Induction of Early Growth Response-1 Gene Expression by
Calmodulin Antagonist Trifluoperazine through the Activation of Elk-1
in Human Fibrosarcoma HT1080 Cells*
Soon Young
Shin
§,
Seong-Yong
Kim
,
Jung-Hye
Kim
,
Do Sik
Min¶,
Jesang
Ko
,
Ung-Gu
Kang**,
Yong Sik
Kim**,
Taeg Kyu
Kwon
,
Mi Young
Han§,
Young Ho
Kim§§, and
Young Han
Lee
¶¶
From the
Department of Biochemistry and Molecular
Biology, College of Medicine, Yeungnam University, Daegu 705-717, the
¶ Department of Physiology, College of Medicine, The Catholic
University of Korea, Seoul 137-701, the
Immunomodulation
Research Center, University of Ulsan, Ulsan 138-736, the ** Department
of Psychiatry, College of Medicine, Seoul National University, Seoul
151-742, the 
Department of Immunology,
School of Medicine, Keimyung University, Daegu 700-712, the
§ Cell Biology Laboratory, Korea Research Institute of
Bioscience and Biotechnology, P. O. Box 115, Yusung, Taejon 305-600,
and the §§ Department of Microbiology, Kyungpook
National University, Daegu 702-701, South Korea
Received for publication, October 17, 2000, and in revised form, December 19, 2000
 |
ABSTRACT |
The early growth response gene-1 (Egr-1) is a
transcription factor that plays an important role in cell growth and
differentiation. It has been known that Egr-1 expression is
down-regulated in many types of tumor tissues, including human
fibrosarcoma HT1080 cells, and introduction of the Egr-1
gene into HT1080 cells inhibits cell growth and tumorigenic potential.
Trifluoperazine (TFP), a phenothiazine class calmodulin antagonist, is
known to inhibit DNA synthesis and cell proliferation and potentially
important in antitumor activities. To understand the regulatory
mechanism of Egr-1, we investigated the effect of TFP on
expression of Egr-1 in HT1080 cells. Herein, we report that
Egr-1 expression was increased by TFP in synergy with serum
at the transcriptional level. Both the
Ca2+/calmodulin-dependent protein kinase II
inhibitor KN62 and the calcineurin inhibitor cyclosporin A enhanced
TFP-dependent increase of Egr-1, suggesting that the
Ca2+/calmodulindependent pathway plays a role in
regulation of Egr-1 expression in HT1080 cells. The
TFP-stimulated increase of the Egr-1 protein was preferentially
inhibited by the MEK-specific inhibitor PD98059. In addition,
activation of human Egr-1 promoter and the transcriptional
activation of the ternary complex factor Elk-1 induced by TFP were
inhibited both by pretreatment of PD98059 and by expression of the
dominant-negative RasN17. These results indicate that the
Ras/MEK/Erk/Elk-1 pathway is necessary for TFP-induced Egr-1 expression. We propose that the calmodulin antagonist
TFP stimulates Egr-1 gene expression by modulating
Ras/MEK/Erk and activation of the Elk-1 pathway in human fibrosarcoma
HT1080 cells.
 |
INTRODUCTION |
Ca2+ is an important intracellular messenger in many
biological processes. Calmodulin is a ubiquitous
Ca2+-binding protein that acts as a Ca2+
mediator (1). Ca2+-bound calmodulin regulates the
activities of a large number of enzymes, including
calmodulin-dependent protein kinases
(CaMK)1 such as CaMK-II and
CaMK-IV. Calmodulin is involved in the Ca2+-mediated
regulation of gene expression and in the regulation of cellular
proliferation process, including DNA synthesis and cell cycle
progression (2-6). It has been reported that the calmodulin level is
elevated in several tumor cell lines (3, 7). Moreover, in
vivo treatment with calmodulin antagonists has been shown to reduce tumor size (8), indicating that calmodulin plays an important
role in control of cell growth. It is now known that calmodulin
antagonists are cytotoxic against tumor cells and can restore
sensitivity to drug-resistant cells, thus addressing the possibility
that calmodulin antagonists may be valuable chemotherapeutic agents in
the treatment of certain cancers (9, 10). However, little information
is available regarding the mechanisms responsible for regulation of
cell growth by calmodulin antagonists.
Trifluoperazine (TFP) is a phenothiazine derivative antipsychotic drug.
TFP is a well known calmodulin antagonist that has been used for
studying the function of calmodulin (11, 12). Previous reports have
demonstrated that TFP inhibits DNA synthesis and cell proliferation
and, thereby, is potentially an important antitumor agent, as well as
having antipsychotic properties (13).
The product of the early growth response gene Egr-1 (14),
which is also known as NGFI-A (15), zif268 (16), krox24 (17), or
Tis8 (18), is a transcription factor that has three
Cys2-His2-type zinc finger-containing
DNA binding domains in the C-terminal portion of the molecule. Egr-1
preferentially binds to GC-rich regulatory elements with the consensus
sequence of GCGGGGGCG or TCCTCCTCCTCC (16, 19), leading to induction or
repression of its target genes. It has been demonstrated that
Egr-1 is important in regulating cell growth,
differentiation, and development (20). Expression of Egr-1
is significantly reduced in a number of tumor cells (21, 22), and loss
of expression is closely associated with tumor formation in mammalian
cells and tissues (22). On the other hand, stable expression of
Egr-1 inhibited cell proliferation and soft agar growth in
NIH3T3 cells transformed with v-sis, indicating that
Egr-1 functions as a tumor suppressor (23).
We examined the effect of TFP on expression of the tumor suppressor
Egr-1 in human fibrosarcoma HT1080 cells. Our results revealed that treatment of cells with serum and TFP increased levels of
both mRNA and the protein of Egr-1. The increment was associated with the increase of Egr-1 promoter activity
through activation of the ternary complex factor (TCF) Elk-1.
 |
EXPERIMENTAL PROCEDURES |
Plasmids and Reagents--
Dulbecco's modified Eagle's medium
(DMEM), fetal calf serum, and LipofectAMINE 2000 were purchased from
Life Technologies, Inc. (Gaithersburg, MD). Cyclosporin A, KN62,
and ionomycin were obtained from Calbiochem (San Diego, CA).
Trifluoperazine, chlorpromazine, and GF109203X were products
of RBI (Natick, MA). Antibodies against Egr-1 and ERK were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA).
Trans-activator plasmid pFA2-Elk1 (amino acids 307-427), which contains the GAL4 DNA binding domain (amino acids 1-147), and
the reporter plasmid pFR-Luc, which contains five repeats of GAL4
binding elements and the luciferase gene, were obtained from Stratagene
(La Jolla, CA). The pCMV/
-gal plasmid and assay kits for luciferase
and
-galactosidase activity were purchased from Promega (Madison,
WI). The cDNA probe for Egr-1 was provided by Dr.
I. K. Lim of the Ajou University School of Medicine, South Korea.
Western Blot Analysis--
Cells were lysed in 20 mM
HEPES, pH 7.2, 1% Triton X-100, 10% glycerol, 150 mM
NaCl, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride (24). Protein samples (20 µg of each) were separated by
SDS-polyacrylamide gel electrophoresis (10%) and transferred to
nitrocellulose filters. The blots were incubated with anti-Egr-1 antibodies and developed with the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Piscataway, NJ). The same blot was
stripped and reprobed with anti-ERK antibody for use as an internal
control. In some experiments, the scanning densitometry was performed
after exposure to x-ray film. The relative band intensities of Egr-1
and ERK1/2 in each lane were measured by quantitative scanning
densitometer and image analysis software, Bio-1D version 97.04.
Northern Blot Analysis--
Total RNA was isolated according to
the methods described previously by Chomczymski and Sacchi (25). 10 µg of total RNA was separated on 1.2% agarose gel containing 6%
formaldehyde in 0.02 M MOPS, pH 7.0, 8 mM
sodium acetate, and 1 mM EDTA, then transferred to a Hybond
N+ nylon membrane (Amersham Pharmacia Biotech) by the
capillary method. Cross-linking was then performed by UV irradiation.
The membrane was incubated overnight at 42 °C in Northern-Max
hybridization solution (Ambion, Inc., Austin, TX) containing
[
-32P]dCTP-labeled probes. The probes used were
Egr-1 (the 1.4-kb-long EcoRI fragment purified
from the pGEM/TIS8 plasmid) and GAPDH (the 0.5-kb
XbaI-HindIII fragment from the pUC/GAPDH
plasmid). The membranes were then washed with 2× SSC/0.1% SDS for 20 min at room temperature, 2× SSC/0.1% SDS at 42 °C for 30 min, and 0.5× SSC/0.1% SDS for 30 min at 65 °C. For rehybridization, the probes were stripped from the membrane by boiling in 0.1× SSC/0.5% SDS.
Human Egr-1 Promoter Construction--
Human Egr-1
promoter fragments (26) spanning from nucleotides
668 to +1 and
454
to +1 were synthesized by PCR in a reaction containing 0.5 µg of
human Egr-1 genomic clone TIS8 (obtained from ATCC) as a
template with the following upstream primers:
668,
5'-CCCGCACTCCCggtaccCTCTCAC-3', and
454,
5'-TCCCGGCTTggtaccAGGGAGGA-3'. A KpnI restriction site is
indicated by lowercase letters. A single downstream primer
(5'-CTCTCGaagcttCCCGGATCCGC-3') containing a HindIII site,
which is indicated by lowercase letters, was used in each
PCR amplification. The PCR fragments were then digested with the
restriction enzymes KpnI and HindIII. The
fragments were extracted from the agarose gel and inserted into the
KpnI and HindIII sites of the pGL2-basic
luciferase-encoding reporter vector (Promega, Madison, WI). Plasmid
p-233egrLuc was constructed by digestion of p-688egrLuc with the
restriction enzymes SacI and HindIII, followed by
ligation into the pGL2 basic vector digested by SacI and
HindIII. The resultant constructs were verified by DNA sequencing.
Cell Culture, Transient Transfection, and Reporter Gene
Assay--
HT1080 cells were grown in DMEM with 10% heat-inactivated
fetal calf serum. For Egr-1 promoter analysis, HEK293T cells
were grown in DMEM supplemented with 10% heat-inactivated fetal calf serum. One day after seeding cells into 35-mm dishes (6 × 105 cells), the cells were cotransfected with 0.5 µg of
5'-deletion constructs of the Egr-1 promoter and 0.2 µg of
pCMV/
-gal plasmid using LipofectAMINE 2000 reagents according to the
manufacturer's instructions. Where indicated, mammalian expression
vectors of dominant-active Ras61L, pZip/Ras61L, and dominant-negative
RasN17, pMT/RasN17, (0.5 µg each) were included. For analysis of
Elk-1 activity, pFA2-Elk1 (50 ng) and pFR-Luc (0.5 µg) were
cotransfected into HEK293T cells. Plasmid pCMV/
-gal was included to
monitor the transfection efficiency. The total amount of DNA was
maintained at 1-2 µg with an empty vector pCDNA3.0. Twenty-four
hours post-transfection, cells were treated with TFP. Cells were
harvested after 6-12 h of TFP treatment, and protein extracts were
prepared by three cycles of freezing and thawing. 1-5 µg of protein
was assayed for luciferase and
-galactosidase activities.
Luminescence was measured using a luminometer model TD 2020 (Berthold,
Tubingen, Germany). Transfection efficiencies were normalized by a
ratio of luciferase activity to
-galactosidase activity obtained
from the same sample.
 |
RESULTS |
Egr-1 Expression by Serum Is Rescued in the Presence of TFP or CPZ
in Human Fibrosarcoma HT1080 Cells--
It has been reported that
stable expression of Egr-1 leads to decreased DNA synthesis
and tumorigenesis in fibrosarcoma HT1080 cells (23). We investigated
whether inhibition of DNA synthesis by calmodulin antagonists is
related to a gain in Egr-1 expression. Phorbol ester
(phorbol 12-myristate 13-acetate (PMA)) was used as a positive control.
When cells in the log phase were treated with TFP for 2 h, a
slight increase in the amount of the Egr-1 protein was observed (Fig.
1A). Serum-starved cells
showed a marginal increase in the Egr-1 protein level when the cells
were treated with 20% serum. However, an increase was more evident in
cells treated with 25 µM TFP. Costimulation with serum
and TFP resulted in a dramatic increase in the level of Egr-1
expression. Another calmodulin antagonist chlorpromazine (CPZ) also
increased the level of the Egr-1 protein (Fig. 1B).
Reprobing the blots with anti-ERK1/2 antibodies revealed that similar
amounts of proteins were present in all lanes. These results indicate
that costimulation with serum and calmodulin antagonist has a
synergistic effect on expression of Egr-1 in serum-starved HT1080
cells.

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Fig. 1.
Effect of calmodulin antagonists on Egr-1
expression. A, mid-log cultured or serum-starved (with
0.5% serum for 24 h) HT1080 cells were pretreated with the
indicated concentrations of TFP for 30 min and treated with 20% serum
for an additional 2 h. 50 nM PMA was used as a
positive control. The Egr-1 level was detected in whole cell lysates
(20 µg/lane) by Western blotting against rabbit anti-Egr-1 antibodies
(1:1000). The lower panel shows the same blot stripped and
reprobed with rabbit anti-ERK1/2 antibodies for an internal control of
the protein contents per lane. B, serum-starved HT1080 cells
treated with TFP (50 µM) or CPZ (50 µM) for
30 min before addition of 20% serum. After 2 h, whole cell
lysates were prepared and Western blotting was performed as in
A.
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|
Kinetics of TFP-dependent Egr-1 Expression--
To
determine the effect of TFP on the increase in the Egr-1 expression
level, HT1080 cells were serum-starved for 24 h, then treated with
20% serum in the presence of various concentrations of TFP. The Egr-1
protein was detected in cells treated with serum and 10 µM TFP. The protein level was markedly increased at a
concentration of 50 µM TFP (Fig.
2A). When the effect of this
concentration of TFP on cell viability was examined by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay, the
cell survival was about 50% of untreated control (data not shown).
Time course studies showed that an increase in the Egr-1 protein level
due to TFP was detected at 2 h with a continual increase through
3 h after addition of TFP (Fig. 2B). Reprobing the
blots with anti-ERK antibodies revealed that similar amounts of
proteins were present in all lanes.

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Fig. 2.
Dose- and time-dependent increase
of the Egr-1 protein by TFP. Serum-starved HT1080 cells (0.5%
serum for 24 h) were treated with various concentrations of TFP
for 2 h (A) or 50 µM TFP for the
indicated times (B). PMA (50 nM) was used as a
positive control. Whole cell lysates were analyzed by Western blotting
against rabbit anti-Egr-1 antibodies (1:1000). The lower
panel shows the same blot stripped and reprobed with rabbit
anti-ERK1/2 antibodies (1:6000) as an internal control of the protein
contents per lane.
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Northern blot analysis was performed to determine whether the increase
in the expression level of the Egr-1 protein due to TFP occurs at the
mRNA level. The GAPDH mRNA level was used as an internal
control. Expression of Egr-1 mRNA was markedly increased by treatment with 50 µM TFP (Fig.
3A). This result was
consistent with results of Western blot analysis. Time course studies
showed that the Egr-1 mRNA level was increased at
0.5 h after addition of serum and TFP with a maximum increase at
1 h, followed by a slow decline (Fig. 3B).

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Fig. 3.
Dose- and time-dependent
induction of Egr-1 mRNA expression by TFP.
HT1080 cells were serum-starved, treated with various concentrations of
TFP for 2 h (A) or 50 µM TFP for the
indicated times (B). PMA (50 nM) was
used as a positive control. Total RNA (10 µg) was isolated from
cells, electrophoresed on 1% agarose-gel, and capillary transferred to
a nylon filter. The blot was hybridized with the
32P-labeled Egr-1 probe. GAPDH mRNA was
determined as a control to verify the amount of RNA in each lane. The
lower panel shows ethidium bromide stained total RNA.
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Egr-1 Promoter Activity Is Stimulated by Serum and TFP--
To
determine whether TFP causes an increase in Egr-1 mRNA
at the transcription level, we constructed a series of luciferase reporter plasmids containing serial deletions of 5'-flanking sequences and the TATA motif of the human Egr-1 promoter (26). Each
promoter-reporter construct was transfected with pCMV/
-gal for
transfection efficiency into HEK293T cells. Twenty-four hours after
transfection, cells were treated without or with 50 µM
TFP (Fig. 4). Results were expressed as
-fold increases in luciferase activity normalized for
-galactosidase
activity. The
668 and
454 constructs exhibited 20- and 14-fold
increased luciferase activity, respectively, due to TFP treatment. A
further deletion between
454 to
233 (p-233egrLuc), which removed
the SRE cluster, displayed 4.2-fold increased promoter activity,
suggesting that SRE cluster region is important for the promoter
activity increment. These results indicate that TFP stimulates
Egr-1 gene expression at the transcription level.

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Fig. 4.
Effect of TFP on human Egr-1
promoter activity. The 5' boundaries of plasmids containing
various truncations of the Egr-1 promoter fused to
luciferase reporter genes are shown. The numeric designations of each
construct refer to the 5'-deletion end points derived from the
transcription start site at +1. The positions of putative regulatory
elements (SP1, CRE, and SRE) are
indicated by circles. A, human HEK293T cells
cultured on 35-mm dishes were cotransfected with each construct of the
Egr-1 promoter (0.5 µg) and the pCMV/ -gal plasmid (0.2 µg). After 24 h of transfection, cells were treated without
(NT) or with 50 µM TFP. B, human
HEK293T cells cultured on 35-mm dishes were cotransfected with
p-233egrLuc (0.5 µg) and the pCMV/ -gal plasmid (0.2 µg). After
24 h of transfection, cells were treated with the indicated
concentrations of TFP. After incubation for an additional 12 h,
cells were harvested and protein extracts were prepared by three cycles
of freezing and thawing. 1 µg of protein was assayed for luciferase
and -galactosidase activities. Results are shown as the amount of
induction after correcting for -galactosidase activity.
Bars represent the mean of a single experiment performed in
triplicate ± S.D. Similar results were observed in three
independent experiments.
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The Ca2+/CaM-dependent Pathway Is Involved
in Serum and TFP-stimulated Egr-1 Expression--
Calmodulin modulates
the action of various calmodulin-binding proteins, including CaMK-II
and calcineurin. Therefore, we investigated whether the
Ca2+/CaM/CaMKII/calcineurin pathway is involved in
TFP-dependent Egr-1 induction. HT1080 cells were
treated in both the presence and absence of TFP with the
Ca2+ ionophore ionomycin and KN62, a selective inhibitor of
CaMK-II. After exposure to x-ray film, the relative intensities of
Egr-1 and ERK1/2 bands in each lane were determined by quantitative scanning densitometer. As shown in Fig.
5A, ionomycin alone had no
effect on Egr-1 induction, whereas serum- and TFP-dependent Egr-1 expression was significantly attenuated by the addition of
ionomycin. In addition, KN62 and cyclosporin A, a calcineurin inhibitor, enhanced TFP-dependent Egr-1 expression (Fig. 5,
B and C). These results suggest that the
Ca2+/CaM/CaMK-II pathway negatively regulates Egr-1
expression.

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Fig. 5.
Effect of ionomycin, KN62, and CsA on
TFP-mediated Egr-1 expression. A, HT1080 cells were
serum-starved, pretreated with TFP (50 µM) for 30 min,
then exposed to 20% serum in the absence or presence of ionomycin (0.5 µM). B and C, serum-deprived HT1080
cells were pretreated with TFP (25 µM) for 30 min
together with various concentrations of either KN62 (B) or
CsA (C), then exposed to 20% serum. After an additional
incubation for 2 h, cells were harvested and whole cell lysates
were prepared for Western blot analysis against rabbit anti-Egr-1
antibodies (1:1000). The middle panel shows the same blot
stripped and reprobed with rabbit anti-ERK1/2 antibodies (1:6000) as an
internal control of the protein contents per lane. The Egr-1 band is
indicated by an arrow. The bottom panel shows the
relative intensity of the fluorographic data quantified by
densitometry, and the ratio of Egr-1 to ERK1/2 was determined.
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PKC Is Not Required for Egr-1 Accumulation Induced by Serum and
TFP--
It has been reported that calmodulin antagonists modulate
protein kinase C (PKC) activity (27) and that the PKC activator PMA
stimulates Egr-1 gene induction (Figs. 1-3). We
investigated the effect of PKC inhibitors on TFP-dependent
Egr-1 expression. Serum-starved HT1080 cells were incubated with
specific inhibitors of PKC (rottlerin and GF109203X) before addition of
serum and TFP. Results showed that PKC inhibitors did not have any
significant effect on TFP-dependent Egr-1 expression (Fig.
6A). To further determine the
role of PKC, the effect of down-regulation of PKCs by prolonged
treatment with high concentrations of PMA was examined (Fig.
6B). Down-regulation of PKCs was determined by Western
blotting using anti-PKC
antibody. After prolonged treatment with 1 µM PMA, PKC
was no longer detected. Because Egr-1 was
not efficiently induced by retreatment with 50 nM PMA, full
suppression of PKC activity is indicated. Under these conditions, Egr-1
remained inducible by stimulation with serum and TFP (compared with
control cells). These results demonstrate that PKCs are not involved in TFP-dependent Egr-1 expression.

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Fig. 6.
PKC is not involved in TFP-mediated Egr-1
induction. A, HT1080 cells were serum-starved,
pretreated with either GF109203X (2 µM) or rottlerin (20 µM) for 30 min, then treated with TFP (50 µM). After incubation for 30 min, cells were exposed to
20% serum. After an additional incubation for 2 h, cells were
harvested and whole cell lysates were prepared for Western blot
analysis against rabbit anti-Egr-1 antibodies (1:1000). The lower
panel shows the same blot stripped and reprobed with rabbit
anti-ERK1/2 antibodies (1:6000) as an internal control of the protein
contents per lane. The Egr-1 band is indicated by an arrow.
B, HT1080 cells were serum-starved in the presence
(PKC depletion) or absence (control) of 1 µM PMA for 24 h. After incubation, cells were
pretreated with TFP (50 µM) for 30 min, then exposed to
20% serum. PMA (50 nM) was used as a positive control.
After an additional incubation for 2 h, cells were harvested and
whole cell lysates were prepared for Western blot analysis against
anti-PKC (upper panel), anti-Egr-1 (middle
panel), and anti-ERK1/2 (lower panel) antibodies. An
arrow indicates the Egr-1 protein.
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The MEK Pathway Is Necessary to Stimulate Egr-1 Expression by
TFP--
To determine the signal pathway involved in the TFP response,
several kinase inhibitors were tested (Fig.
7A). Protein-tyrosine kinase
inhibitor (genistein), phosphatidylinositol 3-kinase inhibitor (LY294002), and PLC inhibitor (U73122) had no effect on Egr-1 expression due to serum and TFP. In contrast, PD98059, a specific MEK
inhibitor, efficiently inhibited TFP-stimulated Egr-1 expression. For
confirmation, the effect of PD98059 concentration dependence was
examined (Fig. 7B). Addition of increasing concentrations of
PD98059 progressively lowered the level of Egr-1 expression due to
serum and TFP, indicating that the MEK signaling cascade is required
for TFP-dependent Egr-1 expression.

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Fig. 7.
Effect of Ras/MEK cascades on TFP-mediated
Egr-1 expression. A, HT1080 cells were serum-starved
for 24 h, preincubated with U73122 (10 µM),
genistein (100 µM), PD98059 (50 µM), or
Ly294002 (50 µM) for 30 min, then treated with TFP (50 µM). After incubation for 30 min, cells were exposed to
20% serum. After an additional incubation for 2 h, cells were
harvested and whole cell lysates were prepared for Western blot
analysis against rabbit anti-Egr-1 antibodies (1:1000). The lower
panel shows the same blot stripped and reprobed with rabbit
anti-ERK1/2 antibodies (1:6000) as an internal control of the protein
contents per lane. The Egr-1 band is indicated by an arrow.
B, HT1080 cells were serum-starved, pretreated with the
indicated concentrations of PD98059 for 30 min, then treated with TFP
(50 µM). After incubation for 30 min, cells were exposed
to 20% serum. After an additional incubation for 2 h, cells were
harvested and whole cell lysates were prepared for Western blot
analysis against rabbit anti-Egr-1 antibodies (1:1000). The lower
panel shows the same blot stripped and reprobed with rabbit
anti-ERK1/2 antibodies (1:6000) as an internal control of the protein
contents per lane. The Egr-1 band is indicated by an arrow. C, HEK293T cells
were transfected with the p-688EgrLuc construct (0.5 µg) together
with expression vectors for dominant-active Ras61L (0.5 µg), and
dominant-negative RasN17 (0.5 µg). The pCMA/ -gal plasmid (0.2 µg) was included as an internal control for normalization of
transfection efficiency. The total amount of DNA was maintained at 1.2 µg with the pcDNA3.0 plasmid. After transfection, cells were
serum-starved and treated with either PMA (20 nM) or serum
in the absence or presence of PD98059 (50 µM). After
12 h the luciferase and -galactosidase activities were
measured. Normalization for transfection efficiency was determined by
-galactosidase expression. Results are presented as the amount of
induction considering the luciferase activity of the cells that were
not treated (NT) transfected with the empty pCDNA3.0
vector. Bars represent the mean of a single experiment
performed in triplicate ± S.D. Similar results were observed in
three independent experiments.
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TFP-dependent Activation of the Egr-1 Promoter Is
Inhibited by Dominant-negative Mutants of Ras--
To demonstrate that
the Ras/MEK pathway responds to TFP-stimulated activation of the
Egr-1 promoter, HEK293T cells were transiently cotransfected
with a p-688egrLuc construct and either a dominant-negative mutant
(pMT/RasN17) or a dominant-active mutant (pZip/Ras61L) of the Ha-Ras
expression plasmid. As shown in Fig. 7C, TFP-stimulated Egr-1 promoter activity was efficiently inhibited by
dominant-negative RasN17 or by pretreatment with PD98059. However, the
expression of dominant-active Ras61L continuously stimulated
Egr-1. These data indicate that the Ras/MEK pathway plays a
role in TFP-dependent Egr-1 gene expression.
ERK1/2 Are Not Directly Activated by TFP--
To investigate
whether TFP stimulates ERK, a downstream target of MEK, we first
examined the kinetics of ERK activation in response to serum
stimulation in HT1080 cells using antibodies specific for
phosphorylated forms of ERK1 and ERK2. In a control experiment for ERK1
and ERK2 phosphorylation, quiescent 3Y1 fibroblasts treated with serum
were included. HT1080 cells were serum-starved for 24 h, then
treated with 20% serum for various times. Phosphorylation of ERK was
analyzed by Western blot analysis. As shown in Fig. 8A, increased phosphorylation
of ERK1/2 was detected after 30 min of serum stimulation in 3Y1
fibroblasts. In contrast, a relatively high level of ERK1/2
phosphorylation was detected in unstimulated HT1080 cells and a further
increase of phosphorylation was not observed until 120 min after serum
stimulation, demonstrating that ERK1/2 are continuously activated
regardless of serum stimulation in HT1080 cells. These results are
consistent with previous reports that HT1080 cells are classified as
type III tumor cells with regard to the constitutive strong activation
of 41- and 43-kDa MAPKs (28). Fig. 8B shows that serum and
TFP had no effect on the increase of ERK1/2 phosphorylation, which was
inhibited by pretreatment with PD98059. Based on these observations we
hypothesize that downstream of ERK1/2 is involved in activation of the
Egr-1 promoter in response to TFP.

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Fig. 8.
TFP has no effect on serum-stimulated ERK
activation. A, 3Y1 fibroblast and HT1080 cells were
serum-starved for 24 h, then treated with 20% serum for the
indicated times. The cells were then harvested and lysed. The protein
samples (20 µg of each) were separated by 10% SDS-polyacrylamide gel
electrophoresis. Activation of ERK was analyzed by Western blotting
against antibodies (1:1000) specific for the ERK phosphorylated form
(anti-phospho-ERK). Arrows indicate the phosphorylated forms
of ERK1 and ERK2. B, HT1080 cells were serum-starved for
24 h, pretreated with the indicated concentrations of PD98059 for
30 min, then stimulated with TFP (50 µM). After 30 min of
incubation, cells were added with 20% serum for an additional 30 min.
After treatment, cells were lysed and protein extracts (20 µg of
each) were separated by 10% SDS-polyacrylamide gel electrophoresis and
analyzed by Western blotting against anti-phospho-ERK antibodies
(upper panel). The same blot was reprobed with anti-GAPDH
antibodies without stripping as an internal control of the protein
contents per lane (lower panel). Arrows indicate
the phosphorylated forms of ERK1 and ERK2 in the upper
panel, and GAPDH in the lower panel.
|
|
Elk-1 Is Involved in TFP-dependent Egr-1
Expression--
Previous studies have demonstrated that
Ca2+/calmodulin-dependent phosphatase
calcineurin negatively regulates the transcription activation of Elk-1
(29, 30). Serum-induced Egr-1 transcription is controlled
through a serum response element (SRE), which binds a complex of the
serum response factor and a member of the ternary complex factor (TCF).
The TCF family includes Elk-1 (31), Sap-1 (32), and Sap-2/Net/Erp
(33-35). The classical Ras/MEK/ERK cascade is responsible for the
phosphorylation of Elk-1, and Elk-1 phosphorylation by ERK correlates
with increased transcription activation (36).
To examine whether Elk-1 is involved in TFP-dependent
Egr-1 expression, a pFA-Elk1 construct that encodes the
fusion protein of the Gal4 DNA binding domain and the activation domain
of Elk-1 (amino acids 307-428) was transfected into HEK293T cells.
pFR-Luc, which contains five repeats of the Gal4 binding element
upstream of the luciferase reporter gene, was also transfected. This
assay system allows direct assessment of Elk-1-mediated transcription activation in response to stimuli. When cells were treated with serum
and TFP, activation of Elk-1 was observed, which was inhibited in a
dose-dependent manner by pretreatment with PD98059 (Fig. 9A). In addition,
TFP-stimulated Elk-1 activation was inhibited by expression of
dominant-negative RasN17 (Fig. 9B). Dominant-active Ras61L
constitutively stimulated Elk-1 transactivation, which was partially
inhibited by PD98059, confirming that MEK is necessary for
TFP-stimulated Elk-1 activation. Together, these results support a
model in which the calmodulin-dependent pathway stimulates
Egr-1 expression by modulating Ras/MEK/ERK/Elk-1
cascades.

View larger version (21K):
[in this window]
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|
Fig. 9.
Effect of TFP on Elk-1-mediated
transcriptional activation. A, HEK293T cells were
cotransfected with trans-activator pFA2-Elk1 (50 ng) and the
pFR-Luc reporter plasmid (0.5 µg). After transfection, cells were
pooled, split into 35-mm dishes, and serum-starved for 24 h. Cells
were pretreated without and with the indicated concentrations of
PD98059 for 30 min, then stimulated with TFP (50 µM).
After an additional incubation for 30 min, cells were added with 20%
serum. After 12 h, cells were harvested and the protein extracts
were prepared by three cycles of freezing and thawing. 1 µg of
protein was assayed for luciferase activity. Bars represent
the mean of a single experiment performed in triplicate ± S.D.
Similar results were observed in three independent experiments.
B, HEK293T cells were transfected with the pFA2-Elk1 (50 ng)
and the pFR-Luc reporter plasmids (0.5 µg) together with either
expression vectors for dominant-active Ras61L (0.5 µg) or
dominant-negative RasN17 (0.5 µg) as indicated. The pCMA/ -gal
plasmid (0.2 µg) was included as an internal control for
normalization of transfection efficiency. The total amount of DNA was
maintained at 1.25 µg by addition of empty pcDNA3.0 plasmid.
After transfection, cells were serum-starved and treated with 20%
serum and 50 µM TFP in the absence or presence of PD98059
as indicated. After 12 h, the luciferase and -galactosidase
activities were measured. Results are presented as the amount of
induction after normalization for transfection efficiency with
-galactosidase activity. Bars represent the mean of a
single experiment performed in triplicate ± S.D. Similar results
were observed in three independent experiments.
|
|
 |
DISCUSSION |
It is well known that Egr-1 is an immediate-early
response transcription factor that is rapidly induced in many different cell types by a variety of growth factors, cytokines, and injurious stimuli (37). Although Egr-1 is rapidly expressed due to
mitogenic signals, evidence is accumulating that suggests that
Egr-1 negatively regulates cell growth. Egr-1 is
little or not expressed in a number of tumor cells, including breast
carcinoma ZR-75-1, glioblastoma U251, osteosarcoma Saos-2, and
fibrosarcoma HT1080 cells (23). Expression of antisense
Egr-1 mRNA in v-sis-transformed NIH3T3 cells
leads to an enhanced transformed phenotype (38), indicating that loss
of Egr-1 gene expression is important for tumorigenic potential. On the contrary, introduction of Egr-1 into
HT1080 fibrosarcoma cells inhibits cell growth and tumorigenesis
(21-23) by induction of transforming growth factor-
1, fibronectin,
and the plasminogen activator inhibitor-1 (20, 39), supporting the
notion that Egr-1 does possess a growth suppressor activity.
TFP is a calmodulin antagonist that binds to calmodulin and inhibits
Ca2+/calmodulin-regulated enzyme activities. Calmodulin is
a Ca2+-binding protein present in all eukaryotic cells that
plays a role as a Ca2+ mediator (1). Recent evidence
suggests that calmodulin is involved in regulation of cellular
proliferation, and calmodulin antagonists offer the potential for new
therapeutic approaches in the treatment of certain cancers. However,
the mechanisms responsible for the antitumor activities of calmodulin
antagonists remain to be determined.
We found that the calmodulin antagonists TFP and CPZ stimulate
Egr-1 gene expression in human fibrosarcoma HT1080 cells.
Stimulation of Egr-1 expression by TFP was more evident in
serum-starved HT1080 cells than in mid-log phase cells (Fig.
1A). We also found that costimulation with serum and TFP
synergistically enhances Egr-1 expression by transcriptional
events (Figs. 3 and 4). We hypothesize that serum-induced
Egr-1 transcription in HT1080 cells is suppressed by some
unknown factors, and this repression can be reversed by treatment with
calmodulin antagonists.
To investigate whether the calmodulin-dependent pathway is
involved in suppression of serum-induced Egr-1, we used the inhibitors KN62 (inhibitor of CaMKII) and cyclosporin A (inhibitor of
calcineurin). In the presence of serum, TFP-mediated Egr-1 expression
was decreased by treatment with calcium ionophore and ionomycin,
whereas expression was enhanced by KN62 and cyclosporin A (Fig. 5).
These data indicate that
Ca2+/calmodulin-dependent protein kinase II and
calcineurin negatively regulate serum-stimulated Egr-1 expression in
HT1080 cells. These findings are consistent with previous studies,
which have shown that expressions of Ca2+-induced
Egr-1 and c-fos are enhanced by inhibition of
calcineurin with cyclosporin A in the PC12 and ELM-I-1 murine
erythroleukemia cells types (40, 41). Calcineurin is a ubiquitously
expressed serine/threonine protein phosphatase under the control of
Ca2+/calmodulin (42). Calcineurin is the main component of
the calcium signaling pathway in T lymphocytes, regulating the
translocation of transcription factor NF-AT by dephosphorylation (43).
As it does in T cells, calcineurin may play an important role in the
regulation of gene expression in other cell types. It has been reported
that activation of calcineurin inhibits epidermal growth factor-induced
stimulation of the Ets family of transcription factor Elk-1 and plays a
negative role in induction of c-fos transcription (30).
However, it remains to be established whether calmodulin/calcineurin constantly inhibits Elk-1 activity in HT1080 cells.
It has been reported that expression of the Egr-1 gene in
response to several mitogenic stimuli and the oncogene v-src
and v-raf is mediated primarily through the serum response
elements (SREs) in the 5'-flanking region of the gene (44-46). Once
activated, the two transcription factors, serum response factor and
ternary complex factor (TCF), form ternary complexes on the SRE and
mediate transcription activation. Phosphorylation of TCF, including
Elk-1, Sap-1, and Sap-2/Net/Erp, causes increased DNA binding and
formation of ternary complexes and thereby plays a primary role in
regulating early response gene expression, such as c-fos and
Egr-1 (47, 48). It is well known that mitogens, including
serum and growth factors, rapidly activate signal transduction pathways
involving the Ras/MEk/ERK pathway. The classic Ras/MEK/ERK cascade is
responsible for phosphorylation of Elk-1, which is a well characterized
TCF. Elk-1 phosphorylation correlates well with increased transcription activation of c-fos and Egr-1 (36).
We found that serum- and TFP-induced Egr-1 expression was inhibited
efficiently by PD98059, a specific MEK inhibitor, but not by the PLC
inhibitor U73122, the phosphatidylinositol 3-kinase inhibitor LY294002,
or the tyrosine kinase inhibitor genistein (Fig. 7, A and
B). We also found that TFP-dependent activation of the Egr-1 promoter was repressed by transient expression
of the dominant-negative mutant of RasN17 and by pretreatment with PD98059 (Fig. 8C). These findings indicate that the Ras/MEK
cascades are necessary to activate Egr-1 expression due to
serum and TFP. Elk-1 was activated by treatment with TFP, and
TFP-stimulated Elk-1 trans-activation was inhibited by both
PD98059 and the dominant-negative mutant RasN17 (Fig. 9). Because TFP
did not directly activate ERK1/2 (Fig. 8B), TFP probably
acts downstream of ERK1/2, at least at the level of Elk-1. There is
evidence that Ca2+/calmodulin-dependent
calcineurin is a major phosphatase of Elk-1. Sugimoto et al.
(29) and the Tian and Karin group (30) reported that constitutive
activated expression of calcineurin or activation of endogenous
calcineurin by Ca2+ ionophore decreased epidermal growth
factor-induced phosphorylation of Elk-1 and that treatment with
cyclosporin A prevented the effect of calcineurin on Elk-1
phosphorylation and Ca2+-induced c-fos
induction. These results are consistent with the idea that TFP acts
upstream of calcineurin to participate in stimulation of Elk-1. Two
other MAPKs, JNK and p38, are also reported to phosphorylate Elk-1 (26,
49). Although the present data did not preclude the possible
involvement of JNK and p38 pathways in TFP-mediated Elk-1 activation,
the efficient inhibition of TFP-mediated Elk-1 activation by the
dominant-negative RasN17 and PD98059 (Fig. 9) indicates that
calcineurin is the major target of TFP, because it is known that the
MEK inhibitor PD98059 does not directly inhibit JNK or p38 activity. We
suggest that the calmodulin antagonist TFP stimulates Egr-1
gene induction via modulation of the Ras/MEK/ERK/Elk-1 cascade. Thus,
the convergence of the
Ca2+/calmodulin-dependent pathway and the
MEK/ERK/Elk-1 cascades coordinates suppression of Egr-1 gene
expression in HT1080 cells.
It has been reported that mutation of the ras oncogene is
commonly found in a variety of human tumors, and it is now clear that
mutated ras expression plays a pivotal role in controlling neoplastic transformation. Several lines of evidence suggest a role for
the constitutive activation of the Ras/MEK/ERK pathway in a mutated
Ras-mediated malignant transformation (28, 50). In human fibrosarcoma
HT1080 cells, a point mutation at codon 61 of N-Ras that converts
Gln61 to Lys61 has been found, which may be
needed to transform these cells to transformation states (51).
If loss of Egr-1 expression plays a role in the transformed
state of HT1080 cells (23) and the transformation states (51). If loss
of Egr-1 expression plays a role in the transformed state of
HT1080 cells (23) and the classic Ras/MEK/ERK pathway mediates the
transcription activation of Egr-1 via Elk-1, how
Egr-1 is down-regulated in oncogenic Ras-expressed HT1080
cells is still unknown. One intriguing possibility is negative feedback
regulation of Egr-1 expression by oncogenic Ras. In this regard, several studies have demonstrated that oncogenic Ras is involved in negative feedback suppression mechanisms as part of the
regulation of redundant incoming signals (52-54). Chen et
al. (55) reported that expression of activated Ras negatively
regulates calcium-dependent c-fos and
Egr-1 gene induction in lymphocytes. They also found that
the negative effect of Ras on Egr-1 and c-fos gene induction by calcium is abolished by pretreatment with cyclosporin A, indicating involvement of calcineurin in oncogenic Ras-mediated negative feedback regulation. Recently, Sugimoto et al. (56) reported that a kinase suppressor of Ras (KSR) specifically blocks epidermal growth factor and Ras-induced transcription activation of all
members of the TCF family, including Elk-1, Sap1a, and Sap2, without
affecting the MAPK activity. Moreover, they found that the effect of
KSR on the inhibition of Elk-1 transcription activation is mediated by
accumulation of dephosphorylated Elk-1 via the action of calcineurin.
Based on these reports, one can speculate that KSR plays a critical
role in suppressing expression of tumor suppressor Egr-1,
thereby promoting Ras-induced transformation in HT1080 cells. In future
studies it will be of interest to examine the possible roles of KSR in
the progression of Ras-induced transformation. We cannot, however, rule
out the possibility that Egr-1 transcription can be
regulated by other proteins that are sensitive to oncogenic Ras expression.
Our study demonstrates that TFP as a calmodulin antagonist
synergistically stimulates induction of tumor suppressor
Egr-1 gene expression in the presence of serum in HT1080
human fibrosarcoma cells. TFP probably plays a role in modulating
Ras/MEK/ERK/Elk-1 cascades, presumably through inhibition of the
Ca2+/calmodulin-dependent pathway. It will be
of interest to determine whether TFP-mediated Elk-1 activation of
transcription through SRE is the only pathway by which TFP induces
expression of SRE-regulated genes. The use of calmodulin antagonists
may provide a useful approach to suppression of neoplastic growth.
 |
FOOTNOTES |
*
This work was supported by grants from the Korean Science
and Engineering Foundation (961-0100-001-2), the Ministry of Health and
Welfare (HMP-96-M-2-1053), and the Ministry of Science and Technology.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶¶
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, College of Medicine, Yeungnam
University, Daemyung-Dong, Nam-Gu, Daegu 705-717, South Korea. Tel.:
82-53-620-4344; Fax: 82-53-654-6651; E-mail:
younglee@med.yu.ac.kr.
Published, JBC Papers in Press, December 19, 2000, DOI 10.1074/jbc.M009465200
 |
ABBREVIATIONS |
The abbreviations used are:
CaMK, calmodulin-dependent protein kinase;
TFP, trifluoperazine;
TCF, ternary complex factor;
DMEM, Dulbecco's modified Eagle's
medium;
MOPS, 4-morpholinepropanesulfonic acid;
kb, kilobase(s);
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
PCR, polymerase chain
reaction;
CMV, cytomegalovirus;
PMA, phorbol 12-myristate 13-acetate;
CPZ, chlorpromazine;
SRE, serum response element;
PKC, protein kinase
C;
MAPK, mitogen-activated protein kinase;
ERK, extracellular
signal-regulated kinase;
MEK, MAPK/ERK kinase;
JNK, c-Jun N-terminal
kinase;
KSR, kinase suppressor of Ras.
 |
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