1 Department of Internal Medicine I, University of Ulm, 89070 Ulm, Germany; and 2 Veterans Affairs Greater Los Angeles Healthcare System, University of California, Los Angeles, California 90073
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ABSTRACT |
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Pancreatic acinar cells depend on the integrity of the cytoskeleton for regulated secretion. Stimulation of isolated rat pancreatic acini with the secretagogue CCK serves as a model for human acute edematous pancreatitis. It induces the breakdown of the actin filament system (F-actin) with the consecutive inhibition of secretion and premature activation of digestive enzymes. However, the mechanisms that regulate F-actin breakdown are largely unknown. Plectin is a versatile cytolinker protein regulating F-actin dynamics in fibroblasts. It was recently demonstrated that plectin is a substrate of caspase 8. In pancreatic acinar cells, plectin strongly colocalizes with apical and basolateral F-actin. Supramaximal secretory stimulation of acini with CCK leads to a rapid redistribution and activation of caspase 8, followed by degradation of plectin that in turn precedes the F-actin breakdown. Inhibition of caspase 8 before CCK hyperstimulation prevents plectin cleavage, stabilizes F-actin morphology, and reverses the inhibition of secretion. Thus we propose that the caspase 8-mediated degradation of plectin represents a critical biochemical event during CCK-induced secretory blockade and cell injury.
cholecystokinin; cytoskeleton; pancreas; secretion
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INTRODUCTION |
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PLECTIN IS A MEMBER OF
THE plakin family of cytoskeletal proteins that act as molecular
bridges within the cytoskeleton and between the cytoskeleton and
membrane adhesion complexes (10, 43, 49). Whereas some
plakins exhibit a specificity for certain cytoskeletal elements,
plectin interacts with a wide range of cytoskeletal systems (18,
20, 29, 51). Plectin is a large molecule (~500 kDa) with a
multidomain structure composed of a central -helical rod structure
and large COOH- and NH2-terminal globular domains
(48). The intermediate filament binding site is localized
on the COOH-terminal domain. The NH2-terminal domain of
plectin harbors an actin-binding domain that is highly conserved in the
spectrin superfamily of actin-binding proteins (11, 14, 23).
The regulation of plectin function remains poorly understood. Plectin is a substrate of p34cdc2 kinase (25). Interactions of plectin with intermediate filaments are regulated by protein kinases A and C (PKC) (9). The binding of plectin to actin can be modulated by phosphatidylinositol-4,5-bisphosphate, which also interacts with other actin-regulating proteins (3, 52). It was recently shown that caspase 8 cleaves plectin at an ILRD motif in the center of the molecule during tumor necrosis factor (TNF)- and CD95-induced apoptosis (41). This finding adds plectin to the small group of caspase 8 substrates, which was previously comprised of BH3 interacting domain death agonist, receptor interacting protein, and caspase 3 (21, 22, 42).
Plectin is expressed in almost all mammalian cells investigated (48). At the subcellular level, plectin is predominantly found at the subplasma membrane cytoskeleton, thus participating in cell-cell and cell-substratum interactions (35, 39). Due to its versatile cytolinker properties, the major function so far assigned to plectin was the maintenance of cytoarchitecture. This hypothesis was confirmed by reports that showed a lack of plectin expression in skin and muscle cells of patients with epidermolysis bullosa simple-muscular dystrophy (EBS-MD) due to mutations in the plectin gene leading to premature stop codons (12, 28, 40). The main clinical features of EBS-MD consist of dystrophy of muscle cells and skin blistering on exposure to mechanical stress.
Consequently, plectin-deficient mice (2) exhibit severe skin blistering and altered myofibrils in skeletal and heart muscles. These mice die from fluid loss and heart malfunction within the first 3 days after birth, thus preventing the analysis of plectin function in other tissues. Interestingly, dermal fibroblasts derived from plectin-deficient mice did not show alterations of the microtubular or intermediate filament network architecture. However, actin stress fibers and focal adhesion contacts were increased, and the response of the actin cytoskeleton to stimuli of the Rho/Rac/Cdc42 pathways was substantially delayed (3). These data indicate that the function of plectin exceeds a simple mechanical stabilization of cellular architecture. Plectin seems to represent an important regulator of actin cytoskeleton dynamics.
Pancreatic acinar cells depend on the integrity of the filamenteous actin (F-actin) cytoskeleton for regulated secretion (16, 30). Acinar cell F-actin is concentrated in the apical and in the basolateral subplasma membrane regions. As in other secretory cells, the F-actin mesh below the apical cytoplasma membrane is thought to act as a physical barrier for exocytosis and, at the same time, seems to be a necessary component for membrane fusion (33, 46). Blocking F-actin remodeling by phalloidin leads to inhibition of exocytosis (30). Reduced actin filament assembly due to introduction of actin monomer-binding proteins into acinar cells facilitates, whereas complete depolymerization of the actin web blocks, regulated secretion (30). Contrary to the latter observation, Valentijn et al. (45) described the occurrence of stimulated exocytosis after the disruption of F-actin by cytochalasin D. However, the recycling of membranes, which is a prerequisite for the generation of new secretory vesicles, was compromised of cytochalasin D. Although partially conflicting, current data strongly suggest a pivotal role of F-actin for stimulated secretion in pancreatic acinar cells.
A disruption of the apical actin web similar to that caused by actin depolymerizing agents is observed in response to stimulation of acinar cells with supramaximal secretory concentrations of CCK or its amphibian analog cerulein (5, 31). This process is followed by inhibition of secretion and is associated with missorting of zymogen granules, i.e., the fusion of zymogen granules with lysosomes or with basolateral membranes (1, 36). These alterations lead to acinar cell injury in vitro and cause an acute pancreatitis in animal models (1) that resembles acute edematous pancreatitis of man (15). This model has been widely used and well characterized (15). It has been suggested that the breakdown of apical F-actin is the first morphological event after supramaximal stimulation with CCK (31). Consequently, F-actin is thought to be one of the key players responsible for the structural remodeling of acinar cells during the initial phase of pancreatitis. However, the precise series of events following supramaximal stimulation of acinar cells with CCK leading to F-actin breakdown is still elusive.
Another morphological phenomenon of supramaximal CCK stimulation in isolated pancreatic acini is the formation of basolateral protrusions of the cytoplasm that contain RER, free ribosomes, and mitochondria (1). The molecular processes leading to the formation of these blebs remain largely unknown. An involvement of F-actin has been discussed because cytochalasin B, an actin microfilament disrupting agent, can block blebbing (5).
In the present study, we demonstrated that plectin is associated with F-actin predominantly in the apical and basolateral regions of acinar cells. Supramaximal concentrations of CCK activate caspase 8 and induce the cleavage of plectin that precedes the breakdown of F-actin. In contrast, inhibition of caspase 8 leads to the rescue of cytoskeletal morphology and prevents the secretory blockade and the consecutive cell damage observed after hyperstimulation with CCK.
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METHODS |
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Materials.
Caspase 3 substrate Z-DEVD-AMC, caspase 3 inhibitor Z-DEVD-FMK, and
caspase 8 inhibitor Z-IETD-FMK (26) were purchased from Calbiochem (La Jolla, CA). Broad-spectrum caspase inhibitor Z-VAD-FMK (26) and caspase 8 substrate Ac-IETD-AMC were from Alexis
(San-Diego, CA). Benzamidine was from Sigma (St. Louis, MO).
Recombinant caspases 3 and 8 were purchased from Biomol (Hamburg,
Germany). Monoclonal anti-plectin antibodies (clone 7A8), recognizing
an epitope located in the central section on the rod domain
(8), monoclonal anti--actin antibodies, and
anti-
-actinin antibodies were from Sigma. Monoclonal anti-caspase 8 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA).
Anti-tubulin antibodies were from NeoMarkers (Fremont, CA). Oregon
Green phalloidin was from Molecular Probes (Eugene, OR). Protein assay
kit was from Bio-Rad Laboratories (Hercules, CA). Enhanced
chemiluminescence (ECL) reagents were obtained from Pierce (Rockford,
IL). Sulfated CCK-8 was from Bachem (Bubendorf, Switzerland). Male
Wistar rats (100-150 g) were bred at the animal care facility of
the University of Ulm.
Preparation of isolated rat pancreatic acini. Preparation of acini was performed as previously described (19). Isolated acini were washed in oxygenated Krebs-Ringer-HEPES buffer, consisting of (in mM) 104 NaCl, 5 KCl, 1 KH2PO4, 1.2 MgCl2, 2 CaCl2, 10 glucose, and 25 HEPES/NaOH, pH 7.4, 0.2% (wt/vol) bovine serum albumin, and 0.01% (wt/vol) soybean trypsin inhibitor, supplemented with essential amino acid solution and glutamine. Cell viability, as assessed by Trypan blue exclusion, exceeded 95%. All preincubation and incubation steps were carried out at 37°C.
Localization of plectin and F-actin in acinar cells.
Acini were stimulated with CCK doses ranging from 1010 to
10
8 M. In this dose range, 10
10 M CCK
induces the maximal secretory response, whereas 10
8 M CCK
represents a supramaximal concentration leading to inhibition of
secretion. For inhibition experiments, acini were preincubated for 15 min with the indicated inhibitors. Stimulation was stopped by adding an
excess volume of ice-cold Krebs-Ringer-HEPES buffer. Acini were placed
on microscope slides and fixed with 4% formaldehyde for 10 min. After
permeabilization with 0.5% Triton X-100 for 5 min, anti-plectin
antibodies (7A8) or anti-caspase 8 antibodies were added overnight in
PBS at 4°C. Slides were washed in PBS, incubated with Cy3-coupled
secondary antibodies together with Oregon Green phalloidin for 3 h, and embedded in Mowiol (Calbiochem). Imaging was performed
with a confocal laser scanning microscope (TCS4D, Leica,
Heidelberg, Germany) equipped with a 25-mW krypton-argon laser
and a 63× objective (NA 1.4). Simultaneous two-channel image acquisition was performed at 488- and 568-nm excitation wavelengths. Laser energy and parameters of intensity detection were kept constant for all slides. The images in this paper represent typical acini from
at least three cell preparations.
In situ quantitation of plectin and F-actin. Acini were stained for plectin and F-actin as described above. All slides for quantitative measurements were prepared in parallel to avoid variations due to the staining procedures. Three-dimensional (3D) images of acini representing a volume of ~50 × 50 × 40 µm3 were reconstructed from stacks of consecutive confocal images scanned as described above. Laser energy and detection parameters were kept constant for all slides. The final resolution of 3D images was 100 nm/pixel in the x and y directions and 500 nm/pixel in axial direction. Ten 3D images were scanned for each experimental setting, resulting in a total of ~300 acinar cells per setting. The Scion Image software (Scion, Frederick, MD) was used for image processing. The quantity of F-actin and plectin was determined as the total fluorescence intensity for Oregon green (FOG-phalloidin) and Cy3 (FCy3-plectin), respectively. To compare the quantities of plectin and F-actin between different images with varying numbers of acinar cells, the ratio (R) between measured fluorescence intensities (R = FCy3-plectin/FOG-phalloidin) was calculated for each image. Statistical analysis was based on the t-test.
Immunoblotting.
For inhibition experiments, acini were preincubated with the indicated
inhibitors for 15 min. CCK was added for indicated time periods and
concentrations, and the incubation was terminated by suspending the
acini in an excess volume of ice-cold Krebs-Ringer-HEPES buffer. The
acini were then pelleted by centrifugation at 300 g.
Cellular protein was extracted by trituration in lysis buffer consisting of 8 M urea supplemented with protease inhibitors [0.5 mM
phenylmethylsulfonyl fluoride (PMSF), 1 mg/ml soybean trypsin inhibitor, 10 µg/ml leupeptin, 10 µg/ml aprotinin]. The lysate was
cleared by centrifugation at 10,000 g at 4°C. The protein content of the supernatant was measured using the Bradford method (BioRad, Munich, Germany). Soluble proteins were separated by SDS-PAGE.
Gel-resolved proteins were electrophoretically transferred to
polyvinylidene difluoride membranes (Immobilon P, Millipore, Bedford,
MA). Membranes were incubated overnight in blocking buffer [50 mM
Tris · HCl, pH 7.8, 100 mM NaCl, 0.05% Tween 20, 2% (wt/vol) bovine serum albumi]. The membranes were then incubated with
anti-plectin (7A8), anti--actin, or anti-
-actinin antibodies in
blocking buffer for 1 h. After washing with Tris-buffered saline
(TBS)/0.1% Tween 20, antigen-antibody complexes were visualized using
peroxidase-conjugated secondary antibody and the ECL system.
Measurement of caspase activities.
Isolated acini were incubated with 1010 to
10
7 M CCK and caspase inhibitors for the indicated times.
Acini were collected, washed with ice-cold PBS, and resuspended in
lysis buffer containing 0.5% Igepal CA-630, 0.5 mM EDTA, 150 mM NaCl,
and 50 mM Tris at pH 7.5. Cell lysates were placed for 30 min on a
rotator at 4°C and then centrifuged for 15 min at 15,000 g. Supernatant was collected, protein concentration was
determined with a BioRad protein assay kit, and the extracts were
aliquoted and stored at
80°C. Proteolytic reactions for caspases 3 and 8 were carried out at 37°C in 25 mM HEPES (pH 7.5), 10% sucrose,
0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and 10 mM 1,4-dithiothreitol (DTT), with 800 µg cytosolic protein and 20 µM of specific fluorogenic substrate. For caspase 3, the substrate was Z-Asp-Glu-Val-Asp-AMC (Z-DEVD); for caspase 8, the
substrate was Ac-Ile-Glu-Thr-Asp-7-amino-4-methylcoumarin (AMC;
Ac-IETD). Cleavage of the substrate releases AMC, which emits a
fluorescence signal at 440 nm when excited at 380 nm. The reaction was
started by the addition of caspase substrate. The readings were taken
at 0, 60, 90, and 120 min. Fluorescence was calibrated using a standard
curve for AMC. The data are expressed as means ± SE for at least
three cell preparations.
In situ plectin cleavage by recombinant caspases. Acini were incubated with 5 mg/ml digitonin (Sigma) for 1 min, washed, and placed on slides. Acini were incubated with recombinant caspases 3 and 8 in 50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% glycerol, and 10 mM DTT at 37°C for 30 min. Caspase activity ranged from 2-8 U/µl. Plectin and F-actin were visualized as described above. The images represent typical acini from three independent cell preparations.
Amylase secretion studies.
Acini were stimulated for 30 min at 37°C with 1012 to
10
8 M CCK or incubated in buffer control after
preincubation with inhibitors for 15 min. A sample of each cell
suspension was transferred to microcentrifuge tubes containing a 2:1
mixture (vol/vol) of dibutyl phtalate and di-'isononyl'-phtalate
(Fluka, Buchs, Switzerland) with a specific weight of 1.063. Cells were
spun through the oil to separate supernatant from cells. Secreted
amylase was measured using the AMYL MPR2 method (Roche). Total cellular
amylase content was determined after lysis of cells in 10 mM
NaH2PO4, pH 7.8, 0.1% sodium dodecyl sulfate.
Amylase secretion is given as the percentage of total amylase content.
The data are expressed as means ± SE for at least three cell preparations.
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RESULTS |
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Supramaximal CCK stimulation causes plectin breakdown.
Immunofluorescence studies of plectin distribution in acinar
cells revealed that plectin is predominantly located close to the
plasma membrane where it colocalizes with F-actin. The strongest enhancement of plectin and F-actin signals was detected in the apical
part of acinar cells (Fig. 1,
A and B).
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CCK hyperstimulation induces activation of caspase 8.
To determine whether caspase 8 might be responsible for the observed
plectin cleavage in acinar cells, we first examined the activation of
caspase 8. Figure 4 depicts the time and
dose-dependent activation of caspase 8 after stimulation of isolated
rat acini with CCK. Activation kinetics of caspase 8 showed a 3.5-fold
increase after 30 min of supramaximal CCK-stimulation (Fig.
4A). Enzymatic activities reached their maximum at
108 M CCK (Fig. 4B). These effects were
specific for supramaximal CCK concentrations (10
8 M),
because caspase 8 was not activated by maximal secretory concentrations
(10
10 M) of CCK (Fig. 4B) or by supramaximal
stimulation with CCK-JMV180 (Fig. 4A), a partial agonist of
CCK that elicits a complete secretory response but does not induce
acinar cell injury or pancreatitis (13, 32). Caspase 3 activity was determined for control purposes. It showed a 1.8-fold
increase after 30 min of supramaximal CCK stimulation.
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Breakdown of plectin and F-actin depends on caspase 8 activity.
We next examined whether caspase 8 could be responsible for the
observed effects on cytoskeletal morphology. Preincubation of acini
with the caspase 8 inhibitor Z-IETD-FMK prevented the breakdown of
plectin and F-actin, as shown by quantitative immunofluorescence studies (Figs. 2 and 5, A-D) and the cleavage of
plectin as demonstrated by Western blotting (Fig. 3A) even
after 30 min of supramaximal CCK stimulation. Plectin degradation was
also at least partially prevented by the broad-spectrum caspase
inhibitor Z-VAD-FMK (Fig. 5,
E-H).
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Recombinant caspase 8 induces cytoskeletal changes resembling those
observed after CCK hyperstimulation.
Incubation of permeabilized acini with activated recombinant caspase 8 for 30 min dose dependently induced plectin degradation in situ with a
consecutive remodeling of the F-actin cytoskeleton. The pattern of
cytoskeletal alterations induced by 2 U/µl recombinant caspase 8 within 30 min was virtually identical with the changes observed after
stimulation of acini with supramaximal concentrations of CCK.
Recombinant caspase 8 (8 U/µl) had even greater effects on plectin
and F-actin (Fig. 6
A-F). By contrast, activated recombinant caspase
3 at maximum concentrations (8 U/µl) did not show any effect within
the investigated time period (Fig. 6, G and H).
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Inhibition of caspase 8 reverses the secretory blockade at
supramaximal concentrations of CCK.
Apical F-actin is a central component for the regulation of
exocytosis in acinar cells (30). The role of caspase 8 activation for the functionality of this microfilament structure, i.e.,
secretion of digestive enzymes, was assessed next. Figure
7A shows a typical biphasic
dose-response curve for pancreatic acinar cell secretion, with its
maximum at 1010 M CCK. Release of amylase was <4% of
the total cellular amylase under basal conditions. Over 21% of total
cellular amylase was released at 10
10 M CCK after 30 min.
Preincubation with the caspase 8 inhibitor Z-IETD-FMK prevented the
downslope of the curve at CCK concentrations >10
10 M and
thus led to reversal of the secretory blockage at supramaximal secretory concentrations of CCK (Fig. 7, A and
B). Although caspase 3 was activated by supramaximal CCK
concentrations, preincubation with the caspase 3 inhibitor Z-DEVD-FMK
had no effect on secretion (Fig. 7B). To verify that the
caspase 8 inhibitor Z-IETD-FMK did not interfere with CCK-receptor
signaling, we investigated the CCK-induced activation of
extracellular signal-regulated kinase (ERK)-1 and -2, which
is mediated by a PKC-dependent pathway (7). Indeed,
the activation of ERK-1 and -2 by CCK was not altered in the presence
of Z-IETD-FMK, as shown by phosphospecific antibodies (data
not shown).
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DISCUSSION |
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The disruption of the apical F-actin network in pancreatic acinar cells is one of the central mechanisms of CCK-induced cell injury and thus is likely to be involved in the initiation of cell damage during CCK-induced pancreatitis (16, 31). However, the mechanisms that regulate F-actin in acinar cells are largely unknown. The structure of F-actin is the result of a balance between assembly and disassembly of monomeric actin into multimeric actin filaments. Potential regulatory mechanisms include the modification of the rate of assembly or disassembly by membrane-bound actin-anchoring complexes, actin-capping or -severing proteins, and the stabilization of actin filaments by cross-linking proteins (24, 27).
In this study, we identify plectin as a functionally important actin cross-linking protein in pancreatic acinar cells. A strong plectin signal colocalizes with the apical F-actin, suggesting a primary role for plectin as an F-actin scaffold in this region. In response to stimulation with supramaximal concentrations of CCK, plectin immunoreactivity rapidly disappears, an effect that is accompanied by plectin protein degradation. Of note, the decrease of the plectin immunoreactivity after supramaximal concentrations of CCK occurs before the desintegration of F-actin, as confirmed by quantitative measurements in situ. In contrast, maximal secretory concentrations of CCK do not cause a visible alteration of plectin distribution nor measurable changes of the plectin-to-F-actin ratio. These data suggest a role of plectin as an early response protein maintaining cytoskeletal integrity in acinar cells.
Previous studies defined a second pool of F-actin located at the basolateral plasma membrane of acinar cells, i.e., the cellular region where supramaximal CCK stimulation initiates the formation of blebs (44, 45). Prominent accumulations of plectin colocalize with this F-actin pool under resting conditions. Because blebs still contain cortical F-actin and myosin, and because cytochalasin D inhibits blebbing, Torgerson and McNiven (44) postulated an actin-based force-generating mechanism as the cause for blebbing. On the basis of our data, plectin degradation precedes blebbing and, subsequently, plectin cannot be detected in blebs. It therefore seems conceivable that the breakdown of plectin leads to an alteration of the mechanical properties of F-actin and thus alters the balance of forces toward blebbing.
In this study, caspase 8 was identified as the mediator of plectin degradation in pancreatic acini during CCK hyperstimulation. Caspase 8 is regarded as an early regulator of a proteolytic cascade leading to apoptosis (6, 37, 50). Apoptosis of acinar cells can be detected during the initial phase of pancreatitis (17). Interestingly, in this particular case, apoptosis may prevent extensive inflammation and thus limit organ damage (4). Caspase 8 in acinar cells is rapidly activated by supramaximal concentrations of CCK. In addition, caspase 8 is translocated to the apical and basolateral cytoskeleton after CCK hyperstimulation. A similar redistribution of caspase 8 has been described during CD95-mediated apoptosis in MCF7 cells (41). To our knowledge, this is most likely the first time that CCK acting on the G protein-coupled CCKA receptor has been demonstrated to activate caspases. The precise mechanism of caspase 8 activation with subsequent cleavage of downstream effectors, such as caspase 3 by supramaximal CCK stimulation, requires further studies. Our preliminary data suggest the involvement of a cytochrome c-dependent pathway.
Caspase 8 is an initiator caspase, stimulating the activation of
several effector caspases, which, in turn, regulate the proteolytic degradation of various cellular proteins including components of the
cytoskeleton. Recently, Stegh et al. (41) identified plectin as a novel substrate of caspase 8 in established cell lines
incubated with TNF- or anti-CD95. Here we describe, for the first
time, that caspase 8 cleaves plectin also during CCK hyperstimulation
in a model system for acute pancreatitis. Cleavage of plectin was
specific for caspase 8, because plectin cleavage could be prevented by
preincubation of acini with selective caspase 8 inhibitors
(26). Furthermore, plectin degradation is not due to
intracellular activation of secretory enzymes such as trypsin, as
demonstrated by incubation of acini with benzamidine (34). We did not observe any degradation of
-actinin or monomeric actin in
parallel to plectin cleavage in response to CCK hyperstimulation, indicating that plectin cleavage is not due to a nonspecific
proteolytic degradation of cytoskeletal components. Furthermore,
incubation of acinar cells with activated recombinant caspase 8 but not
with its downstream effector caspase 3 can reproduce the cytoskeletal changes induced by supramaximal concentrations of CCK.
The finding that plectin is a caspase 8 substrate implies an important regulatory role of this cytolinker for the stabilization as well as for the degradation of the cytoskeleton in acinar cells. During apoptosis of epithelial tumor cells, plectin cleavage by caspase 8 precedes the proteolytic degradation of all cytoskeletal proteins examined such as gelsolin, cytokeratin 18, and lamin B (41). Furthermore, plectin was shown to regulate F-actin dynamics in fibroblasts (3), and plectin-deficient fibroblasts do not exhibit the typical reorganization of F-actin during caspase 8-mediated apoptosis (41). These data suggest that plectin might also be important for the integrity of the F-actin cytoskeleton in pancreatic acini, which is paramount for stimulated secretion in these cells.
If plectin would regulate F-actin in acinar cells, inhibition of plectin degradation by caspase 8 should have an impact on the apical F-actin web and on the related cellular function, i.e., the secretory response. Indeed, we did not find F-actin breakdown, which is typically observed after supramaximal CCK stimulation when caspase 8 activity and, consequently, cleavage of plectin were inhibited. The rescue of the apical F-actin was associated with a restored normal secretion even in the presence of supramaximal secretory concentrations of CCK. Importantly, inhibition of caspase 3, an effector caspase, did not prevent the secretory blockade, suggesting that the effect of the caspase 8 inhibitor is specific and that caspase 8 activation and plectin cleavage are directly linked to the processes that eventually lead to the inhibition of secretion. Furthermore, the specificity of the caspase 8 inhibitor is stressed by the fact that the inhibitor did not interfere with CCK-induced activation of the ERK cascade (7).
Of course, we cannot exclude the possibility that in addition to plectin, other actin-binding proteins are involved in the regulation of F-actin breakdown and inhibition of secretion during CCK hyperstimulation. Unfortunately, the use of plectin-deficient mice as a gold standard to clarifiy this point is compromised by the fact that these mice die very early after birth (2).
In conclusion, we identify, for the first time, caspase 8 and plectin as potential mediators of both disruption of the F-actin cytoskeleton and inhibition of secretion in pancreatic acini exposed to supramaximal concentrations of CCK. On the basis of these data, we speculate that uncontrolled depolymerization of F-actin is the final event in the loss of the apical F-actin web during CCK hyperstimulation. To further investigate the interactions between plectin and F-actin, two models of plectin function are currently examined in our laboratory: plectin, as a static scaffolder of the F-actin web, could act as a controlling factor that limits transient depolymerization of F-actin. Cleavage of plectin in response to supramaximal CCK stimulation would then permit depolymerization beyond that observed during stimulated secretion. Alternatively, plectin could transiently dissociate from F-actin with the subsequent disassembly of the F-actin web during stimulated secretion. Intact plectin would then be required for the controlled reassembly after exocytosis.
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ACKNOWLEDGEMENTS |
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M. Beil and J. Leser contributed equally to this work.
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FOOTNOTES |
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We thank U. Nolte, S. Kohler, and U. Gern for expert technical assistance.
This work was supported by grants from Deutsche Forschungsgemeinschaft to M. Beil and M. P. Lutz (SFB518), and from the Dept. of Veterans Affairs and National Institutes of Health to S. J. Pandol.
Address for reprint requests and other correspondence: G. Adler, Dept. of Internal Medicine I, Univ. of Ulm, 89070 Ulm, Germany (E-mail: guido.adler{at}medizin.uni-ulm.de).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpgi.00042.2001
Received 26 January 2001; accepted in final form 6 November 2001.
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