Proteasome- and p38-dependent Regulation of ERK3
Expression*
Johann
Zimmermann,
Nathalie
Lamerant,
Rita
Grossenbacher, and
Peter
Fürst
From Novartis Pharma AG, Oncology Research, WKL-125.13.14,
CH-4002 Basel, Switzerland
Received for publication, September 19, 2000, and in revised form, December 28, 2000
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ABSTRACT |
Proteasome inhibition leads to accumulation of
transcription factors, heat shock proteins, cyclins, and other
proteasome substrate proteins by blocking their proteolytic
degradation. An increase in gene transcription upon proteasome
inhibition was found for a group of proteins, including
p21WAF1/CIP1, ubiquitin, and transcription factors.
In this study, we have demonstrated selective up-regulation of
extracellular signal-regulated kinase 3 (ERK3) mRNA and
protein expression upon treatment with peptide-based proteasome
inhibitors or lactacystin. ERK3 is a family member of the
mitogen-activated protein kinases (also called ERK) that are key
mediators of signal transduction from the cell surface to the nucleus.
ERK3 up-regulation is independent of the p53, Bcl2, and caspase 3 status of cells. p38 pathway kinase inhibitors prevent
proteasome-dependent ERK3 induction and enhance
the antiproliferative effect of proteasome inhibitors. MCF-7 cells
expressing ERK3 ectopically show increased resistance toward proteasome
inhibition. The results indicate that ERK3 expression is a consequence
of p38 pathway activation and most probably represents an intracellular
defense or rescue mechanism against cell stress and damage induced by proteasome inhibition.
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INTRODUCTION |
Proteasome inhibition leads to an increase in the expression of a
large number of proteins by both posttranslational and transcriptional mechanisms. Various proteins crucial for cell cycle regulation and
stress response, such as cyclins and transcription factors, are
substrates of the ubiquitin-proteasome proteolytic pathway and directly
accumulate in a polyubiquitinylated state after proteasome inhibitor
treatment. On the other hand, proteasome inhibition has been reported
to increase transcription and translation of a group of genes,
including p21WAF1/CIP1 (1), and a number of proteins
involved in heat shock response (2) and transcriptional regulation,
such as Gadd153, ATF3, and MAD1 (3).
As reported previously, we have used a quantitative fluorescent
cDNA hybridization approach to identify genes regulated
transcriptionally in response to proteasome inhibitor treatment.
Changes in mRNA expression of 7900 genes from the UniGene
collection were compared upon exposure of cells to the proteasome
inhibitors lactacystin, lactacystin-
-lactone, or MG132 by
means of microarray-based cDNA hybridization (3). All these
inhibitors block the proteolytic activity of the 26 S proteasome
without influencing its ATPase or isopeptidase activities. The three
gene expression profiles were very similar, revealing 52 genes that are
induced more than 5-fold and 34 genes that are repressed more than
5-fold by at least one of the inhibitors.
A gene raising interest and found to be up-regulated on all three
cDNA array chips was ERK3, a
MAP1 kinase of the
extracellular signal-regulated kinase (ERK) subfamily. MAP kinases are
serine/threonine kinases that are phosphorylated and thereby activated
in response to a wide array of stimuli and are part of sequentially
acting complex protein kinase cascades. They modulate many cellular
events including mitogen-induced cell cycle progression through the
G1 phase, regulation of embryonic development and cell and
neuronal differentiation (4, 5). A controlled regulation of the MAP
kinase cascade is involved in cell proliferation and differentiation,
whereas an unregulated activation can result in oncogenesis. In
vertebrate cells, two additional, distinct kinase cascades have been
discovered to date targeting Jun N-terminal kinases/stress-activated
protein kinases and p38 (6). The Jun N-terminal kinases and p38 kinases
are preferentially transferred to the nucleus in their active
phosphorylated form by cellular stresses including proteasome
inhibition (7).
Based on characteristic features of its sequence, ERK3 is
categorized as one of six members of the ERK subfamily (8). It retains
a much greater similarity to ERK1 and ERK2, particularly in the
phosphorylation lip, than to the other known family members. ERK3 is ubiquitously expressed in a variety of tissues and
mammalian cell lines. ERK3 mRNA, like ERK1
and ERK2 mRNA, is detectable in numerous tissues,
including heart, brain, placenta, lung, liver, skeletal muscle, kidney,
and pancreas tissues and human skin fibroblasts, with its highest level
in skeletal muscle (8). Several properties of ERK3, however,
distinguish it from all other known ERK homologs, indicating that ERK3
appears to represent a distinct evolutionary branch of ERK/MAP kinases
and that it could be controlled by other cell signaling pathways. ERK3
is constitutively nuclear (9), is not translocated in response to a
stimulus, and does not retain the activating tyrosine phosphorylation
site, a hallmark of the ERK family. Upstream ERK3-activating mechanisms
are not yet known, and concerning ERK3 substrates, studies are
contradictory (10, 11).
In this study, we have characterized time- and
dose-dependent ERK3 mRNA and protein overexpression
upon treatment with different proteasome inhibitors in a variety of
cell lines. ERK3 overexpression was independent of the intracellular
p53, caspase 3, and Bcl-2 statuses but was accompanied by activation of
the p38 and MEK3/6 kinases. Moreover, ERK3 induction was completely
blocked upon preincubation with selective inhibitors of the p38
pathway. We demonstrate that cells ectopically expressing ERK3 are more
resistant toward proteasome inhibitors. We further demonstrate that
cells treated with a specific inhibitor of the p38 pathway are more sensitive toward proteasome inhibitor treatment, suggesting that ERK3
overexpression protects cells from the anti-proliferative effect
induced by proteasome inhibition.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
The human adenocarcinoma cell lines MCF-7, MDA
MB231, MDA MB468, and the human prostate cell line DU145 (all from the
American Tissue Culture Collection) were cultured at 37 °C in a
5.5% CO2 atmosphere in minimal essential medium
with
ribonucleosides and deoxyribonucleosides, supplemented with 10% fetal
calf serum, 2 mM L-glutamine, 1 mM
sodium pyruvate, 0.1 mM nonessential amino acids, and 0.01 mg/ml gentamycin (Life Technologies, Inc.). The human leukemic
cell line HL-60 and the human non-small cell lung cancer cell line
NCI-H1299 (obtained from American Tissue Culture Collection) were
cultured in RPMI 1640 medium containing 10% fetal calf serum in a
5.5% CO2 atmosphere at 37 °C. Stable Bcl-2-transfected MDA-MB-453 cells were generated and supplied by Markus Wartmann (Novartis Pharma AG, Basel, Switzerland). Subconfluent cells were treated with proteasome or kinase inhibitors, staurosporine, rotenone, or doxorubicin, respectively, for indicated times and doses.
Z-Ile-Glu(O-t-butyl)-Ala-Leu-H (PSI), Z-Leu-Leu-Nva-H
(MG115), Z-Leu-Leu-Leu-H (MG132), lactacystin, and
lactacystin-
-lactone were purchased from AFFINITI
Research Products Ltd. (Mamhead Castle, UK). Staurosporine was obtained from Kamiya (Seattle, WA). PD169316, SB202190, and SB203580 were purchased from Alexis (Lausen, Switzerland). Doxorubicin was
obtained from Sigma. PS341 was a gift of Carlos Garcia-Echeverria
(Novartis Pharma AG, Basel, Switzerland). Cell proliferation was
measured using the MTS assay kit from Promega (Wallisellen,
Switzerland) according to the recommendations of the manufacturer. Fig.
1 shows the chemical structures of the
compounds utilized in this study.

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Fig. 1.
Structures of utilized compounds. The
structures of PS341, PSI, MG132, lactacystin, lactacystin- -lactone
(proteasome inhibitors), trapoxin A (histone deacetylase inhibitor),
and SB203580 and SB202190 (p38 inhibitors) are represented.
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Stable Transfection--
MCF-7 cells were stably transfected
with expression vector pcDNA3.1/GS-ERK3(His)6 (clone H-L77964M)
carrying a zeocin resistance gene (Invitrogen, Groningen, Netherlands)
using the Effectene transfection kit (Qiagen AG, Basel, Switzerland).
pGreenLantern (Life Technologies, Inc.) was cotransfected for
green fluorescent protein expression to control for transfection
efficiency by means of fluorescence microscopy. Stable transfected
clones (100 µg/ml zeocin resistance) were picked and cultured, and
histidine-tagged ERK3 expression was confirmed by Western blot.
Real Time Reverse Transcription-Polymerase Chain
Reaction--
The PCR primers and TaqMan probes to amplify and detect
ERK3 mRNA were designed using Primer Express software
version 1.0 (PerkinElmer Life Sciences) based on the sequence
reported in GenBankTM as follows: ERK3
forward primer, 5'-GAATGGCAAATCTGGCTCAATT-3'; ERK3 reverse
primer, 5'-ACAGTCCTCCCCACCACTCA-3'; ERK3 probe,
6- carboxyfluorescein-5'-CCTTGTACCAGTCTTCTTGGGACAGCCA-3'6-carboxytetramethylrhodamine. The PCR primers were purchased from BIG Biotech (Freiburg, Germany). The TaqMan probes were labeled with 6-carboxyfluorescein as the reporter dye and 6-carboxytetramethylrhodamine as the quencher fluorescent (PerkinElmer Life Sciences). A TaqMan-GAPDH control was
used to amplify and detect GAPDH as control as recommended by
the manufacturer (PerkinElmer Life Sciences). The following primers
were used: GAPDH forward primer, 5'-GCACCGTCAAGGCTGAGAAC; GAPDH reverse
primer, 5'-GAGGGATCTCGCTCCTGGA; GAPDH probe,
6-carboxyfluorescein-5'-CTTGTCATCAATGGAAATCCCATCACCATC-3'-6-carboxytetramethylrhodamine. Total RNA was obtained from cells with the RNeasy mini kit
(Qiagen, Hilden, Germany) as described in the manufacturer's protocol. Poly(A)+ RNA was isolated from 250 µg of total RNA with
the Oligotex mRNA mini kit (Qiagen, Hilden, Germany). For real time
reverse transcription-PCR, an RNA concentration of 5 ng per reaction
was used, and a one-step, two-enzyme system was chosen according to
recommendations of the manufacturer composed of TaqMan PCR core reagent
kit, reverse transcriptase murine leukemia virus, and RNase
inhibitor (all from PerkinElmer Life Sciences). Thermal cycling
conditions included a reverse transcription step (48 °C, 30 min), followed by Ampli Taq Gold activation (95 °C, 10 min), and a PCR step of 40 cycles (95 °C, 15 s;
60 °C, 1 min). Accumulation of PCR products was detected in real
time directly by monitoring the probe cleavage-induced increase in
fluorescence of the reporter dye with the ABI PRISM 7700 Sequence
detector. Relative quantitation was performed using the comparative
CT method as described by the manufacturer (12).
Western Blot Analysis--
Cells were washed twice with
phosphate-buffered saline (Life Technologies, Inc.), lysed with M-PER
(Pierce), and boiled for 5 min. The protein concentration was
normalized by determination of the total protein amount in the
supernatant using the BCA protein assay kit (Pierce). 40 µg of total
protein per lane was separated by electrophoresis in SDS-polyacrylamide
gels (Bio-Rad) and transferred to Protran nitrocellulose membranes
(Schleicher & Schuell). All primary antibodies and secondary
horseradish peroxidase-conjugated antibodies were purchased from Santa
Cruz Biotechnology Inc. (Santa Cruz, CA), except an anti-polyhistidine
antibody, which was from Sigma. Proteins were detected by
chemoluminescence with SuperSignal substrate (Pierce).
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RESULTS |
Proteasome Inhibition Leads to ERK3 mRNA and ERK3 Protein
Overexpression--
To systematically elucidate pathways linked to
proteasome inhibition, we treated MCF-7 breast carcinoma cells for
14 h with the three chemically different proteasome inhibitors
Z-Leu-Leu-Leu-H (MG132), lactacystin, or lactacystin-
-lactone
at 10 µM compound concentration or with
Me2SO as a control (3). Less than 10% residual
proteasome activity was measurable in protein extracts derived from
MCF-7 cells treated with either one of the proteasome inhibitors under
those conditions (data not shown). In addition, to compare expression
profiles of proteasome inhibitors with those of
apoptosis-inducing compounds, not the proteasome-targeting compounds, a control experiment with trapoxin A was performed. NCI-H1299 cells were treated for 24 h with 1 µM
trapoxin A. Trapoxin A is a histone deacetylase inhibitor that has been
shown to have an impact on gene expression (13) and to have antitumor
activity (14). The transcriptional response was examined by means of microarray-based differential fluorescent cDNA hybridization. In
principle, the technology has been described (15, 16). Comparison of
the three expression profiles of proteasome inhibitors revealed a high
degree of similarity, indicating that the observed changes in gene
expression were indeed a consequence of blocked proteasome activity.
Most of the genes whose expression was influenced by proteasome
inhibitors were not influenced by trapoxin A. ERK3 was one of the genes
found to be significantly up-regulated (Fig. 2A): 2.9-fold by MG132,
3.4-fold by lactacystin, and 2.1-fold by lactacystin-
-lactone.
ERK3 mRNA levels, however, did not change after trapoxin
A treatment, suggesting that ERK3 is not generally up-regulated upon apoptosis induction. Induction of ERK3
gene expression upon MG132 treatment was further confirmed by means of
real time reverse transcription-PCR (Fig. 2B). A fast
response in MCF-7 cells was observed. ERK3 mRNA reached
maximal induction between 12 h (3.4-fold) and 24 h
(3.9-fold), peaked at 16 h (4.2-fold), and started to decrease
from 24 h onwards. Several factors, including the site of mRNA
synthesis, its stability and half-life, the translation efficiency, and
the half-life of translated ERK3, determine intracellular ERK3 protein
levels. The up-regulation of the mRNA was confirmed to be
accompanied by an overexpression at the protein level. A dose-dependent increase in the ERK3 protein level is shown
in Fig. 2C for MCF-7 cells that were incubated for 7 h
with MG132. ERK3 protein was 6.6-fold increased after treatment with 50 µM MG132 compared with cells treated with
Me2SO (Fig. 2D).

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Fig. 2.
ERK3 induction upon proteasome
inhibition. A, comparison of MG132- (10 µM), lactacystin- (10 µM),
lactacystin- -lactone- (10 µM), and trapoxin
A (1 µM)-mediated alternations in ERK3 gene
expression on high density DNA chips. The induction ratio for
ERK3 gene expression is displayed for each compound with
respect to Me2SO-treated cells. Unregulated genes (relative
to Me2SO) appeared as 1-fold induced or repressed.
B, ERK3 mRNA induced by 10 µM
MG132 in MCF-7 cells. The mRNA levels were determined using the
comparative CT method (12). Three independent
experiments were performed. DMSO, dimethylsulfoxide.
C, dose-dependent induction of ERK3 protein.
MCF-7 cells were treated for 7 h with the indicated concentrations
of MG132, and the ERK3 protein level was visualized by Western
blotting. D, quantitation of ERK3 induction. The ERK3
protein levels in Fig. 1C were quantitated by a digital
Kodak Image Station 440CF and 1D Image Analysis software (Eastman Kodak
Co.).
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Selectivity of ERK3 Induction--
To prove that ERK3
overexpression was due to proteasome inhibition and was not an
unrelated side effect of the peptide-based inhibitor MG132, MCF-7 cells
were treated with different proteasome inhibitors for 3.5 and 14 h. ERK3 protein expression was analyzed by immunoblotting (Fig.
3A). Protein expression of
ERK3 was markedly increased after exposure to proteasome inhibitors for
14 h but not for the shorter incubation time of 3.5 h,
suggesting that ERK3 transcription and translation required
more than 3.5 h. The observed ERK3 overexpression was independent
of the proteasome inhibitor type. Despite their chemical
divergence, MG132, MG115, PS341, lactacystin,
lactacystin-
-lactone, and PSI caused a similar degree of
ERK3 up-regulation after 14 h. To address specificity, other apoptosis-inducing compounds were tested for their ability to
induce ERK3 expression in MCF-7 cells. In contrast to the proteasome inhibitor MG132, trapoxin A, doxorubicin, staurosporine, and rotenone did not induce ERK3 even after 14 h (Fig. 3B).

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Fig. 3.
Selectivity of ERK3 protein induction.
A, ERK3 protein expression was analyzed by Western blotting
after treatment of MCF-7 cells for 3.5 h (upper panel)
or 14 h (lower panel) with Me2SO, MG132 (10 µM), MG115 (10 µM), PS341 (2 µM), PSI (10 µM), lactacystin (10 µM), or lactacystin- -lactone (10 µM). B, ERK3 protein expression was analyzed
by Western blotting after treatment of MCF-7 cells for 14 h with
Me2SO, MG132 (10 µM), doxorubicin (1 or 10 µM), or rotenone (0.1 or 0.5 µM), or with
Me2SO, MG132 (10 µM), trapoxin A (1 or 10 µM), or staurosporine (0.1 or 0.5 µM).
C, ERK1/2 protein expression was analyzed by Western
blotting after treatment of MCF-7 cells for 3.5 h (upper
panel), 7 h (middle panel), or 14 h
(lower panel) with Me2SO, MG132 (10 µM), MG115 (10 µM), PS341 (2 µM), PSI (10 µM), lactacystin (10 µM), or lactacystin- -lactone (10 µM). DMSO, dimethylsulfoxide.
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Selectivity of ERK3 Induction within the ERK Family--
Based on
the homology of its protein sequence, ERK3 belongs to the ERK family, a
group of proteins involved in cell signaling, proliferation, and
differentiation control. Overexpression of ERK3 could represent a
general response to cell stress, and other members of the ERK family
might be induced by proteasome inhibitions as well. Immunoblotting of
protein extracts from MCF-7 cells treated with different proteasome
inhibitors revealed no induction of ERK1/2 (Fig. 3C) and no
induction of ERK5 or ERK6 (data not shown) after 3.5, 7, or 14 h
of exposure.
ERK3 Induction Is Independent of p53, Caspase 3, and Ectopic Bcl-2
Expression--
Apoptosis induced by inhibition of the proteasome
involves mechanisms independent of p53 (1) and Bcl-2 (17). In this context it was important to establish a possible relationship between
proteasome inhibitor-induced ERK3 expression and the genetic background
of the cell lines used to study the induction. ERK3 induction was
studied in a number of cell lines with different p53 statuses. MCF-7
cells have wild type p53 (18); HL-60 cells are p53 null (19); and the
other cell lines, MDA MB231 (20), MDA MB468 (21), and DU145 (22), have
mutations in p53. Moreover, MCF-7 cells, but not the other cell lines
tested, are deficient in caspase 3 (23). MG132 and lactacystin
treatment increased the ERK3 protein level in all cell lines tested
(Fig. 4A), independent of the
p53 and caspase 3 statuses.

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Fig. 4.
ERK3 protein induction in various cell
lines. A, ERK3 expression in cell lines with p53 mutant
or null status. ERK3 protein expression was analyzed by Western
blotting after treatment of MDA MB231 (p53 mutant), DU145 (p53
mutant), MDA MB468 (p53 mutant), and HL-60 (p53 null) cells for 14 h with Me2SO, MG132 (10 µM), or lactacystin
(10 µM). B, ERK3 expression in MDA MB453 cells
ectopically expressing Bcl-2. ERK3 protein expression (upper
panel) and Bcl-2 protein expression (lower panel) were
analyzed by Western blotting after treatment of MDA MB453-Bcl-2 or MDA
MB453-neo cells for 14 h with Me2SO, MG132 (10 µM), or lactacystin (10 µM). The
asterisk indicates a protein in MDA MB453 cell extracts
nonspecifically interacting with the ERK3 antibody. DMSO,
dimethylsulfoxide.
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Recently, a functional link has been discussed between Bcl-2 and the
proteasome (17). Moreover, Bcl-2 appears to be a substrate for the
ubiquitin-proteasome pathway (24). To test the effect of Bcl-2
overexpression on ERK3 induction, MDA MB453 cells were stably
transfected either with Bcl-2, producing MDA MB453-Bcl-2 cells, or with
a control vector (MDA MB453-neo). Bcl-2 was present at very low levels
in MDA MB453-neo cells but was readily detected by Western blotting in
MDA MB453-Bcl-2 (Fig. 4B). Following exposure to MG132 or
lactacystin, ERK3 was induced in both cell lines regardless of the
Bcl-2 expression level. Moreover, the induction of ERK3 appears not to
be dependent on the presence of functional Bcl-2. MDA MB468 cells are
deficient in Bcl2 expression (25), but ERK3 was induced upon proteasome
inhibition (Fig. 4A).
ERK3 Is Controlled by the p38 Pathway--
It has been reported
previously that proteasome inhibition leads to activation of p38 (7).
Moreover, p38 is activated by the mitogen-activated/extracellular
response kinase kinases MEK3 and MEK6 (26). As shown in Fig.
5A, by Western blotting with activation state-specific antibodies, p38 and MEK3/6, but not ERK1/2,
are phosphorylated and thereby activated in a
time-dependent manner in MCF-7 cells treated with 10 µM MG132, in agreement with published data (7). Overall
p38 expression levels were not affected (Fig. 5B). Is there
a link between p38 activation and ERK3 induction? To address this
question we tested whether ERK3 induction could be blocked by
preincubation with selective p38 kinase inhibitors. The kinase
inhibitors SB202190, SB203580, and PD169316 are specific inhibitors of
the p38 pathway (26, 27). MCF-7 cells were pretreated for 3 h with
these inhibitors to disable p38 activation and were subsequently
incubated with the proteasome inhibitor MG132 for an additional 14 h. Immunoblotting revealed no induction of ERK3 in cotreated cells
(Fig. 5C), suggesting that ERK3 is controlled by the p38
pathway. Recently, it has been published that two transcription
factors, GADD153 and ATF3, are induced by
inhibition of the proteasome (3). To prove that the kinase inhibitors
selectively block ERK3 overexpression, but not the induction of other
proteasome-controlled proteins, the protein levels of Gadd153 and ATF3
were analyzed by immunoblotting (Fig. 5D). In contrast to
ERK3, pre-incubation with the p38 kinase inhibitors did not block
induction of Gadd153 and ATF3. To exclude the possibility that
the inhibitors of the p38 pathway induce Gadd153 and ATF3 per
se, an additional control experiment was performed. As shown by
Western blot analysis in Fig. 5E, neither Gadd153 nor ATF3
were induced in MCF-7 cells that had been exposed to p38 inhibitors
alone.

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Fig. 5.
p38 kinase activity and ERK3 expression.
A, the amounts of phosphorylated p38, MEK3/6, and ERK-1/2
were determined by Western blotting with activation state-specific
antibodies in MCF-7 cells after treatment for 0, 0.5, 1, 2, 4, 6, or
8 h with Me2SO or MG132 (10 µM).
B, the amounts of p38 protein (upper panel) and
phosphorylated p38 (p-p38, lower panel) were
determined by Western blotting with activation state-specific
antibodies (lower panel) or non-activation state-specific
antibodies (upper panel) in MCF-7 cells. DMSO,
dimethylsulfoxide. C, ERK3 expression in the presence of
inhibitors of p38 activation. ERK3 expression was analyzed by Western
blotting after treatment of MCF-7 cells for 14 h with
Me2SO, MG132 (10 µM), MG132 (10 µM) and PD169316 (0.5 µM), MG132 (10 µM) and SB202190 (0.5 µM), or MG132 (10 µM) and SB203580 (0.5 µM). PD169316,
SB202190, and SB203580 was added 3 h before addition of MG132 or
Me2SO. D, ATF3 and Gadd153 expression in the
presence of inhibitors of p38 activation and the proteasome inhibitor
MG132. ATF3 (upper panel) and Gadd153 (lower
panel) expression was measured by Western blotting after treatment
of MCF-7 cells for 14 h with Me2SO, MG132 (10 µM), MG132 (10 µM) and PD169316 (0.5 µM), MG132 (10 µM) and SB202190 (0.5 µM), or MG132 (10 µM) and SB203580 (0.5 µM). PD169316, SB202190, and SB203580 were added 3 h
prior to the addition of MG132 or Me2SO. E, ATF3
and Gadd153 expression in the presence of inhibitors of p38 activation.
ATF3 (upper panel) and GADD153 (lower panel)
levels were determined by Western blotting after treatment of MCF-7
cells for 14 h with Me2SO, MG132 (10 µM), PD169316 (0.5 µM), SB202190 (0.5 µM), or SB203580 (0.5 µM).
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ERK3 Protects Cells from the Antiproliferative Effect of Proteasome
Inhibitiors--
To investigate the potential role(s) of ERK3
induction upon proteasome inhibition, MCF-7 cells were stably
transfected either with ERK3, producing MCF-7-ERK3 cells, or
with a control vector (MCF-7-neo). ERK3 was detected by Western
blotting in MCF-7-ERK3 cells but was not present in MCF-7-neo cells
(Fig. 5A). Following 40 h of exposure to MG132 or
doxorubicin, the proliferation rates of MCF-7-ERK3 and MCF-7-neo were
measured (Fig. 6). As shown in Fig.
5A, MCF-7-ERK3 cells were more resistant to the
antiproliferative effect of the ERK3 inducer MG132 than to that of
doxorubicin, which did not induce ERK3 (Fig. 3B).
These results implicate ERK3 in defense mechanisms protecting MCF-7
cells from adverse effects induced by proteasome inhibition.

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Fig. 6.
ERK3 expression and
proliferation. Proliferation of MCF-7 cells ectopically
expressing ERK3. Proliferation of MCF-7-neo ( , ) or
MCF-7-ERK3 ( , ) cells was measured with the MTS/formazan
assay in the presence of increasing concentrations of MG132 (left
panel) or doxorubicin (right panel). ERK3 expression of
the stably transfected cell lines was confirmed by Western blotting
(middle panel).
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To further prove this hypothesis, MCF-7 cells were treated for 40 h either with increasing concentrations of the p38-specific kinase
inhibitor SB203580 or with combinations of MG132 and SB203580, PSI and
SB203580, or rotenone and SB203580, respectively (Fig. 7). The concentrations of MG132, PSI, and
rotenone were chosen to yield a half-maximal inhibition (data not
shown) of the proliferation rate (0.25 µM MG132, 0.5 µM PSI, and 0.5 µM rotenone). MCF-7 cells treated with MG132 or PSI were sensitive toward increasing
concentrations of the ERK3-blocking compound SB203580 and showed a
strong SB203580-dependent decrease in the proliferation rate.
In contrast, the decrease in the proliferation rate was less pronounced
in cells treated with SB203580 alone or in combination with rotenone.
Similar results were obtained in MDA MB231, MDA MB468, and DU145 cells,
indicating that the protective effect of ERK3 is not restricted to
MCF-7 cells.

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Fig. 7.
ERK3 expression, proliferation, and p38
inhibition. Proliferation in the presence of combinations of MG132
or rotenone and p38 inhibitors. Proliferation of MCF-7, MDA MB231, MDA
MB468, or DU145 cells was measured with the MTS/formazan assay
in the presence of 0.25 µM MG132 ( ), 0.5 µM rotenone ( ), 0.5 µM PSI ( ), or
Me2SO ( ) and increasing concentrations of SB203580 or
SB202190. SB203580 or SB202190 was added to the cells 3 h prior to
the addition of MG132, PSI, rotenone, or Me2SO.
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DISCUSSION |
A cDNA array hybridization approach as a high throughput
screening method for differential gene expression has been applied to
develop a comprehensive picture of pathways linked to proteasome inhibition (3). Similar approaches were taken to characterize genotoxic
stress patterns (28), transformation-related genes in oral cancer
cells (29) and fibroblasts (30), and early steps in immortalization of
Burkitt lymphoma (31), to name just a few.
The approach chosen did point toward a large number of pathways
affected by the proteasome. In the present study, we focussed on ERK3
because MAP kinases represent key regulators of intracellular signaling
pathways. ERK3 regulation seems to involve at least two mechanisms: i)
at the transcriptional level, as shown here, and upon ganglioside
treatment, as illustrated by Kleines et al. (32),
and ii) at the post-translational level, corroborated for instance by
Camarillo et al. (33).
ERK3 mRNA induction has been shown to occur as a
consequence of proteasome inhibition. The induction was independent of
p53, Bcl-2, or caspase 3, all three of them representing important regulatory mechanisms of apoptosis. p38 inhibitors of different chemical structure all abrogated ERK3 induction by proteasome inhibition. ERK3 overexpression occurred downstream of p38 MAPK activation, which was essential for the induction to take place. However, alternative pathways leading to ERK3 overexpression and/or activation by other triggers cannot be excluded.
Activation of p38 typically is accomplished via activation of MEK3/6
(26). Indeed, we were able to confirm this in MCF-7 cells as well.
Moreover, we do not see an increase in MEK3/6 protein or p38 protein
levels but rather an accumulation of phosphorylated species. At least
one component further upstream of MEK3/6 is postulated to be regulated
at the expression/stability level, linking proteasome inhibition to
ERK3 induction. Expression levels of a range of other transducers of
the mitogenic signal (ERK1, 2, 5, 6, MEK1, and MKP1) all remained
unchanged. This putative factor remains to be identified.
A large set of transcription factors including Elk-1, c-Myc, c-Jun,
c-Fos, and C/EBP
has been described as being activated by MAP
kinases and p38 in particular (34). The nature of the transcription
factor that undergoes p38-dependent activation and, as a
consequence thereof, induces ERK3 transcription is still elusive. Wang and Ron (35) show that Gadd153 undergoes phosphorylation on two adjacent serine residues, most probably by p38. When considering the kinetics of ERK3 versus Gadd153 induction, we cannot
definitely exclude the possibility that phosphorylated Gadd153 indeed
regulates ERK3 transcription and that Gadd153 activity is
regulated in a dual manner by transcriptional induction and by
phosphorylation. This hypothesis, however, is at odds with the notion
that Gadd153 overexpression is associated with apoptosis (35, 36).
Ectopic overexpression of ERK3, on the other hand, was shown to
partially protect cells from growth inhibition by proteasome inhibitor
compounds. This protection was not manifested when cells were
challenged with other cytotoxic compounds (rotenone and doxorubicin),
indicating a divergent mechanism of cell toxicity of those compounds
and a very specific mode of protection by ERK3.
Inversely, p38 inhibition specifically sensitized various cells to
proteasome inhibitors. We would therefore conclude that ERK3 triggers,
or is part of, a rescue mechanism. Combined use of p38 inhibitors with
proteasome inhibitors such as PS341, which is in Phase II clinical
trials for prostate tumor indications, should represent a successful
strategy to treat solid tumors.
 |
ACKNOWLEDGEMENTS |
We are grateful to the staff at Incyte Inc.
(Palo Alto, CA) for performing the array-based hybridizations. We thank
Lidia Sambucetti for trapoxin A and Doriano Fabbro for comments on the manuscript.
 |
FOOTNOTES |
*
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. Tel.:
41-61-696-2636; Fax: 41-61-696-6381; E-mail:
peter.fuerst@pharma.novartis.com.
Published, JBC Papers in Press, January 8, 2001, DOI 10.1074/jbc.M008567200
 |
ABBREVIATIONS |
The abbreviations used are:
MAP, mitogen-activated protein;
ERK, extracellular signal-regulated kinase;
PCR, polymerase chain reaction;
GAPDH, glyceraldehyde-3-phosphate
dehydrogenase.
 |
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