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1998 Oxford University Press
486–489 Nucleic Acids Research, 1998, Vol. 26, No. 2
Activation of stress-activated MAP protein kinases
up-regulates expression of transgenes driven by the
cytomegalovirus immediate/early promoter
W. Bruening+, B. Giasson1, W. Mushynski1 and H. D. Durham*
Department of Neurotoxicology, Montreal Neurological Institute and Department of Neurology/Neurosurgery,
3801 University Street, Montreal, Quebec H3A 2B4, Canada and 1Department of Biochemistry, McGill University,
Montreal, Quebec, Canada
Received September 25, 1997; Revised and Accepted November 20, 1997
ABSTRACT
The immediate/early promoter/enhancer of cytomega-
lovirus (CMV promoter) is one of the most commonly
used promoters for expression of transgenes in
eukaryotic cells. In practice, the CMV promoter is often
thought of as a constitutively active unregulated
promoter. However, we have observed that transcription
from the CMV promoter can be up-regulated by a variety
of environmental stresses. Many forms of cellular
stress stimulate MAP kinase signalling pathways,
resulting in activation of stress-activated protein
kinases [SAPKs, also called Jun N-terminal kinases
(JNKs)] and p38 kinases. We have found that the same
conditions that lead to activation of SAPK/JNKs and
p38 kinases can also dramatically increase expression
from the CMV promoter. Inhibitors of p38 kinases
abolished basal transcription from the CMV promoter
and completely blocked stress-induced up-regulation of
the CMV promoter. Overexpression of a dominant
negative JNK kinase had no effect on basal transcription,
but significantly reduced up-regulation caused by
stress. These results have grave implications for use of
the CMV promoter. If the CMV promoter can be up-
regulated by cellular stresses, inadvertent activation of
the stress kinase pathways may complicate, if not
invalidate, the interpretation of a wide range of
experiments.
INTRODUCTION
It has recently been discovered that a variety of environmental
insults, such as UV irradation, osmotic shock and oxidative stress,
mediate some of their effects on the cell through activation of
MAP kinase signalling pathways (1,2). These kinase cascades
ultimately activate the c-Jun N-terminal kinases (JNKs), also
called stress-activated protein kinases (SAPKs, here referred to as
SAPK/JNKs), a family of kinases which increase the activity of
transcription factors such as c-Jun and ATF-2 (3,4). A parallel
pathway which responds to many of the same stimuli results in
activation of the p38 MAP kinases (5), a family of kinases that
increase the activity of MAPKAP kinase 2 (6), ATF-2 (7) and
Max (8).
During the course of our work we observed that many of the
same stresses that activate the SAPK/JNK pathway could also
dramatically increase expression of transgenes regulated by the
immediate/early cytomegalovirus promoter/enhancer complex
(CMV promoter). This is one of the most commonly used
promoters in eukaryotic expression vectors. Its popularity derives
from its ability to drive high levels of expression in nearly all
mammalian cells. If the CMV promoter can be up-regulated by
stress, its use may complicate a wide range of experiments,
particularly those involving components of the stress-activated
kinase pathways.
MATERIALS AND METHODS
Cell culture
All cell lines were originally from ATCC. Cell lines were maintained
in DMEM supplemented with 10% fetal calf serum. Cells were
transfected with Lipofectamine (Gibco-BRL, Burlington, Ontario)
following the manufacturer’s instructions. Equal amounts of
expression vector were added in each transfection; when necessary
an empty CMV expression vector (pcDNA-3; Invitrogen,
San Diego, CA) was added to equalize the total amount of plasmid
DNA. Under these conditions ∼80% of NIH 3T3 cells are
transfected.
β-Galactosidase activity assays
Forty-eight hours after transfection cells were lysed in PBS, 0.5%
Nonidet P-40. Insoluble debris was pelleted and the supernatant
diluted 10-fold with Z buffer [140 mM sodium phosphate, pH 7.4,
1 mM magnesium sulfate, 140 mM potassium chloride, 0.27%
β-mercaptoethanol, 1 mg/ml o-nitrophenyl-β-D-galactopyranoside
(ONPG)]. After incubation at 37C for 30 min, the absorbance at
420 nm was measured with an LKB Ultrospec 4050
*To whom correspondence should be addressed. Tel: +1 514 398 8509; Fax: +1 514 398 1509; Email: mddm@musica.mcgill.ca
+Present address: FoxChase Cancer Center, Philadelphia, PA 19111, USA
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Nucleic Acids Research, 1998, Vol. 26, No. 2 487
Figure 1. NIH 3T3 cells exposed to arsenite have elevated levels of heat
shock/stress proteins. Western blots were probed with an antibody that
recognizes only the inducible form of HSP70 (K20; Santa Cruz Biotechnology)
and an antibody against HSP27 (M20; Santa Cruz Biotechnology). Lane 1,
untreated control; lane 2, 1 µM arsenite; lane 3, 10 µM arsenite; lane 4, 50 µM
arsenite. Cells were treated with sodium arsenite for 6 h before harvesting and
blotting as described in Materials and Methods.
spectrophotometer. Protein concentration of the extracts was
measured with a bicichoninic acid–copper assay (Sigma, St Louis,
MO). β-Galactosidase activity is defined as (OD420 nm × 1000)
(time of incubation)(mg extract). All assays were done in duplicate.
Immunoblots
Cells were lysed in RIPA buffer (50 mM Tris, pH 8.0, 150 mM
NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS).
Insoluble debris was pelleted and the protein concentration of the
supernatant determined using a bichichoninic acid–copper assay
(BioRad, Mississauga, Ontario). Samples of 20 µg were
electrophoresed by standard SDS–PAGE and transferred to
Immobilon-P membranes (Millipore). Blots were probed with the
indicated antibodies using standard techniques and developed with
ECL chemiluminescence reagents (Amersham).
Northern blots
Forty-eight hours after transfection, RNA was extracted from the
cells with Trizol (Gibco-BRL), following the manufacturer’s
instructions. The RNA was quantified by measuring optical density
at 260 nm and the values were confirmed by ethidium staining after
gel electrophoresis. Five micrograms of RNA were separated on a
MOPS/formaldehyde–agarose gel and transferred to Zetabind
membrane (BioRad) by capillary blotting. The blot was probed with
full-length lacZ cDNA using standard techniques.
Immunoprecipitation kinase assay of JNK1
Activation of JNK1 was assayed as described previously (9).
Briefly, following cell lysis and centrifugation to remove cell
debris the enzyme was immunoprecipitated using anti-JNK1
polyclonal antibody (C-17; Santa Cruz Biotechnology, Santa
Cruz, CA) and GST–cJun (Santa Cruz Biotechnology) was used
as substrate to determine the activity.
Plasmids and reagents
Unless otherwise stated, chemicals were purchased from Sigma
and cell culture reagents were from Gibco-BRL. The p38
inhibitor SB203580 was from Calbiochem. The pCMV-lacZ
plasmid was pUT535 (Cayla, Toulouse, France). Plasmid
pJNKK(–) encodes a JNK kinase (also called SEK1) mutated at
the sites of MEKK1 phosphorylation, such that activated
Figure 2. Activation of stress-activated MAP kinases up-regulates expression
from the CMV promoter. (A) NIH 3T3 cells were transfected with pCMV-lacZ as
described in Materials and Methods. Forty hours after transfection some of the cells
were treated with 50 µM sodium arsenite for 6 h. (B) The indicated cell lines were
transfected with pCMV-lacZ or co-transfected with pCMV-lacZ and pCMV-MEK-
K(Act). Forty-six hours after transfection the cells were harvested and assayed for
β-galactosidase activity as described in Materials and Methods. Each experiment
was done three to five times, in duplicate, and the average was taken.
MEKK1 can bind to the sites but not phosphorylate them, thus
preventing some of the activated MEKK1 from activating
endogenous JNKK. Plasmid pCMV-MEKK(Act) expresses a
constitutively active truncated form of MEKK1 and was a gift
from M.Karin.
RESULTS
Transcription from the CMV promoter can be up-regulated
While performing routine transfection experiments with expression
vectors containing the CMV promoter, we observed that certain
stressors would dramatically increase expression from these
constructs. To study this phenomenon further we employed a
plasmid in which expression of the Escherichia coli gene lacZ,
which encodes β-galactosidase, is regulated by the CMV promoter.
This system allowed us to readily quantify the amount of
CMV-driven expression obtained.
NIH 3T3 cells were transfected with the CMV-lacZ plasmid as
described in Materials and Methods. Forty hours after transfection
the cells were treated with 50 µM sodium arsenite. Six hours were
left between initiation of the treatment and harvesting to allow time
for transcription and translation. The concentration of arsenite used
was chosen to be sublethal but extremely stressful. Under these
conditions of exposure to arsenite induction of a stress response was
also demonstrated by increased levels of two heat shock/stress
proteins, HSP27 and HSP70 (Fig. 1). As shown in Figure 2A,
treatment of transfected NIH 3T3 cells with sodium arsenite, a
reagent which activates SAPK/JNK (10), increased β-galactosidase
activity 3-fold. Other stressors, such as osmotic shock, hydrogen
peroxide and heat shock, were also able to increase β-galactosidase
activity (data not shown).
To confirm that the observed CMV up-regulation was a direct
consequence of stress kinase activation, rather than some
side-effect of drug treatment, we employed a plasmid that
expresses a cDNA encoding an active MEKK1, one of the initial
kinases in the MAP kinase cascade. Low levels of active MEKK1
will preferentially activate SAPK/JNKs, but high levels of
expression, such as are found here, will also activate the p38
kinases (2,11). Co-transfection of NIH 3T3 cells with pCMV-lacZ
Nucleic Acids Research, 1998, Vol. 26, No. 2
488
Figure 3. Stress-induced up-regulation of the CMV promoter occurs at the level
of transcription. NIH 3T3 cells were transfected with pCMV-lacZ or
co-transfected with pCMV-lacZ and pCMV-MEKK(Act). Some cells were
treated with 50 µM sodium arsenite 40 h after transfection for 6 h. The cells
were harvested 46 h after transfection. mRNA was extracted, separated by gel
electrophoresis and blotted as described in Materials and Methods. The relative
amount of β-galactosidase mRNA was measured by phosphorimaging. Lane 1,
mock transfection; lane 2, transfected only with pCMV-lacZ; lane 3, transfected
only with pCMV-lacZ and treated with sodium arsenite; lane 4, transfected with
pCMV-lacZ and pCMV-MEKK(Act). The experiment was repeated several
times and a representative result is shown.
and pCMV-MEKK(Act) resulted in dramatically increased β-ga-
lactosidase activity (Fig. 2A). To see if this effect was cell type
specific a number of different cell lines were tested. Each cell line
was co-transfected with pCMV-lacZ and pCMV-MEKK(Act).
Forty-eight hours later, β-galactosidase levels were measured.
For all cell types tested co-expression of activated MEKK1
caused a significant increase in β-galactosidase levels (Fig. 2B).
The inductive effect was not limited to β-galactosidase, as other
proteins expressed from the CMV promoter were also found to be
up-regulated by stressors (data not shown).
To confirm that up-regulation was at the level of transcription
NIH 3T3 cells were transfected with pCMV-lacZ and treated with
sodium arsenite or co-transfected with pCMV-MEKK(Act).
Levels of β-galactosidase mRNA were examined by Northern
blotting. As can be seen in Figure 3, both sodium arsenite
treatment and co-expression of activated MEKK1 up-regulated
the amount of mRNA driven by the CMV promoter.
Both SAPK/JNKs and p38 kinases are involved in
regulating transcription from the CMV promoter
As mentioned briefly, the stressors that induce CMV up-regulation
are known to activate the SAPK/JNK pathways. To confirm that
SAPK/JNK activation correlates with CMV up-regulation we
assayed SAPK/JNK activity directly. As shown in Figure 4, treating
NIH 3T3 cells with either arsenite or transfection with pCMV-MEK-
K(Act) strongly increases the kinase activity of JNK1. Although
MEKK1 does not activate JNK1 as strongly as does arsenite,
MEKK1 up-regulates expression from the CMV promoter to a
much greater extent than does arsenite (Fig. 1A). However, this
apparent contradiction can easily be explained if we consider that
cells transfected with pCMV-MEKK(Act) are stimulated for 46 h,
allowing a steady accumulation of products expressed from the
CMV promoter, while arsenite treatment only allows 6 h worth
of products to accumulate.
To further investigate the role of SAPK/JNK in CMV
up-regulation we employed a dominant negative JNK kinase. The
Figure 4. JNK1 is activated by the same conditions that up-regulate CMV
expression. NIH 3T3 cells were transfected with pCMV-MEKK(Act) (A, lane 2),
mock transfected (A, lane 1), untreated (B, lane 1) or treated with sodium arsenite
as described in the legend to Figure 1 (B, lane 2). The cells were harvested and
assayed for JNK1 activity as described in Materials and Methods. The relative
JNK1 activity was measured by phosphorimaging and is indicated under each lane.
The experiment was repeated several times and a representative result is shown.
plasmid pJNKK(–) expresses a JNKK (also called SEK1)
mutated such that it partially blocks activation of SAPK/JNKs.
When pCMV-lacZ and pJNKK(–) were co-expressed together in
NIH 3T3 cells basal levels of β-galactosidase were unaltered.
However, when cells co-transfected with pCMV-lacZ and
pJNKK(–) were treated with sodium arsenite the presence of
JNKK(–) reduced up-regulation of β-galactosidase activity by
half (Fig. 5). Since p38 kinases and SAPK/JNKs are both
activated by the same or similar stresses (2), we also investigated
the role of p38 kinases in CMV stress-induced up-regulation. To
study their function we used a specific inhibitor that blocks
activity of the p38 kinases (12). We found that if p38 inhibitor was
added at the time of transfection almost no product was expressed
from the CMV promoter (Fig. 5), indicating that basal levels of
p38 kinase activity are essential for basal CMVpromoter activity.
If, however, p38 inhibitor was added 40 h after transfection, at the
same time sodium arsenite was added, the presence of p38
inhibitor completely blocked stress-induced up-regulation of the
CMV promoter (Fig. 5).
DISCUSSION
Activation of the stress-activated MAP kinase cascades has many
effects on the cell that appear to be mediated primarily through
regulation of transcription. Activated SAPK/JNK is known to
phosphorylate and thereby activate key transcriptional factors such
as c-Jun and ATF-2 (3,4) and the p38 kinases also regulate ATF-2
(7). The immediate/early CMV promoter/enhancer complex
contains AP-1 and ATF binding sites (13). It is, therefore, not
particularly surprising that activity of the CMV promoter should
be altered in a stressed cell. The stress-induced activation of the
CMV promoter that we have observed may simply be a
side-effect caused by a general up-regulation of transcription, but
it is also possible that cytomegalovirus could have evolved to take
advantage of the stress response. Like many other viruses,
cytomegalovirus evades the immune system by remaining
quiescent within the cell until conditions become appropriate for
viral replication (14). Stimulation of cytomegalovirus replication
appears to be primarily mediated by inducing transcription from
the cytomegalovirus immediate/early promoter (15) and many of
the conditions known to stimulate CMV replication, such as
oxidative stress, immunosuppressive drugs and irradiation, are
also conditions that can activate SAPK/JNKs and p38 kinases
(1–4,13,16). These conditions are also likely to result in
immunosuppression of the host, allowing cytomegalovirus to
replicate only under conditions which it is able to survive. Thus
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Nucleic Acids Research, 1998, Vol. 26, No. 2 489
Figure 5. MAP kinases regulate expression from the CMV promoter. NIH 3T3
cells were mock transfected (Mock), transfected with pCMV-lacZ alone (βGal),
co-transfected with pCMV-lacZ and pJNKK(–) [βGal + JNKK(–)] or transfected
with pCMV-lacZ and treated with 0.5 µM SB203580 (βGal + p38-I). Forty hours
after transfection some of the cells were treated with arsenite as described in the
legend to Figure 1. Some cells were treated with SB203580 for the entire 46 h
(added at t = 0), while others were only treated with SB203580 40 h after
transfection for 6 h (added at t = 40). All cells were harvested 46 h after transfection
and assayed for β-galactosidase activity as described in Materials and Methods.
Each experiment was done three times in duplicate and the average was taken.
viral promoters may have been selected to be able to maximize
transcription under stressed conditions.
In addition to variation in transcriptional activity of the CMV
promoter according to cell type and developmental age (17), two
previous reports have suggested that the CMV promoter can be
up-regulated under specific conditions. In one the major immediate/
early enhancer of cytomegalovirus was found to be up-regulated by
cAMP in lymphoid cell lines (18). This up-regulation was mediated
through a CRE element contained within the enhancer. In the other
report NF-κB was shown to play a central role in activation of the
CMV promoter by cytomegalovirus gene products (13). Sambucetti
et al. were also able to artificially activate transcription from the
CMV promoter by stimulating the cells with phorbol esters (13).
Our results are the first to show that stress-activated MAP kinases
are involved in regulating transcription from the CMV promoter. We
find that without basal p38 kinase activity the CMV promoter is
transcriptionally silent (Fig. 4). This may be mediated by lower
ATF-2 activity, as p38 kinases have been shown to directly stimulate
ATF-2 activity (7), or it may be mediated indirectly through loss of
NF-κB function. Wesselborg et al. were able to demonstrate that p38
kinases are necessary for NF-κB-dependent gene expression (19).
p38 kinase inhibitors were also able to completely block stress-
induced up-regulation of the CMV promoter (Fig. 4). It is unclear
whether this blockage is simply due to complete shut-off of the
CMV promoter in the absence of p38 kinases or whether p38 kinases
are also essential for stress-induction of the CMV promoter.
It is unlikely that NF-κB activity is involved in stress induction
of the CMV promoter. Although some stresses are able to activate
NF-κB, sodium arsenite does not (19). Thus the observed
up-regulation of the CMV promoter is probably mediated by
increased activity of c-Jun and ATF-2.
The pertinence of the data presented in this paper stems from the
widespread use of the CMV promoter in expression vectors.
Experimental protocols that might activate the stress response could
be complicated to interprete due to up-regulation of the CMV
promoter. For example, the pCMV-MEKK(Act) plasmid used in our
experiments sets up a self-stimulating loop, expressing increasing
amounts of active MEKK1 until the cell dies. Experiments which
use inhibitors of p38 kinases would be devastated by simultaneous
use of the CMV promoter. On the other hand, protocols that only
require high levels of expression could exploit stress activation of the
CMV promoter to generate even more transgenic protein and
perhaps p38 kinase inhibitors could be used to switch the CMV
promoter on and off.
ACKNOWLEDGEMENTS
This work was supported by grants from the ALS Society of
Canada, the Muscular Dystrophy Association of Canada and the
Medical Research Council of Canada. H.D.D. is an MNI Killam
Scholar. We thank M.Karin for his kind gifts of expression vectors.
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