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INTRODUCTION |
Research from many laboratories has shown that Ca2+
influx through ion channels can regulate gene expression by multiple
and diverse mechanisms. Activation of gene expression by
Ca2+ regulates fundamental biological responses, including
cell cycle, hormone secretion, and cell morphology (1). A
cell-permeable, tumor-promoting thapsigargin can cause a rise in
intracellular Ca2+ levels (2).
Src family kinases are involved in a broad range of cellular responses
ranging from cell division and cytoskeletal rearrangement in
fibroblasts to the differentiation of neuronal PC12 cells (3-5). v-Src
is a constitutively active protein-tyrosine kinase that results in
cellular transformation, and v-Src mutants deficient in kinase activity
do not transform cells, suggesting that phosphorylation of specific
cellular targets is important for the transformed phenotype induced by
v-Src (6). Many putative substrate proteins have been identified. Ras
and Raf have been reported to be required for the transformed phenotype
induced by v-Src (7, 8). Although Ras is not a substrate of v-Src, and
Raf is phosphorylated on tyrosine, Raf functions downstream of Ras
(9).
The immediate early gene pip92 was cloned from activated T
lymphocytes treated with cycloheximide (10) and serum-stimulated BALB/c
3T3 fibroblasts (11). pip92 is rapidly and transiently induced by stimulation with serum and growth factors in fibroblasts. In
addition to cell growth, pip92 is also induced during
neuronal differentiation by differentiating factors (12). Although
pip92 encodes a short-lived, proline-rich protein with no
significant sequence similarity to any known protein, little is known
about the function of its encoded protein.
Previously it was shown that thapsigargin-induced release of
intracellular Ca2+ activated mitogen-activated protein
kinase (MAPK)1 and
extracellular signal-regulated protein kinase (ERK) (13). This
activation is independent of protein kinase C or Ca2+
influx but requires the presence of Raf-1 (14). Src-tyrosine kinase
mediates the stimulation of Raf-1 and ERK by thapsigargin (15).
Recently, we have observed that pip92 is expressed in the
mouse brain after a single intraperitoneal injection of excitatory amino acid NMDA (16). Many actions of NMDA are coupled to the influx of
extracellular Ca2+ mediated directly or indirectly by its
receptor present in neurons, and transient changes in intracellular
Ca2+ levels are known to trigger a number of cellular
responses, including change of gene expression (17). Ca2+
is known to activate ERK signaling in neuronal PC12 and H19-7 cells
(15, 18). On the basis of those findings, it was suggested that the
calcium ion and Src kinase could regulate the pip92
expression. To resolve the mechanism of thapsigargin-induced cell
growth, we analyzed whether pip92 expression is induced by
thapsigargin subsequently followed by intracellular free
Ca2+ mobilization and whether the activation of ERK and Src
kinase is involved. The results indicated that an increase of
intracellular calcium ion levels by thapsigargin stimulates
pip92 expression via ERK- as well as Src
kinase-dependent signaling pathways, suggesting that
pip92 is likely to play a role in thapsigargin-induced
neuronal cell growth.
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EXPERIMENTAL PROCEDURES |
Materials--
Fetal bovine serum, Dulbecco's modified Eagle's
medium (DMEM), and G418 were purchased from Life Technologies, Inc.
Thapsigargin, ionomycin, and PD98059 were purchased from
Calbiochem. All other used chemicals were commercial products of
analytical grade from Sigma. The pip92
promoter-chloramphenicol acetyltransferase (CAT) reporter fusion
construct (
1281pip92/CAT) was provided by L. F. Lau.
Plasmids encoding v-Src and kinase-inactive mutant Src were kindly
provided by D. Foster, and plasmids for kinase-inactive MAPK kinase
(MEK-2A) and constitutively active MEK (MEK-2E) were obtained from G. Johnson. The plasmid encoding a dominant inhibitory Raf-1 kinase
(Raf-KR) was given by C. Marshall. Plasmid GST-ElkC, expressing
glutathione S-transferase fused to the C-terminal peptide (amino acids 307-428) of wild type Elk1 was provided by R. Treismann.
Cell Culture--
The rat neuronal hippocampal cell line H19-7
was generated by transduction with the retroviral vectors containing
the temperature-sensitive simian virus 40 large T antigen that is
functionally active at 33 °C and inactive at 39 °C (19). The
cells were cultured at 33 °C in DMEM containing 10% fetal bovine
serum and 200 µg/ml G418 to maintain selection pressure on the
transduced immortalization vector. When specified, cells were
pretreated with 30 µM synthetic MEK inhibitor PD98059 for
30 min before drug stimulation.
Determination of DNA Synthesis--
DNA synthesis was measured
by use of [3H]thymidine incorporation. The H19-7 cells
were suspended by trypsinization, counted, and replated into 24-well
plates at a density of 30,000 cells/well in media containing 10% fetal
bovine serum. The cells were starved in 0.5% fetal bovine serum for
48 h to decrease the cell proliferation and to induce quiescence.
Then the media were changed to DMEM plus 10% fetal bovine serum with
or without thapsigargin. The cells were incubated with thymidine (1 µCi/well) during the last 4 h. At the end of the incubation, the
medium was aspirated, and the cells were washed three times with cold
DMEM and then solubilized with Protosol (NEN Life Science Products).
Radioactivity was measured with a Beckman scintillation counter.
Measurement of Intracellular Calcium Levels--
For
Ca2+ measurement, Fura-2 loading and fluorescence analyses
were carried out as described previously (20). The cells were maintained in DMEM containing 10% fetal bovine serum and plated on a
sterile 22 × 22-mm coverslip at a density of 2.5 × 105 cells/cm2. At the confluence of 70-80%,
the cells were washed twice with a HEPES-buffered solution (solution A)
and loaded with Fura-2 by a 30-min incubation at room temperature in
solution A containing 5 µM Fura-2/AM (Molecular Probes,
Eugene, OR). Solution A contained 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM glucose, and 10 mM HEPES, pH 7.4. After the loading, the coverslips were washed once with solution A and assembled to form the bottom of perfusion chamber. The chamber was continuously perfused with either a
Ca2+-containing solution (solution A) or a
Ca2+-free solution (solution B). Ca2+-free
solution B was prepared by replacing CaCl2 with 3 mM EGTA. The osmolarity of all solutions was adjusted to
310 mOsm with the major salts before use. Fluorescence was measured and
calibrated using a PTI system (PTI Delta Ram, New Brunswick, NJ) as
described previously (20). Fura-2 fluorescence was excited at 355 and 380 nm and calibrated by exposing the cells to solutions containing high and low concentrations of Ca2+ and 10 µM ionomycin.
DNA Transfection and CAT Assay--
Transient transfection was
performed by using LipofectAMINE Plus reagents (Life Technologies) as
described by the manufacturer's protocol. Plasmid pCMV-GAL, which
contains the Escherichia coli
-galactosidase gene driven
by the cytomegalovirus, promoter was used as an internal control to
determine transfection efficiency. The CAT assay was done with an
enzyme-linked immunosorbent assay CAT assay kit (5 Prime
3 Prime,
Inc., Boulder, CO).
RNA Preparations and Northern Blot Analysis--
Total cellular
RNAs from H19-7 cells were isolated by the single-step extraction
procedure using guanidium isothiocyanate as described elsewhere (21).
Northern blot analysis to measure pip92 mRNA levels was
done as described previously (12).
Src Kinase Assay--
Src-tyrosine kinase activity was measured
by phosphorylation of rabbit muscle enolase as described previously
(15). The samples were resolved on a 12.5% SDS-polyacrylamide gel, and
the phosphorylation level of enolase was measured by scintillation counting of excised protein bands.
Immunoprecipitation and Assay of Hemagglutinin (HA)-tagged
ERK2--
ERK2 kinase activity was measured by immunoprecipitation of
the epitope-tagged Erk2, followed by an in vitro
phosphorylation assay as described previously (12). Transfected cells
were stimulated and lysed with solution C, consisting of 20 mM Tris, pH 7.9, 137 mM NaCl, 5 mM
Na2EDTA, 10% glycerol, 1% Triton X-100, 0.2 mM p-methylsulfonylfluoride, 1 µg/ml
aprotinin, 20 µM leupeptin, 1 mM sodium
o-vanadate, pH 10.0, 1 mM EGTA, 10 mM NaF, 1 mM tetrasodium pyrophosphate, 1 mM
-glycerophosphate, pH 7.4, and 0.1 g/ml
p-nitrophenylphosphate. The cell lysates were then incubated
with protein A-Sepharose coupled with the anti-HA antibody (Santa Cruz
Biotechnologies) for 24 h at 4 °C. The immune complexes were
washed with lysis buffer and with kinase reaction buffer containing 20 mM HEPES, pH 7.4, 10 mM MgCl2, 1 mM dithiothreitol, 200 mM
o-vanadate, and 10 mM
p-nitrophenylphosphate. The assay of
immunoprecipitated HA-tagged ERK2 was done using myelin basic protein
as a substrate as described previously (12).
Electrophoretic Mobility Shift Analysis--
Nuclear extracts
were prepared from H19-7 cells as described elsewhere (22). An
electrophoretic mobility shift analysis assay was performed as
described previously (23).
In Vitro Assay of Elk1 Phosphorylation--
In vitro
Elk1 phosphorylation by using gluthathione-Sepharose 4B beads (Amersham
Pharmacia Biotech) was examined as described previously (23).
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RESULTS |
Thapsigargin Stimulates DNA Synthesis in Rat Hippocampal Neuronal
Cells--
Immortalized H19-7 cells were generated from rat
hippocampal neurons. Initially, we investigated how the stimulation of
the H19-7 cells with thapsigargin affects cell growth. Thapsigargin in
the range of 0.1-10 µg/ml induced a
concentration-dependent increase in DNA synthesis measured
by [3H]thymidine incorporation (Fig.
1). Stimulation of the cells with 10 µg/ml thapsigargin for 24 h increased the DNA incorporation ~1.6-fold compared with that of control cells in the absence of thapsigargin. These data indicated that thapsigargin stimulates the
proliferation of neuronal H19-7 cells.

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Fig. 1.
Thapsigargin stimulates DNA synthesis in
H19-7 cells. Cells were incubated in DMEM including 10% fetal
bovine serum with the indicated concentration of thapsigargin for
24 h. Cell cultures were labeled with [3H]thymidine
during the last 4 h of incubation and analyzed for DNA synthesis,
as described under "Experimental Procedures." Data are shown as
percentage of control (vehicle in media), and all histograms
represent the mean of three replicates.
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Thapsigargin and Ionomycin Cause Mobilization of Intracellular
Calcium Levels in H19-7 Cells--
To examine whether thapsigargin
causes a change in intracellular calcium levels, cells were preloaded
with Fura-2 and then monitored for changes in intracellular calcium
after exposure to thapsigargin in medium containing 1 mM
calcium. The time courses of calcium mobilization by thapsigargin are
shown in Fig. 2. By ~5 min after
addition of 10 µg/ml thapsigargin, cytosolic free calcium levels had
increased from ~70 to 250 nM, followed by a long plateau.
Although there was some variation in the shape of the calcium profile,
depending on the particular cell being monitored, in general
thapsigargin caused a gradual increase followed by a long plateau
phase. Ionomycin also increases the calcium levels by channeling
through membranes from a nonspecific source (24). In the same way,
addition of 1 µM ionomycin increased the intracellular calcium levels up to ~1500 nM, reaching a peak by 1-2
min. Addition of EGTA to the cells during the plateau phase dropped the
calcium down to basal levels.

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Fig. 2.
Thapsigargin and ionomycin triggered calcium
mobilization in H19-7 cells. The cells were preloaded with Fura-2
and perfused with calcium-containing HEPES-buffered solution A to
establish basal Ca2+ levels at 33 °C. Subsequently, the
cells were treated with 10 µg/ml thapsigargin (Tg;
A) or 1 µM ionomycin (Iono;
B), followed by the addition of Ca2+-free EGTA,
as indicated. Each trace is from an individual cell in a
field of cells and represents a typical response of three independent
experiments.
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Induction of pip92 by Intracellular Calcium Mobilization in H19-7
Cells--
Next we examined whether intracellular calcium mobilization
induced by thapsigargin causes the stimulation of pip92
expression in H19-7 cells. We first measured the time course of
pip92 mRNA expression by thapsigargin. Northern blot
analysis showed that pip92 is expressed rapidly and
transiently within 30-60 min after 10 µg/ml thapsigargin stimulation
(Fig. 3A) but not with
vehicle. Transcriptional activation of the pip92 gene was
examined by using a CAT reporter plasmid linked to a
1281-base pair
pip92 promoter fragment (
1281pip92/CAT)
transiently expressed in H19-7 cells. The
1281-base pair
pip92 promoter fragment was shown to mediate serum induction
in a manner that closely mimics the endogenous gene (25). Treatment of
the cells with 10 µg/ml thapsigargin caused rapid stimulation of
pip92 transcriptional activity, which reached a plateau by
1 h as monitored by CAT assay (Fig. 3B). Mock-transfected control cells did not activate the pip92
promoter (data not shown). A similar level of pip92 mRNA
and its transcription were induced with 1 µM calcium
ionophore ionomycin, and an additive increase of pip92
transcription was not observed by the cotreatment with thapsigargin and
ionomycin (data not shown).

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Fig. 3.
Induction of immediate early gene
pip92 by thapsigargin in neuronal H19-7
cells. A, H19-7 cells were treated with 10 µg/ml
thapsigargin (Thap) or vehicle (Veh) at 33 °C
for the indicated times. A 10-µg aliquot of total RNA was extracted
from cells, applied to each lane, and hybridized to a 125-base pair
32P-labeled pip92 cDNA fragment. As a
control for RNA loading, total RNA was hybridized to
32P-labeled glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) cDNA probe. B, 2 µg of CAT reporter
plasmid linked to the 1,281-base pair pip92 promoter
fragment ( 1281pip92/CAT) was transiently transfected into
H19-7 cells. After treatment of the cells with 10 µg/ml thapsigargin,
the activity of the expressed CAT enzyme in 40-60 µg of cell lysates
was measured at the indicated times. Data are plotted as the mean plus
the range of samples from three independent experiments performed in
triplicate.
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Kinase-inactive Src or MEK Inhibitor Blocks Thapsigargin-induced
Activation of pip92 Induction--
Many studies demonstrated a role
for pp60SRC-tyrosine kinase (Src) in Ca2+-induced
cell cycle progression and mitogenesis (26-28). To test that Src
kinase functions as a mediator for thapsigargin and calcium signaling
in H19-7 cells, we assayed the Src kinase activity in response to
thapsigargin. Fig. 4A shows
that thapsigargin activates Src-tyrosine kinase 2.5-fold. Transfection
of kinase-deficient Src mutant resulted in the inhibition of
thapsigargin-induced Src kinase activity to the basal level in H19-7
cells, suggesting that thapsigargin may use Src kinase for downstream
signaling events. To test whether MAP kinase is also a downstream
target of thapsigargin-induced calcium mobilization, the cells were
transiently transfected with a kinase-inactive MEK mutant (MEK-2A),
which encodes a protein with 2 alanine residues substituted at the
sites of the activating serine residues, together with an HA
epitope-tagged ERK2. The addition of thapsigargin resulted in the
significant increase of ERK activity, measured by using an
epitope-tagged ERK2-immune complex kinase assay (Fig. 4B),
and kinase-deficient MEK-2A completely blocked the thapsigargin-induced
HA-ERK2 activity. To further verify the role of MEK-ERK activation by
thapsigargin, we measured the ERK2 activity by thapsigargin after
pretreating the cells with 30 µM MEK inhibitor PD98059
for 30 min. Previously we have shown that activation of ERK by
fibroblast growth factor (FGF) is completely blocked by 30 µM PD98059 in H19-7 cells (29). Addition of MEK inhibitor
also significantly blocked the thapsigargin-induced HA-ERK2 activity.
In all samples, ERK2 enzymes were present at the same levels. These
results suggest that calcium mobilization by thapsigargin activates
both ERK and Src kinase in H19-7 cells.

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Fig. 4.
Dominant-negative Src kinase mutant,
kinase-inactive MEK, or chemical MEK inhibitor blocks
thapsigargin-induced pip92
expression. A, where specified, H19-7
cells were transfected with 10 µg of kinase-deficient Src mutant
plasmid (mSrc) or parental vector (control). The cells were
stimulated with 10 µg/ml thapsigargin (Thap) or vehicle
for 1 h, and then Src kinase activity was measured as the
phosphorylation of rabbit muscle enolase. B, the cells were
transfected with 2.5 µg of an HA-tagged ERK2 plasmid
(HA-ERK) or together with 10 µg of a kinase-inactive MEK
mutant (MEK-2A), as indicated. After transfection, the cells
were stimulated with 10 µg/ml thapsigargin (Thap) for
1 h. Where indicated, cells were pretreated with 30 µM MEK inhibitor (MI) PD98059 for 30 min
before cell harvest. The HA-tagged ERK2 was assayed for kinase activity
by using myelin basic protein (MBP) as a substrate. As a
control for equal protein loading, HA-tagged ERK2 levels were measured
by Western analysis using anti-HA antibodies. C, where
indicated, 2 µg of 1281pip92/CAT plasmid DNA was
transiently cotransfected into H19-7 cells with 10 µg of a
kinase-inactive Src (mSrc) or MEK mutant
(MEK-2A). After H19-7 cells were either untreated or
pretreated with 30 µM MEK inhibitor (MI)
PD98059 for 30 min, the cells were stimulated with vehicle
(Control) or 10 µg/ml thapsigargin (T) for
1 h. The transcriptional activity of pip92 was measured
by CAT assay. Data are plotted as the mean plus the range of samples
from two independent experiments performed in duplicate. D,
where indicated, the cells were transiently transfected with 10 µg of
a kinase-inactive Src (mS) or MEK-2A mutant (mM).
Where specified, H19-7 cells were pretreated with 30 µM
MEK inhibitor (MI) PD98059 for 30 min. The cells were then
stimulated with 10 µg/ml thapsigargin (Thaps) or vehicle
(Con) for 1 h, and Northern blot analysis of
pip92 mRNA was done. GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
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To test whether pip92 is activated via the activation of Src
kinase and ERK in response to thapsigargin, the H19-7 cells were transiently transfected with the combination of
1281pip92/CAT and a kinase-inactive Src or MEK-2A, and
transcriptional activity of pip92 by thapsigargin was
measured. As shown in Fig. 4C, thapsigargin-induced activation of the pip92 promoter was significantly blocked
by the Src kinase mutant. In the presence of kinase-inactive MEK-2A, transcriptional activation of pip92 by thapsigargin was also
significantly decreased (Fig. 4C). In an effect similar to
that of MEK-2A, pretreatment of the cells with the MEK inhibitor
remarkably suppressed pip92 expression by thapsigargin. The
induction of pip92 mRNA levels by thapsigargin was also
greatly reduced by mutant Src, MEK-2A, or PD98059, measured by Northern
blot analysis (Fig. 4D), suggesting that the regulation of
the episomally expressed pip92 reporter does reflect
mRNA expression from the endogenous gene. Overall these results
indicated that the activation of Src kinase and ERK is necessary for
thapsigargin-induced activation of pip92.
Constitutively Active Src Kinase and MEK Increase Basal pip92Expression in Neuronal H19-7 Cells--
v-Src is a constitutively active
tyrosine kinase. To test whether Src kinase is sufficient for mediating
thapsigargin-induced activation of MAP kinase and pip92
expression, v-Src was transfected into H19-7 cells together with
1281pip92/CAT or HA-ERK2 plasmid. Expression of
constitutive v-Src kinase in H19-7 cells resulted in a significant
increase in pip92 transcriptional activity (Fig. 5) and activation of ERK in a
constitutive manner, which is comparable with that by thapsigargin
(Fig. 6B). In a similar way,
transient transfection of the constitutively active MEK mutant
(MEK-2E), which encodes a protein with 2 glutamic acid residues
substituted at the sites of the activating serine residues, markedly
induced pip92 transcription without thapsigargin treatment
(Fig. 5). It was previously shown that expression of MEK-2E is
effective at generating constitutively active MAPK, and the activity is
comparable with that induced by growth factor stimulation of the cells
(30). Pretreatment of the cells with 30 µM MEK inhibitor
also blocks the pip92 expression induced by v-Src or MEK-2E
treatment (Fig. 5). Previously, it was also shown that dominant
negative Src blocks thapsigargin-induced activation of ERK (15). Taken
together, these results confirmed that ERK activation is necessary for
the Src kinase-induced pip92 activation.

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Fig. 5.
Effect of constitutively active MEK and Src
kinase on pip92 expression in H19-7
cells. Where indicated, 2 µg of 1281pip92/CAT
plasmid DNA was transiently transfected into H19-7 cells with 5 µg of
a parental vector (Control), constitutively active Src
(v-Src) or MEK mutant (MEK-2E). When specified,
cells were treated with 30 µM PD98059 for 30 min
(MI), and the transcriptional activity of pip92
was measured by CAT assay. Data are plotted as the mean plus the range
of samples from two independent experiments performed in
duplicate.
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Fig. 6.
Transcriptional activation of
pip92 by thapsigargin and v-Src is
inhibited by dominant-inhibitory Raf-1. A, two
micrograms of 1281pip92/CAT reporter plasmids or with 10 µg of a kinase-inactive Raf mutant (T+Raf-KR) were
transiently transfected into H19-7 cells, as indicated, and the cells
were stimulated with vehicle (Control) or 10 µg/ml
thapsigargin (Thap) for 1 h. Where specified, 2 µg of
1281pip92/CAT plasmid was transiently transfected into the
cells with 10 µg of oncogenic v-Src or a combination with 5 µg of
Raf-KR (S+Raf-KR). The transcriptional activity of
pip92 was measured at 33 °C as described under
"Experimental Procedures." Data are plotted as the percentage of
pip92 transcriptional activity by thapsigargin and represent
the mean plus the range of samples from two independent experiments.
B, the cells were transiently transfected with 1 µg of
HA-ERK2 plasmid with or without 5 µg of kinase-inactive Raf
(Raf-KR) and/or constitutively active src (v-Src)
plasmid. After transfection, the cells were stimulated with 10 µg/ml
thapsigargin (Thap) for 1 h, and the kinase activity of
HA-ERK2 was measured as described in Fig. 3B. As a control
for equal protein loading, HA-tagged ERK2 levels were measured by
Western analysis using anti-HA antibodies. MBP, myelin basic
protein.
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Dominant Inhibitory Raf-1 Mutant Blocks Thapsigargin-induced pip92
Expression--
To further characterize the effect of Raf-1 activity
on the thapsigargin-induced signaling pathway leading to the induction of pip92, we transiently transfected the cells with the
kinase-inactive Raf-1 mutant (Raf-KR) and measured the induction of
pip92 transcriptional activity by thapsigargin. As shown in
Fig. 6A, the dominant negative Raf-1 mutant suppressed
pip92 induction by thapsigargin significantly. In a similar
way, when the cells were cotransfected with constitutively active v-Src
and kinase-deficient Raf-KR, the pip92 activation was
blocked remarkably. To further determine the requirement of Raf-1 for
thapsigargin-induced MAP kinase signaling, we transiently transfected
the H19-7 cells with Raf-KR with an HA-tagged ERK2 plasmid. As analyzed
by an epitope-tagged ERK2 immune complex assay (Fig. 6B),
the dominant negative Raf-KR mutant blocks the epitope-tagged ERK2
activity induced by thapsigargin and v-Src kinase. The data indicate
that Raf-1 kinase is required for thapsigargin-induced MAP kinase
activation and pip92 expression.
Activation of Transcription Factor Elk1 during Thapsigargin-induced
pip92 Expression--
Previous studies have shown that serum response
element (SRE) present in the pip92 promoter can interact with
recombinant serum response factor (SRF) and Elk1 proteins
forming a tertiary complex (16, 25). Elk1 can be phosphorylated by ERK,
and it is critical for transcriptional activation of pip92
and c-fos. Next we examined whether Elk1 can be
phosphorylated by thapsigargin. After cells were stimulated with 10 µg/ml thapsigargin, nuclear extracts and cell lysates were prepared.
Electrophoretic mobility shift analysis using pip92 SRE
oligonucleotide and anti-phospho-Elk1 antibodies showed that endogenous
Elk1 does not bind to the pip92 SRE element without stimulation and is
activated by thapsigargin (Fig.
7A). The cell lysates were
incubated in the presence of radiolabeled ATP with the wild-type
GST-Elk1 C terminus (GST-ElkC) prebound to Sepharose 4B beads. As shown
in Fig. 7B, wild-type GST-ElkC was phosphorylated by
extracts from the cells stimulated with thapsigargin. To demonstrate
that the phosphorylation of GST-ElkC by nuclear extracts from
thapsigargin-treated cells is specifically mediated by ERK and
Src kinase, the effect of the inhibition of ERK or Src kinase activity
was examined. Pretreatment of the cells with PD98059 before stimulation
or removal of ERK by the incubation of cell lysates with
agarose-ERK-immunoglobulin after stimulation resulted in a complete
block of GST-ElkC phosphorylation by cell lysates. Similarly,
transfection of kinase-inactive MEK and the Src mutant remarkably
inhibited GST-ElkC phosphorylation by thapsigargin-induced cell
lysates. These results implied that Src kinase and ERK mediate
thapsigargin-induced activation of pip92 via the phosphorylation of
Elk1.

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Fig. 7.
Activation of endogenous Elk1 by thapsigargin
and inhibition of in vitro Elk1
phosphorylation by kinase-inactive Src kinase and ERK.
A, nuclear extracts from control H19-7 cells or cells
stimulated with 10 µg/ml thapsigargin (Thaps) were
incubated with end-labeled SRE oligonucleotide, and the resulting
protein-DNA complexes were resolved by nondenaturing polyacrylamide gel
electrophoresis. For Elk1 supershift analysis, after binding, 0.5 µg
of phospho-Elk1 antibodies were added, and the reaction mixture was
incubated at 4 °C for 2 h before gel electrophoresis. In
lanes 2-4, 100-fold excesses of various oligonucleotides
(SRE, B, and AP-1) were included as
competitors, as indicated above each lane. In lane
5, antibodies against phospho-Elk1 (pElk1) were
included. One SRE-protein complex and a supershifted SRE-phospho-Elk1
binding complex are denoted are denoted by open and
filled arrows, respectively. Lane 6, control
cells without stimulation. Nonspecific complex (NS) and free
probe (probe) are indicated on the left.
B and C, where specified, 5 µg DNA of a
kinase-inactive Src mutant (mS) or MEK-2A (M2A)
was transiently transfected into H19-7 cells. Where indicated, the
cells were pretreated with 30 µM PD98059 for 30 min
(PD), followed by the stimulation with 10 µg/ml
thapsigargin (Thaps). With the cell extracts, prepared from
vehicle-treated (Veh), thapsigargin-stimulated cells or
incubated with agarose-ERK-antibody (A-ERK) after
stimulation, containing 50-60 µg of proteins and 75 µl of
Sepharose 4B beads, in vitro Elk1 phosphorylation was
performed as described under "Experimental Procedures."
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DISCUSSION |
Thapsigargin, a non-phorbol ester-type tumor promoter, discharges
intracellular Ca2+ stores by specific inhibition of the
endoplasmic reticulum Ca2+-ATPase. Although the mechanism
of tumor promotion by thapsigargin is not well understood, treatment of
mouse NIH 3T3 fibroblasts with thapsigargin induced rapid expression of
the c-fos and c-jun protooncogenes (31).
Furthermore, thapsigargin could synergize with another tumor promoter,
phorbol 12-myristate 13-acetate, to induce c-fos but not
c-jun. The stress-inducible glucose-regulated proteins, a
class of calcium binding molecular chaperones localized in the
endoplasmic reticulum, have been implicated in the development of
thapsigargin-induced tumorigenicity (32). In addition, the present
study suggests that Elk1-dependent activation of pip92 might contribute the tumorigenesis of thapsigargin in neuronal cells.
It has been well documented that thapsigargin and ionomycin mobilize
Ca2+ through distinct mechanisms, thapsigargin by
inhibiting microsomal Ca2+-ATPases and ionomycin by
channeling calcium through the membrane from nonspecific sources (33).
On the basis of the finding that both thapsigargin and ionomycin are
able to induce pip92 expression, and there was no
synergistic effect by the coaddition of the two agents (Fig. 2),
Ca2+ appears to be a common mediator for the
pip92 induction. Although the two drugs produced a 6-7-fold
differential increase of intracellular calcium levels, similar levels
of pip92 induction were observed, suggesting that a slight
increase of intracellular calcium mobilization by thapsigargin is
likely to be sufficient to induce pip92.
Previously we demonstrated that pip92 is expressed rapidly and
transiently during NMDA-induced cell death in H19-7 cells (16). In the
present study we have shown that pip92 is also expressed during
thapsigargin-induced cell proliferation. Both processes are commonly
mediated by the increase of intracellular Ca2+ influx. Many
of the same immediate early genes such as c-fos and
pip92 are induced in a variety of biological contexts as
diverse as mitogenic responses and the cellular response to cytotoxic stimuli. It will be interesting to investigate the molecular mechanism of variable biological responses resulting from the use of genes of
which products are used in many contexts. The mechanism may be
triggered as a consequence of differential quantitation of different
ligands or different kinetics of the induction of immediate early genes
and their products. Because Ca2+ signals are short-lived
when compared with alterations in differentiated gene expression, it is
generally considered that genes coding for short-lived transcription
factors (e.g. c-fos and c-jun) are the
immediate targets of Ca2+ signaling (34, 35). On the basis
of the previous finding that Pip92 is selectively expressed in the
nucleus as a fusion with green fluorescent protein (23), Pip92 seems to
be a second messenger, for example, as a transcription factor, rather
than a direct effector of a variety of cell responses, such as cell growth, differentiation, and transformation. We are currently investigating the functional roles of the Pip92 protein in the nucleus.
Previously we have shown that both FGF and Raf activate
MAPK-independent kinases that can stimulate Elk1 phosphorylation and pip92 transcription (12). An increase of intracellular
calcium levels by thapsigargin or ionomycin caused a rapid and
transient increase of pip92 gene expression, and the signals
for intracellular calcium mobilization are transmitted through the
activation of Src kinase- and Raf-MEK-ERK-dependent
signaling pathways in H19-7 cells. Elk1 becomes a transcriptional
activator to induce pip92 in response to thapsigargin. However, because
of a previous finding that kinase-inactive Src kinase did not
completely block the activation of ERK by FGF and neuronal
differentiation by Raf-1 activation (29), Src-tyrosine kinases appear
to be differentially activated via distinct signaling pathways by FGF
and thapsigargin in H19-7 cells.
Many studies demonstrated a role for Src kinase in intracellular
calcium signaling. For example, functional calcium-sensing receptors in
rat fibroblasts are required for activation of Src kinase and
mitogen-activated protein kinase in response to extracellular calcium
(36). The Src kinase pathway is involved in
Ca2+-dependent pancreatic exocytosis (37), and
angiotension II-induced phosphorylation of p130Cas by Src family
tyrosine kinases is dependent on intracellular calcium and protein
kinase C (38). Considerable evidence also supports the role of Src
family kinases in the stimulation of ERK. Many G protein-coupled
receptors initiate Ras-dependent activation of the ERK
cascade by inducing the tyrosine phosphorylation of proteins that serve
as scaffolds for the plasma membrane recruitment of Ras guanine
exchange factors. Activation of Src kinase by the
-thrombine (39),
lysophosphatidic acid (40), angiotension II (41), N-formyl
peptide chemoattractant (42),
2A-adrenergic (39, 40), and M1
muscarinic receptors (41) has been reported.
A number of studies have suggested that Src cooperates with Ras to
activate MAP kinase in some signaling events. For example, activation
of Src family kinases and Ras is required for activation of ERKs by
angiotension II in smooth muscle cells (43). Oxidative stress, such as
H2O2, activates ERKs through Src kinase in
cultured cardiac myocytes of neonatal rats (44). However, conflicting results also have been reported to indicate that both can activate independent pathways in certain instances. In fibroblast NIH3T3 cells,
Myc, but not Fos or Jun, was able to rescue the inhibition of
platelet-derived growth factor-stimulated DNA synthesis by dominant
negative Src, whereas Fos and Jun, but not Myc, rescued the cell growth
block given by dominant negative Ras (45). Src appears to activate a
Ras- and Raf-independent pathway during the differentiation of H19-7
cells by FGF (29). Studies using dominant inhibitory Raf-1 mutants also
indicate that Src can be an upstream activator for Raf-1, and Raf-1 may
be required for the Src-dependent activation of MAP kinase
(15).