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Am J Physiol Gastrointest Liver Physiol 290: G970-G979, 2006. First published January 6, 2006; doi:10.1152/ajpgi.00461.2005
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MUCOSAL BIOLOGY

Parietal cell hyperstimulation and autoimmune gastritis in cholera toxin transgenic mice

Lymari Lopez-Diaz,1,2,* Karen L. Hinkle,2,* Renu N. Jain,2 Yana Zavros,4 Cynthia S. Brunkan,2 Theresa Keeley,2 Kathryn A. Eaton,3 Juanita L. Merchant,1,2,4 Catherine S. Chew,5 and Linda C. Samuelson1,2

1Cellular and Molecular Biology Program, 2Department of Molecular and Integrative Physiology, 3Unit for Laboratory Animal Medicine, and 4Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan; and 5Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia

Submitted 3 October 2005 ; accepted in final form 30 December 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The stimulation of gastric acid secretion from parietal cells involves both intracellular calcium and cAMP signaling. To understand the effect of increased cAMP on parietal cell function, we engineered transgenic mice expressing cholera toxin (Ctox), an irreversible stimulator of adenylate cyclase. The parietal cell-specific H+,K+-ATPase beta-subunit promoter was used to drive expression of the cholera toxin A1 subunit (CtoxA1). Transgenic lines were established and tested for Ctox expression, acid content, plasma gastrin, tissue morphology, and cellular composition of the gastric mucosa. Four lines were generated, with Ctox-7 expressing ~50-fold higher Ctox than the other lines. Enhanced cAMP signaling in parietal cells was confirmed by observation of hyperphosphorylation of the protein kinase A-regulated proteins LASP-1 and CREB. Basal acid content was elevated and circulating gastrin was reduced in Ctox transgenic lines. Analysis of gastric morphology revealed a progressive cellular transformation in Ctox-7. Expanded patches of mucous neck cells were observed as early as 3 mo of age, and by 15 mo, extensive mucous cell metaplasia was observed in parallel with almost complete loss of parietal and chief cells. Detection of anti-parietal cell antibodies, inflammatory cell infiltrates, and increased expression of the Th1 cytokine IFN-{gamma} in Ctox-7 mice suggested that autoimmune destruction of the tissue caused atrophic gastritis. Thus constitutively high parietal cell cAMP results in high acid secretion and a compensatory reduction in circulating gastrin. High Ctox in parietal cells can also induce progressive changes in the cellular architecture of the gastric glands, corresponding to the development of anti-parietal cell antibodies and autoimmune gastritis.

stomach; gastric acid secretion; cAMP signaling; mucous neck cell; gastrin

AGONIST STIMULATION OF ACID secretion from parietal cells involves both calcium and cAMP intracellular signaling cascades. The primary acid agonists are the hormone gastrin, the neurotransmitter acetylcholine, and the paracrine factor histamine. Both gastrin binding to the cholecystokinin-2 (CCK2) receptor and acetylcholine binding to the muscarinic-3 (M3) receptor primarily couple to Gq to stimulate increased intracellular calcium (7, 10, 39). In contrast, histamine binding to the histamine-2 (H2) receptor couples primarily to Gs, which activates adenylate cyclase and evokes an increase in cAMP (8, 9), although it has been shown in some species, including the mouse, to also increase calcium (10, 17). It is not clear what independent roles these signaling pathways have for acid secretion in vivo. Eating a meal normally results in release of all three agonists. Thus parietal cell activation is generally associated with both calcium and cAMP signaling, with synergistic interactions between these two pathways enhancing acid secretion (34).

Elevation of intracellular calcium and cAMP activates signaling cascades that lead to cellular changes involved in acid secretion, including the movement of the proton pump, H+,K+-ATPase, from an intracellular tubulovesicular compartment to the canalicular membrane at the apical surface. Increased cAMP leads to activation of protein kinase A (PKA) and phosphorylation of effector proteins, several of which have been associated with the cytoskeleton and tubulovesicular or apical membranes (40). One proposed effector is LASP-1 ("LIM and SH3 domain-containing protein-1"), which is phosphorylated upon histamine stimulation, with increased phosphorylation closely correlated with acid secretion in isolated parietal cells (12, 13). Upon PKA phosphorylation, LASP-1 is translocated to the actin-rich canalicular membrane, the site of active HCl secretion (11, 12).

Several genetically engineered mouse models with decreased agonist stimulation have been described, including gastrin- and CCK2 receptor-deficient mice (15, 21, 23, 26), H2 receptor-deficient mice (20), and M3 receptor-deficient mice (1). In general, loss of an acid secretagogue or its receptor results in reduced stimulation of the parietal cell and reduced acid secretion, although the details differ markedly depending on the pathway that is affected (31). Fewer mouse models of constitutive agonist activation have been generated. The "INS-GAS" transgenic model exhibits increased circulating gastrin resulting from the expression of a human gastrin transgene in pancreatic beta-cells through use of the rat insulin I promoter (38). Initially, constitutively elevated gastrin increased both parietal cell number and acid secretion, which is consistent with hyperstimulation of the parietal cell. However, as the transgenic mice aged, the increased acid secretion was lost as gastric atrophy developed, resulting in the loss of parietal cells and the development of mucous cell hyperplasia (36). The basis for the atrophy and abnormal mucous cell expansion is unclear; however, a similar phenotype was observed in a second gastrin transgenic mouse model that exhibited a sixfold elevation of amidated gastrin (22). Increased gastrin would be expected to also increase histamine due to gastrin-stimulated release of histamine from enterochromaffin-like cells. Thus it is not clear what component of the phenotype is due to gastrin signaling and what is due to histamine signaling in the parietal cell.

Our objective in this study was to understand the effects of increased intracellular cAMP on parietal cells and acid secretion in vivo. Transgenic mice with constitutively elevated cAMP were generated using the H+,K+-ATPase beta-subunit promoter to direct expression of the A1 subunit of cholera toxin (CtoxA1) to parietal cells. As an irreversible stimulator of adenylate cyclase, Ctox effectively increased intracellular cAMP in the parietal cells of the transgenic mice. We report here the consequences of constitutively high intracellular cAMP on gastric acid secretion, gastrin levels, and gastric histology. Surprisingly, hyperstimulation of the parietal cells in a highly expressing Ctox transgenic line resulted in the development of atrophic gastritis with anti-parietal cell antibodies and increased expression of IFN-{gamma}, thus defining a new mouse model of autoimmune gastritis (AIG).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Generation of Ctox transgenics. The H/K-Ctox transgene construct contained the CtoxA1 subunit driven by the mouse H+,K+-ATPase beta-subunit promoter (–1,035 to +24) followed by human growth hormone sequences (hGH), which provided introns and a poly A+ site (Fig. 1A). To construct the transgene, a 2.1-kb hGH-NotI/BamHI fragment from plck-hGH (25) was inserted into pHKATP-hGH1 (24) digested with BamHI and EcoRV. A 0.6-kb Ctox-BamHI fragment was excised from the GHCT plasmid (6) and subcloned into the BamHI site downstream of H+,K+-ATPase. Following DNA sequencing and verification of the construct, the 3.8-kb transgene was excised from the vector with HindIII and SacII and submitted to the University of Michigan Transgenic Animal Model Core for microinjection into F2 zygotes from C57BL/6 x SJL parents.


Figure 1
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  		Fig. 1. Generation of Ctox transgenic mice. A: diagram of the H/K-Ctox transgene. The cholera toxin A1 subunit (CtoxA1) was placed under the control of the mouse H+,K+-ATPase beta-subunit promoter (–1,035 to +24). The human growth hormone (hGH) cassette provided intron (open boxes) and polyadenylation sequences. The approximate locations of primers for genotyping transgenic mice are indicated (arrowheads) together with the PCR product. B: Ctox expression in the stomach was determined by quantitative RT-PCR (QRT-PCR) in lines Ctox (CT) -1, -7, -8 and -14. Transgene mRNA signals in transgenic lines were compared with background levels in nontransgenic (Ntg) mice and displayed as fold-increase over the Ntg negative control. Values are shown as means ± SE with n = 3, and mean for n = 2. C: RT-PCR analysis of Ctox expression in various tissues of Ctox-7 transgenics. Amplification of c-abl was used as a control to assess sample quality.

 
Transgenic mice were identified by PCR amplification of a 503-bp product from genomic DNA extracted from tail biopsies using the following primers: CtoxF, 5'-GAGCCGTGGATTCATCATGCA and hGHR, 5'-GCGCGGAGCATAGCGTTGTCA (Fig. 1A). PCR assays (25 µl) included Promega Buffer B with 400 nM of each primer, 200 µM dNTPs, 2.5 mM MgCl2, and 1 U Taq polymerase (Promega) using the following cycle parameters: 94°C, 5 min; 35 cycles at 94°C, 65°C, and 72°C, each for 45 s; and 72°C, 5 min.

Ctox transgenic lines were housed in a specific pathogen-free environment as hemizygotes by continued breeding to C57BL/6J. Nontransgenic (Ntg) littermates were used for controls. Before analysis, mice were fasted overnight with access to water ad libitum. Mouse use was approved by the University of Michigan Committee on Use and Care of Animals.

QRT-PCR/RT-PCR analysis. Ctox transcript levels were determined by quantitative RT-PCR (QRT-PCR). RNAs were isolated from various tissues using TRIzol reagent (Invitrogen), followed by DNase treatment and purification with the RNeasy Mini kit (Qiagen). RT reactions (50 µl) used 1 µg RNA and TaqMan RT (Applied Biosystems) or the Iscript cDNA synthesis kit (Bio-Rad), as recommended by the manufacturer. Quantitative PCR to generate a 244-bp product was performed using the Icycler (Bio-Rad) with a 20-µl reaction in PCR buffer (Invitrogen) containing 2 µl of cDNA (RT product), 5.5 mM MgCl2, 100 nM primers (CtoxF and hGHR), 200 nM dNTPs, 0.1x SYBR Green, 10 nM fluorescein, and 0.025 U Platinum Taq (Invitrogen). Expression levels were normalized to GAPDH expression, which was determined by amplification with the primers 5'-TCAAGAAGGTGGTGAAGCAGG and 5'-TATTATGGGGGTCTGGGATGG. The cycle parameters were as follows: 95°C, 1 min; 33 cycles at 95°C, each for 9 s; 60°C, 1 min; and final elongation at 55°C, 1 min. After amplification was completed, the samples were subjected to melt-curve analysis by increasing the temperature from 60°C to 100°C, in 0.5°C intervals every 10 s for 80 steps to assess product purity. No signal was detected with control samples that were not treated with RT (not shown).

For QRT-PCR analysis of IFN-{gamma} mRNA abundance, fundic RNA was isolated from mice aged 6–12 mo, as described above. Three groups of mice were analyzed, including Ntg, Ctox-7 mice without autoantibodies but with inflammation, and Ctox-7 mice with both autoantibodies and severe inflammatory infiltrates (n = 3 mice/group). RT reactions (20 µl) used 2 µg RNA and the Iscript cDNA synthesis kit (Bio-Rad), and quantitative PCR was performed with IFN-{gamma} primer sequences from Overbergh et al. (29) in a 20-µl reaction containing 4 µl of RT product with the cycle parameters described above.

For RT-PCR, RNA samples from a variety of tissues (5 µg) were treated with RNase-free DNase (1 U; Roche) for 30 min at 37°C, followed by heat inactivation for 5 min at 75°C, and RT using the SuperScript II RT Kit (Life Technologies). Ctox PCR reactions used primers 5'-GGCACGATGATGGATATGTTTCCACCTCAA and 5'-TACTGGGCTTACATGGCGATACTCATTCAG. The c-abl transcript was amplified as a positive control with primers 5'-TTTATGGGGCAGCAGCCTGGAAAAGTA and 5'-CCAGCGAGAAGGTTTTCCTTGGAGTT. RT-PCR products for Ctox (569 bp) and c-abl (239 bp) were detected by agarose gel electrophoresis. No signal was detected with control samples that were not treated with RT (not shown).

Ctox immunodetection. Dispersed fundic mucosal cells were prepared from Ctox-7 and Ntg mice by pronase digestion as described (17), fixed and permeabilized with the CytoFix/CytoPerm kit (Pharmingen), and cytospun onto slides. Cells were immunostained with a Ctox antibody that had been preabsorbed on paraffin sections from Ntg stomach to reduce nonspecific binding. For preabsorption, Ntg sections were deparaffinized, rehydrated, fixed in 4% paraformaldehyde in PBS for 5 min, rinsed in PBS, incubated with 50 mM ammonium chloride for 30 min, rinsed, and blocked with 10% donkey serum in 0.5% Triton X-100 in PBS (TPBS) for 30 min at room temperature, followed by incubation for 3 h at room temperature with rabbit antiserum against Ctox (1:50 in 2% donkey serum in 0.01% TPBS; Accurate, BYA21101). The absorbed antiserum was collected and used (diluted an additional 1:5 in TPBS) to costain dispersed mucosal cells from Ctox-7 and Ntg with a mouse monoclonal H+,K+-ATPase {alpha}-subunit antibody (1:500; Medical and Biological Laboratories). Slides were costained for 1.5 h, as described above, followed by secondary antibody staining for 30 min using a donkey anti-rabbit Cy3 (1:200; Jackson ImmunoResearch Laboratories) and donkey anti-mouse Cy2 (1:200; Jackson ImmunoResearch Laboratories), placed on a coverslip with Fluoromount-G (Southern Biotechnology Associates) containing 1 µg/ml 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), and imaged on a Nikon Eclipse E800 microscope equipped with a Spot camera (Diagnostic Instruments).

Phosphoprotein analysis. For LASP-1 analysis, protein extracts were prepared from fundic mucosal scrapings of Ctox-7 and Ntg mice (6–10 wk old) following ranitidine (10 mg/kg) or histamine (20 mg/kg) treatment. The ranitidine group was injected intraperitoneally 24, 14, and 2.5 h before death, while the histamine group was injected with PBS at those times followed by histamine 30 min prior to death. Mucosa was isolated by scraping the tissue with a scalpel blade and then homogenized by passing through a syringe and progressively smaller needles (18–25 gauge) in 0.1 ml buffer [50 mM beta-glycerophosphate (pH 7.3), 1.5 mM EGTA, 5 mM EDTA, 1 mM DTT, 0.2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride] and a protease-inhibitor cocktail containing leupeptin (2 µg/ml), antipain (2 µg/ml), benzamidine (20 µg/ml), aprotinin (0.01–0.02 U/ml trypsin inhibitor), chymostatin (2 µg/ml), and pepstatin A (2 µg/ml). An aliquot was saved for protein determination (Lowry assay; Bio-Rad), and two-dimensional (2D) Western blot analysis was performed as previously described (11). In brief, an equal volume of 3% SDS and 10% beta-mercaptoethanol was added, and samples were snap-frozen and stored at –80°C. After precipitation of protein with acetone at room temperature, pellets (0.25 mg protein) were dissolved in rehydration buffer {8 M urea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 18 mM DTT, 0.5% IPG buffer (pH 3–10; Amersham-Pharmacia), and 0.001% bromphenol blue} and subjected to isoelectric focusing (IPG strips; pH 3–10, nonlinear) with an IPGPhor isoelectric focusing unit (Amersham Pharmacia Biotech) followed by SDS-PAGE. Resolved proteins were transferred to nitrocellulose for Western blot analysis using the LASP-1 MAb (clone 8C6) (12) and enhanced chemiluminescence detection (Amersham Pharmacia Biotech). Changes in LASP-1 phosphorylation were defined based on the acidic shift of entrained spots (13), which were quantitated with a CCD-based Syngene GeneGnome system (11).

For analysis of cAMP response element binding protein (CREB) phosphorylation, fundic homogenates were prepared in buffer containing 10 mM Tris·HCl (pH 7.5), 140 mM NaCl, 5 mM EDTA, 1% SDS, 100 mM Na3VO4, DMSO (0.2%), and the protease-inhibitor cocktail listed above. Protein (20 µg/lane) was separated on 12% polyacrylamide gels and electrotransferred to PVDF membrane (Osmonics). Membranes were incubated overnight at 4°C with phospho-CREB or CREB antibodies (1:1,000; Upstate Biotechnology), followed by incubation in anti-rabbit IgG horseradish peroxidase (1:10,000; Amersham), treated with chemiluminescence substrates, and visualized and quantitated on a Fluor-S MultiImager system with Quantity One 4.2 software (Bio-Rad). Phospho-CREB data was normalized to CREB levels for each sample and reported as fold increase over Ntg levels.

Basal gastric acid content. Gastric acid content was measured in Ctox-7 (4-wk-old) and Ctox-8 (8-wk-old) transgenic mice and age-matched Ntg littermates. Stomachs were removed and cut along the greater curvature, and the contents were rinsed in 2 ml 0.9% NaCl (pH 7.0). Samples were centrifuged for 10 min at 5,000 rpm to collect clear supernatants, and hydrogen ion concentration and pH were measured with a PHM290 pH-Stat Controller titration system (Radiometer) using 0.005 N NaOH. The results were normalized to body weight (kg).

Gastrin RIA. Blood was collected from fasted mice by terminal eye bleeds into heparinized tubes, and plasma was collected by centrifugation at 4°C and stored at –20°C until assayed. Gastrin RIA was performed using [125I]15Met human G-17 label and antiserum 1296 (CURE, UCLA), as previously described (41). This antiserum recognizes carboxy-terminal gastrin peptides and measures sulfated and nonsulfated G-17 identically.

Flow-cytometric analysis of parietal cells. Parietal cells from 8-wk-old Ctox-7 or Ntg mice were quantitated with a Becton-Dickinson FACSVantage SE cell sorter as described in Hinkle et al. (17). Briefly, cells in the fundic mucosa were dispersed with pronase, fixed, and permeabilized using the Cytofix/Cytoperm kit (Pharmingen), and immunostained with either an H+,K+-ATPase {alpha}-subunit MAb (1:100; Medical and Biological Laboratories) or a cytokeratin-18 polyclonal antibody (1:10; ICN) and appropriate secondary antibodies labeled with Cy2 (Jackson ImmunoResearch Laboratories). The proportion of H+,K+-ATPase-positive parietal cells in the cytokeratin-18-positive epithelial cell population was calculated after analysis of 10,000 cells. Three mice for each genotype were analyzed.

Histochemical analysis and detection of autoantibodies. Stomachs were dissected, opened along the greater curvature, pinned flat on dental wax, and fixed in Carnoy's solution or 4% paraformaldehyde. Paraffin sections (3–5 µm) were immunostained with an H+,K+-ATPase alpha subunit MAb (1:500; Medical and Biological Laboratories), a polyclonal antibody to intrinsic factor (1:2,000 rabbit anti-human intrinsic factor, gift from Dr. David Alpers, Washington University, St. Louis, MO), or a polyclonal antibody to trefoil factor 2 [TFF2 (spasmolytic protein 1)] (1:50 rabbit anti-TFF2/SP, gift from Dr. Andrew Giraud, University of Melbourne, Melbourne, Australia). For staining, deparaffinized and rehydrated sections were subjected to antigen retrieval for 10 min in Antigen Unmasking solution (Vector Labs) at 100°C, and, after cooling, endogenous peroxidase activity was quenched with 3% H2O2 in methanol, followed by blocking in 20% goat serum in TPBS (0.01% Triton X-100 in PBS) before incubation with primary antibody in TPBS. Slides were rinsed and subsequently treated with biotin-conjugated secondary antibodies (1:200; Vector Labs) for 30 min at room temperature. Mucous neck cells were stained for 1.5 h at 37°C with biotin-conjugated Griffonia (Bandeiraea) simplicifolia lectin II (GS II) (1:1,000, Vector Labs) on sections that had been blocked as described above. To visualize biotin staining, the Vectastain Elite ABC kit was used with diaminobenzidine as substrate (Vector Laboratories), and counterstained with hematoxylin. Paraffin sections were also stained with hematoxylin and eosin (H&E) to visualize cellular morphology, and periodic acid-Schiff (PAS)/Alcian blue stain was used to visualize neutral (pink) or acidic (blue) mucin, respectively. A group of H&E-stained Ctox-7 paraffin sections were scored blind by a comparative pathologist for inflammatory cell infiltrates and epithelial cell changes in the oxyntic mucosa. Sections were scored according to extent (percent affected mucosa) and severity (mild, moderate, or severe). The scoring was a modification of a previously described method (14).

Ctox and Ntg plasma was collected as described above and tested for anti-parietal cell antibodies by incubation on paraffin sections from wild-type mouse stomach using a modification of the method described in Alderuccio et al. (2). Sections were subjected to antigen retrieval followed by blocking in 10% donkey serum in TPBS containing 1% BSA. Plasma was diluted in PBS (1:10–1:100) and incubated for 30 min at room temperature, followed by donkey anti-mouse Cy2-conjugated secondary antibody (1:500, Jackson ImmunoResearch Labs) staining. Anti-parietal cell antibody titers were estimated as low, medium, or high based on the intensity of fluorescence and dilution range for detection.

Statistical analysis. Data are presented as means ± SE and analyzed using Student's t-test with P < 0.05 considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Generation of Ctox transgenic lines. To study the role of cAMP for parietal cell function, a transgenic strategy was utilized to constitutively elevate parietal cell cAMP with the CtoxA1 driven by the mouse H+,K+-ATPase-beta-promoter (Fig. 1A). Microinjection of the H/K-Ctox construct resulted in 14 founder mice that contained the transgene, 4 of which were bred to establish transgenic lines (Ctox-1, -7, -8, and -14). Analysis of transgene expression by QRT-PCR showed that these four transgenic lines expressed Ctox mRNA in the parietal cell-rich fundic region of the stomach, with 40- to 70-fold higher levels in Ctox-7 (Fig. 1B). Tissue specificity was determined by RT-PCR analysis of nine tissues, with abundant Ctox mRNA detected in the fundus (Fig. 1C). Further QRT-PCR analysis of thymus, kidney, liver, and fundus showed that among these tissues, expression was limited to the acid-secreting portion of the stomach (data not shown). Analysis of cellular expression specifically localized Ctox to the parietal cells by immunocolocalization of cholera toxin with the parietal cell-specific H+,K+-ATPase (Fig. 2). Quantitation of coexpression of the two antibodies showed that 100% of the Ctox-7 parietal cells expressed the transgene (60/60 cells), and Ctox expression was not observed in H+,K+-ATPase-negative cells. Ctox transgenic mice appeared grossly normal, and the body and wet stomach weight of the transgenics did not differ from control Ntg mice (data not shown).


Figure 2
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  		Fig. 2. Immunolocalization of Ctox protein in transgenic parietal cells. Dispersed fundic mucosal cells from Ctox-7 (A–C) and Ntg (D–F) mice were costained with antibodies for H+,K+-ATPase (A and D; green) and cholera toxin (B and E; red). Merging the images (C and F) demonstrated colocalization of Ctox and H+,K+-ATPase staining in Ctox-7 parietal cells, with no Ctox staining observed in H+,K+-ATPase-negative cells (C) or Ntg controls (F). Nuclear 4,6-diamidino-2-phenylindole (DAPI) staining (blue) was used to visualize all of the cells in the preparation.

 
Increased cAMP signaling in Ctox-transgenic parietal cells. CtoxA1 catalyzes the ADP ribosylation of the {alpha}-subunit of Gs, locking Gs in its activated form, resulting in constitutively increased levels of cAMP (30). To test whether the cAMP pathway is hyperstimulated in the parietal cells of Ctox transgenic mice, we analyzed phosphorylation of the downstream target LASP-1. Ctox-7 and Ntg mice were stimulated with histamine, and LASP-1 phosphorylation was determined by 2D Western blot analysis of fundic mucosal protein extracts. The stimulated samples were compared with samples prepared from unstimulated mice treated with the H2-receptor antagonist ranitidine (Fig. 3). Three different LASP-1 isoforms were detected, with the abundance of the more acidic, phosphorylated form ("c") increased in Ntg controls after histamine stimulation. In contrast, LASP-1 was hyperphosphorylated in unstimulated Ctox-7 mice. Quantification of two independent samples showed that the hyperphosphorylated LASP-1 form c was increased 4.8-fold in unstimulated Ctox-7 mice compared with unstimulated Ntg controls (Fig. 3B). Moreover, histamine stimulation did not further increase LASP-1 phosphorylation in Ctox-7 transgenics. Increased LASP-1 phosphorylation in unstimulated Ctox-7 transgenic mice suggests that PKA is constitutively activated in the transgenic parietal cells. This was also shown by increased CREB phosphorylation in Ctox-7 mice. Quantitative Western blot analysis of fundic protein homogenates showed that although total CREB levels were unchanged, phospho-CREB was increased twofold in Ctox-7 compared with Ntg controls.


Figure 3
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  		Fig. 3. Hyperphosphorylation of LASP-1 ("LIM and SH3 domain-containing protein-1") in Ctox-7 transgenic stomach. A: fundic mucosal protein extracts were analyzed by two-dimensional (2D) Western blot using a LASP-1 monoclonal antibody. Ntg and Ctox-7 mice stimulated with histamine (His) were compared with mice treated with the H2-receptor antagonist ranitidine (Ran). Representative blots from individual mice are shown. LASP-1 was detected in three isoforms (a, b, c; circled), with the more acidic, hyperphosphorylated form "c" increasing in abundance in Ntg mice after histamine stimulation. B: graphic representation of quantification of LASP-1 2D Western blots. Two mice for each treatment group and genotype were analyzed. The averages are shown as bars with individual sample values displayed as a scatter plot.

 
Increased basal acid content and decreased gastrin in Ctox transgenics. To determine whether increased cAMP signaling resulted in increased gastric acid production, basal acid content was measured in transgenic mice from the highly expressing line Ctox-7 and the lower expressing line Ctox-8. Increased acid was observed in both transgenic strains compared with Ntg controls, with basal acid content increased 60% in Ctox-7 mice and 50% in Ctox-8 (Fig. 4A). Increased gastric acid secretion would be expected to result in a compensatory down regulation of circulating gastrin, since gastrin secretion is regulated by gastric acid content (35). Accordingly, plasma gastrin levels were analyzed by RIA. As would be predicted from high gastric acid secretion, both Ctox-7 and Ctox-8 had low circulating gastrin at ~25% of Ntg controls. This result indicates that the increased acid in Ctox transgenics resulted in a negative feedback for gastrin release into the bloodstream.


Figure 4
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  		Fig. 4. Higher basal gastric acid content and reduced plasma gastrin detected in Ctox transgenic mice. A: stomach contents were collected from fasted mice, and hydrogen ion concentrations were determined by automated titration. Acid content was normalized to body weight, and values are shown as means ± SE (n = 13–27; *P < 0.05). B: plasma gastrin concentration was measured in 8-wk-old Ntg, Ctox-7, and Ctox-8 mice by RIA. Values are shown as means ± SE (n = 6–12; *P < 0.0001).

 
Normal parietal cell numbers in young Ctox-7 transgenic mice. To determine whether Ctox expression and increased acid secretion in Ctox-7 mice are associated with cellular changes in the gastric mucosa, paraffin sections were examined after H&E staining. At 2 mo of age, the cellular architecture and height of the mucosa appeared grossly normal (Fig. 5, A and E). In addition, the major cell types recognizable in H&E-stained sections, including parietal cells, chief cells, and surface mucous cells, appeared to have similar distributions in young Ctox transgenics and Ntg mice. Since agonist stimulation can be associated with increased cell proliferation, parietal cell number was determined in the fundic mucosa of 2-mo-old Ctox-7 and Ntg mice by flow cytometry. The transgenic mice had normal proportions of parietal cells, with Ctox-7 and Ntg having 10.7 ± 1.5% and 12.5 ± 2.0% parietal cells, respectively, in the fundic mucosa. Thus parietal cell number and gross fundic histology were not altered by Ctox expression.


Figure 5
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  		Fig. 5. Development of atrophic gastritis in aging Ctox-7 transgenics. Representative gastric paraffin sections from Ntg (A–D) and Ctox-7 transgenics (E–H). Hematoxylin and eosin stained sections from 2-mo-old (A and E) and 15- to 16-mo-old (B and F) mice demonstrate the normal fundic histology in young Ctox-7 mice and the marked changes as they age, including inflammatory cell infiltrates (arrowhead), loss of parietal and chief cells, and expansion of an aberrant mucous cell type. One patch of light-stained aberrant cells in the older Ctox-7 sections is indicated with an arrow (F). C and G: parietal cells were visualized in older Ntg (16 mo) and Ctox-7 (15 mo) fundic sections by immunostaining with a monoclonal antibody to H+,K+-ATPase and counterstained with Harris hematoxylin. D and H: chief cells were visualized in older Ntg (16 mo) and Ctox-7 (15 mo) fundic sections by immunostaining with an antiserum to intrinsic factor and counterstained with hematoxylin.

 
Atrophic gastritis and mucous neck cell expansion in aging Ctox-7 transgenics. In contrast to the normal fundic morphology in young transgenics, there were marked cellular changes in some Ctox-7 mice as they aged, with the emergence of large patches of light-staining cells in the central and basal regions of the gastric gland. By 15 mo of age the pathology was quite remarkable; with the exception of surface mucous cells, the mucosa was taken over by this aberrant cell type (Fig. 5F). Accordingly, normally abundant cell types, including parietal and chief cells, were almost absent in affected older Ctox-7 transgenics as determined by immunostaining for the {alpha}-subunit of H+,K+-ATPase and intrinsic factor, respectively (Fig. 5). Inflammatory cells in the fundic mucosa were also associated with the presence of the aberrant mucosal cells (Figs. 5 and 6, arrowheads).


Figure 6
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  		Fig. 6. Mucous neck cell expansion and expression of Alcian blue-positive mucus in Ctox-7 transgenics. Replicate gastric paraffin sections from older Ntg (A–C) and Ctox-7 transgenic (D–F) mice were stained with Harris hematoxylin (A and D), periodic acid-Schiff (PAS)/Alcian blue (B and E), and Griffonia (Bandeiraea) simplicifolia lectin II (GS II) (C and F). E: the aberrant Ctox-7 cells stained strongly with PAS/Alcian blue, with both pink (gastric-type) and blue (intestinal-type) mucus apparent. F: Ntg and Ctox-7 sections were stained for mucous neck cells with GS II and counterstained with hematoxylin. Inflammatory cells were evident in the mucosa of the Ctox-7 transgenic (arrowheads; D and F).

 
PAS/Alcian blue staining showed that the aberrant cells were filled with mucus, with both neutral (pink) and acidic (blue) mucus observed in the 15-mo-old Ctox-7 transgenic (Fig. 6). Alcian blue-stained mucus is normally characteristic of more distal regions of the gastrointestinal tract, including the intestine. The aberrant cells also stained with GS II and TFF2 (data not shown), which in normal fundus are markers of mucous neck cells (Fig. 6, C and F).

To determine the time line for the mucosal changes, we examined Ctox-7 mice at various ages (Fig. 7). Small patches of aberrant mucous cells were observed as early as 3 mo of age in some mice (data not shown). There was a progressive expansion of these aberrant mucous cells, starting from the midsection of the gland, and then expanding to the deep base of the glands and up toward the surface mucous cells. Associated with the expanded mucous cell patches was a progressive atrophy as shown by the loss of parietal cells detected by immunostaining for H+,K+-ATPase (Fig. 7). Expression of Alcian blue-positive mucus develops with a slower time line, with staining first detected at 12 mo of age.


Figure 7
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  		Fig. 7. Progressive atrophy associated with mucous neck cell expansion in Ctox-7 transgenics. Paraffin sections from Ctox-7 transgenic mice aged 2, 7, 12, and 15 mo are shown after immunostaining for parietal cells with an antibody to H+,K+-ATPase (top) or staining with PAS/Alcian blue (bottom). Aberrant mucous cells (arrows) are seen in small patches in the midsection of the gastric glands by 7 mo of age, and larger patches containing mixed pink (gastric-type) and blue (intestinal-type) mucus are seen by 12 mo (arrowheads). Loss of parietal cells occurs in concert with expansion of the aberrant mucous cells.

 
Development of anti-parietal cell antibodies. One of the mechanisms for development of atrophic gastritis in mouse and human involves autoimmune destruction of the gastric tissue. AIG is associated with the development of antibodies directed against the parietal cell and an inflammatory cell infiltrate reactive to parietal cells. To determine if atrophic gastritis in Cox-7 mice is due to autoimmunity, we tested Ctox-7 plasma for anti-parietal cell antibodies by immunostaining normal stomach sections. We confirmed the presence of anti-parietal cell antibodies in transgenic mice containing the histopathology (Fig. 8). We screened a total of 93 Ctox-7, 36 Ctox-8, and 58 Ntg mice of various ages. There were no autoantibodies detected in Ntg and Ctox-8 mice, whereas 16 of the Ctox-7 mice had anti-parietal cell antibodies (17%). Older Ctox-7 transgenics had the highest incidence of autoantibodies with 7 of 21 mice greater than 12 mo of age testing positive for anti-parietal cell antibodies (33%).


Figure 8
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  		Fig. 8. Development of autoimmune gastritis in Ctox-7 transgenic mice. A–C: anti-parietal cell antibodies were detected by immunohistochemistry. Wild-type paraffin stomach sections were immunostained with different mouse plasmas followed by donkey anti-mouse Cy2-conjugated secondary antibody and fluorescence microscopy to visualize immunoreactivity. A: control staining with a mouse monoclonal antibody to H+,K+-ATPase {alpha}-subunit (diluted 1:100) showed the pattern of parietal cells in the tissue sections. B: plasma from a 9-mo-old Ctox-7 transgenic mouse (diluted 1:100) stained parietal cells similar to the control, indicating the presence of anti-parietal cell antibodies. C: plasma from a 12-mo-old Ntg mouse (diluted 1:10) did not stain the cells in the section. D–F: lymphoid follicles were observed in Ctox-7 transgenics containing anti-parietal cell antibodies, including a high titer (9 mo; D) and two medium titer (6 mo; E; 12 mo, F) mice. G–I: less severe inflammatory infiltrates were observed in the fundic mucosa of Ctox-7 transgenic mice (6–9 mo) that did not contain detectable anti-parietal cell antibodies.

 
A group of 29 Ctox-7 transgenic mice 6–14 mo of age were examined for both autoantibodies and histological evidence of inflammation. Anti-parietal cell antibodies were detected in four mice in this group. Large lymphoid follicles penetrating the oxyntic mucosa were observed in three of these mice, which were those transgenics that contained the highest anti-parietal cell antibody titers (Fig. 8, D–F). Plasma cells were also readily observed in the inflammatory infiltrates in these three mice. The lymphoid follicles were observed in Ctox-7 transgenics with anti-parietal cell antibodies, but not in transgenic mice without autoantibodies or in 28 Ntg mice of similar age. Thus morphological evidence of a severe immune response in the fundic mucosa correlated with the development of autoantibodies. However, a few Ctox-7 transgenic mice with less severe inflammatory infiltrates were identified that did not have anti-parietal cell antibodies (Fig. 8, G–I), suggesting that the cellular response can be dissociated from the humoral response and may precede autoantibody development. Gastric atrophy and mucous cell metaplasia were limited to mice that contained autoantibodies.

Anti-parietal cell antibodies and immune infiltrates in Ctox-7 mice suggest an autoimmune mechanism for the atrophic gastritis. AIG is characterized by a predominant Th1 response. Thus we measured expression of the Th1 cytokine IFN-{gamma} in Ctox-7 mice with immune infiltrates compared with Ntg mice. QRT-PCR analysis showed that Ctox-7 mice with anti-parietal cell antibodies had a 8.3-fold increase in IFN-{gamma} expression over Ntg controls (Fig. 9). The Ctox-7 mice with less severe inflammatory infiltrates and no anti-parietal cell antibodies had a 4.5-fold increased IFN-{gamma} expression, although this was not statistically significant. The detection of increased expression of the Th1 cytokine IFN-{gamma} is consistent with an AIG mechanism for destruction of the tissue.


Figure 9
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  		Fig. 9. Increased expression of the Th1 cytokine IFN-{gamma} in Ctox-7 transgenics with gastritis. Quantitative reverse transcription-PCR analysis of IFN-{gamma} mRNA abundance in fundic RNA samples. Ctox-7 mice with moderate inflammation and no detectable anti-parietal cell antibodies (Ab–), and Ctox-7 mice with large lymphoid follicles and anti-parietal cell antibodies (Ab+) were compared with Ntg controls. Three independent samples were analyzed for each group of mice, and values were normalized to Gapdh expression and shown as fold-change (means ± SE) compared with Ntg levels (*P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study we report a new transgenic mouse model with constitutively high parietal cell cAMP resulting from cell-specific expression of CtoxA1. Ctox catalyzes the ADP-ribosylation of Gs, to lock it in an activated form, which results in continuous activation of adenylate cyclase and constitutively high cAMP (16). Normally, the acid secretagogue histamine binding to the H2 receptor couples through Gs to increase intracellular cAMP in the parietal cell (8, 9). The important contribution of histamine for stimulating acid secretion in vivo has been demonstrated by the efficacy of H2-receptor antagonists to block acid (4, 5). In addition, treatment of isolated parietal cells with histamine and agents that increase cAMP are known to be potent inducers of acid secretion. Our results are in accordance with these previous studies with increased basal acid content observed in the stomachs of transgenic lines expressing both high and low Ctox levels. Thus increased intracellular cAMP appears to hyperstimulate the parietal cell to increase acid secretion in Ctox transgenic mice.

Normally, acid secretion is highly regulated to respond to the ingestion of a meal and to maintain homeostasis of stomach pH. A primary regulator is the acid secretagogue gastrin. Gastrin levels are under feedback control with secretion into the blood regulated by changes in luminal pH (35). When the pH is high, gastrin release from G cells is increased, thereby stimulating acid secretion from parietal cells and lowering gastric pH. Alternatively, when the pH of gastric contents is low, gastrin release is decreased, which results in reduced stimulation of the parietal cell and decreased acid secretion. We measured markedly lower plasma gastrin in Ctox transgenics. This result is consistent with high acid secretion and correspondingly low luminal pH in these mice inhibiting gastrin release, resulting in low-circulating gastrin. The observation of increased basal acid content in Ctox transgenics, in spite of low gastrin, suggests that 1) intrinsic stimulation due to constitutively high intracellular cAMP was sufficient to activate acid secretion in the parietal cell; and 2) gastrin regulatory mechanisms could not compensate for the cAMP hyperstimulation to normalize luminal pH. Crossing the Ctox transgene onto a gastrin-deficient background further demonstrated that gastrin is not required to see the effect of constitutively high parietal cell cAMP on acid secretion. Basal acid content was increased in both Ctox-7 and Ctox-8 transgenic mice on a gastrin-deficient background compared with nontransgenic gastrin-deficient mice (data not shown).

Increased parietal cell cAMP in Ctox transgenic mice resulted in hyperphosphorylation of the F-actin associated adaptor protein LASP-1. In rabbit parietal cells, LASP-1 has been shown to be phosphorylated in response to histamine stimulation and cAMP signaling (13). Furthermore, upon PKA phosphorylation, LASP-1 is translocated to the actin-rich canalicular membrane, the site of active HCl secretion (11, 12). Ctox-7 transgenic mice had increased basal LASP-1 phosphorylation, and phosphorylation was not increased further in response to histamine stimulation. This suggests that the enhanced level of parietal cell cAMP in Ctox-7 transgenic mice results in constitutive LASP-1 phosphorylation similar to histamine-stimulated control mice. Moreover, our data show that mouse LASP-1 phosphorylation status is correlated with cAMP signaling and acid secretion, as had been previously demonstrated in vitro in preparations of isolated gastric glands or purified parietal cells from rabbit (11–13). Observation of increased phospho-CREB in Ctox-7 transgenics confirmed increased PKA activity and suggests that Ctox-7 parietal cells may have changes in transcription of cAMP-regulated genes.

Although there are several genetically engineered mouse models with impaired gastric acid secretion due to mutations in acid secretagogues or their receptors, there are few models of constitutive parietal cell activation. The INS-GAS transgenic mouse model of parietal cell hyperstimulation has several features in common with the Ctox transgenics. INS-GAS transgenic mice have increased circulating gastrin resulting from expression of a human gastrin transgene in pancreatic beta-cells through the use of a rat insulin promoter (38). The increased gastrin results in distinct changes in younger vs. older INS-GAS mice. Similar to Ctox, younger INS-GAS mice have increased basal acid secretion (36). However, in contrast to Ctox, the gastric mucosa of INS-GAS mice is hyperplastic with an increased number of parietal cells (36). This is consistent with the growth factor activity of gastrin on the gastric mucosa. The young Ctox transgenic mice had normal numbers of parietal cells.

Both the Ctox and INS-GAS transgenic models of parietal cell hyperstimulation developed significant gastric mucosal histopathology as they aged. Loss of parietal and chief cells was accompanied by expansion of an aberrant mucous cell type that eventually occupied the majority of the mucosa in the acid-secreting portion of the stomach (36). Similar gastric histopathology was also observed in a second gastrin transgenic mouse model that exhibited a sixfold elevation in gastrin (22). The aberrant mucous cells in Ctox-7 were stained with GS II lectin and TFF2, which are markers of the mucous neck cell in the mouse fundus. As the pathology progresses in Ctox-7 transgenics, the aberrant mucous cells stain with Alcian blue, which is normally seen in more distal portions of the gastrointestinal tract, including the intestine. Mucous neck cell hypertrophy has been reported in both human and mouse in association with a variety of different conditions, including Helicobacter pylori infection (27, 32, 37), DMP-777 chemical ablation of parietal cells (28), autoimmune gastritis (18), IFN-{gamma} administration (19), and gastrin deficiency (41). Thus the appearance and expansion of this aberrant cell type, which has been termed spasmolytic polypeptide-expressing metaplasia (32), appears to be a common response to stress or inflammation of the fundic mucosa.

The cellular changes in the Ctox-7 gastric mucosa are accompanied by inflammation, increased expression of the Th1 cytokine IFN-{gamma}, and the development of anti-parietal cell antibodies. These features identify Ctox-7 as a new mouse model of AIG. The appearance and expansion of aberrant mucus-filled cells is also shared between Ctox-7 and other models of AIG (18). AIG is characterized by the development of autoantibodies and a chronic inflammatory infiltrate reactive to parietal cells. A predominant Th1 inflammatory response that requires IFN-{gamma} (3) causes the eventual destruction of parietal cells and the associated loss of chief cells. In human, this condition is the underlying pathological cause of pernicious anemia due to the loss of the parietal cell product intrinsic factor, which is required for the absorption of vitamin B12 and the formation of red blood cells. A number of experimental animal models of AIG have been described that result from manipulation of the immune system, with the predominant model generated by disruption of self-tolerance by removal of the thymus in neonatal mice (18). Thymectomy-induced AIG is strain dependent, with BALB/c strains readily developing gastritis, whereas other strains, including C57BL/6, are resistant (33). Our demonstration of the development of autoantibodies in Ctox-7 transgenics on a C57BL/6 background suggests differences in this transgenic model compared with thymectomy-induced AIG.

In summary, we have described a new transgenic mouse model with hyperstimulation of the parietal cell due to constitutively high cAMP. These mice have increased gastric acid secretion with a compensatory reduction in circulating gastrin. The Ctox transgenics will be a useful model to dissect the affect of high cAMP on other parietal cell functions, including morphological transformation, phosphoprotein regulation, and regulation of gene expression. Ctox-7 is also a new mouse model of AIG. This transgenic line develops anti-parietal cell antibodies with atrophic gastritis and the expansion of an aberrant mucous neck cell lineage. This is an exciting new animal model that may prove useful for studies of pathogenesis and treatment of AIG.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1-DK-56882 (to L. C. Samuelson), PO1-DK-062041 (to L. C. Samuelson and J. L. Merchant), and P30-DK-34933. L. Lopez-Diaz was supported by the Cellular and Molecular Biology Training Grant (T32-GM-07315), and K. L. Hinkle was supported by the Cellular and Molecular Aspects of Systems and Integrative Biology Training Grant (T32-GM-08322) and the Organogenesis Training Grant (T32-HL-97505).


   ACKNOWLEDGMENTS
 
We acknowledge the expertise of the University of Michigan Transgenic Animal Model Core (http://www.med.umich.edu/tamc/) for microinjection of the transgene construct. We thank David Alpers for providing intrinsic factor antibodies, Andrew Giraud for the TFF2 antibody, and Frank Burton for the CtoxA1 subunit clone.

Present address of K. L. Hinkle: Dept. of Biology, Norwich University, Northfield, VT 05663.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. C. Samuelson, Dept. of Molecular and Integrative Physiology, Univ. of Michigan, 7761 Medical Science II Building, Ann Arbor, MI 48109 (e-mail: lcsam{at}umich.edu)

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.

* L. Lopez-Diaz and K. L. Hinkle contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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