Regulation of c-Jun NH2-terminal kinases in isolated canine gastric parietal cells

A. Nagahara, L. Wang, J. Del Valle, and A. Todisco

Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0682

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-alpha 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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Plasmids. Gal4-c-Jun and 5×Gal-Luc (18) were gifts from M. Karin (San Diego, CA), pCMV-beta 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 rho  = 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-alpha (TNF-alpha ; 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 beta -galactosidase activity. beta -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 beta -glycerophosphate, 0.1 mM Na3VO4, 20 mM p-nitrophenylphosphate, 2 mM DTT, 20 µM ATP, and 5 µCi of [gamma 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-(1---79). 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 [gamma 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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.


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Fig. 1.   Expression of c-Jun NH2-terminal kinases (JNKs) in gastric parietal cells. JNK expression in parietal cell lysates was studied by Western blots using an anti-JNK1 antibody. These data represent results from a single parietal cell preparation. Identical results were obtained in experiments with one other separate parietal cell preparation.

We then demonstrated, using solid-state kinase assays, that carbachol (100 µM) activates JNK activities that specifically phosphorylate c-Jun-(1---79) 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.


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Fig. 2.   Effect of carbachol on JNK activity in gastric parietal cells. A: solid-state kinase assay demonstrating that carbachol (100 µM) stimulates phosphorylation of glutathione S-transferase (GST)-c-jun-(1---79) but not of GST. B: Coomassie blue stain demonstrating that equal amounts of GST-c-jun-(1---79) were loaded on the gel. Data represent results from a single parietal cell preparation.

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-(1---79), whereas no effect was observed in the presence of either the nonimmune serum or the anti-ERK2 antibody.


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Fig. 3.   JNK1 immunoprecipitation and in-gel JNK1 assay in gastric parietal cells. JNK1 in lysates from parietal cells stimulated with carbachol (100 µM) was immunoprecipitated, and its activity was measured by in-gel kinase assays. GST-c-jun-(1---79) phosphorylation was detected in the presence (+) of an anti-JNK1 antibody (anti-JNK1 Ab) but not in the presence (-) of either nonimmune serum or an anti-extracellular signal-regulated kinase 2 antibody (anti-ERK2 Ab). Data represent results from a single parietal cell preparation.

We studied the dose-response effect of carbachol, EGF, TNF-alpha , 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-alpha , 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).


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Fig. 4.   Concentration-dependent effect of carbachol on JNK1 activity in gastric parietal cells. JNK1 in lysates from parietal cells stimulated with increasing concentrations of carbachol was immunoprecipitated, and its activity was measured by in-gel kinase assays. A: representative assays obtained with a single parietal cell preparation. B: linear transformation of the densitometric analysis of autoradiograms is shown. Data are expressed as degree of induction over control. O.D., optical density. Identical results were obtained in experiments with 1 other separate parietal cell preparation.

We then investigated the kinetics of JNK1 induction in response to TNF-alpha , carbachol, EGF, and histamine stimulation. As shown in Fig. 5A, TNF-alpha 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-alpha , 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).


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Fig. 5.   Time course of tumor necrosis factor-alpha (TNF-alpha )- and carbachol-induced activation of JNK1 in gastric parietal cells. JNK1 in lysates from parietal cells stimulated for different time periods with TNF-alpha (10 ng/ml) (A) and carbachol (100 µM) (B) was immunoprecipitated, and its activity was measured by in-gel kinase assays. Top: representative assays obtained with a single parietal cell preparation. Bottom: graphs depicting a linear transformation of the densitometric analysis of the autoradiograms. Data are means ± SE, expressed as degree of induction over control. * P < 0.05.


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Fig. 6.   Time course of epidermal growth factor (EGF)- and histamine-induced activation of JNK1 in gastric parietal cells. JNK1 in lysates from parietal cells stimulated for different time periods with EGF (10 nM) (A) and histamine (100 µM) (B) was immunoprecipitated, and its activity was measured by in-gel kinase assays. Top: representative assays obtained with a single parietal cell preparation. Bottom: linear transformation of the densitometric analysis of the autoradiograms. Data are means ± SE, expressed as degree of induction over control. * P < 0.05.

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).


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Fig. 7.   Effect of the protein kinase C (PKC) inhibitor GF109203X on carbachol- and 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated JNK1 activity in gastric parietal cells. JNK1 in lysates from parietal cells stimulated with either TPA (100 nM) or carbachol (100 µM) in presence or absence of GF109203X (5 µM) was immunoprecipitated, and its activity was measured by in-gel kinase assays. A: representative assays obtained with a single parietal cell preparation. B: linear transformation of the densitometric analysis of the autoradiograms. Data are means ± SE, expressed as % of JNK1 activity; ns, not significant. * P < 0.05.

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.


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Fig. 8.   Effect of intra- and extracellular Ca2+ on carbachol-stimulated JNK1 activity in gastric parietal cells. JNK1 in lysates from parietal cells stimulated with either thapsigargin (1 µM) or carbachol (100 µM) alone, in presence or absence of Ca2+-free medium containing 1 mM EGTA (no Ca2+) or in combination with the cell-permeable Ca2+ chelator BAPTA-AM (100 µM), was immunoprecipitated, and its activity was measured by in-gel kinase assays. A: representative assays obtained with a single parietal cell preparation. B: linear transformation of the densitometric analysis of the autoradiograms is shown. Data are means ± SE, expressed either as degree of induction over control (left) or as % of 100 µM carbachol-stimulated JNK1 activity in absence of BAPTA-AM (right). * P < 0.05.

We then examined whether JNK1 is a signaling pathway specifically activated by inflammatory cytokines. Accordingly, we examined the effect of TNF-alpha on ERK2 activation. As shown in Fig. 9, whereas carbachol potently induced ERK2 activity after 5 min of incubation, TNF-alpha had no effect, suggesting that JNK1 but not ERK2 is involved in TNF-alpha signaling. Identical results were obtained when the cells were incubated for 30 min in the presence of either carbachol or TNF-alpha (data not shown).


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Fig. 9.   Effect of TNF-alpha and carbachol on ERK2 activation in gastric parietal cells. ERK2 in lysates from parietal cells stimulated for 5 min with either TNF-alpha (10 ng/ml) or carbachol (100 µM) was immunoprecipitated, and its activity was measured by in-gel kinase assays. A representative assay obtained with a single parietal cell preparation is shown. Identical results were obtained in experiments with 3 other separate parietal cell preparations.

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-beta 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).


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Fig. 10.   Effect of carbachol on c-jun transcriptional activity in gastric parietal cells. Canine gastric parietal cells were transfected with the Gal4-c-Jun expression vector, the 5×Gal luciferase reporter plasmid, and the pCMV-beta Gal vector and treated with 10 µM carbachol. Data are means ± SE, expressed as degree of induction over control; n = 8. * P < 0.05. RLU, relative light units.

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.


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Fig. 11.   Effect of carbachol on c-jun gene expression in gastric parietal cells. Aliquots of total RNA extracted after exposure of the cells for 30 min to 100 µM carbachol were examined by Northern blot analysis using a 32P-labeled cDNA probe for c-jun. Autoradiograms were controlled for RNA quantity by hybridization of the RNA with a cDNA probe encoding the ubiquitous enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). These data were reproduced in 2 other separate experiments.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-alpha , 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-alpha 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-alpha , which had no effect on ERK2 induction (31). Because both histamine and TNF-alpha 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-alpha , 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-alpha , 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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Gastroint Liver Physiol 275(4):G740-G748
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