H9c2心肌纖維母細胞中NFIL3與CREB於缺氧情況下可藉由調控IGF2R誘導之細胞凋亡以達到保護心臟的效果

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题名:	H9c2心肌纖維母細胞中NFIL3與CREB於缺氧情況下可藉由調控IGF2R誘導之細胞凋亡以達到保護心臟的效果 NFIL3 and CREB exert cardioprotective effect through regulation of IGF2R-induced apoptosis in H9c2 cardiomyoblast cells under hypoxia
作者:	林冠合;Kuan-Ho Lin
贡献者:	臨床醫學研究所博士班
关键词:	NFIL3;CREB;缺氧;凋亡;IGF2R訊息;CREB;hypoxia;apoptosis;IGF2R signaling
日期:	2015-07-29
上传时间:	2015-11-04 17:03:22 (UTC+8)
出版者:	中國醫藥大學
摘要:	類胰島素生長因子2/甘露糖-6-磷酸受體(IGF2R)的過度表達與心臟疾病發展有關。IGF2R的作用如同G蛋白偶聯受體，可能會引起病理性肥大或粒線體調控凋亡途徑的活化。IGF2R不僅是IGF2的受體蛋白，還可觸發下游訊息傳遞，導致心臟肥大、凋亡、與纖維化。目前的研究在探討核因子IL-3(NFIL3)與環磷酸腺?序列結合蛋白(CREB)對IGF2R誘導之細胞凋亡途徑中之效果與可能之分子機制。個別在H9c2心肌細胞中表現NFIL3與CREB，並缺氧處理6-24小時後，進行西方墨點法分析。在出血性休克的大鼠心臟，IGF2和IGF2R蛋白的表現量高度增加。而對H9c2心肌細胞進行缺氧處理，同樣也造成IGF2R蛋白的表現量上升。在H9c2心肌纖維母細胞中過度表達NFIL3或CREB，可抑制缺氧誘導細胞的凋亡，並造成IGF2R表現量下調。以凝膠阻滯分析法發現NFIL3與CREB可個別結合到IGF2R啟動子區域的EBPRE與CRE序列，以雙股DNA沉澱法也得到這樣的結果。染色體免疫沉澱分析法的結果也證實NFIL3可直接結合到IGF2R啟動子區域。利用熒光素?分析法，我們進一步觀察到，NFIL3與CREB皆可抑制IGF2R基因啟動子的活性。我們的研究結果證實，NFIL3與CREB皆是IGF2R重要的轉錄抑制因子，藉由結合IGF2R的啟動子，抑制IGF2R基因表現，進而抑制缺氧誘導的H9c2心肌纖維母細胞中IGF2R訊息引起的細胞凋亡。 Elevated insulin-like growth factor-II/mannose 6-phosphate receptor (IGF2R) expression correlates with heart disease progression. IGF2R acts like a G protein-coupled receptor might cause pathological hypertrophy or activation of the mitochondria-mediated apoptosis pathway. The IGF2R is not only an IGF2 clearance receptor, but also triggers signal transduction, resulting in cardiac hypertrophy, apoptosis and fibrosis. The present study was performed to investigate the effect of transcription factor nuclear factor IL-3 (NFIL3) or cAMP responsive element-binding protein (CREB) in IGF2R-induced apoptosis and the possible molecular mechanism. CREB or NFIL3 were overexpressed in H9c2 cardiomyoblast cells, and treated with hypoxia for 6-24 hr, then analyzed with Western blot assays. IGF2 and IGF2R protein expression were highly increased in rat hearts subjected to hemorrhagic shock. IGF2R protein expression was also up-regulated in H9c2 cells exposed to hypoxia. Over-expression of NFIL3 or CREB in H9c2 cardiomyoblast cells inhibited the induction of hypoxia-induced apoptosis and down-regulated IGF2R expression levels. Gel shift assay, NFIL3 and CREB bound to the EBPRE and CRE site respectively in the IGF2R promoter region in vitro, as well as the DNA pull-down assay. The results of chromatin immune-precipitation analyses indicated that NFIL3 binds directly to the IGF2R promoter region. Using a luciferase assay, we further observed NFIL3 and CREB both repress IGF2R gene promoter activity. Our results demonstrate that NFIL3 and CREB are both important negative transcription factors for IGF2R, which through binding to the promoter of IGF2R suppresses the apoptosis induced by IGF2R signaling in H9c2 cardiomyoblast cells under hypoxic conditions.
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