Promoting effects of isobavachin on neurogenesis of mouse embryonic stem cells were associated with protein prenylation

Abstract

Aim: Some small molecules can induce mouse embryonic stem (ES) cells to differentiate into neuronal cells. Here, we explored the effect of isobavachin (IBA), a compound with a prenyl group at position 8 of ring A, on promoting neuronal differentiation and the potential role of its protein prenylation.

Methods: The hanging drop method was employed for embryonic body (EB) formation to mimic embryo development in vivo. The EBs were treated with IBA at a final concentration of 10(-7) mol/L from EB stage (d 4) to d 8+10. Geranylgeranyltransferase I inhibitor GGTI-298 was subsequently used to disrupt protein prenylation. Neuronal subtypes, including neurons and astrocytes, were observed by fluorescence microscopy. Gene and protein expression levels were detected using RT-PCR and Western blot analysis, respectively.

Results: With IBA treatment, nestin was highly expressed in the neural progenitors generated from EBs (d 4, d 8+0). EBs then further differentiated into neurons (marked by β-tubulin III) and astrocytes (marked by GFAP), which were both up-regulated in a time-dependent manner on d 8+5 and d 8+10. Co-treatment with GGTI-298 selectively abolished the IBA-induced neuronal differentiation. Moreover, in the MAPK pathway, p38 and JNK phosphorylation were down-regulated, while ERK phosphorylation was up-regulated after IBA treatment at different neuronal differentiation passages.

Conclusion: IBA can facilitate mouse ES cells differentiating into neuronal cells. The mechanism involved protein prenylation and, subsequently, phos-ERK activation and the phos-p38 off pathway.

Figure 1

Chemical structure of isobavachin (IBA).

Figure 2

Differentiation potential of mouse ES cells into neurons and astrocytes after IBA treatment. All the morphological images were taken on d 8+10 EBs culture. DMSO: vehicle control; RA: 10⁻⁷ mol/L; IBA: 10⁻⁷ mol/L. (A) Morphological evaluation of neurongenesis of mouse ES cells induced by IBA. A typical axon with three times longer than the size of neuronal body (indicated with square) by phase contrast microscope. (B) Immunostaining with β-tubulin III (neuronal marker in red) or GFAP (astrocytes marker in green). DAPI staining (in blue) shows nuclear morphology. (C) RT-PCR analysis at different passages of mouse ES cells by IBA treatment. Stem cell marker gene (OCT3/4), neuronal genes (nestin for early differentiation, β-tubulin III and GFAP for late differentiation). The expression levels of genes were normalized with those of GAPDH gene. (D) Western blot analysis at different passages of mouse ES cells differentiation induced by IBA. β-tubulin III, NEFM for neurons, GFAP for astrocytes. The expression levels of proteins were normalized with those of GAPDH protein.

Figure 3

GGTI-298 declined mouse ES cell derived neurons and astrocytes promoted by IBA. (A) Immunofluorescent staining of neurons co-localization with nuclei (DAPI staining) differentiated from mouse ES cells with or without GGTI-298 treatment. The morphological images were taken on day 8+10; 0.1% DMSO: (vehicle control), RA: 10⁻⁷ mol/L, IBA: 10⁻⁷ mol/L, GGTI-298: 10⁻⁶ mol/L. (B) Immunofluorescent staining for astrocytes with or without GGTI-298, and co-localization with nuclei (DAPI staining). (C) Neuronal marker proteins expressions in various stages during neurogenesis with or without GGTI-298. Neuronal proteins (β-tubulin III for neurons and GFAP for astrocytes) were examined by Western blot analysis. Results are presented as the ratio of the target protein compared with GAPDH. The treatments were the same as (A).

Figure 4

Effects of IBA on phosphorylation of MAPK pathway in ES cells-derive neurons and astrocytes by Western blot analysis. Samples were harvested at ES, EB, d 8+0, d 8+5, d 8+10 incubation with the treatment of various compounds. 0.1% DMSO: (vehicle control); RA: 10⁻⁷ mol/L; IBA: 10⁻⁷ mol/L. (A) Phos-ERK; (B) phos-p38; (C) Phos-JNK; (D) The semi-quantified levels of phos-ERK, phos-JNK and phos-p38 ratio were represented by histograms.