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Am J Physiol Gastrointest Liver Physiol 293: G347-G354, 2007. First published April 26, 2007; doi:10.1152/ajpgi.00076.2007
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HORMONES AND SIGNALING

Helicobacter and gastrin stimulate Reg1 expression in gastric epithelial cells through distinct promoter elements

Islay A. Steele,1 Rod Dimaline,1 D. Mark Pritchard,2 Richard M. Peek, Jr.,3 Timothy C. Wang,4 Graham J. Dockray,1 and Andrea Varro1

1Physiological Laboratory, School of Biomedical Sciences, and 2Division of Gastroenterology, School of Clinical Sciences, University of Liverpool, Liverpool, United Kingdom; 3Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee; and 4Department of Medicine, Columbia University, New York, New York

Submitted 12 February 2007 ; accepted in final form 19 April 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The gastric pathogen Helicobacter pylori accelerates the progression to gastric cancer but the precise mechanisms that mediate carcinogenesis remain unidentified. We now describe how Helicobacter and gastrin stimulate the expression of a putative growth factor, Reg1, in primary gastric epithelial cells. RT-PCR and Western immunoblotting of human gastric corpus and antrum showed significantly increased Reg1{alpha} in H. pylori-infected patients. Similarly, Reg1 was increased in the stomachs of H. felis-infected INS-GAS mice. To study transcriptional regulation of the Reg1 gene, we transfected primary mouse gastric glands with –2111 bp and –104 bp Reg1 promoter-luciferase reporter constructs. Expression of both constructs was detected in pepsinogen- and VMAT-2-expressing cells, which corresponds to the normal pattern of expression of human and mouse endogenous Reg1. The expression of both constructs was increased in response to gastrin and H. pylori, and there were potentiating interactions between them; in contrast, only the –2111 bp construct responded to H. felis. Mutation of a C-rich putative regulatory element within the –104 bp sequence abolished the response to gastrin but not to H. pylori whereas mutation of the proximal –98 to –93 region of the promoter reduced the response to H. pylori but not to gastrin. Stimulation of Reg1 by H. pylori required the virulence factor CagA. These data indicate that expression of the putative growth factor Reg1 is controlled through separate promoter elements by gastrin and Helicobacter.

primary gastric epithelial cells; Helicobacter felis and pylori; CagA

EPITHELIAL GROWTH, DIFFERENTIATION, AND REPAIR depend on complex patterns of interaction between many different growth factors. In the stomach, the hormone gastrin is a well-recognized physiological regulator of epithelial cell proliferation (45). In experimental models, gastrin appears to be responsible for the increase in proliferation that occurs in the postprandial period (33), and patients with hypergastrinemia exhibit increased mucosal thickness and increased parietal and enterochromaffin-like (ECL) cell numbers (9, 11). In addition, it is now clear that infection with Helicobacter pylori, especially strains containing the virulence factor CagA, is also associated with hyperproliferation in the stomach. Interest in this condition stems from the fact that infection is associated with a progression to gastric cancer, which is the second commonest cause of death from malignancy worldwide (36). There is modest hypergastrinemia in H. pylori-infected subjects (26), but the extent to which interactions between gastrin and H. pylori might contribute to increased proliferation remains uncertain.

A growing body of evidence indicates that both gastrin and H. pylori regulate the production of a range of growth factors including members of the epidermal growth factor (EGF) family notably heparin-binding EGF, transforming growth factor-{alpha}, amphiregulin, and fibroblast growth factor (32, 46). Importantly, there is also emerging evidence for a role of the Reg family of putative growth factors in mediating the proliferative actions of gastrin (14). The Reg family consists of a number of proteins discovered independently by several different groups and also variously called pancreatic stone protein, pancreatic thread protein, and lithostathine (1, 6, 15, 30, 35, 39, 41). Members of this family are expressed in many tissues (gastrointestinal tract, pancreas, neurons, heart), and there is abundant evidence for increased expression in response to damage, inflammation, and infection (8, 24, 25, 27, 31, 38, 40). In human stomach, Reg1{alpha} has been localized to ECL and chief cells (19), and in the rat Reg1 has been localized to ECL cells (3). There is increased Reg expression in hypergastrinemia (19) and in gastric inflammation (49), and overexpression of Reg in transgenic mice is associated with stimulation of gastric epithelial cell proliferation and elevated chief and parietal cell numbers, but interestingly not ECL or mucous cells (29).

In a previous study we showed that a C-rich region of the proximal promoter sequence of Reg1 was required for gastrin-stimulated expression in AGS cells (4). In the present study we asked whether there was increased gastric Reg1{alpha} expression with Helicobacter infection in patients and in an animal model of gastric cancer (46), and we explored the cellular mechanisms regulating Reg1 expression in primary mouse gland cells by gastrin and different Helicobacter strains.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Plasmids and drugs. Plasmids consisting of –2111 and –104 bp of the rat Reg promoter and five mutants of the latter (m1–m5, Table 1), coupled to firefly luciferase (i.e., –2111 and –140 bp Reg-luc), have been described previously (4). Heptadecapeptide amidated gastrin (G17) was purchased from Peninsula (St. Helens, Merseyside, UK). All other chemicals were obtained from Sigma (Poole, Dorset, UK).


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  		Table 1. Plasmids and mutants

 
Patients. Six endoscopic pinch biopsies of gastric corpus and antrum were obtained with 2.4-mm-tip diameter, single-use, biopsy forceps (Diagmed Healthcare, Thirsk, UK) from 40 patients with H. pylori infection confirmed by serology, antral urease test (Prontodry; Medical Instruments, Solothurn, Switzerland), and antral and corpus histology. Dyspeptic patients (n = 40) with normal endoscopy, normal plasma gastrin concentrations (<30 pM), negative H. pylori status by serology, antral urease test, and antral and corpus histology were used as controls. Patients with plasma gastrin concentrations higher than 30 pM were excluded on the grounds that gastrin is a confounding variable in testing the hypothesis that H. pylori infection is associated with increased Reg1{alpha} infection. The study was approved by the Ethics Committee of the Royal Liverpool and Broadgreen University Hospitals National Health Service Trust. All patients gave informed consent.

Animals. Male FVB and INS-GAS mice (47) were killed at 3 mo old ~4 h after lights on, and the gastric corpus was extracted for RNA as previously described. Male INS-GAS mice were infected with Helicobacter felis as previously described (46), and H. felis infection was verified by positive antral urease test (Prontodry) and histology. Gastric corpus was taken at 3, 6, and 9 mo postinfection for histology and extraction for RNA.

RT-PCR. Corpus RNA was extracted in TRIzol (Invitrogen) and DNase treated (Promega, Southampton, UK), and 5 µg of RNA were reverse transcribed with AMV reverse transcriptase (Promega) and random hexamers (Promega). Real-time PCR was carried out using primers specific for human and mouse Reg1 (Table 2) and an 18S rRNA control kit (Eurogentec, Southampton, UK). Results are expressed as Reg1 mRNA expressions relative to 18S.


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  		Table 2. Primers specific for human and mouse Reg1

 
Western blots. Protein extracts of human gastric biopsies were prepared and Western blotting was performed as previously described (16, 28). Samples were probed with antibodies to human Reg1 (Th9, gift from Susanne de la Monte, Boston, MA) followed by horseradish peroxidase-conjugated secondary antibody and detection by incubation with SuperSignal West Pico Chemiluminescent Substrate (Pierce) and HyperFilm (Amersham) as previously described (44). Samples were reprobed for GAPDH (Biodesign, Saco, ME).

Gastrin RIA. Plasma samples from humans and mice were assayed for total amidated gastrin concentrations by using antibody L2 (which reacts with G17 and G34 but not progastrin or Gly-gastrins) as previously described (10).

Cultured gastric gland cells. Adherent mouse gastric glands were prepared using a modification of the method previously described (48). Briefly, the stomach was everted, ligated at both ends, washed in ice-cold HBSS, and injected with 0.5 ml of 0.5 mg/ml collagenase A (Roche Molecular Biochemicals, East Sussex, UK). Glands were liberated by shaking after 45 min, collected by sedimentation, washed, and cultured in DMEM-Ham's F-12 supplemented with 10% FBS and 2% antibiotic-antimycotic solution (Sigma) at 37°C in 5% CO2-95% O2.

Transient transfection and bacterial infection of primary mouse gland cultures. Primary mouse glands cultured for 48 h on 12-well plates were transfected with 2 µg of DNA per well together with a constitutively active Renilla luciferase reporter, phRL-SV40 (5 ng/ well, Promega), when appropriate, by using CombiMag (Oz Biosciences, Marseille, France) according the manufacturer's instructions. Cells were incubated with G17 for 8 h or infected with H. felis strain 49179 (American Type Culture Collection) or H. pylori rodent-adapted strain 7.13 and its isogenic mutants (12) at a range of multiplicities of infection (MOI) of 1:50–200 for 16 h as described previously (48). Luciferase activity was measured by dual luciferase assay (Promega) in a Lumat LB9507 luminometer (Berthold, Redbourne, Herts, UK). Results are presented as fold increase over unstimulated control, so a value of 1.0 signifies no change in luciferase activity.

Cellular targeting of Reg-luc in primary mouse adherent glands. To study cellular targeting of the luciferase vectors, Reg-luc-transfected mouse gastric glands were double immunostained with goat anti-luciferase antibody (Rockland Immunochemicals, Gilbertsville, PA) together with one of the following: rabbit anti-pepsinogen (gift from Mike Samloff, Center for Ulcer Research, Los Angeles, CA), anti H+-K+-ATPase (Calbiochem), or anti-VMAT-2 (20), together with the appropriate FITC or Texas red-conjugated secondary antibodies (Jackson Immunoresearch, Soham, UK), by using Vectashield mounting medium with DAPI (Vector Laboratories, Peterborough, UK) for counterstaining of nuclei. Wild-type Reg1 expression was studied by using a rabbit anti-mouse Reg1 antibody (gift from Catherine Figarella, Faculty of Medicine, Marseille, France) and costaining with antibodies described above except that mouse anti-pepsin and guinea pig anti-VMAT-2 (RDI, Flanders, NJ) were used. Slides were examined with a Zeiss Axioplan-2 microscope (Zeiss Vision, Welwyn Garden City, UK) and images were captured by using a JVC-3 charge-coupled device camera and KS300 software combined with a deconvolution software (Imaging Associates, Bicester, Oxfordshire, UK). Results are expressed as immunoreactive cells as a percentage of total cell number as reported earlier (28).

Statistics. Results are presented as means ± SE; comparisons were made using one-way ANOVA or Student's t-tests where appropriate and were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
H. pylori increases Reg1{alpha} expression in both human gastric corpus and antrum. Using RT-PCR and Western blotting, we first determined that both Reg1{alpha} mRNA and protein were significantly increased in human gastric corpus and antrum in H. pylori-infected patients. Plasma gastrin concentrations in the patients used for this study were within the normal range and were not significantly different from the control group. Thus, although gastrin has been previously shown to increase Reg expression, it was not considered responsible for increased Reg1{alpha} expression noted in H. pylori-infected patients (Fig. 1, A–D).


Figure 1
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  		Fig. 1. Increased gastric Reg1{alpha} expression in Helicobacter pylori (H.p.)-infected patients. A: real-time PCR normalized to 18S shows elevated Reg1{alpha} mRNA expression in both the gastric corpus and antrum in patients with H. pylori infection compared with controls (n = 10). B: plasma gastrin concentrations for the relevant groups of patients. C: quantification of Western blots normalized to GAPDH shows an increase in Reg1 in both the gastric corpus and antrum of H. pylori-infected patients compared with uninfected individuals (n = 10). D: representative Western blots of gastric corpus and antrum from infected and uninfected patients probed for Reg1 and GAPDH.

 
Both gastrin and H. felis increase Reg1 expression in a transgenic mouse model of hypergastrinemia. We next extended our findings in humans to a rodent model in which Helicobacter infection is associated with atrophic gastritis that eventually progresses to gastric cancer (28, 46). When sampled up to 9 mo after infection, i.e., in the preneoplastic phase, RT-PCR indicated that Reg1 mRNA was significantly increased in transgenic (INS-GAS) mice overexpressing gastrin, compared with wild-type (FVB/N) mice, consistent with the findings in hypergastrinemic humans (Fig. 2A). However, although serum gastrin concentrations in uninfected INS-GAS mice were significantly higher at 6 and 9 mo compared with 3 mo, there were no significant differences in relative Reg1 mRNA abundance in these animals (Fig. 2B). Nevertheless, when INS-GAS mice were infected with H. felis, Reg1 mRNA abundance was significantly increased compared with uninfected mice at both 3 and 6 mo, although there was no further increase after 9 mo (Fig. 2B). Together these data indicate that in mouse as in human, Helicobacter infection stimulates Reg1 expression; gastrin also stimulates expression up to, but not above, a plasma concentration of ~500 pM (Fig. 2B). Immunocytochemistry indicated that Reg1 in mice, as in humans, was mainly expressed in a subset of pepsin-positive cells (39.7 ± 12.6% of all Reg-positive cells), and VMAT-2-positive cells (52.4 ± 12.3% of all Reg1-positive cells) (Fig. 2, C and D), indicative of chief and ECL cells, respectively. In both cases, Reg1-immunoreactivity was localized to vesicles compatible with its status as a secretory protein.


Figure 2
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  		Fig. 2. Increased Reg1 expression in mice with hypergastrinemia and Helicobacter felis infection. A: real-time PCR normalized to 18S shows elevated Reg1 mRNA expression in the gastric corpus of INS-GAS mice compared with FVB/N mice (n = 6). There is also around threefold increase in plasma gastrin in these mice. B: real-time PCR normalized to 18S shows elevated Reg1 mRNA expression (*P < 0.05) in the gastric corpus of INS-GAS (n = 6–7) mice after H. felis infection at all time points compared with uninfected mice (n = 5–7). There is also increase in plasma gastrin in uninfected mice at 6 and 9 mo compared with the uninfected 3-mo-old mice (^). Reg1 (green) is expressed in a subset of pepsin (red)-positive cells (C) and in a subset of VMAT-2 (red)-positive cells (D) of wild-type uninfected mice. DAPI (blue) counterstains nuclei of the cells.

 
Basal expression driven by the Reg1 promoter in primary gastric epithelial cells. In pilot experiments, we examined the expression in primary gastric epithelial cells of the –2111 bp and five mutants of the –104 bp sequence of the Reg promoter described previously (4). Basal expression of the Reg-luc vectors (as indicated by immunocytochemical localization of luciferase) was identified in pepsinogen-positive (chief) and VMAT-2 positive (ECL) cells (Fig. 3, A and B) in transfected gastric glands. Expression was not identified in parietal cells although in other experiments using different vectors we have found that all the major differentiated cell types of the epithelium can be transfected by using the present protocol (43). Interestingly, there appeared to be differences between the –2111 and –104 bp sequences in their capacity to target chief and ECL cells. Thus chief cells accounted for 75–80% of cells expressing either the wild-type or –104 bp m3 constructs, whereas the –2111 construct was expressed in approximately similar proportions of chief and ECL cells (Fig. 3C). On the basis of total luciferase activity in cell extracts, there was similar basal expression of the –2111 and –104 bp constructs and in contrast to AGS cells where basal expression of a mutant of the C-rich gastrin-response element (–104 bp m3) was dramatically reduced, there was no decrease in basal expression in primary cells of this mutant (100 ± 16.7% vs. 115.7 ± 21.2%, respectively). Other mutants of the –104 bp sequence did not differ from wild type in basal expression (not shown).


Figure 3
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  		Fig. 3. Both –2111 and –104 bp Reg1 promoter sequences target expression of luciferase to chief and (ECL) cells in primary mouse gastric glands. A: –104 bp Reg1-luc (green) expression in a pepsinogen (red)-positive cell. B: –104 bp Reg1-luc (green) expression in VMAT-2 (red)-positive cells. DAPI counterstains nuclei of the cells. C: proportion (%) of luciferase-positive cells also expressing pepsinogen or VMAT-2 (n = 3).

 
Differential actions of gastrin and H. felis in stimulating Reg1 promoter activity in primary gastric epithelial cells. In response to G17 (1 nM, 8 h) there was an increase in luciferase expression of ~2.6-fold with both the –2111 and the –104 bp constructs. Importantly, however, as in AGS cells, there was no response to G17 of the –104m3 construct (Fig. 4, A and B). Other mutants of the –104 bp sequence did not differ from wild type in gastrin-stimulated expression (not shown). Expression of the –2111 bp Reg-luc also increased ~2.5-fold in response to H. felis (MOI of 100–200, 16 h). In contrast, there was no increase in –104 bp Reg-luc expression after H. felis infection, indicating that different cis-acting response elements mediate the responses to gastrin(Fig. 4, C and D).


Figure 4
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  		Fig. 4. Regulation of Reg-luc in primary gastric epithelial cells by gastrin and H. felis (H.f.). A: in gastric epithelial cells transiently transfected with Reg-luc, G17 (8 h) produces a concentration-dependent increase in luciferase activity of both the –2111 bp (n = 7). B: the wild-type (wt) –104 bp (n = 9) constructs but had no effect on the m3 mutant of the latter. C: H. felis (16 h) stimulates expression of the –2111 bp of Reg construct (n = 9) at multiplicities of infection (MOI) of 1:100 and 1:200. D: there is no stimulation of the –104 bp Reg-luc construct (n = 4) by H. felis. E: gastrin and H. felis act synergistically on the –2111 bp Reg promoter (n = 6) at concentrations that individually had no effect on Reg-luc expression. F: addition of the same combination had no effect on the –104 bp construct (n = 4).

 
To investigate whether gastrin and H. felis acted synergistically to stimulate Reg expression we used a concentration of gastrin (0.1 nM) and a MOI of H. felis (1:50) that individually had no effect on Reg-luc expression. Interestingly, subthreshold stimulation by G17 and H. felis together increased luciferase expression from the –2111 bp but not in the –104 bp Reg-luc promoter constructs, indicating that interactions between gastrin and H. felis occur within the full length of the Reg1 promoter (Fig. 4, E and F). Neither G17 nor Helicobacter treatment altered the cellular localization of the Reg-luc constructs (data not shown).

A rodent-adapted H. pylori strain stimulates both the –2111 and the –104 bp Reg1 promoter activity in primary epithelial cells. H. felis colonizes the mouse stomach readily and induces similar gastric pathology to H. pylori but does not contain the cag pathogenicity island or the VacA cytotoxin. Next, therefore, we examined Reg-luc expression in response to a rodent adapted H. pylori wild-type strain (7.13) that is capable of colonizing and infecting mouse stomach and its isogenic mutants, lacking cagA, cagE, and vacA. The rodent-adapted H. pylori wild-type strain increased expression of the –2111 bp Reg-luc reporter construct but, unlike H. felis, also stimulated –104 bp Reg-luc expression (Fig. 5, A and B). The cag pathogenicity island plays a role in the H. pylori mediated increased Reg-luc expression since the cagA– mutant was significantly less active than the wild type or the vacA cytotoxin-deficient mutant in stimulating both –2111 and –104 bp Reg-luc (Fig. 5B).


Figure 5
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  		Fig. 5. A rodent-adapted H. pylori strain stimulates both –2111 and –104 bp Reg1-luc promoters partly by cagA and targets a distinct promoter element to gastrin. A: the rodent-adapted H. pylori strain, 7.13, stimulates both the –2111 bp and –104 bp Reg1-luc. –2111 bp Reg1-luc promoter (B) and –104 bp Reg1-luc promoter (C) exhibit decreased responses after deletion of cagA but not cagE and vacA compared with the wt (n = 6–8). D: mutation within –98 to –92 (m1) of the –104 bp Reg1 promoter construct virtually abolishes luciferase responses to H. pylori (n = 6–12); other mutations (m2–m5) have no effect.

 
H. pylori and gastrin act on distinct response elements within the 104 bp Reg1 promoter. Finally we examined the effect of H. pylori on expression of a variety of mutants of 104 bp Reg1 as previously described (4). In contrast to gastrin, H. pylori stimulated expression of the C-rich mutant of the –104 bp Reg-luc (m3) promoter. Moreover, H. pylori also stimulated expression of three other 104 bp mutants. However, mutation of the sequence –98 to –93 (m1) yielded a construct that was virtually insensitive to H. pylori (Fig. 5C) but, importantly, exhibited a response to gastrin that was comparable (2.6-fold) to the wild-type sequence.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study shows that the putative gastric growth factor Reg1 is increased by Helicobacter infection in both human patients and in a mouse model leading to gastric cancer. Previous work established that gastrin stimulates Reg expression and identified an important regulatory element in the promoter region (4, 19). The dose-response relationship between gastrin and Reg expression is not straightforward. Although there was an increase in plasma gastrin concentration with age (6 and 9 mo) in uninfected mice, there was no increase in Reg expression at either time point, suggesting that after a threshold plasma gastrin concentration there is no further role for gastrin in regulating Reg expression. Moreover, increased Reg expression was seen with H. felis infection (3 mo) prior to additional increase in plasma gastrin.

The present data suggest that H. pylori infection in patients is able to increase Reg expression independently of gastrin. Moreover, we show that gastrin and H. pylori stimulate expression of Reg through distinct regulatory elements in the promoter. For the first time, we also show that a minimal sequence of the Reg promoter of just 104 bp is able to selectively target expression to the same cell types (chief and ECL cells) that express the endogenous protein. Interestingly, we found that luciferase protein was localized to vesicles in chief and ECL cells, which we attribute to a cryptic signal peptide in the amino-terminal sequence of luciferase.

The finding of increased gastric Reg1 with Helicobacter infection extends previous studies showing that members of the Reg family in stomach are increased by stress (3), injury (22), inflammation (13, 49), and cancer (7, 34). However, the relevant cellular and molecular mechanisms have not been established and, insofar as transcriptional mechanisms have been examined, it has been at the level of gastric cancer cell lines (4). We have developed protocols for transfection of primary gastric cells to facilitate physiological studies of more biologically relevant mechanisms. In this context, it is important to note that both –2111 and –104 bp luciferase vectors were expressed in cell types normally associated with expression of wild-type Reg. It appears then that a sequence within the proximal –104 bp of the Reg1 promoter is sufficient for cell restricted expression to ECL and chief cells. However, whereas basal expression of a mutant of the gastrin-response element (–79 to –76 relative to the transcriptional start site) was substantially reduced in AGS cells compared with wild-type sequence (4), in primary cells basal expression of the wild type and mutants were similar so that care is need in interpreting the physiological significance of studies in cancer cell lines. The precise regulatory elements that drive cell-restricted expression of the Reg gene are still uncertain, and further work will be needed to elucidate them.

The present data indicate that distinct promoter elements are required for H. pylori- and H. felis-stimulated Reg1 expression and that gastrin stimulates expression from a site different to H. pylori. In the case of H. pylori, CagA appears to contribute to stimulation of Reg1 expression from the –104 bp promoter, and the fact that this virulence constituent is absent in H. felis presumably accounts for the failure of the latter to activate the minimal promoter. Cytosolic injection of CagA via a type IV secretory system leads to its tyrosine phosphorylation and stimulation of a number of intracellular signaling pathways including activation of SHP-2 phosphatase, the COOH-terminal Src kinase, activation of c-Met and EGF receptors, and reduced focal adhesion kinase phosphorylation (17, 18, 23, 42). Further work will be needed to dissect the precise signaling pathways leading to H. pylori control of Reg expression. However, notwithstanding the similar phenotypes and cellular responses to H. pylori and H. felis in vivo, the present data strongly indicate that at a cellular and molecular level it cannot be assumed that the two are activating the same mechanisms.

The sequence –98 to –93 of the Reg promoter has not previously been identified as a regulatory motif. In pancreatic beta-cells, the increased Reg expression that occurs with partial pancreatectomy has been attributed to poly(ADP-ribose) polymerase binding to a sequence similar to the gastrin-response element (2), and in AGS cells gastrin drives expression via Sp-family transcription factors binding to this sequence. Even though there have been many other studies demonstrating the remarkable capacity for increased expression of Reg in response to injury, acute stress, inflammation (37), and cancer (see above for references), the relevant transcriptional mechanisms have not been examined. The H. pylori response element identified here is also well conserved between species, and further studies on this element are likely to be rewarding in understanding cellular responses to infection and damage.

An emerging body of evidence suggests that induction of Reg proteins are part of the response to tissue damage and infection in many organs and that they function both as growth factors and as components of the innate immune system (5, 21). Our data suggest that there are likely to be multiple converging pathways on the proximal promoter sequence that regulate expression in a cell type-restricted manner in the gastric epithelium. Progression to cancer, through extensive epithelial remodeling, is a clinically important consequence of H. pylori infection, particularly with CagA-positive strains. The association of H. pylori with a putative growth factor that is upregulated early in infection and persists thereafter in the progression to neoplasia provides an attractive target for future studies in the elucidation of oncogenic mechanisms


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work is funded by The Medical Research Council.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Varro, Physiological Laboratory, School of Biomedical Sciences, Univ. of Liverpool, Crown St., Liverpool L69 3BX, UK (e-mail: avarro{at}liverpool.ac.uk)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Abe M, Nata K, Akiyama T, Shervani NJ, Kobayashi S, Tomioka-Kumagai T, Ito S, Takasawa S, Okamoto H. Identification of a novel Reg family gene, Reg IIIdelta, and mapping of all three types of Reg family gene in a 75 kilobase mouse genomic region. Gene 246: 111–122, 2000.[CrossRef][ISI][Medline]
  2. Akiyama T, Takasawa S, Nata K, Kobayashi S, Abe M, Shervani NJ, Ikeda T, Nakagawa K, Unno M, Matsuno S, Okamoto H. Activation of Reg gene, a gene for insulin-producing beta –cell regeneration: poly(ADP-ribose) polymerase binds Reg promoter and regulates the transcription by autopoly(ADP-ribosyl)ation. Proc Natl Acad Sci USA 98: 48–53, 2001.[Abstract/Free Full Text]
  3. Asahara M, Mushiake S, Shimada S, Fukui H, Kinoshita Y, Kawanami C, Watanabe T, Tanaka S, Ichikawa A, Uchiyama Y, Narushima Y, Takasawa S, Okamoto H, Tohyama M, Chiba T. Reg gene expression is increased in rat gastric enterochromaffin-like cells following water immersion stress. Gastroenterology 111: 45–55, 1996.[CrossRef][ISI][Medline]
  4. Ashcroft FJ, Varro A, Dimaline R, Dockray GJ. Control of expression of the lectin-like protein Reg-1 by gastrin: role of the Rho family GTPase RhoA and a C-rich promoter element. Biochem J 381: 397–403, 2004.[CrossRef][ISI][Medline]
  5. Cash HL, Whitham CV, Behrendt CL, Hooper LV. Symbiotic bacteria direct expression of an intestinal bactericidal lectin. Science 313: 1126–1130, 2006.[Abstract/Free Full Text]
  6. De Caro AM, Bonicel JJ, Rouimi P, De Caro JD, Sarles H, Rovery M. Complete amino acid sequence of an immunoreactive form of human pancreatic stone protein isolated from pancreatic juice. Eur J Biochem 168: 201–207, 1987.[ISI][Medline]
  7. Dhar DK, Udagawa J, Ishihara S, Otani H, Kinoshita Y, Takasawa S, Okamoto H, Kubota H, Fujii T, Tachibana M, Nagasue N. Expression of regenerating gene I in gastric adenocarcinomas: correlation with tumor differentiation status and patient survival. Cancer 100: 1130–1136, 2004.[CrossRef][ISI][Medline]
  8. Dieckgraefe BK, Crimmins DL, Landt V, Houchen C, Anant S, Porche-Sorbet R, Ladenson JH. Expression of the regenerating gene family in inflammatory bowel disease mucosa: Reg Ialpha upregulation, processing, and antiapoptotic activity. J Investig Med 50: 421–434, 2002.[ISI][Medline]
  9. Dockray G, Dimaline R, Varro A. Gastrin: old hormone, new functions. Pflügers Arch 449: 344–355, 2005.[CrossRef][ISI][Medline]
  10. Dockray GJ, Hamer C, Evans D, Varro A, Dimaline R. The secretory kinetics of the G cell in omeprazole-treated rats. Gastroenterology 100: 1187–1194, 1991.[ISI][Medline]
  11. Dockray GJ, Varro A, Dimaline R, Wang T. The gastrins: their production and biological activities. Annu Rev Physiol 63: 119–139, 2001.[CrossRef][ISI][Medline]
  12. Franco AT, Israel DA, Washington MK, Krishna U, Fox JG, Rogers AB, Neish AS, Collier-Hyams L, Perez-Perez GI, Hatakeyama M, Whitehead R, Gaus K, O'Brien DP, Romero-Gallo J, Peek RM Jr. Activation of beta-catenin by carcinogenic Helicobacter pylori. Proc Natl Acad Sci USA 102: 10646–10651, 2005.[Abstract/Free Full Text]
  13. Franic TV, Judd LM, Nguyen NV, Samuelson LC, Loveland KL, Giraud AS, Gleeson PA, van Driel IR. Growth factors associated with gastric mucosal hypertrophy in autoimmune gastritis. Am J Physiol Gastrointest Liver Physiol 287: G910–G918, 2004.[Abstract/Free Full Text]
  14. Fukui H, Kinoshita Y, Maekawa T, Okada A, Waki S, Hassan S, Okamoto H, Chiba T. Regenerating gene protein may mediate gastric mucosal proliferation induced by hypergastrinemia in rats. Gastroenterology 115: 1483–1493, 1998.[CrossRef][ISI][Medline]
  15. Gross J, Carlson RI, Brauer AW, Margolies MN, Warshaw AL, Wands JR. Isolation, characterization, and distribution of an unusual pancreatic human secretory protein. J Clin Invest 76: 2115–2126, 1985.[ISI][Medline]
  16. Hemers E, Duval C, McCaig C, Handley M, Dockray GJ, Varro A. Insulin-like growth factor binding protein-5 is a target of matrix metalloproteinase-7: implications for epithelial-mesenchymal signaling. Cancer Res 65: 7363–7369, 2005.[Abstract/Free Full Text]
  17. Higashi H, Tsutsumi R, Fujita A, Yamazaki S, Asaka M, Azuma T, Hatakeyama M. Biological activity of the Helicobacter pylori virulence factor CagA is determined by variation in the tyrosine phosphorylation sites. Proc Natl Acad Sci USA 99: 14428–14433, 2002.[Abstract/Free Full Text]
  18. Higashi H, Tsutsumi R, Muto S, Sugiyama T, Azuma T, Asaka M, Hatakeyama M. SHP-2 tyrosine phosphatase as an intracellular target of Helicobacter pylori CagA protein. Science 295: 683–686, 2002.[Abstract/Free Full Text]
  19. Higham AD, Bishop LA, Dimaline R, Blackmore CG, Dobbins AC, Varro A, Thompson DG, Dockray GJ. Mutations of RegIalpha are associated with enterochromaffin-like cell tumor development in patients with hypergastrinemia. Gastroenterology 116: 1310–1318, 1999.[CrossRef][ISI][Medline]
  20. Hussain I, Bate GW, Henry J, Djali P, Dimaline R, Dockray GJ, Varro A. Modulation of gastrin processing by vesicular monoamine transporter type 1 (VMAT1) in rat gastrin cells. J Physiol 517: 495–505, 1999.[Abstract/Free Full Text]
  21. Kadowaki Y, Ishihara S, Miyaoka Y, Rumi MA, Sato H, Kazumori H, Adachi K, Takasawa S, Okamoto H, Chiba T, Kinoshita Y. Reg protein is overexpressed in gastric cancer cells, where it activates a signal transduction pathway that converges on ERK1/2 to stimulate growth. FEBS Lett 530: 59–64, 2002.[CrossRef][ISI][Medline]
  22. Kazumori H, Ishihara S, Hoshino E, Kawashima K, Moriyama N, Suetsugu H, Sato H, Adachi K, Fukuda R, Watanabe M, Takasawa S, Okamoto H, Fukui H, Chiba T, Kinoshita Y. Neutrophil chemoattractant 2 beta regulates expression of the Reg gene in injured gastric mucosa in rats. Gastroenterology 119: 1610–1622, 2000.[CrossRef][ISI][Medline]
  23. Keates S, Sougioultzis S, Keates AC, Zhao D, Peek RM Jr, Shaw LM, Kelly CP. cag+ Helicobacter pylori induce transactivation of the epidermal growth factor receptor in AGS gastric epithelial cells. J Biol Chem 276: 48127–48134, 2001.[Abstract/Free Full Text]
  24. Kiji T, Dohi Y, Takasawa S, Okamoto H, Nonomura A, Taniguchi S. Activation of regenerating gene Reg in rat and human hearts in response to acute stress. Am J Physiol Heart Circ Physiol 289: H277–H284, 2005.[Abstract/Free Full Text]
  25. Lawrance IC, Fiocchi C, Chakravarti S. Ulcerative colitis and Crohn's disease: distinctive gene expression profiles and novel susceptibility candidate genes. Hum Mol Genet 10: 445–456, 2001.[Abstract/Free Full Text]
  26. Levi S, Beardshall K, Haddad G, Playford R, Ghosh P, Calam J. Campylobacter pylori and duodenal ulcers: the gastrin link. Lancet 1: 1167–1168, 1989.[ISI][Medline]
  27. Livesey FJ, O'Brien JA, Li M, Smith AG, Murphy LJ, Hunt SP. A Schwann cell mitogen accompanying regeneration of motor neurons. Nature 390: 614–618, 1997.[CrossRef][Medline]
  28. McCaig C, Duval C, Hemers E, Steele I, Pritchard DM, Przemeck S, Dimaline R, Ahmed S, Bodger K, Kerrigan DD, Wang TC, Dockray GJ, Varro A. The role of matrix metalloproteinase-7 in redefining the gastric microenvironment in response to Helicobacter pylori. Gastroenterology 130: 1754–1763, 2006.[CrossRef][ISI][Medline]
  29. Miyaoka Y, Kadowaki Y, Ishihara S, Ose T, Fukuhara H, Kazumori H, Takasawa S, Okamoto H, Chiba T, Kinoshita Y. Transgenic overexpression of Reg protein caused gastric cell proliferation and differentiation along parietal cell and chief cell lineages. Oncogene 23: 3572–3579, 2004.[CrossRef][ISI][Medline]
  30. Miyashita H, Nakagawara K, Mori M, Narushima Y, Noguchi N, Moriizumi S, Takasawa S, Yonekura H, Takeuchi T, Okamoto H. Human REG family genes are tandemly ordered in a 95-kilobase region of chromosome 2p12. FEBS Lett 377: 429–433, 1995.[CrossRef][ISI][Medline]
  31. Namikawa K, Fukushima M, Murakami K, Suzuki A, Takasawa S, Okamoto H, Kiyama H. Expression of Reg/PAP family members during motor nerve regeneration in rat. Biochem Biophys Res Commun 332: 126–134, 2005.[CrossRef][ISI][Medline]
  32. Noble PJ, Wilde G, White MR, Pennington SR, Dockray GJ, Varro A. Stimulation of gastrin-CCKB receptor promotes migration of gastric AGS cells via multiple paracrine pathways. Am J Physiol Gastrointest Liver Physiol 284: G75–G84, 2003.[Abstract/Free Full Text]
  33. Ohning GV, Wong HC, Lloyd KC, Walsh JH. Gastrin mediates the gastric mucosal proliferative response to feeding. Am J Physiol Gastrointest Liver Physiol 271: G470–G476, 1996.[Abstract/Free Full Text]
  34. Oue N, Mitani Y, Aung PP, Sakakura C, Takeshima Y, Kaneko M, Noguchi T, Nakayama H, Yasui W. Expression and localization of Reg IV in human neoplastic and non-neoplastic tissues: Reg IV expression is associated with intestinal and neuroendocrine differentiation in gastric adenocarcinoma. J Pathol 207: 185–198, 2005.[CrossRef][ISI][Medline]
  35. Patthy L. Homology of human pancreatic stone protein with animal lectins. Biochem J 253: 309–311, 1988.[ISI][Medline]
  36. Peek RM Jr, Blaser MJ. Helicobacter pylori and gastrointestinal tract adenocarcinomas. Nat Rev Cancer 2: 28–37, 2002.[CrossRef][ISI][Medline]
  37. Resnick MB, Sabo E, Meitner PA, Kim SS, Cho Y, Kim HK, Tavares R, Moss SF. Global analysis of the human gastric epithelial transcriptome altered by Helicobacter pylori eradication in vivo. Gut 55: 1717–1724, 2006.[Abstract/Free Full Text]
  38. Sekikawa A, Fukui H, Fujii S, Nanakin A, Kanda N, Uenoyama Y, Sawabu T, Hisatsune H, Kusaka T, Ueno S, Nakase H, Seno H, Fujimori T, Chiba T. Possible role of REG Ialpha protein in ulcerative colitis and colitic cancer. Gut 54: 1437–1444, 2005.[Abstract/Free Full Text]
  39. Stephanova E, Tissir F, Dusetti N, Iovanna J, Szpirer J, Szpirer C. The rat genes encoding the pancreatitis-associated proteins I, II and III (Pap1, Pap2, Pap3), and the lithostathin/pancreatic stone protein/regeneration protein (Reg) colocalize at 4q33–>q34. Cytogenet Cell Genet 72: 83–85, 1996.[ISI][Medline]
  40. Takaishi S, Wang TC. Gene expression profiling in a mouse model of Helicobacter-induced gastric cancer. Cancer Sci 98: 284–293, 2007.[CrossRef][Medline]
  41. Terazono K, Yamamoto H, Takasawa S, Shiga K, Yonemura Y, Tochino Y, Okamoto H. A novel gene activated in regenerating islets. J Biol Chem 263: 2111–2114, 1988.[Abstract/Free Full Text]
  42. Tsutsumi R, Takahashi A, Azuma T, Higashi H, Hatakeyama M. Focal adhesion kinase is a substrate and downstream effector of SHP-2 complexed with Helicobacter pylori CagA. Mol Cell Biol 26: 261–276, 2006.[Abstract/Free Full Text]
  43. Varro A, Kenny S, Hemers E, McCaig C, Przemeck S, Wang TC, Bodger K, Pritchard DM. Increased gastric expression of MMP-7 in hypergastrinemia and significance for epithelial-mesenchymal signaling. Am J Physiol Gastrointest Liver Physiol 292: G1133–G1140, 2007.[Abstract/Free Full Text]
  44. Varro A, Noble PJ, Pritchard DM, Kennedy S, Hart CA, Dimaline R, Dockray GJ. Helicobacter pylori induces plasminogen activator inhibitor 2 (PAI-2) in gastric epithelial cells through NF-kB and RhoA: implications for invasion and apoptosis. Cancer Res 64: 1695–1702, 2004.[Abstract/Free Full Text]
  45. Walsh JH. Gastrin. In: Gut Peptides, edited by Walsh JH and Dockray GJ. New York: Raven, 1994, p. 75–121.
  46. Wang TC, Dangler CA, Chen D, Goldenring JR, Koh T, Raychowdhury R, Coffey RJ, Ito S, Varro A, Dockray GJ, Fox JG. Synergistic interaction between hypergastrinemia and Helicobacter infection in a mouse model of gastric cancer. Gastroenterology 118: 36–47, 2000.[CrossRef][ISI][Medline]
  47. Wang TC, Koh TJ, Varro A, Cahill RJ, Dangler CA, Fox JG, Dockray GJ. Processing and proliferative effects of human progastrin in transgenic mice. J Clin Invest 98: 1918–1929, 1996.[ISI][Medline]
  48. Wroblewski LE, Noble PJ, Pagliocca A, Pritchard DM, Hart CA, Campbell F, Dodson AR, Dockray GJ, Varro A. Stimulation of MMP-7 (matrilysin) by Helicobacter pylori in human gastric epithelial cells: role in epithelial cell migration. J Cell Sci 116: 3017–3026, 2003.[Abstract/Free Full Text]
  49. Yoshino N, Ishihara S, Rumi MA, Ortega-Cava CF, Yuki T, Kazumori H, Takazawa S, Okamoto H, Kadowaki Y, Kinoshita Y. Interleukin-8 regulates expression of Reg protein in Helicobacter pylori-infected gastric mucosa. Am J Gastroenterol 100: 2157–2166, 2005.[CrossRef][ISI][Medline]



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S. Kenny, C. Duval, S. J. Sammut, I. Steele, D. M. Pritchard, J. C. Atherton, R. H. Argent, R. Dimaline, G. J. Dockray, and A. Varro
Increased expression of the urokinase plasminogen activator system by Helicobacter pylori in gastric epithelial cells
Am J Physiol Gastrointest Liver Physiol, September 1, 2008; 295(3): G431 - G441.
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