(Received for publication, December 10, 1996, and in revised form, May 30, 1997)
From the Oral and Pharyngeal Cancer Branch, NIDR,
National Institutes of Health, Bethesda, Maryland 20892-4330, ¶ Instituto de Investigaciones Biomedicas, Arturo
Duperier 4, 28029 Madrid, Spain, and the
Department of
Biological Sciences, Columbia University,
New York, New York 10027
The c-Jun amino-terminal kinases (JNKs) are a
subfamily of mitogen-activated protein kinases that phosphorylate c-Jun
and ATF2, and it has been postulated that phosphorylated c-Jun enhances its own expression through AP-1 sites on the c-jun
promoter. In this study, we asked whether signals activating JNK
regulate the c-jun promoter. Using NIH 3T3 cells expressing
G protein-coupled m1 acetylcholine receptors as an experimental model,
we have recently shown that the cholinergic agonist carbachol, but not
platelet-derived growth factor, potently elevates JNK activity.
Consistent with these findings, carbachol, but not platelet-derived
growth factor, increased the activity of a
c-jun promoter-driven reporter gene (for
chloramphenicol acetyltransferase). However, coexpression of JNK kinase kinase (MEKK) effectively increased JNK activity, but
resulted in surprisingly limited induction of the c-jun
promoter. This raised the possibility that pathway(s) distinct from JNK control the c-jun promoter, and prompted us to explore
which of its regulatory elements participate in transcriptional
control. We observed that deletion of the 3 AP-1 site diminished
chloramphenicol acetyltransferase activity in response to carbachol,
but only to a limited extent. In contrast, deletion of a MEF2 site
dramatically reduced expression, and deletion of both the MEF2 and 3
AP-1 sites abolished induction. Furthermore, cotransfection with MEF2C and MEF2D cDNAs potently enhanced the activity of the
c-jun promoter in response to carbachol, and stimulation of
m1 receptors, but not direct JNK activation, induced expression of a
MEF2-responsive plasmid. Taken together, these data strongly suggest
that MEF2 mediates c-jun promoter expression by G
protein-coupled receptors through a yet to be identified pathway,
distinct from that of JNK.
The jun and fos gene families are nuclear proto-oncogenes whose expression is induced in quiescent cells by the addition of serum or other growth promoting stimuli (1, 2). The expression of these genes is rapid and does not require newly synthesized proteins, thus suggesting that their induction involves posttranslational modification of pre-existing transcription factors. For example, c-jun is stimulated in a rapid and transient fashion by a variety of growth factors (1, 2), including those acting on G protein-coupled receptors (3). The protein product of the c-jun gene, c-Jun, then homodimerizes or heterodimerizes with various Jun and Fos family members to form the AP-1 transcription factor (1, 2, 4). In turn, AP-1 binds a palindromic DNA sequence, known as the TPA1-responsive element or TRE, that is present within the regulatory region of a variety of genes. Interestingly, c-jun itself has been described among the genes that display TRE sequences in their promoter regions, thus suggesting that the product of the c-jun gene regulates its own expression (5). The induction of c-jun expression appears to be critical for cell proliferation, as microinjection of neutralizing anti-c-Jun antibodies blocks cell cycle progression upon serum stimulation (6).
Molecules affecting the function of nuclear transcription factors have just begun to be identified. One such example is the family of extracellular signal-regulated kinases or MAPKs (7, 8). The role of MAPKs is to phosphorylate and regulate the activity of key molecules which ultimately control the expression of genes essential for many cellular processes, including cell growth, differentiation, programmed cell death, and neoplastic transformation (9). Recently, it has been shown that a novel family of kinases structurally related to MAPK, termed stress-activated protein kinases or c-Jun NH2-terminal kinases (JNKs), phosphorylate in vivo Ser-63 and Ser-73 in the NH2-terminal transactivating domain of the c-Jun protein, thereby positively regulating its transcriptional activity (10).
In our laboratory, we have genetically engineered NIH 3T3 murine fibroblasts to express subtypes of the human muscarinic acetylcholine receptors (mAChRs), and have utilized these cells as a model system for the study of proliferative signaling through G protein-coupled receptors (11). In this biological system, the m1 class of mAChR can effectively transduce mitogenic signals and, if persistently activated, can behave as a potent agonist-dependent oncogene (12). Recently, we have shown that activation of m1 G protein-coupled receptors by its agonist, carbachol, induces the expression of a clearly distinct set of nuclear transcription factors when compared with those expressed in response to platelet-derived growth factor (PDGF) (3), a mitogen acting on endogenous tyrosine kinase receptors. In particular, carbachol caused a much greater induction of c-jun mRNA expression and AP-1 activity (3). Interestingly, we have observed that the m1 agonist, carbachol, but not PDGF, can induce a remarkable increase in JNK activity, following a temporal pattern distinct from that of MAPK activation (3).
In view of these observations, we set out to investigate whether signals activating JNK regulate expression from the c-jun promoter, using NIH 3T3 cells expressing m1 G protein-coupled receptors (NIH-m1.2 cells) as an experimental model. In these cells, we observed that carbachol, but not PDGF, potently increased the expression from a reporter plasmid containing the chloramphenicol acetyltransferase (CAT) gene under the control of the murine c-jun promoter. However, this effect was not mimicked by expression of activated molecules acting downstream from m1 receptors in the JNK pathway, although they activated JNK to an extent much greater than that caused by the m1 agonist alone. This prompted us to explore the existence of JNK-independent signaling pathways regulating the expression from the c-jun promoter. Using deletion and point mutational analysis of the c-jun promoter, we found a critical role for a DNA sequence that binds the MEF2 family of transcription factors. Furthermore, cotransfection with plasmids carrying the MEF2C and MEF2D cDNAs potently enhanced expression from the c-jun promoter in response to carbachol. In addition, we found that signaling from m1 receptors, but not direct JNK activation, induced expression from a MEF2-responsive plasmid. Taken together, these findings strongly suggest that signaling from G protein-coupled receptors to the c-jun promoter involves a novel, JNK-independent pathway acting on the MEF2 family of transcription factors.
NIH 3T3 fibroblasts expressing approximately 20,000 human m1 mAChRs/cell, designated NIH-m1.2 cells (11), were maintained in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Inc.) supplemented with 10% calf bovine serum. Transfections were performed by the calcium phosphate precipitation technique, adjusting the total amount of plasmid DNA to 5-10 µg/plate with vector alone.
Reporter Gene AssaysNIH-m1.2 cells were transfected with
different expression plasmids together with 1 µg of pCMV-gal, a
plasmid expressing the enzyme
-galactosidase, and 1 µg of each of
the reporter plasmids. After overnight incubation, the cells were
washed with serum-free DMEM, and kept for 24 h in DMEM
supplemented with 0.5% fetal bovine serum. Cells were then stimulated
with agonists for an additional 6 h and lysed using reporter lysis
buffer (Promega).
CAT activity was assayed in the cell extracts by incubation for 6-16 h
in the presence of 0.25 µCi of [14C]chloramphenicol
(100 mCi/mmol) and 200 µg/ml butyryl-CoA in 0.25 M
Tris-HCl, pH 7.4. Labeled butyrylated products were extracted using
a mixture of xylenes (Aldrich) and counted as described (3). Luciferase
activity present in cellular lysates was assayed using
D-luciferin and ATP as substrates, and light emission was quantitated with the aid of a luminometer, Monolight 2010, as specified
by the manufacturer (Analytical Luminescence Laboratory). -Galactosidase activity present in each sample was assayed by a
colorimetric method, and used to normalize for transfection efficiency.
Phosphorylating activity of endogenous MAPK, JNK, and p38, or of transfected, epitope-tagged MAPK, JNK, and p38 was determined as follows. Cells were seeded at 10% confluence, and the following day cells were left untreated for endogenous activity, or transfected with pcDNA3-HA-MAPK, pcDNA3-HA-JNK, and pcDNA3-HA-p38 and additional DNAs by the LipofectAMINE technique (Life Technologies, Inc.). Total amount of plasmid DNA was adjusted to 5-10 µg/plate with vector DNA (pcDNA3, Invitrogen) when necessary. Two days later, NIH-m1.2 cells were incubated in serum-free medium overnight for MAPK or for 2 h for JNK and p38, respectively. Cells were then stimulated with agonists for different periods of time at 37 °C, washed with cold phosphate-buffered saline, and lysed as described (3). Cleared lysates were immunoprecipitated on ice for 2 h with an antibody against ERK-2 (SC-154, Santa Cruz Biotechnology) for endogenous MAPK, with an antibody against JNK-1 (15701A, PharMingen) for endogenous JNK, and with the anti-HA monoclonal antibody (HA.11, Berkeley Antibody Co.) for the epitope-tagged MAPK, JNK, and p38. The immunoprecipitates were washed, and in vitro kinase assays were performed using 1.5 µg/µl myelin basic protein (Sigma) as substrate for MAPK or 1 µg of purified, bacterially expressed, GST-ATF2 protein as substrate for JNK and p38. Samples were analyzed by SDS-gel electrophoresis on 12% acrylamide gels, and autoradiography was performed with the aid of an intensifying screen.
DNA ConstructsA reporter plasmid containing the MEF2 DNA
binding sequence (pGL3MEF2) was engineered by inserting the sequence
5-TCGACGGGCTATTTTTAGGGCCG-3
in the SalI site of the pGL3
plasmid DNA (Promega), upstream of an SV40 minimal promoter and a
luciferase gene. The mutant sequence 5
-TCGACGGGCGATTTTTCGGGCCG-3
,
that does not bind MEF2 proteins (13), was used to generate
pGL3MEF2mut. pJC6, pJC8, pJC9, pJTX, pJSX, and pJSTX have been
described previously (14). MEF2C and MEF2D cDNAs were kindly
provided by Dr. Bruce Paterson (National Cancer Institute, National
Institutes of Health, Bethesda, MD) and subcloned in the pcDNAIIIB
expression vector (Invitrogen). Other cDNA expression vectors have
already been described (15).
In previous studies, we have observed that stimulation of G
protein-coupled receptors of the m1 class by the cholinergic agonist, carbachol, potently elevates JNK activity (3, 16). In turn, JNK has
been shown to phosphorylate the NH2-terminal
transactivating domain of c-Jun, thereby stimulating AP-1-mediated
transcription (17). We have also reported that, consistent with these
observations, stimulation of quiescent NIH-m1.2 cells with carbachol
induces AP-1 activity and greatly enhances the expression of
c-jun mRNA (3). As an approach to explore the
relationship between the enzymatic activity of JNK and the regulation
of c-jun expression, we transfected NIH-m1.2 cells with a
reporter construct containing murine c-jun promoter
sequences from 225 to +150 fused to the CAT gene (pJC6) (14) (see
below). In agreement with our previous observations (3), we show in
Fig. 1 that, whereas carbachol, the
tyrosine kinase-receptor ligand PDGF, and the phorbol ester TPA induced
MAPK activity to similar extents, only carbachol elevated the
phosphorylating activity of JNK in these cells. When reporter gene
assays were performed in parallel, we observed that c-jun promoter activity was strongly enhanced by addition of carbachol, whereas treatment with PDGF and TPA did not result in demonstrable activation. Thus, these data suggest that the activity of the c-jun promoter correlated with JNK, but not MAPK
activation.
To further explore the roles of JNK and MAPK in the regulation of
expression from the c-jun promoter, we employed a
constitutively active mutant of MEK, MEKEE, which results in a
remarkable increase in MAPK activity (15, 18), and a truncated JNK
kinase kinase, MEKK, which potently activates JNK (19, 20).
Accordingly, we cotransfected expression plasmids for MEKEE and MEKK
together with plasmids expressing epitope-tagged MAPK and
epitope-tagged JNK, or the c-jun promoter containing
reporter plasmid. As shown in Fig. 2,
whereas MEKEE strongly induced MAPK activity, it had no effect on JNK
or on the expression of the c-jun promoter-driven reporter
plasmid. On the other hand, MEKK did not elevate MAPK activity but
potently stimulated JNK-phosphorylating activity, to an extent much
greater than even that elicited by m1 receptor stimulation.
Surprisingly, however, activation of CAT expression in response to
carbachol was severalfold higher than that induced by MEKK (Fig. 2).
These observation raised the possibility that signaling pathways in
addition to JNK, may participate in the activation of the
c-jun promoter by m1 G protein-coupled receptors.
The lack of a strict correlation between JNK activation and the
expression from the c-jun promoter-containing reporter
plasmid prompted us to search for those regulatory elements in the
c-jun promoter that participate in the m1-induced response.
In this regard, binding sites for Sp1, AP-1, CTF, and MEF2
transcription factors have been identified in the c-jun
promoter at nucleotides 117,
92,
72, and
59, respectively, and
two GATAA elements are found at
33 and
11 (Fig.
3A) (14). In addition, a
putative site for AP-1, referred to here as AP-1-like, is located at
180 (4). To assess the contribution of the AP-1-like and Sp1 sites, and the CAAT box in the response to m1 receptors, we used two deletion
mutants, pJC8 and pJC9, containing fragments
115 to +150 and
80 to
+150 of the c-jun promoter, respectively (Fig. 3A). As shown in Fig. 3B, carbachol addition to
cells transfected with pJC8 or pJC9 caused an even higher level of CAT
expression than that observed for pJC6. These data suggest that the
AP-1-like and the Sp1 sites that are absent in both constructs, as well as the CAAT box that is deleted in pJC9, are dispensable for signaling from m1 receptors to the c-jun promoter.
Regulatory elements controlling the activity
of the c-jun promoter. A, pJC6 is a
pBLCAT3-based reporter construct that contains bases 225 to +150 of
the murine c-jun promoter. pJC8 and pJC9 contain bases
115
to +150 and
80 to +150, respectively. The sites of point mutations in
the AP-1 binding site that abolish AP-1 binding (pJTX) and point
mutations at the MEF2 binding site that abolish MEF2 binding (pJSX) are
indicated by double vertical lines. The plasmid pJSTX
contains mutations for both the AP-1 and MEF2 sites. B,
NIH-m1.2 cells were cotransfected with the reporter plasmids pJC6,
pJC8, pJC9, pJTX, pJSX, or pJSTX (1 µg/plate) as indicated. In each
case, 1 µg of pCMV-
-gal reporter plasmid DNA was included in the
transfection mixtures. 40 h later, cells were left untreated or
exposed for 4 h to 100 µM carbachol. Cells were
collected, and the lysates were assayed for CAT and
-galactosidase activity. The data represent CAT activity normalized by the
-galactosidase activity present in each sample, and are the
average ± S.E. of triplicate samples from a typical experiment. Similar results were obtained in four independent
experiments. The autoradiogram corresponds to a typical experiment
resolved on a thin layer chromatography (TLC) plate. The
arrows indicate the position of the butyrylated forms of
[14C]chloramphenicol.
To explore the role of the AP-1 and MEF2 sites, we used a plasmid,
pJTX, that harbors four point mutations in the TRE element located at
72 thereby abolishing AP-1 binding (5, 14); a plasmid, pJSX,
containing two point mutations, at nucleotides
51 and
58, that have
been shown to abolish MEF2 binding (13, 14); and a double mutant,
pJSTX, that lacks both AP-1 and MEF2 binding sites (14) (Fig.
3A). As expected, in control experiments we observed that
mutation of the AP-1 site, but not of the MEF2 site, nearly abolishes
the expression from the reporter plasmid elicited by cotransfection of
the activated JNK kinase kinase, MEKK (data not shown). However, the
TRE-deficient mutant, pJTX, showed only a relatively small reduction in
the response to m1 stimulation (Fig. 3B). In contrast, the
MEF2-binding mutant, which contains an intact TRE site (pJSX), showed a
drastic reduction in the m1-induced response (70-80%). Furthermore,
as shown in Fig. 3B, mutation of both MEF2 and TRE sites in
the c-jun promoter (pJSTX) nearly abolished the expression
of CAT elicited by carbachol in m1-expressing cells. These results
suggest that both the MEF2 and AP-1 regulatory sites are critical for
the regulation of expression from the c-jun promoter in
response to intracellular signals transmitted by G protein-coupled
receptors. In addition, these data suggest that, in these cells, the
majority of the stimulatory response on the c-jun
promoter-containing reporter plasmid is exerted through the MEF2
binding site and not through the TRE, as we had initially hypothesized.
MEF2 proteins bind the consensus sequence motif
CTA(A/T)4TAG (13, 14), which is present in the
c-jun promoter between positions 50 and
59. As an
attempt to further investigate if that sequence participates in the
transcriptional response triggered by stimulation of m1 receptors, we
engineered a reporter construct by introducing oligonucleotides that
match exactly the sequence CTATTTTTAG found in the c-jun
promoter, upstream of a minimal promoter in the pGL3promoter plasmid
(Promega). The resulting construct expresses a luciferase reporter gene
and was designated pGL3MEF2. As a control, we generated a similar
reporter plasmid containing the mutant sequence
CGATTTTTCG, which does not bind MEF2 (13, 14)
and therefore is not able to respond to MEF2 (pGL3MEF2mut). Fig.
4 shows that upon transfection in
NIH-m1.2 cells with pGL3MEF2, carbachol induced a remarkable increase
in the luciferase activity when compared with the unstimulated control cells. Furthermore, coexpression of MEF2C (Fig. 4) or MEF2D (data not
shown) also elevated the reporter gene activity. However, no
demonstrable response was observed upon transfection with MEKK, thus
suggesting that the MEF2 binding site is insensitive to the activation
of the JNK pathway. On the other hand, cells transfected with the
construct pGL3MEF2mut exhibited the same level of luciferase activity
in each of these conditions, supporting that an intact MEF2 binding
site is required to elicit a transcriptional response. Taken together,
these findings strongly suggest that the MEF2 binding site could be the
target for signaling pathways linking G protein-coupled receptors to
the c-jun promoter.
To address directly whether proteins of the MEF2 family could regulate
the activity of the c-jun promoter, we next cotransfected pJC6 with expression plasmids harboring the cDNAs for MEF2C or c-Jun in NIH-m1.2 cells. As shown in Fig.
5, CAT expression driven by the
c-jun promoter was elevated by cotransfection with the c-Jun
and MEF2C expression plasmids. A similar result was obtained by
overexpressing MEF2D (data not shown). When cells were stimulated with
carbachol, we observed that coexpression of c-Jun potentiated the
response by only 30-40%, while both MEF2C (Fig. 5) and MEF2D (data
not shown) nearly tripled CAT activity in response to the cholinergic
agonist. Taken together, these findings support a critical role for
MEF2 proteins in signaling from G protein-coupled receptors to the
c-jun promoter.
A recent study (29) showed that one of the members of the MEF2 family
(MEF2C) is phosphorylated by a novel member of the MAPK superfamily,
p38, in myeloid lineage cells. Therefore we explored whether p38, by
regulating the activity of MEF2, might mediate in the induction of the
c-jun promoter by G protein-coupled receptors. To that end,
we transfected NIH-m1.2 cells with pJC6 and measured CAT activity upon
carbachol stimulation in the presence or absence of the specific p38
inhibitor, SB203580 (30). As shown in Fig.
6, incubation with SB203580 nearly
abolished p38 activation by anisomycin or carbachol but did not have
any demonstrable effect on transcriptional activation by the
c-jun promoter. Similar results were obtained using pJTX,
the mutant of the c-jun promoter lacking the AP-1 site but
containing a MEF2-responsive element (Fig. 6). These results suggest
that p38 does not regulate MEF2 activation of the c-jun
promoter in NIH-m1.2 cells.
Positive autoregulation has been considered the major regulatory event in the response of the c-jun promoter to proliferative stimuli. Two steps have been described as participating in that process: 1) post-translational modification of preexisting c-Jun proteins and 2) transcriptional activation of c-jun that leads to an increase in the pool of c-Jun protein present in the cell (1, 4, 5, 21). c-Jun is a constitutive part of the AP-1 transcription factor, and binding of such a factor to the TRE element in the c-jun promoter has been shown to result in the stimulation of transcription (5). Using NIH-3T3 cells expressing m1 G protein-coupled receptors, we had previously observed that addition of the cholinergic agonist, carbachol, results in a remarkable increase in JNK activity and in the level of c-jun expression. Thus, we expected a major participation of the JNK pathway on the regulation of c-jun promoter activity. Indeed, we observed a positive correlation between the c-jun promoter activity and JNK, but not with MAPK. Surprisingly, direct activation of the JNK pathway by overexpression of MEKK resulted in activation of the c-jun promoter but to a much more limited extent than that induced upon m1 stimulation. This observation prompted us to ask whether other biochemical routes, in addition to the JNK pathway, participate in the transduction of signals from G protein-coupled receptors to the promoter of c-jun.
Mapping of the regulatory elements in the c-jun promoter has yielded the existence of several consensus binding sites for different transcription factors such as Sp1, CTF, AP-1, and MEF2 (5, 14). Using a series of c-jun promoter mutants fused to a reporter gene, we observe that deletion of the binding sites for Sp1 or CTF did not affect reporter gene activity. Furthermore, we found that mutations that abolish AP-1 binding prevent expression from the reporter plasmid when induced by MEKK, but display only a partial reduction in the response to carbachol. In contrast, a more dramatic decrease was observed when mutations that inhibit binding of MEF2 factors were introduced, thus suggesting a role for the MEF2 factors in regulating c-jun promoter activity, perhaps even more critical than that played by AP-1 itself. Furthermore, the fact that the double mutant that does not bind AP-1 or MEF2 fails to respond to carbachol, indicates that, for signals initiated at the level of m1 G protein-coupled receptors, the AP-1 and MEF2 sites are sufficient to account for the transcriptional response of the c-jun promoter.
While extensive studies have established the role of AP-1 complexes in transcriptional control, very limited information is available about the functions of MEF2 proteins. The myocyte enhancer family (MEF) of transcription factors comprises a group of transcriptional activators MEF2A, -B, -C, and -D (22-25). MEF2 binding sites have been found in the promoters and enhancers of many skeletal and cardiac muscle structural genes (26). Structurally, MEF2 proteins contain a common motif that was named the MEF2 domain, this feature being unique to all MEF2 family members described so far (26). Despite the importance of MEF2 factors in the regulation of muscle gene expression, very little is known about the mechanism by which these proteins promote transcription or the signal transduction pathways controlling their activity. Recently, MEF2C, but not the other members of the family, has been shown to be a substrate for the MAPK-related p38 (29). While the expression of MEF2A, -B, and -D proteins is ubiquitous, the expression of MEF2C seems to be restricted to cortical neurons, monomyelocytic and muscle cells (23, 24, 27). This wide distribution suggests that their function is not limited to the regulation of muscle specific gene expression (27). Interestingly, they share with other transcription factors, such as serum response factor, the presence of a common structural domain termed MADS box, and due to that characteristic they were originally named RSRFs (related to serum response factors) (13).
In this study, we provide evidence for a critical role for MEF2 in the
regulation of c-jun promoter activity by stimulation of G
protein-coupled receptors. In addition, we show that overexpression of
the MEF2 protein potentiates carbachol-induced expression of the
c-jun promoter, strengthening the concept of MEF2 proteins as the targets of a yet uncharacterized signal transduction pathway. We
also report here that an isolated MEF2 binding site stimulates transcription of a reporter gene when m1-expressing cells are stimulated by carbachol, but not by cotransfection with the
JNK-inducing construct MEKK. Taken together, our data support the
existence of two distinct signaling pathways that initiating on the
same receptor at the level of the plasma membrane converge on the
c-jun promoter: a JNK-dependent pathway, acting
on the AP-1 responsive element; and a JNK-independent pathway, acting
on the MEF2 regulatory sequence. In this regard, a recent report showed
that the transactivating activity of MEF2C can be regulated by p38
(29). However, we observed that SB205380, a potent p38 and p38
blocker, fails to inhibit c-jun promoter activity, thus
ruling out these p38 proteins as mediators of the carbachol-induced
signal to MEF2. Nevertheless, NIH 3T3 cells express MEF2A, -B, and -D
but not MEF2C (23, 24, 27), and it has been recently shown that p38
and p38
are not inhibited by SB205380 (31). Thus, it is likely that
these or other members of the MAPK superfamily might mediate MEF2
activity in these cells. The identity of such regulatory molecule
acting on other members of the MEF2 family of transcription factors is under current investigation.
Our results assign a critical role for MEF2 in the regulation of c-jun expression by the G protein-linked class of cell surface receptors. Interestingly, Han et al. provided evidence that the MEF2 binding site in the c-jun promoter also participates in the activation of c-jun by epidermal growth factor receptors and serum in HeLa cells (14, 28). Thus, the elucidation of the molecular entities that participate in the transduction of signals from the membrane to MEF2 proteins, as well as of the mechanisms whereby those signaling molecules control the transcriptional activity of MEF2, is expected to be central for understanding the regulation of c-jun expression by growth promoting factors. Furthermore, considering the ubiquitous expression of MEF2 proteins and their resemblance to other transcription factors such as serum response factor, we can envision a growing interest for dissecting the signaling pathways that converge on MEF2.