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The Journal of Neuroscience, September 1, 2001, 21(17):6544-6552
Myocyte Enhancer Factor 2A and 2D Undergo Phosphorylation and
Caspase-Mediated Degradation during Apoptosis of Rat Cerebellar Granule
Neurons
Mingtao
Li1,
Daniel A.
Linseman1,
Melissa P.
Allen2,
Mary Kay
Meintzer1,
Xiaomin
Wang1,
Tracey
Laessig1,
Margaret E.
Wierman2, and
Kim A.
Heidenreich1
Departments of 1 Pharmacology and
2 Medicine, University of Colorado Health Sciences Center
and the Denver Veterans Affairs Medical Center, Denver, Colorado 80262
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ABSTRACT |
Myocyte enhancer factor 2 (MEF2) proteins are important regulators
of gene expression during the development of skeletal, cardiac, and
smooth muscle. MEF2 proteins are also present in brain and
recently have been implicated in neuronal survival and differentiation. In this study we examined the cellular mechanisms regulating the activity of MEF2s during apoptosis of cultured cerebellar granule neurons, an established in vitro
model for studying depolarization-dependent neuronal survival. All four MEF2 isoforms (A, B, C, and D) were detected by immunoblot analysis in
cerebellar granule neurons. Endogenous MEF2A and MEF2D, but not MEF2B
or MEF2C, were phosphorylated with the induction of apoptosis. The
putative sites that were phosphorylated during apoptosis are
functionally distinct from those previously reported to enhance MEF2
transcription. The increased phosphorylation of MEF2A and MEF2D was
followed by decreased DNA binding, reduced transcriptional activity,
and caspase-dependent cleavage to fragments containing N-terminal DNA
binding domains and C-terminal transactivation domains. Expression of
the highly homologous N terminus of MEF2A (1-131 amino acids)
antagonized the transcriptional activity and prosurvival effects of a
constitutively active mutant of MEF2D (MEF2D-VP16). We conclude that
MEF2A and MEF2D are prosurvival factors with high transcriptional
activity in postmitotic cerebellar granule neurons. When these neurons
are induced to undergo apoptosis by lowering extracellular potassium,
MEF2A and MEF2D are phosphorylated, followed by decreased DNA binding
and cleavage by a caspase-sensitive pathway to N-terminal fragments
lacking the transactivation domains. The degradation of MEF2D and MEF2A
and the generation of MEF2 fragments that have the potential to act as
dominant-inactive transcription factors lead to apoptotic cell death.
Key words:
MEF2; neurons; apoptosis; transcription; caspase; cerebellum
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INTRODUCTION |
Myocyte enhancer factor 2 (MEF2)
transcription factors are members of the MADS
(MCM1-agamous-deficiens-serum response factor) family of transcription
factors (Yu et al., 1992 ; Naya and Olson, 1999). A hallmark of MADS-box
proteins is their combinational association with other MADS domain
factors, as well as other heterologous classes of transcriptional
regulators (Shore and Sharrocks, 1995). Mammalian MEF2 proteins are
encoded by four genes (MEF2A, MEF2B, MEF2C, and MEF2D), each of which
gives rise to alternatively spliced transcripts (Yu et al., 1992;
Leifer et al., 1993; Martin et al., 1994). The MEF2 family of genes is
highly expressed in cells of muscle lineage, where they have been shown
to be important regulators of gene expression during the development of
skeletal, cardiac, and smooth muscle (McDermott et al., 1993; Martin et
al., 1994; Molkentin et al., 1996). In these tissues the MEF2 proteins
interact with myogenic basic helix-loop-helix transcription factors
such as MyoD to activate myogenesis (Molkentin and Olson, 1996;
Ornatsky et al., 1997).
All MEF2 family members also are highly expressed in neurons of the
CNS (Leifer et al., 1993, 1994; McDermott et al., 1993; Ikeshima
et al., 1995; Lyons et al., 1995; Mao et al., 1999). Recent in
vitro findings support the hypothesis that MEF2 transcription factors regulate neuronal survival and development. In cultures of
cerebral cortical neurons in which proliferating precursor cells and
postmitotic differentiating neurons can be distinguished, MEF2C is
expressed selectively in newly generated postmitotic neurons and is not
detectable in BrdU-positive precursor cells (Mao et al., 1999).
Transfection of postmitotic cortical neurons with different MEF2C
mutants demonstrated that MEF2C is required for the survival of these
neurons. In postnatal day 19 (P19) neuronal precursor cells, the
expression of MEF2 induces a mixed neuronal/myogenic phenotype (Okamoto
et al., 2000). During retinoic acid-induced neurogenesis of these
cells, a dominant-negative form of MEF2C enhances apoptosis but does
not affect cell division. On the other hand, P19 cells induced to
undergo apoptosis can be rescued from cell death by the expression of
constitutively active MEF2C. In addition, overexpression of MEF2C in
P19 cells results in induction of neurofilament protein, the nuclear
antigen NeuN, and MASH-1, a neural-specific transcription factor known
to interact with MEF2s (Skerjanc and Wilton, 2000). These data suggest
that MEF2 proteins regulate neuronal development by promoting survival
and inducing differentiation.
In the present study we examined the mechanisms regulating the activity
of MEF2 proteins during apoptosis of cultured rat cerebellar granule
neurons, a well established model of depolarization-dependent neuronal
survival. We provide evidence that MEF2A and MEF2D are prosurvival
factors with high DNA binding and transcriptional activity in
postmitotic cerebellar granule neurons. When these neurons are induced
to undergo apoptosis by lowering extracellular potassium, MEF2A and
MEF2D are phosphorylated. The phosphorylation of MEF2D and MEF2A is
followed by decreased DNA binding and cleavage by a caspase-sensitive
pathway to N-terminal MEF2 fragments that lack the transactivation
domain. The decreased DNA binding of MEF2s and the formation of MEF2
fragments that can act as dominant-inactive transcription factors are
sufficient to block the prosurvival effects of MEF2s and induce
apoptosis in mature cerebellar granule neurons.
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MATERIALS AND METHODS |
Materials. The MEF2A antibody is an affinity-purified
rabbit polyclonal antibody that was raised to a peptide corresponding to codons 487-507 of human MEF2A purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). According to the manufacturer, this
antibody may cross-react to a small extent with MEF2C. The
affinity-purified rabbit polyclonal MEF2C antibody, a gift from John
Schwarz (University of Texas Medical School, Houston, TX), was raised
against an isoform-specific peptide representing codons 300-316 of
human MEF2C and is specific for MEF2C (Firulli et al., 1996). The
rabbit polyclonal antibody to MEF2B was raised against a polyhistidine
fusion protein corresponding to codons 234-365 of human MEF2B and was
kindly provided by Dr. Ron Prywes (Columbia University, NY). Antibody
to MEF2D is a monoclonal antibody raised against a peptide
corresponding to codons 346-511 of mouse MEF2D purchased from
Transduction Laboratories (Lexington, KY). The dominant-inactive MEF2
mutant pcDNA3-MEF2A131 was kindly provided by Dr. Prywes. The
dominant-active MEF2 mutant pCMV-MEF2D-VP16 was a gift from Dr. John C. McDermott (York University, Toronto, Ontario, Canada). The
pGL2-MEF2-Luc reporter plasmid (Lemercier et al., 2000) was provided by
Dr. Saadi Khochbin (INSERM, France). The caspase-3 antibody was
purchased from Santa Cruz Biotechnology, and the polyclonal
anti- -galactosidase (-gal) antibody was purchased from 5 Prime 3 Prime (Boulder, CO). Cy3-conjugated goat antibody to rabbit
IgG was purchased from Chemicon (Temecula, CA). The caspase inhibitors
YVAD-CHO, DEVD-FMK, and ZVAD-FMK were obtained from Calbiochem (La
Jolla, CA). [ -32P]CTP (3000 Ci/mmol)
was purchased from Amersham Pharmacia Biotechnology (Piscataway, NJ).
Neuronal cell culture. Rat cerebellar granule neurons were
prepared from 7- to 8-d-old Sprague Dawley rat pups (15-19 gm) as
described previously (Li et al., 2000). Briefly, neurons were seeded at
a density of 2.0 × 106 cells/ml in
basal modified Eagle's (BME) medium containing 10% fetal bovine
serum, 25 mM KCl, 2 mM glutamine, and
penicillin (100 U/ml)/streptomycin (100 µg/ml). Cytosine arabinoside
(10 µM) was added to the culture medium 24 hr after
plating to limit the growth of non-neuronal cells. With the use of this
protocol, 95-99% of the cultured cells were granule neurons.
Transfections were performed at day 5-6 in culture, and experiments
were performed after 7 d in culture. Apoptosis was induced by
removing the serum and reducing the extracellular potassium
concentration from 25 to 5 mM. Control cultures were
treated identically but were maintained in serum-free medium
supplemented with 25 mM KCl.
Western blot analysis. Western blot analysis was performed
as described previously (Li et al., 2000). Briefly, neurons were lysed
by adding SDS sample buffer [62.5 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, 10% glycerol, 50 mM DTT, and 0.1% (w/v)
bromphenol blue]. The samples were resolved by SDS-PAGE with the use
of either 7.5 or 12% acrylamide gels, as indicated in the legends.
Proteins were transferred to Hybond-P membranes (polyvinylidene
difluoride). The membranes were incubated with anti-MEF2A (1:5000),
anti-MEF2D (1:1000), anti-MEF2C (1:1000), and anti-MEF2B (1:500). After
incubation with the primary antibodies the filters were washed and then
incubated with the respective horseradish peroxidase (HRP)-conjugated
anti-rabbit or anti-mouse antibody (Amersham Pharmacia Biotech). Then
the blots were washed and subsequently were developed with an enhanced chemiluminescence system (Amersham Pharmacia Biotech) and exposed to
Kodak autoradiographic film. Quantitation was performed with the
Bio-Rad Quantity One software (Hercules, CA).
Preparation of nuclear extracts from cerebellar granule neurons.
After 7 d in culture the cerebellar granule neurons were rinsed two times in serum-free BME containing 25 mM KCl and
then maintained in 25 or 5 mM KCl medium in the absence or
presence of caspase inhibitors. Neurons that did not receive inhibitors received the control vehicle dimethyl sulfoxide (DMSO). After the
indicated times the neurons (100 mm dishes) were washed with ice-cold
PBS and detached from culture dishes by a cell scraper in 0.5 ml of
buffer A [0.25 M sucrose and (in mM) 15 Tris,
pH 7.9, 60 KCl, 2 EDTA, pH 8.0, 0.5 EGTA, 15 NaCl, 1.0 Na3VO4, 50 NaF, 0.5 spermidine, 1 DTT, 1 benzamidine, and 0.5 PMSF plus 20 µg/ml
leupeptin, 0.76 µg/ml pepstatin, and 2 µM aprotinin].
The cells were centrifuged at 250 × g for 5 min. The
pellets were washed twice in buffer A and then homogenized with 15 strokes of a tight-fitting Dounce homogenizer to release the nuclei.
Then the homogenate was centrifuged at 14,000 × g for
15 sec to pellet the nuclei. The supernatants were removed, and the
pellets were resuspended in buffer C [(in mM) 20 HEPES, pH 7.9, 500 KCl, 1.5 MgCl2, 1 EDTA, pH
8.0, 1.0 Na3VO4, 50 NaF,
1.0 DTT, 1 benzamidine, and 0.5 PMSF plus 20 µg/ml leupeptin, 0.76 µg/ml pepstatin, 25% glycerol, and 10 µM
aprotinin]. Nuclear proteins were extracted at 4°C for 45 min, and
insoluble nuclei were precipitated by centrifugation at 14000 × g for 15 min. Supernatants were dialyzed against a buffer
containing 10% glycerol and (in mM) 10 Tris, pH
7.9, 5 MgCl2, 50 KCl, 1 EDTA, pH 8.0, 1.0 Na3VO4, 50 NaF, 1 DTT, 1 PMSF, and 1 benzamidine for 3 hr at 4°C. The extracts were quantified for protein content by the BCA method (Pierce, Rockford, IL) and frozen
in small aliquots at  70°C.
Electrophoretic mobility shift assays. Nuclear extracts from
cerebellar granule neurons (10 µg) were incubated with
double-stranded oligonucleotides corresponding to the muscle creatine
kinase MEF2 site
5'-CGGATCGCTCTAAAAATAACCCTGTCG-3' (Amacher
et al., 1993) or to a mutant oligonucleotide containing CG and AC
substitutions at the two italicized residues. The oligomers were
end-labeled with the Klenow fragment of DNA polymerase I (Life
Technologies, Gaithersburg, MD) and
[-32P]CTP to a specific activity of
10,000-30,000 cpm/ng. Binding reactions were performed for 20 min at
4°C in 1 mM dithiothreitol, 2.5 mM MgCl2, 10% glycerol,
0.1 mg/ml bovine serum albumin, 30 ng/µl of poly(dI-dC), 20 mM HEPES, pH 7.9, and 50-100,000 cpm of oligomer
in a total volume of 20 µl. For the supershift analysis, 1 µl of
specific antisera or preimmune serum was added to the nuclear extracts
for 20 min at 4°C, followed by another 20 min in the presence of
labeled oligomers. The protein-DNA complexes were analyzed on 5%
nondenaturing polyacrylamide gels containing 3% glycerol and 0.25×
TBE (90 M Tris borate, 1 mM
EDTA) in the cold room. After electrophoresis the gels were dried and
exposed to film at 70°C.
Transfection assays and reporter gene expression. Cerebellar
granule neurons were transfected by a calcium phosphate coprecipitation method described previously (Li et al., 2000). Neurons were transfected with 1 µg of MEF2-luciferase expression plasmids (pGL2-MEF2-luc) and/or 1-3 µg of MEF2D expression plasmids (pCMV-MEF2D-VP16,
pcDNA3-MEF2A131) and 1 µg of pCMV--gal as an internal control for
transfection efficiency. The total amount of DNA for each transfection
was kept constant (7 µg/ml) by using the empty vector pcDNA3. Neurons were kept in conditioned medium after transfection for 2 hr; then the
medium was replaced with BME containing 25 or 5 mM KCl.
After 4 hr the cell extracts were prepared with reporter lysis buffer (Promega, Madison, WI), and the activities of luciferase and
-galactosidase were measured with the Luciferase Assay System
(Promega) and the -galactosidase Enzyme Assay System (Promega), respectively.
Quantitation of apoptosis in transfected neurons. Neurons
were transfected as described previously (Li et al., 2000).
Plasmids (pCMV-MEF2D-VP16, pCMV-MEF2D-VP16 plus pcDNA3-MEF2A131, and
pCMV-LacZ) were added to the transfection media at a final
concentration of 4-5 µg/ml. The total amount of DNA for each
transfection was kept constant by using the empty vector pcDNA3. After
transfection the neurons were switched to a medium containing 25 or 5 mM KCl. Then 16 hr later the cells were immunostained with
a polyclonal antibody to -galactosidase (1:500 dilution), followed
by a Cy3-conjugated goat antibody to rabbit IgG (1:500) to identify
cells expressing -galactosidase. To visualize the nuclei of
transfected neurons, we included the DNA dye Hoechst 33258 (5.0 µg/ml) in the wash after the secondary antibody incubation. Apoptosis
was quantified by scoring the percentage of cells in the
-galactosidase-expressing cell population with condensed or
fragmented nuclei. So that we could obtain unbiased counting, cells
(~500) were scored blindly without knowledge of their previous
treatment. Experiments were performed in triplicate.
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RESULTS |
MEF2 protein expression and phosphorylation state in control and
apoptotic cerebellar granule neurons
Primary cerebellar granule neurons represent a widely used
in vitro model system that mimics the trophic action of
neuronal activity that is seen in the developing nervous system
(D'Mello et al., 1993; Miller et al., 1997). Thus, elevated levels of
extracellular potassium promote neuronal survival by opening L-type
voltage-sensitive calcium channels, leading to an influx of calcium
into neurons. Lowering of extracellular potassium decreases calcium
influx and promotes neuronal cell death by an apoptotic mechanism.
Western analysis indicated that all four members of the MEF2
superfamily of transcription factors were present in cerebellar granule
cells (Fig. 1A). When
neurons were induced to undergo apoptosis by lowering extracellular
potassium, there were selective shifts in the mobility of MEF2A (Fig.
1A, top) and MEF2D (Fig. 1A, bottom) on SDS gels. The mobility
shifts were detected in neuronal cell lysates as early as 30 min and
sustained to 8 hr. The shifts in mobility of MEF2A and MEF2D were
enhanced when the samples were electrophoresed for longer times through
higher resolution gels (Fig. 1B). Treatment of
neuronal protein extracts with calf intestinal alkaline phosphatase
before electrophoresis reversed the mobility shift of MEF2A and MEF2D
seen in the low potassium conditions, confirming that the shifts in
mobility of MEF2A and MEF2D were attributable to enhanced
serine/threonine phosphorylation (Fig. 1C). The
phosphorylation sites responsible for the increase in phosphorylation
seen on lowering intracellular calcium are functionally different from
those previously reported to enhance transcription. Both a p38
inhibitor (10 µM SB203580) and a MEK inhibitor
(10 µM PD98059) failed to block the increase in
phosphorylation seen with the induction of apoptosis (data not shown).
The relative intensities of the slower-migrating MEF2A and MEF2D
proteins decreased after 4 and 8 hr in low potassium medium (Fig.
1A, lanes 6, 8), whereas the
faster-migrating MEF2 proteins observed under depolarizing conditions
remained constant, suggesting a link between MEF2 phosphorylation and
degradation.
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Figure 1.
MEF2D and MEF2A, but not MEF2B and MEF2C, are
phosphorylated and degraded during the apoptosis of cerebellar granule
neurons. A, Cerebellar granule neurons (day 7) were
placed in serum-free medium containing 25 or 5 mM KCl for
the indicated times. Neuronal cell lysates were resolved on 7.5%
SDS-acrylamide gels and subjected to Western analysis with the use of
specific antibodies to MEF2A, MEF2B, MEF2C, and MEF2D. Data are
representative of three separate experiments. B, Cell
extracts were prepared as described above and analyzed on higher
resolution gels. C, Cell extracts were incubated in the
absence or presence of calf intestinal alkaline phosphatase
(CIAP; 10 U/ml) before gel electrophoresis.
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MEF2D transcriptional activity is regulated by
extracellular potassium
The transcriptional activity of MEF2 proteins in cerebellar
granule neurons was measured with a luciferase reporter plasmid that
contains two MEF2 consensus sites, followed by the luciferase reporter
gene (Lemercier et al., 2000). Cerebellar granule neurons grown in the
presence of depolarizing potassium (25 mM) demonstrated high endogenous MEF2-driven luciferase activity (Fig.
2A). After the
potassium was lowered to 5 mM for 4-8 hr, the
transcriptional activity of the MEF2 reporter was decreased by ~90%
(p < 0.05) (Figure 2A).
Incubation in 5 mM potassium for 4 hr, followed
by the readdition of 25 mM potassium for an
additional 4 hr, restored ~50% of the MEF2 transcriptional activity
(Fig. 2A). Similarly, the readdition of 25 mM potassium also reversed the mobility shift in
MEF2D (Fig. 2B), suggesting that phosphorylation of
MEF2D during apoptosis is associated with the observed decrease in MEF2
transcriptional activity. The fact that only a partial recovery of
MEF2-driven luciferase activity was observed in the above experiment
could be accounted for by the significant degradation of MEF2D that had
occurred after 4 hr in 5 mM potassium (Fig.
2B, lane 1 vs lane 3). Finally,
transfection of neurons with MEF2D-VP16, a constitutively active mutant
of MEF2D, attenuated the decline of MEF2 luciferase activity during
potassium withdrawal (Fig. 2C). The decrease in MEF2
transcriptional activity was not attributable to a nonselective effect
of cell death, because the transcriptional activity of AP-1 measured
with an AP-1 luciferase reporter construct was increased by 30% under
the same conditions by which the MEF2 transcriptional activity
decreased (data not shown).
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Figure 2.
Neurons switched to 5 mM KCl show
enhanced MEF2D phosphorylation and decreased MEF2 transcriptional
activity; the readdition of 25 mM KCl promotes the
dephosphorylation of MEF2D and the partial recovery of MEF2
transcriptional activity. A, Cerebellar granule neurons
(day 6) were transfected with a MEF2-responsive luciferase reporter and
pCMV--gal. After transfection (2 hr) the neurons were placed in
serum-free medium containing either 25 or 5 mM KCl for 8 hr. In addition, some cells were incubated for 4 hr in 5 mM
KCl, followed by the readdition of 25 mM KCl for an
additional 4 hr. Then luciferase and -galactosidase activities were
determined as described in Materials and Methods. B,
Cerebellar granule neurons were incubated as described in
A, and the phosphorylation status and relative quantity
of MEF2D were determined by immunoblot (IB) analysis.
C, Cerebellar granule neurons were transfected with a
MEF2-responsive luciferase reporter and pCMV--gal in the absence or
presence of pCMV-MEF2D-VP16. After 4 hr the luciferase and
-galactosidase activities were determined. Data are expressed as a
percentage of control neurons grown in 25 mM KCl and are
the mean ± SEM (n = 3).
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DNA binding of MEF2A and MEF2D is regulated by
extracellular potassium
To determine whether the DNA binding activity of MEF2 proteins was
regulated during induction of the apoptosis of cerebellar granule
neurons, we performed electrophoretic mobility shift assays (EMSAs) by
using wild-type and mutant MEF2 double-stranded oligonucleotides. Nuclear extracts from cerebellar granule neurons cultured in medium containing 25 mM KCl demonstrated one specific
high-mobility protein-DNA complex, HMC (Fig.
3). Extracts from neurons cultured in 5 mM KCl revealed a decrease in the HMC that was detected as
early as 2 hr after the potassium was lowered. In addition to the
decrease in the HMC associated with the apoptotic response, a new
lower-mobility MEF2-DNA binding complex, LMC, was detected in extracts
isolated 4 and 6 hr after the media change. Neither the HMC nor the LMC was detected by using a mutant MEF2 oligonucleotide.
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Figure 3.
MEF2 DNA binding activity is decreased in
apoptotic neurons. Cerebellar granule neurons (day 7) were placed in
serum-free medium containing 25 or 5 mM KCl. After the
indicated times, nuclear extracts were prepared, and gel mobility shift
assays were performed with a double-stranded 32P-labeled
consensus (wt) or mutant (mut) MEF2
oligomer. NS, Nonspecific protein/DNA complex;
HMC, high-mobility complex; LMC,
low-mobility complex.
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EMSAs that used antibodies against MEF2A, B, C, and D proteins were
performed to determine which MEF2 proteins were bound to DNA under
control and apoptotic conditions (Fig.
4). In control neurons, antibodies
against MEF2A and MEF2D shifted the HMC to a slower migrating band
(SSB), whereas antibodies against MEF2B and MEF2C had little effect on
the MEF2-DNA complex. Similar results were obtained in apoptotic
neurons, although the amount of high-mobility complex was significantly
lower than that present in healthy neurons. Interestingly, the LMC was
not shifted by any of the MEF2 antibodies. Because each of the
antibodies was raised against the C terminus, but not the N terminus
DNA binding domain of MEF2 proteins (Fig. 5A), we questioned whether
MEF2A and MEF2D were degraded during apoptosis to yield MEF2 fragments
that bound DNA but did not interact with the antibodies.
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Figure 4.
MEF2A and MEF2D are the major MEF2s in the
high-molecular-weight DNA binding complex. Cerebellar granule neurons
(day 7) were placed in serum-free medium containing 25 or 5 mM KCl. After 4 hr, nuclear extracts were prepared, and
supershift gel mobility shift assays were performed with a
double-stranded 32P-labeled consensus MEF2 oligomer in the
absence or presence of MEF2 antibodies. Note that the
lower-molecular-weight complex did not shift with MEF2 antibodies.
SSB, Supershifted band; HMC,
high-mobility complex; LMC, low-mobility complex;
NS, nonspecific protein/DNA complex.
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Figure 5.
MEF2D is cleaved by a caspase-mediated pathway
during apoptosis. A, Domain structure of the MEF2
proteins, including the MEF2A and MEF2D antibody recognition motifs and
the putative caspase cleavage region. B, Cerebellar
granule neurons (day 7) were placed in serum-free medium containing 25 or 5 mM KCl. At the indicated times, Western analysis was
performed with 12% SDS-acrylamide gels and specific MEF2D antibodies.
C, Neurons were placed in serum-free medium containing
25 or 5 mM KCl (4 hr) in the absence or presence of various
concentrations of the caspase-3-specific inhibitor DEVD or the
pan-caspase inhibitor Z-VAD. Brackets indicate a
lower-molecular-weight cleavage product recognized by the C-terminal
MEF2D antibody.
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Degradation of MEF2A and MEF2D during apoptosis of cerebellar
granule neurons
To test the hypothesis that MEF2 proteins were degraded during
apoptosis, we performed Western analysis and exposed the transferred proteins to film for longer periods of time than in previous
experiments. The results showed that, within 1 hr of inducing
apoptosis, MEF2D was phosphorylated, as shown previously, and a
smaller-molecular-weight MEF2D fragment appeared (Fig. 5B).
The fragment, usually a broad band, had an apparent molecular weight of
40-45 kDa, ~15 kDa less than full-length MEF2D.
Analysis of various MEF2 sequences (rat MEF2D and mouse MEF2D1a and
MEF2A) revealed several putative caspase cleavage sites (DXXD) between
the DNA binding domain and the antibody recognition domain (amino acids
100-346) (Fig. 5A). To determine whether the MEF2 fragment
that was generated during apoptosis resulted from caspase cleavage, we
performed experiments to assess whether caspase inhibitors blocked
formation of the MEF2D fragment. DEVD, a caspase-3-specific inhibitor,
and Z-VAD, a nonselective caspase inhibitor, blocked the formation of
the MEF2D fragment in a dose-dependent manner (Fig. 5C).
Z-VAD was much more effective and potent than DEVD in preventing
cleavage of MEF2D, suggesting that an upstream caspase rather than the
downstream caspase-3 might be involved in cleaving MEF2D. Consistent
with this idea was the finding that the cleavage of MEF2D occurred much
earlier than the activation of caspase-3, which was maximal at 5-6 hr
after the induction of apoptosis (Fig. 6).
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Figure 6.
Caspase-3 activation in rat cerebellar granule
neurons. Cerebellar granule neurons (day 7) were placed in serum-free
medium containing 25 or 5 mM potassium. At the indicated
times, Western analysis was performed with 15% SDS-acrylamide gels and
an antibody that detects pro-caspase-3 and an active caspase-3 cleavage
product.
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Caspase inhibitor blocks formation of the low-mobility
protein-DNA complex
Blockade of MEF2D cleavage by caspase inhibitors supported the
hypothesis that during apoptosis MEF2D is cleaved to generate a
N-terminal DNA binding fragment, which is not recognized by antibodies
directed against the regulatory domain, and a C-terminal fragment that
is recognized by Western blotting. To test this hypothesis directly, we
treated neurons with and without a caspase inhibitor before the EMSAs
(Fig. 7). As shown previously in nuclear extracts from control neurons grown in 25 mM KCl, the LMC
was very low (Fig. 7, lane 1). After 4 hr of apoptosis
induced by lowering the extracellular potassium to 5 mM, the LMC was abundant (Fig. 7, lane
2). The caspase inhibitor Z-VAD, added at the time of the medium
change, prevented the formation of the lower-molecular-weight complex
(Fig. 7, lane 3). In addition, DVED and YVAD (a
caspase-1-selective inhibitor) were only partially effective at
inhibiting the formation of the low-molecular-weight complex (data not
shown). Together, these data suggest that MEF2D and MEF2A are cleaved
by a caspase-sensitive pathway to generate N-terminal fragments that
bind to DNA but are not recognized by antibodies directed to the C
terminus of each of these proteins.
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Figure 7.
Formation of the lower-molecular-weight
protein/DNA binding complex is prevented by caspase inhibition.
Cerebellar granule neurons (day 7) were placed in serum-free medium
containing 25 or 5 mM KCl in the absence or presence of 100 µM pan-caspase inhibitor (Z-VAD). After 4 hr, nuclear extracts were prepared, and gel mobility shift assays were
performed with a double-stranded 32P-labeled consensus MEF2
oligomer. HMC, High-mobility complex;
LMC, low-mobility complex; NS,
nonspecific protein/DNA complex.
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The N-terminal MEF2 fragment can act as a dominant-negative
transcription factor
Cleavage of MEF2 between the DNA binding domain and the antibody
recognition domain would separate the DNA binding domain from the
transactivation domain. To test the possibility that the N-terminal
truncated fragment could act in a dominant-negative manner to block
both the DNA binding and transcriptional activity of MEF2, we
transfected neurons with MEF2D-VP16 in the absence or presence of
increasing amounts of truncated MEF2A131. The expression plasmid
MEF2D-VP16 encodes the DNA binding domain of mouse MEF2D (amino acids
1-92) fused to the transcriptional activation domain of VP16 (amino
acids 412-490) under control of a CMV promoter. Its use as a
constitutively active transcription factor has been reported previously
(Han and Prywes, 1995). MEF2A131 encodes mouse MEF2A that is truncated
at position 131, leaving the DNA binding domain intact, and is highly
homologous to the corresponding region of MEF2D. After cotransfection
of these two expression plasmids, transcriptional activity was
determined with the MEF2 luciferase reporter plasmid (Fig.
8A). As observed
previously, neurons incubated in 5 mM potassium
had low MEF2 transcriptional activity. However, the expression of a
constitutively active mutant of MEF2D (MEF2D-VP16) maintained high MEF2
transcriptional activity even in the presence of 5 mM potassium. When the VP16 mutant of MEF2D (1 µg) was expressed in the presence of increasing concentrations of
truncated MEF2A131 (1-3 µg of DNA), the N-terminal MEF2 fragment
blocked MEF2 luciferase activity in a dose-dependent manner (Fig.
8A). These data indicate that a truncated MEF2 can
block the DNA binding and activity of MEF2 competitively. The
consequence of the expression of truncated MEF2 on neuronal apoptosis
is seen in Figure 8B. In these experiments, neurons
were transfected with the control vector, MEF2D-VP16, or MEF2D-VP16 in
the presence of increasing amounts of truncated MEF2A131. In neurons
maintained in 5 mM potassium for 16 hr the level
of apoptosis was 56%. Transfection of the neurons with MEF2D-VP16 reduced the amount of apoptosis to ~22%. When the truncated MEF2A131 mutant (1-3 µg of DNA) was introduced with MEF2D-VP16 into neurons, the ability of MEF2D-VP16 to block apoptosis was attenuated in a
dose-dependent manner. These data indicate that the MEF2 N-terminal fragment can act as a dominant-negative transcription factor and antagonize the prosurvival function of MEF2D.
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Figure 8.
The N terminus of MEF2 antagonizes MEF2 activity
and MEF2-mediated neuronal survival. A, Cerebellar
granule neurons (day 6) were transfected with a MEF2-responsive
luciferase reporter and pCMV--gal in the absence or presence of 1 µg of MEF2D-VP16 and MEF2A131 (1-3 µg). After transfection (2 hr)
the neurons were placed in serum-free medium containing 5 mM KCl. After 4 hr, luciferase and -galactosidase
activities were determined. Luciferase activity was normalized with
respect to that of -galactosidase. Data are expressed as percentage
of control neurons grown in 25 mM potassium. Data are the
mean ± SEM (n = 3). B,
Cerebellar granule neurons (day 5) were cotransfected with pCMV--gal
and the indicated expression vector at the concentrations given in
A. After transfection the neurons were placed in
serum-free medium containing 5 mM KCl for 16 hr and then
fixed and immunostained with a -gal antibody. The neurons also were
stained with Hoechst 33258. Apoptosis was quantified by scoring the
percentage of transfected neurons with condensed or fragmented nuclei.
Data are the mean ± SEM (n = 3).
Vector, Empty pcDNA vector.
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| |
DISCUSSION |
Examination of the temporal and spatial localization of MEF2
proteins in brain has revealed that MEF2 expression coincides with the
initiation of postmitotic neuronal maturation (Leifer et al., 1993,
1994; McDermott et al., 1993; Ikeshima et al., 1995; Lyons et al.,
1995; Mao et al., 1999). For example, in the developing cerebral
cortex, MEF2C immunoreactivity is present in the cortical plate and is
not found in the intermediate zone or ventricular zone (Mao et al.,
1999). At 14 weeks of gestation, MEF2C immunoreactivity is present in
cell nuclei throughout the cortical plate. Subsequently, MEF2C
immunoreactivity develops a bilaminate and then a trilaminate distribution and ultimately is expressed preferentially in layers II,
IV, and VI of mature neocortex (Leifer et al., 1994). These findings
suggest a role for MEF2C in postmitotic neuronal differentiation, in
particular in the development of certain cortical layers.
In the cerebellum, MEF2A and MEF2D mRNA levels dramatically increase at
~P9, reach a peak at P15-P18, and stay high in adults (Leifer et
al., 1994; Ikeshima et al., 1995; Lin et al., 1996; Mao and Wiedmann,
1999). This time course of MEF2 expression coincides with the
expression of the GABAA receptor 6 subunit
mRNA, a marker for the differentiation of mature cerebellar granule
neurons. Immunohistochemical staining reveals that MEF2 protein
expression occurs primarily in the internal granule cell layer of the
cerebellum (Ikeshima et al., 1995). During postnatal development,
differentiated granule neurons generated in the external germinal layer
migrate to the internal granule layer where they are innervated by
mossy fiber axons. There is considerable loss of granule neurons during this process and it is thought that the survival of granule neurons is
regulated by depolarization-induced mechanisms during this time period.
Postmitotic granule neurons derived from neonatal rat can be maintained
readily in vitro in their fully differentiated state if they
are depolarized with a high extracellular concentration of potassium
(D'Mello et al., 1993; Miller et al., 1997). If the extracellular
potassium concentration is reduced, granule neurons undergo programmed
cell death with classic morphological and biochemical features of
apoptosis. These characteristics, along with the abundance and high
degree of homogeneity, make cultured granule neurons an excellent model
to examine the role of MEF2 proteins in depolarization-dependent neuronal survival.
In this report we have defined a novel mechanism by which the activity
and levels of MEF2 proteins are regulated during apoptosis of
cerebellar granule neurons. We also have shown that MEF2 proteins are
regulated in an isotype-specific manner. All four MEF2 isoforms (A, B,
C, and D) were detected by immunoblot analysis in rat cerebellar granule neurons. However, in agreement with results from
immunocytochemistry and in situ hybridization (Ikeshima et
al., 1995; Lyons et al., 1995), MEF2A and MEF2D were the most prominent
MEF2 proteins detected in the cultured cerebellar granule neurons when
equal amounts of total protein were analyzed by Westerns (data not
shown). MEF2A and MEF2D appear to be responsible for most, if not all,
of the MEF2 DNA binding activity in viable cerebellar granule neurons. When granule neurons are induced to undergo apoptosis by lowering potassium, endogenous MEF2A and MEF2D, but not MEF2B and MEF2C, are
phosphorylated. Phosphorylation is accompanied by a decrease in MEF2
transcriptional activity, and both effects are reversed by the
readdition of depolarizing potassium. The most novel findings of the
present study show that the phosphorylation of MEF2D that is induced by
decreasing calcium influx not only correlates with decreased DNA
binding but also is associated with a direct or indirect
caspase-dependent cleavage of MEF2D (Fig.
9). The cleavage of MEF2D results in a
N-terminal fragment (~100 amino acids long) that retains its DNA
binding capacity, is not recognized by C-terminal antibodies, and lacks
the transactivation domain. The MEF2 N-terminal fragment is capable of
blocking the activity of MEF2D, thus acting as a dominant-negative
transcription factor. The decline in MEF2 activity resulting from
decreased DNA binding and formation of a dominant-inactive MEF2
fragment appears to be sufficient to mediate execution of the apoptotic
process. Overexpression of a constitutively active MEF2D that does not
get cleaved prevents the loss in MEF2 transcriptional activity during
apoptosis and protects against apoptosis that is induced by lowering
membrane depolarization and calcium influx.
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|
Figure 9.
The regulation of MEF2 proteins during apoptosis
of rat cerebellar granule neurons. When granule neurons are induced to
undergo apoptosis by lowering extracellular potassium to 5 mM, endogenous MEF2D and MEF2A (data not shown) are
phosphorylated. Phosphorylation of MEF2D induced by decreasing calcium
influx not only leads to decreased DNA binding but also is associated
with a caspase-dependent cleavage of MEF2D. The caspase involved in
cleaving MEF2D remains to be identified. The cleavage of MEF2D results
in an N-terminal fragment (~100 amino acids long) that retains its
DNA binding capacity, is not recognized by C-terminal antibodies, and
lacks the transactivation domain. The MEF2 N-terminal fragment is
capable of blocking the activity of MEF2D, thus acting as a
dominant-negative transcription factor. The decline in MEF2 activity
because of decreased DNA binding and formation of a dominant-inactive
MEF2 fragment leads to apoptosis.
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The signaling pathways responsible for the changes in the
phosphorylation state of MEF2 could involve an increase in the activity of a calcium-sensitive kinase, a decrease in the activity of a calcium-sensitive phosphatase, or both. Mao and Wiedman (1999) recently
reported similar data that MEF2A is hyperphosphorylated when calcium
influx is decreased or when the protein phosphatase calcineurin is
inhibited in cerebellar granule neurons. Although other isoforms were
not examined in the previous study, the data suggest that enhanced
phosphorylation of MEF2A and MEF2D seen on lowering extracellular
potassium is likely to be attributable to decreased activity of the
calcium-dependent phosphatase calcineurin. Furthermore, because MEF2B
and MEF2C did not undergo hyperphosphorylation in response to lowering
extracellular potassium, the data indicate that MEF2A and MEF2D are
regulated post-translationally in an isotype-specific manner in
cerebellar granule neurons.
The putative phosphorylation sites described in this study are
functionally distinct from the previously described phosphorylation sites that enhance MEF2 transcriptional activity. Some MEF2 isoforms directly interact with p38 MAP kinase and ERK5/BMK1 and are
phosphorylated by both protein kinases (Kato et al., 1997; Yang et al.,
1998; Ornatsky et al., 1999; Zhao et al., 1999). Phosphorylation of MEF2 proteins by p38 MAP kinase and ERK5 stimulates transcriptional activity. The increased transcriptional activity could involve changes
in protein conformation that enhance interaction with the
transcriptional machinery. Alternatively, phosphorylation might be
required for the recruitment of an essential transcriptional cofactor
or release of a repressor. In support of the latter mechanism, histone
deacetylases (HDACs) have been shown to repress the transcriptional activity of MEF2s (Miska et al., 1999; Lemercier et al., 2000; Lu et
al., 2000a,b; Youn et al., 2000). Phosphorylation of HDACs by the
calcium-sensitive protein kinase CaMK results in the dissociation of
HDACs and the unmasking of transcriptional activity.
Other studies in T-cells have mapped a calcineurin-dependent induction
of the nur77 promoter to a putative MEF2 DNA binding site (Youn et al.,
1999). Although the transcriptional activity of MEF2 in activated
T-cells requires calcium signals, its DNA binding activity seems to be
constitutive and insensitive to changes in calcium. Again, the data are
in contrast to the results of this study in which decreases in
intracellular calcium signaling resulted in decreased DNA binding and
transcriptional activity. The antibodies used in T-cells examining
nur77 promoter activity did not distinguish between MEF2 isoforms. This
raises the possibility that the discrepancy may be attributable to
differential regulation of the various MEF2 isoforms and/or the
presence of cell type-specific accessory proteins.
In summary, the complexities in MEF2-regulated gene expression have
been advanced primarily from studies in muscle and T-cells. In the
present study we have delineated a novel phosphorylation signaling
pathway associated with DNA binding, transcriptional activity, and
degradation of neuronal MEF2 proteins. The data in this study also
support the hypothesis that MEF2A and MEF2D regulate neuronal survival
in the cerebellum, particularly in response to depolarization-induced
signals that are important during development.
| |
FOOTNOTES |
Received Feb. 9, 2001; revised June 15, 2001; accepted June 21, 2001.
This research was supported by United States Army Medical Research and
Materiel Command Grant DAMD17-99-1-9481, by National Institutes
of Health Grant NS38619-01A1, and by a Veterans Affairs Merit Award and
Research Enhancement Award Program Award to KA.H.
M.L. and D.A.L. contributed equally to this manuscript.
Correspondence should be addressed to Dr. Kim A. Heidenreich, Denver
Veterans Affairs Medical Center-111H, 1055 Clermont Street, Denver, CO
80220. E-mail: kim.heidenreich{at}uchsc.edu.
| |
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B. Gaudilliere, Y. Shi, and A. Bonni
RNA Interference Reveals a Requirement for Myocyte Enhancer Factor 2A in Activity-dependent Neuronal Survival
J. Biol. Chem.,
November 22, 2002;
277(48):
46442 - 46446.
[Abstract]
[Full Text]
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H. Bryant and P. J. Farrell
Signal Transduction and Transcription Factor Modification during Reactivation of Epstein-Barr Virus from Latency
J. Virol.,
September 11, 2002;
76(20):
10290 - 10298.
[Abstract]
[Full Text]
[PDF]
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S.-i. Okamoto, Z. Li, C. Ju, M. N. Scholzke, E. Mathews, J. Cui, G. S. Salvesen, E. Bossy-Wetzel, and S. A. Lipton
Dominant-interfering forms of MEF2 generated by caspase cleavage contribute to NMDA-induced neuronal apoptosis
PNAS,
March 19, 2002;
99(6):
3974 - 3979.
[Abstract]
[Full Text]
[PDF]
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ABSTRACT
INTRODUCTION
Gene
