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INTRODUCTION |
Cardiac hypertrophy is broadly defined as an abnormal enlargement
of the heart characterized by an increase in cardiomyocyte cell
volume and the re-expression of genes encoding fetal protein isoforms.
Current pharmacologic treatment strategies for cardiac hypertrophy
utilize general inhibitors of certain neuroendocrine stimulatory
pathways (such as those mediated by angiotensin II, endothelin-1, and
catecholamines), which are thought to mediate hypertrophic growth of
cardiac myocytes, in part, by activating specific intracellular signal
transduction cascades (1).
In recent years, a number of intracellular signaling pathways have been
implicated as important downstream transducers of neuroendocrine
stimuli and generalized stress stimuli in cardiac myocytes.
Specifically, activation of certain G-protein-coupled receptors and
receptor tyrosine kinases have been shown to promote Ras
activity, the three major branches of the
MAPK1 signaling cascade
(ERKs, JNKs, and p38s), protein kinase C, and calcineurin (reviewed in
Refs. 2 and 3). Activation of these various intracellular signaling
pathways/factors has, in many instances, been directly associated with
the induction of the cardiac hypertrophic response in either the hearts
of transgenic mice or in cultured neonatal rat cardiac myocytes (2,
3).
Data implicating p38 MAPK as a regulator of the hypertrophic response
have largely been obtained in cultured neonatal rat cardiomyocytes. The
G-protein-coupled receptor agonists PE and endothelin-1 are
potent activators of p38 MAPK in cardiomyocytes (4). Overexpression of
activated MKK3 or MKK6 (upstream activators of p38) in neonatal
cardiomyocytes was shown to induce hypertrophy and atrial natriuretic
factor expression in vitro (5-7). Pharmacologic inhibition
of p38 kinase activity with the antagonists SB203580 or SB202190 was
shown to attenuate agonist-stimulated cardiomyocyte hypertrophy in
culture (4, 5, 7). In addition, pharmacologic or dominant
negative inhibition of p38 signaling significantly reduced
agonist-induced b-type natriuretic peptide promoter activity in
vitro (8, 9). However, three additional studies reported that p38
inhibition was not sufficient to attenuate agonist-induced cardiomyocyte hypertrophy under certain conditions, suggesting a
more specialized role for p38 MAPK signaling in vitro (4, 10, 11).
Within the last 2 years, a number of reports have implicated a
calcineurin-dependent signaling pathway in the cardiac
hypertrophic response. Calcineurin is a
calcium/calmodulin-dependent phosphatase that directly
regulates the nuclear translocation of a family of transcription
factors referred to as NFAT (reviewed in Ref. 2). Cardiac-specific
expression of either constitutively active calcineurin or
constitutively nuclear NFATc4 mutant protein in transgenic mouse hearts
resulted in a dramatic hypertrophy response that quickly transitioned
to heart failure (12). Inhibition of calcineurin activity with either
cyclosporin A (CsA) or by infection with a specific
calcineurin-inhibitory adenovirus was sufficient to block
agonist-induced cardiomyocyte hypertrophy (12, 13). While these and
other studies have implicated calcineurin as a hypertrophic regulator,
it is uncertain how calcineurin is interconnected with other
intracellular signaling pathways.
Recently, we reported that calcineurin activation was associated with
activation of protein kinase C
, protein kinase C
, JNK, and ERK
in vitro and in vivo (14). However, the
involvement of p38 MAPK was not characterized. In this report, we show
that calcineurin activation is associated with a down-regulation of p38
MAPK activity in calcineurin transgenic mouse hearts and in calcineurin
adenovirus-infected cardiomyocytes. Calcineurin does not directly
dephosphorylate p38 MAPK but rather is associated with enhanced
expression of the dual specificity phosphatase MKP-1. Characterization
of the MKP-1 upstream regulatory region demonstrates that calcineurin
enhances promoter activity, suggesting potential signaling cross-talk
between calcineurin and p38 MAPK pathways through MKP-1.
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EXPERIMENTAL PROCEDURES |
Plasmids--
The activated MEK1 expression vector was kindly
provided by Dr. C. J. Marshall, while activated MKK6 and MKK7 were
gifts of Dr. J. Han and Dr. E. Nishida, respectively. The activated
calcineurin A
expression vector consisted of amino acids 1-398 as
described previously (15). The full-length NFATc4 expression vector and its N-terminal truncation were described previously (12, 16). The
full-length NFATc3 expression vector was described previously (17),
while the N-terminal truncation was generated at amino acid 315, and
the remaining coding region was cloned into the pECE-FLAG expression
vector by polymerase chain reaction. The expression vector encoding the
C-terminal calcineurin-inhibitory fragment was described previously
(13). The MKP-1 upstream regulatory region was previously sequenced,
and the transcription initiation site was identified (18). Polymerase
chain reaction was used to clone a mouse genomic fragment spanning from
633 to +100 base pairs (relative to the transcription start site) for
insertion into the luciferase reporter pGL3 (Promega, Madison, WI).
Cell Culture--
Primary cultures of neonatal rat
cardiomyocytes were generated and cultured as described previously
(13). Hearts were collected from 1-2-day-old Harlan Sprague-Dawley rat
neonates; the ventricles were cut in four equal parts in a balanced
salt solution and enzymatically digested through multiple rounds in
0.05% pancreatin (Sigma) and 84 units/ml collagenase (Worthington).
The cells were centrifuged at 700 × g for 5 min and
resuspended in M199 media (Life Technologies, Inc.) supplemented
with 15% fetal bovine serum, penicillin/streptomycin (100 units/ml),
and L-glutamine (2 mmol/L). Afterward, the cells were
differentially plated for 1 h to remove contaminating nonmyocytes. The enriched cardiomyocyte fractions were then plated on gelatinized dishes and cultured the following day in serum-free M199 media containing 100 units/ml penicillin/streptomycin and 2 mmol/liter L-glutamine. Cardiomyocytes were maintained in serum-free
M199 media for 48 h before adenoviral infections and/or treatments.
Culturing of 10T1/2 fibroblasts and COS-7 cells was described
previously (12). All transfections employed Fugene (Roche) in 6-cm
tissue culture dishes, after which cells were harvested for Western
blot or luciferase determinations (24 h later). The calcineurin or MKK
expression plasmids were transfected at one-fourth the DNA content
relative to the MKP-1 reporter construct. Protein extracts were
generated for luciferase determinations as described previously
(12).
Replication-deficient Adenovirus
Infections--
Characterization of replication-deficient adenovirus
expressing constitutively active calcineurin (AdCnA) and
-galactosidase (Ad
gal) was recently described in detail (14). All
recombinant adenoviral vectors were plaque-purified in HEK293 cells,
expanded, and titered by plaque assays in HEK293 monolayers embedded in agarose. Cardiomyocytes were infected with each virus at a multiplicity of infection of 100 plaque-forming units for 2 h at 37 °C in a humidified 6% CO2 incubator. Afterward, the cultures were
placed in serum-free M199 media for an additional 24 h prior to
treatments. Under these conditions, ~98% of the cells showed
expression of the viral gene insert.
Western Blot Analysis--
Protein extracts were generated from
cardiomyocytes or COS-7 cells using extraction buffer, and Western
blotting was performed as described previously (14). Quantitative
chemiluminescent detection was performed with Vistra enhanced
chemifluorescence (Amersham Pharmacia Biotech) and scanned utilizing a
Storm 860 (Molecular Dynamics, Inc., Sunnyvale, CA). Antibodies used
included anti-phospho-p38, anti-p38, anti-phospho- ERK1/2, anti-ERK1/2, anti-phospho-MEK1/2, anti-phospho-MKK3/6 (Cell Signaling, Beverly, MA),
anti-MKP-1, and anti-MKP-2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
In Vitro Coupled Kinase Assay--
Both His-tagged MKK6
(E) and p38 were purified as described previously (19). Briefly,
0.1 µg of MKK6 (E) and 0.2 µg of p38 bacterial produced and
purified proteins were incubated with 20 units of calcineurin (BioMol)
or 8 units of calf intestinal alkaline phosphatase (New England
Biolabs, Beverly, MA) and incubated in 1× kinase buffer containing 10 mCi of [32P]ATP at 37 °C for 30 min. Reactions
containing calcineurin enzyme were incubated with excess calcium and
calmodulin as directed by the manufacturer (BioMol). Reactions were
stopped by the addition of 2× loading buffer and 5 min of boiling
before electrophoretic separation on 12% SDS-polyacrylamide gel
electrophoresis. All images were visualized and quantified by
PhosphorImager analysis.
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RESULTS |
Calcineurin Transgenic Hearts Show Reduced p38 MAPK
Activation--
Expression of activated calcineurin in the hearts of
transgenic mice induces a striking hypertrophy response that is
detectable by 9-14 days after birth and results in a 3-fold increase
in heart size by 6 weeks of age (12). It was of interest to examine the status of other intracellular signaling pathways in calcineurin transgenic hearts to characterize the mechanisms that might underlie this dramatic increase in heart size. We focused an analysis on the p38
branch of the MAPK signaling pathway, since it has been shown to play a
modulatory role in the cardiac hypertrophic process (3). Hearts from
calcineurin transgenic or wild-type littermate mice were analyzed at 4 days of age before any signs of cardiac hypertrophy were observed,
guarding against secondary effects associated with hypertrophy itself.
Western blot analysis of cardiac protein extracts from 4-day old hearts
demonstrated a 2.9-fold down-regulation of p38 MAPK phosphorylation
from calcineurin transgenic mice without a change in total p38 MAPK
protein (Fig. 1A,
asterisks). ERK1/2 phosphorylation status was not affected
at this time point (Fig. 1A). A similar down-regulation of
p38 MAPK was also seen at 8 and 14 days of age, but such
down-regulation was gradually lost by day 24 and older (data not shown;
see "Discussion"). These results suggest that early neonatal
expression of activated calcineurin in the heart promotes p38 MAPK
inactivation before the onset of significant cardiac hypertrophy.

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Fig. 1.
Expression of activated calcineurin promotes
p38 MAPK inactivation in vivo. A, day 4 mouse hearts from calcineurin transgenic (TG) and wild-type
littermates (NTG) were subject to Western blot analysis with
a phosphospecific p38 MAPK antibody, demonstrating reduced
phosphorylation (asterisks; 2.9-fold decrease) in the
transgenic littermate, while phospho-ERK1/2, total p38 MAPK, and total
ERK1/2 protein levels were invariant. B, Western blot of
MKP-1 and MKP-2 protein levels demonstrates increased MKP-1 at day 4 (asterisks; 2.3-fold increase) but not MKP-2. C,
phosphorylation of MEK1/2 and MKK3/4 were not significantly different
between TG and NTG. Each lane (four in total) represents a
pool of two hearts.
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To characterize a potential mechanism whereby calcineurin might promote
p38 MAPK inactivation, Western blotting was performed to characterize
the dual specificity phosphatases MKP-1 and MKP-2 as well as the
upstream MAPK kinase factors MEK1/2 and MKK3/6 (Fig. 1, B
and C). MEK1/2 or MKK3/6 phosphorylation status was not
significantly affected, suggesting that the upstream activation cascade
was not influenced by calcineurin. However, a 2.3-fold increase in
MKP-1 protein levels, but not MKP-2, was observed in 4-day-old
calcineurin transgenic hearts. These data suggest that p38 MAPK
inactivation is associated with augmented MKP-1 protein levels in
calcineurin transgenic hearts.
Calcineurin Indirectly Promotes p38 MAPK
Inactivation--
MAPK activation occurs through the dual
phosphorylation of a threonine and adjacent tyrosine residue
(TXY). A large family of at least nine individual
dual specificity protein phosphatases (MKPs) has been shown to
specifically recognize this unique phosphorylation motif in MAPK
factors, causing their dephosphorylation (reviewed in Ref. 20). Each of
these dual specificity MKPs differs in substrate specificity (p38
versus ERKs versus JNKs), tissue distribution, subcellular localization, or inducible expression profile (20). In
addition to the dual specificity phosphatases, protein phosphatase 2A,
a serine/threonine phosphatase has also been shown to specifically dephosphorylate ERK1/2 (21-23). This observation suggested the possibility that the serine/threonine phosphatase calcineurin might
directly inactivate p38 MAPK. To test this possibility, bacterial
generated p38 MAPK or a kinase-dead mutant of p38 (KM) was
phosphorylated with purified MKK6 and subsequently incubated with
purified calcineurin protein in an excess of calmodulin and calcium. The data demonstrate that calcineurin is unable to directly dephosphorylate p38 MAPK in vitro, while a control utilizing
calf intestinal alkaline phosphatase efficiently dephosphorylated p38 MAPK (Fig. 2A). These data
indicate that calcineurin is unlikely to directly dephosphorylate p38
MAPK.

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Fig. 2.
A, calcineurin fails to directly
dephosphorylate p38 MAPK in vitro as measured after
SDS-polyacrylamide gel electrophoresis. Recombinant p38 MAPK protein or
a kinase inactivate mutant protein (KM) were labeled with
MKK6 and incubated with excess calcineurin (CnA) or calf
intestinal alkaline phosphatase (CIP). B, Western
blot analysis of phosphorylated p38 MAPK and MKP-1 protein levels from
cultured neonatal cardiomyocytes infected with AdCnA or Ad gal and
treated with PE for 1 h. Under these conditions, AdCnA
significantly reduced p38 MAPK phosphorylation (0.54×) and
increased MKP-1 protein levels (6.29×). Similar results
were observed in two independent experiments.
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Dual specificity phosphatases, in general, are regulated at the
transcriptional level so that stress stimuli or agonist stimulation augment mRNA and protein content (20). MKP-1 is most selective for
p38 MAPK, less selective for JNK, and less selective again for ERK1/2
(20, 24, 25). In contrast, MKP-2 is more selective for ERK1/2 and JNK
compared with p38 (20). Since both MKP-1 and MKP-2 are prominently
expressed in cardiac myocytes, we examined their potential association
with reduced p38 MAPK phosphorylation in response to calcineurin
activation (24, 26, 27).
Cultured cardiomyocytes were infected with either AdCnA or
Ad
gal (24 h) and treated with PE for 1 h to induce p38 MAPK
phosphorylation. The data demonstrate that PE augmented p38 MAPK
phosphorylation by ~1.8- and 1.4-fold in uninfected and
Ad
gal-infected cultures, respectively (Fig. 2B). In
contrast, AdCnA infection was associated with a 3.5-fold reduction in
p38 MAPK phosphorylation in cardiac myocytes compared with myocytes
stimulated with PE alone (Fig. 2B). Interestingly, MKP-1
protein expression was most potently induced by the combination of
AdCnA infection and PE treatment (Fig. 2B). While these data
suggest that calcineurin induces MKP-1 protein expression, control
experiments consisting of either AdCnA or Ad
gal alone (no PE
stimulation) did not show induction of MKP-1 expression at 12, 24, 48, or 72 h postinfection (data not shown). These results suggest that
calcineurin requires an additional signal to promote efficient MKP-1
expression. MKP-2 levels were also stimulated by PE, but AdCnA
infection did not promote a further increase.
CsA Reverses p38 MAPK Inactivation and Decreases MKP-1 Protein
Levels--
To examine the association between calcineurin and p38
MAPK activation in more detail, CsA was used to
pharmacologically inhibit calcineurin in cultured cardiomyocytes. CsA
treatment promoted greater p38 MAPK phosphorylation at both 30 min and
1 h of PE stimulation compared with PE stimulation alone (Fig.
3). These data indicate that calcineurin
inhibition is associated with greater p38 MAPK phosphorylation. CsA
treatment also significantly decreased MKP-1 protein expression at 30 min and 1 h (Fig. 3). Specifically, MKP-1 protein levels were
decreased by ~2.5-fold after 1 h of PE stimulation in the
presence of CsA compared with PE stimulation alone (Fig. 3). In
contrast, PE-induced MKP-2 protein levels were unaffected by CsA (Fig.
3). These data suggest that inhibition of endogenous calcineurin with
CsA decreases basal and/or inducible MKP-1 protein levels in cardiac
myocytes, which is associated with enhanced p38 MAPK
phosphorylation.

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Fig. 3.
Endogenous calcineurin regulates p38 MAPK
phosphorylation. Western blot analysis of p38 MAPK phosphorylation
and MKP-1 and MKP-2 levels at 5 min, 15 min, 30 min, and 1 h after
PE agonist stimulation of cultured neonatal cardiomyocytes. CsA
enhanced p38 MAPK activation at 30 min and 1 h and prevented
up-regulation of MKP-1 protein levels. This effect was not seen at 5 or
15 min; nor was MKP-2 altered by CsA. Similar results were obtained in
two independent experiments.
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MKP-1 is transcriptionally regulated in response to stress or mitogen
stimulation, resulting in at least a 30 min delay in measurable
phosphatase activity (20). Consistent with this time course of MKP-1
expression and turnover, PE and CsA treatment did not affect p38 MAPK
phosphorylation within 5 or 15 min of treatment; nor was there a change
in basal MKP-1 at 5 or 15 min (Fig. 3). However, by 30 min, increased
MKP-1 protein was detectable, and this increase was sensitive to CsA,
which was associated with greater p38 MAPK phosphorylation (Fig. 3).
Indeed, after 1 h of PE and CsA treatment, a more pronounced
modulation of p38 MAPK phosphorylation and MKP-1 expression was
observed. Collectively, these data suggest that calcineurin activity
enhances MKP-1 protein expression in response to agonist stimulation
through a transcriptional mechanism.
p38 MAPK phosphorylation status and MKP-1 protein levels were
also examined after calcium ionophore stimulation. Calcium ionophores enhance intracellular calcium concentration, which stimulates calcium-sensitive signaling pathways, including calcineurin. The calcium ionophore A23187 promoted a decrease in p38 MAPK
phosphorylation after 30 min and 3 h of treatment without
affecting total p38 MAPK protein levels (Fig.
4). Likewise, A23187 treatment was also
associated with increased MKP-1 protein levels, while MKP-2 protein
levels were unaffected. CsA treatment also reversed the decrease in p38
MAPK phosphorylation mediated by A23187, which was further associated
with a detectable decrease in MKP-1 protein levels (Fig. 4).
Collectively, these data indicate that activation of endogenous
calcineurin with calcium ionophore results in decreased p38 MAPK
activation and increased MKP-1 protein content in cardiac myocytes.
Such an interpretation is also consistent with a recent report
demonstrating that the calcium ionophore, ionomycin, negatively regulated MAPK activity in a neuronal-like cell line by specifically inducing MKP-1 (28).

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Fig. 4.
Western blots showing that stimulation of
endogenous calcineurin with A23187 decreased p38 MAPK phosphorylation
at 30 min and 3 h, which was rescued by CsA treatment. A23187
also increased MKP-1 but not MKP-2 protein levels at 30 min and 3 h. Similar results were observed in two independent experiments and
at 1- and 4-h time points.
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Calcineurin Promotes p38 MAPK Inactivation in COS-7 Cells--
To
determine if the identified association between calcineurin and p38
MAPK inactivation was specific to cardiac myocytes, we performed
transient transfection experiments in another cell type, COS-7. An
expression vector encoding the upstream activator of p38 MAPK, MKK6,
was transfected into COS-7 cells, resulting in significant p38 MAPK
phosphorylation assessed by Western blotting (Fig.
5A). However, co-transfection
of an activated calcineurin expression vector along with the MKK6
encoding vector significantly reduced p38 MAPK phosphorylation (Fig.
5A).

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Fig. 5.
Calcineurin is associated with p38 MAPK
inactivation and increased MKP-1 expression in COS-7 cells.
A, Western blot from COS-7 cells transfected with an MKK6
expression vector alone or in combination with an activated calcineurin
expression vector. B, Western blot of MKP-1 protein levels
from COS-7 cells transfected with the indicated expression vectors.
Similar results were obtained in two independent experiments.
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While expression of activated calcineurin in COS-7 cells promoted p38
MAPK inactivation, the potential association with MKP-1 expression in
this cell type was uncertain. Accordingly, calcineurin transfection by
itself did not result in a significant increase in MKP-1 protein in
COS-7 cells (Fig. 5B). However, MKP-1 protein levels
increased when calcineurin was co-transfected with MAPK kinase
expression vectors (MEK1, MKK6, or MKK7), suggesting that calcineurin
requires MAPK co-stimulation to promote efficient MKP-1 expression
(Fig. 5B). Such results are consistent with the observations
in cardiomyocytes whereby calcineurin promoted the greatest increase in
MKP-1 protein levels when co-stimulated by PE (Fig. 2B).
Taken together, these data suggest that efficient MKP-1 induction
requires coordinate input from calcineurin and MAPK signaling pathways.
Calcineurin Augments MKP-1 Promoter Activity--
The mouse MKP-1
genomic organization was previously characterized, including the
upstream regulatory region and transcription start site (18). A
633
base pair fragment of the MKP-1 promoter was cloned and fused to a
luciferase reporter (Fig. 6A).
The MKP-1 promoter contains a TATA box, Sp1 element, CAAT box, serum
response factor binding site, and multiple AP1 and cAMP-response
element-binding protein stress-responsive elements (29). However,
consensus or near consensus NFAT binding elements were not identified
within the minimal MKP-1 promoter (see "Discussion"). The
633
base pair MKP-1 promoter was transiently co-transfected with an
expression vector encoding activated calcineurin or wild-type and
activated NFAT expression vectors. We determined that calcineurin
augmented promoter activity 4-5-fold in multiple experiments,
suggesting that calcineurin directly regulates MKP-1 promoter activity
(Fig. 6B). Calcineurin-mediated induction of the MKP-1
promoter was blocked by co-transfection with an expression vector
encoding a 194-amino acid calcineurin-inhibitory domain from the
protein chain (13).

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Fig. 6.
Calcineurin regulates the MKP-1
promoter. A, the MKP-1 promoter contains multiple
consensus sites including elements associated with inducible
expression, (AP1/cAMP-response element-binding protein, and serum
response factor binding sites). B, co-transfection of the
MKP-1 promoter plasmid in 10T1/2 fibroblasts demonstrates increased
luciferase activity mediated by calcineurin but not by cotransfection
with NFAT expression vectors. C, the MKP-1 reporter
construct demonstrated induction by MEK1, MKK6, and MKK7, which was
further increased by calcineurin. Results represent three independent
experiments, each performed in duplicate. Error
bars represent S.E.
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NFAT transcription factors are direct downstream mediators of
calcineurin-induced alterations in gene expression in multiple cell
types. However, not all calcineurin-induced transcriptional effects are
mediated by NFAT factors. Likewise, we observed that co-transfection of
the MKP-1 promoter construct with full-length NFATc4 or NFATc3
expression vectors or constitutively nuclear forms of these factors
(
NFATc4 and
NFATc3) did not significantly augment promoter
activity (Fig. 6B). We also failed to identify any
additional increase in promoter activity when calcineurin was
co-transfected in conjunction with NFATc4 or NFATc3 (data not shown),
which is consistent with the lack of an identifiable consensus NFAT
binding site within the
633 base pair MKP-1 promoter. Collectively,
these data indicate that calcineurin regulates the minimal MKP-1
promoter in an NFAT-independent manner.
Last, we also investigated the potential cooperation between
calcineurin and MAPK signaling in the activation of the MKP-1 promoter.
Expression vectors encoding activated forms of the MAPK kinase
signaling factors MEK1 (activates ERKs), MKK6 (activates p38), and MKK7
(activates JNK) each stimulated MKP-1 promoter activity in
co-transfection experiments, similar to calcineurin (Fig.
6C). Furthermore, co-transfection experiments between
calcineurin and individual MAPK kinase factors promoted an additive or
greater than additive increase in MKP-1 promoter activity (Fig.
6C). These data indicate that the calcineurin regulates
MKP-1 promoter activity in coordination with MAPK stress-responsive
signaling pathways. This interpretation is consistent with the
coordinated increase in MKP-1 protein level that was observed between
calcineurin and MAPK signaling responses in cardiomyocytes and COS-7 cells.
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DISCUSSION |
A wide array of intracellular signaling pathways have been
implicated in the regulation of cardiac myocyte hypertrophic growth. Intracellular signaling pathways such as calcineurin/NFAT and MAPK
represent central mediators of reactivity in cardiac myocytes as well
as other diverse cell types. For example, calcineurin and MAPK
signaling pathways are critical mediators of T-cell reactivity where
they regulate proliferation and cytokine production in response to
antigen. A challenge that remains is to elucidate the potential interconnectivity between calcineurin and MAPK signaling pathways in
cardiac myocytes as well as other cell types. Here we show that
calcineurin activation promotes a decrease in p38 MAPK activity in both
transgenic mice and cultured cardiomyocytes through a potential
mechanism involving the dual specificity phosphatase MKP-1.
Interconnection between Calcineurin and p38 MAPK in Cardiac
Hypertrophy--
Transgenic mice expressing an activated calcineurin
cDNA in their hearts were characterized by a significant reduction
in basal p38 MAPK phosphorylation at early developmental times.
However, this decrease in p38 MAPK activation gradually diminished with age so that by 24 days postnatal, altered activation was less obvious.
Such regulation might be explained by the following observations. First, p38 MAPK activation is itself dynamically regulated during postnatal development such that greater activation is observed during
early developmental periods when the heart is undergoing developmental
hypertrophy (30). In older hearts, basal p38 MAPK activation status is
probably reduced so that a further reduction in activity (as mediated
by calcineurin) would be less obvious. Second, as calcineurin
transgenic hearts progressively hypertrophy with age, secondary effects
associated with pathologic hypertrophy might influence the activity of
various intracellular signaling pathways. In this respect, the observed
reduction in p38 MAPK activity in very young calcineurin transgenic
hearts suggests a more direct interconnection between calcineurin and
p38 MAPK, without the potential secondary effects associated with
cardiac hypertrophy itself.
In vitro, we observed that prolonged stimulation of cardiac
myocytes with PE, which potently activates calcineurin (13), promoted a
gradual reduction of p38 phosphorylation (Fig. 3). This reduction in
p38 phosphorylation after just 1 h of PE treatment was largely
prevented with CsA, a calcineurin inhibitor (Fig. 3). These data
suggest that prolonged agonist stimulation can lead to p38
inactivation, in part, through a calcineurin-regulated pathway. This
interpretation is consistent with a recent report by Hines et
al. (11) in which prolonged electrical pacing of cardiac myocytes
in culture resulted in p38 inactivation despite a progressive
hypertrophy response. Interestingly, electrical pacing of cultured
cardiomyocytes was also recently shown to directly activate
calcineurin/NFAT signaling (31). Taken together, these data suggest
that calcineurin activation can promote p38 MAPK inactivation in
cultured cardiomyocytes.
Calcineurin Promotes p38 MAPK Inactivation in Association with
MKP-1 Expression--
Calcineurin is a serine/threonine phosphatase
that has not been reported to act on MAPK proteins. In contrast, a
specialized family of immediate early genes encoding dual specificity
phosphatases have been identified that act as specific negative
regulators of ERKs, JNKs, and p38. MKPs are largely regulated at the
transcriptional level such that stress stimuli or mitogen stimulation
induce gene expression (reviewed in Ref. 20). MKP-1 is most selective
for p38 MAPK at physiologic levels of expression, although high levels of expression are also associated with JNK and even mild ERK1/2 inactivation (24, 25, 32). Indeed, at physiologic expression levels
MKP-1 directly binds to p38 kinases but not ERKs or JNKs (24, 25).
These reports are consistent with our observation that an approximate
2-fold increase in MKP-1 protein in day 4 calcineurin transgenic hearts
is associated with p38 MAPK inactivation, but not ERKs or JNKs (Fig. 1,
and data not shown).
The inability of purified calcineurin to directly
dephosphorylate p38 MAPK in vitro suggested that calcineurin
might potentially act indirectly through MKP-1. Indeed,
calcineurin transgenic hearts at 4 days of age and AdCnA infection
of PE-stimulated cardiac myocytes demonstrated a dramatic increase in
MKP-1 protein levels but no change in MKP-2 (Fig. 2B). In
addition, inhibition of calcineurin with CsA led to a loss of MPK-1
induction and even promoted a decrease in basal MKP-1 protein levels in
cardiac myocytes (Fig. 3). These data are consistent with the recent
observation that MAPK transcriptional responses in a neuronal-like cell
line were inhibited with a calcium ionophore through a mechanism
involving enhanced MKP-1 expression (28).
To examine the ability of calcineurin to directly regulate
MKP-1 expression, we characterized the mouse minimal MKP-1 promoter. Co-transfection of an activated calcineurin expression vector with an
MKP-1 promoter-luciferase fusion reporter resulted in a 4-5-fold
increase in promoter activity compared with reporter alone, suggesting
that calcineurin might regulate MKP-1 at the transcriptional level.
This interpretation is further supported by the observation that
MKP-1 protein levels were not altered by 5 or 15 min of PE stimulation
in cardiac myocytes, but levels were augmented in a CsA-sensitive
manner by 30 and 60 min. Despite a potential direct role for
calcineurin in regulating MKP-1 expression, we also observed that
calcineurin acted in concert with MAPK signaling pathways to more
efficiently up-regulate MKP-1 expression, suggesting that calcineurin
and MAPK coordinate maximal MKP-1 induction.
To more carefully evaluate the transcriptional mechanism whereby
calcineurin might regulate MKP-1 promoter activity, we co-transfected full-length or constitutively nuclear expression vectors for NFATc4 and
NFATc3. However, NFAT factors did not significantly induce MKP-1
promoter activity; nor were good consensus sites present within the
633 base pair region that was analyzed. We did identify a site at +25
base pairs (5'-AGGAAAGCG-3') in the 5'-untranslated region that weakly
resembled a NFAT element, but this site failed to interact with
in vitro translated NFATc4 protein by gel shift assay, while
a control NFAT site from the IL-4 promoter demonstrated significant
binding (data not shown). While it is reasonable to conclude that the
MKP-1 minimal promoter is regulated in an NFAT-independent manner, it
is formally possible that one or more relevant NFAT sites lie upstream
of the area analyzed.
Implications for p38 MAPK and Cardiac Hypertrophy--
A large
number of reports have demonstrated that the MKK3/6-p38 MAPK signaling
branch is sufficient to initiate the hypertrophic program in cultured
cardiomyocytes. For example, transfection of cultured cardiomyocytes
with a MKK6-encoding expression vector or infection with a
MKK6-encoding adenovirus produced a profound hypertrophy response
in vitro (5-7). In addition, Zhang et al. (33)
generated transgenic mice expressing an activated form of TAK1 (MAPK
kinase kinase signaling factor), which resulted in p38 MAPK activation
and hypertrophic cardiomyopathy and neonatal lethality in
vivo.
While p38 MAPK signaling is undoubtedly sufficient to induce a
hypertrophic response, evidence supporting a necessary role for this
signaling factor in the hypertrophic response is less certain. Indeed,
electrical pacing of cultured cardiomyocytes leads to a down-regulation
of p38 MAPK activity, despite progressive hypertrophy (11), and
pharmacologic p38 MAPK inhibition was reported to be ineffective in
blocking endothelin-1-induced hypertrophy in vitro (10).
Despite these reports, others have observed a complete or partial
attenuation of agonist-induced cardiac myocyte hypertrophy with p38
MAPK inhibitors (4, 5, 7). Collectively, these various reports suggest
that p38 MAPK plays a role in the hypertrophic response, although its
function may be more specialized or associated with acute temporal effects.
Calcineurin transgenic mice are characterized by a dramatic hypertrophy
response, despite a down-regulation of p38 MAPK signaling. In
vitro, calcineurin adenovirus efficiently induces a hypertrophy response characterized by increased cell size, increased sarcomeric organization, and increased atrial natriuretic factor expression despite a down-regulation in p38 signaling (see Ref. 34 and Fig.
2B). These results indicate that p38 MAPK activation is not required for the initiation or progression of a calcineurin-induced hypertrophy response in vitro or in vivo.
However, it is likely that p38 MAPK plays a role in other forms of
reactive hypertrophy or is involved in aspects of myocyte survival
(apoptosis). This possibility is supported by the observation that p38
MAPK activation can promote apoptosis in cultured cardiomyocytes
(6), while calcineurin signaling was shown to be cardioprotective (34). These relationships are of particular interest, since calcineurin (antiapoptotic) promotes p38 MAPK inactivation. Accordingly, sustained p38 MAPK activation in the heart might be predicted to promote decompensation and cardiomyopathy, consistent with the phenotype of
TAK1 transgenic mice (33). In this respect, p38 MAPK activation might
represent a deleterious signaling pathway in the heart and is a
desirable target for pharmacologic inhibition.