www.elsevier.com/locate/yexcr

Experimental Cell Research 297 (2004) 11–26

Keratin 8/18 breakdown and reorganization during apoptosis

Bert Schutte,a,* Mieke Henfling,a Wendy Kolgen,a Maartje Bouman,a Stephan Meex,a

Mathie P.G. Leers,b Marius Nap,b Viveka Bjorklund,c Peter Bjorklund,c

Bertil Bjorklund,d E. Birgitte Lane,e M. Bishr Omary,f

Hans Jornvall,d and Frans C.S. Ramaekersa

aDepartment of Molecular Cell Biology (Box 17), Research Institute Growth and Development (GROW), University of Maastricht, The NetherlandsbDepartment of Pathology, Atrium Medical Centre, Heerlen, The Netherlands

cPeviva AB, Stromkarlsvagen 82, SE-167 62 Bromma, SwedendDepartment of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77, Stockholm, SwedeneCancer Research UK, Cell Structure Research Group, School of Life Sciences, University of Dundee, UK

fPalo 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 helical NH -terminal (head) and COOH terminal (tail)

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-

0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.yexcr.2004.02.019

* 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: bert.schutte@molcelb.unimaas.nl (B. Schutte).

2

domains. The head and tail domains contain the sites for

several posttranslational modifications, including phosphor-

ylation and glycosylation [3].

Keratins are highly dynamic and become reorganized

during various cellular events such as differentiation, mito-

sis, 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 interac-

tion with non-keratin proteins [9].

During apoptosis, a cell undergoes dramatic changes in

morphology due to a complete reorganization of its cyto-

B. Schutte et al. / Experimental Cell Research 297 (2004) 11–2612

plasmic and nuclear skeleton [10,11]. These alterations

probably facilitate the rapid, but securely orchestrated break-

down of the cell into apoptotic bodies, ensuring the mainte-

nance of an intact cellular membrane and efficient clearance

by phagocytes [12–14].

Many components of the cytoplasmic and nuclear cyto-

skeleton 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]. Interfer-

ence 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 proteoly-

sis, 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 respon-

sible 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-polymer-

ase (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 proteol-

ysis 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 as393DALD/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 reorga-

nize 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 apopto-

sis [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]. Al-

ternatively, 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 reorganiza-

tion 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 hyper-

phosphorylation 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 M30-

ApoptoSensek 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 aggre-

gates. Phosphorylation facilitates formation of these aggre-

gates, 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 medi-

um (Cat. no. 12565-024; GIBCO, Paisley, Scotland, Unit-

ed Kingdom), supplemented with 1% non-essential amino

acids (GIBCO), 1% HEPES (GIBCO), 2 mM L-glutamine

(Cat. no. 22942; Serva, Heidelberg, Germany), 10% heat-

inactivated 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 Nether-

lands) 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-

B. Schutte et al. / Experimental Cell Research 297 (2004) 11–26 13

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%).

Cell harvesting and fixation

Cells growing in monolayers were harvested quantita-

tively 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% parafor-

maldehyde for 15 min at RT, followed by permeabiliza-

tion for 10 min at room temperature (RT) in PBS

containing 0.005% SDS.

Kinase inhibitors

For inhibition of keratin hyperphosphorylation, stauro-

sporine (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 cas-

pase 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,

interconnecting solid lines. Phosphorylation sites, caspase cleavage sites, as well as

this study are indicated.

cultures at the following concentrations unless stated differ-

ently: Ac-DEVD-CHO or Ac-LEHD, 20 AM; z-VEID-fmk or

z-VAD-fmk, 100 AM.

Antibodies

The following primary antibodies were used: (1)

Monoclonal antibody IIB5 (diluted 1:50) to bromodesox-

yuridine (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 anti-

caspase 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, Maas-

tricht, The Netherlands; (6) monoclonal antibody LE61,

recognizing a shared epitope on K8/18 [34]. (7) Polyclon-

al rabbit antiserum CA1, raised against the carboxy-

terminal 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 mono-

clonal 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

and rod domains are indicated by cylinders and linker region domain as

the location of the different epitopes of K8 and 18 of the antibodies used in

B. Schutte et al. / Experimental Cell Research 297 (2004) 11–2614

against Lamin A/C was kindly provided by Dr. G Warren

(Heidelberg, Germany).

As secondary antibodies, R-Phycoerythrin (RPE)-conju-

gated F(ab’)2 fragment of affinity-purified goat anti-mouse

immunoglobulins (R0480, DAKO A/S, Glostrup, Den-

mark; 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 red-

conjugated goat anti-rabbit immunoglobulins (Southern

Biotechnology Associates (SBA), Birmingham, USA; di-

luted 1:80), FITC- and Texas red-conjugated goat anti-

mouse 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 expo-

sure of phosphatidylserine at the outer leaflet of the mem-

brane, annexin V biotin (APOPTEST-BIOTIN, Nexins Re-

search 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 (Mo-

lecular 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 m-

chlorophenylhydrazon (CCCP, Sigma, The Netherlands).

In vitro keratin cleavage

Cytoskeletal keratin extracts were prepared by resuspend-

ing approximately 5 � 107 MR65 cells for 10 min in 10 ml

cold RSB/PMSF/TritonX100 buffer (1.5 mMMgCl2, 10mM

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 incu-

bated for 20min at RT. Five milliliters RSB/PMSFwas added

to the suspension and a pellet fraction was recovered after

centrifugation for 5 min at 1500 � g at 4jC. The pellet wasrinsed once in 1 ml RSB/PMSF buffer, and finally resus-

pended in 500 Al caspase working buffer (20 mMHepes, 100

mMNaCl, 0.1%CHAPS, 10mMdithiothreitol (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 medi-

um 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 anti-

body. The cells were counterstained with 5 Ag/ml propi-

dium 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 anti-

mouse and anti-rabbit Ig or monoclonal antibodies of

different isotype, which were detected by anti-isotype-

specific secondary antibodies. Antibody binding was vi-

sualized by incubating the cells with FITC- and TxRed-

conjugated 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 con-

trol, mouse anti-BrdU (clone IIB5) was used as primary

antibody.

Immunocytochemistry for flow cytometric analysis

After harvesting and fixation, the cells were subse-

quently 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 resus-

pended in PBS, containing 20 Ag/ml propidium iodide

(Calbiochem) and 100 Ag/ml RNase (Serva). For double

labeling experiments with M30 and anti-keratin antibod-

ies, 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 visual-

ized using FITC-conjugated anti-mouse IgG1 (SBA).

B. Schutte et al. / Experimental Cell Research 297 (2004) 11–26 15

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 immedi-

ately 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.

Fig. 2. Temporal order of K18 cleavage during apoptosis in MR65 cells. Apop

monoclonal antibody alone (a, b) or in combination with other known markers of

microscopy. (a) A shadow projection of a confocal stack of images of M30 reacti

(green) combined with a fragmented and condensed chromatin (red); (b) a shadow

cell. This early apoptotic cell is characterized by a fine filamentous M30 sta

fragmentation; (c) linear projection of a confocal stack of images of a triple labeled

counterstained with DAPI (blue). Note that PS exposure does not occur in the cell

cell, which shows a membranous annexin V staining pattern. (d) Double labeling

antibodies. Note that in one cell showing a filamentous M30 staining pattern har

pattern is observed in very few M30-positive (green) cells after loading of the cel

showing a typical mitochondrial labeling pattern with CMXRos were always devo

accumulation of CMXRos in the mitochondria was observed. (f) Double labeling ex

M30-reactive cells, including those with a filamentous type of staining, showed a

apoptotic cells always showed a discrete mitochondrial labeling with the anti-cyt

Fifty microliters of substrate mix (50 mM Tris/HCl,

pH7.5, containing 2.5 mM DTT and 40 AM Ac-DEVD-

AMC (Biomol), Ac-WEHD, Ac-IETD, AcVEID, Ac-

VDAD 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 (SLM-

Aminco) spectrofluorometer.

Western blotting

Apoptosis was induced by replacement of the culture

mediumwith 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-

tosis was induced using roscovitine and cells were stained with the M30

apoptosis (c– f). Stained cells were analyzed using confocal scanning laser

vity in a typical late apoptotic cell. Note the granular M30 staining pattern

projection of a confocal stack of images of a M30-reactive early apoptotic

ining pattern without any obvious signs of chromatin condensation or

apoptotic cells with annexin V (green) and M30 (red) antibody. Nuclei are

showing a filamentous M30 staining pattern, in contrast to the late apoptotic

of early apoptotic cells with anti-active caspase 3 (green) and M30 (red)

dly any active caspase 3 is detected. (e) A discrete mitochondrial staining

ls with the mitochondrial-specific dye CMXRos (red); the majority of cells

id of M30 staining; in apoptotic cells showing a dot-like M30 reactivity no

periment with the M30 (red) and an anti-cytochrome c (green) antibody. All

diffuse distribution of cytochrome c in the cytoplasm. M30-negative, non-

ochrome c antibody.

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.

B. Schutte et al. / Experimental Cell Research 297 (2004) 11–2616

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), con-

taining 2.3% SDS, 62.5 mMTris–HCl pH 6.8, 10% glycerol,

5% h-mercaptoethanol, and 0.05% bromophenol blue. Sam-

ples were boiled for 5 min.

One-dimensional sodium dodecylsulphate polyacryl-

amide gel electrophoresis (SDS-PAGE) was performed

according to Laemmli [37] using gels containing 10%,

13%, or 15% polyacrylamide (Bio-Rad Laboratories, Her-

cules, 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 (Bio-

Rad 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 chemilumines-

cence (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, Ger-

many). The laser-scanning microscope was used in the

dual-parameter setup, according to manufacturer’s specifi-

cation, 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 Dick-

inson, 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. Elec-

tronic 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-pro-

cessed 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 apopto-

sis, the majority of immunoreactive cells show a dot-like

staining pattern with M30, as well as chromatin fragmenta-

tion (Fig. 2a). However, in a minority of the cells, all still

attached to the glass surface, a filamentous M30 staining

B. Schutte et al. / Experimental Cell Research 297 (2004) 11–26 17

pattern was observed without obvious chromatin condensa-

tion 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

Fig. 4. (a, b) Western blotting analysis of in vitro caspase cleaved cytoskeletal pre

RCK106 antibody (a) and M30 (b). Note that with RCK106 (a) a specific 30 kDa c

seen in caspase 6-treated samples. Caspases 3-, 7-, or 9-treated samples show a

untreated sample. The M30 antibody (b) shows a 45 kDa reactive only in caspases 3

immunoreactivity. When samples were simultaneously treated with caspases 3 and

control lysate, are observed. These cleavage products correspond to the K18 molec

(c) Western blotting analysis of an apoptotic cell lysate (lane 1) and a crude cyt

RCK106 and anti procaspase 3 antibodies. Note that the cytoskeletal preparation i

roscovitine in the presence or absence of different caspase inhibitors, probed with

and the caspase 6 inhibitor prevent the appearance of the 30-kDa RCK106-reacti

could not completely prevent the appearance of a 45-kDa M30-reactive fragm

Apoptosis in the presence of caspases 3 or 9 inhibitor resulted in the accumulatio

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 cleav-

age of K18 at the 393DALD/S site precedes loss of mem-

brane phospholipid asymmetry [8]. Early after induction of

parations of MR65 cells treated with different caspases and probed with the

leavage product, identical to the product present in the control lysate, is only

n additional fragment with slightly lower Mr (arrowhead) than that of the

-, 7- and 9-treated samples. Untreated or caspase 6-treated samples show no

6, two characteristic cleavage products of 45 and 20 kDa, also found in the

ule lacking either the tail or the tail and the rod 2 domain of K18 (see Fig. 1).

oskeletal preparation (lane 2) of MR65 cells. The blots were probed with

s devoid of procaspase 3. (d, e) Western blotting of MR65 cells treated with

RCK106 (d) or the M30 antibody (e). The general caspase inhibitor z-VAD

ve cleavage product, in contrast to the caspases 3 and 9 inhibitors. z-VAD

ent. Caspase 6 inhibitor failed to inhibit this cleavage event completely.

n of the 20-kDa M30 reactive fragment.

B. Schutte et al. / Experimental Cell Research 297 (2004) 11–2618

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 antise-

rum 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 M30-

positive cells showing an aggregated type of staining,

active caspase 3 was clearly present. Active caspase 3

immunoreactivity was quantified in M30-negative cells,

B. Schutte et al. / Experimental Cell Research 297 (2004) 11–26 19

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 over-

lapped 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 potential-

sensitive 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, cytoskel-

eton 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

Fig. 5. (a–g) Keratin phosphorylation and cleavage. Non-treated MR65 cells (a–c

LJ4 (K8 pSer73, c) 5B3 (K8 pSer431). All cells at all stages were phosphorylated

Ser73 to a much lower extent (b), but massive hyperphosphorylation of K8 on Se

the latter two antibodies. Treatment of MR65 cells with roscovitine (d–g) resulted

seen by comparison of figures d (control) and e (roscovitine treated). To analyze

apoptosis was induced in MR65 cells using roscovitine in the presence (f) or absen

labeled with M30 (red) and LJ4 (green), display a filamentous M30 staining pat

apoptotic cells that show K18 aggregates (f). Induction of apoptosis in the presenc

prevent the collapse of the K8/18 network into aggregates (g). (h–n) Dependenc

labeling with M30 (green) and propidium iodide (red) in roscovitine treated MR65

caspase 6 resulted in an almost complete absence of keratin aggregates, but also ha

condensed but not fragmented chromatin. As shown in figure j, the C-terminal K18

these roscovitine and caspase 6 inhibitor-treated cells. Inhibition of caspase 6 activ

for lamin A/C (k, l) and lamin B2 (m, n). In the absence of caspase 6 inhibitor (k

cells, whereas a complete nuclear lamina in combination with a filamentous M30

observation is made for lamin A/C, except for the fact that in the absence of caspas

Fate of the different K8/18 cleavage products. (o) The C-terminal K18 fragment

cytoplasm, in contrast to the M30 reactive fragments (red) which accumulate as

recognized by the RCK106 antibody (red) in relation to the diffuse distribution o

RCK106 (green) and M30 (red) reactivity colocalize in the large cytoplasmic gran

(red) reactive fragments, indicating that caspase cleaved K18 still forms stable di

reactivity (green), loss of M30-reactive K8/18 granules (red) is observed.

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 approxi-

mately 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 approx-

imately 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 recom-

binant caspase 9 but residual procaspase 3 activity in the

cytoskeleton preparation is indirectly responsible for C-

terminal 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 the393DALD/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.

) stained with phosphokeratin-specific antibodies: (a) IB4 (K18 pSer33, (b)

at K18 Ser33 (a), whereas mitotic cells showed phosphorylation of K8 on

r431 (c). Spontaneously occurring apoptotic cells were clearly positive with

in rapid hyperphosphorylation of K8 Ser431 in virtually all cells as can be

the relationship between phosphorylation and caspase cleavage of keratins,

ce (g) of the kinase inhibitor staurosporine. Early apoptotic cells (f), double

tern and show hardly any phosphorylation of K8 Ser73, in contrast to late

e of staurosporine completely inhibited phosphorylation of K8, but did not

e of K8/18 network collapse on cleavage and/or phosphorylation. Double

cells in the absence (h) or presence (i) of caspase 6 inhibitor. Inhibition of

d a profound effect on nuclear morphology. Affected nuclei showed heavily

fragment was diffusely distributed in the cytoplasm and the nucleoplasm of

ity (l, n) also resulted in the absence of nuclear lamina breakdown as shown

, m), lamin B2 (green) is completely absent in apoptotic M30-reactive (red)

staining is observed in the presence of caspase 6 inhibitor (l, n). A similar

e 6 inhibition some residual lamin A/C staining can be observed (m). (o–s)

, recognized by the CA-1 antiserum (green) is diffusely distributed in the

large granules; (p) aggregated distribution of N-terminal fragments of K18

f C-terminal K18 fragments recognized by the CA1 antiserum (green); (q)

ules of apoptotic MR65 cells. (r) Colocalization of LE61 (green) and M30

mers with K8; (s) in some late apoptotic cells, identified by anti-caspase 3

Table 1

K8/18 phosphorylation of apoptotic MR65 cells

Antibody Staining intensity Roscovitine Roscovitine +

Staurosporine

K18 pSer 33, n.s. weakly positive 18 19

strongly positive 30 31

K8 pSer431, n.s. weakly positive 17 21

strongly positive 27 21

K8 pSer73, P < 0.01 weakly positive 8 32

strongly positive 35 13

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.

l Cell Research 297 (2004) 11–26

Role of keratin phosphorylation in apoptosis

Having established the role of caspases in the reorga-

nization of the keratin cytoskeleton during apoptosis, we

embarked on a series of experiments to determine to what

extent (hyper)phosphorylation contributes to this reorga-

nization and to the final collapse of the cytoskeleton.

Some studies suggest that hyperphosphorylation might be

a very early physiological stress signal preceding prote-

olysis. 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 phosphor-

ylation of K8 on pSer73 and pSer431.

Treatment of cells with roscovitine induced rapid phos-

phorylation 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 phosphoepi-

tope-specific antisera tested. To test whether phosphoryla-

tion 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.

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 pheno-

type persisted (Fig. 5g).

B. Schutte et al. / Experimenta20

Phosphorylation of pSer33 and pSer431 on K18 and

8, respectively, was not sensitive to staurosporine treat-

ment. Apoptosis was not inhibited but, more importantly,

neither the level of phosphorylation in both non-apopto-

tic and apoptotic cells nor the filamentous versus aggre-

gated 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 cleav-

age 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

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/PI-

positive 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.

Table 2

The keratin staining pattern of apoptotic (M30-positive) MR65 cells were

analyzed in the presence or absence of caspase 6 inhibitor

Antibody Phenotype Roscovitine Roscovitine +

caspase 6 inhibitor

M30, P < 0.001 filamentous 13 95

dot-like 5 87

Lamin A/C, P < 0.001 intact lamina 2 29

irregular lamina 34 20

loss of lamina 14 1

Lamin B2, P < 0.001 intact lamina 13 47

loss of lamina 37 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.

B. Schutte et al. / Experimental Cell Research 297 (2004) 11–26 21

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 filamen-

tous staining was seen with the CA1 antiserum. CA-1

immunoreactivity was never associated with the keratin

aggregates (Figs. 5o, p). These aggregates showed immu-

noreactivity 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 dimin-

ished 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 prote-

olysis, 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

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 max-

imum level of apoptosis was reached after approximately

8 h, at which time the number of annexin V-positive/PI-

positive (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 experi-

ments 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. 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.

B. Schutte et al. / Experimental Cell Research 297 (2004) 11–2622

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 orches-

trated, 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 interme-

diate 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 the393DALD/S site, resulting in the exposure of a neo-

epitope 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 bind-

ing 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 23

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 indi-

vidual 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] sup-

ports this notion. As a consequence of caspase 3 activa-

tion, 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

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 suffi-

cient 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 phosphorylat-

ed 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 phosphor-

ylation 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 epi-

dermal 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 acti-

vated 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

B. Schutte et al. / Experimental Cell Research 297 (2004) 11–2624

phosphorylation at this site by induction of apoptosis in the

presence of staurosporine. These results indicate that phos-

phorylation 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 obser-

vation of Ku et al. [6] showed that keratin hyperphosphor-

ylation does not render the keratins to be better substrates

for caspases, but can protect from caspase-mediated degra-

dation 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 sep-

arate 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 phosphory-

lation 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 execu-

tion 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 informa-

tive 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 M30-

ELISA 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 cytoskele-

ton during apoptosis. Firstly, a cell responds to an apopto-

genic 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 execu-

tion 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 hyperphosphor-

ylation 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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