in the presence of either NADPH oxidase inhibitor DPI or antioxidant Trolox. Meanwhile, expression of NOX4, a membrane combined enzyme responsible for ROS generation, was promoted by icariin in a dose-dependent manner. Although p38MAPK (mitogen-activated protein kinase), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal protein kinase (JNK) were spontaneously activated in early differentiation, only the phosphorylation of p38MAPK was enhanced and prolonged when icariin was present, whereas both ERK and JNK showed no response to icariin treatment. Moreover, the inducible effect of icariin was blunted by SB203580, a specific inhibitor of p38MAPK. On the contrary, neither UO126 nor SP600125, the specific inhibitor of ERK and JNK, could abolish icariin-stimulated differentiation. Nuclear location of MEF2C, which played a critical role in cardiomyocyte differentiation and could be activated by p38MAPK, was stimulated after icariin exposure. Taken together, these results suggest that ROS generation and the subsequent activation of p38MAPK are essential for the inducible function of icariin on cardiomyocyte differentiation of murine embryonic stem cells in vitro.
Ding
Ling
L
Institute of Pharmacology-Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
Liang
Xing-Guang
XG
Hu
Ying
Y
Zhu
Dan-Yan
DY
Lou
Yi-Jia
YJ
D021581:Active Transport, Cell Nucleus / Q000187:drug effects / Q000502:physiology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects* / Q000502:physiology; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology; D002839:Chromans / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D053595:Embryonic Stem Cells / Q000166:cytology / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology*; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D064326:MEF2 Transcription Factors; D051379:Mice; D032383:Myocytes, Cardiac / Q000166:cytology / Q000201:enzymology*; D018006:Myogenic Regulatory Factors / Q000037:antagonists & inhibitors / Q000378:metabolism; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013997:Time Factors; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000975:Antioxidants; D002839:Chromans; D004791:Enzyme Inhibitors; D005419:Flavonoids; D064326:MEF2 Transcription Factors; C578041:Mef2c protein, mouse; D018006:Myogenic Regulatory Factors; D017382:Reactive Oxygen Species; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C422943:Nox4 protein, mouse; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; C056599:icariin