Ras Regulates NFAT3 Activity in Cardiac Myocytes*
Masaru
Ichida and
Toren
Finkel
From the Laboratory of Molecular Biology, NHLBI, National
Institutes of Health, Bethesda, Maryland 20892
Received for publication, May 18, 2000, and in revised form, October 2, 2000
 |
ABSTRACT |
Multiple distinct signal transduction
pathways have been implicated in the development of cardiac myocyte
hypertrophy. These hypertrophic pathways include those regulated by the
Ras superfamily of small GTPases and a separate calcineurin-regulated
pathway that culminates in the activation of the transcription factor NFAT3. In this report, we demonstrate a functional interaction between
Ras-regulated and calcineurin-regulated pathways. In particular, expression in neonatal myocytes of a constitutively active form of Ras
(V12ras), but not activating mutants of Rac1, RhoA, or Cdc42, results
in an increase in NFAT activity. Similarly, expression of an activated
Ras, but not other small GTPases, results in the nuclear translocation
of an NFAT3 fusion protein. Expression of a dominant negative
ras gene product blocks phenylephrine-stimulated NFAT transcriptional activity and the ligand-stimulated NFAT3 nuclear
localization. Ras proteins appear to function upstream of
calcineurin, because cyclosporin A blocks the ability of V12ras to
stimulate NFAT-dependent transcription and nuclear
localization. Similarly, expression of a dominant negative
ras gene inhibits phenylephrine-stimulated calcineurin
activity. Pharmacological inhibition of MEK1 or expression of a
dominant negative form of c-Raf or ERK2 inhibits
phenylephrine-stimulated NFAT3 activation. Conversely, NFAT activity
was stimulated by expression of constitutively active forms of c-Raf or
MEK1. Taken together, these results imply that, in cardiac myocytes, a
Ras-regulated pathway involving stimulation of mitogen-activated
protein kinase regulates NFAT3 activity.
 |
INTRODUCTION |
Due to their inability to divide, terminally differentiated
cardiac myocytes respond to a diverse range of stimuli, including hypertension, myocardial infarction, or inborn mutations in sarcomeric gene products by undergoing hypertrophy. Initially this increase in
myocyte size leads to an increase in contractile power allowing the
heart to meet the increased demands induced by these precipitating genetic or acquired conditions. However, with time this initially beneficial response turns maladaptive. Indeed, the presence of cardiac
hypertrophy carries with it an approximate 2-fold increase in overall
cardiovascular mortality (1).
Laboratory analysis of cardiac hypertrophy has often involved the use
of primary cultures of neonatal rat cardiac myocytes. Studies over the
last decade have demonstrated that these cells undergo an increase in
size following ligand stimulation with agents such as endothelin-1,
angiotensin II, or phenylephrine (2, 3). In cultured ventricular
myocytes, treatment with hypertrophic stimuli also increases expression
of a host of immediate early genes, alters sarcomeric protein
expression, and reactivates expression of atrial natriuretic factor
(ANF)1 and the related B-type
natriuretic peptide (BNP).
Analysis of the signaling pathways activated in these in
vitro models has yielded considerable insight into the process of hypertrophy. Early studies demonstrated a role for Ras proteins in the
hypertrophic process. In particular, microinjection of constitutively
active forms of Ras recapitulated the hypertrophic program with an
increase in cell size and the enhanced expression of hypertrophic
markers such as ANF (4). In addition, stimulation with hypertrophic
agents such as phenylephrine leads to an increase in Ras-GTP levels
(5). These in vitro studies have been complemented with
in vivo experiments that demonstrated increased left
ventricular mass and increased ANF expression in cardiac-targeted
transgenics expressing a constitutively active ras gene
product (6).
Recently, other members of the Ras superfamily of small GTPases have
been implicated in hypertrophic signaling. Studies with activating and
dominant negative mutants of RhoA have demonstrated a role for this
GTPase in certain aspects of ligand-stimulated myofibrillogenesis and
hypertrophic gene expression (7-11). Similarly, expression of
activated forms of the related small GTPase Rac1 also leads to a
hypertrophic response, whereas expression of dominant negative Rac1
blocks phenylephrine-stimulated hypertrophy (11, 12). Recently, both
activated forms of RhoA and Rac1 have been expressed in a
cardiac-targeted fashion in transgenic mice. Mice that overexpress
constitutively active RhoA appear to have an increase in atrial but not
ventricular mass and significant atrial-ventricular conduction
abnormalities (13). These conduction abnormalities may relate more to
the ability of RhoA to regulate ion channel activity then to any direct
effect of the GTPase on cardiac myocyte growth (14). Cardiac expression
of constitutively active forms of Rac1 results in a pattern of
ventricular dilatation in some animals whereas left ventricular
hypertrophy is evident in other animals (15).
Recently, intense interest has focused on another pathway that appears
to contribute to the hypertrophic response. Analysis of the BNP
promoter demonstrated a crucial role for NFAT3 in the transcriptional
activation of this promoter following hypertrophic stimulation (16).
NFAT3 is one of four known NFAT family members. Expression of NFAT3
occurs in the heart and in a number of other tissues, whereas
expression of the other NFAT family members appears to be restricted to
immune cells or skeletal muscle (17). Elegant studies in immune cells
have demonstrated that T cell activation results in an increase in
intracellular calcium leading to the subsequent activation of the
cytoplasmic phosphatase calcineurin. Once activated, calcineurin
dephosphorylates NFAT resulting in nuclear translocation of NFAT. Once
in the nucleus, NFAT binds DNA in a sequence-specific fashion to
activate expression of a host of gene products involved in T cell
activation. The best studied example of NFAT-mediated gene expression
involves interleukin-2 (IL-2) production following T cell activation.
The importance of the pathway described above is evident by the
discovery that the immunosuppressant cyclosporin A functions by
inhibiting calcineurin and, hence, subsequent NFAT activation and T
cell responsiveness. The relevance of this pathway to cardiac hypertrophy is underscored by the observation that cardiac-targeted transgenic animals expressing constitutively activated forms of either
calcineurin or NFAT3 produced ventricular hypertrophy (16). Similarly,
pharmacological inhibition of calcineurin by the administration of
cyclosporin A inhibited some (16, 18, 19) but not necessarily all
(19-21) forms of genetic or acquired ventricular hypertrophy.
Given the expanding list of pathways implicated in the hypertrophic
signaling cascade, it seems possible that some degree of crosstalk or
interactions might exist between these seemingly diverse members. In
this report, we have specifically attempted to understand whether there
is any potential interaction between hypertrophic pathways regulated by
the various small GTPases and the more recently described
NFAT-dependent pathway.
 |
EXPERIMENTAL PROCEDURES |
Myocyte Preparation--
Neonatal ventricular myocytes were
prepared from hearts of 2-day-old Harlan Sprague-Dawley rats. Excised
hearts were treated overnight with cold trypsin digestion in calcium
and magnesium free Hepes-buffered salt solution followed by collagenase
digestion in Leibovitz's L-15 media (Worthington Biochemical, NJ).
Myocytes were subsequently isolated by 40 trituration strokes, and
debris was removed by passage through a 70-µm filter. The
cells were pelleted by low speed centrifugation and subject to two
30-min rounds of preplating to allow for the purification of myocytes from contaminating fibroblasts. Previous immunohistochemical staining has verified that this procedure results in a population of cells that
is over 90% myocytes (12). Cells were plated on laminin-coated 6-well
dishes at a density of 3 × 105 cells/cm2
in plating media that consisted of Dulbecco's modified Eagle's medium/Ham's F-12 (1:1, v/v) with 15 mM Hepes, pH 7.5, 2 mg/ml bovine serum albumin, 10 mg/liter insulin, 5.5 mg/liter
transferrin, 5 µg/liter selenium, 2 mg/liter ethanolamine, 10,000 units/ml penicillin, and 10,000 µg/ml streptomycin supplemented with
5% horse serum (Life Technologies, MD). Cells were transfected 24 h after plating. Eighteen hours after transfection, the medium was
changed to serum free plating medium, and phenylephrine (Sigma Chemical
Co., St. Louis, MO), cyclosporin A (Sigma), or PD98059 (Calbiochem, La
Jolla, CA) was added for the remaining 48 h prior to harvest.
Plasmids and Transfection--
Plasmids encoding activated forms
of Ras (V12ras), Rac1 (V12rac), RhoA (V14rhoA), Cdc42H (V12cdc42H),
c-Raf (Raf BXB), or MEK1 (MEK-E) or plasmids encoding dominant negative
forms of Ras (N17ras), Rac1 (N17rac), RhoA (N19RhoA), c-Raf (Raf301),
and ERK2 (ERK185) have been described previously (22-24). All plasmids
were generous gifts of S. Gutkind (National Institutes of Health). The
Ras effector mutant (V12/C40), which exclusively activates phosphatidylinositol 3-kinase (PI3K) but not c-Raf or other Ras effector molecules, has also been described previously (25). For
analysis of NFAT activity, a luciferase reporter containing three
tandem repeats of a 30-bp fragment of the IL-2 promoter was routinely
used. To confirm these results, an authentic NFAT3 reporter was
constructed containing two tandem copies of the sequence 5'-CGCGTCTATCCTTTTGTTTTCCATCCTG-3' derived from the BNP promoter and
previously demonstrated to bind NFAT3 (16). A fusion protein encoding
green fluorescence protein (GFP) and NFAT3 (a gift of J. Molkentin) was
constructed by ligation of full-length NFAT3 into pEGFP-C1
(CLONTECH). The construct was confirmed by
nucleotide sequencing.
For luciferase assays, except where indicated, myocytes were
transfected using 4 µg of the indicated small GTPase or corresponding empty vector and 1 µg of the indicated NFAT reporter construct and
0.1 µg of the pPRL-TK renilla transfection control plasmid. Cells
were harvested 66 h after transfection, and the ratio of luciferase to renilla activity was measured using the dual luciferase assay in accordance with the manufacturer's recommendation (Promega, WI). Where indicated, cells were treated with either phenylephrine, cyclosporin A, wortmannin, or the MEK1 inhibitor PD98059 (20 µM). All experiments were performed at least three times,
and one representative experiment performed in triplicate is routinely
shown. Statistical significance (*) is determined by a paired
t test with a value of p < 0.05 considered significant.
NFAT3 Localization--
Following transfection with GFP-NFAT3
fusion protein construct (2.5 µg) and a construct encoding the
indicated small GTPase, dominant negative c-Raf, ERK2, or empty vector
control (2.5 µg), cells were visualized by a Nikon TE300 fluorescence
microscope. For quantification purposes, cells were considered positive
if the nuclear fluorescence was the sole or predominant localization of
GFP-NFAT3. Values represent the mean percentage ± S.D. of cells demonstrating nuclear predominant fluorescence obtained from three wells of a single representative experiment (~200 cells).
Kinase and Phosphatase Assays--
For analysis of c-Jun
N-terminal (JNK) kinase activity, myocytes were transiently transfected
with mammalian expression vectors encoding an epitope-tagged form of
JNK (HA-JNK) with or without the indicated small GTPase. Twenty-four
hours after transfection, cells were washed three times and maintained
in serum-free medium for an additional 12 h. Unstimulated cells or
cells stimulated for 30 min with phenylephrine (50 µM)
were subsequently harvested in lysis buffer as described previously
(26). Transfected HA-JNK was immunoprecipitated from 30 µg of protein
lysate using an anti-HA mouse monoclonal antibody (Santa Cruz
Biotechnology, Santa Cruz, CA). The activity of the immune complex was
assayed using 2 µg of GST-ATF-2 as a substrate (26). The reactions
were terminated with SDS-polyacrylamide gel electrophoresis sample
buffer, and samples were then subjected to electrophoresis on 12%
polyacrylamide gels followed by autoradiography.
For assessment of calcineurin activity, cells were infected with a
recombinant adenovirus encoding N17ras (Ad.N17ras) or a control
adenovirus lacking a transgene (Ad.dl312). Both viruses have been
described previously (27), and the infection was performed at a
multiplicity of infection of 25. Twelve hours after infection, the
viral particles were removed and the media was replaced with serum free
media with or without phenylephrine. Forty eight hours later,
cardiomyocytes were harvested in lysis buffer (50 mM
Tris-HCl (pH 7.5), 0.5% Nonidet P-40, 1 mM EDTA, 1 mM CaCl2) supplemented with protease inhibitor
mixture (Roche Molecular Biochemicals), and clarified by
centrifugation. The Quantizyme assay system AK-804 was performed by
using 1 µg of protein lysate according to the manufacturer's
procedure (BIOMOL, Plymouth Meeting, PA). Calcineurin phosphatase
activity was measured spectrophotometrically by detecting free-phosphate released from the calcineurin-specific RII
phosphopeptide as described previously (28). Results are from one of
three similar experiments each performed in triplicate.
 |
RESULTS |
To understand the relationship between the Ras family of GTPases
and the regulation of NFAT3, we transfected cultures of neonatal rat
myocytes with plasmids encoding constitutively active forms of Ras,
Rac1, RhoA, and Cdc42 along with an NFAT-dependent reporter plasmid. As demonstrated in Fig.
1A, expression of an activated ras gene (V12ras) but not similar forms of Rac1, Cdc42, or
RhoA resulted in an increase in NFAT activity. In six separate
experiments, each performed in triplicate, expression of V12ras
resulted in anywhere from a 2- to 6-fold increase in NFAT activity.
This degree of increased NFAT activation seen with V12ras was
comparable to what we observed with phenylephrine stimulation (see Fig.
3). Because separately, activated forms of Rac1 and RhoA were
incapable of activating NFAT, we next tested whether these small
GTPases could synergize with V12ras. As seen in Fig. 1B,
addition of activated forms of RhoA or Rac1 to V12ras tended to inhibit
activation rather than potentiate V12ras-stimulated reporter gene
activity. The basis for this inhibition is unknown but may reflect
competition for shared downstream molecules.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 1.
V12ras expression activates NFAT
activity. A, cardiac myocytes were transfected with
plasmids encoding constitutively active forms of Ras, Rac1, RhoA, or
Cdc42 along with a NFAT-dependent reporter gene derived
from the IL-2 promoter. All activity was normalized to an internal
renilla transfection standard and is expressed as -fold change in NFAT
activity compared with an empty vector control ( ). B,
addition of activated forms of Rac1 or RhoA does not potentiate the
effects seen with V12ras alone. Amount of total transfected empty
vector or small GTPase was maintained at 6 µg. C, effects
of V12ras or V12rac on NFAT activity using a reporter plasmid
containing two tandem repeats of the NFAT3-binding sites derived from
the BNP promoter.
|
|
The NFAT reporter used in Fig. 1 (A and B)
represents a sequence derived initially from the promoter of IL-2, a
known NFAT-responsive gene in T cells. The predominant form of NFAT in
cardiac myocytes is NFAT3. Recent evidence suggests that BNP expression
in myocytes is regulated by NFAT3, and an authentic consensus binding
site has been found in the BNP promoter (16). To confirm the effects of
V12ras using an authentic NFAT3 binding site, we constructed a reporter
plasmid containing two tandem NFAT3 sites, derived from the BNP
promoter, upstream of luciferase. As demonstrated in Fig.
1C, expression of V12ras stimulated NFAT activity using this
reporter. Again activation of NFAT activity was specific for Ras and
not shared by other small GTPases such as V12rac. Using this reporter,
the level of activation observed with V12ras expression, as well as
with phenylephrine stimulation (data not shown), tended to be slightly
less robust then with the standard NFAT reporter. These differences may
relate to the spacing of the tandem NFAT3 sites or due to the fact that
NFAT proteins predominantly bind in a cooperative fashion with other
transcription factors (17).
Activation of NFAT involves the dephosphorylation of the transcription
factor by calcineurin, resulting in its translocation from the cytosol
to the nucleus. To confirm the effects of V12ras on NFAT activity in
neonatal myocytes, we constructed a GFP-NFAT3 fusion construct.
Transfection of this construct into myocytes revealed that, under basal
conditions, the fusion protein was either distributed in the cytosol
with nuclear exclusion of GFP or a combination of both cytosol and
nucleus (Fig. 2, A and
B). A nuclear-predominant fluorescence was routinely
observed in less than 5% of control cells. As demonstrated in Fig.
2C, expression of V12ras resulted in a shift in GFP-NFAT3
localization with a significant number of cells now displaying nuclear
predominant fluorescence. Quantification of these observations
confirmed that activated Ras expression, but not activated forms of
other small GTPases, resulted in a 5- to 10-fold increase in the number
of cells expressing a nuclear predominant form of the GFP-NFAT3 fusion protein (Fig. 2D).

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 2.
Activated Ras proteins induce nuclear
localization of NFAT3. A, transfection of a GFP-NFAT3
fusion protein results in a cytosolic or, B, combined
nuclear and cytosolic distribution of GFP-NFAT3 in control transfected
cells. C, the distribution of GFP-NFAT3 becomes
predominantly nuclear in a significant percentage of cells following
V12ras expression. D, quantification of the nuclear
predominant fluorescence observed under control conditions or following
expression of various activated small GTPases.
|
|
We next sought to understand the role of Ras proteins in physiological
activation of NFAT in cardiac myocytes. To begin to address this, we
transfected myocytes with dominant negative forms of either Ras, Rac1,
or RhoA proteins and stimulated cells with phenylephrine. As previously
noted, phenylephrine led to an ~3-fold increase in NFAT activity
(Fig. 3). Expression of N17rac had no effect on phenylephrine-stimulated NFAT activity. The functional effects of N17rac expression in cardiac myocytes was confirmed by
analyzing phenylephrine-stimulated c-jun N-terminal kinase (JNK)
activity. As seen in Fig. 3B, N17rac expression inhibited the activation of JNK by phenylephrine under conditions in which it had
no effect on phenylephrine-stimulated NFAT activity. Similarly, in
a separate series of three experiments performed in triplicate, expression of N19RhoA did not effect phenylephrine-stimulated NFAT
activity (control = 2.2 ± 0.8-fold and N19RhoA = 2.8 ± 1.2-fold, p = 0.57). In contrast,
expression of N17ras reduced phenylephrine-stimulated NFAT reporter
activity (Fig. 3A). Similarly, treatment of cells with
phenylephrine led to an increased nuclear localization of the GFP-NFAT3
fusion protein (Fig. 4A). The
ability of phenylephrine to stimulate this nuclear translocation was
significantly inhibited by N17ras expression (Fig.
4B).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 3.
Phenylephrine-stimulated NFAT activity is
inhibited by N17ras expression. Myocytes were transfected with
dominant negative forms of Ras or Rac1 and stimulated with
phenylephrine (10 µM). Levels of NFAT activity were
determined under basal (open bars) and ligand-stimulated
(shaded bars) conditions using the dual luciferase assay.
B, effects of N17rac on JNK activity. Myocytes were
transfected with an epitope-tagged JNK construct along with either a
plasmid encoding N17rac or empty vector DNA. Kinase activity was
measured in an immune complex assay under basal and phenylephrine
(PE)-stimulated conditions.
|
|

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 4.
Dominant negative Ras inhibits the
translocation of NFAT3. A, nuclear fluorescent
localization of GFP-NFAT3 following phenylephrine stimulation.
B, quantification of nuclear predominant fluorescence under
basal conditions or following phenylephrine stimulation in control
(open bars) or N17ras (shaded bars) expressing
cells.
|
|
We next sought to analyze at what level Ras intersects with the NFAT3
pathway. To understand whether this occurs upstream or downstream of
calcineurin, we transfected myocytes with V12ras and subsequently
treated cells with the calcineurin inhibitor cyclosporin A. As
demonstrated in Fig. 5A, the
addition of cyclosporin A inhibited in a
concentration-dependent fashion the ability of V12ras to
stimulate NFAT activity. Similarly, treatment of cells with cyclosporin
A blocked V12ras-stimulated NFAT3 nuclear localization (Fig.
5B). These results argue that the effects of Ras are most likely upstream of calcineurin activation. To further pursue this notion, we infected cells with a recombinant adenovirus encoding N17ras
or with a control adenovirus lacking a transgene. As demonstrated in
Fig. 6, in control infected cells
stimulation of myocytes with phenylephrine led to an increase in
calcineurin activity consistent with previous results (28). In cells
expressing N17ras, although basal calcineurin activity was not
significantly effected, the rise in calcineurin activity seen following
ligand stimulation was inhibited.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 5.
Inhibition of V12ras stimulation of NFAT
activity by cyclosporin A. A, myocytes were transfected
with an activated Ras construct and subsequently treated with the
indicated amounts of cyclosporin A (CSA) for 48 h prior
to harvest. Levels of normalized luciferase activity is shown.
B, cyclosporin A treatment blocks V12ras-induced nuclear
localization of GFP-NFAT3.
|
|

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 6.
Expression of N17ras blocks
phenylephrine-stimulated calcineurin activity. Myocytes were
infected with a control adenovirus ( ) or with a recombinant
adenovirus encoding N17ras (Ad.N17ras). Where indicated,
cells were stimulated with phenylephrine (PE; 10 µM) for 48 h prior to lysis. Calcineurin activity in
the lysate was measured by phosphate release using a calcineurin
specific phosphopeptide as substrate.
|
|
Ras proteins activate a number of downstream effector pathways. Perhaps
the best characterized pathway involves the activation of MAPK
that proceeds through c-Raf and MEK1 activation (29). To test whether
this pathway was involved in NFAT activation in cardiac myocytes, we
treated cells with the specific MEK1 inhibitor PD98059. As seen in Fig.
7A, treatment of cells with
this compound inhibited NFAT transcriptional activity induced by either
V12ras expression or by phenylephrine stimulation. Similarly, PD98059 treatment inhibited nuclear translocation of GFP-NFAT3 induced by
either V12ras expression or phenylephrine stimulation (Fig. 7B).

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 7.
Pharmacological inhibition of MEK1 activation
blocks NFAT activity. A, NFAT-dependent
luciferase activity following V12ras expression or phenylephrine (10 µM) stimulation in the presence or absence of 20 µM PD98059 treatment. B, quantification of
nuclear localization of a GFP-NFAT3 fusion protein following V12ras
expression or phenylephrine stimulation in the presence or absence of
20 µM PD98059.
|
|
Consistent with the results obtained by pharmacological inhibition, as
demonstrated in Fig. 8, expression of a
dominant negative form of c-Raf or ERK2 prevented
phenylephrine-stimulated NFAT3 nuclear localization. To further address
whether the activation of the MAPK pathway was sufficient to activate
NFAT, we expressed constitutively activated forms of c-Raf or MEK1. As
seen in Fig. 9, these constructs
activated NFAT activity to a similar degree as V12ras. In addition, the
ability of both the activated c-Raf and MEK1 constructs to stimulate
NFAT activity was inhibited by cyclosporin treatment.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 8.
Expression of a dominant negative c-Raf or
ERK2 inhibits NFAT3 nuclear localization. The percentage of cells
demonstrating nuclear predominant staining was assessed following
phenylephrine stimulation in the presence or absence of a vector
control or a dominant negative form of c-Raf or ERK2.
|
|

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 9.
Expression of an activated form of c-Raf
(Raf-BXB) or MEK1 (MEK-E) activates
NFAT in a calcineurin-dependent fashion. The level of
NFAT transcriptional activity was determined in myocytes transfected
either with empty vector or with activated forms of c-Raf or MEK1 and
then treated with or without cyclosporin (CSA;10
ng/ml).
|
|
Although these results suggest a significant role for a pathway
involving Ras-Raf-MEK1 and MAPK, it does not exclude a role for other
Ras-regulated pathways. One downstream effector of Ras implicated in
cardiac hypertrophy is PI3K (30). To ascertain whether this
Ras-regulated pathway participated in V12ras-stimulated NFAT
activation, we asked whether the ability of V12ras to stimulate NFAT
activity could be inhibited by the PI3K inhibitor wortmannin. As
demonstrated in Fig. 10A,
treatment of V12ras-expressing cells with wortmannin produced a modest
but concentration-dependent inhibition of NFAT activity.
Similarly, expression of the Ras effector mutant V12/C40 capable of
specifically activating PI3K but not other Ras targets such as c-Raf
produced a small increase in NFAT activity (Fig. 10B). These
results suggest that, although activation of
Raf-MEK1-MAPK-dependent pathways is necessary and sufficient for NFAT activation in cardiac myocytes, other Ras effector
pathways may also participate.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 10.
A role for PI3K in Ras activation of
NFAT. A, effects of increasing concentrations
(nanomolar) of the PI3K inhibitor wortmannin on V12ras-stimulated NFAT
activity. B, comparison of the NFAT stimulatory activity of
V12ras with the effector mutant V12/C40 Ras that activates only
PI3K-dependent pathways.
|
|
 |
DISCUSSION |
Our results demonstrate an essential role for Ras proteins in the
regulation of NFAT3 activity in cardiac myocytes. In particular, we
demonstrate that activated Ras stimulates NFAT activity and nuclear
translocation and a dominant negative form of Ras blocks phenylephrine-stimulated NFAT activation. The ability of V12ras or
phenylephrine to stimulate NFAT activity is significantly inhibited by
treatment with the pharmacological MEK1 inhibitor PD98059, suggesting
that activation of MAPK is necessary for NFAT activation. This is also
supported by the inhibition of phenylephrine-stimulated NFAT3
translocation seen with expression of a dominant negative form of c-Raf
and ERK2. Our results also place the Ras/Raf/MEK1/MAPK pathway upstream
of calcineurin, because cyclosporin blocks the ability of
constitutively active mutants in this pathway to activate NFAT. In
addition, expression of N17ras inhibits the rise in calcineurin activity seen following phenylephrine stimulation.
Previous studies in immune cells has also implicated Ras proteins in
the activation of NFAT. Expression of V12ras can mimic in part T cell
receptor activation, although expression of activated Ras is not by
itself sufficient to activate NFAT (31). Coexpression of an activated
form of calcineurin or treatment with calcium ionophores appears
however to synergize with a Ras signal to produce full NFAT activation
(31-34). In both T cells and other immune cells a role for Rac
proteins has also been demonstrated with experiments demonstrating that
a dominant negative form of Rac can inhibit NFAT activation (35, 36).
The activation of NFAT3 in cardiac myocytes therefore appears to differ
in significant fashion from immune cells. In particular, our studies
suggest that V12ras is sufficient alone to activate NFAT3. In addition, we observed no effects of N17rac or other small GTPases on
phenylephrine-stimulated NFAT activity. The level of activation seen
with V12ras was comparable to what was observed with phenylephrine
stimulation, and thus there is no evidence that an additional
calcium-dependent stimulus was needed. These latter
differences may relate to the large differences in calcium handling
between immune cell and contractile cardiac myocytes.
The elucidation of multiple pathways that contribute to cardiac
hypertrophy represent an important advance in our understanding of this
condition. The development of therapeutic options to treat hypertrophy
will undoubtedly be dependent on a deeper understanding of the
inter-relations and hierarchy of the various pathways. The results
presented here place Ras and subsequent MAPK activation upstream of
calcineurin and NFAT activation in cardiac myocytes. This underscores a
central role for Ras in the development of hypertrophy. Our preliminary
evidence suggests that an MAPK-dependent pathway represents
the major pathway through which Ras proteins activate NFAT.
Nonetheless, other Ras effectors may also contribute as we do observe a
small but noticeable contribution from PI3K-dependent pathways in the activation of NFAT (Fig. 10).
Although this study increases our understanding of the molecular
mechanisms underlying cardiac hypertrophy, many questions remain. For
instance, it is unclear to what degree the activation of NFAT by Ras
proteins contributes to the ability of Ras proteins to induce
hypertrophy in vivo (6). Similarly, the mechanism through
which MAPK activation leads to NFAT activation is unknown. Recent
evidence suggests that a Raf/MEK1/MAPK-dependent pathway is
involved in the regulation of intracellular calcium transients in
cardiac myocytes (37). As such, these results are consistent with what
is described here and suggest MAPK is upstream of calcineurin and may
regulate the phosphatase by altering intracellular calcium levels.
The ability of Rac proteins to produce hypertrophy in vitro
and in vivo (11, 12, 15) but their apparent inability, as demonstrated here, to regulate NFAT activity suggests that not all
described pathways leading to hypertrophy overlap. The activation of
c-jun N-terminal kinase (JNK) and the p38 MAPK family has recently been
implicated in cardiac hypertrophy (38-45). In myocytes and other cell
types, these kinases are regulated by Rac and related small GTPases
(46). The activity of Rac, RhoA, and Cdc42 is also regulated in part by
Ras proteins suggesting that Ras might be an important regulator of a
number of critical and distinct pathways leading to hypertrophy.
Similarly, given the existence of these separate pathways, it would
seem reasonable that inhibition of calcineurin activity may not abolish
hypertrophy induced by all stimuli. This is apparently supported by
data suggesting that cyclosporin A is ineffective in preventing
pressure-overload-induced hypertrophy in rats (20, 21) and only
partially effective in reversing the hypertrophy induced by
G
q overexpression (19).
In summary, we demonstrate the requirement for Ras activity and
subsequent MAPK activation in the regulation of NFAT3 in cardiac myocytes. These results are distinct from what is observed in the
immune activation of NFAT, but in both cells, there appears to be at
least a partial requirement for Ras proteins. We cannot demonstrate a
role for other small GTPases in NFAT activation in myocytes, although
previous evidence suggests that these proteins can participate in
hypertrophic signaling. These results further our understanding of the
hypertrophic process and suggest a critical and expanded role of Ras
proteins in this response.
 |
ACKNOWLEDGEMENTS |
We thank S. Gutkind, J. Molkentin, J. Downward, and J. Bruder for reagents and I. Rovira for help with
preparation of the graphics.
 |
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) .
To whom correspondence should be addressed: Laboratory of
Molecular Biology, NHLBI, National Institute of Health, Bldg.
10/6N-240, 10 Center Dr., Bethesda, MD 20892-1622. Tel.: 301-402-4081;
Fax: 301-402-9311; E-mail: finkelt@nih.gov.
Published, JBC Papers in Press, October 23, 2000, DOI 10.1074/jbc.M004275200
 |
ABBREVIATIONS |
The abbreviations used are:
ANF, atrial
natriuretic factor;
BNP, B-type natriuretic peptide;
IL-2, interleukin-2;
JNK, c-Jun N-terminal kinase;
HA-JNK, epitope-tagged form of JNK;
MAPK, mitogen-activated protein kinase ERK,
extracellular signal-regulated kinase;
MEK, MAPK/ERK kinase;
PI3K, phosphatidylinositol 3-kinase;
bp, base pair(s);
GFP, green
fluorescence protein.
 |
REFERENCES |
1.
|
Levy, D.,
Garrison, R. J.,
Savage, D. D.,
Kannel, W. B.,
and Castelli, W. P.
(1990)
N. Engl. J. Med.
322,
1561-1566[Abstract]
|
2.
|
Sugden, P. H.,
and Clerk, A.
(1998)
J. Mol. Med.
76,
725-746[CrossRef][Medline]
[Order article via Infotrieve]
|
3.
|
Hunter, J. J.,
and Chien, K. R.
(1999)
N. Engl. J. Med.
341,
1276-1283[Free Full Text]
|
4.
|
Thorburn, A.,
Thorburn, J.,
Chen, S. Y.,
Powers, S.,
Shubeita, H. E.,
Feramisco, J. R.,
and Chien, K. R.
(1993)
J. Biol. Chem.
268,
2244-2249[Abstract/Free Full Text]
|
5.
|
Chiloeches, A.,
Paterson, H. F.,
Marais, R.,
Clerk, A.,
Marshall, C. J.,
and Sugden, P. H.
(1999)
J. Biol. Chem.
274,
19762-19770[Abstract/Free Full Text]
|
6.
|
Hunter, J. J.,
Tanaka, N.,
Rockman, H. A.,
Ross, J., Jr.,
and Chien, K. R.
(1995)
J. Biol. Chem.
270,
23173-23178[Abstract/Free Full Text]
|
7.
|
Sah, V. P.,
Hoshijima, M.,
Chien, K. R.,
and Brown, J. H.
(1996)
J. Biol. Chem.
271,
31185-31190[Abstract/Free Full Text]
|
8.
|
Thorburn, J.,
Xu, S.,
and Thorburn, A.
(1997)
EMBO J.
16,
1888-1900[Abstract/Free Full Text]
|
9.
|
Hoshijima, M.,
Sah, V. P.,
Wang, Y.,
Chien, K. R.,
and Brown, J. H.
(1998)
J. Biol. Chem.
273,
7725-7730[Abstract/Free Full Text]
|
10.
|
Hines, W. A.,
and Thornburn, A.
(1998)
J. Mol. Cell Cardiol.
30,
485-494[CrossRef][Medline]
[Order article via Infotrieve]
|
11.
|
Aikawa, R.,
Komuro, I.,
Yamazaki, T.,
Zou, Y.,
Kudoh, S.,
Zhu, W.,
Kadowaki, T.,
and Yazaki, Y.
(1999)
Circ. Res.
84,
458-466[Abstract/Free Full Text]
|
12.
|
Pracyk, J. B.,
Tanaka, K.,
Hegland, D. D.,
Kim, K. S.,
Sethi, R.,
Rovira, I. I.,
Blazina, D. R.,
Lee, L.,
Bruder, J. T.,
Kovesdi, I.,
Goldshmidt-Clermont, P. J.,
Irani, K.,
and Finkel, T.
(1998)
J. Clin. Invest.
102,
929-937[Abstract/Free Full Text]
|
13.
|
Sah, V. P.,
Minamisawa, S.,
Tam, S. P.,
Wu, T. H.,
Dorn, G. W.,
Ross, J., Jr.,
Chien, K. R.,
and Brown, J. H.
(1999)
J. Clin. Invest.
103,
1627-1634[Abstract/Free Full Text]
|
14.
|
Cachero, T. G.,
Morielli, A. D.,
and Peralta, E. G.
(1998)
Cell
93,
1077-1085[Medline]
[Order article via Infotrieve]
|
15.
|
Sussman, M. A.,
Welch, S.,
Walker, A.,
Klevitsky, R.,
Hewett, T. E.,
Price, R. L.,
Schaefer, E.,
and Yager, K.
(2000)
J. Clin. Invest.
105,
875-886[Abstract/Free Full Text]
|
16.
|
Molkentin, J. D.,
Lu, J. R.,
Antos, C. L.,
Markham, B.,
Richardson, J.,
Robbins, J.,
Grant, S. R.,
and Olson, E. N.
(1998)
Cell
93,
215-228[Medline]
[Order article via Infotrieve]
|
17.
|
Rao, A.,
Luo, C.,
and Hogan, P. G.
(1997)
Annu. Rev. Immunol.
15,
707-747[CrossRef][Medline]
[Order article via Infotrieve]
|
18.
|
Sussman, M. A.,
Lim, H. W.,
Gude, N.,
Taigen, T.,
Olson, E. N.,
Robbins, J.,
Colbert, M. C.,
Gualberto, A.,
Wieczorek, D. F.,
and Molkentin, J. D.
(1998)
Science
281,
1690-1693[Abstract/Free Full Text]
|
19.
|
Mende, U.,
Kagen, A.,
Cohen, A.,
Aramburu, J.,
Schoen, F. J.,
and Neer, E. J.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
13893-13898[Abstract/Free Full Text]
|
20.
|
Zhang, W.,
Kowal, R. C.,
Rusnak, F.,
Sikkink, R. A.,
Olson, E. N.,
and Victor, R. G.
(1999)
Circ. Res.
84,
722-728[Abstract/Free Full Text]
|
21.
|
Ding, B.,
Price, R. L.,
Borg, T. K.,
Weinberg, E. O.,
Halloran, P. F.,
and Lorell, B. H.
(1999)
Circ. Res.
84,
729-734[Abstract/Free Full Text]
|
22.
|
Coso, O. A.,
Chiariello, M., Yu, J. C.,
Teramoto, H.,
Crespo, P.,
Xu, N.,
Miki, T.,
and Gutkind, J. S.
(1995)
Cell
81,
1137-1146[Medline]
[Order article via Infotrieve]
|
23.
|
Cacace, A. M.,
Ueffing, M.,
Philipp, A.,
Han, E. K.,
Kolch, W.,
and Weinstein, I. B.
(1996)
Oncogene
13,
2517-2526[Medline]
[Order article via Infotrieve]
|
24.
|
Widmann, C.,
Gibson, S.,
Jarpe, M. B.,
and Johnson, G. L.
(1999)
Physiol. Rev.
79,
143-180[Abstract/Free Full Text]
|
25.
|
Rodriquez-Viciana, P.,
Warne, P. H.,
Khwaja, A.,
Marte, B. M.,
Pappin, D.,
Das, P.,
Waterfield, M. D.,
Ridley, A.,
and Downward, J.
(1997)
Cell
89,
457-467[Medline]
[Order article via Infotrieve]
|
26.
|
Minden, A.,
Lin, A.,
McMahon, M.,
Lange-Carter, C.,
Derijard, B.,.,
Davis, R. G.,
Johnson, G. L.,
and Karin, M.
(1994)
Science
266,
1719-1723[Medline]
[Order article via Infotrieve]
|
27.
|
Kim, K. S.,
Takeda, K.,
Sethi, R.,
Pracyk, J. B.,
Tanaka, K.,
Zhou, Y. F., Yu, Z. X.,
Ferrans, V. J.,
Bruder, J. T.,
Kovesdi, I.,
Irani, K.,
Goldschmidt-Clermont, P.,
and Finkel, T.
(1998)
J. Clin. Invest.
101,
1821-1826[Abstract/Free Full Text]
|
28.
|
Taigen, T.,
De Windt, L. J.,
Lim, H. W.,
and Molkentin, J. D.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
1196-1201[Abstract/Free Full Text]
|
29.
|
Shields, J. M.,
Pruitt, K.,
McFall, A.,
Shaub, A.,
and Der, C. J.
(2000)
Trends Cell Biol.
10,
147-154[CrossRef][Medline]
[Order article via Infotrieve]
|
30.
|
Shioi, T.,
Kang, P. M.,
Douglas, P. S.,
Hampe, J.,
Yballe, C. M.,
Lawitts, J.,
Cantley, L. C.,
and Izumo, S.
(2000)
EMBO J.
19,
2537-2548[Abstract/Free Full Text]
|
31.
|
Cantrell, D.,
Pastor, M. I.,
and Woodrow, M.
(1994)
Adv. Exp. Med. Biol.
365,
73-79[Medline]
[Order article via Infotrieve]
|
32.
|
Woodrow, M. A.,
Rayter, S.,
Downward, J.,
and Cantrell, D. A.
(1993)
J. Immunol.
150,
3853-3861[Abstract/Free Full Text]
|
33.
|
Genot, E.,
Cleverley, S.,
Henning, S.,
and Cantrell, D.
(1996)
EMBO J.
15,
3923-3933[Abstract]
|
34.
|
Woodrow, M.,
Clipstone, N. A.,
and Cantrell, D.
(1993)
J. Exp. Med.
178,
1517-1522[Abstract]
|
35.
|
Turner, H.,
and Cantrell, D. A.
(1997)
J. Exp. Med.
185,
43-53[Abstract/Free Full Text]
|
36.
|
Turner, H.,
Gomez, M.,
McKenzie, E.,
Kirchem, A.,
Lennard, A.,
and Cantrell, D. A.
(1998)
J. Exp. Med.
188,
527-537[Abstract/Free Full Text]
|
37.
|
Ho, P. D.,
Zechner, D. K.,
He, H.,
Dillmann, W. H.,
Glembotski, C. C.,
and McDonough, P. M.
(1998)
J. Biol. Chem.
273,
21730-21735[Abstract/Free Full Text]
|
38.
|
McDonough, P. M.,
Hanford, D. S.,
Sprenkle, A. B.,
Mellon, N. R.,
and Glembotski, C. C.
(1997)
J. Biol. Chem.
272,
24046-24053[Abstract/Free Full Text]
|
39.
|
Ramirez, M. T.,
Sah, V. P.,
Zhao, X. L.,
Hunter, J. J.,
Chien, K. R.,
and Brown, J. H.
(1997)
J. Biol. Chem.
272,
14057-14061[Abstract/Free Full Text]
|
40.
|
Clerk, A.,
Michael, A.,
and Sugden, P. H.
(1998)
J. Cell Biol.
142,
523-535[Abstract/Free Full Text]
|
41.
|
Nemoto, S.,
Sheng, Z.,
and Lin, A.
(1998)
Mol. Cell. Biol.
18,
3518-3526[Abstract/Free Full Text]
|
42.
|
Thuerauf, D. J.,
Arnold, N. D.,
Zechner, D.,
Hanford, D. S.,
DeMartin, K. M.,
McDonough, P. M.,
Prywes, R.,
and Glembotski, C. C.
(1998)
J. Biol. Chem.
273,
20636-20643[Abstract/Free Full Text]
|
43.
|
Wang, Y.,
Su, B.,
Sah, V. P.,
Brown, J. H.,
Han, J.,
and Chien, K. R.
(1998)
J. Biol. Chem.
273,
5423-5426[Abstract/Free Full Text]
|
44.
|
Wang, Y.,
Huang, S.,
Sah, V. P.,
Ross, J., Jr.,
Brown, J. H.,
Han, J.,
and Chien, K. R.
(1998)
J. Biol. Chem.
273,
2161-2168[Abstract/Free Full Text]
|
45.
|
Choukroun, G.,
Hajjar, R.,
Fry, S.,
del Monte, F.,
Haq, S.,
Guerrero, J. L.,
Picard, M.,
Rosenzweig, A.,
and Force, T.
(1999)
J. Clin. Invest.
104,
391-398[Abstract/Free Full Text]
|
46.
|
van Aelst, L.,
and D'Souza-Schorey, C.
(1997)
Genes Dev.
11,
2295-2322[Free Full Text]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.