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Supported by National Institutes of Health Grant DK48077. To whom correspondence should be addressed: Massachusetts General Hospital, Gastrointestinal Unit, GRJ 724, 32 Fruit St., Boston, MA 02114. Tel.: 617-726-9228; Fax: 617-726-3673;
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Supported by Grant DFG HO 1288/2-1 from the German Research Council and by a grant from the Forschungsschwerpunkt Molekulare Endokrinologie, Universitätsklinikum Benjamin Franklin. ‖ Supported by the Verum Stiftung, the Mildred Scheel Stiftung, and the Deutsche Forschungsgemeinschaft. ‡ Supported by a grant from the Deutsche Forschungsgemeinschaft (Ro 674/10-1). §§ Supported by National Institutes of Health Grant DK41557.
Oxidant stress is thought to play a role in the pathogenesis of many gastric disorders. We have recently reported that histidine decarboxylase (HDC) promoter activity is stimulated by gastrin through a protein kinase C- and extracellular signal-regulating kinase (ERK)-dependent pathway in gastric cancer (AGS-B) cells, and this transcriptional response is mediated by a downstream cis-acting element, the gastrin response element (GAS-RE). To study the mechanism through which oxidant stress affects gastric cells, we examined the effects of hydrogen peroxide (H2O2) on HDC promoter activity and intracellular signaling in AGS-B cells. H2O2(10 mm) specifically activated the HDC promoter 10–12-fold, and this activation was blocked by both mannitol and N-acetylcysteine. Hydrogen peroxide treatment of AGS-B cells increased the phosphorylation and kinase activity of ERK-1 and ERK-2, but did not affect Jun kinase tyrosine phosphorylation or kinase activity. In addition, treatment of AGS-B cells with H2O2 resulted in increasedc-fos/c-jun mRNA expression and AP-1 activity, and also led to increased phosphorylation of epidermal growth factor receptor (EGFR) and Shc. H2O2-dependent stimulation of HDC promoter activity was completely inhibited by kinase-deficient ERKs, dominant-negative (N17 and N15) Ras, and dominant-negative Raf, and partially blocked by a dominant-negative EGFR mutant. In contrast, protein kinase C blockade did not inhibit H2O2-dependent induction of the HDC promoter. Finally, deletion analysis demonstrated that the H2O2 response element could be mapped to the GAS-RE (nucleotides 2 to 24) of the basal HDC promoter. Overall, these studies suggest that oxidant stress activates the HDC promoter through the GAS-RE, and through an Ras-, Raf-, and ERK-dependent pathway at least partially involving the EGFR.
ROM
reactive oxygen metabolites
HDC
histidine decarboxylase
hHDC
human HDC
EGFR
epidermal growth factor receptor
ERK
extracellular signal-regulated kinase
JNK
Jun kinase
GAS-RE
gastrin response element
PKC
protein kinase C
Luc
luciferase
CAT
catecholamine acetyltransferase
PMA
phorbol 12-myristate 13-acetate
MAP
mitogen-activated protein
DN
dominant-negative
AP-1
activator protein 1
kb
kilobase pair(s).
Reactive oxygen metabolites (ROMs),1 such as superoxide anion O⨪2, hydroxyl radical OH, and H2O2, are generated in cells as physiological by-products of electron transfer reactions and arachidonic acid metabolism (
). An elevated level of ROMs reflecting oxidative stress is observed during a number of acute physiologic and pathological states, including sepsis, exposure to ionizing radiation, ischemia and reperfusion, and diverse inflammatory conditions (
); despite constant exposure to luminal oxidants, the gastric epithelium remains unaffected. However, enhanced production of ROMs due to acute and chronic inflammation of the gastrointestinal tract may contribute to the mucosal injury. Inflammatory and ulcerative diseases of the gastric mucosa, such as those associated with ethanol, nonsteroidal anti-inflammatory drugs, cold stress, burn stress, ischemia-reperfusion, and Helicobacter pylori, are associated with increased oxidative stress (
), but it can also modulate the expression of a variety of genes that are involved in the immune and inflammatory response, such as the nuclear transcription factor kB (NF-κB) (
). In addition, ROMs at low levels have been shown to induce the expression of growth factor-regulated genes, such as AP-1 (c-fos and c-jun), c-myc, and egr-1 (
). Oxidative stress appears to affect numerous signaling pathways through protein phosphorylation and induce selectively a number of genes, and thus chronic exposure to low levels of ROM may conceivably influence cell growth and differentiation.
Histidine decarboxylase (HDC) is the major enzyme involved in the production of histamine from the amino acid l-histidine, a key step in the stimulation of gastric acid secretion (
). Several previous observations have suggested that the HDC gene may be activated in the stomach under conditions of oxidative stress. For example, HDC activity has been shown to be increased during ischemia-reperfusion injury to the gastrointestinal tract (
). Experiments from our laboratory have shown that the HDC promoter is transcriptionally regulated by gastrin and the phorbol ester (phorbol 12-myristate 13-acetate, PMA) through a protein kinase C (PKC)- and MAP kinase- dependent pathway, acting on a 23-nucleotide cis-acting element, the gastrin response element (GAS-RE) (
). More recently, we have shown that activation of AP-1 is essential for gastrin-stimulated HDC transcription, although the mechanism is most likely indirect (
), indicating that HDC may be an important downstream target of AP-1. Thus, we decided to study the effects of oxidative stress on gastric epithelial cells, AP-1 activity, and HDC promoter activity. These studies suggest that H2O2 can selectively stimulate HDC promoter activity, most likely through an EGFR-dependent signaling pathway that leads to activation of the MAP kinase/ERK pathway.
EXPERIMENTAL PROCEDURES
Materials
Dulbecco's modified Eagle's medium, fetal calf serum, penicillin-streptomycin, and EDTA-trypsin were obtained from Bio Whittaker, Walkerville, MA. Sulfated gastrin-17 was purchased from Peninsula Laboratories, San Diego, CA. The protein kinase inhibitor 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7) and the phorbol ester PMA were obtained from BIOMOL Research Laboratories, Plymouth Meeting, PA. H2O2, N-acetylcysteine, mannitol, and dibutyryl cAMP were obtained from Sigma.
DNA Constructs and Reporter Plasmids
The human 1.8-kb HDC-luciferase construct and the GAS-RE/thymidine kinase-Luc construct have been described elsewhere (
). The GAS-RE/thymidine kinase-Luc construct is derived from thymidine kinase-Luc but also contains the human HDC GAS-RE sequences ligated upstream of the herpes simplex virus 1 thymidine kinase promoter. The 4XTRE-CAT construct has been previously reported (
). The EGFR dominant-negative expression (HER653) construct was a gift of Murray Korc and contains the kinase-deleted mutant of the human EGFR cDNA (dominant-negative (DN)-EGFR) under the control of the cytomegalovirus (CMV) promoter. Transfection of this DN-EGFR construct has been shown to block EGFR signaling (
). The ornithine decarboxylase-luciferase construct was a gift of Juanita L. Merchant, and contained 450 base pairs of human ornithine dcarboxylase 5′-flanking DNA. The GAL4-c-Jun wild type construct contains the GAL4 DNA binding domain linked to amino acids 1–246 of human c-Jun, while the GAL4-c-Jun mutant (AA) has Ser-63 and Ser-73 of c-Jun mutated to alanines. These constructs and the 5XGAL-Luc system have been described previously (
The ERK cDNAs and mutants were subcloned into the expression vector pCMV5 (in which cDNA expression is driven by a CMV promoter) and have been described previously (
). Wild type ERK constructs contained full-length human cDNAs, whereas the mutants K71R-ERK-1 and K52R-ERK-2 encode kinase-deficient, interfering proteins with kinase activities <5% of the corresponding wild type kinases (
). The constructs Ha-ras-ASN17 (RasN17) and Ha-ras-Ala15 (RasN15) represent dominant-negative Ras expression constructs in which mutated Ras genes are under control of the murine mammary tumor virus promoter (
AGS-B, the cell line used in these studies, was prepared by stable transfection of the human gastric adenocarcinoma cell line AGS (ATCC CRL 1739) with the CCK-B/gastrin receptor and has been reported (
). AGS-B and AGS-B/1.8-kb hHDC-Luc cells were grown in Dulbecco's modified Eagle's medium plus 10% bovine calf serum, 100 IU/ml penicillin, and 100 IU/ml streptomycin in a humidified atmosphere (5% CO2). Transient transfections of cultured cells were carried out using the calcium phosphate precipitation technique (DNA transfection kit, 5 Prime → 3 Prime, Inc.) as described previously (
). H2O2 stimulation was performed for 10 min, and cells were harvested 6 h thereafter. In time course studies, cells were harvested 0, 2, 4, and 6 h after a 10-min H2O2 pulse. Gastrin and PMA were added at stimulatory concentrations (12–24 h after transient transfection), cells were harvested, and luciferase assays were performed at 48 h. Luciferase assays were carried out using luciferin, ATP, coenzyme A (Promega system) with a Monolight Luminometer (Analytical Luminescence Laboratory). CAT assays were performed as described previously (
). Experiments were performed in triplicate or quadruplicate, and results were calculated as mean ± S.D. Values for HDC luciferase were expressed as a fold increase in luciferase compared with untreated controls. Activities in all transfection experiments represented the mean ± S.D. of at least 4–6 independent transfections. Activities varied <15% among transfection experiments. Statistical differences were calculated using the Student's t test (Microsoft Excel).
RNA Isolations and Northern Blot Analysis
Total RNA from AGS-B cells was prepared using TRIzol R reagent (Life Technologies, Inc.) according to the manufacturer's instructions. 20 μg of total RNA were separated on 2.2 m formaldehyde, 1.2% agarose gels. Ethidium bromide staining confirmed that equal amounts of RNA were loaded per lane (data not shown). Probes for c-jun and c-fos probes were excised from expression constructs which have previously been reported (
) and were labeled with the Ready-to-go kit (Amersham Pharmacia Biotech) according to the manufacturer's instructions. Membranes were prehybridized for 30 min and hybridized for 60 min using Rapid-Hyb buffer (Amersham Pharmacia Biotech) at 65 °C. Following hybridization the membranes were washed twice for 20 min each in 2× SSC, 0.1% SDS at 25 °C, then once in 2× SSC, 0.1% SDS at 55 °C for 20 min. Membranes were exposed to x-ray film for 16 h at −80 °C.
Immunoprecipitation and Western Blotting
AGS-B cells were grown to subconfluence and then starved for 36 h in Dulbecco's modified Eagle's medium without serum. Cells were then stimulated with H2O2 (10 mm final concentration) for 10 min at 37 °C. After stimulation, cells were washed in ice-cold phosphate-buffered saline and lysed in lysis buffer (20 mm Tris, pH 7.8, 150 mm NaCl, 2 mmEDTA, 0.5% Nonidet P-40, 10 mm NaF, 10 mmsodium orthovanadate, 2 mm phenylmethylsulfonyl fluoride, and 10 μg/ml each of leupeptin, pepstatin, and aprotinin). The lysates were precleared for 1 h by incubating them with normal rabbit serum and protein A-Sepharose beads (Pharmacia). Antibodies to ERK-1, JNK-1, EGF-R, and Grb2 (Santa Cruz Biochemicals), and Shc (Transduction Laboratories) were used to immunoprecipitate these proteins from cells lysates. Immune complexes were collected on protein A-Sepharose beads and washed three times with ice-cold lysis buffer. SDS-sample buffer was added, and the beads were boiled for 5 min. Proteins were separated by SDS-polyacrylamide gel electrophoresis on 7.5% polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore). The membrane was then probed with anti-phosphotyrosine antibody (4G10) (1 μg/ml) (Upstate Biotechnology Inc.). Bands were visualized with the enhanced chemiluminescence system (Amersham). Membranes that were probed with anti-phosphotyrosine antibody were reprobed after stripping the membrane. Stripping was performed by washing extensively with TTBS (20 mm Tris pH 7.6, 150 mm NaCl plus 0.05% Tween 20) for 4 h, then incubating the membrane in a solution of 62.5 mm Tris-HCl, pH 6.8, 2% SDS, and 100 mm 2-mercaptoethanol for 30 min at 55 °C followed by washing in TTBS for 2 h changing the buffer every 30 min.
MAP Kinase Activity Assays
To determine ERK activity in response to H2O2, cell-free extracts were prepared after treatment of subconfluent AGS-B cells with H2O2 (10 mm). Extracts were boiled with Laemmli sample buffer, and aliquots (10 μg protein) were separated on a 10% SDS-polyacrylamide gel containing 0.5 mg/ml ERK substrate myelin basic protein (
). Gels were subsequently submitted to denaturation-renaturation and kinase activity was measured by incubating the gels in kinase buffer containing 25 μm ATP and 100 μCi [γ-32P]ATP for 90 min at room temperature. Kinase reaction was terminated by washing the gels in 5% trichloroacetic acid, 10 mm sodium pyrophosphate. Dried gels were exposed to x-ray films, and signal intensity was determined by scanning densitometry using a Molecular Dynamics personal densitometer with Imagequant version 3.22 software.
RESULTS
Hydrogen Peroxide Activates the HDC Promoter
To determine whether oxidative stress stimulates HDC promoter activity, AGS-B cells stably transfected with the 1.8-kb hHDC-Luc construct were exposed to varying concentrations of hydrogen peroxide for 10 min, and cell extracts were assayed for luciferase activity 6 h later (Fig. 1A). Hydrogen peroxide stimulated HDC promoter activity in a dose-dependent fashion, with a 10–12-fold increase seen when using 10 mmH2O2. Stimulation by 10 mmH2O2 resulted in a measurable increase in HDC promoter activity as early as 3 h post-stimulation, with a maximal increase seen at 5–7 h after treatment (Fig. 1B). The effect of H2O2 appeared to be selective, since no activation of the ornithine decarboxylase promoter nor of the basal thymidine kinase promoter was observed under similar conditions (data not shown).
Figure 1H2O2 stimulates the HDC promoter in AGS-B cells. Cells stably transfected with the 1.8-kb hHDC-Luc construct (
) were treated with H2O2 as indicated. Luciferase activity is expressed as increase in activity compared with untreated controls and represents means ± S.E. from four separate transfections.A, dose response curve showing HDC promoter activity in response to various concentrations (0–10 mm) of H2O2 for 10 min. B, time course showing rate of increase of HDC promoter activity in response to 10 mm H2O2. Cells were exposed to H2O2 for 10 min and then harvested and assayed for luciferase activity after the indicated time periods.
To confirm that induction of HDC promoter activity was due to increased oxidant stress and free radical generation, we examined the effect of pretreatment with N-acetylcysteine and mannitol on stimulation by H2O2.N-Acetylcysteine is a glutathione precursor known to enhance cellular antioxidant potential, while mannitol is a free radical scavenger with relative specificity for the hydroxyl radical (
). BothN-acetylcysteine and mannitol completely abolished HDC promoter activation by H2O2, consistent with a mechanism involving oxidant stress and a hydroxyl radical-like species (Fig. 2).
Figure 2Induction of HDC promoter activity by H2O2 is blocked by N-acetylcysteine and mannitol. AGS-B cells were transiently transfected with 1.8-kb hHDC-Luc. N-Acetylcysteine (20 mm) or mannitol (100 mm) was added where indicated 45 min before the addition of H2O2 (10 mm, where indicated) for 10 min. Cells were harvested and assayed for luciferase activity 6 h later. Luciferase activity represents the mean ± S.E. of four separate experiments and is expressed as the -fold increase over untreated controls. The inhibitors alone had no effect on HDC-Luc activity.
). Therefore, we examined the effect of H2O2 on the expression of c-fos and c-jun mRNA in AGS-B gastric cells using Northern blot analysis. Treatment of AGS-B cells with 10 mm H2O2 led to a marked increase in mRNA expression of both c-fos and c-jun, with initial increases seen at 30 min and the largest increases observed at 120 min (Fig. 3A). We also examined the effect of H2O2 on c-Jun-dependent transactivation, using the GAL4-c-Jun (wild type and mutant) expression vectors and the 5XGAL-Luc reporter gene (see “Experimental Procedures”). Both H2O2and PMA stimulated transactivation by GAL4-c-Jun wild type but had no effect on the mutant construct, GAL4-c-Jun mutant (AA) (Fig. 3B). Hydrogen peroxide also stimulated AP-1-dependent transactivation, as measured using AGS-B cells transiently transfected with the 4XTRE-CAT construct (Fig. 3C). The effect of H2O2 on c-fos gene expression was most likely due to transcriptional induction, as evidenced by a 10-fold stimulation effect of H2O2 on c-fospromoter activity in transiently transfected AGS-B cells (Fig. 3D). Finally, H2O2 also stimulated c-Myc-dependent transactivation, as shown in studies using wild type and mutant GAL4-c-Myc expression vectors and the 5XGAL-Luc reporter gene (Fig. 3E). These studies indicate that H2O2 transactivates c-Myc, and that the AP-1 pathway is activated both transcriptionally and post-translationally by oxidative stress in gastric cells.
Figure 3H2O2 activates AP-1 and Myc in AGS-B cells.A, Northern blot analysis showing the time course for induction of c-fos and c-jun mRNA after treatment with 10 mmH2O2 for 10 min. B, H2O2 stimulates c-Jun-dependent transactivation in AGS-B cells. H2O2 and PMA stimulate GAL4-c-Jun wild type but not GAL4-c-Jun mutant (MT) (AA). AGS-B cells were transfected with 5XGAL-Luc and either GAL4-c-Jun wild type or GAL4-c-Jun mutant, and treated with either H2O2 (10 mm for 10 min) or PMA (10−7m for 4 h) where indicated. Luciferase activity represents the mean ± S.E. of four separate experiments, and is expressed as -fold increase of untreated controls.C, effect of H2O2 on AP-1-dependent transactivation. AGS-B cells were transfected with the 4XTRE-CAT construct and treated with either H2O2 or PMA as above. CAT activity represents the mean ± S.E. of four separate transfections, and is expressed as -fold increase over untreated controls. D, stimulation of c-fos promoter activity by H2O2 in AGS-B cells. Top, schematic diagram of the c-fospromoter-luciferase construct. Bottom, AGS-B cells were transiently transfected with the c-fos-luciferase construct, and treated with either H2O2 or PMA as in B. Luciferase activity represents the mean ± S.E. of four separate experiments and is expressed as -fold increase of untreated controls. E, H2O2stimulates c-Myc-dependent transactivation in AGS-B cells. H2O2 and PMA stimulate GAL4-c-Myc wild type but not GAL4-c-Myc mutant (MT). AGS-B cells were transfected with 5XGAL-Luc and either the wild type or mutant GAL4-c-Myc construct, and treated with either H2O2 or PMA as in B. Luciferase activity represents the mean + S.E. of 4 separate experiments, and is expressed as -fold increased of untreated controls. * indicates statistically significant (p < 0.05) differences compared with controls or mutants using Student'st test.
Since both HDC and c-fos (and to a lesser extent, c-jun) promoter activity are known downstream targets of ERK activation, we investigated the effect of H2O2 on the phosphorylation and kinase activity of the ERKs. As shown in Fig. 4A, H2O2 stimulated both the phosphorylation and kinase activity of the ERKs. H2O2 stimulated ERK kinase activity to a greater degree (>15-fold) than either gastrin or PMA (5–10 fold) or dibutyryl cAMP (3–5-fold) in AGS-B cells (Fig. 4B). In contrast, the JNKs were not activated by H2O2, although they were stimulated (and phosphorylated) by UV light exposure (Fig. 4C). Taken together, these findings suggest selective activation of ERKs by H2O2 in the AGS-B system.
Figure 4H2O2 stimulates ERK phosphorylation and kinase activity, but not JNK.Top, scanning densitometry of the MBP kinase assay blot (middle), showing activation of ERK activity by H2O2, gastrin, PMA, and dibutyryl (db) cAMP. Middle, upper panel, induction of ERK tyrosine phosphorylation by H2O2, gastrin, and PMA. AGS-B cells were treated for 5 min with gastrin (10−7m), PMA (10−7m), dibutyryl cAMP (10−7m), H2O2 (10 mm), or left untreated (control). Cell lysates were immunoprecipitated with anti-ERK-1 antibody, and then subjected to immunoblot analysis by anti-phosphotyrosine antibody. Lower panel, myelin basic protein kinase assay. AGS-B cells were stimulated with H2O2, gastrin, or PMA as above, or left untreated (control). To determine ERK activity in response to H2O2, cell-free extracts were prepared after treatment of subconfluent AGS-B cells with H2O2(10 mm). Aliquots (10 μg of protein) of extracts were separated on an 10% SDS-polyacrylamide gel containing 0.5 mg/ml ERK substrate myelin basic protein. Gels were submitted to denaturation-renaturation and subsequently incubated in kinase buffer containing [γ-32P]ATP. After termination of kinase reaction, dried gels were exposed to x-ray films, and signal intensity was determined by scanning densitometry (Molecular Dynamics densitometer; Imagequant 3.22 software). Bottom, H2O2 does not stimulate JNK phosphorylation. AGS-B cells were stimulated with UV light (1 min), gastrin, PMA, H2O2, or left untreated (control). Upper panel, Western blot of AGS-B cell lysates immunoprecipitated with anti-JNK antibody and then probed with the same anti-JNK antibody. Treatment with UV light led to a slower (lower) migrating JNK band compared with JNK present in H2O2-stimulated and the other lanes. The upper band represents cross-reactivity of the secondary antibody with mouse immunoglobulin heavy chain. Lower panel, the same blot probed with anti-phosphotyrosine antibody.
H2O2 Treatment Leads to Shc and EGFR Phosphorylation
Previous studies have suggested that in some cell systems H2O2 activates the ERKs through a Ras pathway that involves tyrosine phosphorylation of EGFR, followed by phosphorylation of Shc and the induction of Shc-Grb2-SOS complexes (
). To investigate the pathways utilized by gastric cells in response to oxidant stress, we analyzed protein extracts from H2O2-treated AGS-B cells for phosphotyrosine proteins by Western blotting. H2O2 induced a marked increase in tyrosine phosphorylation of multiple proteins (Fig. 5A, lane 4) with molecular masses that ranged from 60 to 200 kDa. In order to examine the effect of H2O2 on the phosphorylation of Shc and its association with the adaptor protein Grb2, Grb2 and Shc proteins were first immunoprecipitated with specific antibodies, and phosphorylation levels were assessed using an antiphosphotyrosine antibody. As shown in Fig. 5A (lanes 5–12), treatment of AGS-B cells with H2O2 resulted in an increase in tyrosine phosphorylation of both the 52- and 46-kDa isoforms of Shc. The increase was greater than that seen with PMA or gastrin stimulation. In addition, multiple bands representing tyrosine phosphorylated proteins, not seen with PMA or gastrin stimulation, were co-immunoprecipitated when using antibodies to Grb2 or Shc (Fig. 5A).
Figure 5H2O2 stimulates tyrosine phosphorylation of multiple proteins.A, H2O2 stimulates tyrosine phosphorylation of Shc and its association with Grb2. Lanes 1–4 show a Western blot of AGS-B cell lysates probed with the anti-phosphotyrosine antibody. Prior to lysis, AGS-B cells were stimulated for 5 min with either gastrin (10−7m), PMA (10−7m), H2O2 (10 mm), or left untreated (control). Lanes 5–12show a Western blot (probed with anti-phosphotyrosine antibody) of the same lysates that had been first immunoprecipitated with antibodies to either Grb2 or Shc. Both the 52- and 46-kDa forms of phosphorylated Shc are apparent in the gastrin and PMA lanes (predominantly 52 kDa). Additional bands are present in the H2O2 lanes.B, H2O2 stimulates tyrosine phosphorylation of the EGFR. AGS-B cells were unstimulated (control) or stimulated with either PMA or H2O2. After immunoprecipitation with antibodies to Shc or EGFR, the immunoprecipitates were run on SDS-polyacrylamide gel electrophoresis, 10% gel, and transferred to polyvinylidene difluoride membrane. The blot was then probed with anti-phosphotyrosine antibody. Thearrows show either the Shc band (predominantly 52 kDa) or EGF-R (170-kDa band) with H2O2.
Several of the proteins that were phosphorylated in response to H2O2 ranged in size from 100 to 200 kDa. Since the EGFR protein is 170 kDa, we examined whether EGFR associates with Shc and undergoes phosphorylation in response to H2O2. As shown in Fig. 5B, treatment of AGS-B cells resulted in tyrosine phosphorylation of EGFR and association with Shc, while PMA treatment did not. Gastrin stimulation also did not lead to EGFR phosphorylation (data not shown).
Effect of PKC Blockade on H2O2-stimulated hHDC Promoter Activity
To define the functional role of PKCs for H2O2-dependent hHDC transactivation, we down-regulated PKCs using pretreatment with 10−8m phorbol ester PMA as described previously (
). These studies revealed that blockade of PKCs using PMA pretreatment had no effect on the transactivating effect of H2O2, whereas PMA-stimulated activity of the 1.8-kb hHDC-Luc construct was completely abolished (Fig. 6). These data strongly suggest that the effect of H2O2 on the hHDC promoter is preferentially mediated through activation of pathways independent of PKCs.
Figure 6Effect of PKC on H2O2-stimulated hHDC promoter activity.AGS-B cells transfected with the 1.8-kb hHDC-Luc construct were pretreated with the phorbol ester PMA (10−8mfor 24 h = PMA pretreatment (PMA-PT) and subsequently stimulated with a maximally effective concentration of H2O2 (10 mm) for 6 h. Luciferase activity is expressed as a -fold increase relative to control transfectants and represents the mean ± of four separate experiments. The asterisk (*) indicates a statistically significant difference (p < 0.05) compared with no PMA-PT.
The Effect of H2O2 on the hHDC Promoter Is Mediated through MAP Kinase/ERK Signaling Pathways
To evaluate the importance of ERK-dependent signaling pathways for H2O2 -dependent regulation of the hHDC promoter, AGS-B cells were co-transfected with 1.8-kb hHDC-Luc, and expression constructs encoding dominant-negative ERK mutants and subsequently stimulated with a maximal concentration of H2O2. Expression of dominant-negative ERK proteins abolished the effect of H2O2 on hHDC promoter activity (Fig. 7), demonstrating that H2O2-dependent transactivation of the hHDC promoter is mediated through activation of MAP kinase/ERKs.
Figure 7Role of ERKs for H2O2-stimulated regulation of hHDC promoter activity. 1.0 μg of kinase-deficient ERK-1 (K71R-ERK-1 (ERK-1-KR)) or ERK-2 (K52R-ERK-2 (ERK-2-KR)) was cotransfected with the 1.8-kb hHDC-Luc construct, and cells were stimulated with a maximally effective concentration of H2O2 (10 mm) for 10 min and assayed for luciferase activity at 24 h. Luciferase activity is expressed as a -fold increase relative to control transfectants and represents the mean ± of four separate experiments. The asterisk(*) indicates significant (p < 0.05) inhibition compared with H2O2 treatment alone.
The Effect of H2O2 on the hHDC Promoter Is Mediated through Ras- and Raf-mediated Pathways
To define the initial steps in activation of intracellular signaling cascades transmitting the effect of H2O2 on the hHDC promoter, we analyzed the role of Ras and Raf for H2O2-stimulated hHDC promoter activity. Expression of the interfering Ras mutants Ha-ras-ASN17 (RasN17) or Ha-ras-Ala15 (RasN15) abolished the effect of H2O2 on the hHDC construct (Fig. 8). In contrast, no effect on PMA-stimulated hHDC promoter transactivation could be observed (
) (data not shown). Similarly, inhibition of Raf by expression of a dominant-negative Raf-1 mutant abolished the H2O2 effect (Fig. 8). Raf blockade also inhibited hHDC promoter transactivation in response to PMA (
) (data not shown). Taken together, our data demonstrate transmission of H2O2-dependent hHDC transactivation through a Ras- and Raf-dependent proximal pathway.
Figure 8H2O2-dependent transactivation of the hHDC promoter is mediated through a Ras- and Raf-dependent pathway. AGS-B cells were transfected with 1.0 μg of the 1.8-kb hHDC-Luc construct along with the dominant-negative Ras-mutants (RasN15 or RasN17), or a dominant-negative Raf-1 mutant where indicated. After transfection, cells were stimulated with a maximally effective concentration of H2O2 (10 mm for 10 min) or 10−8m PMA for 24 h. Luciferase activity is expressed as a -fold increase relative to controls which were transfected with empty vectors. Data shown represent the mean ± of four separate experiments. The asterisk (*) indicates significant (p < 0.05) inhibition compared with H2O2 treatment alone.
H2O2 Induction of the HDC Promoter Is Partially Blocked by Dominant-Negative EGFR
The role of EGFR in mediating stimulation of the HDC promoter was further studied using a kinase-deficient EGFR mutant lacking the cytoplasmic domain, which inhibits EGFR downstream signaling by formation of signaling-defective heterodimers with the wild-type receptor. This DN-EGFR construct was co-transfected into AGS-B cells along with the 1.8-kb hHDC-Luc construct, and the response to H2O2 and PMA was assessed. The DN-EGFR partially blocked H2O2-mediated induction of HDC promoter activity, but had no effect on the PMA response (Fig. 9). In addition, the DN-EGFR construct had no effect on the response to gastrin (not shown).
Figure 9Dominant-negative EGFR partially inhibits H2O2 stimulation of the HDC promoter.AGS-B cells were transfected with the 1.8-kb hHDC-Luc construct, and with the DN-EGF-R construct where indicated. Transfected cells were stimulated with PMA (10−7m) or H2O2 (10 mm) or left untreated (C). Luciferase activity is expressed as -fold increase of unstimulated controls, and represents the mean ± S.E. of four separate transfections. The asterisk (*) indicates significant (p < 0.05) inhibition compared with H2O2 treatment alone.
The GAS-RE Functions as an H2O2-responsive Element
To investigate further the downstream mechanism by which H2O2 activates the HDC promoter, we analyzed a number of HDC deletion constructs for activation by H2O2 treatment. These deletion studies showed that the H2O2 responsive element was located within the basal (−59 to +125) human HDC promoter (data not shown). In previous reports, we mapped the HDC GAS-RE to a 23-bp element located just downstream of the transcriptional start site (
) (Fig. 10A). Ligation of the GAS-RE enhancer upstream of the gastrin -insensitive thymidine kinase promoter in the thymidine kinase-Luc construct resulted in a 4-fold response to H2O2, slightly less than that seen with PMA (Fig. 10B).
Figure 10H2O2 stimulates the HDC promoter through the GAS-RE in AGS-B cells.A, schematic diagram of the HDC-GAS-RE construct, which contains the 2 to 24 sequences of human HDC upstream of the thymidine kinase promoter driving luciferase expression. B, AGS-B cells were transiently transfected with the GAS-RE-thymidine kinase-luciferase construct, and stimulated with either H2O2 (10 mm) or PMA (10−7m). Lucifease activity is expressed as fold increase relative to unstimulated controls and represents mean ± S.E. from five separate transfections. The asterisk (*) indicates significant (p < 0.05) stimulation compared with untreated controls. Activity was normalized compared with stimulation of thymidine kinase-Luc (pTK-Luc).
Recent studies have suggested that H2O2can selectively activate a number of genes involved in a variety of biologic responses to oxidant stress, including immunologic, proliferative, and apoptotic responses (
). We now show that HDC promoter activity can be stimulated by H2O2. The responses were seen at a slightly higher H2O2 concentration (10 mm) than that used in previous studies (
), but may reflect the greater resistance of gastric cells to oxidative stress. The responses were specific for oxidative stress pathways and reactive oxygen intermediates (such as the hydroxyl radical) as shown by the inhibition seen with N-acetylcysteine and mannitol. In addition, the activation of HDC was selective, since no effect of H2O2 was observed on the ODC promoter under similar conditions. Further, we have shown that the downstream target of oxidant stress is the GAS-RE element, which we have previously shown to mediate the HDC transcription response to gastrin and PMA.
Treatment of AGS-B cells with H2O2 led to significant increases in ERK-1 and AP-1 activity. Since earlier studies by our group demonstrated that activation of HDC transcription by gastrin and PMA occurred through ERK- and AP-1-dependent pathways (
), these results strongly suggested that H2O2-induced HDC promoter activation might be mediated through a similar pathway. A role for ERKs in H2O2-dependent HDC promoter activation was confirmed through studies using kinase-deficient ERK constructs. Previous studies by Guyton et. al. (
) reported that the ERKs and AP-1 could be activated by 200 μmH2O2 in both NIH 3T3 cells and PC12 cells through a Ras-dependent pathway, and that ERK activation mediated resistance to H2O2 toxicity. This earlier study also suggested that 200 μmH2O2 treatment moderately stimulated (3–5-fold) JNK activity. In contrast, our results indicated that the JNK pathway was not activated by H2O2 in AGS-B gastric cells, suggesting the possibility of cell-specific differences in the response to oxidant stress. H2O2treatment stimulated a dramatic increase in the mRNA expression of c-jun and c-fos, similar to that previously observed in NIH 3T3 cells (
). However, in contrast to this earlier study, the induction of c-jun and c-fos gene expression in AGS-B gastric cells was more sustained. Indeed, the greatest levels of c-fos and c-jun gene expression in H2O2-treated AGS-B cells occurred at 120 min, suggesting a more prolonged growth factor response in gastric cells under conditions of oxidant stress.
Gastrin-dependent activation of the HDC promoter in AGS-B cells has previously been shown by our group to occur through a signaling pathway that involves both Raf-1 and MAP kinase (
). In contrast, the stimulation of ERK activity by H2O2 in AGS-B cells occurs at least in part through activation of the Ras/EGFR pathway (Fig. 11). The increase in EGFR phosphorylation was observed only with H2O2stimulation and was not seen with gastrin or PMA stimulation. In addition, expression of the dominant-negative Ras and dominant-negative EGFR construct significantly inhibited H2O2stimulation of the HDC promoter, but did not affect gastrin- or PMA-stimulated HDC expression. In contrast, PKC blockade or down-regulation effectively blocked both PMA and gastrin stimulation of the HDC promoter, but did not inhibit stimulation of HDC transcription by H2O2. Thus, while both gastrin/PMA and H2O2 pathways leading to induction of HDC appear to converge on Raf-1 kinase and the MAP kinases, the H2O2 pathway is distinguished by its utilization of EGFR and Ras, and the lack of dependence on PKCs (Fig. 11). The mechanism by which H2O2 stimulates EGFR phosphorylation is unclear, although some have speculated that it may involve inhibition of a protein tyrosine phosphatase or the activation of intracellular protein tyrosine kinases such as Src (
). Oxidant-induced EGFR tyrosine phosphorylation may be involved in protective responses to oxidant-induced cell injury in the gastric mucosa, possibly through activation of MAP kinases (
Figure 11Putative H2O2-dependent signaling pathways leading to HDC promoter activation.H2O2-induced proximal signals activate the EGFR and Ras, resulting in activation of ERK cascades and HDC transactivation. In contrast, gastrin activates the CCK-B/gastrin receptor and PKC; both pathways converge on the level of Raf. (Gα, β, and γ, G proteins of the α, β, and γ subfamily;AP1, activating protein 1).
Deletion analysis of the human HDC promoter showed that the majorcis-acting region mediating responsiveness to H2O2 could be mapped to the GAS-RE element (2 to 24) which mediates response to gastrin and PMA (
). Thus, the GAS-RE appears to belong to the growing family of H2O2-responsive elements.
The activation of HDC transcription by H2O2 may have some human biologic significance, given the importance of oxidant stress in the pathogenesis of diverse stomach disorders including gastritis, peptic ulcer disease, and gastric cancer. In particular,H. pylori-associated gastritis has been associated with activated neutrophils and oxidant stress (
). The role of HDC activation and histamine generation in the response to oxidant stress of the gastrointestinal tract remains unclear, although several studies have suggested that histamine synthesized by HDC may facilitate healing of the gut mucosa (
). In addition, a role in acid secretion has been suggested by recent studies showing that H2O2 at low concentrations is capable of stimulating acid secretion by sevenfold in isolated gastric mucosa (
). Analysis of HDC promoter activation by H2O2 should provide insight into the molecular events characterizing the adaptive response of gastric cells to oxidant stress.
ACKNOWLEDGEMENT
Oligonucleotides were supplied by the Center for the Study of Inflammatory Bowel Disease.
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