(Received for publication, October 30, 1995)
From the
Stimulation of pancreatic acini from male Sprague-Dawley rats by
both cholecystokinin (CCK)-8 and anisomycin caused an increase in
p46 and p55
activities.
Both forms of c-Jun amino-terminal kinase (JNK) were slightly activated
at 5 min, reached a maximum at 30 min, and remained significantly
increased at 60 min of CCK stimulation. By contrast,
p42
was activated fully by 5 min. In pancreatic
acini stimulated with different concentrations of CCK for 30 min, the
minimal and maximal JNK responses were observed at 30 pM and
100 nM CCK, respectively; p42
activation was, as previously reported, much more sensitive,
with maximal activation by 1 nM CCK. Carbachol and bombesin
also stimulated JNK activity, while vasoactive intestinal peptide did
not. Neither activating protein kinase C nor increasing intracellular
Ca
significantly activated JNK. In in vivo experiments, rats were infused intravenously for 5 and 15 min with
a secretory (0.1 µg/kg/h) or supramaximal (10 µg/kg/h) dose of
the CCK analog caerulein (CER). Secretory doses of CER induced a 4-fold
increase of both forms of JNK in pancreatic tissue at 5 and 15 min,
while at the same time points, supramaximal stimulation with CER caused
4- and 27-fold increases, respectively, of these kinase activities. The
secretory dose of CER slightly increased the activities of both forms
of mitogen-activated protein kinase, while the supramaximal dose
induced a 10-fold increase of p42
at 5 min. In
conclusion, JNKs and mitogen-activated protein kinases are rapidly
activated in rat pancreatic acini stimulated with CCK as well as in
pancreatic tissue during in vivo stimulation with CER. The
large response to supramaximal CER stimulation may be of importance in
the early pathogenesis of acute pancreatitis.
Mitogen-activated protein kinases (MAPKs), ()also
known as extracellular signal-regulated kinases, are serine/threonine
protein kinases that are rapidly activated by a variety of cell-surface
receptors(1, 2, 3) . They function in signal
cascade pathways that control the expression of genes involved in many
cellular processes, including cell growth and
differentiation(3, 4) . Blocking the function of MAPK
prevents cell proliferation in response to a number of
growth-stimulating agents(5, 6) .
Recently, a novel
signal cascade of mammalian enzymes closely related to that culminating
in MAPK activation has been identified. The kinases related to MAPKs
were identified by virtue of their ability to phosphorylate the amino
terminus of the c-Jun transcription factor, and they were therefore
termed c-Jun amino-terminal kinases
(JNKs)(7, 8, 9) . Two forms were identified
with molecular masses of 46 and 55 kDa, both of which are activated by
dual phosphorylation on threonine and tyrosine residues, similar to the
activation of MAPKs. JNKs can be potently activated by inhibitors of
protein synthesis such as cycloheximide and anisomycin, inflammatory
cytokines such as interleukin-1 and tumor necrosis factor , heat
shock, changes in osmolarity, and ultraviolet irradiation and are also
referred to as stress-activated protein
kinases(8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) .
JNKs are believed to be responsible for phosphorylating the
transactivating domain of c-Jun protein in
vivo(8, 9, 10) , which can then dimerize
or bind with fos as a heterodimer and can then control the
expression of a number of genes, including c-jun itself(4, 21, 22, 23) .
Cholecystokinin (CCK) regulates a variety of pancreatic functions,
including secretion of pancreatic juice(24) , stimulation of
pancreatic growth(25, 26) , digestive enzyme
synthesis(27) , and enhancement of expression of
transcriptional factors such as c-myc, c-jun, and
c-fos(28) . Stimulation with a supramaximal dose of
the CCK analog caerulein (CER) is known to induce acute pancreatitis in
the rat(29) . The trigger and exact mechanism of this
phenomenon are not completely understood. Recently, we found that CCK
activates p42 and p44
as well as other upstream components of the MAPK signaling
cascade, including Ras and MEK, in isolated rat pancreatic
acini(30, 31) . The aim of this study was therefore to
evaluate whether JNKs are activated in pancreatic acini as well as in
pancreatic tissue during in vivo stimulation with CER and to
compare their activation to that of MAPK.
Previously, we demonstrated that CCK rapidly activates MAPK
in rat pancreatic acini(30, 31) . Since anisomycin is
known as a strong activator of another member of the MAPK family, JNKs,
we decided to compare the effects of CCK and anisomycin in pancreatic
acini. Using an in-gel kinase assay, we found that both agents
significantly stimulated the activities of two kinases with apparent
molecular masses of 46 and 55 kDa, which were able to phosphorylate
GST-c-Jun-(1-79) (Fig. 1, left panel). The
response to 10 nM CCK was in the same range of magnitude as
observed after 30 min of stimulation with anisomycin (50 µg/ml).
The same samples were subjected to the in-gel assay with GST
copolymerized into the gel (Fig. 1, right panel). In
the positions corresponding to 46 and 55 kDa, only very faint bands
were occasionally visible, indicating slight phosphorylation of GST or
minimal autophosphorylation of the kinases. Because of the mass,
substrate specificity, and activation by anisomycin, these kinases can
be identified as p46 and p55
, respectively.
In-gel MAPK assay of the samples using myelin basic protein as
substrate showed strong activation of both forms of MAPK (p42
and p44
) after CCK stimulation, while anisomycin
had much less effect (data not shown).
Figure 1: Identification of JNKs in rat pancreatic acini by in-gel kinase assay. The extracts of pancreatic acini that had been treated for 30 min with 10 nM CCK-8 or 50 µg/ml anisomycin were subjected to SDS-polyacrylamide gel electrophoresis with GST-c-Jun-(1-79) (left panel) or GST (right panel) copolymerized into the gel. In the left panel, two major phosphorylated bands were visualized in the positions corresponding to 46 and 55 kDa after the kinase reaction. The density of these bands, an indicator of JNK activity, was significantly increased in extracts of cells treated with CCK-8 or anisomycin.
Fig. 2presents the
time course of CCK-induced activation of JNKs and MAPKs in pancreatic
acini. In the upper panel of Fig. 2A, the
radioactivity of the p46 and p55
bands is
shown. The integrated density of the bands was calculated and is
illustrated in the lower panel of Fig. 2A.
CCK-8 slowly increased the activities of both JNKs, reaching a maximum
at 30 min, when 2.5-fold increases of p46
and p55
activities were noted. The activity remained elevated at 45 min.
In another experiment, activity was similar at 30 and 60 min. The same
samples were subjected to in-gel MAPK assay. CCK-8 rapidly increased
the activities of both MAPKs, reaching a maximum within 5 min, when
7.5- and 6-fold increases of p44
and p42
activities, respectively, were noted over the activities at time
0 (Fig. 2B). The activities of both kinases remained
elevated at 45 min of CCK-8 stimulation.
Figure 2: Time course of CCK-induced activation of JNKs (A) and MAPKs (B) in rat pancreatic acini. Acini were incubated with or without 10 nM CCK-8 for the indicated times and then sonicated in lysis buffer and boiled for 5 min in stop solution. Samples were run separately on gels containing either GST-c-Jun-(1-79) or myelin basic protein, and in-gel kinase assays were performed in duplicate. The intensity of phosphorylation was measured with the use of a Model GS-250 molecular imager and is expressed as a percentage of the average value at time 0. The data presented are from one experiment that is representative of three different experiments.
Pancreatic acini were then
incubated for 30 min with different doses of CCK-8 (Fig. 3). The
minimal response of JNKs to CCK stimulation was observed at a 30 pM concentration of the hormone, while half-maximal and maximal
responses were observed at 3 and 100 nM, respectively (Fig. 3, A and C). The activities of both
kinases remained elevated at 1 µM CCK. In the same
samples, p42 and p44
activities were much
more elevated at 30 pM CCK-8 compared with the effect on JNKs,
reached a maximum at 1 nM, and remained at the similar level
of activation up to 1 µM CCK-8 (Fig. 3, B and C).
Figure 3:
Concentration-dependent effect of CCK-8 on
JNK (A and C) and MAPK (B and C)
activities in rat pancreatic acini. Acini were incubated with CCK-8 at
various concentrations for 30 min. Cell extracts were prepared as
described under ``Experimental Procedures'' and submitted to
in-gel kinase assays. A and B, representative
autoradiographs; C, diagram showing the CCK-stimulated
activity of p55 (
) and
p42
(
). The data are expressed as a
percentage of maximal activity stimulation. Each point in C represents the mean ± S.D. of four independent experiments,
each performed in duplicate.
It is known that CCK, after binding its
receptor, triggers hydrolysis of polyphosphoinositide, generating
inositol 1,4,5-triphosphate and diacylglycerol, which mobilize
intracellular calcium and activate protein kinase C,
respectively(35) . We investigated whether one of these signal
transduction pathways was responsible for activation of JNKs by
determining the effects of various agonists on these kinase activities
in comparison with the effect on the MAPK signaling pathway (Fig. 4). CCK, bombesin, and carbachol significantly stimulated
the activities of both JNKs (Fig. 4A). The efficacy of
CCK was 2 times higher than that of bombesin and carbachol.
12-O-Tetradecanoylphorbol-13-acetate, a potent stimulator of
protein kinase C, as well as the Ca
-ATPase inhibitor
cyclopiazonic acid had only a minimal effect on JNK activity, while
vasoactive intestinal peptide, which stimulates pancreatic acini via a
pathway related to cyclic AMP, had no effect. As shown before (30) , CCK, bombesin, carbachol, and
12-O-tetradecanoylphorbol-13-acetate significantly stimulated
MAPK activity in pancreatic acini, while the effect of cyclopiazonic
acid was much less. Incubation of acini with a combination of
cyclopiazonic acid and 12-O-tetradecanoylphorbol-13-acetate
resulted in additive effects on MAPK activity. Vasoactive intestinal
peptide had no effect on MAPK activity in pancreatic acini.
Figure 4: Effects of various agonists on JNK and MAPK activities in isolated rat pancreatic acini. Acini were stimulated for 30 min with the concentrations of the indicated agonists. The upper panels of A and B show representative autoradiographs. In the lower panels, the results are expressed as a percentage of control values, which were obtained in acini incubated without any stimulator for 30 min at 37 °C. The data are presented as the mean ± S.D. of eight experiments for CCK and three experiments for the other agonists, with each experiment performed in duplicate. TPA, 12-O-tetradecanoylphorbol-13-acetate; VIP, vasoactive intestinal peptide; CCh, carbachol; BBS, bombesin; CPA, cyclopiazonic acid.
To
examine the regulation of these kinases in the intact pancreas, we
examined the effect of in vivo stimulation with the CCK analog
CER on the activities of JNKs and MAPKs in pancreatic tissue (Fig. 5). Stimulation of rats with a secretory dose of CER (0.1
µg/kg) resulted in a significant 3-fold increase of both forms of
JNK activity as early as 5 min of intravenous infusion, while at 15
min, the activities of these kinases were slightly higher. In the same
animals, pancreatic p42 activity was increased 2-fold at
5 min, while at 15 min of stimulation, the levels returned to those
seen in control animals infused with 0.9% NaCl. Stimulation with a
supramaximal dose of CER (10 µg/kg), which is known to induce acute
pancreatitis, induced a 4-fold increase of both JNKs at 5 min, while 15
min of stimulation resulted in a 27-fold elevation of these kinase
activities. In the same animals, a 9-fold increase of p42
was observed in pancreatic tissue at 5 min, while at 15 min, it
was significantly decreased, but remained at 2.5 times above control
levels. It is noteworthy that basal levels of both JNKs and MAPKs
appear much lower in the intact pancreas versus dispersed
acini. Previously, we had found that basal levels of MAPK and JNK
declined after a 2-h preincubation in vitro, suggesting that
the preparation of acini increases the activities of both kinases from
those found in the in situ pancreas.
Figure 5: Effect of in vivo stimulation with CER on the activities of JNKs and MAPKs in rat pancreatic tissue. A and B show representative autoradiographs following infusion of saline (Control), 0.1 µg/kg/h CER (CER secr), or 10 µg/kg/h CER (CER supr). C shows the results expressed as a percentage of control values, which were obtained in rats intravenously injected with 0.9% NaCl and sacrificed after 5 or 15 min. The data for the 5-min groups of animals were obtained from two independent experiments (two rats each group), while the data for 15 min are presented as the mean ± S.D. of three independent experiments (two rats each group).
We recently reported that CCK activates p42 and p44
as well as other upstream components of
the MAPK signaling cascade, including Ras and MEK, in isolated rat
pancreatic acini(30, 31) . In the present study, we
have demonstrated for the first time that CCK activates two forms of
JNK (p46
and p55
) in isolated rat
pancreatic acini. Interestingly, the effectiveness of CCK was the same
or even higher than that of anisomycin, which is known as one of the
strongest JNK activators(2, 13) . In response to CCK,
both JNKs were slowly activated in pancreatic acini, with maximum
activation at 30 min, compared with p42
and
p44
, which reached a maximum at 5 min. Furthermore, the
minimal CCK concentration that activated JNKs was 10 times higher than
that which activated the MAPKs (p42
and
p44
) (30) . Correspondingly, a 100 times greater
CCK concentration was necessary to induce the maximal response of JNKs
in pancreatic acini when compared with MAPKs. Thus, CCK activation of
JNKs occurs slower and requires higher concentrations of CCK compared
with the activation of MAPKs.
Since JNKs are known to be activated
by a signal cascade distinct from that of MAPKs, it is not surprising
that there were some differences in the activation of JNKs and MAPKs in
acini in response to intracellular messengers. In acini, CCK,
carbachol, and bombesin are all known to interact with heterotrimeric G
proteins and thereby activate a phospholipase C that hydrolyzes
phosphatidylinositol bisphosphate, generating inositol
1,4,5-triphosphate and diacylglycerol, which mobilize intracellular
Ca and activate protein kinase C, respectively.
Increasing intracellular Ca
with cyclopiazonic acid
failed to activate either JNKs or MAPKs. Activation of protein kinase C
with active phorbol ester, which is known to stimulate MAPK activity in
acini(30) , had its expected effect, but showed a minimal
effect on JNKs. Although CCK stimulates cAMP formation in acini at high
concentrations, this does not appear to be important in activating JNKs
as vasoactive intestinal peptide, which potently increases cAMP, had no
effect. Thus, JNK activation cannot be explained by the intracellular
mediators known to activate digestive enzyme secretion. Since CCK,
carbachol, and bombesin all activate a variety of heterotrimeric G
proteins, it seems likely that activation of the JNK cascade involves G
proteins and diverges at that level from pathways activating enzyme
secretion. It was recently reported that stimulation with carbachol
activates JNKs and MAPKs in NIH 3T3 cells expressing muscarinic
acetylcholine receptor(36) , which led the authors to suggest
the existence of pathways leading to the activation of either MAPK or
JNK. Recently, two independent groups have found that the monomeric
GTP-binding proteins Rac and Cdc42 regulate the activity of the cascade
leading to JNK activation(37, 38) . The authors of
these studies suggested that G protein- and tyrosine kinase-linked
receptors may activate Rac and Cdc42 directly or through activation of
Ras. This then results in activation of a kinase cascade with MEK
kinase-phosphorylating and -activating JNK kinase, which dually
phosphorylates
JNK(39, 40, 41, 42, 43) .
Since MEK kinase and JNK kinase are expressed in most tissues, it seems
likely that such a pathway occurs in acinar cells. An uncertainty in
all types of cells is how MEK kinase is activated. Thus, how JNKs are
activated in pancreatic acini remains to be determined, but our data
clearly support the existence of a pathway distinct from that
activating the MAPKs.
In the in vivo part of our study,
rats were stimulated intravenously with different doses of the CCK
analog CER for 5 or 15 min. Stimulation with the lower dose of CER,
known to stimulate pancreatic secretion, produced a mild increase of
JNK activity in pancreatic tissue at 5 and 15 min. Under the same
conditions, p42 and p44
were only
transiently activated at 5 min. These data suggest that both MAPKs and
JNKs may be activated physiologically by CCK. Stimulation with a
supramaximal dose of CER, known to induce acute pancreatitis, produced
significant increases of p42
and p44
at 5
min of stimulation. This increased activity of both MAPKs was transient
in that they were markedly decreased at 15 min. In the same group of
animals, JNK activity was moderately elevated at 5 min and dramatically
increased (27-fold) following 15 min of hyperstimulation. In our study,
hyperstimulation both in vitro and in vivo was
accompanied by significant activation of JNKs that may reflect the
response to cellular stress. It is noteworthy that after CCK or CER
stimulation in vitro as well as in vivo, the
activation of p42
and p44
preceded the
activation of JNKs. A similar phenomenom, in response to cellular
stress, was recently observed by others in a rat model of kidney
ischemia-reperfusion(44) . These authors suggested that strong
activation of the stress-activated protein kinase family of
serine/threonine kinases very early after reperfusion of ischemic
kidney may transduce an important signal to the nucleus and trigger the
complex genetic response to ischemia. An important role of oxygen
radicals is suggested in the early pathogenesis of both experimental
models: CER-induced acute pancreatitis and organ ischemia-reperfusion (45) . Since it is also known that reactive oxygen species are
capable of different protein kinase
activation(46, 47) , it is possible that they may be
partially responsible for the activation of MAPKs and JNKs in these
experimental models.
Taken together, CCK stimulation of dispersed acini or the intact pancreas stimulates two separate signaling cascades leading to activation of JNKs and MAPKs. The extensive activation of both MAPKs and JNKs during supramaximal in vivo stimulation with CER may be of importance in the early pathogenesis of acute pancreatitis and/or may reflect a universal reaction of the organism responding to cellular stress.