Introduction

Embryonic stem (ES) cells are pluripotent cells that have the capacity to continuously proliferate while retaining the potential to differentiate into three germ cells (cells derived from the end-, mes-, and ectoderm).

Mouse ES cells or their derivatives have been widely studied in the field of developmental biology, drug discovery and regenerative medicine1, 13, 34. It has also been reported that the sustained activation or phosphorylation of ERK is associated with differentiation35. In our study, the level of ERK phosphorylation was high in ES cells and was low in the early periods of d 4 and d 8+0 EB culture, while it increased greatly during late neuronal differentiation at d 8+5 and d 8+10 culture. These data indicate that ERK phosphorylation is a key event required for the survival of ES cells and for neurite formation and neuron survival during mouse ES cell differentiation16. At d 4 and d 8+0 culture, nestin was highly expressed in EBs, while ERK phosphorylation was inhibited, suggesting that the inhibition of ERK phosphorylation is associated with neural progenitor formation in mouse ES cells. p38 MAPK phosphorylation was shown to be a trigger in determining whether ES cells differentiated into neuronal cells or cardiomyocytes15. The control of p38 MAPK activity constitutes an early switch in ES cell commitment into cardiomyocytes (phos-p38 on) and neurons (phos-p38 off)18. In our study, p38 phosphorylation was expressed in the period of ES and EBs (d 4, d 8+0 culture), suggesting that p38 phosphorylation plays a critical role in maintaining mouse ES cell pluripotency and appears to promote neuroectoderm formation, while IBA can prevent p38 phosphorylation and subsequently promote neuronal differentiation. In the late period of neuronal differentiation, p38 phosphorylation was rare, indicating that p38 phosphorylation is associated with the trigger of mouse ES-cell differentiation and that its inhibition is important to neuronal differentiation. JNK phosphorylation was inhibited during neuronal differentiation compared with early stages of ES and EBs. It is noteworthy that the inhibitory effect of IBA on p38 is larger than on JNK phosphorylation. These results suggest that the regulation of the p38 and JNK MAPK signaling cascade may involve two mechanisms underlying the promoting effect of IBA on mouse ES cell neuronal differentiation19. Thus, a phosphatase-mediated crosstalk may exist between protein prenylation and the MAPK pathway in the regulation of neuronal differentiation from mouse ES cells promoted by IBA. Moreover, there may be a negative feedback regulation of their phosphorylation17, 36. Otherwise, ERK, p38, and JNK may be downstream targets of protein prenylation. However, how IBA regulates the ERK/JNK/p38 MAPK pathways and the specific relationship with protein prenylation needs to be further investigated.

In conclusion, this study suggests that IBA (prenyl group at position 8 of ring A) can facilitate the differentiation of mouse ES cells into multiple kinds of neuronal cell subtypes, such as neurons and astrocytes. The mechanism involved protein prenylation and, subsequently, phos-ERK activation and the phos-p38 off pathway.

Author contribution

Yi-jia LOU: Conception and design, financial support, final approval of manuscript; Dan-yin WANG: Provision of study material, acquisition of data and data analysis, manuscript writing; Yu-zhe HU and Si-si KONG: Partial acquisition of data; Yong-** YU: Partial provision of study molecule; Dan-yan ZHU: Partial conception and new skills; all authors have read and approved the final manuscript.