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INTRODUCTION |
Type I diabetes is an inflammatory disease that is characterized
by apoptotic destruction of pancreatic
-cells. It is thought to be
mediated at least in part by cytokines such as interleukin (IL)1-1
, tumor necrosis
factor, and interferon-
released from infiltrating macrophages.
Among other effects, these cytokines induce the expression of iNOS and
the production of NO within
-cells, which is proposed to be a
significant trigger for apoptosis (1-4).
IL-1
induces the expression of iNOS protein in
-cells by
activating the stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase as well as the
NF-
B pathway, which in turn activate ATF2, AP-1, and NF-
B transcription factors, respectively (5-9). These translocate to the
nucleus and bind to specific regions within the promoter to initiate
iNOS message transcription (10). Gene regulation is also controlled
post-transcriptionally at the level of message stability (11), and this
is particularly true of iNOS mRNA, which is targeted for rapid
degradation because of signals in the 3'-untranslated region (12,
13).
PKC is a family of serine/threonine protein kinases consisting of 11 isoforms, comprising conventional, novel, and atypical subgroups, each
with varying cofactor requirements and cellular distribution (14).
Studies using pharmacological PKC activators or PKC overexpression have
established that PKC activation is necessary for enhanced gene
expression in response to proinflammatory stimuli (15-17) and in a
limited number of cells is sufficient for iNOS induction (18, 19). PKC
is capable of activating AP-1 (20), ATF2 (21), and NF-
B (22-26) and
may also act upstream of the mitogen-activated protein kinase cascade
to modulate activity of extracellular signal-regulated kinase 1/2, p38,
and JNK/SAPK (27, 28), so it could mediate its effects on cytokine
signaling by acting at any of these sites.
PKC
and PKC
are the predominant PKC isozymes expressed in islets
and insulinoma cells (29), but their role in IL-1
-induced gene
expression has not been defined. Determination of that role was the aim
of the current study. Using adenoviral constructs for overexpression of
wild-type and kinase-dead PKC isoforms in the INS-1 cell line, we
demonstrate that PKC
is necessary for IL-1
-stimulated iNOS
protein expression and NO production and that the underlying mechanism
is control of iNOS mRNA stability.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
The rat insulinoma cell line INS-1 was grown
in RPMI medium containing 10% heat-inactivated fetal calf serum,
penicillin G sodium (500 units/ml), streptomycin (50 µg/ml), and 2 mM glutamine (reagents from Life Technologies, Inc.) and
passaged by trypsinization into T150 flasks (Becton and Dickinson,
Franklin Lakes, NY). INS-1 cells (passage 15-30) were routinely seeded
at 0.5 × 106/well of a 24 well plate for experiments,
and used on day 4 at around 80% confluency.
Isolation of Rat Pancreatic Islets--
Pancreatic islets were
isolated from 230-270-g male Wistar rats by ductal infusion of
collagenase, purified on Histopaque 1077 gradient, and then hand-picked
under a binocular microscope. Islets were maintained in culture for
48 h in RPMI containing 5.5 mM glucose, 10%
heat-inactivated fetal calf serum, penicillin G sodium (500 units/ml),
streptomycin (50 µg/ml), and 2 mM glutamine. Islets were
incubated with 300 pg/ml IL-1
in the presence or absence of
rottlerin (10 µM) for the final 24 h.
Generation and Expression of PKC
and PKC
Recombinant
Adenovirus--
cDNA for mouse PKC
and rat PKC
(generous
gifts from Fredrick Mushinski and Peter Parker, respectively) were
first subcloned into pALTER (Promega, Annandale, New South Wales,
Australia) using conventional molecular biological techniques (30).
Mutagenesis was performed according to the protocol using the mutagenic
primers 5'-TACGCCATCAGGATCCTGAAG-3' and 5'-CTTTGCAATCAGGTACCTGAAGAAG-3' (for PKC
and PKC
, respectively) (Beckman Instruments,
Gladesville, Australia) targeted to conserved lysine residues in the
ATP binding domain. Mutants were designated PKC
KD (K368R
substitution) and PKC
KD, (K376R substitution). Overexpression of
kinase-dead forms have previously been shown to act in an
isoenzyme-specific dominant negative fashion (31, 32), while our data
(not shown) and others have confirmed that the PKC
KD mutant is
catalytically inactive (20, 33). Wild type and mutant PKC cDNAs
were then subcloned from pALTER into pXCMV, an adenoviral shuttle
vector constructed in this laboratory by subcloning the pRcCMV
(Invitrogen) expression cassette into pXCX3, derived from pXCX2 (34).
Recombinant adenovirus was then prepared by recombination with the
adenovirus plasmid pJM17, essentially as described by Graham and Prevec
(35). The MX17 control is an adenovirus that contains just the virus backbone (pJM17) and the expression cassette from pXCX3, but without cDNA. Adenovirus-mediated expression of PKC
and PKC
proteins was determined by infecting INS-1 cells at 10-20
plaque-forming units/cell, in 200 µl of medium and then incubating
for 1 h at 37 °C, mixing every 15 min. Virus was then removed,
and fresh medium was applied. At 48 h postinfection, proteins were
separated by 10% SDS-PAGE, and PKC
or PKC
expression was
determined by immunoblotting as described below.
Analysis of IL-1
-induced PKC
Translocation to the Plasma
Membrane--
At 48 h of culture, INS-1 cells were exposed to 300 pg/ml IL-1
(R & D Systems, Minneapolis, MN) for the times indicated. Cells were then washed in ice-cold PBS and cytosol, and membrane fractions were prepared, essentially as described (36). Proteins in
individual fractions were separated by SDS-PAGE on 10% gels (all
reagents from Bio-Rad) and immunoblotted as described below.
Immunoblot Analysis--
Whole cell lysate fractions were
prepared from INS-1 cells by washing monolayers in ice-cold PBS and
resuspending in modified Laemmli sample buffer (37) containing 1% SDS
and 10% mercaptoethanol. Membrane and cytosolic fractions prepared
above were also resuspended in this buffer. Proteins were separated by
SDS-PAGE on 10% gels, transferred to nitrocellulose membranes
(Trans-blot; Bio-Rad) by electroblotting, blocked with 5% milk powder,
and probed with appropriate antibodies, i.e. mouse
anti-PKC
antibody from Transduction Laboratories (San Diego, CA),
rat anti-PKC
and iNOS antibodies from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA), and cRel, I
B
, I
B
, phospho-p38, JNK, and
extracellular signal-regulated kinase 1/2 antibodies from New England
Biolabs (Beverly, MA). Donkey anti-rabbit horseradish
peroxidase-conjugated secondary antibody was from Jackson
ImmunoResearch Laboratories Inc. (West Grove, PA), and goat anti-mouse
horseradish peroxidase-conjugated secondary antibody was from Caltag
Laboratories (Burlingame, CA). The antigen-antibody complexes were
visualized by ECL detection (Amersham Pharmacia Biotech) and exposure
to x-ray film (Fuji, Tokyo, Japan). Signal intensities were determined
by densitometric analysis (on a Molecular Dynamics Personal
Densitometer SI and software by IPLabGel, Signal Analytics, VA).
Measurement of IL-1
-induced iNOS Expression and NO
Production--
INS-1 and islets cells were either infected with
recombinant adenovirus as described above or exposed to PKC inhibitors
(Calbiochem) 30 min prior to the application of 300 pg/ml IL-1
.
After 24-h exposure to IL-1
, 100 µl of medium was analyzed for
nitrite via the Griess diazo reaction (38). Analysis of iNOS expression in INS-1 cells was performed by washing the remaining cells in PBS and
immunoblotting cell lysates as described above, using a rat anti-iNOS antibody.
Isolation of Nuclear Extracts for Immunoblot Analysis--
INS-1
cells were treated appropriately, harvested by washing with ice-cold
PBS, and resuspended in 1 ml. Cells were pelleted by centrifugation at
13,000 × g for 20 s. The PBS was removed, and
cells were resuspended in 175 µl of ice-cold buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM
MgCl2, 0.1 mM EDTA, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 100 µg/ml aprotonin, 100 µg/ml leupeptin). Cells were incubated for 10 min on ice before the addition of 9 µl of 10% (v/v) Nonidet P-40
(Sigma) and vortexed for 20 s. The nuclear fraction was pelleted
at 13,000 × g for 20 s and washed once in buffer
A, without resuspending the pellet. 40 µl of buffer C (10 mM Hepes, pH 7.9, 420 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl
fluoride, 100 µg/ml aprotonin, 100 µg/ml leupeptin) was then
applied, and the pellet was resuspended by vortexing and vigorous
agitation for 30 min at 4 °C. This nuclear fraction was then
pelleted for 15 min at 13,000 × g, and supernatant was
removed for analysis by SDS-PAGE and immunoblotting.
Luciferase Reporter Assays--
INS-1 cells were transfected
with pCIneo (Promega) and
B, API (kind gifts from Malcom
Handel, Garvan Institute) and ATF2 (Promega) luciferase reporter DNA
using Superfectamine (Life Technologies, Inc.) according to the
manufacturer's instructions. The
B luciferase plasmid has six
NF-
B sites and a
-fibrinogen basal promoter, while the AP-1
plasmid has three AP-1 sites also driven by the
-fibrinogen basal
promoter. Stably transfected populations were selected using neomycin
(400 µg/ml) over 6 weeks. Stable cell lines were then seeded in
96-well plates at 5 × 104 cells/well and exposed to
adenovirus after a 24-h culture. 48 h postinfection, cells were
exposed to 300 pg/ml IL-1
for 4 h, and luciferase activity was
measured using the LucLite luciferase assay kit (Amersham Pharmacia
Biotech) according to the manufacturer's instructions. Luminescence
was measured using a Canberra Packard (Sydney, Australia) TopCount
microplate scintillation counter.
RNA Isolation and Semiquantitative RT-PCR of iNOS
Message--
INS-1 cells were cultured in six-well plates (at 1 × 106 cells/well) and infected with adenovirus as
described above. At 48 h postinfection, cells were exposed to 300 pg/ml IL-1
for either 6 or 12 h. To measure message
stabilization, cells were exposed to actinomycin D (1 µM)
for the indicated times. Cells were then washed in PBS and resuspended
in 1 ml of Trizol/well (Life Technologies, Inc.). RNA was extracted
according to the manufacturer's instructions and quantified using
absorbance at 260 nm. cDNA synthesis was performed using a
preamplification kit (Life Technologies, Inc.) according to the
manufacturer's instructions, using 5 µg of RNA in each reaction. PCR
was then performed using 2 µl of cDNA, oligonucleotides specific
for either
-actin or iNOS (39), and AmpliTaq Gold (PerkinElmer Life
Sciences). The PCR amplification protocol was designed to allow
quantification of iNOS and
-actin PCR product while in the
exponential phase of amplification. Products were amplified
using a 5-min hot start at 95 °C and 18 (
-actin) or 24-28 cycles
(iNOS) of 30 s at 95 °C, 30 s at 55 °C, and 60 s at 74 °C. Products were analyzed by gel electrophoresis on 1.5% agarose gels, stained with ethidium bromide, visualized on a GelDoc 1000 illuminator (Bio-Rad), and analyzed using IPLabGel software.
Statistics--
All results are presented as mean ± S.E.
Statistical significance was determined using unpaired Student's
t test.
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RESULTS |
Role of PKC
in IL-1
Signaling in INS-1 Cells--
Pancreatic
islets and
-cell lines have been well documented to contain PKC, of
which the predominant isoenzymes expressed are PKC
and PKC
(29,
40). To determine whether these isozymes are involved in IL-1
signaling in the clonal insulin secreting INS-1 cell line, we first
measured PKC translocation to the plasma membrane as an indicator of
activation. In response to IL-1
, PKC
translocates from the
cytosolic to the membrane fraction of INS-1 cells as early as 2 min
after stimulation with IL-1
(Fig.
1A). No such translocation
with PKC
was observed. Densitometric analysis (Fig. 1B)
revealed that accumulation of PKC
in the membrane fraction was more
than doubled by 2 min stimulation and significantly elevated for at
least 7.5 min.

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Fig. 1.
IL-1 induces
PKC translocation in INS-1 cells.
A, INS-1 cells were treated with IL-1 (300 pg/ml) for 0, 2, and 7.5 min. Cytosolic and membrane fractions were then prepared (as
described under "Experimental Procedures"), and proteins were
separated by SDS-PAGE on 10% gels. Proteins were transferred to
nitrocellulose membranes and blotted with anti-PKC and anti-PKC
antibodies. A representative blot from three separate experiments is
shown in A, while the mean density (arbitrary values) of
membrane-associated PKC from these experiments is shown in
B (*, p < 0.05; **, p < 0.01 versus unstimulated control).
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Role of PKC
in IL-1
-induced iNOS Expression and NO
Production--
To determine whether PKC activation participated in
iNOS induction by IL-1
in
-cells, we measured iNOS expression and
NO production in the presence of specific PKC inhibitors (41). IL-1
stimulated the induction of NO production and iNOS expression at
24 h in INS-1 cells (Fig. 2,
A and B). When incubated with 30 nM
Go6976, a PKC inhibitor selective for PKC
(41, 42), iNOS expression
and NO production were not inhibited (but instead were enhanced).
However, rottlerin (10 µM), which selectively inhibits
PKC
, almost completely inhibited production of NO. Most importantly,
rottlerin also completely inhibited IL-1
-stimulated NO accumulation
in isolated rat pancreatic islets (Fig. 2C), confirming that
this effect is not limited to the INS-1 cells.

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Fig. 2.
Inhibition of PKC by
rottlerin inhibits IL-1 induced NO production
and iNOS expression in INS-1 cells and rat islets.
A, INS-1 cells were treated with Go6976 (30 nM) or Rottlerin (10 µM) for 30 min prior to
the addition of IL-1 (300 pg/ml). After 24 h, medium was taken
to determine levels of NO production by the Griess reaction. Data are
the mean of four separate experiments (**, p < 0.01 when compared with control INS-1 cells with IL-1 ). B,
whole cell lysates were prepared in parallel, and proteins were
separated by SDS-PAGE on 10% gels and transferred to nitrocellulose
membranes, which were blotted with anti-iNOS antibody. The immunoblot
shown is representative of four separate experiments. C,
whole rat islets were also exposed to IL-1 for 24 h with and
without rottlerin, and NO production was measured as above.
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These results therefore implicate PKC
in IL-1
signaling and
prompted us to investigate its role more directly. Recombinant adenovirus capable of overexpressing WT and KD mutant forms of PKC
were generated and used to infect INS-1 cells. Recombinant PKC
adenovirus induced a high degree of overexpression by 72 h relative to MX17 control virus (Fig.
3A). A direct role for PKC
in IL-1
-induced NO production and iNOS expression was confirmed using the adenoviral constructs as shown in Fig. 3, B and
C, respectively. IL-1
stimulated a robust induction of
iNOS and a 6-fold increase in NO production in INS-1 cells infected
with control virus. Overexpression of PKC
WT, however, significantly
potentiated the NO response to IL-1
without affecting basal levels.
Similar effects were seen on iNOS expression. Effects of PKC
KD
overexpression were reciprocal to those of PKC
WT, with
IL-1
-stimulated iNOS expression markedly reduced and NO production
levels almost completely abolished.

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Fig. 3.
Inhibition of
IL-1 -induced iNOS expression and NO production
by PKC KD overexpression. INS-1 cells were
infected with MX17 control virus and PKC WT and PKC KD recombinant
adenoviruses at 10-20 plaque-forming units/cell for 1 h. At
48 h postinfection, cells were treated with IL-1 (300 pg/ml)
for a further 24 h. Immunoblot analysis of overexpressed PKC is
shown in A, while effects on NO production (B)
and iNOS expression (C) were measured as in Fig. 2. Data are
the mean of five individual experiments (*, p < 0.05;
***, p < 0.001 when compared with the MX17 control
cells with IL-1 ). The immunoblot shown is representative of four
separate experiments.
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PKC
Modulates IL-1
-induced iNOS mRNA Levels--
The
results above suggest that PKC
activation is required for
IL-1
-stimulated iNOS protein expression. To assess the underlying mechanism, we first measured iNOS mRNA levels after a 12-h exposure to IL-1
. Semiquantitative RT-PCR analysis (Fig.
4A) showed that IL-1
robustly induces iNOS message from virtually undetectable levels.
Overexpression of PKC
WT significantly potentiated message abundance,
over 2-fold compared with the MX17 control virus. Conversely, overexpression of PKC
KD reduced iNOS message levels to 40% of control. The results confirm that PKC
plays a major role in
controlling IL-1
induced iNOS expression by effects mediated at the
level of mRNA.

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Fig. 4.
Reciprocal modulation of
IL-1 -induced iNOS mRNA levels by
PKC KD and PKC WT.
INS-1 cells were infected with MX17 control virus and PKC WT and
PKC KD recombinant adenoviruses as described under "Experimental
Procedures," and at 48 h postinfection they were exposed to 300 pg/ml IL-1 for 12 h. RNA was extracted using Trizol reagent,
and RT-PCR was performed using oligonucleotides specific for rat iNOS.
-Actin PCR products were isolated at 18 cycles, and iNOS PCR
products were isolated at 24 cycles (known to be the exponential phase
for iNOS cDNA amplification). These were separated on a 1.5%
agarose gel, stained with ethidium bromide, and analyzed using Bio-Rad
and IPLabGel software. The top shows a plot of mean
data ± S.E. from four individual experiments (where *,
p < 0.05; **, p < 0.01), while the
bottom shows a representative gel.
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Signal Transduction Pathways Controlling iNOS Gene
Transcription--
The transcription factor AP-1 plays a role in
regulating iNOS gene expression in pancreatic
-cells (2, 5). Because PKCs are important regulators of AP-1 activity in many cells, acting
upstream of JNK/SAPK, we first examined the potential for PKC
to act
in this pathway. Fig. 5A shows
the results of Western blot analysis to measure JNK/SAPK activation
using phosphospecific antibodies that recognize only the activated form
of these kinases. IL-1
clearly activated this kinase but in a manner
that was unaffected by the presence of PKC
WT or PKC
KD. In Fig.
5B, we show activation of AP-1-mediated luciferase
expression in INS-1 stably expressing the AP-1 reporter plasmid and
exposed to IL-1
for 4 h. Clearly, overexpression of either
PKC
WT or PKC
KD had no effect on AP-1 activity stimulated by
IL-1
.

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Fig. 5.
JNK/SAPK activation and AP-1 transcriptional
activity is not affected by PKC .
A, INS-1 cells were infected with MX17 control virus and
PKC WT and PKC KD recombinant adenoviruses as described under
"Experimental Procedures," and at 48 h postinfection they were
exposed to 300 pg/ml IL-1 for 30 min. Whole cell lysate was prepared
in sample buffer, and proteins were separated by 10% SDS-PAGE.
Proteins were transferred to nitrocellulose membrane and blotted for
phospho-JNK/SAPK. The immunoblot shown is representative of four
separate experiments. B, INS-1 cells stably transfected with
an AP-1-luciferase reporter were infected with adenovirus as described
under "Experimental Procedures," and at 48 h postinfection
they were exposed to 300 pg/ml IL-1 for a further 4 h.
Luminescence was measured according to the manufacturer's
instructions. Results are the mean data ± S.E. of five separate
experiments.
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We next analyzed the ATF2 transcription factor and the role of PKC upon
this pathway, since ATF2 has been shown to be regulated by PKC in other
systems (21). We again used phosphospecific antibodies to measure the
effect of PKC
WT and PKC
KD protein on IL-1
-induced activation
of p38 (Fig. 6A) and a
specific luciferase reporter to measure ATF2 activity in INS-1 cells
exposed to IL-1
. While the IL-1
-induced phosphorylation of p38
was unaltered by PKC
overexpression, there were effects at the level
of ATF2-mediated luciferase expression. In fact, PKC
WT clearly and
significantly augmented both the basal and IL-1
response. This
suggests that PKC
might be sufficient for up-regulating ATF2
reporter activity. However, this effect is independent of IL-1
,
since the response to PKC
WT and IL-1
appeared additive, and the
PKC
KD mutant did not affect stimulated reporter activity.

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Fig. 6.
p38 activation and ATF2 transcriptional
activity is not dependent on PKC .
A, INS-1 cells were infected with MX17 control virus and
PKC WT and PKC KD recombinant adenoviruses as described under
"Experimental Procedures," and at 48 h postinfection they were
exposed to 300 pg/ml IL-1 for 30 min. Whole cell lysate was prepared
in sample buffer, and proteins were separated by 10% SDS-PAGE.
Proteins were transferred to nitrocellulose membrane and blotted for
phospho-p38. The immunoblot shown is representative of four separate
experiments. B, INS-1 cells stably transfected with a
ATF2-luciferase reporter were infected with adenovirus as described
under "Experimental Procedures," and at 48 h postinfection
they were exposed to 300 pg/ml IL-1 for a further 4 h.
Luminescence was measured according to the manufacturer's
instructions. Results are mean data ± S.E. of five separate
experiments (**, p < 0.01; ***, p < 0.001 when compared with the MX17 control cells without or with
IL-1 , respectively).
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We next investigated the effect of PKC
on various aspects of the
NF-
B signaling cascade. NF-
B exists as a cytosolic heterodimer, where activation is mediated by degradation of an inhibitory (I
B) subunit, which thereby facilitates translocation of the active subunit
(p65) to the nucleus. The time course of degradation and resynthesis of
I
B
and I
B
in response to IL-1
are shown in Fig.
7A. We also show the
appearance of p65 in the nucleus over this time. Both I
B
and
I
B
are degraded in response to IL-1
with the concomitant
appearance of p65 in the nucleus indicating activation of the NF-
B
pathway. There is, however, no effect of either PKC
WT or PKC
KD on
the kinetics of these events. Transcriptional activity of NF-
B in
INS-1 cells stably transfected with the
B luciferase reporter
construct was also investigated (Fig. 7B). IL-1
induced a
20-fold increase in luciferase expression over 4 h, which was
significantly enhanced by overexpression of the PKC
WT construct, as
was basal luciferase expression. There was, however, no inhibition of
IL-1
-induced
B-mediated luciferase expression by PKC
KD. As
with the ATF2 data presented above, these results suggest that the
PKC
is sufficient to increase basal NF-
B reporter activity but
acts independently of IL-1
.

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Fig. 7.
NF- B activation and
transcriptional activity is not dependent on PKC
activity. A, INS-1 cells were infected with MX17
control virus and PKC WT and PKC KD recombinant adenoviruses as
described under "Experimental Procedures," and at 48 h
postinfection they were exposed to 300 pg/ml IL-1 for the indicated
time course. Whole cell lysates and nuclear extracts were separated by
10% SDS-PAGE, and proteins were transferred to nitrocellulose
membrane. Whole cell lysates were probed using anti-I B and
anti-I B antibodies. Nuclear extracts were probed using anti-p65
antibodies. Representative blots from four separate experiments are
shown. B, INS-1 cells stably transfected with a
-luciferase reporter were infected with adenovirus as described
under "Experimental Procedures," and at 48 h postinfection
they were exposed to 300 pg/ml IL-1 for a further 4 h.
Luminescence was measured according to the manufacturer's
instructions. Results are mean data of five separate experiments (**,
p < 0.01; ***, p < 0.001 when
compared with the relevant MX17 control).
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Role of PKC
in iNOS mRNA Stability--
Since we did not
observe a requirement for PKC
activity in the regulation in the
three major signaling pathways known to control IL-1
-induced iNOS
gene expression in pancreatic
-cells, we next assessed whether
PKC
might exert effects at the level of iNOS mRNA stability.
This was assessed by stimulating INS-1 cells for 6 h with IL-1
and then halting transcription with actinomycin D. RNA was harvested at
2, 4, and 6 h after this point. The results demonstrate that in
control cells iNOS mRNA is degraded following actinomycin D
treatment, with iNOS mRNA having a half-life
(t1/2) of ~6 h (Fig.
8). PKC
WT clearly reduces the
rate of degradation such that at 6 h postapplication of
actinomycin D, iNOS message levels are less than 30% below the peak
levels prior to actinomycin D treatment. Conversely, PKC
KD
overexpression increased the rate of iNOS mRNA degradation such
that more then 80% of the message is degraded after 6 h in the
presence of actinomycin D, and its t1/2 is reduced to less than
3 h. These observations strongly implicate a role for PKC
in
iNOS mRNA stabilization and provide a mechanism by which PKC
can
regulate IL-1
induced iNOS mRNA levels and protein
expression.

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Fig. 8.
PKC controls iNOS
message stability. INS-1 cells were infected with MX17 control
virus and PKC WT and PKC KD recombinant adenoviruses as described
above, and at 48 h postinfection they were exposed to 300 pg/ml
IL-1 for 6 h. Actinomycin D (1 µM) was applied at
6 h, and RNA was extracted using Trizol reagent at 2, 4, and
6 h after. RT-PCR was performed over 28 cycles using
oligonucleotides specific for rat iNOS. Products were separated on a
1% agarose gel and analyzed using Bio-Rad and IPLabGel software. Shown
is a plot with mean data ± S.E. from three individual experiments
(*, p < 0.05; **, p < 0.001). ,
MX17; , PKC WT; , PKC KD. The insert shows a
representative gel. Mean mRNA levels were not significantly
different for PKC WT at 6 h with IL-1 compared with MX17,
while PKC KD reduces iNOS mRNA at 6 h to 77 ± 6% that
of MX17.
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|
 |
DISCUSSION |
We have described a novel role for PKC
in regulating NO
generation in IL-1
-stimulated pancreatic
-cells. This was
elucidated using specific PKC inhibitors and recombinant adenoviruses
for overexpression of PKC
WT or -KD. Our results indicate that PKC
activation was absolutely required for up-regulation of iNOS mRNA and protein levels, as well as generation of NO, in cells stimulated for 12-24 h with IL-1
. However, overexpression of PKC
KD did not
inhibit any of the three major transcriptional pathways (AP-1, ATF-2,
and NF-
B) known to be important for iNOS induction in IL-1
-stimulated
-cells (1-6). Moreover, when transcription was
inhibited using actinomycin D, iNOS mRNA was shown to degrade from
stimulated levels with a t1/2 of ~6 h. This was
shortened to less than 3 h in cells overexpressing PKC
KD,
whereas iNOS mRNA degradation was markedly inhibited in the
presence of PKC
WT.
IL-1
has previously been demonstrated to generate a rapid (<10-min)
increase in the PKC activator diacylglycerol in pancreatic islets (43)
and
-cell lines (5) and to induce the phosphorylation of known PKC
substrates (43). Our results now suggest that it is PKC
that is
specifically activated by IL-1
in INS-1 cells under these
circumstances. Importantly, it is well documented that even short term
exposure to IL-1
is sufficient to trigger much longer acting
proinflammatory and proapoptotic events in pancreatic
-cells (2, 3).
However, regulation of PKC
is complex. For example, it is known to
be targeted by upstream kinases, both on a region known as the
activation loop and on certain tyrosine residues, and
phosphorylation of these sites augments PKC
activity (44).
Therefore, we do not exclude the possibility that PKC
is activated
in response to IL-1
, and thereby stabilizes iNOS mRNA, by
mechanisms other than a rapid rise in diacylglycerol.
Irrespective of the actual mechanism, our results clearly suggest that
IL-1
initiates a bifurcating signaling cascade in the pancreatic
-cells; one arm controls iNOS gene transcription through a
relatively well characterized signaling pathway, whereas a second arm,
poorly understood but possessing a requirement for PKC
, regulates
iNOS mRNA stabilization. Both arms act in concert to ensure
efficient regulation of NO generation. This is consistent with the
observation that overexpression of PKC
WT was not in itself
sufficient to up-regulate iNOS expression in the absence of IL-1
stimulation. This also explains previous findings that activation of
PKC with phorbol esters was insufficient to stimulate NO generation in
clonal
-cells but that the phorbol esters could potentiate the
response to IL-1
(45).
A similar dual control mechanism has been recently observed in other
cell types for IL-1
-induced genes such as cyclooxygenase-2 (46) and
IL-8 (47) (48). However, in the published studies, mRNA
stabilization appeared to involve activation of p38 rather than PKC,
although the latter was not specifically addressed. Our data are the
first to describe a role for PKC
in this bifurcating cascade, by
which iNOS mRNA is stabilized. In our studies, p38 did not lie
downstream of PKC
in a linear signaling pathway, since p38
activation was not affected by overexpression of either of the PKC
constructs. However, it is not excluded that both kinases might
phosphorylate, and thereby regulate, a common substrate that acts as
trans-acting factor in the control of mRNA stabilization.
The full importance that message stabilization has in regulating iNOS
gene expression has only recently become apparent. Two studies (12, 13)
have now shown that iNOS mRNA is highly transcribed, but the
3'-untranslated region severely destabilizes the RNA structure such
that it is efficiently degraded in an unstimulated cell. ATTTA motifs
are thought to contribute to structural instability that targets the
RNA for degradation, but these are specifically silenced in the
presence of certain proinflammatory stimuli, the message is stabilized,
and iNOS mRNA levels increase. In this context, a role for the RNA
binding protein HuR has recently been invoked (13), but the upstream
pathways linking this to receptor occupation are not defined. On the
other hand, there is a limited literature to suggest that PKC does
promote stabilization of some gene transcripts such as those of lactate
dehydrogenase. An AT-rich region in the 3'-untranslated region of the
lactate dehydrogenase mRNA sequence has been identified as the
PKC-stabilizing region (49). The trans-acting factors that target this
region are unknown. However, since iNOS and lactate dehydrogenase
mRNA share similar AT-rich sequence motifs, it is clearly possible
that PKC might regulate stability of both transcripts through a common mechanism.
It is well documented that PKC activation is sufficient for induction
of NF-
B-responsive genes in many cell types, but a demonstration
that PKC is necessary for up-regulation of iNOS expression by
proinflammatory stimuli has been limited to studies using macrophages
and astrocytes (15-17). In only one instance was a specific
requirement of PKC
reported, and even this was not selective, since
PKC
and PKC
I were also implicated (15). A role for PKC in
stabilizing iNOS mRNA has only been proposed once previously, but
this was based on the use of phorbol esters in macrophages and so could
not discriminate between PKC isoforms (50). The PKC requirement in the
earlier studies (15-17) was generally explained at the level of
NF-
B activation. In contrast, our results suggest that PKC
is not
necessary for IL-1
-stimulated NF-
B activation in pancreatic
islets. We did also observe an IL-1
-independent up-regulation of
NF-
B (and ATF2) reporter activity in cells overexpressing PKC
WT,
but in both instances PKC
did not appear to be acting in the
upstream signaling cascades. This suggests a site of action at the
level of phosphorylation of p65 (or ATF-2) or of other proteins with
which they interact to regulate transcription. However, since the
effects of PKC
on NF-
B and ATF2-mediated transcription were
independent of IL-1
, we have not further pursued the underlying mechanisms.
In addition to iNOS described here, several other genes have also been
shown to be regulated by PKC
, including manganese superoxide
dismutase (51), a regulator of inflammation, and genes responsible for
cell growth such as p21 and p27 (52, 53) and differentiation,
i.e. involucrin (54). The expression of many other
regulatory genes can also be modulated in the presence of phorbol
esters and/or PKC inhibitors, thus implicating various PKC isozymes
(55, 56). While in many instances regulation by PKC is likely to be
transcriptional, our data and the recent finding that genes like iNOS
are highly regulated at a post-transcriptional stage (12, 13) and that
some other gene transcripts may contain PKC recognition sequences (49)
clearly suggest that post-transcriptional mRNA stabilization could
provide a second mechanism by which PKC modulates gene expression. In
the context of pancreatic
-cells, it will be of great interest to
determine whether a PKC
-mediated mRNA stabilization is also
involved in regulation of other genes known to be induced by
IL-1
.
In conclusion, we have demonstrated a novel requirement for PKC
in
IL-1
-induced iNOS induction in INS-1 and islet
-cells and shown
this to be mediated by mRNA stabilization. IL-1
-induced NO
produced by iNOS has been implicated in
-cell dysfunction, secretory
defects, and eventually cell death by apoptosis. Therefore, an obvious
focus of future studies will be to determine whether inhibition of
PKC
abrogates any aspect of the cellular dysfunction initiated by
IL-1
in pancreatic
-cells.