Adenosine diphosphate-ribosylation factor 6 is required for epidermal growth factor-induced glioblastoma cell proliferation.
BACKGROUND
: 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.


METHODS
: 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.


RESULTS
: 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.


CONCLUSIONS
: 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.
Li
Ming
M
Department of Chemistry, Open Laboratory of Chemical Biology, the University of Hong Kong, Hong Kong, China.
Wang
Jide
J
Ng
Samuel S M
SS
Chan
Chu-Yan
CY
He
Ming-Liang
ML
Yu
Fang
F
Lai
Lihui
L
Shi
Chao
C
Chen
Yangchao
Y
Yew
David T
DT
Kung
Hsiang-Fu
HF
Lin
Marie Chia-Mi
MC
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D049109:Cell Proliferation; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism*; D005909:Glioblastoma / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction; D016329:Sp1 Transcription Factor / Q000378:metabolism
D000089982:ADP-Ribosylation Factor 6; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human