Caspase Proteolysis of Desmin Produces a Dominant-negative Inhibitor of Intermediate Filaments and Promotes Apoptosis*

Feng ChenDagger , Roger ChangDagger , Marcus TrivediDagger , Yassemi Capetanaki§, and Vincent L. CrynsDagger

From the Dagger  Division of Endocrinology, Metabolism, and Molecular Medicine, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611 and the § Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030

Received for publication, November 25, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (VEMDdown-arrow 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-alpha -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 VEMDdown-arrow 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-alpha . 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-alpha -induced apoptosis, thereby indicating that caspase cleavage of desmin is a functionally relevant proteolytic event during the induction of apoptosis.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-alpha 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-alpha 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 Cdelta polyclonal antibody (Santa Cruz Biotechnology). For inhibitor studies, H9c2 myoblasts were treated with 10 ng/ml TNF-alpha 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-alpha 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-alpha 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 VEMDdown-arrow 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-alpha 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-alpha . 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-alpha -induced proteolysis of the well characterized caspase-3 substrate protein kinase Cdelta (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-alpha 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-alpha -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 Cdelta (PKCdelta ) cleavage in murine HL-1 cardiac myocytes induced to undergo apoptosis by treatment with TNF-alpha . Differentiated HL-1 cells were treated with 10 ng/ml TNF-alpha 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-alpha -induced desmin proteolysis. Rat H9c2 cardiac myoblasts were treated with 10 ng/ml TNF-alpha 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-alpha . HEK 293 cells transiently transfected with WT or mutant D263E desmin cDNAs were treated with 10 ng/ml TNF-alpha 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-alpha -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-alpha -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-alpha -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-alpha -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-alpha 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (VEMDdown-arrow 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 alpha 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 alpha B-crystallin result in similar appearing cytoplasmic aggregates composed of desmin and alpha 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-alpha -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-alpha apoptotic signaling pathway, such as TRADD or TNFR2 (or others), that have been shown to interact with cytokeratins, thereby negatively regulating TNF-alpha -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.

    ACKNOWLEDGEMENT

We are indebted to Dr. Robert Talanian for the recombinant caspases used in these experiments.

    FOOTNOTES

* This work was supported in part by a grant from the Muscular Dystrophy Association (to V. L. C.), Grant NS31957 from the National Institutes of Health (to V. L. C.), an institutional research grant to Northwestern University from the Howard Hughes Medical Institute (to V. L. C.), and the Elizabeth Boughton Trust (to V. L. C.).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.

To whom correspondence should be addressed: Division of Endocrinology, Tarry 15-755, Feinberg School of Medicine, Northwestern University, 303 E. Chicago Ave., Chicago, IL 60611. Tel.: 312-503-0644; Fax: 312-908-9032; E-mail: v-cryns@northwestern.edu.

Published, JBC Papers in Press, December 10, 2002, DOI 10.1074/jbc.M212021200

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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