- 學位論文
探討纖維母細胞生長因子第一型參與神經分化與多形性膠質母細胞瘤之自我更新的機制
FGF1 and regulation of FGF-1B transcriptional promoter－ Implications for neuronal differentaition and regulation of Gliblastoma stem cells.
摘要
纖維母細胞生長因子第一型(FGF-1)在許多生物反應中扮演重要的角色，如細胞分化、傷口修復、血管新生、及組織生長等。FGF-1B 是人類FGF-1基因在腦部組織中主要產生的轉錄片段，先前的研究指出由FGF1-1B 啟動子的-540到 +31所驅動的GFP報導基因可以被用來偵測細胞內FGF-1的表現並且可應用於分離具有自我更新能力(self-renewal)神經幹細胞及神經前驅細胞。丙戊酸 (Valproic acid, VPA）是一種第一線稳定情绪的精神科用藥，先前研究顯示VPA具有保護神經細胞的能力，在臨床上常被用於治療躁鬱症(bipolar disorder)。在這個研究種，我們提出了幾個證據說明VPA具有活化FGF-1B啟動子的能力與其機制：(一) 處理VPA可以顯著提升細胞內FGF-1B mRNA的量; (二) 處理VPA會改變調控因子X (RFX) 聚合體與FGF-1B啟動子結合的能力並且FGF-1B啟動子附近的組蛋白H3的乙醯化; (三) 我們處裡其他種類的HDAC抑制劑，丁酸鈉 (Sodium Butyrate)或trichostatin A，其結果顯示HDAC抑制後會增加FGF-1B、調控因子X2與X3的表現; (四) GSK3抑制劑或GSK3 siRNA的處理也會增加FGF-1B啟動子的活性; (五) VPA的處理會增加胚胎幹細胞與神經幹細胞中FGF1B(+)細胞群往神經細胞分化的能力。此研究指出VPA透過抑制HDAC與GSK3來活化FGF-1B啟動子。更進一步，我們發現VPA促進神經分化時會伴隨Aurora A (AurA)激酶活性的降低，在這個研究中，我們進一步發現FGF1/FGFR的訊息傳遞參與調控AurA的基因表現與激酶活性，並且進一步影響人類多形性膠質母細胞瘤 (GBM) 與神經幹細胞的細胞自我更新。首先，在經過FGF1處理後，AurA的表現量會在GBM細胞中大量增加; 第二，我們利用流式細胞分選儀發現，F1BGFP(+)的GBM細胞中的AurA與FGFR的表現量會大於F1BGFP(-)的GBM細胞; 第三，處裡FGFR抑制劑會降低細胞中AurA激酶活性; 第四，當AurA激酶活性被抑制後，細胞形成神經球的能力會大幅降低同時伴隨神經分化能力的上升; 第五，流式細胞儀分析顯示，F1BGFP(+)的GBM細胞會表現神經幹細胞的表面標記; 最後，當AurA激酶活性被抑制後，在不同種類的神經幹細胞都會發現神經球形成能力降低的現像。我們結果顯示，FGF1/FGFR訊息傳遞會透過活化AurA來維持與穩定神經幹細胞與GBM幹細胞的幹細胞特性。
並列摘要
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.