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Physiol. Genomics 29: 1-12, 2007. First published November 14, 2006; doi:10.1152/physiolgenomics.00176.2006
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Received 10 August 2006; accepted in final form 9 November 2006.
Physiological Genomics 29:1-12 (2007)
1094-8341/07 $8.00 © 2007 American Physiological Society

Transcriptional profiling of gastrin-regulated genes in mouse stomach

Renu N. Jain and Linda C. Samuelson

Department of Molecular and Integrative Physiology, The University of Michigan, Ann Arbor, Michigan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Gastrin, a potent stimulator of gastric acid secretion, primarily targets the acid-secreting parietal cells and histamine-secreting enterochromaffin-like (ECL) cells in the stomach. Accordingly, gastrin-deficient (GAS-KO) mice have a severe impairment in acid secretion. The aim of this study was to characterize changes in gene expression in GAS-KO mice to identify gastrin-regulated genes and to gain insight into how gastric cell types are regulated by gastrin and acid secretion. Affymetrix microarray analysis of GAS-KO and wild-type mice identified numerous differentially expressed transcripts. The results were compared with GAS-KO mice treated with gastrin to identify genes that were gastrin responsive. Finally, genes that were primarily changed due to gastrin and not hypochlorhydria were identified by comparison to mice that are deficient in both gastrin and cholecystokinin (GAS/CCK-KO), since these mice have restored basal acid secretion. The data were validated by quantitative reverse transcriptase polymerase chain reaction analysis. Interestingly, a number of inflammatory response genes were induced in GAS-KO mice and normalized in GAS/CCK-KO mice, suggesting that they were increased in response to low gastric acid. Moreover, a number of parietal cell transcripts that were downregulated in GAS-KO mice were similarly restored in GAS/CCK-KO mice, suggesting that parietal cell changes were also primarily associated with hypochlorhydria. In contrast, ECL cell genes that were markedly downregulated in GAS-KO mice continued to be reduced in GAS/CCK-KO mice, demonstrating that gastrin coordinately regulates a number of ECL cell genes, including several involved in histamine synthesis and secretion.

parietal cell; enterochromaffin-like cell; cholecystokinin; parathyroid hormone-related protein; inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
THE GASTRIC HORMONE GASTRIN plays a key role in the physiological regulation of acid secretion. Secreted by G cells in the distal stomach in response to a meal, circulating gastrin stimulates acid secretion by binding to gastrin/cholecystokinin (CCK)-B receptors on parietal and enterochromaffin-like (ECL) cells in the fundic region of the stomach. Gastrin stimulates ECL cells to secrete the potent acid secretagogue histamine, which binds to histamine 2 receptors on parietal cells. Thus the acid-secreting parietal cells are both directly and indirectly activated by gastrin. Another stimulatory pathway for acid secretion involves activation of the neurotransmitter acetylcholine, which binds to muscarinic 3 receptors on the parietal cell. Thus, a complex network of hormones, neurotransmitters, and paracrine signals regulates the process of gastric acid secretion (3).

The in vivo importance of gastrin for acid secretion was established by analysis of gastrin-deficient (GAS-KO) and CCK-B receptor-deficient mice (11, 19, 22, 24). These mouse mutants have marked reductions in both basal and induced acid secretion (11, 22). Parietal cells, ECL cells, and the other major cell lineages in the acid-secreting fundic mucosa are formed, indicating that gastrin is not required for cellular development of the gastric glands. However, careful examination of the two gastrin cell targets, parietal and ECL cells, demonstrated that gastrin is required for their normal function (13, 16, 26). Gastrin administration using osmotic minipumps can partially restore basal acid secretion in GAS-KO mice, indicating that the acid secretory machinery is capable of repair once gastrin is made available (5, 11). The cellular changes resulting from gastrin replacement that restore acid secretion have not been elucidated.

The effects of gastrin are mediated in part by changes in gene expression. Previous studies have shown that hypergastrinemia increases the expression of ECL cell genes encoding proteins involved in histamine production, including histidine decarboxylase, vesicular monoamine transporter-2, and the secretory granule marker chromogranin A (7, 12, 14). Thus, in addition to stimulating ECL cell histamine secretion, gastrin increases histamine synthesis by coordinately increasing the expression of a number of ECL cell genes. Hypergastrinemia has also been shown to increase expression of regenerating gene protein 1 in ECL cells (18). The effect of loss of gastrin signaling on ECL cell function appears to be quite severe since studies of ECL cell morphology in CCK-B receptor-deficient mice showed a dramatic loss of secretory granules as well as histamine (4). The extreme changes in ECL cell morphology and function suggests that expression of a large number of genes will be altered with loss of gastrin signaling.

In addition to gastrin regulating ECL cell genes, previous studies have shown that gastrin can regulate parietal cell gene expression (2, 15). Gastrin increases the expression of H+,K+-ATPase and carbonic anhydrase II mRNAs in isolated canine parietal cells and in animal models with elevated gastrin (2, 17, 30). Detailed microarray gene expression analysis of purified GAS-KO parietal cells showed reductions in several acid secretion-related genes, which were normalized by gastrin replacement, including genes encoding the {alpha}- and ß-subunits of the H+,K+-ATPase proton pump, the potassium channel KCNQ1 on the apical membrane, and the water channel aquaporin (AQP) 4 on the basolateral membrane (15). This comprehensive study also brought to light some new gastrin-regulated genes, including parathyroid hormone-related protein (Pthlh), which was markedly downregulated in GAS-KO parietal cells. In addition, this study noted increased expression of a large number of Wnt and Myc target genes, suggesting a possible role of Wnt signaling in parietal cell maturation (15). Thus, although parietal cells in gastrin-deficient mice are formed, there is a general decrease in expression of markers of differentiated cell function with increased expression of markers of immature cells, suggesting that gastrin is critical for the functional maturation of these cells.

The intestinal hormone CCK belongs to the same peptide hormone family as gastrin, with both members binding CCK-B receptor with similar affinity (33). CCK-deficient (CCK-KO) mice do not have apparent stomach defects; however, acid secretion has not been studied (20). Circulating CCK levels are normal in GAS-KO mice (6, 11), suggesting that CCK is not upregulated to compensate for loss of gastrin. However, acid secretion was significantly increased in gastrin-CCK double knockout (GAS/CCK-KO) mice compared with GAS-KO mice (6). This surprising finding suggests that net acid output is dependent on balancing stimulatory gastrin signaling at CCK-B receptors with inhibitory CCK signaling through the CCK specific CCK-A receptor (27).

The present study was undertaken to more fully understand how gastrin deficiency and low acid secretion regulate gene expression in the stomach. We focused on mucosal gene expression in the acid-secreting fundus using Affymetrix microarray. Gene expression of GAS-KO mice and GAS-KO mice treated with gastrin were compared with wild-type (WT) and GAS/CCK-KO mice. A large number of differentially expressed genes were identified, including genes that appeared to be primarily regulated by gastrin and genes that were more closely associated with low gastric acid secretion. Selected genes were further studied by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) to validate the microarray findings. These studies also included CCK-KO mice to determine whether loss of CCK had an effect on expression of the gastrin-regulated genes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice.
The generation of GAS-KO and CCK-KO mice by gene targeting in embryonic stem cells was described previously (11, 20). Gastrin/CCK-KO mice were generated by intercrossing GAS-KO with CCK-KO mice, and GAS-KO, GAS/CCK-KO, and WT mice were maintained by homozygous breeding on strain background 129X1/SvJ in a specific pathogen-free facility. CCK-KO mice used for qRT-PCR analysis were on a C57BL/6 strain background. Experiments were performed on male mice 8–10 wk of age. Before death mice were fasted overnight with free access to water. All animal care and protocols were reviewed and approved by The University of Michigan Committee on Use and Care of Animals.

Gastrin replacement.
Gastrin (5 µg·kg–1·h–1 rat gastrin-17, Bachem) or vehicle control (1% BSA in 0.9% NaCl) was infused into GAS-KO mice (n = 4/group) for 7 days with 0.2-ml capacity miniosmotic pumps (1 µl/h, 2001 model; Alzet), as previously described (11). After 7 days the animals were killed with the pumps intact and processed similarly to the untreated mice.

RNA isolation and microarray analysis.
We isolated the fundic mucosa by scraping stomach tissue with a scalpel, and RNA was extracted with 1 ml of TRIzol per sample (Invitrogen) and purified by RNeasy (Qiagen). Three or four independent RNA samples were isolated from individual GAS-KO mice infused with saline, GAS-KO infused with gastrin [gastrin replacement (GR)], CCK-KO and GAS/CCK-KO mice. WT mucosal scrapings were pooled from four or five mice, with three independent pooled RNA samples analyzed. For microarray analysis, three independent RNA samples from each of the four groups (WT, GAS-KO, GR, and GAS/CCK-KO) were hybridized to the Affymetrix 430A chip, which contains 22,626 probe sets representing 14,000 genes. Microarray Suite 5.0 software (Affymetrix) was used to characterize probe pair hybridization signals. Data analysis was performed as described previously (15). Detailed data analysis methods are available at http://dot.ped.med.umich.edu:2000/ourimage/pub/shared/Affymethods.html. Data were quantile normalized to adjust for differences in the probe intensity distribution across the 12 chips. Fold change was calculated by replacing mean expression values <50 units with 50 to avoid negative values or spuriously large fold changes. Our complete data set is deposited in the Gene Expression Omnibus database (accession no. GSE 4810) at the following site: http://www.ncbi.nlm.nih.gov/geo/.

qRT-PCR analysis.
RNA from fundic mucosal scrapings (100 µg) was DNase treated (Qiagen), and 2 µg were reverse transcribed (RT) in a 20-µl volume using Iscript (Bio-Rad), following manufacturers' protocols. The RT product was brought to 100-µl volume by addition of 80 µl of H2O, and qRT-PCR was performed on 2 µl of RT product in duplicate using an iCycler with SYBR green dye (Molecular Probes). Each 20-µl reaction contained Bio-Rad PCR buffer, 5.5 mM MgCl2, 100 nM each primer, SYBR green, 1 nM fluorescein, 200 µM dNTPs, and 0.025 U Platinum Taq polymerase (Invitrogen). The following amplification conditions were used: 3 min at 95°C, 35 cycles of 9 s at 95°C, and 1 min at 60°C, followed by 1 min at 55°C. Data for each gene were normalized to the expression of Gapdh, which remained the same in WT and mutant samples. Melt curve analysis was used to assess product purity. Beacon software (Bio-Rad) was used to design primer sequences (Table 1). Reaction specificity was validated by sequencing the amplicons.


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  		Table 1. qRT-PCR primer sequences

 
Statistical analysis.
Data are presented as means ± SE (n = 3–4) and analyzed by Student's t-test with P < 0.05 considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Gastrin-responsive genes in the fundic mucosa.
Affymetrix microarray analysis of genetically engineered mouse models was used to identify fundic genes that changed expression due to the loss of gastrin. GAS-KO signals were compared with WT mice and to GAS-KO mice that had been treated with gastrin by osmotic minipump infusion. In addition we analyzed GAS/CCK-KO mice, as this mouse mutant has relatively normal acid secretion in spite of the loss of both gastrin and CCK (6). This comparison allowed us to distinguish genes that were regulated by gastrin from genes that were associated with the low acid in GAS-KO mice. A large number of differentially expressed genes (≥2-fold, P < 0.05) were identified, including 469 probe sets differing between GAS-KO and WT; 471 between GAS-KO and GR; 605 between GAS/CCK-KO and WT; and 627 between GAS/CCK-KO and GAS-KO. Thus 2–3% of the probe sets on the 430A chip were differentially expressed in each of the four different comparisons.

Responsive genes were identified as probe sets that were changed in GAS-KO mice compared with WT and recovered after gastrin infusion. By this definition, 172 probe sets were responsive as they were significantly changed in the GAS-KO:WT comparison and returned toward normal in the GR:GAS-KO comparison (Fig. 1). Most of these were downregulated in GAS-KO mice (132/172, 77%) (Supplemental Table S1; the online version of this article contains supplemental material). The 172 responsive probe sets were further categorized as acid responsive (A) or gastrin responsive (G) or acid/gastrin responsive (A/G), depending on whether they were normalized in the GAS/CCK-KO mice. Approximately half of the responsive probe set signals were normalized in GAS/CCK-KO, suggesting that they were primarily acid responsive and not directly gastrin regulated (Supplemental Table S1).


Figure 1
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  		Fig. 1. Identification of gastrin-responsive genes that were differentially expressed in gastrin-deficient (GAS-KO) mice and restored after treating GAS-KO mice with gastrin [gastrin replacement (GR)]. Comparison of the Affymetrix signal strengths of probe sets in GAS-KO to wild-type (WT) mice identified 469 sets with significant differences (≥2-fold changed, P < 0.05). A similar comparison of GR to GAS-KO identified 471 differentially expressed probe sets. There were 172 probe sets in common in these 2 lists, as represented in the Venn diagram (probe sets are listed in Supplemental Table 1). The overlapping probe sets identified genes that were affected by loss of gastrin and corrected after exogenous gastrin administration.

 
Induction of inflammatory genes in GAS-KO Mice.
Initial analysis of the 172 responsive genes that were differentially expressed in GAS-KO mice and recovered after GR identified a large number of inflammatory response genes. This was interesting since our study was performed on young mice (8–10 wk of age) to minimize the effect of the gastritis that occurs as the GAS-KO mice age (10, 34, 35). Despite no obvious histological signs of inflammation at this age (data not shown), several inflammatory genes were increased in expression in GAS-KO mice (Table 2). Among these genes were several interferon (IFN)-responsive genes, such as defensin-ß3 (Defb3), interferon-induced protein with tetratricopeptide repeats 1 (Ifit1), and the GTPases IFN-{gamma}-induced GTPase (Igtp), and guanylate nucleotide binding proteins 1 and 2 (Gbp1, Gbp2), among many others. The microarray findings were validated for several of these inflammation genes by qRT-PCR analysis of five groups of mice: GAS-KO, WT, gastrin-infused GAS-KO, CCK-KO, and GAS/CCK-KO (Fig. 2). The CCK-KO mice were included with the four groups of mice examined by microarray to determine whether the responsive genes were affected by the loss of CCK, the other CCK-B receptor ligand.


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  		Table 2. Induction of inflammation-related genes in GAS-KO mice

 

Figure 2
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  		Fig. 2. Increased expression of inflammation-related genes in GAS-KO mice. Transcript abundance was determined by quantitative RT-PCR (qRT-PCR) analysis with fundic mucosal RNA samples from GAS-KO mice (open bars), WT mice (solid bars), GR mice (gray bars), CCK-deficient (CCK-KO) mice (hatched bars), and gastrin-CCK double knockout (DKO) mice (cross-hatched bars). Data were normalized to Gapdh expression in the same samples and reported as fold change (means ± SE) from levels in WT mice. Significant changes in mRNA abundance compared with GAS-KO are indicated (*P < 0.05), with n = 3–4 mucosal RNA preparations per group.

 
The qRT-PCR analysis showed that induction of Defb3 and the cytokine interleukin 1 family member 6 (Il1f6) in GAS-KO mice was particularly large, with >10-fold increased mRNA abundance compared with WT. Moreover, the increased expression was completely normalized after 1 wk of gastrin infusion (Fig. 2, A and B). Furthermore, expression of these two genes as well as the polymeric immunoglobulin receptor (Pigr) remained high in GAS/CCK-KO mice, suggesting that they were primarily regulated by gastrin and not low acid (Fig. 2, A–C). In contrast, the majority of the inflammation-related genes in GAS-KO were significantly normalized in GAS/CCK-KO mice, suggesting that low acid secretion was associated with the induction of these genes (Table 2 and Fig. 2, D–G). Thus the inflammation genes are regulated by gastrin through at least two different mechanisms.

Other highly differentially expressed genes.
In contrast to the inflammatory genes, which were uniformly upregulated in GAS-KO, the vast majority (>80%) of the remaining highly responsive genes (≥3-fold, P < 0.05) were expressed at lower levels in GAS-KO mice, with expression increasing after gastrin treatment (Table 3). These genes fell into several functional categories and included genes not previously known to be gastrin responsive. One of the most highly regulated genes encoded the morphogen Pthlh, which was 10-fold decreased in GAS-KO mice. Interestingly, the transcription factor SRY-box containing gene 4 (Sox4), which is regulated by Pthlh, was also decreased in GAS-KO, suggesting that gastrin regulates Pthlh signaling in the fundic mucosa. In addition to downregulation of transcripts encoding various signaling molecules, several transcripts encoding molecules involved with cell adhesion or extracellular matrix were downregulated in GAS-KO mice, including vitronectin (Vtn), which was fivefold reduced in GAS-KO, and Sparc, which encodes a matrix-associated protein that can interact with Vtn (Table 3). There were also genes in this category that were upregulated in GAS-KO, including desmocollin 1 (Dsc1) and matrix metalloproteinase 23 (Mmp23). A large number of genes associated with secretory granules or vesicular trafficking were downregulated in GAS-KO mice. This is likely due to gastrin regulation of these functions in ECL cells (see below). Finally, transcripts encoding transporters, including the cation and anion carriers Slc41a3 and Slco3a1, were of lower abundance in the GAS-KO stomach. These data suggest that gastrin regulates many different physiologic processes in the fundic mucosa. A few of the genes are known to be enriched in specific epithelial cell types in the fundic mucosa, including parietal and ECL cells, but the cellular localization of the majority of these genes is unknown (Table 3).


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  		Table 3. Noninflammatory, highly responsive genes in the fundic mucosa

 
Major ECL cell defect in GAS-KO and GAS/CCK-KO mice.
To gain some understanding of the physiological changes in GAS-KO stomach, we examined differentially expressed transcripts that are enriched in specific gastric cell types (Table 4). Notably there was reduced expression of a large number of ECL/endocrine cell genes, including many encoding proteins involved with histamine synthesis and secretion [histidine decarboxylase (Hdc), CCK-B receptor (Cckbr)], secretory granules [chromogranin A (Chga), Chgb, Resp18, Pcsk4, secretogranin V (Scg5)] and secreted factors expressed in ECL cells (Nov, Vtn). Validation of these changes in gene expression was performed by qRT-PCR analysis (Fig. 3). The results confirmed the marked reduction in expression of several of these genes in GAS-KO mice, and the recovery of expression after 1 wk of GR. The gene encoding the histamine biosynthetic enzyme Hdc was reduced fourfold in GAS-KO, and the histamine transporter vesicular monoamine transporter 2 (Vmat2) was downregulated threefold in GAS-KO mice. Genes encoding the secretory granule proteins Chga and Chgb, and Scg5, were two- to fourfold downregulated in GAS-KO mice. Finally the gene encoding the gastrin receptor (Cckbr), which stimulates histamine secretion from ECL cells, was threefold decreased in GAS-KO mice. Further analysis of expression in CCK-KO and GAS/CCK-KO mice supports the hypothesis that these ECL/endocrine cell genes are coordinately upregulated by gastrin and not associated with low acid since expression for most of them was reduced in GAS/CCK-KO mice similar to GAS-KO mice (Fig. 3). The data show that there are a large number of ECL cell genes that are coordinately upregulated by gastrin and thus are markedly reduced in GAS-KO mice.


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  		Table 4. Differential expression of genes enriched in specific gastric cell types

 

Figure 3
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  		Fig. 3. Decreased expression of enterochromaffin-like (ECL)/endocrine cell genes in GAS-KO mice. Transcript levels were determined by qRT-PCR analysis of fundic mucosal RNA from GAS-KO mice (open bars), WT mice (solid bars), GR mice (gray bars), CCK-KO mice (hatched bars), and DKO mice (cross-hatched bars). Data were normalized to Gapdh and shown as fold change (means ± SE) from WT mice. Significant changes to GAS-KO are indicated (*P < 0.05), with n = 3–4 mucosal RNA preparations per group.

 
Acid-regulated parietal cell transcripts.
A number of genes known to be specific for, or enriched in, the acid-secreting parietal cell were also reduced in expression in GAS-KO mice (Table 4). This included genes encoding proteins involved with water or ion transport, such as the alpha and beta subunits of the H+,K+-ATPase proton pump (Atp4a and Atp4b), the potassium channel Kcnq1, and the water channel Aqp4. Some parietal cell genes encoding proteins regulating intracellular metabolism were also decreased in GAS-KO mice, including the energy-coupling protein creatine kinase b (Ckb) and the regulator of intracellular calcium stores inositol 1,4,5-triphosphate receptor 2 (Itpr2). The secreted growth regulators Pthlh, vascular endothelial growth factor B (Vegfb), and insulin-like growth factor binding protein 2 (Igfbp2) were also downregulated in GAS-KO mice. In contrast to the long list of parietal cell genes that were downregulated in GAS-KO mice, three key transcripts remained unchanged, including histamine 2 receptor, muscarinic 3 receptor, and amphiregulin (data not shown).

The microarray results were validated by qRT-PCR, with the results confirming reduced expression of a large group of parietal cell genes in GAS-KO mice and restored expression after GR (Fig. 4). Moreover, in contrast to the regulated ECL cell genes, expression of most of these parietal cell genes was normalized in GAS/CCK-KO mice. This result suggests that downregulation of these genes in GAS-KO is associated with reduced acid secretion and not gastrin deficiency, since the GAS/CCK-KO mice restore gene expression despite the loss of gastrin. However, Pthlh gene expression did not follow this pattern. Transcripts were decreased sixfold in GAS-KO mice and were similarly decreased in GAS/CCK-KO mice, suggesting that downregulation is specific for the loss of gastrin (Fig. 4G).


Figure 4
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  		Fig. 4. Decreased expression of parietal cell genes in GAS-KO mice. Transcript levels were determined by qRT-PCR analysis of fundic mucosal RNA from GAS-KO mice (open bars), WT mice (solid bars), GR mice (gray bars), CCK-KO mice (hatched bars), and DKO mice (cross-hatched bars). Data were normalized to Gapdh and shown as fold change (means ± SE) from WT mice. Significant changes to GAS-KO are indicated (*P < 0.05), with n = 3–4 mucosal RNA preparations per group.

 
Changes in other gastric cells in GAS-KO mice.
In addition to changes in ECL cell and parietal cell transcripts, some genes characteristic of other gastric cell types were differentially expressed in GAS-KO stomach (Table 4). The chief cell markers alanyl aminopeptidase (Anpep) and osteopontin [secreted phosphoprotein 1 (Spp1)] were differentially expressed in GAS-KO compared with WT (Table 4 and Fig. 5, A and B). However, the chief cell markers intrinsic factor and pepsinogen C were similar in WT, GAS-KO, gastrin-infused GAS-KO, and GAS/CCK-KO (data not shown), suggesting that changes in chief cell gene expression are limited. Similarly, the mucous cell markers trefoil factors 1 and 2 were unchanged in the microarray analysis (data not shown), although the surface mucous cell chloride channel gene Clca3 was increased fivefold in GAS-KO mice (Table 4 and Fig. 5C). Finally, the D cell transcript somatostatin (Sst) was decreased threefold in GAS-KO mice (Table 4 and Fig. 5D). Gastrin regulation of transcripts in these other gastric cell types are likely to be indirect since only ECL and parietal cells are known to contain gastrin receptors.


Figure 5
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  		Fig. 5. Gastrin regulation of genes expressed in cells without gastrin receptors. Analysis of the chief cell genes alanyl aminopeptidase (Anpep) (A) and osteopontin (Spp1) (B); the mucous cell gene for the calcium-activated chloride channel 3 (Clca3) (C); and D cell gene somatostatin (Sst) (D) by qRT-PCR. Transcript levels in fundic mucosal RNA from GAS-KO mice (open bars), WT mice (solid bars), GR mice (gray bars), CCK-KO mice (hatched bars), and DKO mice (cross-hatched bars) were measured. Data were normalized to Gapdh and shown as fold change (means ± SE) from WT mice. Significant changes to GAS-KO are indicated (*P < 0.05), with n = 3–4 mucosal RNA preparations per group.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study is a comprehensive analysis of gastrin-regulated genes in the stomach of genetically engineered mice. Gastrin is known to be an important physiological stimulator of gastric acid secretion (8, 9, 13, 26). Accordingly, previous studies showed that GAS-KO mice have a severe reduction in both basal and stimulated acid secretion (11, 19) that could be repaired by GR (5, 11). In the present study we observed numerous changes in gene expression in the GAS-KO stomach, demonstrating that the loss of gastrin and the resulting defects in acid secretion led to changes in expression of many genes. We focused on 172 probe sets on the Affymetrix chip that were differentially expressed in GAS-KO and returned toward normal after GR (Fig. 1). Furthermore, to identify genes that were primarily associated with low acid secretion and not by the loss of gastrin, we took advantage of GAS/CCK-KO mice, which have relatively normal basal and histamine-induced acid secretion despite the constitutive loss of both gastrin and CCK (6). Our studies are similar to another recently published analysis of changes in gene expression in GAS-KO mice (10). However, the study by Friis-Hansen and colleagues (10) analyzed whole stomach in contrast to the focused analysis of the fundic mucosa in our study. Despite this difference there were similar findings in both studies.

Our analysis demonstrated a dramatic transcriptional repression of ECL cells in both GAS-KO and GAS/CCK-KO mice, with coordinated reduction of transcripts encoding proteins involved with histamine synthesis (Hdc), histamine transport (Vmat2), secretory granules (Chga, Chgb, and Scg5), and stimulated secretion (Cckbr). This result extends the findings in earlier studies showing some ECL cell defects in mouse strains with loss of gastrin signaling (6, 11, 19, 22, 24). Importantly, the ECL cells of CCK-B receptor-deficient and GAS/CCK-KO mice have a marked reduction in histamine secretory vesicles (4, 6), suggesting that the changes in ECL cell gene expression result in major alterations in cellular morphology and differentiated function. Moreover, we observed that other genes enriched in ECL cells, such as Nov and Vtn (21), were also downregulated in GAS-KO mice, suggesting that gastrin is regulating global ECL cell function.

It is intriguing that despite the extreme reduction of both gastrin and histamine signaling in GAS/CCK-KO mice, acid secretion is largely restored (6). CCK is an inhibitor of stomach function, although its role in the regulation of acid secretion is not well defined (8). It has been proposed that recovery of acid secretion in GAS/CCK-KO mice could be due in part to the loss of CCK-induced inhibitory signals, although the specific mechanism has not been established (27). Circulating CCK is thought to act on CCK-A receptors present on D cells causing somatostatin release, which in turn results in inhibiting acid secretion through the somatostatin 2 receptor (3). In this regard, we observed that CCK-KO and GAS/CCK-KO mice have decreased expression of the negative regulator somatostatin compared with WT mice (Fig. 5). However, increased vagal stimulation may also play a role in increasing acid secretion in GAS/CCK-KO mice since these mice exhibit a blunted acid secretory response to cholinergic stimulation (6).

Our previous microarray study of purified parietal cells showed that several genes involved with acid secretion are downregulated in GAS-KO mice and restored after GR (15). In the present study, we have extended these findings using fundic mucosal RNA on a newer Affymetrix chip. We demonstrate that most of the gastrin-regulated parietal cell transcripts are normalized in GAS/CCK-KO mice, including the proton pump subunits (Atp4a, Atp4b), the ATP supplier to the proton pump (Ckb), as well as apical and basolateral channels (Kcnq1 and Aqp4) (Fig. 4). Thus, the parietal cell genes appear not to be primarily regulated by gastrin, instead they appear to be responsive to low acid and the other complex signals involved in regulating gastric acid secretion.

Parietal cells express a number of gastrin-regulated signaling molecules that have been proposed to act in a paracrine or autocrine fashion in the gastric mucosa (16). Genes encoding several secreted molecules were identified as gastrin responsive in this microarray study, including Pthlh, Vegfb, and Igfbp2. The gene encoding the multifunctional molecule Pthlh, which is reported to be abundantly expressed in parietal cells (15, 23), was the most highly differentially expressed parietal cell gene, with 10-fold lower expression in GAS-KO mice. Although the function of PTHLH in the stomach is not fully understood, it has been reported to play a role in gastric cancer cell proliferation (25) and to be involved with stress-induced changes in gastric motility (1). This hormone is broadly expressed outside of the stomach and has been shown to be important for cartilage differentiation and tooth eruption and to induce branching morphogenesis in mammary gland (29). Reduced expression of the Pthlh gene target Sox4 in GAS-KO stomach further suggests that gastrin regulates PTHLH signaling in the stomach. Moreover, Pthlh is known to be a target of hedgehog signaling (32) and parietal cells have been shown to express hedgehog protein (28, 31). The recent preliminary report of reduced sonic hedgehog production in GAS-KO mice (36) is intriguing and suggests a possible mechanism for Pthlh regulation by gastrin in the stomach. In contrast to the acid secretion-related genes, Pthlh expression was not normalized in GAS/CCK-KO mice, suggesting that it is primarily regulated by gastrin and not low acid.

There were a large number of inflammation-related genes induced in GAS-KO mice. Previous studies have shown that the hypochlorhydric GAS-KO mice develop bacterial overgrowth and with increasing age develop gastric atrophy and inflammation, ultimately resulting in antral tumors by 1 yr of age (10, 34, 35). Although there are no histological signs of inflammation at 2 mo of age, our observation of increased expression of inflammation-related genes suggests that the inflammatory changes occur very early. Expression of some inflammation genes was corrected in GAS/CCK-KO mice (Slurp1, Lgals3, Nfkbia), supporting the hypothesis that differential expression in GAS-KO mice was related to low acid secretion and bacterial overgrowth. The finding that several inflammation-related genes are upregulated in GAS-KO mice is in agreement with the results of another recently published microarray analysis of GAS-KO mice (10). This study of whole stomach identified several immune-defense genes that increased expression in association with low acid secretion. However, our study also identified several inflammatory genes that were not corrected in the GAS/CCK-KO mice (Defb3, Il1f6, and Pigr), indicating that loss of gastrin and not low acid regulates gene expression of many inflammatory response genes.

Interestingly, several of the inflammation-related genes upregulated in GAS-KO mice were also acutely induced after Helicobacter pylori infection of mice (15, 23). Infection by the gastric pathogenic bacteria Helicobacter largely evokes a T helper cell 1 (Th1) immune response, characterized in part by elevated mucosal IFN-{gamma}. GAS-KO mice have been reported to increase IFN-{gamma} expression in association with cellular transformation events leading to gastric cancer (10, 34). Several IFN-{gamma}-responsive genes were identified in our study (Table 2) as well as in the study by Friis-Hansen (10) to be significantly induced in GAS-KO mice. Genes acutely induced by Helicobacter infection that were also upregulated in GAS-KO include Pigr, Igtp, and Ifit1 (23). This correspondence of changes in gene expression suggests that young GAS-KO mice exhibit characteristic changes indicative of a mucosal Th1 immune response before overt inflammation and cellular transformation are evident. These changes are likely to be important for the cellular remodeling that occurs in these mice as they age, including parietal cell atrophy and mucous gland metaplasia (10, 35).

Parietal and ECL cells in the fundic mucosa are known to express gastrin receptors. The observed changes in gene expression in the fundic mucosa of GAS-KO mice reflect a direct effect of loss of gastrin signaling on these target cells, as well as indirect effects due to reduced acid and alterations in other signaling molecules, including PTHLH and histamine. Thus, gastrin is a global regulator of gastric gene expression, affecting several different cell types in the gastric mucosa and not just those expressing gastrin receptors.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-56882 (to L. C. Samuelson) and Pilot Project Award from the University of Michigan Gastrointestinal Peptide Center P30-DK-34933 (to R. N. Jain).


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the assistance of Dr. Thomas Saunders for implantation of the osmotic minipumps; David Misek, James MacDonald, and Rork Kuick for expert gene expression microarray analysis; and Cynthia Brunkan and Theresa Keeley for technical assistance. We thank Andrea Todisco, Lymari Lopez-Diaz, and Kelli VanDussen for careful reading of the manuscript.


   FOOTNOTES
 
Address for reprint requests and other correspondence: L. Samuelson, Dept. of Molecular and Integrative Physiology, Univ. of Michigan, 2041 BSRB, 109 Zina Pitcher Pl., Ann Arbor, MI 48109-2200 (e-mail: lcsam{at}umich.edu).

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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B. J. Capoccia, W. J. Huh, and J. C. Mills
How form follows functional genomics: gene expression profiling gastric epithelial cells with a particular discourse on the parietal cell
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