Apoptosis in activated rat pancreatic stellate
cells
Hanne
Klonowski-Stumpe,
Richard
Fischer,
Roland
Reinehr,
Reinhard
Lüthen, and
Dieter
Häussinger
Department of Gastroenterology, Hepatology and Infectiology,
Heinrich-Heine Universität Düsseldorf, 40225 Düsseldorf, Germany
 |
ABSTRACT |
Proliferation and matrix synthesis
by activated pancreatic stellate cells (PSC) participate in the
development of chronic pancreatitis. Apoptosis of PSC may
terminate this process but has not yet been studied in this particular
cell type and was the aim of the present study. PSC were isolated from
rat pancreas and characterized for expression of glial fibrillary
acidic protein,
-smooth muscle actin, CD95, and tumor necrosis
factor-
-related apoptosis-inducing ligand (TRAIL) receptors.
Apoptosis was determined by TdT-UTP nick end-labeling reaction,
annexin V binding, and caspase-8 activation. Both CD95L and TRAIL
induced apoptosis in PSC. The apoptotic response was minor
in PSC cultured for 7 days but increased markedly thereafter.
Sensitization of PSC with culture duration was accompanied by increased
expression of CD95 and TRAIL receptor 2 and no alterations of Flip
expression or protein kinase B phosphorylation but was paralleled by
the appearance of a COOH-terminal cleavage product of
receptor-interacting protein. PSC apoptosis was also induced by
PK-11195, a ligand of the peripheral benzodiazepine receptor. PSC
apoptosis may be important in terminating the wound-healing response after pancreas injury and exhibits features distinct from
apoptosis induction in hepatic stellate cells.
tumor necrosis factor-
-related apoptosis-inducing
ligand; CD95L; transformation; receptor-interacting protein cleavage; pancreas
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INTRODUCTION |
ABOUT 30 YEARS
AGO Watari and co-workers (29) identified
lipid-storing cells in pancreatic tissue with marked similarities to
hepatic stellate cells (HSC; see Ref. 29). Whereas HSC
were intensively studied, their pancreatic counterparts (pancreatic stellate cells; PSC) were isolated in 1998 for the first time (1, 5).
In normal pancreas, PSC are in a quiescent state and are characterized
by the presence of vitamin A-containing lipid droplets and positive
staining for glial fibrillary acidic protein (GFAP; see Ref.
1). PSC are activated in both experimental and human
pancreatic fibrosis and then represent the major source of collagen in
chronic pancreatitis (9). Transformation of quiescent PSC
to a myofibroblast-like phenotype also occurs during culture. This
activation is associated with an increased expression of
-smooth
muscle actin and a loss of GFAP expression (5). Cultured
PSC proliferate and produce extracellular matrix proteins, such as
collagens I and III, fibronectin, and laminin (5). Most of the experiments performed with isolated PSC focused on the
activation mechanisms of these cells (2, 3, 5, 13, 16, 23,
25), but little is known about the termination of this process.
Because there is no evidence for redifferentiation of
myofibroblast-like cells back into the quiescent state,
apoptosis of activated cells is probably involved. However,
this process has not yet been studied. Here we show that activated PSC
undergo apoptosis after incubation with the two death receptor
ligands CD95L and TRAIL or with the exogenous peripheral benzodiazepine receptor ligand
1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-isoquinoline-3-carboxamide (PK-11195).
In most cases of apoptosis, changes in mitochondrial
permeability precede the major changes in cell morphology and
biochemistry. This change is the result of an opening of a dynamic
multiprotein pore formed in the contact side between the inner and
outer mitochondrial membrane. The peripheral benzodiazepine receptor is
part of this pore and seems to be involved in a wide spectrum of
activities within cells (7). PK-11195 has been shown to
induce apoptosis in thymocytes and more recently in HSC
(6).
CD95L and tumor necrosis factor (TNF)-
-related
apoptosis-inducing ligand (TRAIL) are ligands to receptors
belonging to the TNF receptor family of death receptors
(26). These receptors are characterized by an
intracellular death domain that serves to recruit adapter proteins such
as TRADD and FADD and cysteine proteases such as caspase-8. Activation
of caspase-8 at the activated death receptor complex leads to
apoptosis. Although the TRAIL receptors 1 and 2 are widely
expressed in several tissues (4), most cell types are not
sensitive to TRAIL-mediated cell killing (30).
Normal pancreatic acini, duct cells, and islet cells do not express
CD95 (11), but CD95 expression is observed in chronic pancreatitis in acini and duct cells (11, 28). Various
intracellular molecules affect the sensitivity of a cell to
apoptotic signals. Death domain proteins are recruited to death
receptors and propagate the apoptotic signal (19).
This complex is known as death-inducing signaling complex (DISC).
Activation of caspase-8 follows recruitment of this enzyme to the
complex and leads to activation of effector caspases like caspase-3
through proteolytic cleavage and triggering mitochondrial damage
(24).
Receptor-interacting protein (RIP) is another component of the DISC.
The recruitment of RIP to this complex is responsible for activation of
nuclear factor (NF)-
B and activator protein-1 (14, 15).
Activation of NF-
B is thought to provide an antiapoptotic signal. RIP can be cleaved by caspase-8, whereby its
NF-
B-inducing ability is abolished. In addition, the COOH-terminal
cleavage product RIPc promotes apoptosis
(12).
Nothing is known about induction and signaling pathways of
apoptotic cell death in PSC. We therefore studied the expression of
death receptors in quiescent and activated PSC and the potential of
death receptor ligands for PSC apoptosis.
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MATERIALS AND METHODS |
Materials.
Pronase and collagenase P were from Boehringer (Mannheim, Germany).
Desoxyribonuclease 1, BSA, Percoll, propidium iodide, and rabbit
anti-GFAP antibody were obtained from Sigma (Deisenhofen, Germany).
Soluble CD95L with enhancer protein, TRAIL, and PK-11195 were from
Alexis (Grünberg, Germany). TACS TdT Kit,
Z-IETD-fluoromethylketone (FMK), Z-LHD-FMK, and Z-DEVD-FMK were
purchased from R&D Systems (Wiesbaden, Germany). The enhanced
chemiluminescence (ECL) kit was obtained from Amersham Pharmacia
Biotech (Freiburg, Germany), and mouse anti-RIP antibody and Matrigel
were from BD Transduction Laboratories (Heidelberg, Germany). Rabbit
anti-phospho-PKB antibody and rabbit anti-PKB antibody were from Cell
Signaling Technology. Annexin-V fluorescein isothiocyanate (FITC) was
from Bender Medical Systems (Vienna, Austria). Anti-CD95 antibody and
monoclonal mouse anti-FLIPS/L antibody were from Santa Cruz
Biotechnology (Santa Cruz, CA), and rabbit anti-DR4 and rabbit anti-DR5
antibodies were from Serotec (Eching, Germany). All other chemicals
were obtained from local sources at the highest purity available.
Isolation and culture of PSC.
For preparation of PSC, male Wistar rats (300-350 g body wt) were
decapitated, and the pancreas was removed quickly. After injection of
10 ml Gey's buffered salt solution containing 5,000 units DNase 1, 12.5 units collagenase P, and 21 units pronase in the glandular
parenchyma, the pancreas was minced in small fragments and incubated at
37°C (5% CO2) for 50 min. Cells were mechanically dispersed by pipetting them through a Nalgene pipette. Undigested tissue was removed with a nylon mesh (70 µm). Cells were
washed (747 g; 4°C) and resuspended in DMEM (10% FCS).
The cell suspension was mixed with the 1.5-fold volume of a 90%
Percoll solution in 0.9% NaCl and centrifuged for 1 h at 40,000 g (4°C). The upper part of the gradient, containing the
PSCs and avital cells, was filtered again to remove lumping cell debris
and centrifuged at 747 g for 7 min (4°C). Cells were
resuspended in DMEM (10% FCS and 0.01% gentamicin) and seeded in a
density of 105 cells/ml. At days 5,
12, and 20 after preparation, confluent cultures
were trypsinized, washed with DMEM (747 g, 10 min, 4°C), and again adjusted to 105 cells/ml. The purity of PSC
preparations using the Percoll gradient was 75-85% immediately
after seeding the cells. Contaminating cells were removed by carefully
changing the medium 3 and 24 h after seeding, increasing the
purity of the preparation to 95-99%.
Isolation of pancreatic acinar cells.
Pancreatic acinar cells (PAC) were isolated from male Wistar rats by
collagenase digestion. In brief, the pancreas was removed quickly from
a rat as outlined above, and 5 ml of a HEPES-Ringer (HR) solution
containing 320 U/ml collagenase, 200 U/ml hyaluronidase, and 10 U/ml
chymotrypsin were injected in the glandular parenchyma. The pancreas
was minced in small fragments and incubated for 20 min at 37°C. The
medium was replaced with 10 ml of a Ca2+- and
Mg2+-depleted HR containing 1 mmol/l EDTA, and the
incubation was continued for 10 min. Pancreatic fragments were returned
to 5 ml HR containing 320 U/ml collagenase for a final 20-min
incubation. The fragments were dispersed by repeatedly pipetting
through a Nalgene pipette. The cells were filtered through a 70-µm
nylon mesh, centrifuged for 5 min at 10 g, and resuspended
in DMEM. For immunostaining and detection of apoptotic cell death,
cells were plated on coverslips (12 mm diameter) coated with Matrigel and incubated for 30 min at 37°C before induction of apoptosis.
Immunofluorescence staining of the TRAIL receptor 2 and CD95.
Analysis of CD95 and TRAIL receptor 2 membrane trafficking was
performed with living, unfixed, and unpermeabilized cells either stimulated for 24 h with 0.5 µmol/l cycloheximide alone, for
1 h with 100 ng/ml CD95L and 1 µg/ml CD95L-enhancer protein or
200 ng/ml TRAIL, or preincubated for 24 h with 0.5 µmol/l
cycloheximide and then stimulated for 1 h with CD95L and enhancer
or TRAIL. Control staining was performed with unstimulated cells. The
receptors were stained for 45 min at 4°C with rabbit anti-CD95
antibody or rabbit anti-TRAIL receptor 2 antibody (1:500) and a
Cy3-labeled goat-anti-rabbit antibody (1:500). Cells were washed in PBS
supplemented with 2% FCS and 0.1% NaN3. Immunostained
cells were analyzed using an Axioskop (Zeiss, Jena, Germany) with a
3CCD Camera (Intas) and a Leica TCS-NT confocal laser scanning system
with an argon-krypton laser on a Leica DM IRB inverted microscope
(Leica, Bensheim, Germany). Images were acquired at 568 nm wavelength
to visualize Cy3. CD95 receptor or TRAIL receptor 2 membrane staining
was defined as one or more intensely fluorescent spots on the surface
of the cells.
Annexin V assay.
Annexin V assay was performed with cells plated on glass coverslips and
incubated with various substances for the times indicated. Coverslips
were transposed in a moist chamber, and cells were incubated with
annexin V-FITC and propidium iodide (both diluted 1:1,000 in binding
buffer provided by the manufacturer) for 15 min. Thereafter, coverslips
were rinsed two times with PBS and analyzed using a Zeiss microscope
equipped for fluorescence microscopy. Only fluorescein-positive cells
without nuclear propidium iodide staining were regarded apoptotic
and differentiated from healthy cells by phase-contrast microscopy. At
least 100 cells were counted for each experiment (3-4 different
preparations for each condition).
TdT nick end-labeling reaction.
Labeling and visualization of DNA fragments were performed with the
TACS TdT kit using Mn2+ to enhance the reaction. Cells were
counterstained with 0.5 µg/ml tetramethylrhodamine isothiocyanate
(TRITC)-conjugated phalloidin for detection of cell shape.
Incorporated FITC was detected using a fluorescence microscope (Zeiss)
at an excitation wavelength of 488 nm. TRITC was excited at a
wavelength of 568 nm. The number of apoptotic cells was determined
by counting the percentage of FITC-positive cells. At least 100 cells
were counted for each condition (3-4 different preparations for
each experiment).
Determination of protein expression.
Cells were washed with PBS and lysed, and protein was harvested by
scraping. Samples were then centrifuged at 10,000 g for 10 min, and the supernatant was collected for Western blotting. Protein
content of the cell lysates was measured with the advanced protein
assay reagent (Cytoskeleton) following the manufacturer's instructions
using BSA as the standard. Proteins from each sample (10-40
µg/lane) were separated by gel electrophoresis using a 10%
SDS-polyacrylamide gel. Known molecular weight protein standards were
run with the samples. Separated proteins were transferred to a
nitrocellulose membrane using a semidry blotting apparatus (Multiphor
II; Pharmacia). The membrane was then incubated at 4°C with 5% BSA
in Tris-buffered saline (pH 7.6) for 2 h to prevent nonspecific
binding of antibodies. This was followed by an overnight incubation
with the primary antibody (1:10,000) in TBS with 1% BSA. The membrane
was washed three times and incubated with the secondary antibody for
2 h. Protein bands were detected by the ECL technique using the
ECL kit (Amersham).
Determination of caspase-8 activity.
The caspase-8 assay from R&D Systems measures the colorimetric reaction
of the cleavage of the amino acid motif IETD, thereby releasing the
chromophore p-nitroanilide. The level of caspase enzymatic
activity in the cell lysates is directly proportional to the color
reaction that was quantified spectrophotometrically at a wavelength of
405 nm, using a microplate reader (Pharmacia). Data were corrected for
background (no substrate or no cell lysate) and expressed as a
percentage of the control levels (no induction of apoptosis).
Analysis of results and statistics.
Data are expressed as means ± SE. Each experiment was performed
from at least three different cell preparations. Results were compared
using the Student's t-test. P < 0.05 was
considered statistically significant.
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RESULTS |
Characterization of isolated PSC.
PSC isolated by Percoll density gradient centrifugation exhibited the
fat-storing phenotype with numerous fat droplets located in the
perinuclear region of the cells (data not shown). At 2 days of culture,
these cells stained positive for GFAP, whereas no
-smooth muscle
actin (SMA) was detectable by Western blot analysis (Fig.
1). Within the 1st wk of culture, the
number and size of fat droplets and the GFAP expression decreased, and
the cells developed a typical myofibroblast morphology and expressed
-SMA (Fig. 1). Therefore, PSC isolated by Percoll density
centrifugation show similar characteristics as PSC isolated by other
methods as Nycodenz density centrifugation or outgrowth. Similar to
HSC, PSC are activated by cultivation and transform to
myofibroblast-like cells.

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Fig. 1.
Protein levels of glial fibrillary acidic protein (GFAP)
and -smooth muscle actin (SMA) in pancreatic stellate cells (PSC) in
culture. Cells were harvested after the time period indicated and
subjected to Western blot analysis using antibodies raised specifically
against GFAP or 1-SMA as a parameter of activation. Data
are representative for 3-5 different experiments. d2, d7, d14, and
d28, days 2, 7, 14, and 28,
respectively.
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Induction of apoptosis in PSC by CD95L.
Induction of apoptotic cell death in PSC by addition of CD95L
depended strongly on the culture duration. Whereas CD95L induced neither annexin V staining (Table 1) nor
DNA strand breaks [TdT nick end-labeling (TUNEL) assay; Table
2] in 7-days cultured PSC, cells
cultured for 14 or more days exhibited an apoptotic reaction to
CD95L (Tables 1 and 2). In PSC cultured for 28 days, CD95L induced
apoptotic cell death in almost one-third of the cells within
24 h. The apoptotic response to CD95L was enhanced in the
presence of cycloheximide in the culture medium (Table 2). The number
of TUNEL-positive cells exceeded the number of annexin V-positive
cells. This might in part reflect the fact that fixed cells were used
for TUNEL assay but not for the annexin V assay. In contrast to
activated stellate cells, acinar cells of the pancreas showed no
annexin V staining when incubated with CD95L at concentrations up to 10 ng/ml with or without cycloheximide (data not shown).
Induction of apoptosis in PSC by TRAIL.
PSC exhibited a dose- and activation-dependent sensitivity toward
TRAIL-induced apoptosis, as shown by annexin V binding and TUNEL assay (Tables 3 and
4). As it was true for the
induction of apoptosis by CD95L, the number of apoptotic
cells in cultures incubated with 100-200 ng/ml TRAIL increased
with culture duration. TRAIL-induced cell death did not depend on the
presence of cycloheximide (data not shown). PAC were insensitive to
TRAIL when added at concentrations sufficient to induce
apoptosis in activated PSC (data not shown).
Induction of apoptosis in PSC by PK-11195.
The peripheral benzodiazepine receptor ligand PK-11195 was recently
shown to induce apoptosis in HSC (6). As shown in
Tables 5 and
6, PSC also underwent
apoptosis in response to PK-11195. The effect was most
pronounced in PSC cultured for up to 28 days. On the other hand, no
apoptosis was induced by PK-11195 in PAC; however, at a
concentration of 200 µmol/l, PK-11195 caused necrotic cell death of
acinar cells within 6 h.
A general problem in determining the percentage of apoptotic cells
in PSC cultures was the fact that untreated cultures showed different
levels of basal apoptotic rates (2-10% in 28-day-old cells).
Cultures with high apoptotic background were in general more
sensitive to apoptotic stimuli independent of the method used to
determine the percentage of apoptotic cells. However, in each
experiment, all conditions were performed with cells from one
preparation and are therefore comparable. Different experimental settings were performed with cells from different preparations and are
for that reason hard to compare.
Expression of CD95 and TRAIL receptor.
Death receptor expression was studied in PSC by Western blot analysis
(Fig. 2, A and B).
Expression of CD95 and the TRAIL receptor 2, but not of TRAIL receptor
1, increased with increasing culture time. In unstimulated PSC, which
were kept in culture for 28 days, no CD95 immunostaining was detectable
in nonpermeabilized cells, indicating that CD95 was mainly localized
inside the cells (Fig. 3). However,
treatment of PSC with CD95L induced within 1 h intense CD95
immunostaining at the cell surface, indicating CD95 trafficking to the
plasma membrane. CD95L-induced CD95 membrane targeting occurred
regardless of whether cycloheximide was present or not (Fig. 3). No
translocation of the CD95 receptor was observed in response to
cycloheximide alone (data not shown). Treatment of PSC with TRAIL did
not induce TRAIL receptor 2 translocation to the plasma membrane (Fig.
3).

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Fig. 2.
Protein levels of tumor necrosis factor- -related
apoptosis-inducing ligand (TRAIL) receptors (rec) 1 and 2, CD95, and the peripheral benzodiazepine receptor (PBR) in PSC cultured
for 7-28 days. Cells were harvested after the time period
indicated and subjected to Western blot analysis using antibodies
raised specifically against TRAIL receptor 1 ( ), TRAIL
receptor 2 ( ), CD95 ( ), PBR or
1-SMA ( ) as a parameter of activation.
Data are given as means ± SE (n = 3-6).
* P < 0.05. SE values were omitted when bars were
within the symbol size.
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Fig. 3.
Immunocytochemical staining of PSC incubated with CD95L (100 ng/ml)
or TRAIL (200 ng/ml) in the absence or presence of cycloheximide (CH;
500 nmol/l). Cells were stimulated for 1 h with CD95L and enhancer
(C) or TRAIL (G) or stimulated for 24 h with
cycloheximide alone and then incubated with CD95L and enhancer
(D) or TRAIL (H). Subsequently, cells were
stained with anti-CD95 (A-D) or anti-TRAIL receptor 2 (E-H). Control staining was performed with unstimulated
cells (B and F) or permeabilized (0.1% Triton
X-100, vol/vol in PBS) unstimulated cells (A and
E). Insets: phase-contrast recordings showing the
presence of cells on coverslips, which were negative for receptor
trafficking to the plasma membrane. After incubation with CD95L, PSC
translocate the CD95 receptor to the plasma membrane, whereas no TRAIL
receptor 2 membrane trafficking occurs in response to
TRAIL.
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Studies on the mechanisms of apoptosis in PSC.
As shown in Tables 1-6, PSC in culture for 4 wk are much more
sensitive to apoptosis induction by CD95L, TRAIL, or PK-11195 than PSC, which were kept in culture for 1 wk only. Although this may
in part be explained by an increased expression of CD95 and TRAIL receptors with culture duration (Fig. 2), further mechanisms involving intracellular apoptosis-modulating signals may
contribute. PKB is known to exert antiapoptotic effects. However,
as shown in Fig. 4, PKB phosphorylation
did not significantly change in PSC cultured for 1 or 4 wk, although
there was a slight increase in total PKB expression. Also, expression
of antiapoptotic c-Flip was largely unaffected (Fig. 4).

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Fig. 4.
Protein levels of phospho-protein kinase B (PKB), PKB, c-Flip, and
receptor-interacting protein (RIP) in cultured PSC. Cells were
harvested after the time period indicated and subjected to Western blot
analysis using antibodies raised specifically against p-PKB
( ), PKB ( ), c-Flip ( ),
or RIP ( and ). Expression of
full-length (~75 kDa) RIP ( ) was slightly increased.
In addition to this full-length protein, a smaller protein
( ) was strongly expressed in cells cultured for 28 days
but not in those cultured for shorter time periods. Data are
representative of 3-6 different experiments. AU, arbitrary
units.
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RIP is one of the kinases that is involved in receptor-mediated
apoptosis of many cell types. As shown in Fig. 4, there is a
slight increase in the amount of the uncleaved RIP (molecular mass
~74 kDa) in PSC cultured for 4 wk compared with 1 wk of culture. However, the amount of RIPc, a cleavage product of RIP with
a molecular mass of ~50 kDa, became increasingly apparent after 4 wk
of culture.
Because RIP cleavage is known to depend on caspase-8 activity,
caspase-8 activity was determined in PSC cultured for 28 days. Figure
5 shows that caspase-8 activity was
significantly increased upon addition of CD95L or TRAIL for 24 h.
CD95L-induced caspase-8 activation was further enhanced by
cycloheximide. No increase in caspase-8 activity was measured after
incubating PSC with 100 µM PK-11195 (Fig. 5). Inhibition of caspase-8
activity by 50 µmol/l Z-IETD-FMK completely abolished
apoptosis in PSC induced by TRAIL or CD95L. This was also found
upon inhibition of caspase-3 (50 µmol/l Z-DEVD-FMK) or caspase-9 (50 µmol/l Z-LEHD-FMK; Fig. 6).

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Fig. 5.
Caspase-8 activity in PSC cultured for 28 days. After
isolation (28 days), PSC were incubated with PK-11195 (100 µmol/l),
TRAIL (200 ng/ml), CD95L (10 ng/ml), CD95L with cycloheximide (500 nmol/l), or in control medium. After 24 h, the cells were lysed,
and caspase-8 activity was determined in the cell lysates. Data are
given as means ± SE (n = 3).
* P < 0.05 vs. control cells
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Fig. 6.
Inhibition of apoptosis by caspase inhibitors.
PSC were cultured for 28 days and then incubated with or without TRAIL
or CD95L in the absence or presence of caspase inhibitors (50 µmol/l). Thereafter, the percentage of apoptotic cells was
determined by the annexin V binding assay. Cells were incubated with
either 10 ng/ml CD95L, 1 µg/ml enhancer protein and 500 nmol/l
cycloheximide, 200 ng/ml TRAIL, or left untreated as control. Data are
given as means ± SE (n = 3-6).
P < 0.05 vs. control cells (*) or vs. cells incubated
with CD95L or TRAIL (#). FMK, fluoromethylketone.
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Western blot analyses were performed to investigate the influence of
the TRAIL and CD95L on RIP cleavage. As shown in Fig. 7, both CD95L and TRAIL induced the
cleavage of RIP, however, with different time courses. Whereas CD95L
induced RIP cleavage within 10 min (data not shown), cleaved RIP was
found only after 6 h of TRAIL treatment.

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Fig. 7.
Induction of a cleaved form of RIP in PSC incubated with
CD95L (10 ng/ml + 1 µg/ml enhancer protein and 500 nmol/l
cycloheximide) or TRAIL (200 ng/ml). PSC were cultured for 28 days and
then incubated with or without CD95L or TRAIL for 6 h. Expression
of proteins reacting with an antibody raised specifically against RIP
was monitored by Western blot analysis. CD95L and TRAIL induce the
appearance of an ~50-kDa protein reacting with an antibody against
the COOH-terminal part of RIP. Data are representative of 3-5
different experiments.
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 |
DISCUSSION |
Transformation of PSC to myofibroblast-like cells was paralleled
by an increased sensitivity to CD95L- and TRAIL-mediated apoptosis. This time-dependent sensitization was accompanied by an increased expression of CD95 receptor and the TRAIL receptor 2. An
increasing sensitivity to CD95L parallel to cell transformation was
also reported for HSC (8); however, HSC apoptosis
is induced by CD95L only, when simultaneously cycloheximide is present
(8). Furthermore, HSC do not undergo apoptosis in
response to TRAIL (unpublished observations). This is different to PSC,
which undergo apoptosis in response to CD95L or TRAIL even in
the absence of cycloheximide, although cycloheximide augments
CD95L-induced apoptosis. The mechanism underlying the
sensitizing effect of cycloheximide is not clear (20) but
may involve (8) a modulation of the p53 pathway,
downregulation of Flip, and activation of stress-activated protein
kinases by cycloheximide (20).
In contrast to activated PSC, PAC were not sensitive to death receptor
ligand-mediated cell death. The lacking sensitivity toward CD95L can be
explained by the fact that normal PAC do not express CD95
(11). The insensitivity of acinar cells toward TRAIL is
due to a preferential expression of antagonistic receptors (TRAIL
receptors 3 and 4) as it was shown for other cell types (22,
27).
Induction of PSC apoptosis by both CD95L and TRAIL was
accompanied by an activation of caspase-8 and was sensitive to
inhibition of either caspase-3, -8, or -9. This finding suggests that
apoptosis induction by CD95L or TRAIL may involve amplification
of the death signal through the mitochondrial pathway, as suggested
recently for many other cell types, including hepatocytes (10,
18, 21).
As shown recently for HSC (6), PSC also are susceptible
toward apoptosis induction by PK-11195, a specific ligand of
the peripheral-type benzodiazepine receptor. However, HSC show a
transient sensitivity to PK-11195-induced apoptosis, which is
maximal at about the 7th day of culture and disappears within 4 wk
(6), whereas sensitivity of PSC increased with culture
over a period of 4 wk. This culture time dependence of PK-11195-induced
apoptosis in PSC cannot be explained exclusively by
corresponding alterations of the expression of the peripheral
benzodiazepine receptor, as shown for HSC (6).
The mechanisms underlying the culture-dependent sensitization of PSC
toward CD95L- and TRAIL-induced apoptosis is unclear but may
involve an increased expression of CD95 and TRAIL receptors. Clearly,
culture-dependent changes in PKB phosphorylation or Flip expression do
not provide an explanation. Interestingly, a cleavage product of the
RIP is found in PSC after 4 wk of culture, and its amount further
increases in response to CD95L or TRAIL addition, as expected from the
known caspase-8-triggered RIP cleavage (12, 17). RIP is
thought to be antiapoptotic by activation of the transcription
factor NF-
B (15). On the other hand, a COOH-terminal proteolytic fragment of RIP was shown to inhibit NF-
B activation through inhibition of inhibitory factor-
B-kinase-
(12). The COOH-terminal fragment of RIP (RIPc)
also augments the association between death receptors and death domain
proteins (e.g., TRADD and FADD; see Ref. 14), thereby
favoring caspase-8 activation and stimulation of apoptosis. The
proteolytic RIP fragment found in activated but not quiescent PSC (Fig.
5) stains with an antibody specific raised again the COOH-terminal part
of RIP. It exhibits a molecular mass of ~50 kDa. This 50-kDa
protein may well be the rat counterpart of the 42-kDa RIPc
described in HeLa and Jurkat cells (14). In these cells,
the COOH-terminal cleavage product appears when the cells are incubated
with TNF, CD95L, or TRAIL. A similar cleavage product with a molecular
mass of 38 kDa was also detected in Jurkat T cells exposed to CD95L
(17). As shown in the present study, the appearance of a
COOH-terminal RIP cleavage product during the transformation of PSC to
a myofibroblast-like phenotype is paralleled by an enhanced sensitivity
to apoptotic signals. The amount of this protein was further
enhanced by induction of apoptosis with CD95L or TRAIL. CD95L
induced the protein within 10 min of incubation in the presence of
cycloheximide, which argues against de novo synthesis of the protein.
Probably, the 50-kDa protein is the product of cleaving native RIP by
caspase-8 as it was also described by others (12, 17). In
line with this, CD95L in the presence of cycloheximide increases
caspase-8 activity sevenfold, and under these conditions a rapid and
pronounced increase in the amount of the 50-kDa protein is found.
TRAIL, however, only doubles caspase-8 activity in PSC, and this is
accompanied by a less pronounced and delayed increase of the 50-kDa
protein. The molecular mass of the TRAIL-induced protein is
somewhat higher compared with the strong band induced by incubation
with CD95L. The meaning and the mechanisms leading to this difference
are not clear so far.
The increasing susceptibility of activated PSC to death receptor
ligand-mediated apoptosis may be an efficient way to eliminate transformed PSC in chronic and acute pancreatitis to terminate the
healing response, restore cellular homeostasis, and to prevent the
persistence of cells engaged in excessive matrix production.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
H. Klonowski-Stumpe, Dept. of Gastroenterology, Hepatology and
Infectiology, Heinrich-Heine Universität Düsseldorf,
Moorenstr. 5, 40225 Düsseldorf, Germany (E-mail:
Hanne.Klonowski-Stumpe{at}uni-duesseldorf.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.
May 1, 2002;10.1152/ajpgi.00073.2002
Received 20 February 2002; accepted in final form 25 April 2002.
 |
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