Thapsigargin-Induced Gene Expression in Nonexcitable Cells Is Dependent on Calcium Influx
Karin D. Rodland,
Robert P. Wersto,
Susan Hobson and
Elise C. Kohn
Department of Cell and Developmental Biology (K.D.R., S.H.)
Oregon Health Sciences University Portland, Oregon 97201-3098
Laboratory of Pathology (R.P.W., E.C.K.), National Cancer
Institute Bethesda, Maryland 20892
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ABSTRACT
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Agents such as thapsigargin and endothelin elevate
intracellular calcium levels by a combination of calcium release from
intracellular stores and calcium influx across the plasma membrane;
however, the relative contribution of influx vs. release in
modulating calcium-dependent gene expression is not as well understood
in nonexcitable cells as in excitable cells. In this report we have
been able to separate thapsigargin-induced elevation of intracellular
calcium into release and influx components, using carboxyamido-triazole
(CAI), a known inhibitor of calcium influx with antiproliferative
activity against a number of human carcinomas, to selectively inhibit
influx without affecting release. The results of these experiments
indicate that the ability of thapsigargin to induce calcium-dependent
gene expression in nonexcitable cells is dependent on the induction
of calcium influx, presumably through store-operated calcium
channels. CAI treatment specifically inhibited thapsigargin- or
endothelin-stimulated expression from the c-fos promoter in
Rat-1 cells and in epithelial cell lines derived from ovary and breast.
Use of the VL30 model system confirmed the ability of CAI to inhibit
calcium-dependent gene expression and further demonstrated that the
ability of elevated calcium to synergize with other signaling pathways
required close temporal coupling. In addition to inhibiting
endothelin-induced calcium influx, CAI treatment also resulted in a
partial inhibition of IP3 production and
calcium release. CAI treatment also blocked the increase in ERK1 kinase
activity observed in response to either endothelin or thapsigargin,
suggesting a role for calcium influx in the activation of
mitogen-activated protein kinase pathways.
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INTRODUCTION
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Calcium is an ubiquitous intracellular messenger and regulator of
cellular activities. As both a second messenger and a modulator of
selected signal transduction pathways, calcium has the potential to
modulate a variety of cellular functions, including proliferation,
cell-substratum interaction, cytoskeletal organization, and gene
expression (1, 2, 3). Elevation of intracellular calcium has been
associated with the induction of a variety of proliferation-associated
immediate early genes, including c-fos, c-jun,
and VL30 (4, 5, 6, 7).
In the case of neuronal cells, c-fos expression appears to
be regulated at the transcriptional level by two distinct
calcium-dependent pathways. Calcium influx through voltage-sensitive
L-type calcium channels results in the phosphorylation of serine 133 on
CREB (the cAMP response element binding protein), presumably through a
Ca2+-calmodulin kinase-dependent pathway (8, 9, 10). In
contrast, calcium influx through the
N-methyl-D-aspartate receptor activates
c-fos transcription through the serum response element and
appears to involve phosphorylation of Elk-1 in a Ras-dependent fashion
(11). Growth factor-mediated induction of c-fos gene
expression appears to utilize a similar Ras-dependent phosphorylation
of Elk-1, and potentially CREB (12). Elevated intracellular calcium has
also been shown to promote elongation of c-fos transcripts
via an attenuator sequence in the first intron (13).
The importance of these pathways in regulating c-fos
expression in nonexcitable cells has not been well characterized. The
ability of endothelin-1 to induce c-fos expression in Rat-1
fibroblasts is dependent upon the elevation of intracellular calcium,
as demonstrated by the inhibitory effect of intracellular calcium
chelators (6). Intracellular calcium must be elevated above a threshold
of 200 nM in order for endothelin-1 to induce expression of
the immediate early gene VL30 (7). Endothelin-1 is known to activate
both calcium release and calcium influx in Rat-1 cells (14, 15). The
ability of endothelin-1 to elevate intracellular calcium can be
mimicked by treating cells with thapsigargin, an inhibitor of the
Ca2+-ATPase responsible for sequestering calcium in
intracellular stores (16, 17). Thapsigargin treatment leads to both
increased calcium release and calcium influx across the plasma membrane
(16, 18). The relative contribution of calcium release vs.
calcium influx in the regulation of either c-fos or VL30
expression has not been determined.
Carboxyamido-triazole (CAI) is a novel inhibitor of non-voltage-gated
calcium influx in response to a variety of agonists including carbachol
(19), maitotoxin [a nonionophoretic stimulator of calcium influx
(20)], and the ionophore A23187 (21). In the concentration range that
inhibits calcium influx (110 µM), CAI has demonstrated
antiproliferative and antimetastatic properties (19, 21, 22, 23, 24).
Structure-activity investigations demonstrate that the same moieties of
the CAI molecule required for modulation of calcium influx are also
required for the antiproliferative actions of CAI (23). In this report
we use CAI to selectively inhibit the influx component of the calcium
elevation observed in response to thapsigargin treatment and further
demonstrate that calcium influx is required for the transcriptional
induction of the immediate early genes c-fos and VL30 by
thapsigargin. We also demonstrate a previously undocumented ability of
CAI to inhibit calcium release and inositol triphosphate
(IP3) generation in Rat-1 fibroblasts stimulated with
endothelin-1. These data suggest that the antiproliferative activity of
CAI may be a consequence of CAIs ability to inhibit calcium-dependent
expression of immediate early genes required for cellular
proliferation.
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RESULTS
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Effect of CAI Treatment on Intracellular
Ca2+ Levels in Rat-1 Cells
CAI has been previously characterized as a specific inhibitor of
calcium influx (19, 21). In an attempt to selectively inhibit calcium
influx in response to endothelin-1 and thapsigargin without affecting
calcium release, we determined the effect of CAI pretreatment on
intracellular calcium levels in Rat-1 cells treated with either
thapsigargin or endothelin-1. Thapsigargin treatment produced a gradual
rise in intracellular calcium concentration attributed to release from
internal stores, followed by a slow return to basal levels; this
calcium shoulder is a consequence of calcium influx through
store-operated calcium channels (SOCC1;
Refs. 16 and 18). Exposure to CAI minimally reduced the internal
release component observed in response to thapsigargin treatment but
markedly attenuated the component attributable to calcium influx
through SOCC (Fig. 1
, panels A and B). This is the first
demonstration that CAI inhibits the store-operated calcium channel.

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Figure 1. Effect of CAI Treatment on Thapsigargin-Induced
Changes in Intracellular Calcium Levels
Rat-1 TK3R-3 cells were grown on Pronectin-coated coverslips,
serum-starved, and loaded with Fura-2-AM as described in
Materials and Methods. Cells were exposed to DMSO (panel
A) or 10 µM CAI (panel B) for 4 h, then stimulated
with 2 µM thapsigargin (TG), and changes in intracellular
calcium concentration were determined as described in Materials
and Methods. Results presented are mean ± SD
of n 35 cells per experiment, representative of n = 3
experiments.
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CAI treatment had a pronounced effect on the magnitude of the calcium
transient observed in response to endothelin-1, whether measurements
were made in the presence or absence of extracellular calcium (Fig. 2
, panels A and B). This would indicate that CAI is able
to inhibit intracellular calcium release in response to endothelin-1.
In the presence of extracellular calcium, there is a sustained
elevation of intracellular calcium after endothelin-1 treatment, and
this has been attributed to calcium influx (14, 15). CAI treatment
eliminated this sustained increase (as did calcium-free external
medium), indicating that CAI also inhibited endothelin-1-mediated
calcium influx, possibly via the SOCC.

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Figure 2. Effect of CAI Treatment on Endothelin-1-Mediated
Signaling
Panels A and B, Effects of CAI on intracellular calcium levels. Rat-1
TK3R-3 cells were grown on coated coverslips, serum-starved, and loaded
with Fura-2-AM as described above. Cells were exposed to DMSO
(open circles) or 10 µM
CAI (closed circles) for 4 h, then
stimulated with endothelin-1 at 10-8 M (ET).
Changes in intracellular calcium concentration were measured in medium
containing 2 mM Ca2+ (panel A) or 0
mM Ca2+ 0.5 mM EGTA (panel B).
Results presented are mean ± SD of n 30
cells; the peak Cai in response to CAI + ET is offset
relative to ET alone for clarity. Panel C, Effect of CAI on
IP3 production. Rat-1 cells were grown to confluence in
10-cm plates, then serum-deprived in the presence of 3 µCi
[3H]myo-inositol as described in
Materials and Methods. After a 16-h exposure to either
CAI (black bars) or DMSO (open bars),
Rat-1 cells were exposed to either endothelin-1 (10-8
M) or vehicular control for 20 min in the presence of 100
mM LiCl. Inositol phosphates were extracted in formic acid
and fractionated on Dowex-formate columns as described in
Materials and Methods. Bars represent the
mean ± SD of pooled IP3 fractions
obtained from triplicate plates; similar results were obtained in three
independent experiments. Panel D, Effect of SK&F 96365 on intracellular
calcium levels. Rat-1 TK3R-3 cells were grown on coated coverslips,
serum-starved, and loaded with Fura-2-AM as described above. Cells were
exposed to DMSO (open circles) or 50 µM
SK&F 96365 (closed circles) for 16 h, then
stimulated with endothelin-1 at 10-8 M (ET).
Changes in intracellular calcium concentration were measured in medium
containing 2 mM Ca2+. Results presented are
mean ± SE of n 20 cells.
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To provide a potential explanation for the ability of CAI to inhibit
endothelin-mediated calcium release in Rat-1 cells, we measured the
effects of CAI treatment on IP3 production in response to
endothelin. Rat-1 cells exposed to 10 µM CAI for 16
h displayed a significant inhibition of IP3 production
measured 20 min after endothelin-1 treatment (45% inhibition,
P
0.03; Fig. 2C
).
Although the ability of CAI to inhibit thapsigargin-induced calcium
influx without affecting calcium release provides a useful tool for
distinguishing between these two components of the intracellular
calcium response to thapsigargin, the observation that CAI inhibited
both calcium release and influx in response to endothelin precluded us
from using CAI to separate these two components of endothelin
signaling. However, when the functionally similar but structurally
distinct compound SK&F 96365 was tested in a similar fashion, SK&F
96365 was observed to inhibit only the influx component of the
endothelin response (Fig. 2D
). This result is in accord with the
published effects of SK&F 96365 on calcium influx through refilling
channels (26) and provides a method for inhibiting endothelin-mediated
calcium influx independent of calcium release.
Inhibition of c-fos Expression by CAI
To determine whether the dual ability of CAI to inhibit the
elevation of intracellular calcium in response to endothelin-1 and to
inhibit thapsigargin-stimulated calcium influx could affect
calcium-sensitive gene expression, we tested the ability of CAI
treatment to modulate expression of FC2CAT. These
experiments used Rat-1 cells stably transfected with the
FC2CAT construct containing 1.4 kb of the c-fos
promoter, including the serum response element and calcium response
element/cAMP response element enhancer elements, which are known to be
responsive to calcium-dependent signals (27). Endothelin-1 is a potent
inducer of FC2CAT expression in Rat-1 cells, as illustrated
in Fig. 3
(lanes 3 and 13). As endothelin-1 is known to
signal by both calcium mobilization and activation of protein kinase C,
each of these component parts was tested individually, using
thapsigargin to elevate intracellular calcium and
12-O-tetradecanoyl-phorbol-13-acetate (TPA) to activate
protein kinase C. Although treatment with thapsigargin alone failed to
induce FC2CAT expression over control levels (lanes 5 and
15), cotreatment with TPA plus thapsigargin produced a 2- to 8-fold
increase in FC2CAT expression (Fig. 3
, lanes 9 and 19).
This result suggests that induction of c-fos by endothelin-1
requires concurrent activation of both protein kinase C- and
calcium-dependent pathways.

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Figure 3. Inhibition of c-fos Expression by
CAI in Rat-1 Cells
Panel A, Inhibition of FC2CAT expression after CAI
pretreatment. Rat-1 cells stably transfected with the
FC2CAT plasmid, as described in Materials and
Methods, were grown to confluence in 10-cm plates as described
in Fig. 1 . Cells were serum-deprived for 24 h before addition of
either DMSO (0.5%; open bars) or CAI (10
µM; hatched bars) for a 4-h pretreatment.
The pretreatment medium was replaced with fresh DMEM, and agonists were
added as indicated; cells were harvested for analysis of CAT activity
4 h later. Results are mean ± SD, n = 3,
and similar results were obtained in three experiments. Panel B,
Inhibition of FC2CAT expression after SK&F 96365
pretreatment. FC2CAT Rat1 cells were serum-deprived for
24 h before addition of either DMSO (0.5%; open
bars) or SK&F 96365 (50 µM; hatched
bars) for 4 h pretreatment. The pretreatment medium was
replaced with fresh DMEM, and agonists were added as indicated; cells
were harvested for analysis of CAT activity 4 h later. Results are
mean ± SD, n = 3. Similar fold induction and
percent inhibition were observed in three replicate experiments.
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In the presence of 10 µM CAI, FC2CAT
expression was inhibited to nearly basal levels whether the inducing
agent was endothelin-1 or combined TPA + TG (Fig. 3A
, lanes 4 and 10).
Treatment with SK&F 96365 also inhibited calcium-dependent induction
from the c-fos promoter (Fig. 3B
). In Fig. 2D
, we
demonstrated that SK&F 96365 had little or no effect on calcium release
in response to endothelin-1, but did reduce the sustained calcium
threshold attributed to calcium influx through the SOCC. These results
suggest that the initial elevation of intracellular calcium seen after
either endothelin-1 or thapsigargin treatment is insufficient to induce
c-fos expression, but the sustained increase dependent on
activation of SOCC is essential for induction.
Because elevated calcium has been implicated not only in the
transcriptional induction of c-fos but also in the sustained
elongation of nascent transcripts (13), it is important to determine
independently the effects of CAI on the accumulation of full length
c-fos mRNA. Analysis of c-fos mRNA, as opposed to
chloramphenicol acetyl transferase (CAT) protein, also controls for any
possible effects of thapsigargin on protein synthesis (28, 29).
Endogenous c-fos mRNA accumulation was measured in the
parental Rat-1 cells that had been exposed to either 10
µM CAI or a vehicular control before stimulation with
either endothelin-1 or TPA + thapsigargin for 20 min. Induction of
c-fos by either endothelin-1 or TPA + thapsigargin was
completely inhibited after exposure to CAI for 20 h (Fig. 4
, lanes 7 and 9 compared with lanes 2 and 4); an
approximately 50% reduction in c-fos mRNA levels was seen
after a 4-h exposure (data not shown). CAI treatment had a negligible
effect on the accumulation of cyclophilin mRNA (Fig. 4
, lower
panel; see also Fig. 5A
). Cyclophilin is
peptidyl-prolyl cis-trans isomerase, a ubiquitously
expressed gene required for collagen synthesis (31, 32).

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Figure 4. Inhibition of c-fos mRNA
Accumulation by CAI
Rat-1 cells were grown to confluence in 10-cm plates, then incubated in
serum-free medium containing either 10 µM CAI (lanes
610) or 0.1% DMSO (lanes 15) for 24 h before the addition of
endothelin-1 (10-8 M), TPA (100 ng/ml),
thapsigargin (2 µM), or TPA + thapsigargin where
indicated. RNA was extracted and processed for Northern hybridization
analysis as previously described (30). The 2.2-kb mRNA hybridizing to a
c-fos riboprobe (pGem3Z-cFos, obtained from D. Pribnow)
is indicated. Hybridization to the constitutively expressed gene
cyclophilin indicated that equivalent amounts of RNA were present in
each lane (lower panel). 32P was visualized
by exposure to a PhosphorImager screen (Molecular Dynamics) for 16
h.
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Figure 5. Inhibition of c-fos Expression by
CAI in Epithelial Cell Lines
Panel A, Inhibition of c-fos mRNA accumulation by CAI in
SKOV-3 cells. Confluent cultures of SKOV-3 cells were serum deprived
for 24 h, then exposed to 10 µM CAI for 20 h
before addition of endothelin-1 at 10-8 M. RNA
was harvested 20 min after endothelin-1 addition. RNA processing and
Northern hybridization analysis were conducted as described in Fig. 4 .
Similar results were obtained in two replicate experiments. Panel B,
Inhibition of c-fos mRNA accumulation by CAI in MCF-10F
cells. MCF-10F immortalized but nonmalignant human mammary epithelial
cells were grown to confluence in the presence of 10% bovine calf
serum (HyClone). CAI was added at a final concentration of 10
µM 24 h before harvesting as indicated. RNA was
harvested, processed, and subjected to Northern hybridization analysis
as described in Fig. 4 . Similar results were obtained in two
experiments.
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To determine whether the ability of CAI to inhibit c-fos
expression in murine fibroblasts was a general phenomenon of
nonexcitable cells, we tested the ability of CAI to modulate
c-fos mRNA and protein expression in two human cell lines
representing nonexcitable epithelial cell types. The human ovarian
carcinoma cell line SKOV-3 has low endogenous levels of
c-fos mRNA expression, but responds to 10-8
M endothelin-1 with a substantial induction of
c-fos mRNA (Fig. 5A
). Treatment of SKOV-3 cells with CAI at
10 µM for 20 h before addition of endothelin-1
completely inhibited c-fos mRNA accumulation (Fig. 5A
). A
similar reduction in serum-induced c-fos mRNA levels was
observed after CAI treatment of MCF-10F cells, an immortalized but
nonmalignant line of human mammary epithelial cells (Fig. 5B
).
Inhibition of VL30 Expression after CAI Exposure
To extend our observations to another proliferation-associated
gene with a well characterized induction in response to calcium, we
tested the effects of CAI and SK&F 96365 on the VL30 calcium-sensitive
enhancer. The responsiveness of the VL30 enhancer to various treatments
can be quantitatively studied in the TK3R-CAT cell line (described in
Ref.33), a Rat-1 derivative line containing a stably integrated VL30
enhancer-CAT reporter construct. When TK3R-CAT cells are exposed to
either epidermal growth factor (EGF), TPA, or thapsigargin as single
agonists, a 3-fold or less increase in CAT activity is observed;
simultaneous addition of thapsigargin with either EGF or TPA produces a
marked and statistically synergistic increase in TK3R-CAT expression
(Fig. 6
and Refs. 17 and 33). Endothelin-1, which is
known to elevate intracellular calcium, activate protein kinase C, and
activate the EGF receptor (14, 34), stimulates TK3R-CAT expression even
better than the combination of TPA and thapsigargin (Fig. 6
, lane 3
compared with lane 9). This increased response may be attributed to the
additional ability of endothelin-1 to activate the EGF receptor in
Rat-1 cells (34). To determine whether the known ability of CAI to
modulate changes in intracellular calcium in response to endothelin-1
and thapsigargin (Figs. 1
and 2
) would influence TK3R-CAT expression,
we exposed TK3R-CAT cells to CAI for up to 24 h before the
addition of agonists. Induction of TK3R-CAT expression by endothelin-1
was significantly inhibited by CAI pretreatment (P <
0.01) as shown in Fig. 6
(hatched bars). The ability of
thapsigargin to stimulate TK3R-CAT expression in conjunction with
either TPA, EGF, or cAMP was also significantly inhibited
(P < 0.01, Fig. 6
). These inhibitory effects were
observed even when the CAI-containing medium from the 20-h pretreatment
was replaced with fresh, CAI-free medium concurrent with agonist
addition, indicating that the continuous presence of CAI may not be
required for inhibition. The ability of CAI to inhibit combinations
involving cAMP and either EGF or TPA was also tested to determine
whether the inhibitory effects of CAI were specific for thapsigargin;
CAI failed to inhibit combinations that did not include thapsigargin
(Fig. 6B
). Similar effects were observed when the calcium ionophores
A23187 or ionomycin were substituted for thapsigargin (data not shown).
These results suggest that the inhibitory effects of CAI on VL30
expression are selective for interactions involving elevated
intracellular calcium.

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Figure 6. Inhibition of Calcium-Dependent VL30 Expression by
CAI
The Rat-1-derived TK3R-3 cell line, stably transfected with
pCBTK3R-CAT, was grown to confluency in 12-well plates in the presence
of DMEM + 10% defined calf serum (HyClone). Cells were serum-deprived
in DMEM for 72 h before agonist addition. Cells were exposed to
either 0.5% DMSO (hatched bars) or 10 µM
CAI (in 0.5% DMSO; open bars) for 20 h before
addition of agonists. The CAI and DMSO-containing media were aspirated
and replaced with fresh DMEM (without CAI) before addition of agonists
as indicated. After 4 h, cells were lysed, and CAT activity in the
cell extracts was assayed as described in Materials and
Methods. DMEM, No agonists; ET, 10-8 M
endothelin-1; TG, 2 µM thapsigargin; TPA, 100 ng/ml TPA;
cAMP, 600 µM (Bu)2cAMP. Results presented are
mean ± SD, n = 3. Similar fold induction and
percent inhibition were observed in four replicate experiments.
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Further support for calcium influx as a major requirement for VL30
expression in response to endothelin-1 or thapsigargin was obtained in
experiments in which SK&F 96365 was substituted for CAI. As was
observed for c-fos expression in Fig. 3
, 50 µM
SK&F 96365 inhibited TK3R-CAT expression in response to endothelin-1 or
combinations of thapsigargin and either EGF or TPA (Fig. 7
, light bars), although the magnitude of the
inhibition was less than had been observed after CAI treatment (compare
Fig. 6
, lanes 3, 4 and 9, 10 with Fig. 7
, lanes 3, 4 and 7, 8). This
result may reflect the generally lower potency of SK&F 96365 compared
with CAI (20); in the case of endothelin-induced VL30 expression the
difference may also represent the contribution of endothelin-induced
calcium release, which is not inhibited by SK&F 96365.

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Figure 7. Effect of SK&F 96365 Treatment on Calcium-Dependent
TK3R-CAT Expression
TK3R-3 cells were cultured and serum-starved as described. SKF
treatment (open bars) was achieved by adding 50
µM SK&F 96365 to media for 20 h before agonist
addition. All cells were changed into fresh serum-free DMEM immediately
before addition of agonists, and cells were harvested 4 h later.
Results shown are mean ± SD, n = 3, and similar
results were obtained in three replicate experiments.
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Effects of a Reversible Inhibitor of Calcium Sequestration on
TK3R-CAT Expression
To determine whether the elevation of intracellular calcium must
occur contemporaneously with the other agonists to produce the
synergistic increase in VL30 expression, we used the reversible
Ca2+-ATPase inhibitor 2,5-di-tert-butylhydroquinone (DBHQ)
(35). TK3R-CAT cells were exposed to EGF, TPA, or cAMP either singly
(Fig. 8
, lanes 14), in the continuous presence of 10
µM DBHQ (lanes 58), or 15 min after DBHQ had been
removed from the medium (lanes 912). Exposure to DBHQ alone for
either 15 min or 4 h produced a 5-fold increase in TK3R-CAT
expression, which was sustained throughout a 4-h wash-out period.
Coaddition of either EGF, TPA, or cAMP throughout the DBHQ exposure
produced a greater than additive increase in CAT expression similar to
that observed for the combination of thapsigargin and the same
agonists. However, removal of the DBHQ before addition of the other
agonists resulted in levels of CAT expression indistinguishable from
those induced by DBHQ alone. This result implies that elevation of
intracellular calcium must be coordinated with the activation of other
signaling pathways to produce a synergistic increase in gene
expression.

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Figure 8. Effects of DBHQ on Calcium-Dependent TK3R-CAT
Expression
TK3R-3 cells grown in DMEM as described were exposed to agonists as
indicated and harvested for CAT activity after 4 h. Control cells
received 0.5% DMSO during stimulation in addition to other agonists as
indicated, while co-DBHQ received 10 µM DBHQ added
concurrently with other treatments, and maintained throughout the 4-h
agonist treatment. In the DBHQ-removed group, cells were exposed to 10
µM DBHQ for 15 min, after which the DBHQ was removed and
cells placed in DMEM for 15 min before addition of other agonists:
DMSO, 0.05%, open bars; EGF, 10 ng/ml,
cross-hatched bars; TPA, 160 nM,
striped bars; cAMP, 600 µM, black
bars. Results are mean ± SD, n = 3.
Similar results were obtained with wash-out periods of 30, 60, and 120
min (data not shown).
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Effect of CAI on MAP Kinase Activation
Activation of c-fos expression through the SRE is known
to be influenced by the MAP kinase-mediated phophorylation of Elk-1
(11, 12). To determine whether this pathway of c-fos
induction might be inhibited as a consequence of CAI treatment, we
conducted in vitro kinase assays using immunoprecitated
ERK-1 and synthetic GST-ELK as a substrate. Endothelin-1 acted as a
potent activator of ERK1 kinase activity, which was increased
approximately 15- to 20-fold 20 min after exposure to 10-8
M endothelin-1 (Fig. 9A
, lane 3 compared
with lane 1); in comparison, 10 ng/ml EGF for 20 min produced a 40- to
50-fold increase in ERK1 activity (lane 5 compared with lane 1).
Pretreatment with CAI for 16 h completely inhibited the activation
of ERK-1 by endothelin (Fig. 9
, lane 4 compared with lane 3) but had no
signficant effect on the EGF-induced ERK1 activation (Fig. 9
, lane 6
compared with lane 5). This inhibition of endothelin-mediated, but not
EGF-mediated, ERK1 activation correlates well with the differential
effects of CAI treatment on endothelin-induced vs.
EGF-induced gene expression.

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Figure 9. Effect of CAI Treatment on ERK1 Activity
Rat-1 cells were grown to confluence in 10-cm plates, then
serum-deprived for 24 h in either 10 µM CAI or 0.1%
DMSO, as indicated. Cells were exposed to the indicated agonists (ET-1,
10-8 M; EGF, 10 ng/ml, TPA, 100 ng/ml; TG, 2
µM) for 20 min. Cells were lysed and cell lysates
(normalized to equal amount of protein) incubated overnight with
anti-ERK1 antibodies as described in Materials and
Methods. The immunoprecepitated proteins were used in in
vitro kinase assays and phosphorylated GST-Elk-1 visualized
with a PhosphorImager (panel A, 4 h exposure; panel B, 16 h
exposure). Panel A represents one complete set from triplicate lysates,
while panel B represents one set from duplicate lysates. Similar
results were obtained in three independent experiments.
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Neither TPA alone nor thapsigargin alone was highly effective in
activating ERK1, but the combination of TPA + thapsigargin produced an
increase in ERK-1 activity comparable to that observed in response to
endothelin-1 (Fig. 9B
, lanes 14). CAI treatment had little or no
effect on the TPA-mediated induction (lane 7 compared with lane 3) but
reduced the thapsigargin-mediated response to control levels (lane 8
compared lanes 1 and 4). In the presence of CAI, the combined TPA +
thapsigargin response was inhibited approximately 50% (lane 6 compared
with lane 2), consistent with the relative lack of CAI effect on
TPA-stimulated ERK1 activity.
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DISCUSSION
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Calcium has been shown to be an important messenger in the
regulation of several genes. In this report we show that CAI and SK&F
96365, two structurally unrelated inhibitors of calcium influx, can
inhibit the calcium-dependent induction of the proliferation-associated
genes c-fos and VL30. Neither compound had a significant
effect on the calcium-independent expression of these two genes. This
report also contains the first published demonstration that CAI can
specifically inhibit store-operated calcium channels. CAI has been
previously shown to inhibit non-voltage-gated calcium influx across the
plasma membrane through receptor-operated and ionophoretic channels
(20, 21, 23).
Thapsigargin is a selective activator of SOCC activity subsequent to
the depletion of intracellular calcium stores (16, 36, 37). The ability
of CAI to inhibit thapsigargin-dependent FC2-CAT and
TK3R-CAT expression can be attributed to the marked reduction in
thapsigargin-mediated SOCC activity observed in the presence of CAI
(Fig. 1
). Confirmation that inhibition of calcium influx is responsible
for the reduction in TK3R-CAT activity is provided by studies using
SK&F 96365, an agent known to inhibit both receptor-operated calcium
influx and SOCC activity (28, 38, 39). The inhibition of
thapsigargin-stimulated VL30 and c-fos expression by CAI
and SK&F 96365 suggests that transmembrane calcium influx, most likely
due to SOCC activity in these cells, is an important component of the
calcium-signaling pathways leading to induction of c-fos and
VL30 gene expression. Results with the reversible inhibitor DBHQ would
suggest that a sustained elevation of intracellular calcium concurrent
with the stimulation of other pathways, such as protein kinase C, is
required for the synergistic induction of VL30.
The ability of endothelin-1 to induce both VL30 and c-fos
expression has been linked to elevation of intracellular calcium above
a threshold of 200 nM (6, 7). Endothelin-1 treatment
induces both a rapid release of intracellular calcium, producing peak
values over 700 nM, and a sustained elevation of
intracellular calcium over 200 nM, attributable to calcium
influx (Fig. 8
and Refs. 7, 14, 15, 40, and 41). In this report we
demonstrate that the sustained threshold elevation of intracellular
calcium dependent on calcium influx is required for the induction of
immediate early genes in response to endothelin.
CAI treatment has been previously shown to inhibit
carbachol-induced IP3 production in CHO cells expressing
the m5 acetylcholine receptor by about 40% (20), a reduction that is
similar to that observed in endothelin-stimulated Rat-1 cells exposed
to CAI (Fig. 2C
). In endothelin-stimulated Rat-1 cells treated with
CAI, this 45% inhibition of IP3 release was associated
with an 86% decrease in peak intracellular Ca2+ release
(Fig. 2
, A and B); such a substantial inhibition of intracellular
Ca2+ release in response to CAI has not been reported in
any other system. Dose-response curves for the effect of endothelin on
IP3 production and peak intracellular calcium concentration
in Rat-1 cells indicate that calcium release is somewhat more sensitive
to endothelin concentration than is IP3 production (15). At
an endothelin concentration producing half-maximal IP3
levels (0.3 nM), intracellular calcium concentration is
only 30% of the peak value observed at 10 nM (15). Thus
the partial inhibition of IP3 production observed in
response to CAI treatment may result in a proportionately greater
reduction in Ca2+ release. It is also possible that the
nearly complete inhibition of endothelin-stimulated Ca2+
release observed in the presence of CAI may reflect a previously
undescribed effect of CAI independent of direct effects on calcium
influx.
In this report, we have demonstrated that the calcium-dependent
induction of both VL30 and c-fos gene expression can be
inhibited by cellular CAI exposure, despite the substantial structural
differences in the regulatory elements of these two genes. The
calcium-responsive sequence elements of c-fos and VL30 are
functionally distinct, in that neither sequence will compete for
protein binding by the other (33, 42). While the ability of elevated
intracellular calcium to induce c-fos and VL30 expression
has been well studied, particularly regulation of c-fos in
excitable cells, little is known about the relative importance of
calcium influx vs. calcium release in modulating expression
in nonexcitable cells. The ability of CAI and SK&F 96365 to inhibit the
disparate calcium-responsive enhancers of c-fos and VL30
suggests that calcium influx, including SOCC-mediated influx, may be
effective generally as an inducer of calcium-dependent gene
expression in nonexcitable cells, regardless of the specific
transcription factors ultimately responsible for
trans-activation. The inhibition of ERK1s ability to
phosphhorylate Elk-1 observed after CAI treatment suggests that this
pathway is responsive to changes in intracellular calcium
concentration, particularly the sustained elevation attributed to SOCC
activity and inhibited by both CAI and SK&F 96365. Although the
signaling intermediary directly responsive to changes in intracellular
calcium has not been identified in these studies, inhibition of Elk-1
phosphorylation does provide a a potential model for the inhibition of
c-fos expression by CAI and SK&F and suggests that the
activation of c-fos expression in Rat-1 cells may utilize a
similar Ras-dependent pathway as that activated by calcium influx
through the N-methyl-D-aspartate receptor in
PC-12 cells (11).
Calcium-dependent expression of c-fos has the potential to
modulate expression of other genes such as c-jun (43),
interleukin-2 (44, 45), proenkephalin (46), and many members of the
matrix metalloproteinase family (47, 48). Kohn et al. (24)
have previously demonstrated that expression of matrix
metalloproteinase (MMP)-1/interstitial collagenase and MMP-2/type IV
collagenase can be inhibited by CAI in the same concentration range
that inhibits calcium influx. Expression of MMP-1/interstitial
collagenase is known to have a c-fos-dependent component
(48, 49), and the results of these studies suggest that inhibition of
MMP-1/interstitial collagenase expression by CAI may be attributed to
inhibition of c-fos expression. Other members of this
proteinase family with AP-1 regulatory sites include the newly
described matrix metalloproteinase MT-MMP (50), MMP-9 (51), matrilysin
(52), and stromelysin (53). The broad effect of CAI on the expression
of the MMPs is consistent with its demonstrated antiinvasive and
antiangiogenic effects (19, 24, 54).
The complex signaling pathways regulating cellular function
are now being elucidated by dual strategies focusing, respectively, on
the functional consequences of gene expression and on the propagation
of signaling events from the plasma membrane to the nucleus through
various cytoplasmic cascades. The use of CAI in this study combines
these two concepts in demonstrating that modulation of calcium influx
can be critical in driving the transcription of genes associated
with cell proliferation. The ability to modulate c-fos
expression has broader implications as suggested by the ability of CAI
to inhibit gene expression of MMPs with known AP-1-dependent
regulation. The ability of CAI to inhibit calcium-dependent gene
expression provides a unifying explanation for the biological effects
of CAI on proliferation, invasion, and angiogenesis.
 |
MATERIALS AND METHODS
|
---|
Cell Culture
The FC2-Rat1 cell line was created by stable
transfection of the FC2CAT plasmid (obtained from I. Verma,
Salk Institute, La Jolla, CA) into Rat-1 cells. Clonal isolates of
G418-resistant FC2-Rat1 cells were cultured and tested for
responsiveness to serum; the FC2-Rat1 cell line used in
these experiments displays a 30-fold increase in CAT expression after
serum stimulation. The Rat-1-derived TK3R-3 cell line, stably
transfected with the VL30 enhancer element linked to the
chloramphenicol acetyltransferase reporter gene in the context of the
herpes simplex thymidine kinase promoter, has been described previously
(33). TK3R-3 and FC2-Rat1 cells were maintained in DMEM
plus 10% defined calf serum (Hyclone, Logan UT) in 37 C, 95% air/5%
CO2. Cultures were supplemented with 10 µg/ml gentamicin,
1.75 mg/ml amphotericin B (Fungizone, Sigma, St. Louis MO), and 750
µg/ml G418 (GIBCO BRL, Gaithersburg, MD) was used for selection,
instead of neomycin.
Measurement of CAT Activity
The desired cell lines were grown to confluence in 12-well
plates and subjected to serum deprivation for 36 h
(FC2CAT) or up to 72 h (TK3R-3) to induce quiescence
before use. Agonists were added as described and cells harvested 4
h later, to allow sufficient time for synthesis of newly induced CAT
protein. Cellular proteins were extracted in 10 mM
Tris-HCl, 0.05% Triton X-100, and the cell extracts were incubated for
10 min at 70 C to inhibit cellular acetylases as previously described
(17). CAT activity was measured by the two-phase CAT assay of Neumann
et al. (55), and a rate of enzyme activity was determined
from the increase in [3H]acetyl-coenzyme A incorporated
over time. Only data from the linear portion of the reaction curve were
used.
Measurement of Intracellular Calcium
Intracellular Ca2+ concentrations were measured
using the Ca2+-sensitive fluorescent dye, Fura-2, as
previously described (14, 56). Rat-1 TK3R-3 cells were grown in
gelatin-coated coverslip chambers (Lab-Tek, Naperville IL) and serum
deprived for 48 h before loading with the acetoxyester of Fura-2
(Fura-2/AM, Molecular Probes Inc, Eugene OR; final concentration 1
µM) for 30 min at 37 C. An equal volume of 20% Pluronic
F-127 (Molecular Probes, Inc., Eugene OR) was added to facilitate
Fura-2 incorporation. After loading, cellular fluorescence was measured
at 400x using a using a Nikon inverted microscope coupled to a CCD
camera (Videoscope Int., Herndon VA), and the images were analyzed
using either the Image 1/Fluor software package (Universal Imaging,
West Chester, PA) or the InCa2-double wavelength package (Intracellular
Imaging, Inc., Cincinnati, OH). In each experiment, at least 30 cells
per field were measured. Intracellular calcium concentrations were
quantified in Fura-2-loaded cells by the fluorescence ratio method
(58), using the ratio of fluorescence emission at 510 nm from
excitation at 340 nm and 380 nm and calibrated in situ. All
measurements were acquired at room temperature.
Analysis of c-fos mRNA Expression
Confluent 10-cm plates of Rat-1 cells were serum-deprived for
36 h before the addition of experimental agents. Cell lysates for
RNA analysis were prepared at specified times after agonist addition by
the addition of a LiCl-urea-SDS lysis buffer as previously described
(14). RNA was processed and subjected to Northern hybridization
analysis, using a [32P]CTP-labeled c-fos
riboprobe transcribed from the plasmid pGEM3Z-cFos (obtained from D.
Pribnow, Oregon Health Sciences University, Portland OR, Ref.6).
Measurement of IP3 Levels
Confluent 10-cm plates of Rat-1 cells were labeled with
[3H]myo-inositol (3 µCi/ml) for 36 h,
followed by a 24-h incubation in serum-free DMEM lacking
[3H]myo-inositol and containing either
dimethylsulfoxide (DMSO) (0.1%) or 10 µM CAI. Cells were
exposed to either 10-8 M endothelin-1 or
vehicular control (0.05 mM HCl) for 20 min in the presence
of 100 mM LiCl. Cells were extracted in 0.2 M
formic acid, then diluted to 50 mM formic acid-100
mM ammonium formate by addition of NH4OH and
H2O. Extracts were applied to Dowex 1 ion exchange columns
and IP1 and IP2 were removed by multiple washes
in 0.1 M formic acid/0.5 M NH4
formate. Elutions of IP3 in 0.1 M formic
acid/0.75 M NH4 formate and IP4 in
0.1 M formic acid/1.1 M NH4 formate
were collected and quantified by liquid scintillation counting, as
previously described (14).
Measurement of MAP Kinase Activity
Confluent Rat-1 cells in 10 cm plates were cultured in
serum-free DMEM containing either DMSO (0.1%) or 10 µM
CAI for 24 h before the addition of the desired agonists
(endothelin-1, TPA, thapsigargin, or EGF). After 20 min of agonist
stimulation, the medium was aspirated and cells were immediately lysed
in 750 µl HEPES-KOH lysis buffer [20 mM HEPES-KOH, 2
mM EGTA, 50 mM ß-glycerophosphate, 10%
glycerol, 1% Triton X-100, 1 mM dithiothreitol (DTT), 1
mM vanadate, 0.4 mM phenylmethylsulfonyl
fluoride, 0.5 µg/ml aprotinin, 0.5 µg/ml leupeptin]. Lysates were
cleared by addition of 10 µl protein A agarose and centrifugation.
Aliquots of cleared lysates normalized for protein content were
subjected to immunoprecitation overnight at 4 C using anti-ERK-1
(SantaCruz Biotechnology, Santa Cruz, CA), followed by addition of
protein A agarose and an additional 2 h incubation at 4 C.
Immunoprecipitates were recovered by centrifugation and washed once in
HEPES-KOH lysis buffer, once in LiCl buffer [500 mM LiCl,
100 mM Tris HCl, pH 7.6, 0.1% Triton X-100, 1
mM DTT, 1 mM vanadate, 0.4 mM
phenylmethylsulfonyl fluoride] and once in
3-[N-morpholino]propanesulfonic acid (MOPS) assay buffer
[20 mM MOPS, pH 7.2, 20 mM MgCl2,
2 mM EGTA, 2 mM DTT, 0.2% Triton X-100]. The
pellets were resuspended in 20 µl kinase assay buffer [10
mM MOPS, pH 7.2, 20 mM MgCl2, 1
mM EGTA, 1 mM DTT, 0.1% Triton X-100, 3 µg
GST-Elk1, 1 µCi 32P-
-ATP] and incubated at 30 C for
20 min. Phosphorylated proteins were resolved by SDS-PAGE in 12%
acrylamide gels. Gels were dried and radioactivity visualized and
quantitated with a Molecular Dynamics PhosphorImager (Molecular
Dynamics, Sunnyvale, CA) and IP LabGel software.
Reagents
Thapsigargin (LC Services, Woburn, MA) was dissolved in dimethyl
sulfoxide (DMSO) and stored at -20 C before use. Endothelin-1 was
obtained from Peptides International (Louisville, KY) and was dissolved
in H2O and stored at -70 C before use. TPA, A23187, and
EGF were obtained from Sigma Chemical Co. (St. Louis, MO) and were
dissolved in the appropriate vehicle (DMSO for TPA and A23187; 50
mM HCl for EGF). SK&F 96365 and
2,5-di-tert-butylhydroquinone (DBHQ) were obtained from BioMol
(Plymouth Meeting, PA) and dissolved in DMSO. CAI was obtained from the
Developmental Therapeutics Program, National Cancer Institute. Working
stocks of all reagents were prepared in 50% DMSO immediately before
use, and the final DMSO concentration was held constant throughout each
experiment at either 0.5% or 1%. Control experiments indicated that
neither of these DMSO concentrations affected gene expression or cell
viability.
 |
ACKNOWLEDGMENTS
|
---|
We thank Kirsten Taylor and Thanh-Hoai Dinh for expert technical
assistance, and Drs. Richard Mauer, David Levens, Lance Liotta, and
Bruce Magun for thoughtful discussions.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Karin D. Rodland, Department of Cell and Developmental Biology, L215, Oregon Health Sciences University, Portland, Oregon 97201-3098.
This work was supported by Grant CA-60738 from the NIH-National Cancer
Institute (to K.D.R.).
1 SOCC is used to denote the channel responsible
for calcium influx observed after depletion of intracellular calcium
stores; this channel is also referred to as a refilling or
capacitance channel (18 ). ICRAC is one
example of a SOCC (25 ). 
Received for publication April 26, 1996.
Revision received November 26, 1996.
Accepted for publication November 27, 1996.
 |
REFERENCES
|
---|
-
Cole K, Kohn EC 1994 Calcium-mediated signal
transduction: biology, biochemistry, and therapy. Cancer Metastasis Rev 13:3341
-
Schwartz MA 1993 Spreading of human endothelial cells on
fibronectin or vitronectin triggers elevation of intracellular free
calcium. J Cell Biol 120:10031010[Abstract]
-
Sjaastad M, Angres B, Lewis RS, Nelson WJ 1994 Feedback
regulation of cell-substratum adhesion by integrin-mediated
intracellular calcium signaling. Proc Natl Acad Sci USA 91:82148218[Abstract]
-
Fisch TM, Prywes R, Roeder R 1987 c-Fos sequences necessary
for basal expression and induction by epidermal growth factor,
120-tetradecanoyl phorbol 13-acetate, and the calcium ionophore. Mol
Cell Biol 7:34903502[Medline]
-
Sheng M, Dougan ST, McFadden G, Greenberg ME 1988 Calcium and
growth factor pathways of c-fos transcriptional activation
require distinct upstream regulatory sequences. Mol Cell Biol 8:27872796[Medline]
-
Pribnow D, Muldoon L, Fajardo M, Theodor L, Chen L-YS, Magun
BE 1992 Endothelin induces transcription of
fos/jun family genes: a prominent role for
calcium ion. Mol Endocrinol 6:10031012[Abstract]
-
Rodland KD, Muldoon LL, Lenormand P, Magun BE 1990 Modulation
of RNA expression by intracellular calcium. J Biol Chem 265:1100011007[Abstract/Free Full Text]
-
Thompson MA, Ginty DD, Bonni A, Greenberg ME 1995 L-type
voltage-sensitive Ca2+ channel activation regulates
c-fos transcription at multiple levels. J Biol Chem 270:42244235[Abstract/Free Full Text]
-
Sheng M, McFadden G, Greenberg ME 1990 Membrane
depolarization and calcium induce c-fos transcription via
phosphorylation of transcription factor CREB. Neuron 4:571582[Medline]
-
Sheng M, Thompson MA, Greenberg ME 1991 CREB: a
Ca2+-regulated transcription factor phosphorylated by
calmodulin-dependent kinases. Science 252:14271430[Medline]
-
Bading H, Ginty DD, Greenberg ME 1993 Regulation of gene
expression in hippocampal neurons by distinct calcium signaling
pathways. Science 260:181186[Medline]
-
Xing J, Ginty, DD, Greenberg ME 1996 Coupling of the RAS-MAPK
pathway to gene activation by RSK2, a growth factor-regulated CREB
kinase. Science 273:959963[Abstract]
-
Collart MA, Tourkine N, Belin D, Vassalli P, Jeanteur P,
Blanchard A-M 1991 c-Fos gene transcription in murine
macrophages is modulated by a calcium-dependent block to elongation in
intron 1. Mol Cell Biol 11:28262831[Medline]
-
Muldoon LL, Rodland KD, Forsythe ML, Magun BE 1989 Stimulation
of phosphatidylinositol hydrolysis, diacylglycerol release, and gene
expression in response to endothelin, a potent new agonist for
fibroblasts and smooth muscle cells. J Biol Chem 264:85298536[Abstract/Free Full Text]
-
Muldoon LL, Enslen H, Rodland KD, Magun BE 1991 Stimulation of
Ca2+ influx by endothelin-1 is subject to negative feedback
by elevated intracellular Ca2+. Am J Physiol
260:C1273C1281
-
Thastrup O, Cullen PJ, Drobak BK, Hanley MR, Dawson AP 1990 Thapsigargin, a tumor promoter, discharges intracellular
Ca2+ stores by specific inhibition of the endoplasmic
reticulum Ca2+-ATPase. Proc Natl Acad Sci USA 87:24662470[Abstract]
-
Lenormand P, Muldoon LL, Enslen H, Rodland KD, Magun BE 1990 Two tumor promoters, TPA and thapsigargin, act synergistically via
distinct signalling pathways to stimulate gene expression. Cell Growth
Differ 1:627635[Abstract]
-
Takemura H, Hughes AR, Thastrup O, Putney JWJ 1989 Activation
of calcium entry by the tumor promoter thapsigargin in parotid acinar
cells: evidence that an intracellular calcium pool, and not an inositol
phosphate, regulates calcium fluxes at the plasma membrane. J Biol
Chem 264:1226612271[Abstract/Free Full Text]
-
Kohn EC, Sandeen MA, Liotta LA 1992 In vivo
efficacy of a novel inhibitor of selected signal transduction pathways
including calcium, arachidonate, inositol phosphates. Cancer Res 52:32083212[Abstract]
-
Gusovsky F, Lueders JE, Kohn EC, Felder CC 1993 Muscarinic
receptor-mediated tyrosine phosphorylation of phospholipase C-gamma.
J Biol Chem 268:77687772[Abstract/Free Full Text]
-
Felder CC, Ma AL, Liotta LA, Kohn EC 1991 The
antiproliferative and antimetastatic compound L651582 inhibits
muscarinic acetylcholine receptor-stimulated calcium influx and
arachidonic acid release. J Pharmacol Exp Ther 257:967971[Abstract]
-
Kohn EC, Liotta LA 1990 L651582: a novel antiproliferative and
antimetastasis agent. J Natl Cancer Inst 82:5460[Abstract]
-
Kohn EC, Felder CC, Jacobs W, Holmes KA, Day A, Freer R,
Liotta LA 1994 Structure-function analysis of signal and growth
inhibition by carboxyamido-triazole, CAI. Cancer Res 54:935942[Abstract]
-
Kohn EC, Jacobs W, Kim Y-S, Alessandro R, Stetler-Stevenson
WG, Liotta LA 1994 Calcium influx regulates expression of matrix
metalloproteinase-2 (72 kDa Type IV collagenase, gelatinase
A). J Biol Chem 269:2150521511[Abstract/Free Full Text]
-
Hoth M, Penner R 1993 Calcium release-activated calcium
current in rat mast cells. J Physiol 465:359386[Abstract]
-
Merritt JE, Armstrong WP, Benham CD, Hallam TJ, Jacob R,
Jaxa-Chamiec A, Leigh B, McCarthy SA, Moores KE, Rink TJ 1990 SK&F
986365, a novel inhibitor of receptor-mediated calcium entry. Biochem J 271:515522[Medline]
-
Deschamps J, Meijlink F, Verma IM 1985 Identification of a
transcriptional enhancer element upstream from the proto-oncogene fos.
Science 230:11741177[Medline]
-
Wong WL, Brostrom MA, Kuznetsov G, Gmitter-Yellen D, Brostrom
CO 1993 Inhibition of protein synthesis and early protein processing by
thapsigargin in cultured cells. Biochem J 289:7179[Medline]
-
Magun BE, Rodland KD 1995 Transient inhibition of protein
synthesis induces the immediate early gene VL30: alternative method for
thapsigargin-induced gene expression. Cell Growth Differ 6:891897[Abstract]
-
Rodland KD, Jue SF, Magun BE 1986 Regulation of VL30 gene
expression by activators of protein kinase C. J Biol Chem 261:50295033[Abstract/Free Full Text]
-
Fischer G, Wittmann-Liebold B, Lang K, Kiefhaber T, Schmid F 1989 Cyclophilin and peptidyl-prolyl cis-trans isomerase are probably
identical proteins. Nature 337:476478[CrossRef][Medline]
-
Takahashi N, Hayano T, Suzuki M 1989 Peptidyl-prolyl cis-trans
isomerase is the cyclosporin A-binding protein cyclophilin. Nature 337:473475[CrossRef][Medline]
-
Rodland KD, Pribnow D, Lenormand P, Chen SLY, Magun BE 1993 Characterization of a unique enhancer element responsive to cyclic
adenosine 3', 5'-monophosphate and elevated calcium. Mol Endocrinol 7:787796[Abstract]
-
Daub H, Weiss FU, Wallasch C, Ullrich A 1996 Role of
transactivation of the EGF receptor in signalling by G-protein-coupled
receptors. Nature 379:557560[CrossRef][Medline]
-
Llopis J, Chow SB, Kass GE, Gahm A, Orrenius S 1991 Comparison
between the effects of the microsomal Ca2+-translocase
inhibitors thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone on
cellular calcium fluxes. Biochem J 277:553556[Medline]
-
Putney JJ, Takemura H, Hughes AR, Horstman DA, Thastrup O 1989 How do inositol phosphates regulate calcium signaling? FASEB J 3:18991905[Abstract/Free Full Text]
-
Thastrup O 1990 Role of Ca2+-ATPases in regulation
of cellular Ca2+ signalling, as studied with the selective
microsomal Ca2+-ATPase inhibitor, thapsigargin. Agents
Actions 29:815[Medline]
-
Nordstrom T, Nevanlinna HA, Andersson LC 1992 Mitosis-arresting effect of the calcium channel inhibitor SK&F
96365 on human leukemia cells. Exp Cell Res 202:487494[Medline]
-
Mason MJ, Mayer B, Hymel LJ 1993 Inhibition of
Ca2+ transport pathways in thymic lymphocytes by econazole,
miconazole, and SKF 96365. Am J Physiol 264:C654C662
-
Griendling KK, Tsuda T, Alexander RW 1989 Endothelin
stimulates diacylglycerol accumulation and activates protein kinase C
in cultured vascular smooth muscle cells. J Biol Chem 264:82378240[Abstract/Free Full Text]
-
Hirata Y, Yoshimi H, Takata S, Watanabe TX, Kumagai S,
Nakajima K, Sakakibara S 1989 Cellular mechanism of action by a novel
vasoconstrictor endothelin in cultured rat vascular smooth muscle
cells. Biochem Biophys Res Commun 154:868875
-
Lenormand P, Pribnow D, Rodland KD, Magun BE 1992 Identification of a novel enhancer element mediating calcium-dependent
induction of gene expression in response to either epidermal growth
factor or activation of protein kinase C. Mol Cell Biol 12:27932803[Abstract]
-
Angel P, Karin M 1991 The role of Jun, Fos, and the AP-1
complex in cell-proliferation and transformation. Biochim Biophys Acta 1072:129157[CrossRef][Medline]
-
Boise LH, Petryniak B, Mao X, June CH, Wang CY, Lindsten T,
Bravo R, Kovary K, Leiden JM, Thompson CB 1993 The NFAT-1 DNA binding
complex in activated T cells contains Fra-1 and JunB. Mol Cell
Biol 13:19111919[Abstract]
-
Jain J, McCaffrey PG, Miner Z, Kerppola TK, Lambert JN,
Verdine GL, Curran T, Rao A 1993 The T-cell transcription factor NFATp
is a substrate for calcineurin and interacts with Fos and Jun. Nature 365:352355[CrossRef][Medline]
-
Sonnenberg JL, Rauscher FJ, Morgan JI, Curran T 1989 Regulation of proenkephalin by Fos and Jun. Science 246:16221625[Medline]
-
McDonnell SE, Kerr LD, Matrisian LM 1990 Epidermal growth
factor stimulation of stromelysin mRNA in rat fibroblasts requires the
induction of the protooncogenes c-fos and c-jun,
and the activation of protein kinase C. Mol Cell Biol 10:42844293[Medline]
-
Angel P, Imagawa M, Chiu R, Stein B, Imbra RJ, Ramsdorf HJ,
Jonat C, Herrlich P, Karin M 1987 Phorbol ester-inducible genes contain
a common cis element recognized by a TPA-modulated trans-acting factor.
Cell 49:729739[Medline]
-
Angel P, Baumann I, Stein B, Delius H, Ramsdorf HJ, Herrlich P 1987 12-O-tetradecanoyl-phorbol-13-acetate induction of the human
collagenase gene is mediated by an inducible enhancer element located
in the 5'-flanking region. Mol Cell Biol 7:22562266[Medline]
-
Sato H, Takino T, Okada Y, Cao J, Shinagawa A, Yamamoto E,
Seiki M 1994 A matrix metalloproteinase expressed on the surface of
invasive tumour cells. Nature 370:6165[CrossRef][Medline]
-
Sato H, Seiki M 1993 Regulatory mechanism of 92 kDa type IV
collagenase gene expression which is associated with invasiveness of
tumor cells. Oncogene 8:395405[Medline]
-
Gaire M, Magbanua Z, McDonnell S, McNeil L, Lovett D,
Matrisian L 1994 Structure and expression of the human gene for the
matrix metalloproteinase matrilysin. J Biol Chem 269:20322040[Abstract/Free Full Text]
-
Kerr LD, Holt JT, Matrisian LM 1988 Growth factors regulate
transin gene expression by c-fos-dependent and
c-fos-independent pathways. Science 242:14241427[Medline]
-
Kohn EC, Alessandro R, Spoonster J, Wersto RP, Liotta LA 1995 Angiogenesis: role of calcium-mediated signal transduction. Proc Natl
Acad Sci USA 92:13071311[Abstract]
-
Neumann JR, Morency CA, Russian KO 1987 A novel rapid assay
for chloramphenicol acetyltransferase gene expression. BioTechniques 5:444447
-
Grynkiewicz G, Poenie M, Tsien RY 1985 A new generation of
Ca2+ indicators with greatly improved fluorescence
properties. J Biol Chem 260:34403450[Abstract]