1 Department of Internal Medicine, CHUV-1011 Lausanne, Switzerland
2 Institute of Pharmacology and Toxicology, University Hospital, CHUV-1011
Lausanne, Switzerland
Author for correspondence (e-mail:
jhaeflig{at}chuv.hospvd.ch)
Accepted 7 January 2003
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Summary |
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Key words: Type-I diabetes, ß-Cell line, Islet-brain-1, IB1/JIP-1, INS-1, Pancreatic islets, Apoptosis, Adenovirus, JNK activity
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Introduction |
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The molecular basis of JNK activation and the functional role of IB1/JIP-1 were mostly assessed in the transformed mouse insulin-secreting cell line ßTC3. Accordingly, we investigated the contribution of IB1/JIP-1 to rat ß-cell survival in freshly isolated pancreatic islets and in the differentiated insulin-secreting ß-cell line INS-1. Exposure of primary pancreatic islets and INS-1 cells to cytokines drastically reduced the IB1/JIP-1 content. Using adenovirus-mediated gene transfer of IB1/JIP-1 to increase or reduce the IB1/JIP-1 content, we could modulate the content of the scaffold protein in primary pancreatic islets and in INS-1 cells. Decreasing IB1/JIP-1 content induced an increase in JNK-activity and a concomitant increase in cytokine-mediated apoptosis rate, whereas cells with higher levels of IB1/JIP-1 are protected against the cytokine-induced apoptosis. Last, we assessed the sensitivity of isolated pancreatic islets obtained from haploinsufficient mice (IB1/JIP-1+/) and found that, compared with wild-type pancreatic islets, the reduction of IB1/JIP-1 content is associated with an increase JNK activity and basal apoptosis.
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Materials and Methods |
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For mouse islet isolation, pancreases were excised and placed in a 2 mg
ml1 collagenase solution (collagenase type IV; Worthington)
in Hank's balanced salt solution containing 10 mmol l1
Hepes, pH 7.4. Tissue was minced and incubated for 38 minutes at 37°C.
After washing of the digested tissue, islets were hand picked under a
stereomicroscope. Rodent islets were cultured in RPMI-1640 supplemented with
10% fetal calf serum, 10 mmol l1 Hepes, 1 mmol
l1 sodium pyruvate and 50 µmol l1
ß-mercaptoethanol (Guillam et al.,
2000).
The INS-1 (Asfari et al.,
1992) cells were cultured in RPMI-1640 medium supplemented with
11.1 mmol l1 glucose, 10% fetal calf serum (heat-inactivated
for INS-1), 2 mmol l1 L-glutamine, 1 mmol
l1 sodium pyruvate, 50 mmol l1
ß-mercaptoethanol, 110 U ml1 penicillin and 110 µg
ml1 streptomycin. INS-1 cells were kept at 37°C in a
humidified incubator gassed with air and CO2 to maintain medium pH
at 7.4, fed at 3-day intervals and passed by trypsinization once a week. A
combination of cytokines comprising interleukin 1ß (IL-1ß)
(2x105 units µg1; Alexis) tumor necrosis
factor-
(TNF-
) (105 units µg1;
Alexis) and interferon
(IFN-
) (10 ng ml1;
Alexis) was added for a period of 24 hours.
Hoechst-33342 staining
INS-1 cells and dispersed rat islets were grown on coverslips. After 2
days, the culture medium was changed and cytokines were added. Incubations
were performed for 48 hours and cells were then fixed for 15 minutes with 1%
paraformaldehyde. To evaluate the number of apoptotic cells, cells were
incubated in the presence of Hoechst 33342 (dilution 1:1500) for 7 minutes and
the nuclear morphology analyzed under a fluorescence microscope. The number of
cells displaying a pycnotic (highly condensed) nucleus and/or a fragmented
nucleus was evaluated in a blindly fashion. A minimum of 250 cells in four
separate experiments was counted for each condition
(Bonny et al., 2000;
Hoorens et al., 2001
). Mouse
islets were cultured for 48 hours and then fixed in 1% paraformaldehyde,
stained with Hoechst 33342 and mounted in mowiol (Calbiochem) before counting
the apoptotic nuclei.
Western blotting
Polyclonal IB1/JIP-1 antiserum was described previously
(Bonny et al., 1998). These
antibodies were affinity purified from crude serum using a Hitrap
NHS-activated affinity Sepharose column (Amersham Pharmacia Biotech,
Switzerand) coupled to the immunogenic protein. Polyclonal antibodies against
cleaved caspase-3 (Asp175, CST) and monoclonal antibodies against
-tubulin (T5168, Sigma, Switzerland) were diluted 1:100 and 1:10,000,
respectively. The cells were lysed in 5% SDS. Protein content was determined
using the DC protein assay reagent kit (Bio-Rad Laboratories, Switzerland).
Aliquots were fractionated by electrophoresis in a 10% polyacrylamide gel and
immunoblotted onto Immobilon PVDF membranes (Millipore, MA) overnight, at a
constant voltage of 30 V. The membranes were blocked for 3 hours at room
temperature in PBS containing 3% bovine serum albumin and 0.1% Tween 20, and
then incubated for 24 hours with antibodies diluted in blocking buffer. After
repeated rinsing in PBS and PBS + 0.1% Tween20, immunoblots were incubated
overnight at 4°C with an anti-mouse antibody coupled to alkaline
phosphatase (Dako Diagnostic, Zug, Switzerland), diluted 1:5000. Specific
antigen-antibody complexes were detected using the alkaline phosphatase
development reagent kit (BCIP-NBT) method (AP development reagent, BioRad
Laboratories, Glattburg, Switzerland). For the Phototope-HRP western blot
detection system, membranes were incubated for 1 hour at room temperature in
PBS containing 5% milk and 0.1% Tween 20 (blocking buffer) and then incubated
for 2 hours at room temperature with antibodies directed against IB1/JIP-1.
Specific antigen-antibody complexes were detected with the Phototope-HRP
western blot detection system (Amersham, Switzerland).
Protein-kinase assays
JNK activity was measured using
glutathione-S-transferase/c-Jun(1-79) bound to
glutathione/Sepharose-4B (Bonny et al.,
1998; Tawadros et al.,
2002
). INS-1 cells or rodent islets were lysed in 0.5% Nonidet
P-40, 20 mM Tris-HCl, pH 7.6, 0.25 mM NaCl, 3 mM EDTA, 3 mM EGTA, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM sodium
vanadate, 20 µg ml1 aprotinin and 5 µg
ml1 leupeptin. Lysates were centrifuged at 14,000
g for 10 minutes to remove nuclei, and supernatants (50 µg
of protein) were mixed with 19 µl of
glutathione-S-transferase/c-Jun(1-79)). The mixture was
rotated at 4°C for 1 hour, washed twice in lysis buffer and twice in
kinase buffer (20 mM Hepes, pH 7.5, 10 mM MgCl2, 20 mM
ß-glycerophosphate, 10 mM p-nitrophenylphosphate, 1 mM
dithiothreitol, 50 µm sodium vanadate). Beads were suspended in 40 µl of
kinase buffer with 10 µCi of [
33P] ATP and incubated at
30°C for 25 minutes. Samples were boiled in loading buffer and
phosphorylated proteins were resolved on 10% SDS-polyacrylamide gels. To
verify the selectivity of the JNK assay, cell lysates were fractionated by
Mono-Q ion exchange chromatography and each fraction assayed as described
above. Fractions were immunoblotted with rabbit antiserum recognizing JNK.
Only fractions containing immunoreactive JNK phosphorylated the
glutathione-S-transferase/c-Jun(1-79) protein.
DNA fragmentation assay
INS-1 cells or rat islets were washed in PBS and genomic DNA was isolated
using the InViSorb Apoptosis Detection Kit following the manufacturer's
procedure (InViTek, Germany). Using the ApoAlert ligation-mediated PCR Kit
(LM-PCR, Clonteck, CA), 50 µg genomic DNA was PCR amplified and the
resulting nucleosomal ladder was visualized on an agarose/ethidium-bromide
gel.
Generation of recombinant adenoviruses
We generated recombinant adenoviruses comprising the complete cDNA of rat
IB1/JIP-1 (Bonny et al., 1998;
Tawadros et al., 2002
) in the
sense or antisense orientation to modulate IB1. Adenoviruses expressing green
fluorescent protein (GFP) were used as a control. To generate the
adenoviruses, the cDNAs were inserted into the plasmid pXC15
(Schaack et al., 1995
) and
adenoviruses were then generated by homologous recombination in 293 cells
following co-transfection by the calcium-phosphate procedure of the plasmid
pJM17 and pXC15 constructs. Viruses were further plaque-purified three times
on HR911 cells (IntroGene, Leiden, The Netherlands). A large stock of viruses
was purified by two rounds of CsCl centrifugation. After the second
centrifugation, the virus band (1.5 ml) was collected and dialysed at 4°C
against three changes (at least 200 volumes each) of 10 mM Hepes pH 8.0, 150
mM NaCl in a Slide-A-Lyzer (0.5-3.0 ml capacity)
-irradiated 10K
dialysis cassette (Pierce, Switzerland). The recombinant adenoviruses allowed
expression of IB1/JIP-1, the GFP and/or the IB1/JIP-1 antisense RNA under the
control of the strong immediate early cytomegalovirus (CMV) promoter.
Statistical analysis
Densitometric analysis of signals (autoradiograms) were performed using a
Molecular Dynamics scanner (Sunnyvale, USA), which integrates areas and
corrects for background. Data were expressed as means ± s.e.m. and were
compared using Student's-t test and/or the MannWhitney
U test. Statistical significance was defined at a value
P<0.05 (*), P<0.01 (**) and P<0.001
(***).
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Results |
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IB1/JIP-1 production is regulated by gene transfer in INS-1
cells
IB1/JIP-1 content was experimentally modulated in ß cells using
adenovirus gene transfer. We used recombinant adenoviruses containing the
complete IB1/JIP-1 cDNA inserted in the sense or antisense orientation
(Ad-sIB1 and Ad-asIB1, respectively). A control adenovirus encoding GFP was
used as control. To study the efficiency of adenovirus-mediated transfer of
IB1/JIP-1, western-blot analysis of INS-1 cells infected with Ad-GFP,
Ad-sIB1/JIP-1 or Ad-asIB1/JIP-1 showed that the cells infected with the
Ad-sIB1 produce high amounts of IB1/JIP-1 compared with ß-cell extracts
infected with Ad-GFP. By contrast, the IB1/JIP-1 content was decreased with
the Ad-asIB1 (Fig. 2). Similar
results were observed using freshly dispersed primary rat islets (data not
shown). Quantitative assessment of IB1 content in ß cells demonstrates a
50% decrease in IB1 content in cells infected with Ad-asIB1 and a 300%
increase in IB1 content in cells overproducing IB1, whereas no change was
observed in cells infected with Ad-GFP
(Fig. 2).
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Contribution of IB1/JIP-1 to control of the rate of apoptosis
Representative gel-electrophoresis images of DNA laddering analysis are
shown in Fig. 3A. Cells grown
in the absence of cytokines show a barely visible DNA laddering in control and
cells overproducing IB1. By contrast, cells whose IB1 content is reduced using
antisense RNA showed a significant DNA laddering
(Fig. 3B). Furthermore, cells
incubated in presence of different cytokines show a drastic increase of DNA
laddering. These data indicate that modulation of IB1/JIP-1 content
contributes to DNA cleavage quantified by DNA laddering
(Fig. 3A,B).
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Apoptosis is characterized by morphological changes including the
condensation of nuclear chromatin, cellular shrinkage, membrane blebbing and
the formation of apoptotic bodies. Changes in nuclear morphology are
recognized as a reliable criterion with which to assess apoptosis, and the
DNA-binding dye Hoechst 33342 was used to visualize nuclear shape. The rate of
apoptosis was evaluated by scoring blindly the number of cells displaying a
fragmented nucleus. In INS-1, 48 hours incubation in presence of cytokines
IL-1ß, TNF- and IFN-
induced an increase in the rate of
apoptosis from 2% to 9% (Fig.
3C). INS-1 cells infected with the control virus Ad-GFP increased
their basal apoptosis rate threefold, whereas cells overproducing IB1 had a
similar rate of apoptosis to non-infected cells. ß Cells with reduced IB1
content had, without any stimuli, a 80% increase in the number of apoptotic
cells. These data suggest that IB1/JIP-1 can protect cells from apoptosis
caused by viral load. In the presence of cytokines, the increase in apoptosis
rate in cells infected with a control adenovirus (Ad-GFP) was similar to that
observed in non-infected cells (16% versus 5% instead of 9% versus 2% for
controls). This increase was abolished in cells overproducing IB1 and,
moreover, cells overproducing IB1 have a lower rate of apoptosis (7.5%) than
cells producing GFP (9.5%), indicating a protective effect of IB1 to
apoptosis. By contrast, cells producing lower levels of IB1 have a twofold
increase (29%) in apoptosis compared with cells infected with Ad-GFP (16%)
(Fig. 3C). This protective
effect of IB1/JIP-1 was confirmed using caspase-3 cleavage
(Widmann et al., 1998
) as a
marker for apoptosis (Fig.
3D).
The JNK activity was then evaluated in INS-1 cells incubated in presence of cytokines. The presence of cytokines was associated with an increase JNK activity (Fig. 4A). By contrast, when the IB1/JIP-1 content is experimentally increased by Ad-sIB1, JNK activity is reduced compared with the controls (Ad-GFP) and, conversely, as expected, JNK activity was found to be increased in cells producing lower amounts of IB1 (Fig. 4B). Quantitative assessment demonstrated a twofold increase in c-Jun phosphorylation in INS-1 cells infected with the Ad-asIB1 and, conversely, a 50% decrease in cells infected with the Ad-sIB1 compared with controls (Ad-GFP).
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In primary rat islet cells, a reduction of IB1/JIP-1 content increased the
susceptibility of the pancreatic cells to cytokine-induced apoptosis
(Fig. 5). A trend towards a
reduced cytokine-induced apoptosis was seen in primary pancreatic cells
overproducing IB1/JIP-1 (Fig.
5). To evaluate the potential role of IB1/JIP-1 in the control of
JNK activation in islets, we measured the JNK activity in the islets of mice
in which the gene encoding IB1/JIP-1 had been disrupted. Because the
homozygote IB1/JIP-1 (/) mice are associated with embryonic
lethality (Tawadros et al.,
2002; Thompson et al.,
2001
), we studied heterozygous IB1/JIP-1 (+/) mice. These
mice have a normal phenotype but the IB1/JIP-1 content is decreased
(Fig. 6A) in their islets
compared with their wild-type littermates (+/+). Concomitantly, the JNK
activity was increased twofold (Fig.
6B). These data indicate that the JNK activity in mice islets is
controlled by the IB1/JIP-1 content. The rate of apoptosis was further
evaluated in whole islets by blindly scoring the number of cells displaying a
fragmented nucleus (Fig. 6C).
The basal apoptosis was increased
3.7 times in islets from heterozygous
mice compared with islets from wild-type mice (14.9% in
IB1/JIP-1+/ versus 4.0% in IB1/JIP-1+/+).
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Discussion |
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IB1/JIP-1 binds to JNK, MKK7 and members of the MLK group of MAPK kinase
kinases (Bonny et al., 1998;
Bonny et al., 2000
;
Whitmarsh et al., 1998
). The
specificity and functions of JNK is controlled by the scaffold protein
IB1/JIP-1. IB1/JIP-1 facilitates the signal transduction mediated by the
interacting proteins. IB1/JIP-1 interacts with JNK through the JBD, a domain
able to prevent apoptosis of pancreatic ß-cell lines induced by
IL-1ß. Moreover, it has been demonstrated that producing the JBD in
cortical neurons prevent apoptotic cell death induced by arsenite
(Namgung and Xia, 2000
) and is
sufficient to prevent apoptotic death of nerve growth factor (NGF)-deprived
superior cervical ganglion neurons by inhibiting the phosphorylation of c-Jun
(Harding et al., 2000
).
Recently, it has been shown in sympathetic neurons undergoing apoptosis
following NGF withdrawal, that the IB1/JIP-1 JBD inhibits c-Jun
phosphorylation (Eilers et al.,
1998
).
We have recently investigated a rat model of complete bladder-outlet
obstruction to study in vivo the activity of MAPKs
(Tawadros et al., 2001;
Tawadros et al., 2002
). In
this model, an increased voiding pressure enhances bladder wall stress. A
drastic increase in the phosphorylated state of JNK was observed that was
associated with a reduced IB1/JIP-1 content in the urothelium. We therefore
demonstrated that IB1/JIP-1 is a crucial regulator of JNK activity in vivo and
that increased levels of IB1/JIP-1 prevents the stress-induced activation of
c-Jun.
We also generated mice carrying a targeted disruption of the
MAPK8IP1 gene, which encodes IB1/JIP-1
(Waeber et al., 2000). The
null mutation resulted in early embryonic lethality. The JNK activation was
found to be increased in freshly isolated mouse islets of heterozygous mice
carrying a disruption of the MAPK8IP1 gene exposed to cytokines.
Similarly, we have recently shown that JNK activation was increased in
unstressed urothelial cells of heterozygous mice
(Tawadros et al., 2002
) and
these data further indicating a crucial role for IB1/JIP-1 in islet
homeostasis.
In conclusion, we show that IB1/JIP-1 content within ß cells is crucial to protect cells from the stress-induced apoptosis. IB1 is therefore a regulator of survival in insulin-secreting cells and might represent an alternative therapeutic tool to prevent ß-cell damage observed in diabetes or during ß-cell transplantation.
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Acknowledgments |
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Footnotes |
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