18 breakdown and reorganization during apoptosis

Experimental Cell Research 297 (2004) 11 – 26 www.elsevier.com/locate/yexcr

Keratin 8/18 breakdown and reorganization during apoptosis Bert Schutte, a,* Mieke Henfling, a Wendy Ko¨lgen, a Maartje Bouman, a Stephan Meex, a Mathie P.G. Leers, b Marius Nap, b Viveka Bjo¨rklund, c Peter Bjo¨rklund, c Bertil Bjo¨rklund, d E. Birgitte Lane, e M. Bishr Omary, f Hans Jo¨rnvall, d and Frans C.S. Ramaekers a a

Department of Molecular Cell Biology (Box 17), Research Institute Growth and Development (GROW), University of Maastricht, The Netherlands b Department of Pathology, Atrium Medical Centre, Heerlen, The Netherlands c Peviva AB, Stro¨mkarlsva¨gen 82, SE-167 62 Bromma, Sweden d Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77, Stockholm, Sweden e Cancer Research UK, Cell Structure Research Group, School of Life Sciences, University of Dundee, UK f Palo Alto VA Medical Center and Stanford University School of Medicine, Stanford University, Palo Alto, CA 94304, USA Received 28 October 2003, revised version received 19 February 2004 Available online 27 March 2004

Abstract Monoclonal antibodies that specifically recognize caspase cleaved K18 fragments or specific (phospho)epitopes on intact K8 and K18 were used for a detailed investigation of the temporal and causal relationship of proteolysis and phosphorylation in the collapse of the keratin cytoskeleton during apoptosis. Caspases involved in the specific proteolysis of keratins were analyzed biochemically using recombinant caspases and specific caspase inhibitors. Finally, the fate of the keratin aggregates was analyzed using the M30-ApoptoSensek Elisa kit to measure shedding of caspase cleaved fragments into the supernatant of apoptotic cell cultures. From our studies, we conclude that C-terminal K18 cleavage at the 393DALD/S site is an early event during apoptosis for which caspase 9 is responsible, both directly and indirectly by activating downstream caspases 3 and 7. Cleavage of the L1-2 linker region of the central a-helical rod domain is responsible for the final collapse of the keratin scaffold into large aggregates. Phosphorylation facilitates formation of these aggregates, but is not crucial. K8 and K18 remain associated in heteropolymeric aggregates during apoptosis. At later stages of the apoptotic process, that is, when the integrity of the cytoplasmic membrane becomes compromised, keratin aggregates are shed from the cells. D 2004 Elsevier Inc. All rights reserved. Keywords: M30-CytoDeath; Caspase; Keratin; Phosphorylation; Cytoskeleton

Introduction Keratins, the epithelium-specific intermediate filament proteins, compose of 20 cytoplasmic members in man, excluding the trichocytic keratins [1]. They are subdivided into type I and type II keratins [2], of which non-covalent 1:1 heteropolymers are expressed in a cell type-specific manner. For example, K8 and 18 are co-expressed and form heterodimers in glandular epithelia. Intermediate filament proteins contain a central a-helical rod domain and non-a* Corresponding author. Department of Molecular Cell Biology (Box 17), Maastricht University, P.O. Box 616, 6200 MD Maastricht, The Netherlands. Fax: +31-43-3884151. E-mail address: [email protected] (B. Schutte). 0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2004.02.019

helical NH2-terminal (head) and COOH terminal (tail) domains. The head and tail domains contain the sites for several posttranslational modifications, including phosphorylation and glycosylation [3]. Keratins are highly dynamic and become reorganized during various cellular events such as differentiation, mitosis, and apoptosis. Although many of their functions remain to be established, it is evident that keratins provide structural support to the cell and help cells to cope with stress [4,5]. Periodic filament remodeling is essential to keratin function. Potential mechanisms involved in filament reorganization include phosphorylation [3], proteolysis [6 – 8], and interaction with non-keratin proteins [9]. During apoptosis, a cell undergoes dramatic changes in morphology due to a complete reorganization of its cyto-

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plasmic and nuclear skeleton [10,11]. These alterations probably facilitate the rapid, but securely orchestrated breakdown of the cell into apoptotic bodies, ensuring the maintenance of an intact cellular membrane and efficient clearance by phagocytes [12 – 14]. Many components of the cytoplasmic and nuclear cytoskeleton are targeted for proteolysis, including members of the intermediate filament proteins, and proteins associated with the cytoskeleton such as plectin [15], gelsolin [16], Gas2 and h-catenin [17 –20]. Recently, it was shown that procaspases 3 and 9 are specifically targeted to the K8/18 intermediate filament network by the death effector domain containing DNA binding protein (DEDD) [21,22]. Interference with this apoptosis-dependent targeted breakdown results in severe impairment of the cytoplasmic and nuclear cytoskeleton reorganization and accompanying processes such as chromatin condensation [11]. Aside from proteolysis, targeted phosphorylation of cytoplasmic and nuclear cytoskeleton components also potentiates their disassembly during cell death [23,24]. A key feature during the process of apoptosis is the activation of the proteolytic caspase cascade that is responsible for the stepwise cleavage of many critical cellular proteins. These cleavage processes result in activation of enzymes including kinases and procaspases, proteolytic inactivation of enzymes, such as poly-ADP-ribose-polymerase (PARP), or degradation of structural proteins including intermediate filaments. Many intermediate filament proteins contain a caspase consensus site in the conserved L1-2 linker region of the central a-helical rod domain [25]. For instance, nuclear lamins and type I keratins possess a VEVD/A or VEID/A site that is targeted by caspase 6. In contrast, type II keratins are apparently resistant to proteolysis by caspases and remain associated with fragments of their partner keratins during apoptosis [6]. A second caspase cleavage site was identified in the COOH terminal (tail) domain of K18 [7], which has been further characterized as 393 DALD/S. Cleavage at this site generates a neo-epitope that is specifically recognized by the M30 CytoDeath monoclonal antibody [8]. It has been suggested that caspase cleavage of the K18 COOH terminal domain is an early event in the apoptotic cascade, preceding loss of membrane asymmetry, DNA fragmentation as determined by DNA nick end labeling, and cleavage of the L1-2 linker region of the central a-helical rod domain [8]. During apoptosis, K8/18 intermediate filaments reorganize into granular structures enriched for K18 that has been phosphorylated on serine 52. In fact most of the known K8/ 18 phosphorylation sites are phosphorylated during apoptosis [6,26]. Whether hyperphosphorylation is an early or a late event during apoptosis is still unclear. Some authors suggest that hyperphosphorylation of keratins may represent an early physiologic stress marker for simple epithelia [27,28]. Alternatively, keratins might be phosphorylated during the execution phase of the apoptotic process, facilitating the rapid collapse of the cytoskeletal architecture.

The proteolytic fragments of human type I keratins are stable [6] and persist as large aggregates in the apoptotic bodies [8,10]. Indeed, it has been suggested that these keratin aggregates are shed from the cells and escape the clearance by phagocytes, since keratins can be detected in sera of cancer patients [29 – 33]. In this study, a detailed investigation of temporal and mechanistic aspects of keratin breakdown and reorganization during apoptosis has been undertaken. The fate of the cleaved keratin fragments was studied in relation to each other and to known indicators of the apoptotic process using monoclonal antibodies directed against different epitopes on K8 and K18. Caspases involved in the specific proteolysis of keratins were analyzed biochemically using recombinant caspases and specific caspase inhibitors. The role of hyperphosphorylation during pre-apoptotic reorganization of the cytoskeleton was studied using phosphokeratin-specific antibodies and specific kinase inhibitors. Finally, the fate of the keratin aggregates was analyzed using the M30ApoptoSensek Elisa kit to measure shedding of caspase cleaved fragments into the supernatants of apoptotic cell cultures. From our studies, we conclude that C-terminal K18 cleavage is an early event during apoptosis for which caspase 9 is responsible directly and indirectly by activating downstream caspases 3 and 7. Cleavage of the L1-2 linker region of the central a-helical rod domain is responsible for the final collapse of the keratin scaffold into large aggregates. Phosphorylation facilitates formation of these aggregates, but is not crucial. K18 and K8 remain associated in heteropolymeric aggregates during apoptosis. At later stages of the apoptotic process, that is, when the integrity of the cytoplasmic membrane becomes compromised, keratin aggregates are shed into the culture medium.

Materials and methods Cell lines The human squamous cell lung cancer cell line MR65 was cultured in Eagle’s modified minimal essential medium (Cat. no. 12565-024; GIBCO, Paisley, Scotland, United Kingdom), supplemented with 1% non-essential amino acids (GIBCO), 1% HEPES (GIBCO), 2 mM L-glutamine (Cat. no. 22942; Serva, Heidelberg, Germany), 10% heatinactivated newborn calf serum (Cat. no. 021-6010M; GIBCO) and 50 Ag/ml gentamycin (AUV, Cuyck, The Netherlands). In some experiments, the cells were cultured on eight-well glass slides (Nutacon, Schiphol, The Netherlands) pre-coated with freshly isolated rat-tail collagen I (10 mg/ml). Induction of apoptosis Apoptosis was induced by incubation of cell cultures for various periods of time with 50 AM roscovitine (final con-

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centration of DMSO was 2.5%), a kind gift from Dr. Laurent Meyer (Station Biologique, CNRS, Roscoff, France) or with 50 AM etoposide (final concentration of DMSO was 0.3%).

cultures at the following concentrations unless stated differently: Ac-DEVD-CHO or Ac-LEHD, 20 AM; z-VEID-fmk or z-VAD-fmk, 100 AM.

Cell harvesting and fixation

Antibodies

Cells growing in monolayers were harvested quantitatively by standard trypsinization. The cells were rinsed once in phosphate-buffered saline (PBS) before fixation for 5 min in absolute methanol at 20jC. For double staining with M30 CytoDeath monoclonal antibody and CMXRos (Molecular Probes, Leiden, The Netherlands), or anti-active caspase 3, cells were fixed in 4% paraformaldehyde for 15 min at RT, followed by permeabilization for 10 min at room temperature (RT) in PBS containing 0.005% SDS.

The following primary antibodies were used: (1) Monoclonal antibody IIB5 (diluted 1:50) to bromodesoxyuridine (BrdU) from Euro-Diagnostics BV (Arnhem, The Netherlands) as a negative control; (2) rabbit antiserum directed against active caspase 3, a gift from IDUN Pharmaceuticals (San Diego, CA); (3) monoclonal anticaspase 3 from BD Transduction Laboratories Cat. No. 610322); (4) monoclonal anti-cytochrome C antibody from BD Pharmingen Cat. No. 556432 (San Diego, CA); (5) monoclonal antibody RCK106 directed against an epitope in the NH2-terminus of K18 (see also Fig. 1). The antibody is available from MuBio Products BV, Maastricht, The Netherlands; (6) monoclonal antibody LE61, recognizing a shared epitope on K8/18 [34]. (7) Polyclonal rabbit antiserum CA1, raised against the carboxyterminal peptide of K18; (8) monoclonal antibody M30 CytoDeath (diluted 1:40, Peviva AB, Bromma, Sweden) recognizing the neo-epitope formed by caspase cleavage at the 393DALD/S site of K18. Isotyping of the monoclonal antibody M30 was performed with the mouse monoclonal antibody isotyping kit (GIBCO BRL, Cat. no. 19663020, Cell Biology Products). The isotype of the M30 monoclonal antibody was IgG2b; (9) monoclonal antibodies RCK102 and M20 recognizing K8 (MuBio Products BV) [35]; (10) the phosphokeratin antibodies mAb LJ4 (K8 pSer73), mAb 5B3 (K8 pSer431), mAb IB4 (K18 pSer33) [28]; (11) monoclonal antibody 41CC4 directed

Kinase inhibitors For inhibition of keratin hyperphosphorylation, staurosporine (Sigma) was added to 100 nM final concentration 0.5 h before induction of apoptosis in exponentially growing MR65 cells. Caspase inhibitors The cell-permeable caspase 3 inhibitor (Ac-DEVD-CHO) and z-VAD.fmk were purchased from Biomol (Sanvertech, Heerhugowaard, The Netherlands). The cell-permeable caspase 9 inhibitor (Ac-LEHD-CHO) was purchased from Calbiochem. Caspase 6 inhibitor (z-VEID-fmk) was obtained from Alexis (Kordia, Leiden, The Netherlands). Protease inhibitors were dissolved in DMSO and added to the cell

Fig. 1. Schematic representation of the K8/18 dimer. The different head, tail, and rod domains are indicated by cylinders and linker region domain as interconnecting solid lines. Phosphorylation sites, caspase cleavage sites, as well as the location of the different epitopes of K8 and 18 of the antibodies used in this study are indicated.

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against Lamin A/C was kindly provided by Dr. G Warren (Heidelberg, Germany). As secondary antibodies, R-Phycoerythrin (RPE)-conjugated F(ab’)2 fragment of affinity-purified goat anti-mouse immunoglobulins (R0480, DAKO A/S, Glostrup, Denmark; diluted 1:10), fluorescein isothiocyanate (FITC)conjugated F(ab’)2 fragments of rabbit anti-mouse IgG (F313; DAKO A/S; diluted 1:10), FITC or Texas redconjugated goat anti-rabbit immunoglobulins (Southern Biotechnology Associates (SBA), Birmingham, USA; diluted 1:80), FITC- and Texas red-conjugated goat antimouse IgG1 and goat anti-mouse IgG2b (SBA; diluted 1:80), FITC-conjugated streptavidin (F0422; DAKO A/S; diluted 1:100) were used. Annexin V labeling To quantify the apoptotic frequency based on the exposure of phosphatidylserine at the outer leaflet of the membrane, annexin V biotin (APOPTEST-BIOTIN, Nexins Research BV, Hoeven, The Netherlands) was added to cells. Adherent cells were harvested by mechanical scraping with a disposable cell scraper and stained and analyzed as described previously [36]. Staining for mitochondrial membrane potential MR65 cells were loaded with 500 nM CMXRos (Molecular Probes) at 37jC, 30 min before harvesting. As a control, cells were loaded with CMXRos in the presence of 75 mM of the uncoupling reagent carbonyl cyanide mchlorophenylhydrazon (CCCP, Sigma, The Netherlands). In vitro keratin cleavage Cytoskeletal keratin extracts were prepared by resuspending approximately 5  107 MR65 cells for 10 min in 10 ml cold RSB/PMSF/Triton X100 buffer (1.5 mM MgCl2, 10 mM Tris – HCl, 0.5 mM PMSF, 0.5% Triton X100, pH 7.4). The suspension was centrifuged for 5 min at 1500  g at 4jC and the pellet was resuspended in 200 Al DNAse/RNAse mix (1.5 mM MgCl2, 110 mM NaCl, 10 mM Tris –HCl pH 7.4 to which 1.1 mg/ml DNAse I, 50 ng/ml RNAse, and 0.5 mM PMSF have been added). The resuspended pellet was incubated for 20 min at RT. Five milliliters RSB/PMSF was added to the suspension and a pellet fraction was recovered after centrifugation for 5 min at 1500  g at 4jC. The pellet was rinsed once in 1 ml RSB/PMSF buffer, and finally resuspended in 500 Al caspase working buffer (20 mM Hepes, 100 mM NaCl, 0.1% CHAPS, 10 mM dithiothreitol (DTT), 1 mM EDTA, pH 7.4), and stored at 70jC. For in vitro cleavage studies, recombinant caspases were added to 40 Al keratin extract, diluted four times in caspase working buffer. The final concentration of the recombinant caspases was 0.8 Ag/ml or 1 unit in the case of caspase 9. Recombinant caspases 3, 6, 7, and 8 were

kind gifts from Dr. G. Salvesen (Burnham Institute, San Diego, USA). Recombinant caspase 9 was purchased from BioVision Inc., Mountain View, CA. Immunocytochemistry on glass slides Apoptosis was induced by replacing the culture medium with fresh medium to which etoposide or roscovitine was added. At various time intervals after induction of apoptosis, the cells were washed twice with PBS and fixed in methanol ( 20jC). The cells were rinsed with PBS and incubated with the primary antibody for 1 h in a humidified chamber at RT. The cells were then rinsed three times with PBS/BSA (1 mg/ml BSA). Antibody binding was visualized by incubation for 1 h at RT in the dark with an appropriate FITC-labeled secondary antibody. The cells were counterstained with 5 Ag/ml propidium iodide (PI, Sigma) in PBS containing RNAse (1 mg/ml; Serva) for 7 min and mounted in glycerol/ DABCO (9:1 glycerol, 1 part 0.2 M Tris –HCl pH 8.0, 2% DABCO pH 8.0, 0.02% NaN3). In double staining experiments, the cells were incubated simultaneously with the two primary antibodies, either combinations of antimouse and anti-rabbit Ig or monoclonal antibodies of different isotype, which were detected by anti-isotypespecific secondary antibodies. Antibody binding was visualized by incubating the cells with FITC- and TxRedconjugated antibodies simultaneously. Annexin V biotin was added to the cells before fixation of the cells. Annexin V binding was visualized using FITC-labeled streptavidin. The cells were counterstained with DAPI (5 Ag/ml; Sigma, St. Louis, MO) for 7 min and mounted in glycerol/DABCO (9:1 glycerol, 1 part 0.2 M Tris –HCl pH 8.0, 2% DABCO pH 8.0, 0.02% NaN3). The glass slides were covered and sealed before examination by confocal scanning laser microscopy. As a negative control, mouse anti-BrdU (clone IIB5) was used as primary antibody. Immunocytochemistry for flow cytometric analysis After harvesting and fixation, the cells were subsequently rinsed twice with PBS. The resuspended pellet of approximately 106 cells was incubated for 1 h in the dark with the monoclonal antibody M30. Antibody binding was visualized with FITC-conjugated Fab2 fragments of anti-mouse IgG (DAKO F0313). After incubation for 1 h, the cells were rinsed twice with PBS and finally resuspended in PBS, containing 20 Ag/ml propidium iodide (Calbiochem) and 100 Ag/ml RNase (Serva). For double labeling experiments with M30 and anti-keratin antibodies, both primary antibodies were added to the cells simultaneously. M30 immunoreactivity was visualized by incubation with RPE-conjugated goat anti-mouse IgG2b (SBA), and keratin immunoreactivity was visualized using FITC-conjugated anti-mouse IgG1 (SBA).

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Both secondary antibodies were added simultaneously and the cells were incubated for 1 h at RT. The cells were rinsed twice in PBS/BSA, finally resuspended in PBS/PI/RNase, and kept on ice for at least 15 min before flow cytometric analysis. Measurement of caspase activity MR65 cells were washed three times with PBS and harvested in cold 50 mM Tris – HCl, pH 7.5, with a disposable cell scraper. Subsequently cells (1  107/ml) were sonicated for 5 s using a Soniprep 150 (MSE Scientific Instruments, Beun-De Ronde) sonicator (amplitude 12 Am). Cell lysates were centrifuged at 10,000  g at 4jC in an Eppendorf centrifuge and supernatants were either immediately assayed or frozen at 20jC. Caspase 3 activity was measured in 50 Al aliquots of cell lysate, which is the equivalent of 500,000 cells.

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Fifty microliters of substrate mix (50 mM Tris/HCl, pH7.5, containing 2.5 mM DTT and 40 AM Ac-DEVDAMC (Biomol), Ac-WEHD, Ac-IETD, AcVEID, AcVDAD or Ac-LEHD (Alexis, Kordia)) was added to 50 Al of cell lysate. The substrate mixture was incubated for 60 min at 37jC and then diluted with 1.8 ml PBS. Fluorescence was measured using a SPF-500C (SLMAminco) spectrofluorometer. Western blotting Apoptosis was induced by replacement of the culture medium with fresh medium to which roscovitine or etoposide was added. At various time intervals after induction of apoptosis, cells were harvested. Detached and adherent cells were combined, washed with PBS and resuspended in lysis buffer (62.5 mM Tris – HCl pH 6.8, 12.5% glycerol, 2% Nonidet P40 (NP40), 2.5 mM phenylmethylsulphonyl fluo-

Fig. 2. Temporal order of K18 cleavage during apoptosis in MR65 cells. Apoptosis was induced using roscovitine and cells were stained with the M30 monoclonal antibody alone (a, b) or in combination with other known markers of apoptosis (c – f). Stained cells were analyzed using confocal scanning laser microscopy. (a) A shadow projection of a confocal stack of images of M30 reactivity in a typical late apoptotic cell. Note the granular M30 staining pattern (green) combined with a fragmented and condensed chromatin (red); (b) a shadow projection of a confocal stack of images of a M30-reactive early apoptotic cell. This early apoptotic cell is characterized by a fine filamentous M30 staining pattern without any obvious signs of chromatin condensation or fragmentation; (c) linear projection of a confocal stack of images of a triple labeled apoptotic cells with annexin V (green) and M30 (red) antibody. Nuclei are counterstained with DAPI (blue). Note that PS exposure does not occur in the cell showing a filamentous M30 staining pattern, in contrast to the late apoptotic cell, which shows a membranous annexin V staining pattern. (d) Double labeling of early apoptotic cells with anti-active caspase 3 (green) and M30 (red) antibodies. Note that in one cell showing a filamentous M30 staining pattern hardly any active caspase 3 is detected. (e) A discrete mitochondrial staining pattern is observed in very few M30-positive (green) cells after loading of the cells with the mitochondrial-specific dye CMXRos (red); the majority of cells showing a typical mitochondrial labeling pattern with CMXRos were always devoid of M30 staining; in apoptotic cells showing a dot-like M30 reactivity no accumulation of CMXRos in the mitochondria was observed. (f) Double labeling experiment with the M30 (red) and an anti-cytochrome c (green) antibody. All M30-reactive cells, including those with a filamentous type of staining, showed a diffuse distribution of cytochrome c in the cytoplasm. M30-negative, nonapoptotic cells always showed a discrete mitochondrial labeling with the anti-cytochrome c antibody.

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ride (PMSF), 1.25 mM EDTA, 12.5 Ag/ml leupeptin and 10 Ag/ml aprotinin) to a concentration of 2  107 cells/ml. As a control, non-induced, exponentially growing MR65 cells were rinsed with PBS, scraped, collected, and resuspended in lysis buffer. The cell lysates were kept on ice for 30 min before freezing at 20jC. Before gel electrophoresis, the samples were supplemented with sample buffer (1:1), containing 2.3% SDS, 62.5 mM Tris – HCl pH 6.8, 10% glycerol, 5% h-mercaptoethanol, and 0.05% bromophenol blue. Samples were boiled for 5 min. One-dimensional sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli [37] using gels containing 10%, 13%, or 15% polyacrylamide (Bio-Rad Laboratories, Hercules, CA) and 0.1% SDS (Merck, Darmstadt, Germany). As molecular weight markers, a low molecular weight marker kit (97, 66, 45, 31, 21, and 14 kDa; BioRad Laboratories) was used. Gels were run on the Mini-Protean II system (BioRad Laboratories) for 45 min at 200 V. For Western blotting, the method described by Towbin et al. [38] was used. Detection was performed using enhanced chemiluminescence (ECL; Cat. no. RRN 2106; ECL-kit, Amersham Life Science, Amersham, UK). Confocal scanning laser microscopy Cells grown on glass slides were analyzed using the MRC600 confocal scanning laser microscope (Bio-Rad, Hemel Hempstead, United Kingdom), equipped with an air-cooled Argon-Krypton mixed-gas laser and mounted onto an Axiophot microscope (Zeiss, Oberkochen, Germany). The laser-scanning microscope was used in the dual-parameter setup, according to manufacturer’s specification, using dual wavelength excitation at 488 and 568 nm. Emission spectra were separated by the standard set of dichroic mirrors and barrier filters. All scans were recorded in the Kalman filtering mode. For recording of DAPI staining, mercury arc illumination was used. To this end, the laser pathway was blocked and the filters were removed. Recordings were made, using the Kalman filtering mode. Image deconvolution was performed using Huygens software (Scientific Volume Imaging, The Netherlands) and three-dimensional reconstruction using Imaris software (Bitplane AG, Switzerland). Both software packages were run on a Silicon Graphics workstation. For microscopic quantification of anti-active caspase 3 immunofluorescence, the public domain software Image J was used (Wayne Rasband, National Institutes of Health, USA) on a linear projection of a stack of confocal images. Flow cytometry For flow cytometric analysis, a FACSort (Becton Dickinson, Sunnyvale, CA) equipped with an Argon ion laser and a diode laser was used. Excitation was performed at 488 nm, and the emission filters used were 515 – 545 BP (green;

FITC), 572 –588 BP (orange; PE), and 600 LP (red; PI). A minimum of 10,000 cells per sample was analyzed. FITC and PE signals were recorded as logarithmic amplified data, while the PI signals were recorded as linear amplified data. Electronic compensation was used to eliminate any bleed-through of fluorescence. For bivariate analysis, no compensation was used. Data analysis was performed using the CellQuest 3.1 software (Becton Dickinson, San Jose, CA). As a standard procedure for all analyses, data were gated on pulse-processed PI signals to exclude doublets and larger aggregates. Detection of shedded K18 fragments Keratin fragments in culture supernatants was determined using the M30-ApoptoSensek ELISA kit (Peviva AB). The assay was performed according to the manufacturer’s instructions.

Results In a previous study, we showed that the M30 antibody recognizes a neo-epitope at the C-terminus of K18 [8]. This neo-epitope is generated during the apoptotic process by caspase cleavage at 393DALD/S. After induction of apoptosis, the majority of immunoreactive cells show a dot-like staining pattern with M30, as well as chromatin fragmentation (Fig. 2a). However, in a minority of the cells, all still attached to the glass surface, a filamentous M30 staining

Fig. 3. Quantification of the immunoreactivity of anti-active caspase 3 antibody in M30-positive MR65 cells. M30 staining patterns were scored negative, filamentous or dot like. Anti-active caspase 3 immunoreactivity was measured using the public domain software Image J in linear projections of stacks of confocal images. Note that some cells with a filamentous staining pattern showed background levels of anti-active caspase 3.

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pattern was observed without obvious chromatin condensation or nuclear disintegration (Fig. 2b). These staining patterns suggest that caspase cleavage plays an important role in the reorganization of the keratin cytoskeleton during apoptosis. Therefore, experiments were designed to determine the temporal order of the different caspase-mediated cleavage processes of keratins using known hallmarks of apoptosis as key reference points. Furthermore, we addressed the relationship between keratin hyperphosphorylation and these cleavage processes during apoptosis. Finally, we determined the fate of the keratin

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cleavage products and keratin aggregates generated during apoptosis. Temporal order of K18 cleavage events versus known apoptosis hallmarks C-terminal K18 cleavage precedes phosphatidylserine exposure We demonstrated earlier that C-terminal caspase cleavage of K18 at the 393DALD/S site precedes loss of membrane phospholipid asymmetry [8]. Early after induction of

Fig. 4. (a, b) Western blotting analysis of in vitro caspase cleaved cytoskeletal preparations of MR65 cells treated with different caspases and probed with the RCK106 antibody (a) and M30 (b). Note that with RCK106 (a) a specific 30 kDa cleavage product, identical to the product present in the control lysate, is only seen in caspase 6-treated samples. Caspases 3-, 7-, or 9-treated samples show an additional fragment with slightly lower Mr (arrowhead) than that of the untreated sample. The M30 antibody (b) shows a 45 kDa reactive only in caspases 3-, 7- and 9-treated samples. Untreated or caspase 6-treated samples show no immunoreactivity. When samples were simultaneously treated with caspases 3 and 6, two characteristic cleavage products of 45 and 20 kDa, also found in the control lysate, are observed. These cleavage products correspond to the K18 molecule lacking either the tail or the tail and the rod 2 domain of K18 (see Fig. 1). (c) Western blotting analysis of an apoptotic cell lysate (lane 1) and a crude cytoskeletal preparation (lane 2) of MR65 cells. The blots were probed with RCK106 and anti procaspase 3 antibodies. Note that the cytoskeletal preparation is devoid of procaspase 3. (d, e) Western blotting of MR65 cells treated with roscovitine in the presence or absence of different caspase inhibitors, probed with RCK106 (d) or the M30 antibody (e). The general caspase inhibitor z-VAD and the caspase 6 inhibitor prevent the appearance of the 30-kDa RCK106-reactive cleavage product, in contrast to the caspases 3 and 9 inhibitors. z-VAD could not completely prevent the appearance of a 45-kDa M30-reactive fragment. Caspase 6 inhibitor failed to inhibit this cleavage event completely. Apoptosis in the presence of caspases 3 or 9 inhibitor resulted in the accumulation of the 20-kDa M30 reactive fragment.

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apoptosis the few M30-positive cells, which are observed are all devoid of annexin-V binding. The M30 staining pattern in these few cells is largely filamentous, indicating the keratin network did not yet collapsed (Fig. 2c). C-terminal K18 cleavage precedes activation of caspase 3 The temporal relationship between caspase 3 activation and the C-terminal cleavage of K18 was studied by

double labeling of MR65 cells with M30 and an antiserum directed against the activated caspase 3. Early after induction of apoptosis by roscovitine, many M30-positive cells, showing a filamentous type of staining, were virtually devoid of active caspase 3 (Fig. 2d). In M30positive cells showing an aggregated type of staining, active caspase 3 was clearly present. Active caspase 3 immunoreactivity was quantified in M30-negative cells,

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M30-positive cells with filamentous type of staining and M30-positive cells with an dot-like type of staining. As shown in Fig. 3, active caspase 3 immunoreactivity of M30-positive cells with filamentous type of staining overlapped with that of M30-negative non-apoptotic cells. These experiments show that a caspase upstream of caspase 3 must be responsible for the initial C-terminal K18 cleavage at the 393DALD/S site. C-terminal K18 cleavage follows loss of mitochondrial membrane potential and cytochrome c release To compare the appearance of M30 immunoreactivity with loss of mitochondrial membrane potential, apoptotic cells were harvested at various time intervals after induction with roscovitine. Before harvesting, roscovitine-treated cells were loaded for 30 min with the membrane potentialsensitive probe CMXRos. The majority of M30-reactive cells showed a loss of discrete mitochondrial accumulation of CMXRos fluorescence. Very few cells with a filamentous type of M30-staining pattern showed discrete labeling of mitochondria (Fig. 2e). Similarly, when cells were double labeled for M30 and cytochrome c, all M30-positive cells showed a diffuse cytoplasmic staining for cytochrome c. No M30-positive cells were found with cytochrome c discretely distributed over the mitochondria (Fig. 2f). From these experiments, it is clear that C-terminal cleavage of K18 occurs (immediately) after the mitochondria loose their membrane potential and release cytochrome c into the cytoplasm. The different caspases involved in K18 cleavage To identify the different caspases that are potentially responsible for the generation of the M30 epitope, cytoskeleton preparations were incubated with recombinant initiator caspases 8, 9 and executioner caspases 3, 6, and 7. Western blots were probed with the anti-K18 antibody RCK106 or

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M30. As shown in Fig. 4a, K18 is cleaved in the L1-2 linker region only by caspase 6, resulting in a cleavage fragment of approximately 30 kDa. Fig. 4b shows that caspases 3, 7, and 9 are able to generate an M30-reactive fragment of approximately 45 kDa, corresponding to K18 with the C-terminus removed. When extracts were treated with combinations of caspases 3 and 6, an additional cleavage product of approximately 20 kDa was observed, corresponding to the coil2 region, resulting from cleavage at both the L1-2 linker region and at the C-terminus (Fig. 4b, lane 7). When blots were probed with RCK106, a cleavage fragment with slightly lower Mr than intact K18 was detected in caspases 3-, 7-, or 9-treated samples (arrowhead in Fig. 4a). Unlike the other caspases, recombinant caspase 8 was not able to cleave K18 (data not shown). To exclude the possibility that not recombinant caspase 9 but residual procaspase 3 activity in the cytoskeleton preparation is indirectly responsible for Cterminal K18 cleavage, blots of cytoskeletal preparations were probed with a monoclonal antibody against procaspase 3. Fig. 4c shows that cytoskeleton preparations were devoid of procaspase 3, while the protein could be detected in the total cell extract. To determine which caspases are responsible for cleavage at the DALD site in vivo, cells were treated with roscovitine in the presence or absence of various caspase inhibitors. In the presence of caspase 6 inhibitor or the general caspase inhibitor zVAD-fmk, cleavage at the L1-2 linker region was inhibited (Fig. 4d). The caspases 3 and 9 inhibitors did prevent cleavage of K18 at the 393DALD/S site to some extent, but not completely (Fig. 4e). Also, cleavage at the 393 DALD/S site was only partially inhibited in the presence of zVAD-fmk, although cleavage of the L1-2 linker region was completely inhibited (Figs. 4d and e). These experiments show that caspases 3, 7, and 9 are able to cleave K18 at the 393DALD/S site and that caspase 6 is responsible for cleavage at the L1-2 linker region.

Fig. 5. (a – g) Keratin phosphorylation and cleavage. Non-treated MR65 cells (a – c) stained with phosphokeratin-specific antibodies: (a) IB4 (K18 pSer33, (b) LJ4 (K8 pSer73, c) 5B3 (K8 pSer431). All cells at all stages were phosphorylated at K18 Ser33 (a), whereas mitotic cells showed phosphorylation of K8 on Ser73 to a much lower extent (b), but massive hyperphosphorylation of K8 on Ser431 (c). Spontaneously occurring apoptotic cells were clearly positive with the latter two antibodies. Treatment of MR65 cells with roscovitine (d – g) resulted in rapid hyperphosphorylation of K8 Ser431 in virtually all cells as can be seen by comparison of figures d (control) and e (roscovitine treated). To analyze the relationship between phosphorylation and caspase cleavage of keratins, apoptosis was induced in MR65 cells using roscovitine in the presence (f) or absence (g) of the kinase inhibitor staurosporine. Early apoptotic cells (f), double labeled with M30 (red) and LJ4 (green), display a filamentous M30 staining pattern and show hardly any phosphorylation of K8 Ser73, in contrast to late apoptotic cells that show K18 aggregates (f). Induction of apoptosis in the presence of staurosporine completely inhibited phosphorylation of K8, but did not prevent the collapse of the K8/18 network into aggregates (g). (h – n) Dependence of K8/18 network collapse on cleavage and/or phosphorylation. Double labeling with M30 (green) and propidium iodide (red) in roscovitine treated MR65 cells in the absence (h) or presence (i) of caspase 6 inhibitor. Inhibition of caspase 6 resulted in an almost complete absence of keratin aggregates, but also had a profound effect on nuclear morphology. Affected nuclei showed heavily condensed but not fragmented chromatin. As shown in figure j, the C-terminal K18 fragment was diffusely distributed in the cytoplasm and the nucleoplasm of these roscovitine and caspase 6 inhibitor-treated cells. Inhibition of caspase 6 activity (l, n) also resulted in the absence of nuclear lamina breakdown as shown for lamin A/C (k, l) and lamin B2 (m, n). In the absence of caspase 6 inhibitor (k, m), lamin B2 (green) is completely absent in apoptotic M30-reactive (red) cells, whereas a complete nuclear lamina in combination with a filamentous M30 staining is observed in the presence of caspase 6 inhibitor (l, n). A similar observation is made for lamin A/C, except for the fact that in the absence of caspase 6 inhibition some residual lamin A/C staining can be observed (m). (o – s) Fate of the different K8/18 cleavage products. (o) The C-terminal K18 fragment, recognized by the CA-1 antiserum (green) is diffusely distributed in the cytoplasm, in contrast to the M30 reactive fragments (red) which accumulate as large granules; (p) aggregated distribution of N-terminal fragments of K18 recognized by the RCK106 antibody (red) in relation to the diffuse distribution of C-terminal K18 fragments recognized by the CA1 antiserum (green); (q) RCK106 (green) and M30 (red) reactivity colocalize in the large cytoplasmic granules of apoptotic MR65 cells. (r) Colocalization of LE61 (green) and M30 (red) reactive fragments, indicating that caspase cleaved K18 still forms stable dimers with K8; (s) in some late apoptotic cells, identified by anti-caspase 3 reactivity (green), loss of M30-reactive K8/18 granules (red) is observed.

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Role of keratin phosphorylation in apoptosis

Table 1 K8/18 phosphorylation of apoptotic MR65 cells

Having established the role of caspases in the reorganization of the keratin cytoskeleton during apoptosis, we embarked on a series of experiments to determine to what extent (hyper)phosphorylation contributes to this reorganization and to the final collapse of the cytoskeleton. Some studies suggest that hyperphosphorylation might be a very early physiological stress signal preceding proteolysis. However, since caspases activate certain protein kinases, (hyper)phosphorylation of keratins during the execution phase of apoptosis could facilitate the rapid collapse of the cytoskeletal architecture. To investigate the role of phosphorylation during apoptosis in more detail, we used phosphokeratin-specific antibodies. First, we determined the staining patterns of these antibodies in exponentially growing cell cultures. Staining was carried out 1 day after seeding of the cells, since we found that trypsinization of cells causes rapid phosphorylation of some serine residues (especially keratin pSer431, data not shown). After 1 day, cells had recovered from the stress induced by trypsinization as concluded from the absence of pSer431 staining in the majority of the cells. K18 is phosphorylated on pSer33 in all cells during exponential growth (Fig. 5a), whereas phosphorylation on pSer73 and pSer431 on K8 was absent in the majority of interphase cells (Figs. 5b, c). Mitotic cells and some cells undergoing spontaneous apoptosis showed phosphorylation of K8 on pSer73 and pSer431. Treatment of cells with roscovitine induced rapid phosphorylation of pSer431 in K8 in all cells (Figs. 5d, e), whereas phosphorylation of pSer73 on K8 remained at very low or undetectable levels. Similar results were obtained using etoposide, a toposiomerase inhibitor and inducer of apoptosis (data not shown). In apoptotic cells, the keratin aggregates stained brightly for all phosphoepitope-specific antisera tested. To test whether phosphorylation of pSer73 either preceded or resulted from caspase activation, cells were double labeled with M30 and the phosphokeratin-specific antibodies. As shown in Fig. 5f, cells showing a filamentous staining pattern with M30 were often negative for LJ4, while apoptotic cells showing the aggregated keratin phenotype were positive for this antibody recognizing phosphorylation of Ser73.

Antibody

Staining intensity

Roscovitine

Roscovitine + Staurosporine

K18 pSer 33, n.s.

weakly positive strongly positive weakly positive strongly positive weakly positive strongly positive

18 30 17 27 8 35

19 31 21 21 32 13

Role of caspase cleavage and phosphorylation in keratin collapse Effect of kinase inhibition on keratin reorganization When apoptosis was induced in the presence of the kinase inhibitor staurosporine, we noticed a decrease in pSer431-immunoreactivity in all interphase cells (Table 1). Most strikingly, a complete absence of K8 pSer73 phosphorylation was observed only in apoptotic cells. Despite loss of phosphorylation, the aggregated phenotype persisted (Fig. 5g).

K8 pSer431, n.s. K8 pSer73, P < 0.01

The number of M30-positive cells showing (either weak or strong) staining with the different phosphokeratin-specific antibodies were scored. Only M30-positive cells showing an aggregated keratin staining pattern were included.

Phosphorylation of pSer33 and pSer431 on K18 and 8, respectively, was not sensitive to staurosporine treatment. Apoptosis was not inhibited but, more importantly, neither the level of phosphorylation in both non-apoptotic and apoptotic cells nor the filamentous versus aggregated staining pattern was affected after staurosporine treatment. Apoptotic cells still showed the characteristic aggregated keratin staining with both antibodies. In contrast, phosphorylation of pSer73 on K8 was sensitive to staurosporine treatment. However, a phosphorylation of this serine residue seemed not to be responsible for the collapse of the keratin network, since double labeling with M30 showed apoptotic cells with an aggregate-like staining pattern, devoid of K8 pSer73 immunoreactivity (Fig. 5g). Effect of caspase inhibition on keratin reorganization We tested whether caspase cleavage of K18 is an important trigger for the final collapse of the keratin cytoskeleton. To this end, apoptosis was induced in the presence of various caspase inhibitors. Immunoblots of the cultures show that neither caspase 9 nor caspase 3/7 inhibitors could completely prevent the C-terminal cleavage of K18 (Figs. 4d, e). In contrast, caspase 6 inhibitor was very effective in preventing the L1-2 linker region cleavage. When treated cells were stained for M30, it became evident that caspase 9, and caspase 3/7 inhibitors could not prevent the appearance of the aggregated phenotype. In contrast, cells that were induced to undergo apoptosis in the presence of the caspase 6 inhibitor showed a predominantly filamentous staining pattern (Figs. 5h, i; Table 2). Furthermore, nuclear morphology was also affected. Chromatin fragmentation was absent, but the nuclei of M30-positive apoptotic cells showed prominent perinuclear condensation. The C-terminal K18 fragment was also in the nucleoplasm in between the condensed chromatin (Fig. 5j). Since nuclear lamins belong to the intermediate filament family and also contain a caspase 6 consensus site, we double-labeled these cells with M30 and antibodies directed against lamin A/C and lamin B1, respectively. As expected, in caspase 6 inhibitor-treated cells, the nuclear lamina was

B. Schutte et al. / Experimental Cell Research 297 (2004) 11–26 Table 2 The keratin staining pattern of apoptotic (M30-positive) MR65 cells were analyzed in the presence or absence of caspase 6 inhibitor Antibody M30, P < 0.001

Phenotype

filamentous dot-like Lamin A/C, P < 0.001 intact lamina irregular lamina loss of lamina Lamin B2, P < 0.001 intact lamina loss of lamina

Roscovitine Roscovitine + caspase 6 inhibitor 13 5 2 34 14 13 37

95 87 29 20 1 47 3

The number of cells with either a filamentous or dot-like M30 staining was scored. In double-labeled cells, the percentage of M30-positive cells with different organizations of nuclear lamin A/C or B2 lamin were determined.

still intact in contrast to cells induced to undergo apoptosis in the absence of the caspase 6 inhibitor (Figs. 5k– n; Table 2). Fate of the keratin cleavage fragments Intracellular distribution of keratin cleavage fragments K8 does not contain any of the identified caspase cleavage sites. In contrast K18 is cleaved at two sites, as shown in previous experiments [7,8,39]. K18 forms stable dimers with K8. Therefore, we investigated the fate of the K18 cleavage fragments and their association with K8 during cytoskeletal reorganization. For this purpose, antibodies against known epitopes on the K18 molecule were used (see Fig. 1). Both in early and late apoptotic cells, the cleaved C-terminal fragments were diffusely distributed in the cytoplasm and nucleoplasm. At this stage, caspase cleavage of the keratin cytoskeleton at the 393DALD/S site was complete, since no filamentous staining was seen with the CA1 antiserum. CA-1 immunoreactivity was never associated with the keratin aggregates (Figs. 5o, p). These aggregates showed immunoreactivity with keratin antibodies that recognize both the cleaved coil2 region and the N-terminal fragments of K18 (Fig. 5q). Immunostaining with K8 antibodies revealed similar aggregates. Interestingly, at least part of the cleaved coil2 region was probably still found to be associated with K8, since these aggregates were still immunoreactive with the LE61 antibody (Fig. 5r). At late stages of apoptosis, when the chromatin was heavily fragmented, immunoreactivity with all antibodies diminished and some late apoptotic cells were completely negative (Fig. 5s). Determination of K18 fragments in the culture supernatant Since anti-keratin immunoreactivity is completely lost in late apoptotic cells, in the absence of additional proteolysis, we investigated whether K18 fragments were shed into the culture medium. To correlate loss of K18/M30 immunoreactivity in the cells with appearance of keratin

21

fragments in the culture supernatant, cells were harvested by trypsinization and split into two aliquots. One aliquot was labeled with annexin V-FITC and used for bivariate annexin V/PI analysis. The second aliquot was fixed and analyzed for M30 immunoreactivity. Culture supernatants were analyzed for the presence of caspase cleaved K18 fragments using the ApotoSensek ELISA. As shown in Fig. 6, an increase in M30-positive cells preceded an increase in annexin V-positive/PI-negative cells. The maximum level of apoptosis was reached after approximately 8 h, at which time the number of annexin V-positive/PIpositive (secondary necrotic) cells started to increase. Parallel to this, increase in secondary necrotic cells, the levels of M30-positive K18 cleavage fragments in the culture supernatants increased dramatically. To investigate whether K8 and other K18 fragments were also lost from the dying cells, dual staining experiments were performed using M30 and anti-K8 (M20), anti-N terminal fragment-specific K18 (RCK106) and with the antibody recognizing the shared epitope on K8 and K18 (LE61). As shown in Fig. 7, immunoreactivity of both M30 and the anti-keratin antibodies decreased in

Fig. 6. Shedding of K8/18 by apoptotic MR65 cells. Cells were treated with roscovitine and cell and culture supernatants samples were taken at various periods of time. The increase in the number of M30-positive cells (solid circle) precedes the increase in annexin V-positive/PI-negative apoptotic cells (solid square), indicating that exposure of the M30 epitope precedes loss of membrane phospholipid asymmetry. Concomitant with the decrease of annexin V-positive and M30-positive cells, an increase in the number of cells with a compromised plasma membrane (annexin V-positive/PIpositive secondary necrotic cells, solid triangle) is observed. The appearance of K18 fragments in the culture medium (solid diamonds), measured in U/l, follows similar kinetics as those of the secondary necrotic cells. Three separate experiments were performed showing similar results. Data of a single representative experiment are shown.

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Fig. 7. Bivariate keratin/DNA flow cytometric analysis of control MR65 cells (a) and MR65 cells treated with roscovitine for 7 h (b – d) and double labeled for M30 ( y-axis) and different anti-K8/18 antibodies (x-axis). a) Exponentially growing MR65 control cells double labeled with the M30 and M20 antibody. (b – d) The decrease in M30 staining intensity is paralleled by a similar decrease in staining intensity in roscovitine treated cells for the K8-specific M20 (b), the K18-specific RCK106 (c) and the K8/18-shared epitope LE61 (d) antibodies.

parallel, indicating simultaneous loss of the different types of K8/K18 fragments from the dying cells.

Discussion During apoptosis, a cell undergoes dramatic changes in morphology, partly due to a complete reorganization of its cytoplasmic and nuclear cytoskeletal structures [10,11]. This breakdown process involves the caspases and is well orchestrated, since some of the cytoskeletal components retain a function during apoptosis. For example, actin seems essential for membrane integrity and seems to play a role in the formation of apoptotic blebs [10]. Other components of the cytoplasmic and nuclear cytoskeleton, such as the intermediate filaments, are targeted for rapid proteolysis during apoptosis. These cleavage events are pivotal for the rapid fragmentation of the cell into apoptotic bodies, ensuring rapid clearance by phagocytes [12 –14], as particle size seems to be an important parameter in this process [40]. Monoclonal antibodies that specifically recognize either caspase cleaved keratin fragments or specific phosphoe-

pitopes on K8 and K18 allow a detailed investigation of the temporal and causal relationship of proteolysis and phosphorylation in the collapse of the keratin cytoskeleton during apoptosis. In this study, we have shown that K18 is initially cleaved in the carboxy-terminal domain at the 393 DALD/S site, resulting in the exposure of a neoepitope recognized by the M30-CytoDeath antibody. The appearance of M30 reactivity is a very early event during the caspase cascade, since the first M30-immunoreactive cells exhibit hardly any detectable active caspase 3 immunoreactivity. On the other hand, M30 reactivity is always observed in cells showing loss of cytochrome c from the depolarized mitochondria. However, in a small fraction of the apoptotic cells, polarized mitochondria were found in M30-reactive cells. A likely explanation for this phenomenon is the lag-time between CMXRos loading and fixation of the cells. CMXRos accumulates in polarized mitochondria and is retained by covalent binding with mitochondrial proteins through SH moieties. When cells become apoptotic during labeling with CMXRos, a discrete mitochondrial staining can be expected.

B. Schutte et al. / Experimental Cell Research 297 (2004) 11–26

In experiments using K8/K18-containing cytoskeleton extracts, recombinant caspases 9, 3, and 7, but not caspase 6, could generate the M30 neo-epitope. Indirect activation of procaspase 3 activity by apical caspases could be ruled out by the fact that no cleavage of K18 was observed in the presence of recombinant caspase 8, which is an activator of caspase 3. Furthermore, no procaspase 3 could be detected in our cytoskeleton preparations. In vitro assays using caspase inhibitors were less conclusive. None of the individual caspase inhibitors could prevent the C-terminal K18 cleavage completely. The most probable explanation for this is the redundancy of the caspase cascade or the presence of additional caspase(-like) activities, which are less sensitive to inhibition by the general caspase inhibitor zVAD-fmk [41 – 46]. Based on these observations, it is most likely that K18 is first cleaved at the 393DALD/S site by caspase 9 or another apical caspase [45,46], and at a later stage by caspases 3/7 in a kind of amplification loop. The recent finding that procaspases 3 and 9 are specifically targeted to the K8/18 intermediate filament network [21,22] supports this notion. As a consequence of caspase 3 activation, caspase 6 is also activated, resulting in a second cleavage event in the L12 linker region of K18. This event is responsible for the final collapse of the keratin skeleton into large aggregates (see below) and coincides with loss of intracellular contacts and detachment of cells from their substrates. In a previous report, we could show that in apoptotic cells that were detached from the culture flasks a 20-kDa M30-reactive cleavage product could be detected due to cleavage of K18 at the two caspase consensus sites. On the other hand, in early apoptotic cells, still attached to the culture flasks, an approximately 45-kDa M30 reactive fragment was seen. The biological significance of the C-terminal cleavage step remains obscure. The 393DALD/S site is only present in K18 and is conserved during evolution, since it is also present in murine K18. Obviously, C-terminal cleavage does not seem to affect the keratin organization to a great extend, since cells still show a normal filamentous structure and remain attached to their substrate. We noticed that early during apoptosis, the C-terminal K18 peptide is distributed diffusely throughout the cyto- and nucleoplasm. Whether or not this C-terminal keratin fragment plays a role in changes in nuclear architecture during apoptosis remains to be established. L12 linker region cleavage in K18 Cleavage of the L12 linker region in K18 has a dramatic effect on the organization of the keratin cytoskeleton. In the absence of this cleavage event, for example, after addition of a caspase 6 inhibitor, the apoptotic cells tend to partially round up but still remain attached to their substrates. Breakdown of the keratin filamentous structure into large granules is prevented, although large condensed filament

23

bundles are seen. Caspase 6 seems to be the caspase solely responsible for this cleavage event, since only recombinant caspase 6 was able to cleave keratin in this L12 linker region, while caspases 3, 7, and 9 failed to do so. Secondly, pretreatment of cells with a caspase 6 inhibitor was sufficient to prevent this L12 linker cleavage in vitro. It should be kept in mind that under all these circumstances, K8 remains associated with K18. Phosphorylation of K8/K18 Although all keratin aggregates are highly phosphorylated on serine residues, this hyperphosphorylation seems not to be important for the final collapse of the keratin network as no obvious change in phosphorylation state of K18 Ser33 was noticed during the process of apoptosis. This serine residue on K18 seems to be constantly phosphorylated. It has been shown that K18 Ser33 phosphorylation is essential for the association of K18 with 14-3-3 proteins, and plays a role in keratin organization and distribution [47]. In our study, we could not show a role for K18 Ser33 phosphorylation during apoptosis. In contrast, phosphorylation of either the head or the tail domain of K8 was prominent during apoptosis. It has been suggested that accumulation of hyperphosphorylated K8 might represent a physiologic stress marker for simple epithelia [27]. Our results support the interpretation of K8 phosphorylation at Ser 431 as stress marker, since K8 Ser431 phosphorylation occurred in all cells immediately after treatment with the CDK inhibitor roscovitine, although only some of the cells became apoptotic. Our findings that trypsinization and treatment with other apoptosis-inducing compounds also showed a rapid phosphorylation of K8 Ser431 were in line with this reasoning. Ku and Omary [26], however, showed that K8 phosphorylation at Ser431 increased dramatically upon stimulation of cells with epidermal growth factor (EGF) or after mitotic arrest. These authors also showed that the phosphorylation at this site was likely to be mediated by mitogen-activated protein and cdc2 kinases. In our experiments, stress-induced phosphorylation of K8 Ser431 is not likely to be mediated by cdc2 kinases, since phosphorylation took place in the presence of the general CDK inhibitor roscovitine. However, the stress response was to some extent inhibited in the presence of staurosporine, a kinase inhibitor with high affinity towards PKCs. This finding is supported by previous reports that showed a role for PKC in the phosphorylation of epithelial keratins [48,49]. Many such kinases are specifically activated by caspase-dependent cleavage. They are generally involved in the execution of apoptosis and contribute to the cell death response. Among these kinases are MEKK1 [23,50 – 52], p21-activated kinase 2, and focal adhesion kinase [53], as well as protein kinase C (PKC) family members [24,54,55]. Phosphorylation at K8 Ser73 seems to be a late event during apoptosis. We were able to completely inhibit

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B. Schutte et al. / Experimental Cell Research 297 (2004) 11–26

phosphorylation at this site by induction of apoptosis in the presence of staurosporine. These results indicate that phosphorylation of K8 Ser73 is initiated during the execution phase of apoptosis, by kinases sensitive to staurosporine. Inhibition of K8 Ser73 phosphorylation did not prevent the formation of keratin aggregates, therefore, phosphorylation at this site may facilitate but is not a prerequisite for cytoskeletal collapse. In line with this reasoning, the observation of Ku et al. [6] showed that keratin hyperphosphorylation does not render the keratins to be better substrates for caspases, but can protect from caspase-mediated degradation at the L12 site but not the 393DALD/S site [39]. From our experiments, we conclude that different kinases are responsible for phosphorylation of keratins at the separate serine residues. Firstly, K18 Ser33 is phosphorylated in all cells during exponential growth of cells, whereas K8 Ser 431 and K8 Ser73 are not. Secondly, treatment of cells with the CDK inhibitor roscovitine resulted in phosphorylation of all cells at K8 Ser431, whereas phosphorylation of K8 Ser73 was absent in the majority of the cells. This kinase activity was to a small extent sensitive to inhibition by staurosporine. Thirdly, phosphorylation during the execution phase of apoptosis was prominent only at K8 Ser73 and was almost completely inhibited by staurosporine. This latter kinase activity might be ascribed to PKCs since these kinases are efficiently inhibited by staurosporine. K8/18 fragment shedding The cleaved keratin fragments are apparently stable [6] since no signs of additional proteolysis is observed in the immunoblotting studies. The cleaved fragments are retained in the apoptotic cells as long as the cell membrane remains intact. Loss of membrane phospholipid asymmetry does not affect membrane permeability for these fragments, since phosphatidylserine exposure proceeds shedding of K18 fragments by several hours. Once membrane integrity is lost during secondary necrosis, K18 fragments are shed into the culture medium. In patients, the clearance of apoptotic cells seems not to be very efficient since keratin fragments can be observed in sera of cancer patients. In support of this finding, we observed M30-reactive material in small blood vessels of different tissues using immunohistochemistry [56]. The recently developed M30-ApoptoSensek Elisa assay might therefore be useful in quantitating cell death using sera of patients. M30 serum levels might be informative on growth rate of tumors, since a direct correlation between proliferative activity and apoptosis was found in a series of 300 colorectal tumors [57]. Furthermore, an M30ELISA assay could be used to monitor response to therapy, since most cancer treatments result in rapid induction of apoptosis in tumor cells. In conclusion, we propose the following sequence of events for the rapid reorganization of the K8/18 cytoskeleton during apoptosis. Firstly, a cell responds to an apoptogenic trigger by rapid phosphorylation of K8 Ser431. This

might represent a stress response aimed at protecting the cell from the insult. Secondly, once the apoptotic program is initiated and procaspases 9 and 3 are specifically targeted to the K8/18 intermediate filament network possibly through DEDD, caspase 9 is activated, resulting in C-terminal cleavage of K18 at the 393DALD/S site. During the execution phase of the apoptotic program executioner caspases, such as caspases 3, 7 and 6, are activated, resulting in (i) more efficient C-terminal cleavage of K18 by caspases 3 and 7, (ii) activation of kinases resulting in hyperphosphorylation of K8 Ser73, and (iii) L12 linker cleavage of K18 by caspase 6. Hyperphosphorylation of keratins probably results in the formation of condensed fibers but is not a prerequisite for the final collapse of the filament network. It may even slow down degradation at the L12 site [39] to allow coordination of the execution phase of apoptosis. Cleavage in the L12 linker region of K18 by caspase 6 seems to be solely responsible for the formation of keratin aggregates. In these aggregates, K8 and K18 fragments form stable interactions. At later stages of the apoptotic process, these aggregates can be shed into the surrounding medium or serum.

Acknowledgments The authors acknowledge the Dutch Science Foundation (NWO; project nr. 901-28-134) for the financial support for the Imaris/Huygens software running on a Silicon Graphics workstation. Support by National Institute of Health grant DK47918, Department of Veterans Affairs (to M.B.O.) and Cancer Research, UK (to E.B.L) is also acknowledged.

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