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.
