Li Ming, Wang Jide, Ng Samuel S M, Chan Chu-Yan, He Ming-Liang, Yu Fang, Lai Lihui, Shi Chao, Chen Yangchao, Yew David T, Kung Hsiang-Fu, Lin Marie Chia-Mi
Department of Chemistry, Open Laboratory of Chemical Biology, the University of Hong Kong, Hong Kong, China.
Cancer. 2009 Nov 1;115(21):4959-72. doi: 10.1002/cncr.24550.
: Epidermal growth factor (EGF) signaling plays a pivotal role in gliomagenesis. The authors previously demonstrated that adenosine diphospate-ribosylation factor 6 (ARF6), a member of the Ras-related small guanosine-5'-triphospate-binding protein family, is required for EFA6A-induced glioma cell migration and invasion. However, the role of ARF6 in EGF signaling is unknown.
: The authors analyzed messenger RNA (mRNA) levels of ARF6 and EGF receptor (EGFR) in 16 high-grade glioma samples and in 6 low-grade glioma samples by reverse transcriptase-polymerase chain reaction analysis. To determine whether EGF induces ARF6 expression in human glioblastoma U87 cells through transcriptional regulation and EGFR activation, the levels of ARF6 were assayed in EGF-treated U87 cells that were preincubated with a transcriptional inhibitor (actinomycin D) and an EGFR tyrosine kinase inhibitor (PD153035), respectively. The downstream signaling of EGFR-mediated ARF6 up-regulation also was investigated using specific inhibitors of mitogen-activated protein kinase (MEK), phosphatidylinositol 3' kinase (PI3K), and Janus kinase 2. The involvement of SP1 in the downstream signaling was studied by using an SP1 inhibitor (mithramycin A). Small-interfering RNAs (siRNAs) targeting ARF6 were used to investigate the effects of ARF6 on EGF-mediated glioma cell proliferation.
: The results demonstrated that ARF6 and EGFR mRNA levels were elevated in glioma tissues. Furthermore, EGF stimulated ARF6 expression in U87 cells in a dose-dependent and time-dependant manner. This stimulation was caused by increased transcription of ARF6 and by activation of the MEK/extracellular signal-regulated kinase 1 and 2 (ERK1/2) and PI3K signaling pathways. It is noteworthy that SP1 was essential for EGF-induced ARF6 up-regulation. Finally, EGF-induced glioblastoma cell proliferation depended on ARF6, because the suppression of ARF6 by siRNA or by a dominant-negative mutant significantly inhibited EGF-induced cell proliferation.
: The results of the current study suggested that EGF-induced ARF6 expression plays a significant role in glioma cell proliferation. Cancer 2009. (c) 2009 American Cancer Society.
表皮生长因子（EGF）信号传导在胶质瘤发生过程中起关键作用。作者之前证明，Ras相关小GTP结合蛋白家族成员之一的二磷酸腺苷核糖基化因子6（ARF6）是EFA6A诱导的胶质瘤细胞迁移和侵袭所必需的。然而，ARF6在EGF信号传导中的作用尚不清楚。
作者通过逆转录聚合酶链反应分析，检测了16例高级别胶质瘤样本和6例低级别胶质瘤样本中ARF6和表皮生长因子受体（EGFR）的信使核糖核酸（mRNA）水平。为了确定EGF是否通过转录调控和EGFR激活在人胶质母细胞瘤U87细胞中诱导ARF6表达，分别在预先用转录抑制剂（放线菌素D）和EGFR酪氨酸激酶抑制剂（PD153035）预处理的EGF处理的U87细胞中检测ARF6水平。还使用丝裂原活化蛋白激酶（MEK）、磷脂酰肌醇3'激酶（PI3K）和Janus激酶2的特异性抑制剂研究了EGFR介导的ARF6上调的下游信号传导。通过使用SP1抑制剂（光神霉素A）研究了SP1在下游信号传导中的作用。靶向ARF6的小干扰RNA（siRNA）用于研究ARF6对EGF介导的胶质瘤细胞增殖的影响。
结果表明，胶质瘤组织中ARF6和EGFR mRNA水平升高。此外，EGF以剂量和时间依赖性方式刺激U87细胞中ARF6的表达。这种刺激是由ARF6转录增加以及MEK/细胞外信号调节激酶1和2（ERK1/2）和PI3K信号通路的激活引起的。值得注意的是，SP1对于EGF诱导的ARF6上调至关重要。最后，EGF诱导的胶质母细胞瘤细胞增殖依赖于ARF6，因为siRNA或显性负性突变体对ARF6的抑制显著抑制了EGF诱导的细胞增殖。
当前研究结果表明，EGF诱导的ARF6表达在胶质瘤细胞增殖中起重要作用。癌症2009。（c）2009美国癌症协会。
