Caspase cleavage of key cytoskeletal proteins,
including several intermediate filament proteins, triggers the dramatic
disassembly of the cytoskeleton that characterizes apoptosis. Here we
describe the muscle-specific intermediate filament protein desmin as a novel caspase substrate. Desmin is cleaved selectively at a conserved Asp residue in its L1-L2 linker domain (VEMD
M264)
by caspase-6 in vitro and in myogenic cells undergoing
apoptosis. We demonstrate that caspase cleavage of desmin at
Asp263 has important functional consequences, including the
production of an amino-terminal cleavage product, N-desmin, which is
unable to assemble into intermediate filaments, instead forming large intracellular aggregates. Moreover, N-desmin functions as a
dominant-negative inhibitor of filament assembly, both for desmin and
the structurally related intermediate filament protein vimentin. We
also show that stable expression of a caspase cleavage-resistant desmin
D263E mutant partially protects cells from tumor necrosis
factor-
-induced apoptosis. Taken together, these results
indicate that caspase proteolysis of desmin at Asp263
produces a dominant-negative inhibitor of intermediate filaments and
actively participates in the execution of apoptosis. In addition, these
findings provide further evidence that the intermediate filament
cytoskeleton has been targeted systematically for degradation during apoptosis.
 |
INTRODUCTION |
Caspases are a novel family of pro-apoptotic proteases that
dismantle and reorganize the cytoskeleton and nucleus during the execution of apoptosis by specifically proteolyzing proteins at Asp
residues (1, 2). For example, caspase proteolysis of ROCK1 promotes the
phosphorylation of myosin light chain and triggers membrane blebbing
(3, 4). In addition, caspase cleavage of actin, the actin-severing
protein gelsolin, or Gas2 directly induces the disassembly of
the actin microfilament network (5-8). Similarly, caspase proteolysis
of several intermediate filament proteins, including the nuclear lamins
and the tissue-specific cytokeratins 14, 18, and 19 (epithelial cells)
and vimentin (mesenchymal cells), leads to the disassembly of the
nuclear envelope and the cytosolic intermediate filament network that
links the nucleus to the plasma membrane (9-17). Each of these
intermediate filament proteins is cleaved at a conserved Asp residue in
its L1-L2 linker domain by caspase-6 (and/or other caspases). Because
several other intermediate filament proteins, including desmin, also
contain a consensus caspase-6 cleavage motif (VEXD) (18, 19)
in their L1-L2 domains, we postulated that desmin might be cleaved by
caspase-6 during the induction of apoptotic cell death.
Desmin is an abundant muscle-specific protein that polymerizes to form
an extensive network of 10-nm intermediate filaments that span the
nuclear envelope to the sarcolemma and link myofibrils together
laterally at Z discs (20). Indeed, recent studies indicate that desmin
is essential for both the structural integrity and the survival of
muscle cells. Desmin-deficient mice develop a progressive and
fatal generalized myopathy, which affects cardiac, skeletal, and smooth
muscles and is characterized by myofibril disarray and accelerated
muscle cell death. These abnormalities are particularly prominent in
the myocardium, resulting in heart failure (21-24). In addition,
mutations of the desmin gene have been identified in patients with a
familial generalized myopathy that closely resembles the myopathy seen
in desmin-deficient mice and in patients with idiopathic dilated
cardiomyopathy (25-28). Many of these mutations encode a desmin gene
product that is unable to assemble into intermediate filaments; these
mutant desmin proteins form intracellular aggregates/inclusions in
muscle cells. These findings underscore the importance of desmin in
muscle function and survival.
Here we report for the first time that desmin is cleaved specifically
by caspase-6 at VEMD
M264 in its L1-L2 linker domain
in vitro and in myogenic cells induced to undergo apoptosis
by treatment with tumor necrosis factor
(TNF)1-
. Caspase
proteolysis of desmin at Asp263 produces an amino-terminal
cleavage product that functions as a dominant-negative inhibitor of
intermediate filament assembly. Furthermore, we show that a caspase
cleavage-resistant desmin mutant partly protects against
TNF-
-induced apoptosis, thereby indicating that caspase cleavage of
desmin is a functionally relevant proteolytic event during the
induction of apoptosis.
 |
EXPERIMENTAL PROCEDURES |
Cell Lines and Reagents--
Rat cardiac H9c2 myoblasts, human
MCF-7 breast carcinoma cells, and HEK 293 cells were maintained
in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with
10% fetal bovine serum. Murine HL-1 cells, a well differentiated
cardiac myocyte cell line (29), were provided kindly by Dr. William
Claycomb and were grown in Ex-Cell 320 medium (JRH Biosciences, Lenexa,
KS) supplemented with 10% fetal bovine serum, 1 µM
retinoic acid (Sigma), 10 µg/ml insulin (Invitrogen), 10 µM norepinephrine (Sigma), 1× nonessential amino acids
(Invitrogen), and 50 µg/ml endothelial cell growth supplement
(Upstate Biotechnology, Lake Placid, NY) as detailed elsewhere (29).
TNF-
was purchased from R&D Systems (Minneapolis, MN) and zVAD-fmk
from ICN Biomedicals (Aurora, OH). All other reagents were purchased
from Sigma unless otherwise specified.
Desmin Plasmid Constructs--
A mutant desmin construct
specifically altered at a potential caspase cleavage site (D263E) was
made using the QuikChange site-directed mutagenesis kit (Stratagene)
according to the manufacturer's instructions. Wild-type murine desmin
cDNA was employed as a template, and the following oligonucleotide
primers were used: 5'-CCAGGTGGAGATGGAAATGTCCAAGCCGG-3' and
5'-CCGGCTTGGACATTTCCATCTCCACCTGG-3'. To make FLAG-tagged constructs, full-length desmin and truncated desmin cDNAs encoding the
amino-terminal caspase cleavage product (amino acids 1-263, designated
N-desmin) or the carboxyl-terminal product (amino acids 264-469,
designated C-desmin) were PCR-amplified from murine desmin cDNA
with the following primers: 5'-GGCGGAATTCATGAGCCAGGCCTACTCGTC-3'
and 5'-GTGGGCGGCCGCTTACAGCACTTCATGTTGTT-3' (full-length),
5'-GGCGGAATTCATGAGCCAGGCCTACTC GTC-3' and
5'-GTGGGCGGCCGCCTAGTCCATCTCCACCTGGACC-3' (N-desmin), or
5'-GGCGGAATTCATGTCCAAGCCGGACCTCAC-3' and
5'-GTGGGCGGCCGCTTACAGCACTTCATGTTGTT-3' (C-desmin). The PCR
products then were digested with EcoRI and NotI
and subcloned into these sites in pcDNA3-NFLAG. Each desmin construct was verified by DNA sequencing.
Caspase Cleavage of Desmin in Vitro--
Murine desmin cDNA
was transcribed and translated in vitro with
35S-labeled methionine using the TNT T7 Quick
Coupled transcription/translation system (Promega) according to the
manufacturer's instructions. 35S-Labeled desmin then was
incubated with a control buffer, 2.5 or 25 ng of recombinant caspase-1,
-2, -3, -6, -7, or -8 for 1 h at 37 °C; and the cleavage
products were analyzed as described previously (30, 31). The enzymatic
activity of each caspase was confirmed using a well characterized
substrate for each caspase as a positive control. To determine whether
desmin mutant D263E was resistant to caspase cleavage in
vitro, 35S-labeled wild-type or mutant D263E
desmin was incubated with 25 ng of recombinant caspase-6 and
analyzed as above.
Immunoblot Analyses--
Differentiated HL-1 cardiac
myocytes were treated with 10 ng/ml TNF-
and 1 µg/ml cycloheximide
(CHX) for 0, 3, 6, 9, 12, or 18 h. Whole cell lysates were
analyzed by immunoblotting as described previously (30) using a desmin
mAb (Sigma) or protein kinase C
polyclonal antibody (Santa Cruz
Biotechnology). For inhibitor studies, H9c2 myoblasts were treated with
10 ng/ml TNF-
and 1 µg/ml CHX for 24 h in the presence or
absence of 50 µM zVAD-fmk. To determine whether the
mutant D263E desmin protein was resistant to caspase proteolysis
in vivo, HEK 293 cells were transfected with 1 µg of
wild-type or mutant D263E desmin cDNA using LipofectAMINE Plus
reagent (Invitrogen) according to the manufacturer's instructions. After overnight incubation, transfected HEK 293 cells were treated with
10 ng/ml TNF-
and 1 µg/ml CHX for 24 h, and the cell lysates were analyzed as above.
Indirect Immunofluorescence--
H9c2 myoblasts were grown to
~80% confluence on glass coverslips and then transfected with 1 µg
of pcDNA3-NFLAG plasmid encoding full-length desmin, the
amino-terminal caspase cleavage product (amino acids 1-263), or the
carboxyl-terminal caspase cleavage product (amino acids 264-469) using
LipofectAMINE Plus reagent (Invitrogen). After a 24-h
incubation, the cells were fixed in methanol for 2 min at
20 °C
and were incubated sequentially, first with FLAG M2 mAb (1:500 dilution
in phosphate-buffered saline; Sigma) for 1 h at 37 °C, and
subsequently with a fluorescein-conjugated, goat affinity-purified
antibody to mouse IgG (1:20 dilution; ICN Pharmaceuticals) together
with 10 µg/ml Hoechst 33258 for 30 min at 37 °C. The desmin and
nuclei were visualized by fluorescence microscopy (Nikon Eclipse E400).
Intermediate Filament Assembly Co-transfection
Experiments--
MCF-7 cells were grown to ~80% confluence on glass
coverslips and then co-transfected with varying ratios of FLAG-tagged
wild-type desmin to FLAG-tagged N-desmin (4:1, 2:1, or 1:1) using
LipofectAMINE Plus reagent (Invitrogen). 24 h after transfection,
the desmin was visualized by indirect immunofluorescence using FLAG M2
mAb (Sigma) as detailed under "Indirect Immunofluorescence." Cells were scored as containing normal desmin filaments only, desmin aggregates only, or a mixture of filaments and aggregates. For vimentin
experiments, MCF-7 cells were co-transfected with 0.4 µg of
pcDNA3-NFLAG-N-desmin and 0.4 µg of pEGFPN1-vimentin (17), and
vimentin was visualized by green fluorescent protein expression as described previously (17).
Stable Transfection of MCF-7 Cells with cDNAs
Encoding Wild-type or Mutant D263E Desmin and Apoptosis
Assays--
MCF-7 cells were transfected with 1.2 µg of
pcDNA3-wild-type desmin or pcDNA3-D263E desmin using
LipofectAMINE Plus reagent (Invitrogen). After 48 h, cells stably
expressing these desmin cDNAs were selected by adding 500 µg/ml
G418 (Invitrogen) to the growth medium for 3 weeks. Stable expression
was confirmed by immunoblotting with a desmin mAb (Sigma). MCF-7 cells
stably expressing wild-type or mutant D263E desmin then were treated
with 10 ng/ml TNF-
and 1 µg/ml CHX for 24 h, and the nuclei
were scored for apoptotic morphology (chromatin condensation or nuclear
fragmentation) by staining with Hoechst 33258 as described (17).
 |
RESULTS |
Specific Cleavage of Desmin by Caspase-6 at Asp263 in
Vitro--
Because desmin contains in its L1-L2 domain a
consensus caspase-6 cleavage motif (VEXD) (18, 19) similar
to those present in other intermediate filament proteins previously
shown to be caspase substrates (9-17), we postulated that desmin also
might be cleaved by caspase-6 in vitro. To test this
hypothesis, we incubated 35S-labeled wild-type murine
desmin with a buffer control (C in Fig.
1A), 2.5 or 25 ng of
caspases-1, -2, -3, -6, -7, or -8 (C1-C8) for
1 h at 37 °C. As shown in Fig. 1A,
35S-labeled desmin was proteolyzed specifically by
caspase-6 (C6) into two products, which were ~29 and 27 kDa in size (indicated by arrows). None of the other
caspases tested cleaved desmin, although each caspase was capable of
proteolyzing one or more of its known substrates (latter data not
shown). To identify the Asp residue in desmin cleaved by caspase-6
in vitro, we specifically substituted Asp263 in
the putative caspase-6 cleavage sequence VEMD
M264 with a
Glu residue by site-directed mutagenesis. As demonstrated in Fig.
1B, the resulting 35S-labeled mutant D263E
desmin was resistant to proteolysis by caspase-6 (C6),
whereas WT desmin was cleaved by caspase-6 into the appropriate sized
products. These results indicate that desmin is proteolyzed selectively
by caspase-6 at Asp263 in its L1-L2 linker domain.

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Fig. 1.
Desmin is specifically cleaved by caspase-6
at Asp263 in vitro. A,
35S-labeled wild-type murine desmin was incubated with
buffer control (C), 2.5 or 25 ng of caspases-1, -2, -3, -6, -7, or -8 (C1-C8) for 1 h at 37 °C, and the
reaction products were analyzed by SDS-PAGE, followed by
autoradiography. The desmin cleavage products are indicated by
arrows. B, the desmin D263E mutant is not cleaved
by caspase-6 in vitro. 35S-Labeled WT or mutant
D263E desmin was incubated with buffer control (C) or 25 ng
of caspase-6 (C6) for 1 h at 37 °C. The proteolytic
products of WT desmin are indicated by arrows.
|
|
Proteolysis of Desmin by Caspases at Asp263 in Myogenic
Cells Undergoing Apoptosis--
To determine whether desmin is cleaved
by caspases in apoptotic myogenic cells, we treated differentiated HL-1
cardiac myocytes (29) with 10 ng/ml TNF-
and 1 µg/ml CHX for 0-18
h; CHX was added to sensitize these cells to the induction of
apoptosis. As shown in Fig.
2A, desmin was cleaved into a
product of ~27 kDa that first was observed faintly 9 h
after treatment with TNF-
. The abundance of this proteolytic product
increased with time, corresponding with a reduction in the amount of
full-length desmin. Desmin also was cleaved into a similarly sized
product in differentiated HL-1 myocytes induced to undergo apoptosis by
treatment with staurosporine (data not shown). Furthermore, the
size of the observed proteolytic product was similar to one of the
desmin cleavage products generated by caspase-6 in vitro
(the other product was not detected by the desmin mAb used in these
studies), suggesting that caspase-6 may be the protease cleaving desmin
during apoptosis in vivo. Interestingly, the TNF-
-induced
proteolysis of the well characterized caspase-3 substrate protein
kinase C
(32) into its characteristically sized cleavage product
(indicated by an arrow in Fig. 2A)
preceded that of desmin, consistent with caspase-3 being upstream of
caspase-6 in the execution of apoptosis (33). As demonstrated in Fig. 2B, the broad spectrum caspase inhibitor zVAD-fmk potently
inhibited the production of the apoptotic desmin cleavage product in
H9c2 myogenic cells treated with TNF-
and CHX, indicating that this proteolytic event in apoptotic myoblasts indeed is mediated by one or
more caspases. Finally, WT desmin, but not the D263E mutant, was
cleaved during TNF-
-induced apoptosis in HEK 293 cells
transiently transfected with the corresponding cDNAs (Fig.
2C). These findings demonstrate that desmin is cleaved by
caspases at Asp263 during the induction of apoptosis
in vivo.

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Fig. 2.
Desmin is proteolyzed by caspases at
Asp263 in myogenic cells undergoing apoptosis.
A, immunoblot analysis of desmin and protein kinase C
(PKC ) cleavage in murine HL-1 cardiac myocytes induced to
undergo apoptosis by treatment with TNF- . Differentiated HL-1 cells
were treated with 10 ng/ml TNF- and 1 µg/ml CHX for the indicated
number of hours (h), and whole cell lysates were analyzed by
immunoblotting as detailed under "Experimental Procedures." The
cleavage products are indicated by arrows. B, the
caspase inhibitor zVAD-fmk antagonizes TNF- -induced desmin
proteolysis. Rat H9c2 cardiac myoblasts were treated with 10 ng/ml TNF- and 1 µg/ml CHX for 24 h in the absence ( ) or
presence (+) of 50 µM zVAD-fmk. C, desmin is
cleaved at Asp263 in cells induced to undergo apoptosis by
treatment with TNF- . HEK 293 cells transiently transfected with WT
or mutant D263E desmin cDNAs were treated with 10 ng/ml TNF- and
1 µg/ml CHX for 24 h, and whole cell lysates then were analyzed
by immunoblotting.
|
|
Caspase Cleavage of Desmin at Asp263 Generates an
Amino-terminal Product That Is a Dominant-negative Inhibitor of
Intermediate Filament Assembly--
We next examined the functional
consequences of caspase cleavage of desmin at Asp263 by
transiently transfecting H9c2 myoblasts with cDNAs encoding FLAG-tagged WT desmin, the amino-terminal caspase cleavage product (N-desmin, amino acids 1-263), or the carboxyl-terminal caspase cleavage product (C-desmin, amino acids 264-469). After a 24-h incubation, transfected cells were examined by indirect
immunofluorescence using a FLAG mAb. As demonstrated in Fig.
3A, WT desmin assembled into
an elaborate network of intermediate filaments, whereas N-desmin was
unable to assemble into filaments, instead forming a striking array of
spheroidal aggregates throughout the cell. C-desmin also did not
assemble into well defined filaments, forming a more diffuse pattern of
staining distinct from either WT desmin or N-desmin. Similar results
were obtained in MCF-7 cells, which lack desmin and other type III
intermediate filament proteins (data not shown) (34). Neither caspase
cleavage product was sufficient to induce apoptosis in transiently
transfected myogenic cells under these conditions. The intact nuclei
corresponding to transfected cells are indicated by arrows
in the lower panels (Fig. 3A). As shown in Fig.
3B, each of these desmin cDNAs was expressed at
comparable levels in H9c2 myoblasts. N-desmin also inhibited the
assembly of WT desmin in co-transfection experiments performed in
desmin-deficient MCF-7 cells; C-desmin was not tested in this system
because of its more subtle defect in filament assembly. As demonstrated
in Fig. 3C, even small amounts of N-desmin potently
disrupted the ability of WT desmin to assemble into filaments, instead
promoting the formation of aggregates. Furthermore, N-desmin also
interfered with the assembly of the structurally related intermediate
filament protein vimentin (Fig. 3D) (35). These findings
indicate that caspase cleavage of desmin has dramatic functional
consequences, viz. the production of a truncated protein
that interferes in a dominant-negative fashion with the assembly of
both desmin and vimentin filaments.

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Fig. 3.
Caspase proteolysis of desmin generates an
amino-terminal cleavage product that is assembly-incompetent and
functions as a dominant-negative inhibitor of intermediate filament
assembly. A, N-desmin is unable to assemble into desmin
filaments in myogenic cells. H9c2 myogenic cells were transiently
transfected with FLAG-tagged WT desmin, N-desmin (amino acids 1-263),
or C-desmin (amino acids 264-469) and analyzed by indirect
immunofluorescence with FLAG mAb (upper panels); the nuclei
(lower panels) were visualized as detailed under
"Experimental Procedures" with those nuclei corresponding to the
transfected cells indicated by arrows. B,
immunoblot analysis of H9c2 myogenic cells transiently transfected with
each of these desmin cDNAs using FLAG mAb. C, N-desmin
is a dominant-negative inhibitor of desmin filament assembly. MCF-7
cells, which lack desmin and other type III intermediate filament
proteins (34), were co-transfected with varying ratios of FLAG-tagged
wild-type desmin to FLAG-tagged N-desmin: 4:1, 2:1, or 1:1, and
transfected cells were scored for the presence of intact intermediate
filaments (open bars), desmin aggregates (black
bars), or a mixture of filaments and aggregates (gray
bars). The data are presented as the mean ± S.D. of three
independent experiments. D, N-desmin inhibits vimentin
filament assembly. MCF-7 cells were co-transfected with equal amounts
of FLAG-tagged N-desmin and green fluorescent protein-tagged vimentin,
and the vimentin was visualized by green fluorescent protein
expression.
|
|
Inhibition of TNF-
-induced Apoptosis by Caspase
Cleavage-resistant Desmin--
To determine whether caspase cleavage
of desmin at Asp263 might contribute to the execution of
apoptosis, we stably transfected MCF-7 cells with cDNAs encoding
wild-type or mutant D263E desmin. MCF-7 cells were chosen for these
studies because they lack desmin and vimentin (34), thereby avoiding
the potentially confounding influence of the endogenous caspase
cleavage-sensitive desmin (and vimentin). As shown in Fig.
4, stable expression of the caspase cleavage-resistant D263E mutant in two independent clones
(B1 and C4) partially protected MCF-7 cells from
TNF-
-induced apoptosis compared with MCF-7 cells stably expressing
WT desmin. The immunoblot at the top of Fig. 4 shows that
the mutant and WT proteins were expressed at similar levels. No desmin
expression was detected in the control (C)
vector-transfected cells, which had a similar sensitivity to
TNF-
-induced apoptosis as did WT desmin-expressing cells (latter
data not shown). The partial protection conferred by expression of the
cleavage-resistant D263E desmin mutant indicates that cleavage at this
site is a functionally important event in the execution of
apoptosis.

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Fig. 4.
Stable expression of caspase
cleavage-resistant desmin confers partial protection against
TNF- -induced apoptosis. MCF-7
cells stably expressing wild-type desmin or the caspase
cleavage-resistant mutant D263E desmin (two independent clones
B1 and C4) were treated with 10 ng/ml TNF- and
1 µg/ml CHX for 24 h, and the cells were scored for apoptotic
nuclei by staining with Hoechst 33258 as described (17). The data
represent the mean ± S.D. of three independent experiments. The
expression of desmin in control (C) vector-transfected MCF-7
cells, WT desmin-expressing cells, or mutant D263E desmin-expressing
cells is shown in the immunoblot at the top of the
figure.
|
|
 |
DISCUSSION |
We have identified the muscle-specific intermediate filament
protein desmin as a novel caspase substrate that is selectively proteolyzed by caspase-6 at a highly conserved Asp residue in its L1-L2
linker domain (VEMD
M264) in vitro and in
apoptotic myogenic cells. The P1-P4 positions of this substrate
recognition sequence (VEMD) in desmin have been conserved absolutely
from chicken to man and match the VEXD consensus caspase-6
cleavage motif (18, 19). Interestingly, a similar or identical
caspase-6 cleavage motif is present in each of the intermediate
filament proteins previously demonstrated to be caspase substrates: the
nuclear lamins (VE(I/V)D), cytokeratin 14 (VEMD), cytokeratins 18 and
19 (VEVD), and vimentin (IDVD) (11-17). Of these substrates, all but
the lamins and desmin also are cleaved by other caspases at sites
outside the L1-L2 linker domain. Together with our results, these
findings strongly suggest that caspase-6 may play a specialized role in
the proteolytic dismantling of the intermediate filament network. This
notion is supported by the recent demonstration of defects in lamin
(A/C) cleavage, chromatin condensation, and apoptotic body formation in
nuclei that were added to apoptotic extracts lacking caspase-6
(36).
We also have demonstrated that caspase proteolysis of desmin has
profound effects on its ability to assemble into intermediate filaments. Indeed, the amino-terminal caspase cleavage product (N-desmin encoding amino acids 1-263) is completely unable to assemble
into filaments and forms intracellular aggregates/inclusions. These
desmin aggregates morphologically are very similar to those formed by
caspase-cleaved cytokeratins and vimentin (13, 17, 37). The cytokeratin
aggregates have been studied most extensively and contain activated
caspase-3, the putative scaffold proteins DEDD and DEDD2, and
cytokeratins 8, 18, and 19, in addition to caspase-cleaved cytokeratin
18 (37, 38). Although the other components of the aggregates formed by
N-desmin are currently unknown, they likely contain the small heat
shock protein
B-crystallin, a molecular chaperone that binds to
desmin and other intermediate filament proteins (39). Interestingly,
mutations in either desmin (predominantly in the carboxyl terminus) or
B-crystallin result in similar appearing cytoplasmic aggregates
composed of desmin and
B-crystallin in muscle cells of individuals
with desmin-related myopathies (26-28, 40).
Furthermore, we have shown that N-desmin interferes in a
dominant-negative fashion with the assembly of WT desmin and vimentin into intermediate filaments. In this way, proteolytic production of
even a small amount of N-desmin likely would have a substantial deleterious impact on the intermediate filament network.
Importantly, our results are entirely consistent with earlier
studies using an arbitrarily truncated desmin cDNA (encoding amino
acids 1-272 of hamster desmin); expression of this cDNA in
cultured skeletal myoblasts or in muscles of transgenic animals led to
the formation of aggregates and the disassembly of the desmin and
vimentin intermediate filament networks (41, 42). Moreover, expression
of this truncated hamster desmin cDNA in the PtK2 epithelial cell
line led to the disruption of cytokeratin filaments as well as the
desmin and vimentin cytoskeletal networks (43). Although it seems
plausible that N-desmin likewise may disrupt cytokeratin assembly,
we have not yet examined this potential interaction experimentally.
Nevertheless, our results indicate that the proteolytic removal of the
carboxyl terminus of desmin by caspases generates a dominant-negative
inhibitor of both desmin and vimentin filament assembly, a potent and
parsimonious strategy to potentially dismantle multiple intermediate
filament networks during the induction of apoptosis. As noted, the
concept that truncated desmin can disrupt the intermediate filament
cytoskeleton in a dominant-negative manner (41-43) is not a new one;
however, the findings presented here provide the first evidence for the production of such an inhibitor during apoptosis in
vivo.
In addition, our observation that a caspase cleavage-resistant desmin
mutant (D263E) partially protects cells from TNF-
-induced apoptosis
clearly indicates that caspase proteolysis of desmin is a functionally
relevant event in the execution of apoptotic cell death. How might
caspase cleavage of desmin promote apoptosis? We previously have
demonstrated that caspase proteolysis of vimentin produces a
pro-apoptotic amino-terminal product that is sufficient to induce cell
death (17). In contrast, neither of the desmin cleavage products
significantly induced apoptosis in myogenic H9c2 cells in transient
transfection experiments. Indeed, the vast majority of myoblasts
transfected with N-desmin were still viable at 72 h despite the
presence of extensive aggregates (data not shown). These findings are
in agreement with those of Schultheiss et al. (41), who
demonstrated that expression of a similarly truncated hamster desmin
cDNA (encoding amino acids 1-272) in chicken skeletal myoblasts is
well tolerated for up to 2 weeks in culture, despite the complete
disruption of the intermediate filament network. Although we cannot
exclude the possibility that N-desmin (or C-desmin) might sensitize
muscle cells to the induction of apoptosis by oxidative stress or other
apoptotic signals, our results argue against a potent direct
pro-apoptotic function for either desmin cleavage product.
Instead, the findings presented here suggest that caspase cleavage of
desmin impairs, in some way, its pro-survival function. In
support of this model is the observation that mice deficient in desmin
develop a generalized myopathy, especially prominent in cardiac muscle,
that is characterized by a disruption of the lateral alignment of
myofibrils and a dramatic increase in cardiac myocyte cell death,
ultimately leading to heart failure (21-24). One potential
mechanism linking these structural deficits to cell death is the
observation that the mitochondria of desmin-deficient mice are
morphologically abnormal with matrix swelling and degeneration; furthermore, the mitochondria are defective in ADP-stimulated respiratory function in vivo (44). Given the central role of the mitochondria in stress-induced apoptosis (1), these mitochondrial defects stemming from desmin deficiency or potentially resulting from
caspase cleavage of desmin could promote muscle cell death. Moreover,
desmin (or vimentin in mesenchymal cells) is tightly associated with
the nuclear matrix via binding to lamin B (45-47). Hence, caspase
proteolysis of desmin may be important for the apoptotic disassembly of
the nuclear envelope, as has been previously demonstrated for caspase
cleavage of the nuclear lamins and vimentin (9-12, 16, 36).
Alternatively, desmin may sequester components of the TNF-
apoptotic
signaling pathway, such as TRADD or TNFR2 (or others), that have been
shown to interact with cytokeratins, thereby negatively regulating
TNF-
-induced apoptosis (48, 49). Regardless of the
mechanism(s), the results presented here indicate that caspase cleavage
of desmin is a functionally important molecular event in apoptosis,
producing a truncated desmin protein that disrupts intermediate filaments.
We are indebted to Dr. Robert Talanian for the
recombinant caspases used in these experiments.
Published, JBC Papers in Press, December 10, 2002, DOI 10.1074/jbc.M212021200
The abbreviations used are:
TNF, tumor necrosis
factor;
HEK, human embryonic kidney;
CHX, cycloheximide;
mAb, monoclonal antibody;
WT, wild-type;
zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone.
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