(Received for publication, October 16, 1995; and in revised form, January 18, 1996)
From the
The aim of this investigation was to study the putative
involvement of lipid second messengers, protein kinases, and
transcription factors in interleukin-1 (IL-1
)-induced signal
transduction in insulin-producing cells. For this purpose,
insulin-producing RINm5F cells were exposed to IL-1
(25 units/ml),
and the ceramide, ceramide 1-phosphate, sphingomyelin, diacylglycerol,
and phosphatidic acid contents of the cells were subsequently
determined. It was found that IL-1
induced a transient increase
(2-5 min) in ceramide and diacylglycerol, which was not
paralleled by an increase in ceramide 1-phosphate and phosphatidic
acid. A rapid decrease in the sphingomyelin content of the cells was,
however, observed. The cell-permeable ceramide analogue N-acetylsphingosine and the phorbol ester phorbol 12-myristate
13-acetate (PMA) both induced the phosphorylation and increased the
activities of the protein kinase JNK1 and the transcription factor
ATF2. These effects were, however, not as pronounced as those induced
by IL-1
. The DNA binding activity of transcription factors in
nuclear extracts was determined using the electrophoretic mobility
shift assay method. Transcription factor binding to the
ATF/cAMP-responsive element consensus sequence was increased
4-5-fold by acetylsphingosine, PMA, or IL-1
, whereas binding
to the CCAAT/enhancer-binding protein and AP-1 elements was found to be
only slightly stimulated by these three agents. Binding to the
NF-
B element was strongly induced by IL-1
, but not by
acetylsphingosine or PMA. Finally, acetylsphingosine and PMA did not
mimic the nitric oxide-inducing effects of IL-1
. It is concluded
that IL-1
-stimulated formation of ceramide and diacylglycerol may
contribute to JNK1 and ATF2 transcription factor activation, which may
be a necessary (but not sufficient) step in
-cell nitric-oxide
synthase induction.
It has been demonstrated that interleukin-1 (IL-1
) (
)exerts inhibitory and cytotoxic effects on rodent
pancreatic beta cells in
vitro(1, 2, 3) . This has led to the
suggestion that this cytokine, alone or in combination with other
cytokines, may be an important mediator of the autoimmune destruction
of beta cells during the course of insulin-dependent diabetes
mellitus(4, 5) . The IL-1
effects are thought to
be mediated by, at least in part, induction of nitric-oxide synthase
(iNOS)(6, 7) . Nitric oxide production leads to
inhibition of aconitase, glucose oxidation rates, ATP generation, and
insulin
production(2, 3, 7, 8, 9) .
The intracellular signals generated in insulin-producing cells by the
interaction between IL-1
and its receptor have, however, not yet
been elucidated.
Sphingomyelin hydrolysis and ceramide generation
constitute a signal transduction pathway that mediates some of the
effects of the cytokines IL-1 and tumor necrosis
factor-(10, 11) . Sphingomyelin consists of
sphingosine, a fatty acid, and a phosphocholine head group. Upon
hydrolysis, the phosphocholine head group is released, and ceramide is
formed. The ceramide generated by a cytokine-stimulated
sphingomyelinase is thought to activate a 97-kDa proline-directed
serine/threonine kinase(12) . Moreover, ceramide activation of
this protein kinase has been reported to induce NF-
B, a stress
response transcription factor(13) . In insulin-producing cells,
indirect evidence has been presented suggesting a role of protein
Ser-Thr and Tyr phosphorylation events (14, 15) leading to NF-
B activation (16) and induction of iNOS(17) . Therefore, it was
investigated here whether IL-1
stimulates sphingomyelin hydrolysis
and ceramide generation and whether this putative event may participate
in IL-1
-induced transcription factor activation and the subsequent
induction of iNOS.
Figure 1:
Effects of
IL-1 on DAG and ceramide contents of RINm5F cells. RINm5F cells
were exposed to 25 units/ml IL-1
for 0, 2, 5, and 20 min. Lipids
were extracted, and DAG and ceramide contents were determined as
described under ``Experimental Procedures.'' Results are
means ± S.E. for 9-12 observations. * and **, p < 0.05 and p < 0.01, respectively, for a chance
difference versus corresponding control using Student's
paired t test.
Figure 2:
Effects of IL-1 on sphingomyelin
content of RINm5F cells. RINm5F cells equilibrium-labeled with
[
H]choline were exposed to 25 units/ml IL-1
for 0, 2, 5, and 20 min. Lipids were extracted and quantified as
described under ``Experimental Procedures.'' Results are
means ± S.E. for 9-11 observations. *, p <
0.05 for a chance difference versus corresponding control
using Student's paired t test.
Figure 3:
A, effects of acetylsphingosine, PMA, and
IL-1 on JNK1 phosphorylation. RINm5F cells were
P-labeled for 2 h and then exposed to 10 µM acetylsphingosine (lane 2), 100 nM PMA (lane
3), and 25 units/ml IL-1
(lane 4) for 20 min. JNK1
was immunoprecipitated and analyzed by SDS-polyacrylamide gel
electrophoresis. The position of JNK1 (46 kDa) is indicated by the lower arrow. The upper arrow indicates the position
of the 54-kDa JNK2 phosphoprotein. This figure is representative of
three separate experiments. B, effects of acetylsphingosine,
PMA, and IL-1
on JNK1 in vitro kinase activity. RINm5F
cells were exposed for 20 min to 10 µM acetylsphingosine (lane 2), 100 nM PMA (lane 3), 10 µM acetylsphingosine + 10 nM PMA (lane 4), and
25 units/ml IL-1
(lane 5). Lane 1 in A and B is the control. Homogenates were then
immunoprecipitated for JNK1, and the in vitro kinase activity
was determined using GST-c-Jun fusion protein as substrate. The
position of phosphorylated GST-c-Jun is indicated by the arrow. C, Coomassie Brilliant Blue staining of cell
homogenates separated by SDS-polyacrylamide gel electrophoresis. Lanes
are the same as described for B.
The in vitro kinase activity of
JNK1, measured as phosphorylation of the GST-c-Jun fusion protein, was
weakly increased by 10 µM acetylsphingosine (37 ±
12%, n = 3) and by 100 nM PMA (95 ±
35%, n = 3). The combination of acetylsphingosine and
PMA increased GST-c-Jun phosphorylation by 63 ± 12%, and
IL-1 (25 units/ml) increased GST-c-Jun phosphorylation by 275
± 70% (n = 3) (Fig. 3B). There
was no phosphorylation of a 38-kDa protein when only GST was used as a
phosphorylation substrate (data not shown). Fig. 3C demonstrates that equal amounts of protein were used for
immunoprecipitation and JNK1 activity determination.
Figure 4:
A, immunoblot analysis of ATF2 in RINm5F
cells exposed to acetylsphingosine, PMA, and IL-1. RINm5F cells
were exposed for 20 min to 0, 0.1, 1.0, and 10 µM acetylsphingosine (lanes 1-4, respectively), 100
nM PMA (lane 5), and 25 units/ml IL-1
(lane
6). The lower arrow indicates the position of the 69-kDa
ATF2 form, and upper arrow indicates the position of the
phosphorylated 72-kDa ATF2 form. Positions of molecular mass markers
(in kilodaltons) are given on the right. The figure is representative
of three separate experiments. B, immunoblot analysis of ATF2.
Homogenates from control RINm5F cells (lanes 1 and 2)
or IL-1
-exposed RINm5F cells (lanes 3 and 4)
were either untreated (lanes 1 and 3) or incubated
with alkaline phosphatase (lanes 2 and 4). The upper arrow shows the position of phosphorylated ATF2, and the lower arrow show that of nonphosphorylated
ATF2.
Figure 5:
Autoradiogram showing effects of
acetylsphingosine, PMA, and IL-1 on transcription factor gel
retardation in an electrophoresis mobility shift assay experiment.
Nuclear extracts from RINm5F cells exposed for 20 min to 1 and 10
µM acetylsphingosine (lanes 2 and 3),
100 nM PMA (lane 4), and 25 units/ml IL-1
(lane 5) were incubated with
P-labeled
double-stranded oligonucleotides specific for NF-
B, AP-1, C/EBP,
and ATF/CREB and run on 5% polyacrylamide gels. Lane 1 is the
untreated control, and lane 6 is nuclear extracts from
IL-1
-treated RINm5F cells incubated with a 1000-fold excess of
unlabeled oligonucleotide.
Figure 6:
Effects of acetylsphingosine, PMA, and
IL-1 on transcription factor activation. Data are means ±
S.E. from densitometric scannings of four to five separate experiments
performed as described for Fig. 6. Bar 1, control; bars 2 and 3, 1 and 10 mM acetylsphingosine,
respectively; bar 4, 100 nM PMA; bar 5, 25
units/ml IL-1
.
Figure 7:
Effects of acetylsphingosine on RINm5F
cell nitrite production. RINm5F cells were exposed to acetylsphingosine
(ceramide (Cer.), okadaic acid (Ok. A), sodium
orthovanadate, and IL-1 as shown. After 6 h, medium samples were
taken for nitrite determinations. Results are means ± S.E. for
five to nine observations.
The results of this study suggest that IL-1 receptor
activation in RINm5F cells leads to phospholipase C activation and DAG
generation. This is in line with previous studies demonstrating IL-1
activation of a phosphatidylcholine-specific phospholipase C in Jurkat
T cells, which generates DAG but not inositol trisphosphate, and
showing that DAG was generated in isolated mouse pancreatic islets in
response to IL-1 without any concomitant increase in intracellular
free Ca
concentrations(26, 27) . The
presently observed decrease in sphingomyelin and a concomitant increase
in ceramide imply the activation of a sphingomyelinase by IL-1
in
RINm5F cells. It is unclear whether the sphingomyelinase is stimulated
directly by the activated IL-1 receptor or whether sphingomyelinase
activation is indirectly accomplished by the increased DAG level.
Indeed, DAG is known to trigger sphingomyelinase activity in Jurkat T
cells and in GH3 rat pituitary cells(28, 29) . The
present finding that acetylsphingosine and PMA could not be clearly
differentiated with respect to their activation profiles of JNK1 and
the transcription factors may be explained by DAG- or PMA-induced
sphingomyelinase activation and the concomitant generation of ceramide.
Another possibility to be considered is that arachidonic acid can
increase the activity of sphingomyelinase, a finding described in HL-60
cells(30) . Indeed, increased levels of prostaglandin
E
, a metabolite of arachidonic acid, have been observed in
response to IL-1
in isolated rat islets(31) .
Ceramide
1-phosphate has been suggested to act as a regulator of intracellular
calcium stores and cell proliferation and is thought to be generated by
ceramide kinase activation(20) . Although ceramide 1-phosphate
contents appear to be high in RINm5F cells, they were not increased by
IL-1, which speaks against cytokine-induced activation of ceramide
kinase. The finding that phosphatidic acid was not increased suggests
that IL-1
does not stimulate the activity of phospholipase D in
RINm5F cells.
Addition of ceramide analogues to dermal fibroblast
and EL4 T helper cells mimics some IL-1 and tumor necrosis factor-
actions by inducing cyclooxygenase and IL-2 gene
expression(21, 32) . These effects induced by the
proinflammatory cytokines and ceramide analogues have been proposed to
be mediated by the ceramide-activated protein kinase and downstream
protein kinases belonging to the mitogen-activated protein kinase
family(33) . The c-Jun NH
-terminal kinase (JNK1) (34) and its homologues, MAPKAP kinase-2 reactivating
kinase(35) , stress-activated protein kinase-
(36) , HOG1(37) , p38(38) , and
cytokine-suppressive anti-inflammatory drug-binding
protein(39) , all belong to the mitogen-activated protein
kinase family and are activated by cellular stress agonists such as
proinflammatory cytokines, heat, arsenite, UV light, and osmotic and
chemical stress. The present finding that IL-1
and, to a lesser
extent, acetylsphingosine and PMA induce the phosphorylation and
activation of JNK1, an event possibly mediated by the extracellular
signal-regulated kinase kinase kinase(40) , indicates that this
pathway is active also in insulin-producing cells. In previous reports,
it was demonstrated that JNK1 phosphorylates the stress protein
hsp27(35) , c-Jun(41) , and a transcription factor that
belongs to the ATF/CREB family, namely ATF2, leading to its
transcriptional activation(42) . This is probably also the case
in RINm5F cells since increased phosphorylation of ATF2 in response to
acetylsphingosine, PMA, and IL-1
was presently observed. Moreover,
both PMA and acetylsphingosine induced enhanced transcription factor
binding to the ATF/CREB element. Thus, IL-1
-induced ceramide/DAG
generation may contribute to the phosphorylation and activation of JNK1
and ATF2 in insulin-producing cells.
Even though relatively high
concentrations of acetylsphingosine were presently used (1.0-10
µM), the effects of the ceramide analogue were generally
less pronounced than those induced by IL-1. This may, on one hand,
be due to a lower efficiency of the ceramide analogue in activating the
ceramide-dependent protein kinase(s) compared with endogenously
produced ceramide. On the other hand, it cannot be excluded that
ceramide generation plays a less prominent role in this pathway and
that other signals mediate or are necessary for IL-1
-induced JNK1
and ATF2 activation. Indeed, it has recently been demonstrated that the
GTP-binding proteins Cdc42 and Rac1 initiate a phosphorylation cascade
that activates the stress-activated protein kinase/c-Jun
NH
-terminal kinase signaling pathway (23) .
It
has been proposed that cytokines and ceramide induce indirect
phosphorylation events leading to the activation of the transcription
factor NF-B(13) . IL-1
has been shown to induce
NF-
B activation in RINm5F cells, an event that was necessary for
iNOS gene expression(16) . However, the finding that
acetylsphingosine and PMA did not activate NF-
B argues against a
role for these lipids in this specific pathway. This is in line with
other reports showing a dissociation between ceramide generation and
NF-
B translocation(43, 44) . Interestingly,
activated ATF2 has been demonstrated to interact not only with several
viral proteins, but also with NF-
B and c-Jun (45, 46) . Thus, IL-1
-induced iNOS expression may
involve not only NF-
B activation, which seems to be ceramide- and
PMA-independent, but also a parallel ceramide-dependent pathway leading
to phosphorylation of c-Jun and ATF2, which both act synergistically in
stimulating NF-
B and AP-1 activity.
It is unclear whether
activated ATF2 interacts directly with the promoter region of the iNOS
gene. Instead, ATF2 might induce the transcription of other genes
necessary for iNOS induction. Indeed, the mRNA for iNOS appears as late
as 3 h after IL-1 addition in RINm5F cells, and iNOS mRNA
expression requires protein synthesis since cycloheximide prevents the
induction(47) . Thus, other transcription factors besides
NF-
B and ATF2 are probably necessary for maximal iNOS gene
expression. Since acetylsphingosine did not induce nitrite production
in RINm5F and rat islet cells, either alone or together with the
phosphatase inhibitors okadaic acid and vanadate, which both increase
protein phosphorylation, ceramide generation alone does not appear to
be sufficient for induction of iNOS.
No strong or specific effects
were presently observed on AP-1 DNA binding activity. It should,
however, be pointed out that phosphorylation and transcriptional
activation of nuclear factors, such as c-Jun, do not necessarily lead
to increased binding to specific promoter elements in gel shift
experiments. Weak effects were also observed with the C/EBP element. To
the C/EBP element binds, for example, C/EBP, a transcriptional
activator present in differentiated cells, such as hepatocytes, and
involved in the acute-phase response and IL-1-induced gene
expression(48) . This transcription factor becomes activated by
the protein kinase C pathway, leading to enhancement of its
transcriptional efficacy(49) .
In summary, IL-1
stimulates the formation of ceramide and DAG in insulin-producing
cells. This event may contribute to the phosphorylation of JNK1 and the
transcription factors c-Jun and ATF2. A similar situation has recently
been observed in human promyelocytic cells (HL-60 cells), in which
sphingomyelinase activation and ceramide generation mediate tumor
necrosis factor-
-induced activation of JNK1 (50) .
Activation of this signaling pathway, however, does not lead to the
activation of NF-
B and is not sufficient for induction of nitric
oxide production in insulin-producing cells. Further studies are
warranted for the identification of additional transcription factors
that are activated or induced in response to IL-1
.