Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0682
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
c-Jun
NH2-terminal kinases (JNKs) are
protein kinases that are activated by a wide variety of extracellular
signals. This study investigated the expression and regulation of JNKs
in isolated gastric canine parietal cells. Western blot analysis of
cell lysates from highly purified (>95%) parietal cells with an
antibody recognizing JNK1 and to a lesser degree JNK2 revealed the
presence of two bands of 46 and 54 kDa, respectively. JNK1 activity was
quantitated by immunoprecipitation and in-gel kinase assays. Of the
different agents tested, carbachol was the most potent inducer of JNK1
activity, whereas histamine and epidermal growth factor induced weaker
responses. The proinflammatory cytokine tumor necrosis factor-
stimulated JNK1 but had no effect on extracellular signal-regulated
kinase (ERK2) induction, suggesting that activation of JNK1 might
represent an important event in mediation of the inflammatory response
in the stomach. The action of carbachol was dose (0.1-100 µM)
and time dependent, with a maximal stimulatory effect (fourfold)
detected after 30 min of incubation and sustained for 2 h. Addition of the specific protein kinase C (PKC) inhibitor GF109203X did not affect
the stimulatory action of carbachol. The intracellular Ca2+ chelator
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM inhibited carbachol induction of JNK1 activity by 60%. Thapsigargin (1 µM), an intracellular
Ca2+-rising agent, induced JNK1
activity more than threefold. Carbachol activation of JNK1 resulted in
induction of c-Jun (protein) transcriptional activity and in
stimulation of parietal cell mRNA content of
c-jun. In conclusion, our data
indicate that carbachol induces JNK activity in gastric parietal cells
via intracellular Ca2+-dependent,
PKC-independent pathways, leading to induction of c-jun gene expression via
phosphorylation and transcriptional activation of c-Jun.
early response genes; protein kinases; transcriptional regulation; extracellular signal-regulated kinase
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE GASTRIC PARIETAL CELL is a complex biological structure whose behavior is regulated by a broad variety of extracellular signals that interact with specific receptors present on the cell surface to initiate a flow of information that moves to the cell nucleus along highly organized and complex signal transduction pathways (19). Once in the nucleus, these signals are known to activate specific programs of transcriptional events, which lead in the end to the expression of specialized cellular functions (19). Recent studies have indicated that parietal cells express members of a recently discovered family of protein kinases known as mitogen-activated protein kinases (MAPKs) or extracellular signal-regulated protein kinases (ERKs) (8, 27, 31). The ERKs are important elements in a signaling cascade known to involve upstream protein kinases such as Raf and MAPK/ERK kinase (MEK) (9, 11, 13, 22). Activation of the ERKs is known to target numerous cellular proteins, including downstream protein kinases such as 90-kDa S6 kinase (RSK) (9, 13) and transcription factors such as Elk-1 that regulate the activity of the promoter of the early response gene c-fos (20, 23, 35). Thus the ERKs, via activation of immediate-early gene function and of downstream protein kinases, appear to play a crucial role in the process of amplification, integration, and transmission of the extracellular signals from the cell surface to the nucleus, leading to induction of cellular growth and proliferation and, in some systems, cellular differentiation (9, 13, 19, 24, 25).
Our understanding of the function and physiological role of the ERK pathway in the stomach has been improved significantly by the recent development of PD98059, a highly specific inhibitor of the ERK activator MEK (1). With the use of this compound, we were able to demonstrate that although the acute effect of the ERKs on gastric acid secretion appears to be inhibitory, activation of transcription factors and of early gene expression could be responsible for its chronic stimulatory effects (31).
Recently, other members of the ERK family of protein kinases, c-Jun NH2-terminal kinases 1 and 2 (JNK1 and JNK2), have been cloned and characterized (14, 20). These enzymes are members of a complex signaling cascade that is separate from that leading to activation of the ERKs. In response to a broad variety of extracellular signals, the serine-threonine protein kinase MAPK/ERK kinase kinase 1 (MEKK1) is activated, leading to the phosphorylation of the c-Jun NH2-terminal kinase kinase 1 (JNKK1), a dual-specificity kinase that activates and phosphorylates the JNKs. The JNKs in turn appear to play an important role in the phosphorylation and transcriptional activation of nuclear proteins such as c-Jun (protein), Elk-1, and activating transcription factor II (14, 20). The JNKs are involved in numerous cellular functions such as regulation of cell growth and response to environmental stress (14, 20). These enzymes are activated by cytokines, hormones, and neurotransmitters in a broad variety of physiological systems. To date little is known about the function of these enzymes in the gut. Thus we undertook these studies to investigate the regulation of the JNKs in the parietal cells. Using highly purified parietal cells in primary culture we were able to demonstrate that carbachol induces JNK activity via intracellular Ca2+ concentration ([Ca2+]i)-dependent, protein kinase C (PKC)-independent pathways, leading to induction of c-jun gene expression via phosphorylation and transcriptional activation of c-Jun.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasmids.
Gal4-c-Jun and 5×Gal-Luc (18) were gifts from M. Karin (San
Diego, CA), pCMV-Gal was a gift from M. Uhler (Ann Arbor, MI), and
pGEX-KG-c-Jun was a gift from J. Dixon (Ann Arbor, MI). pGEX-KG-c-Jun was constructed by cloning amino acids 1
79 of c-Jun into pGEX-KG (17)
to generate GST-c-Jun.
Primary parietal cell preparation and culture.
For preparation of primary parietal cells we used the method of Soll
(30), with modifications (4, 5, 31, 32). The mucosal layer of freshly
obtained canine gastric fundus was bluntly separated from the submucosa
and rinsed in Hanks' balanced salt solution containing 0.1% BSA. The
cells were then dispersed by sequential exposure to collagenase (0.35 µg/ml) and 1 mM EDTA, and parietal cells were enriched by centrifugal
elutriation using a Beckman JE-6B elutriation rotor. Our best
preparations contained 70% parietal cells as determined by hematoxylin
and eosin and periodic acid-Shiff reagent staining. The parietal cells
were further purified by centrifugation through density gradients
generated by 50% Percoll (Pharmacia Biotech, Piscataway, NJ) at 30,000 g for 20 min. The cell fraction at = 1.05 consisted of 95%-100% parietal cells. The isolated
parietal cells (0.8 × 106
cells/well) were cultured for 16 h according to the method of Chew et
al. (7) with some modifications (26, 31). Briefly, the cells were
cultured in Ham's F-12 medium-DMEM (1:1) containing 50 µg/ml
gentamycin, 50 U/ml penicillin G, and 2% DMSO (Sigma) on 6-well
culture dishes (Costar, Cambridge, MA) coated with 150 µl of
H2O-diluted (1:5) growth
factor-reduced Matrigel (Becton Dickinson, Bedford, MA). For our
studies, the parietal cells were incubated with carbachol (Sigma) (0.1 µM-100 µM), histamine (Sigma) (0.1 µM-100 µM),
epidermal growth factor (EGF; 0.1-100 nM; Becton Dickinson), tumor
necrosis factor-
(TNF-
; 5-50 ng/ml; Sigma), thapsigargin (1 µM; Calbiochem, La Jolla, CA), and
12-O-tetradecanoylphorbol-13-acetate (TPA; 0.1-100 nM;
Sigma) for various time periods. In some experiments, either
bisindolylmaleimide I (GF109203X; 5 µM; Calbiochem) or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid (BAPTA)-AM (100 µM; Calbiochem) was added before the addition of
the secretagogues. BAPTA-AM, TPA, and GF109203X were dissolved in DMSO.
All other test substances were dissolved in culture medium. Control
experiments with untreated cells were performed by incubating the cells
in either vehicle (0.1% DMSO) or incubation buffer without the test substances.
Transfection of primary cultured parietal cells.
Before transfection the cells were washed once with 1 ml of Opti-MEM I
serum-reduced media (GIBCO BRL) and fed with 400 µl of Opti-MEM I
medium supplemented with 2% DMSO. The cells were transfected with 5 µg of the luciferase reporter plasmids and 0.5 µg of the expression
vectors. Transfections were carried out using Lipofectin (GIBCO BRL) as
previously described (26, 31). The day after transfection the medium
was removed and the cells were fed with serum-free medium for 24 h,
then incubated for 5 h with the test substances. At the end of the
incubation period, the cells were washed twice with cold
Ca2+-free PBS and then 100 µl of
cell lysis buffer (25 mM Tris · HCl, pH 7.8, 2 mM
EDTA, 2 mM
1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10% glycerol, and 1% Triton X-100) were added and incubated at
room temperature for 15 min. The cells were then scraped and transferred to Eppendorf tubes. After quick centrifugation to pellet
large debris, the supernatant was transferred to a new tube. An aliquot
of cell lysate (20 µl) was mixed with 100 µl luciferase assay
reagent [20 mM tricine, 1.07 mM
(MgCO3)4Mg(OH)2 · 5 H2O, 2.67 mM
MgSO4, 0.1 mM EDTA, 33.3 mM
dithiothreitol (DTT), 270 µM coenzyme A, 470 µM luciferin, and 530 µM ATP, final pH 7.8], and luminescent intensity was measured
for 10 s, using a Lumat LB9501 luminometer (Berthold, Germany).
Luciferase activity was expressed as relative light units and
normalized for -galactosidase activity.
-Galactosidase activity
was measured by the luminescent light derived from 10 µl of each
sample incubated in 100 µl of Lumi-Gal 530 (Lumigen, Southfield, MI)
and used to correct the luciferase assay data for transfection
efficiency.
Solid-state c-Jun kinase (JNK) assay.
These experiments were performed according to previously described
methods (33, 36, 37). Briefly, equal aliquots of lysates from parietal
cells treated with and without 100 µM carbachol were incubated with
20 µl of glutathione-agarose suspensions (Sigma) to which 20 µg of
either glutathione S-transferase
(GST)- or GST-c-Jun were bound for 3 h at 4°C. GST-c-Jun fusion
proteins were generated as previously described (17). The mixtures were
pelleted by centrifugation and washed in HEPES binding buffer (20 mM
HEPES at pH 7.7, 50 mM NaCl, 2.5 mM
MgCl2, 0.1 mM EDTA, and 0.05%
Triton X-100). The pelleted beads were resuspended in 30 µl of kinase buffer (20 mM HEPES at pH 7.6, 20 mM
MgCl2, 20 mM -glycerophosphate, 0.1 mM
Na3VO4,
20 mM p-nitrophenylphosphate, 2 mM
DTT, 20 µM ATP, and 5 µCi of
[
32P]ATP), and
after 20 min at 30°C the reaction was terminated by washing with
HEPES binding buffer. Phosphorylated proteins were eluted by boiling in
20 µl of electrophoresis buffer and applied to a 10%
SDS-polyacrylamide gel, followed by staining with Coomassie blue,
destaining, and autoradiography (37).
Immunoprecipitations and in-gel JNK1 and ERK2 assays.
Immunoprecipitations and in-gel JNK1 and ERK2 assays were performed
according to previously described techniques (12, 29, 31, 33, 36) with
minor modifications. The parietal cells were lysed in 500 µl of lysis
buffer [50 mM HEPES, 150 mM NaCl, 10% glycerol, 1% Triton
X-100, 1 mM EDTA, 1.5 mM MgCl2, 1 mM
Na3VO4, 10 mM NaF, 10 mM
Na4P2O7 · 10 H2O, 1 mM
4-(2-aminoethyl)benzenesulfonylfluoride hydrochlorine (ICN-Biomedicals,
Aurora, OH), 1 µg/ml leupeptin, and 1 µg/ml aprotinin]. The
lysates were transferred into Microfuge tubes and spun at 16,000 g for 20 min at 4°C. Equal amounts
of protein from each treatment group (1,000 g) were incubated with either an
anti-JNK1 or an anti-ERK2-specific antibody (Santa Cruz Biotechnology,
Santa Cruz, CA) and mixed on a rotating platform for 3 h at 4°C.
Protein concentrations were measured by the Bradford method (3).
Aliquots of protein A Sepharose (50 µl) (Pharmacia Biotech,
Piscataway, NJ) were then added, and the solutions were mixed for 1 additional hour. After centrifugation the pellets were washed four
times with lysis buffer. The samples were resuspended in 20 µl of
electrophoresis buffer (for 10 ml: 1 ml glycerol, 0.5 ml
2-mercaptoethanol, 3 ml 10% SDS, 1.25 ml 1 M Tris buffer, 2 ml 0.1%
bromphenol blue, 0.6 g urea), boiled for 5 min, and applied to a 10%
SDS-polyacrylamide gel containing either 0.5 mg/ml myelin basic protein
(MBP; Sigma) or 0.1 mg/ml GST-c-Jun-(179). GST-c-Jun was eluted from
the glutathione agarose beads as previously described (17). After
electrophoresis, the gels were washed with two changes of 20%
2-propanol in 50 mM Tris (pH 8.0) for 1 h and then with two changes of
50 mM Tris (pH 8.0) containing 5 mM 2-mercaptoethanol for 1 h. The
enzymes were denatured by incubating the gels with two changes of 6 M
guanidine-HCl for 1 h and then renaturated with five changes of 50 mM
Tris (pH 8.0) containing 0.04% Tween 40 and 5 mM 2-mercaptoethanol for
1 h. The kinase reactions were performed in conditions inhibitory to cyclic nucleotide-dependent protein kinase and
Ca2+-dependent protein kinases by
incubating the gels at 25°C for 1 h with 40 mM HEPES (pH 8.0)
containing 0.5 mM EGTA, 10 mM
MgCl2, 2 µM cAMP-dependent
protein kinase inhibitor peptide (Sigma), 20 µM ATP, and 2.5 µCi/ml
of [
32P]ATP (6,000 Ci/mmol; Amersham, Arlington Heights, IL). After incubation, the gels
were washed with a 5% (wt/vol) trichloroacetic acid solution
containing 1% (wt/vol) sodium pyrophosphate, dried, and subjected to
autoradiography.
Northern blot analysis. After 30 min of incubation with the test substances, the parietal cells were lysed with TRIzol (GIBCO BRL, Grand Island, NY) according to the manufacturer's instructions. Northern blot hybridization assays were performed as previously described (33). Equal amounts of each RNA sample, with ethidium bromide (10 mg/ml) in a final volume of 20 µl, were electrophoresed on a 1.25% agarose gel containing formaldehyde, and the RNA was transferred from the gel to nitrocellulose filters. The ethidium-stained ribosomal RNA bands in the gel were photographed before and after transfer to ensure that equivalent amounts of RNA were loaded onto each lane and that no residual RNA was left on the gel. The human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe was obtained from Clontech (Palo Alto, CA). The cDNA was labeled with [32P]dCTP by the random-priming procedure, and the nitrocellulose filters were hybridized to the 32P-labeled cDNA probes as previously described (33). For analysis of c-jun mRNA we used a 40-base single-stranded c-jun synthetic oligonucleotide probe obtained from Calbiochem (Cambridge, MA). Labeling and hybridizations were carried out according to the manufacturer's instructions.
Western blots. Parietal cell lysates (40 µl) were loaded on a 10% SDS-polyacrylamide mini-gel and run at 20 A for 8 h. The gel was transferred on an Immobilon-P transfer membrane (Millipore, Bedford, MA) in 25 mM Tris, 150 mM glycine, and 20% methanol. After transfer the membrane was blocked in 10 ml of TBST (20 mM Tris, 0.15 M NaCl, 0.3% Tween) and 5% dry milk for 2 h and then incubated for 1 h at 37°C in 10 ml of TBST and 5% dry milk containing 10 µg of a specific anti-JNK1 antibody (Santa Cruz Biotechnology). At the end of the incubation period the membrane was washed in TBST for 30 min at room temperature and then incubated for 1 h in TBST and 5% dry milk containing protein A directly conjugated to horseradish peroxidase (HRP) (1:2,500) (Amersham Life Science, Arlington Heights, IL). The membrane was washed in TBST for 30 min at room temperature and then exposed to the Amersham enhanced chemiluminescence (ECL) detection system according to the manufacturer's instructions.
Data analysis. Data are given as means ± SE; n is equal to the number of separate dog preparations from which the parietal cells were obtained. Statistical analysis was performed using Student's t-test. P values <0.05 were considered to be significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We examined the expression of JNKs in gastric parietal cells. As shown in the Western blot in Fig. 1, the parietal cells express two major isoforms of JNKs of 46 and 54 kDa, corresponding to JNK1 and JNK2, respectively.
|
We then demonstrated, using solid-state kinase assays, that carbachol
(100 µM) activates JNK activities that specifically phosphorylate
c-Jun-(179) without having any effect on GST (Fig. 2A). The
Coomassie blue stain of the gel, shown in Fig.
2B, demonstrates that identical
amounts of protein were loaded on the gel.
|
To study more precisely the regulation of JNK1 in the stomach, we
performed immunoprecipitations and in-gel JNK1 assays. The specificity
of the assay was confirmed by using both nonimmune serum and an
antibody known to immunoprecipitate ERK2. As shown in Fig.
3, only the JNK1 antibody
immunoprecipitated a 46-kDa enzyme capable of phosphorylating
c-Jun-(179), whereas no effect was observed in the presence of either
the nonimmune serum or the anti-ERK2 antibody.
|
We studied the dose-response effect of carbachol, EGF, TNF-, and
histamine on JNK1 activity. Carbachol (0.1-100 µM)
dose-dependently induced JNK1 activity, with a maximal effect detected
between 10 and 100 µM (Fig. 4).
Similarly, EGF, TNF-
, and histamine activated JNK1 in a
dose-dependent manner, with maximal stimulatory effects detected at
doses of 10 nM, 10 ng/ml and 100 µM, respectively (data not shown).
|
We then investigated the kinetics of JNK1 induction in response to
TNF-, carbachol, EGF, and histamine stimulation. As shown in Fig.
5A,
TNF-
induction of JNK1 was modest and of short duration (1.23 ± 0.25-, 2.4 ± 0.25-, 1.14 ± 0.33-, and 0.81 ± 0.18-fold induction over control, n = 4, after
5, 30, 60, and 120 min of incubation with 10 ng/ml TNF-
,
respectively). In contrast, carbachol induced a strong and sustained
induction of JNK1 activity [1.6 ± 0.36- (n
= 4), 5.03 ± 1.08- (n = 4), 4.33 ± 0.97- (n = 5), and 4.13 ± 1.18-fold
(n = 4) induction over control, after
5, 30, 60, and 120 min of incubation with 100 µM carbachol,
respectively] (Fig. 5B). EGF
(10 nM) rapidly activated JNK1, with a maximal stimulatory effect
detected after 5 min of incubation [3.28 ± 0.98- (n = 6), 2.62 ± 0.56- (n = 5), 1.95 ± 0.14- (n = 4), and 1.6 ± 0.15-fold
(n = 4) induction over control, after
5, 30, 60, and 120 min of incubation with 10 nM EGF,
respectively] (Fig. 6A),
whereas histamine exhibited a slower and more sustained effect (1.21 ± 0.3-, 2.41 ± 0.21-, 3.38 ± 0.73-, and 2.92 ± 0.77-fold induction over control, n = 3, after 5, 30, 60, and 120 min of incubation with 100 µM histamine,
respectively) (Fig. 6B).
|
|
PKC is known to mediate numerous physiological functions of gastric parietal cells (15). Thus we undertook studies to examine the role of PKC in carbachol induction of JNK1. TPA (0.1-100 nM) induced JNK1 activity in a dose-dependent manner, with a maximal effect detected at the dose of 100 nM (5.69 ± 2-fold induction over control, n = 6, after 30 min of incubation with 100 nM TPA; data not shown), and this effect was inhibited, although not completely, by preincubation of the cells for 30 min with the selective PKC inhibitor bisindolylmaleimide I (GF109203X; 5.5 µM) (48.17 ± 7.79% of the effect observed in the presence of TPA alone, n = 4). In contrast, carbachol induction of JNK1 was unaffected by GF109203X (104.12 ± 22.7% of the effect observed in the presence of carbachol alone), suggesting the involvement of PKC-independent pathways (Fig. 7). Vehicle (0.1% DMSO) had no effect on JNK1 activity (1.45 ± 0.4-fold induction over control, n = 4) (data not shown).
|
Because Ca2+ is an important mediator of carbachol actions on gastric parietal cells (15, 16), we undertook studies to examine the role of both intra- and extracellular Ca2+ on carbachol induction of JNK1 activity. As shown in Fig. 8, chelation of intracellular Ca2+ by preincubation of the cells for 10 min with the cell-permeable Ca2+ chelator BAPTA-AM (100 µM) significantly inhibited carbachol induction of JNK1 (35.14 ± 6.062% of the response observed in the presence of carbachol alone, n = 4). Treatment of the cells for 30 min with thapsigargin (1 µM), an agent known to release Ca2+ from intracellular stores, induced JNK activity almost fourfold (3.81 ± 0.95-fold induction over control; n = 3) (Fig. 8). In contrast, deprivation of extracellular Ca2+ by incubation of the cells in Ca2+-free Earle's balanced salt solution containing 1 mM EGTA failed to affect the stimulatory action of carbachol on JNK1 activity (4.94 ± 0.1 vs. 6.32 ± 1.9-fold induction over control in absence and presence of Ca2+-free medium, respectively, n = 3) (Fig. 8). Thus carbachol induces JNK1 activation via mobilization of [Ca2+]i through signaling pathways that are not affected by extracellular Ca2+ levels.
|
We then examined whether JNK1 is a signaling pathway specifically
activated by inflammatory cytokines. Accordingly, we examined the
effect of TNF- on ERK2 activation. As shown in Fig.
9, whereas carbachol potently induced ERK2
activity after 5 min of incubation, TNF-
had no effect, suggesting
that JNK1 but not ERK2 is involved in TNF-
signaling. Identical
results were obtained when the cells were incubated for 30 min in the
presence of either carbachol or TNF-
(data not shown).
|
To establish the functional significance of our observations, we sought
to investigate the effects of carbachol on
c-jun transcriptional activation. For
these experiments we used a yeast hybrid system involving
cotransfection of the parietal cells with the
Gal4-c-jun and the pCMV-Gal
expression vectors and the 5×Gal luciferase reporter plasmids. In
this system Gal4-c-jun transactivates
and stimulates luciferase activity only if the
c-jun
NH2 terminus is phosphorylated by
JNK. As shown in Fig. 10, carbachol (10 µM) stimulated a twofold increase in luciferase activity (2.35 ± 0.35-fold induction over control, n = 8).
|
Finally, we examined the effect of carbachol on the induction of the c-jun gene. As shown in Fig. 11, carbachol potently induced c-jun gene expression, whereas no effect was observed when the mRNA was hybridized with a probe encoding GAPDH. Taken together, these data indicate that carbachol activates a cascade of phosphorylation reactions that targets the JNKs, leading to induction of early gene expression via phosphorylation and transcriptional activation of c-jun.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The JNKs are members of the MAPK family of protein kinases, enzymes
known to play an important role in the process of integration and
transmission of the extracellular signals from the cell surface to the
nucleus (11, 19, 22, 24, 25, 28). Each group of MAPKs appears to be
activated by a relatively specific set of extracellular signals. In
fact, whereas the ERKs are potently stimulated by growth factors, the
JNKs are activated by treatment of cells with ultraviolet radiation,
inflammatory cytokines, and environmental stress (9, 13, 14, 20).
Prolonged activation of the JNKs appears to be, at least in some
systems, an important signaling pathway for the induction of programmed
cell death (6). In the gastrointestinal tract, the JNKs are involved in
the process of hepatic regeneration after partial hepatectomy (36) and
in the mediation of some of the intracellular events that are activated during the onset of acute pancreatitis (12). In our study, using a
highly specific assay, we studied both the activation and the kinetics
of JNK1 induction in response to carbachol, TNF-, EGF, and
histamine, agents known to exert a wide variety of physiological actions in the stomach. Of the different agents tested, carbachol and
histamine were the most potent inducers of JNK1 activation, whereas
TNF-
and EGF resulted in weaker stimulatory effects. Interestingly,
carbachol and EGF were also potent inducers of the ERKs in the gastric
parietal cells (31), indicating that these agents stimulate both ERKs
and JNKs, in contrast to histamine and TNF-
, which had no effect on
ERK2 induction (31). Because both histamine and TNF-
are agents
involved in the inflammatory response, it is possible that activation
of JNK1 might represent an important and specific event in the
induction of proinflammatory pathways in the stomach. More broadly,
activation of JNKs by carbachol, TNF-
, EGF, and histamine might
represent an important signaling system mediating biological processes
such as programmed cell death, induction of genes involved in the
inflammatory response, or activation of cellular repair and
proliferation. It is interesting to speculate that the different
patterns of JNK activation observed in response to carbachol, TNF-
,
EGF, and histamine might be responsible for the induction of different
programs of cellular responses. Further studies are needed to elucidate
some of the downstream targets of the JNKs in the gastric parietal
cells.
Because of the lack of specific inhibitors, we were unable to link the activation of JNK1 to a specific cellular response. However, because carbachol and histamine are potent gastric acid secretagogues, it is possible that JNK1 activation could also play a role in the regulation of gastric acid secretion.
In our study, carbachol was the most potent inducer of JNK1 activation. Accordingly, we decided to focus our efforts primarily on understanding the intracellular pathways that target JNK1 in response to carbachol and to analyze the functional relevance of this phenomenon. In the stomach, carbachol is known to interact with specific M3 muscarinic receptors present on the surface of the parietal cells leading to mobilization of both intracellular and extracellular Ca2+ and to the activation of PKC (15). Thus we investigated the role of PKC in carbachol induction of JNK1 and noted that this agent potently induced JNK1 via PKC-independent pathways. This finding is consistent with previous observations indicating that carbachol induces JNK in NIH/3T3 cells stably transfected with M1 muscarinic receptors via PKC-independent pathways (10). In addition, other agents such as angiotensin II and endothelin I, known to activate PKC via interaction with seven transmembrane, G protein-linked receptors, are able to induce the JNKs via PKC-independent pathways in GN4 rat liver epithelial cells and in rat glomerular mesangial cells, respectively (2, 37). The complexity of this finding is further underscored by the observation that, in contrast to the GN4 cells, angiotensin II induces the JNKs via PKC-dependent pathways in neonatal cardiac myocytes (21), suggesting the presence of cell type-specific differences in the requirement of PKC for activation of the JNKs. In our study we observed that TPA induced JNK1 activity. Thus it is possible that in the parietal cells there are PKC-dependent pathways for JNK1 induction but that carbachol might use PKC-independent pathways for its activation. Alternatively, this observation might reflect differences in the degree of PKC induction between carbachol and TPA in the gastric parietal cells. It is possible, in fact, that induction of JNK1 by PKC might require a strong and sustained activation of this kinase that could be achieved only by stimulation of the cells with a maximal dose of phorbol esters and that the degree of PKC activation achieved in the presence of carbachol could be insufficient for induction of JNK1. Indeed, in our system, we noted that TPA (100 nM) caused a greater and more sustained induction of PKC than carbachol (100 µM), whereas a lower dose of TPA (1 nM), able to activate PKC to a similar degree as carbachol, could not induce JNK1 (data not shown). These observations suggest that a threshold level of PKC activation might be required for induction of the JNKs. To study the role of PKC in carbachol induction of JNK1, we used the highly specific PKC inhibitor GF109203X. As previously discussed, this compound did not influence the action of carbachol on JNK1 activity, whereas it inhibited by 50% the stimulatory effect observed in response to TPA. The lack of complete inhibition of TPA stimulation of JNK1 by GF109203X might reflect the relatively low potency of this compound in the presence of the high concentrations of ATP present in the gastric parietal cells (34).
We next investigated the role of Ca2+ signaling in carbachol induction of JNK1. In our study we noted that carbachol targets JNK1 via signaling pathways that require mobilization of intracellular Ca2+ but that are unaffected by changes in extracellular Ca2+ levels. In contrast to this finding, the effect of carbachol on ERK2 induction in the parietal cells appeared to be completely independent from Ca2+ signaling. Thus mobilization of intracellular Ca2+ appears to be a specific signaling event for activation of JNK1 but not of ERK2 in response to carbachol stimulation. Previous reports have shown that Ca2+ signaling is an important intracellular mediator for the activation of JNK in several cell types (2, 21, 38). However, there are mechanistic variations for Ca2+ signaling among different cell types. For example, in the GN4 cells activation of JNK is inhibited by both intra- and extracellular Ca2+ chelation (38), whereas in neonatal cardiac myocytes induction of JNK by angiotensin II requires only mobilization of intracellular Ca2+ without being affected by removal of Ca2+ from the extracellular medium (21). This latter observation shows clear similarities to what we observed in the gastric parietal cells.
We finally demonstrated that, in the parietal cells, induction of JNK1 by carbachol leads to both c-Jun transcriptional activation and stimulation of c-jun gene expression. Thus carbachol activates a complex chain of phosphorylation reactions that in the end targets the JNKs and c-jun. This event in turn could be responsible for the induction of numerous programs of transcriptional activation leading to the expression of highly specialized cellular functions such as regulation of growth or response to environmental stress and inflammation.
In conclusion, our data indicate that carbachol induces JNK activity in gastric parietal cells via [Ca2+]i-dependent, PKC-independent pathways, leading to induction of c-jun gene expression via phosphorylation and transcriptional activation of c-Jun.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. John Williams for helpful advice and Saravanan Ramamoorthy, Kristina Tacey, and Bill Malone for technical assistance.
![]() |
FOOTNOTES |
---|
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1-DK-33500, RO1-DK-47434, and RO1-DK-34306 and by funds from the University of Michigan Gastrointestinal Peptide Research Center (NIH Grant P30-DK-34933). Dr. Todisco is a recipient of an American Gastroenterological Association Industry Research Scholar Award, a Clinical Investigator Award from the National Institutes of Health (NIH grant K08-DK-02336), and a grant from the Charles E. Culpeper Foundation Health Program.
Address for reprint requests: A. Todisco, 6520 MSRB I, University of Michigan Medical Center, Ann Arbor, MI 48109-0682.
Received 20 October 1997; accepted in final form 18 June 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alessi, D. R.,
A. Cuenda,
P. Cohen,
D. T. Dudley,
and
A. Saltiel.
PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo.
J. Biol. Chem.
270:
27489-27494,
1995
2.
Araki, S.,
M. Haneda,
M. Togawa,
and
R. Kikkawa.
Endothelin-1 activates c-Jun NH2-terminal kinase in mesangial cells.
Kidney Int.
51:
631-639,
1997[Medline].
3.
Bradford, M. M.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:
248-254,
1983.
4.
Campbell, V. W.,
J. Del Valle,
M. Hawn,
J. Park,
and
T. Yamada.
Carbonic anhydrase II gene expression in isolated canine gastric parietal cells.
Am. J. Physiol.
256 (Gastrointest. Liver Physiol. 19):
G631-G636,
1989
5.
Campbell, V. W.,
and
T. Yamada.
Acid secretagogue-induced stimulation of gastric parietal cell gene expression.
J. Biol. Chem.
264:
11381-11386,
1989
6.
Chen, Y.,
X. Wang,
D. Templeton,
R. J. Davis,
and
T. H. Tan.
The role of c-Jun N-terminal kinase in apoptosis induced by ultraviolet C and radiation.
J. Biol. Chem.
271:
31929-31936,
1996
7.
Chew, C. S.,
M. Ljungstroem,
A. Smolka,
and
M. R. Brown.
Primary culture of secretagogue-responsive parietal cells from rabbit gastric mucosa.
Am. J. Physiol.
256 (Gastrointest. Liver Physiol. 19):
G254-G263,
1989
8.
Chew, C. S.,
K. Nakamura,
and
A. C. Petropoulos.
Multiple actions of epidermal growth factor and TGF- on rabbit gastric parietal cell function.
Am. J. Physiol.
267 (Gastrointest. Liver Physiol. 30):
G818-G826,
1994
9.
Cobb, M. H.,
and
E. J. Goldsmith.
How MAP kinases are regulated.
J. Biol. Chem.
270:
14843-14846,
1995
10.
Coso, O.,
M. Chiarello,
G. Kalinec,
J. M. Kyriakis,
J. Woodgett,
and
J. S. Gutkind.
Transforming G protein-coupled receptors potently activate JNK (SAPK).
J. Biol. Chem.
270:
5620-5624,
1995
11.
Crespo, P.,
N. Xu,
J. L. Daniotti,
J. Troppmair,
U. R. Rapp,
and
S. Gutkind.
Signaling through transforming G protein-coupled receptors in NIH 3T3 cells involves c-Raf activation.
J. Biol. Chem.
269:
21103-21109,
1994
12.
Dabrowski, A.,
T. Grady,
C. D. Logsdon,
and
J. A. Williams.
Jun kinases are rapidly activated by cholecystokinin in rat pancreas both in vitro and in vivo.
J. Biol. Chem.
271:
5686-5690,
1996
13.
Davis, R. J.
The mitogen-activated protein kinase signal transduction pathway.
J. Biol. Chem.
268:
14553-14556,
1993
14.
Davis, R. J.
MAPKs: new JNK expands the group.
Trends Biochem. Sci.
19:
470-473,
1994[Medline].
15.
Del Valle, J.,
M. R. Lucey,
and
T. Yamada.
Gastric secretion.
In: Textbook of Gastroenterology (2nd ed.), edited by T. Yamada. Philadelphia, PA: J. B. Lippincott, 1995, p. 295-326.
16.
Del Valle, J.,
Y. Tsunoda,
J. A. Williams,
and
T. Yamada.
Regulation of [Ca2+]i by secretagogue stimulation of canine gastric parietal cells.
Am. J. Physiol.
262 (Gastrointest. Liver Physiol. 25):
G420-G426,
1992
17.
Guan, K. L.,
and
J. E. Dixon.
Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase.
Anal. Biochem.
192:
262-267,
1991[Medline].
18.
Hibi, M.,
A. Lin,
T. Smeal,
A. Minden,
and
M. Karin.
Identification of an oncoprotein-and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain.
Genes Dev.
7:
2135-2148,
1993[Abstract].
19.
Hill, C. S.,
and
R. Treisman.
Transcriptional regulation by extracellular signals: mechanisms and specificity.
Cell
80:
199-211,
1995[Medline].
20.
Karin, M.
The regulation of AP-1 activity by mitogen-activated protein kinases.
J. Biol. Chem.
270:
16483-16486,
1995
21.
Kudoh, S.,
I. Komuro,
T. Mizuno,
T. Yamazaki,
and
Y. Zou.
Angiotensin II stimulates c-Jun NH2-terminal kinase in cultured cardiac myocytes of neonatal rats.
Circ. Res.
80:
139-146,
1997
22.
Lev, S.,
H. Moreno,
R. Martinez,
P. Canoll,
E. Peles,
J. M. Musacchio,
G. D. Plowman,
B. Rudy,
and
J. Schlessinger.
Protein tyrosine kinase PYK2 involved in Ca2+-induced regulation of ion channel and MAP kinase functions.
Nature
376:
737-745,
1995[Medline].
23.
Marais, R.,
J. Wynne,
and
R. Treisman.
The SRF accessory protein Elk-1 contains a growth factor-regulated transcriptional activation domain.
Cell
73:
381-393,
1993[Medline].
24.
Marshall, C. J.
Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation.
Cell
80:
179-185,
1995[Medline].
25.
Marx, J. L.
The fos gene as "master switch."
Science
237:
854-856,
1987[Medline].
26.
Muraoka, A.,
M. Kaise,
Y. Guo,
J. Yamada,
I. Song,
J. Del Valle,
A. Todisco,
and
T. Yamada.
Canine H+-K+-ATPase -subunit gene promoter: studies with canine parietal cells in primary culture.
Am. J. Physiol.
271 (Gastrointest. Liver Physiol. 34):
G1104-G1113,
1996
27.
Nakamura, K.,
C. J. Zhou,
J. Parente,
and
C. S. Chew.
Parietal cell MAP kinases: multiple activation pathways.
Am. J. Physiol.
271 (Gastrointest. Liver Physiol. 34):
G640-G649,
1996
28.
Sadoshima, J.,
and
S. Izumo.
The heterotrimeric Gq protein-coupled angiotensin II receptor activates p21ras via the tyrosine kinase-Shc-Grb2-Sos pathway in cardiac myocytes.
EMBO J.
15:
775-787,
1996[Abstract].
29.
Sadoshima, J.,
Z. Qiu,
J. P. Morgan,
and
S. Izumo.
Angiotensin II and other hypertrophic stimuli mediated by G protein coupled receptors activate tyrosine kinase, MAP kinase and 90 K S6 kinase in cardiac myocytes. A critical role of Ca2+-dependent signaling.
Circ. Res.
76:
1-15,
1996
30.
Soll, A. H.
The actions of secretagogues on oxygen uptake by isolated mammalian parietal cells.
J. Clin. Invest.
61:
370-380,
1978[Medline].
31.
Takeuchi, Y.,
J. Yamada,
T. Yamada,
and
A. Todisco.
Functional role of extracellular signal-regulated protein kinases in gastric acid secretion.
Am. J. Physiol.
273 (Gastrointest. Liver Physiol. 36):
G1263-G1272,
1997
32.
Todisco, A.,
V. Campbell,
C. J. Dickinson,
J. Del Valle,
and
T. Yamada.
Molecular basis for somatostatin action: inhibition of c-fos expression and AP-1 binding.
Am. J. Physiol.
267 (Gastrointest. Liver Physiol. 30):
G245-G253,
1994
33.
Todisco, A.,
Y. Takeuchi,
C. Seva,
C. J. Dickinson,
and
T. Yamada.
Gastrin and glycine-extended progastrin processing intermediates induce different programs of early gene activation.
J. Biol. Chem.
270:
28337-28341,
1995
34.
Toullec, D.,
P. Pianetti,
H. Coste,
P. Bellevergue,
T. Grand-Perret,
M. Ajakane,
V. Baudet,
P. Boissin,
E. Boursier,
F. Loriolle,
L. Duhamel,
D. Charon,
and
J. Kirilovsky.
The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C.
J. Biol. Chem.
266:
15771-15781,
1991
35.
Treisman, R.
Journey to the surface of the cell: Fos regulation and the SRE.
EMBO J.
14:
4905-4913,
1995[Medline].
36.
Westwick, J. K.,
C. Weitzel,
H. L. Leffert,
and
D. A. Brenner.
Activation of Jun kinase is an early event in hepatic regeneration.
J. Clin. Invest.
95:
803-810,
1995[Medline].
37.
Westwick, J. K.,
and
D. A. Brenner.
Methods for analyzing c-Jun kinase.
Methods Enzymol.
255:
342-359,
1995[Medline].
38.
Zohn, I. E.,
H. Yu,
X. Li,
A. Cox,
and
H. S. Earp.
Angiotensin II stimulates calcium-dependent activation of c-Jun N-terminal kinase.
Mol. Cell. Biol.
15:
6160-6168,
1995[Abstract].