Abstract
Fibroblast growth factor 1 (FGF1) binds and activates FGF receptors, thereby regulating cell proliferation and neurogenesis. Human FGF1 gene 1B promoter (-540 to +31)-driven SV40 T antigen has been shown to result in tumorigenesis in the brains of transgenic mice. FGF-1B promoter (-540 to +31)-driven green fluorescent protein (F1BGFP) has also been used in isolating neural stem cells (NSCs) with self-renewal and multipotency from developing and adult mouse brains. Valproic acid (VPA) is the primary mood-stabilizing drug to exert neuroprotective effects and to treat bipolar disorder in clinic. In this study, we provide several lines of evidence to demonstrate the underlying mechanisms of VPA in activating FGF-1B promoter activity: (i) VPA significantly increased the FGF-1B mRNA expression and the percentage of F1BGFP(+) cells; (ii) the increase of F1BGFP expression by VPA involves changes of RFX1-3 transcriptional complexes and the increase of histone H3 acetylation on the 18-bp cis-element of FGF-1B promoter; (iii) treatments of other HDAC inhibitors, sodium butyrate and trichostatin A, significantly increased the expression levels of FGF-1B, RFX2 and RFX3 transcripts; (iv) treatments of GSK-3 inhibitor, lithium, or GSK-3 siRNAs also significantly activated FGF-1B promoter; (v) VPA specifically enhanced neuronal differentiation in F1BGFP(+)NSPCs rather than GFP(-) cells. This study suggested, for the first time, that VPA activates human FGF1 gene promoter through inhibiting HDAC and GSK-3 activities. Furthermore, valproic acid not only induces neuronal differentiation of F1BGFP(+) gliblastoma (GBM) cells but also inhibits Aurora A (AurA) activation. Hence it would be interesting to further study the role of AurA in regulating NSPCs behavior. In this study, six lines of evidence were provided to demonstrate that FGF1/FGFR signaling is implicated in the expression of Aurora A (AurA) and the activation of its kinase domain (Thr288 phosphorylation) in the maintenance of GBM cells and NSPCs. First, treatment of FGF1 increases AurA expression in human GBM cell lines. Second, using fluorescence-activated cell sorting, we observed that F1BGFP reporter facilitates the isolation of F1BGFP(+) GBM cells with higher expression levels of FGFR and AurA. Third, both FGFR inhibitor (SU5402) and AurA inhibitor (VX680) could down-regulate F1BGFP-dependent AurA activity. Fourth, inhibition of AurA activity by AurA inhibitor VX680 not only reduce neurosphere formation but also induce neuronal differentiation of F1BGFP(+) GBM cells. Fifth, flow cytometric analyses demonstrated that F1BGFP(+) GBM cells possessed different NSC cell surface markers. Finally, inhibition of AurA by VX680 reduced the neurosphere formation of different types of NSCs. Our results showed that activation of AurA kinase through FGF1/FGFR signaling axis sustains the stem cell characteristics of GBM cells.