From the Department of Environmental Medicine and
¶ Department of Pharmacology and Physiology, University of
Rochester School of Medicine and Dentistry,
Rochester, New York 14642
Received for publication, September 13, 2000, and in revised form, October 27, 2000
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ABSTRACT |
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Sympathetic neurons undergo protein
synthesis-dependent apoptosis when deprived of nerve growth
factor (NGF). Expression of SM-20 is up-regulated in NGF-deprived
sympathetic neurons, and ectopic SM-20 is sufficient to promote
neuronal death in the presence of NGF. We now report that SM-20 is a
mitochondrial protein that promotes cell death through a
caspase-dependent mechanism. SM-20 immunofluorescence was
present in the cytoplasm in a punctate pattern that colocalized with
cytochrome oxidase I and with mitochondria-selective dyes. Analysis of
SM-20/dihydrofolate reductase fusion proteins revealed that the first
25 amino acids of SM-20 contain a functional mitochondrial targeting
sequence. An amino-terminal truncated form of SM-20 was not restricted
to mitochondria but instead localized throughout the cytosol and
nucleus. Nevertheless, the truncated SM-20 retained the ability to
induce neuronal death, similar to the wild type protein. SM-20-induced
death was accompanied by caspase-3 activation and was blocked by a
general caspase inhibitor. Additionally, overexpression of SM-20, under
conditions where cell death is blocked by a general caspase inhibitor,
did not result in widespread release of cytochrome c from
mitochondria. These results indicate that SM-20 is a novel
mitochondrial protein that may be an important mediator of
neurotrophin-withdrawal-mediated cell death.
Sympathetic neurons undergo apoptosis when deprived of their
survival factor, nerve growth factor
(NGF).1 Inhibitors of RNA or
protein synthesis block this death, suggesting that gene expression is
important for apoptosis in these cells (1, 2). Consistent with this
idea, a small number of genes have been shown to increase in expression
in neurons after NGF withdrawal (3-5). Among these, c-jun
and cyclin D1 have been implicated as upstream regulators of neuronal
death (4-9). Recently, we found that expression of the SM-20 gene
is also up-regulated in NGF-deprived sympathetic neurons (10). SM-20
expression is also induced in sympathetic neurons treated with the
anti-tumor agent cytosine arabinoside or with inhibitors of
phosphatidylinositol 3-kinase, agents that cause apoptosis in the
presence of NGF (11, 12). Although a requirement for SM-20 in cell
death remains to be established, overexpression of SM-20 is sufficient
to induce cell death in neurons maintained in the continual presence of NGF (10). These findings suggest that SM-20 may function as a regulator
of neuronal death.
Regulation of SM-20 expression also occurs in muscle cells and in
response to activation of the p53 protein in fibroblasts. SM-20
mRNA levels increase in vascular smooth muscle cells stimulated with growth factors and are up-regulated when muscle cells are induced
to differentiate (13, 14). In vivo, SM-20 mRNA levels are elevated in muscle cells in response to injury to the blood vessel
wall (15). SM-20 expression also increases after activation of a
temperature-sensitive p53 protein in rat embryo fibroblasts (16).
Activation of p53 in these cells results in both growth arrest and
apoptosis. Interestingly, stable expression of SM-20 in tumor cells
lacking functional p53 resulted in greatly reduced numbers of colonies
in colony formation assays, suggesting that SM-20 might act downstream
of p53 in either growth-arrest or apoptosis (16).
Although the biochemical function of the SM-20 protein is unknown, it
contains a 218-amino acid region that is closely related to sequences
in the Caenorhabditis elegans Egl-9 protein (17). Mutations
in the egl-9 gene had been originally shown to disrupt normal egg laying in nematodes (18). More recently, egl-9
was implicated as a mediator of toxin-induced neuromuscular paralysis in C. elegans infected with the pathogenic bacterium
Pseudomonas aeruginosa (17).
To help elucidate the function of SM-20, its subcellular localization
was investigated. We report that the amino terminus of SM-20 contains a
mitochondrial targeting sequence that localizes SM-20 to mitochondria.
Because mitochondria have a prominent role in the regulation of cell
death (19), we tested the significance of the mitochondrial targeting
of SM-20 on its ability to promote neuronal death. Our results indicate
that targeting of SM-20 to mitochondria is not required for
SM-20-induced death. In addition, we find that caspase-3 activation
occurs during SM-20-induced cell death. In contrast to Bax, expression
of SM-20 in neurons maintained in the presence of a general caspase
inhibitor did not lead to detectable cytochrome c release
from mitochondria. These results indicate that SM-20 is a novel
mitochondrial protein that may be an important mediator of
neurotrophin-withdrawal mediated cell death.
Cell Culture--
CV-1 (ATCC; Manassas, VA) and NIH3T3 cells
were maintained in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) supplemented with 10% fetal bovine serum (Hyclone;
Indianapolis, IN), 100 units/ml penicillin, and 100 µg/ml
streptomycin (all from Life Technologies, Inc.). Primary cultures of
sympathetic neurons were prepared from the superior cervical ganglia of
embryonic day 21 rats as described previously (12). Cultures were
maintained in vitro for 5-6 days in media consisting of
90% minimal essential medium, 10% fetal bovine serum (Harlan
Bioproducts; Madison, WI), 2 mM L-glutamine, 20 µM uridine, 20 µM fluorodeoxyuridine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 50 ng/ml NGF (Harlan Bioproducts).
Expression Vectors and Transient Transfections--
The
expression vectors containing the SM-20 open reading frame in
Rc/cytomegalovirus (CMV) (Invitrogen; Carlsbad, CA) and the
SM-20/green fluorescent protein (GFP) cDNA in
pcDNA3 (Invitrogen) were described previously (10). For
expressing T7 epitope-tagged forms of SM-20, dihydrofolate reductase
(DHFR), SM-20(60-355), and SM-20/DHFR fusions,
complementary oligonucleotides encoding the T7 epitope tag (Novagen;
Madison, WI) flanked by restriction sites for
EcoRI-NdeI (5') and EcoRV (3') were
annealed and ligated into Bluescript KS+ (Stratagene; La Jolla, CA)
between the EcoRI and EcoRV sites. The various
cDNAs were inserted into the T7-modified Bluescript vector upstream
of the T7 epitope using EcoRI and NdeI. The SM-20
sequences used to make SM-20(60-355), SM-20
(1-25)/DHFR, and SM-20(1-50)/DHFR encoded an
amino-terminal methionine and were generated by polymerase chain
reaction using Pfu polymerase (Stratagene). The
EcoRI to EcoRV fragments containing each cDNA
fused at its 3' end to the T7 epitope were then subcloned into
pcDNA3. For SM-20(60-355)/GFP, a DNA
fragment consisting of an amino-terminal methionine followed by amino
acids 60-355 of SM-20 was amplified using Pfu polymerase
fused to the 5' end of GFP and inserted into pcDNA3 between the EcoRI and EcoRV sites. All amplified
DNAs were sequenced to confirm that they were correct.
CV-1 cells (2 × 105 cells) were plated onto glass
coverslips in 35-mm plastic tissue culture dishes 24 h before
transfection with LipofectAMINE (Life Technologies, Inc.) as described
by the manufacturer. NIH3T3 cells (1 × 107 cells)
were plated onto 100-mm tissue culture dishes and transfected with
LipofectAMINE. Cells were used for experiments on the second day after transfection.
Mitochondrial Labeling--
CV-1 cells were incubated for 5 h in media containing 1 µM Mitotracker Green FM
(Molecular Probes; Eugene, OR), a mitochondria-selective dye that is
retained in fixed cells. To establish the specificity of this dye for
labeling mitochondria, cells were plated on grid coverslips and then
stained with Mitotracker Green FM as described above. After staining,
the cells were visualized using a fluorescein isothiocyanate (FITC)
filter set ( Immunofluorescence--
CV-1 cells were fixed and permeabilized
with 4% paraformaldehyde containing 0.2% Triton X-100 in
phosphate-buffered saline (PBS) for 10 min and then incubated in block
buffer consisting of 3% goat serum and 0.2% bovine serum albumin in
PBS. To detect SM-20, DHFR, or SM-20/DHFR proteins, cells were
incubated with either a 1:200 dilution of rabbit polyclonal anti-SM-20
antibody (10) or 0.2 µg/ml anti-T7 monoclonal antibody (Novagen) in
block buffer. Coverslips were then rinsed with PBS and incubated with TRITC-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (Jackson ImmunoResearch; West Grove, PA) at 15 µg/ml in
block buffer. The cells were counterstained with Hoechst 33258 (Molecular Probes) and then mounted onto slides for viewing by epi-fluorescence with a Nikon Diaphot 300 microscope. Digital images
were captured using a Dage-MTI CCD camera and Scion Image software.
For dual immunofluorescence, CV-1 cells were first incubated with
IgG-purified anti-SM-20 primary antibody and TRITC-conjugated goat
anti-rabbit secondary antibody (Jackson ImmunoResearch) as described
above. After extensive washing of the coverslips, antibodies to
cytochrome oxidase I (A-6403; Molecular Probes) or cytochrome oxidase
VI (A-6401; Molecular Probes) were added at 1 µg/ml or 4 µg/ml in
block buffer, respectively, followed by FITC-conjugated goat anti-mouse
IgG at 15 µg/ml in block buffer. Secondary antibodies used in the
double-labeling experiments were cross-absorbed against mouse or rabbit IgG.
For visualization of active caspase-3 and cytochrome c,
sympathetic neurons were fixed with 4% paraformaldehyde in PBS for 30 min at 4 °C and then incubated in block buffer consisting of 5%
goat serum and 0.3% Triton X-100. Primary antibodies to active caspase-3 (Pharmingen; San Diego, CA) and cytochrome c
(Pharmingen) were added at 0.5 µg/ml in PBS containing 1% goat serum
and 0.3% Triton X-100, and the neurons were labeled overnight at
4 °C. The appropriate FITC-conjugated secondary antibodies were
diluted to 15 µg/ml in PBS containing 1% goat serum and 0.3% Triton
X-100, and the cultures were incubated for 2 h. Cultures were then
rinsed in PBS and counterstained with Hoechst 33342 (Molecular Probes).
Subcellular Fractionation, Immunoprecipitation, and
Immunoblotting--
CV-1 cells stably expressing SM-20 were
preincubated for 30 min in media lacking cysteine and methionine and
then incubated for 4 h in the same media containing 0.5 mCi/ml
Tran35S-label (ICN; Costa Mesa, CA) and 10%
dialyzed calf serum. For preparing whole cell lysates, cells were
rinsed twice with cold PBS and then lysed in radioimmune precipitation
(RIPA) buffer (50 mM Tris (pH 8.0), 150 mM
NaCl, 1% Nonidet-P40, 1% deoxycholate, 0.1% SDS, 0.1 mM
phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml
aprotinin) for 15 min at 4 °C. Cell lysates were clarified by
centrifugation at 10,000 × g for 10 min, preabsorbed
with protein A-Sepharose (Amersham Pharmacia Biotech), and then
incubated with anti-SM-20 antibody or, in some cases, antibody that had
been preincubated with excess blocking peptide. Immune complexes were precipitated with protein A-Sepharose, washed four times with RIPA
buffer buffer, separated by 12.5% SDS-polyacrylamide gel electrophoresis, and analyzed using a PhosphorImager and ImageQuant software (Molecular Dynamics; Sunnyvale, CA).
Mitochondrial-enriched fractions were prepared essentially as described
by others (21, 22). For these experiments, NIH3T3 cells were used
rather than CV-1 cells because higher levels of SM-20 expression were
obtained (note that both CV-1 and NIH3T3 cells showed the same pattern
of SM-20 immunofluorescence). Cells were transiently transfected with
SM-20/Rc-CMV or an empty control vector and metabolically labeled as
described above. After labeling, each dish was rinsed twice with PBS
before adding ice-cold homogenization buffer (17 mM MOPS,
2.5 mM EDTA, 250 mM sucrose, 0.1 mM
phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, and 5 µg/ml
aprotinin). The cells were then scraped from the dishes, homogenized in
a Dounce homogenizer (Kontes; Vineland, NJ) for 20-25 strokes, and
then centrifuged at 500 × g for 10 min at 4 °C to
pellet nuclei. The supernatants were then centrifuged at 10,000 × g for 15 min. The resultant pellets from this centrifugation
were resuspended in homogenization buffer and centrifuged again at
10,000 × g for 15 min, yielding a
mitochondria-enriched heavy membrane pellet that also contains lysosomes, golgi, and rough endoplasmic reticulum. The
mitochondria-enriched fraction was resuspended in RIPA buffer and
subjected to immunoprecipitation with anti-SM-20 antibody as described
above. A soluble cytosolic fraction was prepared from the supernatants
obtained after the 10,000 × g spin by further
centrifuging the supernatants at 150,000 × g for 60 min. To verify that the heavy membrane fraction was enriched for
mitochondria, 25 µg of protein from the heavy membrane and cytosolic
fractions were separated by SDS-polyacrylamide gel electrophoresis and
transferred to nitrocellulose membranes. The membranes were blocked in
5% nonfat dry milk in Tris-buffered saline before labeling overnight
with primary antibody against cytochrome c (Pharmingen) at 5 µg/ml in 1× Tris-buffered saline containing 1% nonfat dry milk and
0.05% Tween 20. The blots were then incubated with biotinylated goat
anti-mouse IgG (Jackson ImmunoResearch) at 0.3 µg/ml and detected
using a streptavidin-conjugated alkaline phosphatase assay kit
(Bio-Rad).
Intracellular Microinjections and Quantitation of Cell
Death--
Sympathetic neurons were microinjected with expression
plasmids and scored for viability as described previously (12). Neurons were injected directly into the nucleus with each plasmid at 50 µg/ml
in a buffer containing nonfixable rhodamine-dextran (survival experiments) or lysine-fixable rhodamine-dextran (immunofluorescence experiments) to permit visualization of injected cells. For some experiments, the neurons were treated immediately after microinjection by including the general caspase inhibitor BOC-Asp-CH2F
(BAF) (Enzyme Systems Products; Dublin, CA) at 50 µM in
the culture medium. The number of successfully injected
(rhodamine-positive) neurons was determined 12-15 h after injection.
Three days after injection, cells were stained with the fluorescent DNA
binding dye Hoechst 33342 (Molecular Probes) and evaluated for survival by counting the number of rhodamine-positive cells that were
phase-bright with smooth and intact neurites, a discernible nucleus and
diffusely stained chromatin. In contrast, dying cells are characterized by condensed or undetectable chromatin, fragmented neurites, and atrophic cell bodies. Percent survival is reported as the number of
healthy cells divided by the number of rhodamine-positive cells counted
12-15 h after injection.
Localization of SM-20 to Mitochondria--
To determine where the
SM-20 protein is localized in cells, we first performed
immunofluorescence on transfected cells using a polyclonal anti-peptide
antibody directed against the carboxyl terminus of SM-20. This antibody
specifically recognizes recombinant SM-20 protein, and it
immunoprecipitates SM-20 from cells transfected with an SM-20
expression plasmid but not from cells transfected with an empty vector
(10). CV-1 cells were chosen for these experiments because of their
flattened morphology and large cytoplasmic volume and because transient
expression of SM-20 does not lead to death in these cells (use of
NIH3T3 cells yielded identical results, data not shown). SM-20
immunofluorescence occurred in a punctate pattern throughout the
cytoplasm of expressing cells (Fig. 1,
panels B and E). In contrast, neighboring
untransfected cells or cells transfected with an empty expression
vector did not show detectable immunofluorescence.
The distribution of SM-20 immunofluorescence appeared similar to the
distribution of mitochondria described previously in CV-1 cells (23).
Therefore, experiments were performed to determine whether SM-20
expression colocalizes with known mitochondrial markers. In
dual-labeling experiments, SM-20 immunofluorescence completely
coincided with the labeling generated by a monoclonal antibody that
recognizes cytochrome oxidase subunit I, an inner mitochondrial
membrane protein (Fig. 1, panels A-C). SM-20
immunofluorescence also colocalized with a second mitochondrial
protein, cytochrome oxidase subunit VI (data not shown).
The SM-20 localization pattern was also compared with the staining
pattern of the mitochondria-selective dye Mitotracker Green FM. SM-20
immunofluorescence colocalized perfectly with mitochondria stained by
Mitotracker Green FM (Fig. 1, panels
D-F). The colocalization of SM-20 with two
distinct mitochondrial proteins and a mitochondria-selective dye
strongly suggests that SM-20 is localized to mitochondria in these cells.
Identification of a 33-kDa Processed Form of SM-20 in
Mitochondria--
The SM-20 open reading frame predicts a
355-amino acid protein with a calculated molecular mass of 39.8 kDa
(13). When whole cell lysates were prepared from CV-1 cells stably
expressing SM-20 and immunoprecipitated with anti-SM-20 antibody,
proteins of ~33 and 40 kDa were specifically recovered (Fig.
2A). Pre-incubating the
antibody with the cognate peptide blocked immunoprecipitation of both
proteins. Moreover, proteins of the same sizes were detected in cells
expressing a T7 epitope-tagged form of SM-20 (tagged at its carboxyl
terminus) when an antibody specific for the T7 epitope was used for the
immunoprecipitation (data not shown). These results indicate that both
the 33 and 40 kDa proteins are products of the SM-20 cDNA. Because
antibodies specific for the carboxyl terminus of SM-20 recognize the
33-kDa protein, it most likely corresponds to a processed form of SM-20
lacking amino-terminal sequences.
Proteins that are synthesized in the cytosol and then targeted to
mitochondria often contain mitochondrial-targeting sequences at their
amino termini. Mitochondrial targeting sequences are typically 15-35
residues long, rich in hydroxylated and positively charged amino acids,
and devoid of acidic residues (24). Inspection of the SM-20 protein
sequence revealed that the first 59 amino acids contain 13 hydroxylated
residues, 13 basic residues, and no acidic residues. Because
mitochondrial targeting sequences are often proteolytically removed
during import into mitochondria (25), we suspected that the 33-kDa
protein represented a processed form of SM-20 present in mitochondria.
NIH3T3 cells transiently transfected with SM-20 cDNA or a control
plasmid were used to prepare cytosolic and mitochondria-enriched
protein fractions. Immunoprecipitation of these fractions with
anti-SM-20 antibody revealed that the 33-kDa SM-20 protein was the
predominant form present in the mitochondria-enriched fraction
(Fig. 2B). In contrast, the soluble cytosolic fraction
contained almost exclusively the full-length SM-20 protein. Because the
antibody used to detect SM-20 recognizes its carboxyl terminus, these
results provide evidence that an ~7-kDa polypeptide is removed
from the amino terminus of SM-20 when it is targeted to mitochondria.
SM-20 Contains an Amino-terminal Mitochondrial Targeting
Sequence--
To determine whether the amino terminus of SM-20 is
required for its mitochondrial localization, we constructed a truncated form of SM-20 (SM-20(60-355)) expected to encode a protein
similar in size to the 33-kDa form of SM-20 detected by
immunoprecipitation. As predicted, SM-20(60-355) was not
targeted to mitochondria in CV-1 cells but, instead, was diffusely
dispersed throughout the cytosol and nucleus, similar to DHFR (Fig.
3). Thus the loss of 59 amino-terminal
residues apparently disrupted a mitochondrial targeting sequence within SM-20.
To ascertain whether sequences within the amino-terminal end of SM-20
are capable of targeting a normally cytosolic protein to mitochondria,
the first 50 amino acids of SM-20 were fused to the amino terminus of
DHFR. When expressed in CV-1 cells, the fusion protein
(SM-20(1-50)/DHFR) colocalized with Mitotracker Green FM,
indicating that SM-20(1-50)/DHFR was efficiently targeted
to mitochondria (Fig. 4, panels A-C). A second fusion protein consisting of the first
25 amino acids of SM-20 fused to DHFR (SM-20(1-25)/DHFR)
was also targeted to mitochondria (Fig. 4, panels
D-F). These data indicate that the amino-terminal 25 residues of SM-20 can function as a mitochondrial targeting
sequence.
Mitochondrial Targeting of SM-20 Is Not Required for Its Ability to
Induce Cell Death in Sympathetic Neurons--
SM-20 mRNA levels
and SM-20 protein synthesis increase in sympathetic neurons undergoing
apoptosis (10). Despite this, SM-20 protein levels are still relatively
low in these cells, and this has impeded our attempts at confirming the
localization of the endogenous SM-20 in neurons. To determine whether
SM-20 can be targeted to mitochondria in neurons, we microinjected
sympathetic neurons with a cDNA encoding a SM-20/GFP fusion
protein. The distribution of SM-20/GFP in sympathetic neurons
visualized with confocal microscopy coincided with the labeling pattern
of Mitotracker Orange, a mitochondria-selective dye (Fig.
5, panels
A-C). The mitochondrial labeling was specific to
the full-length SM-20 protein; SM-20(60-355)/GFP was
diffusely localized throughout the cytosol and nucleus and did not
colocalize with Mitotracker Orange (Fig. 5, panels D-F).
Expression of SM-20 in sympathetic neurons maintained in the presence
of NGF induces cell death in approximately half of the microinjected
neurons (10). To determine whether the mitochondrial localization of
SM-20 is necessary for its ability to promote neuronal death, we
microinjected sympathetic neurons with expression plasmids for
SM-20(60-355)/GFP or, as a control, GFP. Three days after
microinjection, most GFP-injected neurons appeared healthy with
phase-bright cell bodies and readily discernible nuclei and nucleoli
(Fig. 6A). In contrast, most
of the SM-20(60-355)/GFP-injected neurons displayed
morphological features of apoptosis, including atrophic cell bodies and
condensed or degraded chromatin. Similar results were obtained when the
SM-20(60-355) cDNA fused to the T7 epitope tag was
expressed in the neurons. Quantifying these results showed that greater
than 60% of the SM-20(60-355)-injected neurons contained
condensed or degraded chromatin compared with only 25% of the control
cells injected with SM-20-promoted Death in Sympathetic Neurons Is
Caspase-dependent and Leads to Activation of
Caspase-3--
Sympathetic neurons deprived of NGF undergo
caspase-dependent apoptosis that can be blocked by
the general caspase inhibitor BAF (26). To investigate
whether SM-20-induced death is also caspase-dependent, we
microinjected sympathetic neurons with expression vectors containing
SM-20 or LacZ and then incubated the cells in NGF-containing media with
or without BAF (50 µM). After 3 days, the SM-20-injected
neurons maintained in the absence of BAF displayed morphological
features of apoptosis, including condensed, crescent-shaped or degraded
chromatin (Fig. 7A,
panels a-c). In contrast,
SM-20-injected neurons maintained in the presence of BAF were healthy
with intact cell bodies and diffuse chromatin (Fig. 7A,
panels d-f). Greater than 70% of
SM-20-injected neurons maintained in the presence of BAF survived after
72 h compared with 38% in the absence of BAF (Fig.
7B). LacZ-injected cells, maintained under the same conditions, showed no significant difference in percent survival. Thus
SM-20-induced neuronal death is caspase-dependent and can be blocked by a general caspase inhibitor.
To test whether SM-20-induced death leads to activation of effector
caspases such as caspase-3, sympathetic neurons expressing SM-20, LacZ,
or Bax were analyzed by immunofluorescence using an antibody that
recognizes activated caspase-3. As shown in Fig. 8 (panels a-d), LacZ-injected
neurons appeared healthy and lacked active caspase-3
immunofluorescence. In contrast, both SM-20- and Bax-injected neurons
showed significant staining for active caspase-3 (Fig. 8,
panels e-l). The percentage of injected cells expressing activated caspase-3 at 72 h was significantly greater for both SM-20 (18.5 ± 1.5%) and Bax (47.2 ± 9.8%) when
compared with LacZ-injected cells (6.3 ± 1.7%). For
SM-20-injected neurons (see Fig. 7B) and neurons injected
with Bax or deprived of NGF (data not shown), the percentage of cells
with activated caspase-3 was less than the fraction that underwent cell
death, as assayed by visualizing Hoechst-stained nuclei. Thus,
activated caspase-3 may only accumulate to detectable levels in cells
with advanced chromatin condensation and degradation. Taken together,
these data suggest that SM-20-induced cell death is
caspase-dependent and that it involves activation of
caspase-3.
SM-20 Expression Does Not Cause Cytochrome c Release from
Mitochondria--
The release of cytochrome c from
mitochondria in sympathetic neurons after NGF withdrawal is thought to
contribute to caspase activation (27). Once in the cytosol, cytochrome
c can form a complex with Apaf-1 and caspase-9, resulting in
the activation of caspase-9 and, eventually, downstream caspases such
as caspase-3 (28). To determine whether ectopic expression of SM-20 can
cause release of cytochrome c from mitochondria, we
performed cytochrome c immunofluorescence on NGF-maintained
neurons microinjected with expression plasmids for SM-20, LacZ, or Bax.
For these experiments, the injected neurons were incubated in the
presence of BAF (50 µM), which prevents the final stages
of cell death caused by NGF withdrawal without blocking cytochrome
c release (27). Greater than 85% of SM-20- or LacZ-injected
neurons showed a punctate mitochondrial pattern after labeling with an
anti-cytochrome c antibody, indicating that cytochrome
c was not released from mitochondria in these cells (Fig.
9, A and B). In
contrast, ~75% of Bax-injected neurons displayed a diffuse
cytoplasmic staining pattern that closely resembled the cytochrome
c immunofluorescence observed after NGF deprivation (27),
consistent with a role for Bax as an upstream regulator of cytochrome
c release (29). These data indicate that SM-20 expression,
by itself, is not sufficient to cause cytochrome c
release.
In nonneuronal and neuronal cells, SM-20 immunofluorescence
colocalized with mitochondria-selective dyes as well as the
immunofluorescence for two distinct mitochondrial proteins. Deletion of
59 amino acids from the amino terminus of SM-20 resulted in the
truncated protein being distributed uniformly throughout the cell.
Consistent with its mitochondrial localization, the amino-terminal 25 amino acids of SM-20 were found to be sufficient to target a normally cytosolic protein to mitochondria. In addition to the expected 40-kDa
SM-20 protein, both CV-1 cells and NIH3T3 cells expressed a 33-kDa form
of SM-20. In cell fractionation experiments, the smaller SM-20 protein
was present predominantly in the mitochondria-enriched fraction. This
result, together with the presence of an amino-terminal mitochondrial
targeting sequence in SM-20, suggests that the mitochondrial targeting
sequence is proteolytically removed when SM-20 is imported into
mitochondria. Together, these results identify SM-20 as a novel
mitochondrial protein.
SM-20 immunocytochemistry was previously detected in a punctate pattern
in the cytoplasm of vascular smooth muscle cells (15). The SM-20
immunostaining was shown to be distinct from that of Although SM-20 is predominantly localized to mitochondria, a truncated
form of SM-20 lacking its mitochondrial targeting sequence unexpectedly
retained the ability to induce death in sympathetic neurons, suggesting
that endogenous SM-20 may not need to be localized to mitochondria to
induce cell death. One explanation for this apparent discrepancy is
that SM-20 might be released from mitochondria into the cytosol after
NGF withdrawal, analogous to the release of cytochrome c
(27, 30). Expression of truncated SM-20(60-355) that is
present in the cytoplasm could mimic this event. An alternative explanation is that endogenous SM-20 does not get released from mitochondria during cell death and that it is the mitochondrial form of SM-20 that contributes to apoptosis. Expression of
SM-20(60-355) may result in a small amount of
SM-20(60-355) being imported into mitochondria or binding
to undefined proteins present on the surface of mitochondria, which in
this case may be sufficient to promote death.
To further examine the mechanism of SM-20-induced cell death, we
investigated the role of SM-20 in caspase activation and in cytochrome
c release from mitochondria. As with NGF-withdrawal-induced death, SM-20-induced death was efficiently blocked by the general caspase inhibitor BAF. In addition, we detected activated caspase-3 in
a significant fraction of SM-20-injected neurons, suggesting that SM-20
may directly or indirectly participate in the activation of this
caspase in dying neurons. The release of cytochrome c from
mitochondria occurs after NGF withdrawal and has been suggested to
contribute to cell death in this paradigm (27, 30). In contrast to Bax,
ectopic expression of SM-20 did not cause widespread cytochrome
c release. Since SM-20 does not act by directly causing cytochrome c release, our results suggest that SM-20
functions during cell death in a pathway that is either downstream or
independent of cytochrome c release.
Although SM-20 does not resemble known mammalian proteins, it contains
a 218-amino acid region that is 43% identical (61% similar) to a
portion of the protein encoded by the C. elegans egl-9 gene
(17). Deletions and mutations that disrupt the egl-9 gene
confer resistance to a P. aeruginosa-derived toxin that
causes a lethal neuromuscular paralysis in C. elegans. The
genetics in this model indicate that the toxin results in aberrant
activation of Egl-9 rather than in its inactivation. Consistent with a
role for Egl-9 as a regulator of neuronal signaling or muscle
contraction, the egl-9 promoter is most active in muscle
cells and in neurons (17). Expression of the rat SM-20 gene is also
greatest in muscle-containing tissues and in brain, suggesting that the
function of SM-20/Egl-9 in these tissues may be conserved (13). In
humans, several SM-20-related genes or pseudogenes appear to be present
on distinct chromosomes (31).2 Future studies will
examine the ability of these SM-20-related proteins, including Egl-9,
to induce cell death as well as their subcellular localization.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ex = 450-490 nm,
em = 520-560 nm), and images were captured. The same cells were then
stained with the nonfixable mitochondria-specific dye Rhodamine 123 (20), and images were obtained using a tetramethylrhodamine
isothiocyanate (TRITC) filter set (
ex = 546 ± 10 nm,
em > 590 nm). In every cell, the fluorescence of
Mitotracker Green FM and Rhodamine 123 coincided completely. For
labeling of mitochondria in sympathetic neurons, Mitotracker Orange (25 nM, Molecular Probes) was added to the culture medium for
15 min, after which images of live cells were captured using a Leica
confocal microscope and TCS-NT software.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Localization of SM-20 to mitochondria.
CV-1 cells were transiently transfected with SM-20-Rc/CMV
plasmid DNA. Dual immunofluorescence was performed for SM-20 and
cytochrome oxidase I (panels A-C) 48 h after
transfection. Primary antibodies for cytochrome oxidase I (panel
A) and SM-20 (panel B) were detected using FITC- and
TRITC-conjugated secondary antibodies, respectively. Other cells were
labeled with Mitotracker Green FM and then subjected to
immunofluorescence with anti-SM-20 antibody (panels
D-F). Yellow in the overlay images
(panels C and F) appears where the red
fluorescence of SM-20 colocalizes with the green fluorescence of either
cytochrome oxidase I (panel A) or Mitotracker Green FM
(panel D). Bar = 10 µm.
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Fig. 2.
A 33-kDa processed form of SM-20 is enriched
in mitochondria. A, whole cell extracts prepared from
metabolically labeled CV-1 cells (lane 1) or from CV-1 cells
stably expressing SM-20 (lanes 2 and 3) were
immunoprecipitated with a SM-20 anti-peptide antibody.
Immunoprecipitations were done in the presence (+) or absence ( ) of
excess blocking peptide corresponding to the last 14 amino acids of
SM-20. The positions of molecular mass markers are indicated. The
arrows indicate the presence of ~40- and 33-kDa proteins
in SM-20-expressing cells that were specifically immunoprecipitated in
the absence of the blocking peptide (lane 2). B,
cytosolic (Cyto) and mitochondria
(Mito)-enriched fractions were prepared from NIH3T3 cells
transiently transfected with a SM-20 expression vector (lanes
2 and 4) or with pcDNA3 (lanes
1 and 3) and metabolically labeled for 4 h with
Tran35S-label. Fractions were immunoprecipitated with
anti-SM-20 antibody, and then the immunoprecipitated proteins were
subjected to SDS-polyacrylamide gel electrophoresis and PhosphorImager
analysis. Note that the 33-kDa protein (lower arrow) is
present only in the mitochondria-enriched fraction. The
cytosolic and mitochondrial-enriched fractions were
immunoblotted with anti-cytochrome (cyt.) c
antibody to confirm that a fraction enriched in mitochondrial proteins
was obtained.
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Fig. 3.
An amino-terminal deletion of SM-20 prevents
its mitochondrial localization. Expression vectors encoding DHFR
tagged with a carboxyl-terminal T7 epitope (A),
SM-20(60-355)/T7 (SM-20 minus its first 59 amino acids)
(B), or full-length SM-20 (C) were transfected
into CV-1 cells. Indirect immunofluorescence was performed 48 h
later using anti-T7 antibody or anti-SM-20 antibody. Both DHFR and
SM-20(60-355)/T7 show diffuse cytoplasmic and nuclear
fluorescence in marked contrast to the punctate distribution of the
full-length SM-20. Bar = 10 µm.
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Fig. 4.
SM-20 contains an amino-terminal
mitochondrial targeting sequence. CV-1 cells transfected with
expression vectors for SM-20(1-50)/DHFR (panels
A-C) and SM-20(1-25)/DHFR fusion proteins
(panels D-F) were incubated with Mitotracker Green FM
(panels A and D). Indirect immunofluorescence
using anti-T7 antibody and TRITC-conjugated secondary antibody was
performed to detect the T7-tagged fusion proteins (panels B
and E). Yellow in the overlay images
(panels C and F) appears where the red
fluorescence of the fusion proteins colocalizes with the green
fluorescence of Mitotracker Green FM. Bar = 10 µm.
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Fig. 5.
SM-20, but not SM-20(60-355), is
localized to mitochondria in sympathetic neurons. Sympathetic
neurons were microinjected with expression vectors encoding SM-20/GFP
(panels A-C) or SM-20(60-355)/GFP fusion
proteins diluted to 50 µg/ml in injection buffer without
rhodamine-dextran (panels D-F). Cells were labeled with the
mitochondria-selective dye Mitotracker Orange 24 h after
microinjection (panels A and D). Images of live
cells were acquired by confocal microscopy. The green fluorescence in
cells injected with SM-20/GFP fusion constructs is shown in
panels B and E. These images are
superimposed with the red fluorescence of Mitotracker Orange in
panels C and F. The letter
n represents the position of the nucleus in the injected
neurons. Bar = 10 µm.
-galactosidase (LacZ) (Fig. 6B). The
extent of cell death induced by SM-20(60-355) in these
experiments is comparable with the level of cell death produced by
microinjection of full-length SM-20 (10). Since SM-20(60-355) is not targeted to mitochondria in
sympathetic neurons, the mitochondrial localization of SM-20 does not
appear to be critical for its death promoting activity.
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Fig. 6.
SM-20(60-355) induces cell death
in NGF-maintained sympathetic neurons. A, sympathetic
neurons were microinjected with GFP (panels a-c) or
SM-20(60-355)/GFP (panels d-i) expression
plasmids and, after 72 h, stained with the DNA binding dye Hoechst
33342. Injected cells were identified by the green fluorescence of GFP
(panel b) or SM-20(60-355)/GFP fusion protein
(panels e and h). The Hoechst-stained nuclei of
the same cells are shown in panels c,
f, and i, and phase-contrast images are shown in
panels a, d, and g. The
white arrows indicate the injected cells and their
corresponding phase-contrast and Hoechst-stained nuclei.
Bar = 15 µm. B, NGF-maintained neurons
were microinjected with expression vectors encoding LacZ or
SM-20(60-355). After 72 h, the injected cells were
stained with Hoechst 33342 and scored for viability as described under
"Experimental Procedures." Data are the mean ± S.E. from six
independent experiments with 200-300 neurons scored per injected DNA
in each experiment. The mean survival of SM-20-injected cells was
significantly less than that of LacZ-injected cells (*, two-tailed
t test, p = 0.004).
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Fig. 7.
The cell death induced by SM-20 in
sympathetic neurons is blocked by a general caspase inhibitor.
A, sympathetic neurons were microinjected with a SM-20
expression plasmid and treated with (panels d-f) or without
(panels a-c) 50 µM BAF for 72 h.
Injected cells were identified by inclusion of a non-fixable
rhodamine-dextran dye in the injection buffer (panels b
and e). The white arrows indicate the injected
cells and their corresponding phase-contrast (panels a and
d) and Hoechst-stained nuclei (panels c and
f). Bar = 15 µm. B,
NGF-maintained neurons were microinjected with expression vectors
encoding LacZ or SM-20 and incubated in the presence or absence of 50 µM BAF. After 72 h, the injected cells were stained
with Hoechst 33342 and scored for viability as described under
"Experimental Procedures." Data are the mean ± S.E. from four
independent experiments. The mean survival of SM-20-injected cells in
the absence of BAF was significantly less than that of SM-20-injected
cells in the presence of BAF (two-tailed t test,
p = 0.0013).
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Fig. 8.
Expression of SM-20 in sympathetic neurons
leads to caspase-3 activation. NGF-maintained sympathetic neurons
were microinjected with expression plasmids encoding LacZ (panels
a-d), SM-20 (panels e-h), or Bax (panels
I-l). Injected cells were identified by inclusion of a fixable
rhodamine-dextran dye in the injection buffer (panels b,
f, and j). After 3 days, the cells were fixed,
and immunofluorescence was performed using anti-activated caspase-3
antibody (panels c, g, and k). The
white arrows indicate the injected cells and their
corresponding phase-contrast (panels a, e, and
i) and Hoechst-stained nuclei (panels d,
h, and l). Bar = 15 µm.
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Fig. 9.
Absence of detectable cytochrome c
release from mitochondria in SM-20-injected sympathetic
neurons. A, sympathetic neurons were microinjected with
LacZ (panels a-c), SM-20 (panels d-f), or Bax
(panels g-i) expression vectors and maintained in
BAF-containing media. After 24-48 h, the cells were fixed, and
immunofluorescence was performed using anti-cytochrome c
antibody (panels c, f, and i).
Injected cells were identified by inclusion of a fixable
rhodamine-dextran dye in the injection buffer (panels b,
e, and h). The white arrows indicate
the injected cells and their corresponding phase-contrast images
(panels a, d, and g).
Bar = 15 µm. B, NGF and BAF-maintained
neurons were microinjected with expression vectors encoding LacZ,
SM-20, or Bax. After 48 h, the percentage of injected cells
(rhodamine-positive) showing a loss of punctate mitochondrial
cytochrome c labeling was determined. Data are mean ± S.E. from 3-7 independent experiments. The mean number of
SM-20-injected cells showing release of cytochrome c was not
significantly different than that for LacZ (two-tailed t
test, p > 0.2).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin, but
efforts to further localize SM-20 were not undertaken. The appearance
of SM-20 immunostaining in smooth muscle cells was very similar to the
distribution of SM-20 in neurons and CV-1 cells that we observed. The
identification of a mitochondrial targeting sequence in SM-20 suggests
that the punctate distribution of SM-20 detected previously in smooth
muscle cells reflects targeting of the endogenous SM-20 protein to mitochondria.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Daniel Donoghue for providing the DHFR plasmid, Dr. Eugene Johnson for the Bax plasmid, Dr. Patricia Hinkle for providing NIH3T3 cells, and Dr. Jane Chisholm for expert assistance with confocal microscopy. We also thank Daphne Hasbani and Leah Larocque for excellent technical assistance.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant NS34400.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.
§ Supported by NIEHS, National Institutes of Health institutional training Grant ES07026.
Supported in part by the Paul Stark Endowment at the
University of Rochester. To whom correspondence should be
addressed: Dept. of Pharmacology and Physiology, University of
Rochester, School of Medicine and Dentistry, 601 Elmwood Ave.,
Rochester, NY 14642. Tel.: 716-273-4893; Fax: 716-273-2652; E-mail:
Robert_Freeman@urmc.rochester.edu.
Published, JBC Papers in Press, November 1, 2000, DOI 10.1074/jbc.M008407200
2 D. M. Hasbani and R. S. Freeman, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are:
NGF, nerve growth
factor;
BAF, BOC-Asp-CH2F;
DHFR, dihydrofolate reductase;
GFP, green fluorescent protein;
LacZ, -galactosidase;
PBS, phosphate-buffered saline;
TRITC, tetramethylrhodamine isothiocyanate;
MOPS, 4-morpholinepropanesulfonic acid;
CMV, cytomegalovirus;
FITC, fluorescein isothiocyanate;
RIPA, radioimmune precipitation.
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REFERENCES |
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